Solvent-free synthesis, DNA-topoisomerase II activity and molecular docking study of new asymmetrically N,N′-substituted ureas

Article abstract of DOI:10.3390/molecules171112882

A new series of asymmetrically N,N′-substituted ureas 20-25 was prepared using solvent free conditions, which is an eco-friendly methodology, starting with Schiff bases derived from cinnamaldehyde and p-substituted anilines, which are subsequently submitt

Full text of DOI:10.3390/molecules171112882

Molecules 2012, 17, 12882-12894; doi:10.3390/molecules171112882
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Solvent-Free Synthesis, DNA-Topoisomerase II Activity
and Molecular Docking Study of New Asymmetrically
N,N'-Substituted Ureas
Andressa Esteves-Souza, Claudio E. Rodrigues-Santos, Catarina de Nigris Del Cistia,
Daniel Rosa da Silva, Carlos Maurício R. Sant’Anna and Aurea Echevarria *
Departamento de Química, Instituto de Ciências Exatas, Universidade Federal Rural do Rio de Janeiro,
23890-000, Seropédica, Rio de Janeiro, Brazil
* Author to whom correspondence should be addressed; E-Mail: echevarr@ufrrj.br;
Tel./Fax: +55-21-2682-1201.
Received: 28 August 2012; in revised form: 26 September 2012 / Accepted: 26 October 2012 /
Published: 1 November 2012
Abstract: A new series of asymmetrically N,N'-substituted ureas 20–25 was prepared
using solvent free conditions, which is an eco-friendly methodology, starting with Schiff
bases derived from cinnamaldehyde and p-substituted anilines, which are subsequently
submitted to reduction reactions that afford the corresponding asymmetric secondary
amines. All of the intermediates were prepared using solvent free reactions, which were
compared to traditional methodologies. All of the reactions required a remarkably short
amount of time and provided good yields when solvent free conditions were employed
compared to other methodologies. The DNA-topoisomerase II-α (topo II-α) activity was
evaluated in relaxation assays, which showed that all of the compounds inhibited the
enzyme activity at 10 μM, except for urea 24. Furthermore, a molecular docking study
indicated that the compounds 20–25 binding to the topo II-α are able to interact with the
same binding site as the anticancer drug etoposide, suggesting that the ureas could inhibit
the enzyme by the same mechanism of action observed for etoposide, which prevents
re-ligation of the DNA strands.
Keywords: ureas; solvent-free synthesis; DNA-topoisomerase; molecular docking

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1. Introduction
Recently, chemists have become interested in developing methodologies for solvent-free organic
reactions to minimise the environmental damage due to the disposal of organic solvents, to reduce reaction
times, to increase reactions yields, to reduce costs and to simplify the experimental procedures [1,2]. A
solvent-free or solid-state reaction can be performed using the reactants alone or by incorporating them
into solid supports. This methodology can also be performed using thermal processes as well as
microwave or ultrasound irradiation. Often, the products of solid state reactions may be different from
those obtained by reactions in the presence of solvent, due to specific spatial orientation or packing of
the reacting molecules in the crystalline state. The interaction of reactants is obtained by simple mixing
of the components, allowed to stay at room temperature or heated carefully in an oil bath or exposed to
appropriate radiation until the reaction is complete [3,4].
Ureas are well known to be an important class of compounds with a wide range of biological
activities, such as antiviral [5,6], antitumor [7,8] and antimicrobial [9,10] activities. Due to the
importance of these properties, it is important to synthesise new asymmetrically N,N'-substituted ureas,
preferably under eco-friendly conditions.
The DNA-topoisomerases are ubiquitous nuclear enzymes that play crucial roles in DNA
metabolism events, such as replication and transcription [11]. Because of the crucial role of
topoisomerases in the maintenance and replication of DNA during proliferation, cells become highly
vulnerable when these functions are lost [12].
Our research group has been working on more efficient and cleaner synthetic methods using
microwave irradiation and solvent-free conditions [13–16]. To extend our investigation, we report the
synthesis of a new series of asymmetrically N,N'-substituted ureas using cinnamaldehyde-derived
Schiff bases, which are subsequently reduced and reacted with suitable isocyanates. All the steps were
executed under solvent-free conditions with good yields being obtained after short reaction times,
which represents a good example of an eco-friendly methodology. Furthermore, the DNA-topoisomerase II
inhibitory effect of the final products was evaluated using a relaxation assay, and a molecular docking
study was implemented to identify the molecular cause of the observed enzyme inhibition profile.
2. Results and Discussion
2.1. Synthesis and Spectroscopic Characterisation
A new series of asymmetrically N,N'-substituted ureas 20–25 was synthesised using solvent free
reactions. The synthetic procedure was performed in three steps starting with Schiff bases derived from
cinnamaldehyde with subsequent reduction reactions affording the corresponding allyl amines, which
was followed by coupling with phenyl isocyanate, as outlined in Scheme 1. The syntheses of all of the
compounds, Schiff bases, corresponding amines and ureas were performed under solvent free conditions.
First, the Schiff bases were prepared from cinnamaldehyde (1) and 4-substituted-anilines 2–7,
which were mixed using a mortar and pestle for 15 min. The products 8–13 were obtained in good
yield and were recrystallised from ethanol. The Schiff bases were also prepared with the same
reactants, silica gel and ethanol as solvent using an ultrasonic bath for 20 min. The products were
obtained after silica gel filtration, solvent evaporation and recrystallisation from ethanol.

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Table 1 shows the yields and the corresponding melting points of the Schiff bases prepared under both
reaction conditions.
Scheme 1. Synthesis of asymmetrically N,N'-substituted ureas.
O
NH2
i
N
X
H
+
X
1
2-7
8-13
ii
X
O
N
H
iii
N
X
N
H
14-19
X= H (20), CH3 (21), OCH3 (22), Cl (23), Br (24), F (25)
20-25
Reactions Conditions: (i): ethanol and silica gel, 20 min. (ultrasonic method) or reactants mixture
without silica gel, 15 min (solvent-free method); (ii): NaBH4, ethanol, reflux 8–10 h (traditional
method) or NaBH4/H3BO3 (1:1), 30 min. (solvent free method); (iii): toluene anhydrous,
phenylisocyanate, reflux 4–24 h (traditional method) or phenyl isocyanate (solvent-free method),
30–45 min.
Table 1. Yields and melting points of the Schiff bases (8–13) obtained in solvent free
conditions and under ultrasonic irradiation.
Ultrasonic irradiation
Solvent free condition
Schiff base
X
MP (°C)
Yield (%) 20 min
Yield (%) 15 min
8
H
4-CH3
4-OCH3
4-Cl
107 a
77 b
120–121 c
104–106 d
120–121 d
69–71 d
70
70
85
90
90
75
100
98
9
10
11
12
13
100
97
4-Br
100
4-F
98
a Lit. 107–109 °C [17]; b lit. 76–77 °C [18]; c lit. 119–121 °C [17]; d lit. 105 °C [19]; d lit. 121 °C [19].
The allyl amines 14–19 were obtained by reduction of the Schiff bases using NaBH4 as the reducing
agent via two different methodologies. Under solvent free conditions, the reduction products were
obtained by vigorous mixing of the Schiff bases with NaBH4 and H3BO3 in a mortar and pestle for
30 min. Next, the solids were washed with a NaHCO3 solution, extracted with CH2Cl2 and dried under
vacuum affording the corresponding allyl amines in quantitative yields. The second methodology
involved equimolar quantities of the Schiff bases and NaBH4 with refluxing for 8 to 10 h in ethanol,

Molecules 2012, 17
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which was then evaporated under vacuum. Then, the allyl amines were extracted using the method
described above to afford the products in good yields (45–75%). Table 2 shows the yields obtained for
the allyl amines via both methodologies.
Table 2. Allyl-amine 8–12 yields obtained under solvent free conditions and with the reflux method.
Reflux procedure
Solvent free conditions
Allyl amine
X
MP (°C)
Yield (%) 8–10 h
Yield (%) 30 min
14
15
16
17
18
19
H
4-CH3
4-OCH3
4-Cl
Oil a
Oil b
67 c
60
45
55
72
75
70
96
99
99
98
98
99
83 c
4-Br
86–87 c
60 c
a Lit. [20]; b lit. [21]; c lit. [22].
4-F
The asymmetrically N,N'-substituted ureas 20–25 were synthesised from allyl amines 14–19 in the
presence of phenyl isocyanate via two different methods. The traditional methodology afforded the
target compounds after refluxing for 4 to 24 h in toluene. Under solvent free conditions, the reactants
were mixed vigorously for 30–45 min with a mortar and pestle. The solid products were recrystallised
from ethanol, and the oil products were purified by column chromatography using CHCl3 as the
solvent. The ureas were obtained in excellent yield (98–100%) after remarkably low reaction times
(30 min) compared to those obtained with traditional methodology (25–64% and 4–24 h). Table 3
shows the yields for the asymmetrically N,N'-substituted ureas obtained from both methodologies.
Table 3. Asymmetrically N,N'-substituted urea (20–25) yields obtained in solvent free
conditions and by the traditional reflux method.
Reflux procedure
Solvent free conditions
Urea
X
MP (°C)
Time (h)
Yield (%)
Yield (%) 30 min
20
21
22
23
24
25
H
4-CH3
4-OCH3
4-Cl
oil
65–68
oil
26
30
40
64
30
25
4
4
>99
>99
98
24
4
98–100
oil
>99
>99
>99
4-Br
4
4-F
75–78
4
The formation of the ureas 20–25 was observed via infrared spectra. The C=O stretching vibration
observed at 1654 to 1674 cm−1 (Experimental section) indicates the formation of the products because
the secondary amines (14–19) do not absorb in this region. These values are in good agreement with
the literature values (1623–1647 cm−1) [8].
1
The H-NMR spectra of compounds 20–25 was employed to identify the three aromatic rings.
13
However, other chemical shifts did not change compared to the amines 14–19. The C-NMR spectra
were of fundamental importance in the characterisation because the chemical shifts of the downfield
signals with a δ of 154.29 to 154.43 could be attributed to the carbon atom of the urea carbonyl groups
(Experimental section).

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2.2. DNA-Topoisomerase Assays
The ability of compounds 20–25 to inhibit human DNA topoisomerase II-α (topo II-α) activity was
observed in relaxation assays using supercoiled pBR322 plasmid DNA in the presence of ATP. The
reaction products were analysed by electrophoretic mobility and revealed in ethidium bromide in the
presence of UV light. As shown in Figure 1, all compounds inhibited the enzyme activity at 10 μM;
however the urea 24 presented a weak supercoilled DNA band indicating a smaller inhibitory effect.
Etoposide, a known topo II-α inhibitor, was used as a positive control at 100 μM.
Figure 1. Effect of ureas on topo II-α Line 1: Etoposide (100 μM) + pBR322;
line 2: pBR322 only; line 3: pBR322 + topo II-α; line 4: pBR322 + topo II-α + 23 (10 μM);
line 5: pBR322 + topo II-α + 24 (10 μM); line 6: pBR322 + topo II-α + 21 (10 μM);
line 7: pBR322 + topo II-α + 22 (10 μM); line 8: pBR322 + topo II-α + 20 (10 μM);
line 9: pBR322 + topo II-α + 25 (10 μM).
2.3. Molecular Docking Studies
To analyse the molecular details of the mode of interaction with topo II-α, compounds 20–25 were
docked into the enzyme’s DNA and ATP binding sites. Initially, all of the scoring functions available
in the GOLD 5.1 program (CCDC Software Ltd., Cambridge, UK) were tested through a redocking
procedure with a co-crystallised ligand (i.e., etoposide and phosphoaminophosphonic acid-adenylate
ester, an ATP analogue, the PDB codes for the crystallographic structures are 3QX3 and 1ZXM,
respectively). Although all of the available scoring functions were able to dock both ligands into their
respective binding sites, the best results were obtained with the ChemPLP function [23,24], which
resulted in etoposide and the ATP analogue exhibiting the lowest RMSD values (i.e., 0.250 Å and
0.645 Å, respectively) compared to the crystal structures.
Several poses were obtained for each urea with ChemPLP, and the best-ranked pose for each one
was chosen for analysis of the interactions with the enzyme. In the GOLD docking program, the
docking functions yield fitness scores, which are dimensionless values. In each case, the scale of the
score is a guide of how good the docking pose is with a higher score indicating a better docking result.
The fitness scores for each ligand in topo II-α are presented in Table 4.

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Table 4. Asymmetrically N,N'-substituted urea (20–25), etoposide (ETP) and ATP
analogue docking results (ChemPLP scoring function) with the topo II-α DNA and ATP
binding sites.
Fitness score into DNA
Fitness score into ATP
Compound
X
binding site
79.54
83.94
84.29
82.17
80.10
81.39
111.26
-
binding site
64.65
71.29
69.08
67.01
66.90
69.81
-
20
H
4-CH3
4-OCH3
4-Cl
4-Br
4-F
21
22
23
24
25
ETP
-
ATP analogous
-
143.37
All of the tested ureas were predicted to successfully dock in both binding sites of topo II-α, but the
score was higher for the DNA binding site, indicating that this site was the most probable interaction
site for these compounds. The differences observed between the scores from the ureas and the ATP
analogue also indicated that it would be more difficult for these compounds to effectively compete
with ATP by its binding site. In fact, the ATP analogue established a number of interactions that the
ureas were not able to establish. Therefore, the docking results suggest that the DNA site in topo II-α is
the more probable binding site for this type of molecule, which results in isosteric inhibition.
Etoposide is known to prevent re-ligation of the DNA strands, and by doing so causes DNA strands
to break. Analysis of the co-crystal structure of a fragment of human topoisomerase complexed to
DNA and to the anticancer drug etoposide (PDB code 3QX3) revealed that the inhibitor forms H bonds
with Asp479 residue and also with a guanine fragment (DG13) of DNA; the inhibitor’s aglicone
moiety is inserted between DNA base pairs. Compound 22 was predicted as the best urea ligand for the
DNA binding site; although not all interactions observed for etoposide could be matched by compound
22 in the best-ranked docking pose, its overall structure is able to occupy at least partially the same
subsites occupied by the co-crystallized ligand molecule. As presented in Figure 2, compound 22 is
inserted between the same base pairs as etoposide, which is expected to abolish stacking interactions,
effectively blocking religation of the cleaved phosphodiester bond. The major difference is observed in
the etoposide’s glycosidic group subsite, but this group is not involved in drug-DNA interactions. The
4-hydroxy-3,5-dimethoxyphenyl and the fused four-ring system of the co-crystallized ligand are
mimicked by the methoxy-phenyl and the N-[(2E)-3-phenylprop-2-en-1-yl]urea groups, respectively,
of compound 22, so a mechanism of action similar to that of etoposide can be proposed for topo II-α
inhibition by the urea ligands.

Molecules 2012, 17
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Figure 2. superposition of experimentally observed etoposide (carbon atoms in yellow)
interactions and compound 22 (carbon atoms in cyan) docking interactions into the DNA
(carbon atoms in green) binding site of topo II-α (carbon atoms in white); H atoms were
removed to improve clarity (figure generated with PyMOL software).
3. Experimental
3.1. General
The 1H- and 13C-NMR spectra were obtained on a Bruker Avance II 400 spectrometer (1H, 400 MHz;
13C, 100 MHz) using tetramethylsilane (TMS) as the internal standard and CDCl3 as the solvent, and
1
the abbreviations to indicate the multiplicity of H-NMR spectra signals were: s (singlet), sl (singlet
large), d (doublet), dd (double doublet), t (triplet) and m (multiplet). The melting points were
determined with a Meltemp II apparatus and were uncorrected. The infrared spectra (KBr pellets) were
recorded on a Bruker Vertex 70 spectrophotometer. The ultrasonic-assisted reactions were performed
in a Unique ultracleaner bath, model 1400A.
3.2. Procedure for the Preparation of N-(p-X-Phenyl)-N-[(E, 2E)-3-phenyl-2-propenylidene]imines 8–13
3.2.1. Ultrasonic-Assisted Method Using Ethanol as the Solvent
The Schiff bases 8–13 were prepared according to previously published methods [25]. The
cinnamaldehyde (3.7 mmol), 4-X-aniline (3.7 mmol) and silica gel (1 g) were mixed in ethanol (20 mL).
The reaction mixture was submitted to an ultrasonic bath for 20 min. Then, the silica gel was filtered,
and the products obtained by recrystallised from ethanol and characterised by comparing melting
points to the literature values.

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3.2.2. Solvent Free Method
The Schiff bases 8–13 were prepared by solid-solid reactions according to previously published
methods [26]. The 4-X-aniline (3.7 mmol) and cinnamaldehyde (1) were mixed with a mortar and
pestle for 15 min. The obtained products were recrystallised from ethanol and characterised by
comparing melting points to the literature values.
3.3. Procedure for the Preparation of 4-(p-X-Phenyl)-N-[(2E)-3-phenyl-2-propenyl]benzenamines 14–19
3.3.1. Using Traditional Reflux
NaBH4 (2 mmol) was added to a solution of Schiff base (2 mmol) in ethanol (10 mL), and the
mixture was refluxed for 8–10 h [8]. Next, water (1 mL) was added, and the products were extracted
with CHCl3 (3 × 10 mL). The organic phase was dried with anhydrous Na2SO4, which was followed
by filtration. Then, the products were obtained by evaporation under vacuum. The spectra of the
obtained products were compared with spectroscopic data obtained from the literature.
3.3.2. Solvent Free Reactions
4-X-anilines 2–7 and cinnamaldehyde (1) (2 mmol each) were vigorously mixed for 15 min in a
mortar and pestle. Next, a mixture of NaBH4 and H3BO3 (5 mmol) was added, and the reaction mixture
was vigorously mixed for an additional 30 min [26]. Then, the reaction mixture was washed with a
NaHCO3 solution, and the product was extracted with CHCl3 (3 × 10 mL). The chloroform solution
was dried with anhydrous Na2SO4, filtered and evaporated under vacuum. The spectra of the obtained
products were compared with spectroscopic data obtained from the literature.
3.4. Procedure for the Preparation of Ureas 20–25
3.4.1. Using Traditional Reflux
A secondary amine 14–19 (2 mmol) in anhydrous toluene (30 mL) was mixed with phenyl
isocyanate (2 mmol) and refluxed for 4–24 h [8]. The reaction was monitored by TLC (EtOAc–hexane,
7:3). Next, the toluene was evaporated under vacuum. The solid products were recrystallised from
ethanol, and the oil products were purified by column chromatography using CHCl3 as the eluent.
3.4.2. Solvent Free Reactions
A secondary amine 14–19 (2 mmol) and phenylisocyanate (2 mmol) were vigorously mixed for
30–45 min in a mortar and pestle. The time reaction was monitored by TLC (EtOAc–hexane, 7:3).
Next, the solid products were recrystallised from ethanol, and the oil products were purified by column
chromatography using CHCl3 as the eluent.
N,N'-Diphenyl-N-[(2E)-3-phenylprop-2-en-1-yl]-urea (20). Oil; yield 100%; FT-IR (KBr, υ cm−1):
1
3421 (N-H), 2923/2854 (C-H), 1674 (C=O), 1597/1496 (C=C); H-NMR (DMSO-d6): δ 8.02 (s, 1H),
7.41 (d, J = 5.0 Hz, 1H), 7.40 (dd, J = 5.0 Hz, 2H), 7.38 (d, J = 5.0 Hz, 1H), 7.37 (sl, 1H), 7.33 (dd,

Molecules 2012, 17
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J = 5.0 and 10.0 Hz, 2H), 7.29 (d, J = 5.0 Hz, 2H), 7.26 (t, J = 5.0 Hz, 2H), 7.20 (t, J = 5.0 and 10.0 Hz,
3H), 6.93 (t, J = 5.0 and 10.0 Hz, 1H), 6.43 (d, J = 15.0 Hz, 1H), 6.34 (dd, J = 5.0 Hz and 15.0 Hz)
13
4.45 (d, J = 5.0 Hz, 2H); C-NMR (DMSO-d6): δ 154.43, 142.44, 139.98, 136.46, 131.18, 129.44,
128.65, 128.31, 127.54, 127.28, 126.33, 126.25, 122.14, 119.92, 51.20. Anal. Calcd. for C22H20N2O
(328.4071); C, 80.46; H, 6.14; N, 8.53%. Found: C, 81.05; H, 6.02; N, 8.74%.
N-(4-Methylphenyl)-N-[(2E)-3-phenylprop-2-en-1-yl]urea (21). White solid; m.p. 65–68 °C; yield
1
99%; FT-IR (KBr, υ cm−1): 3419 (N-H), 2920/2854 (C-H), 1658 (C=O), 1595/1519 (C=C); H-NMR
(DMSO-d6): δ 7.39 (m, J = 5.0 and 10.0 Hz, 4H), 7.29 (t, J = 5.0 and 10 Hz, 2H), 7.21 (sl, 5H), 7.19
(d, J = 5.0 Hz, 2H), 6.92 (t, J = 5.0 and 10.0 Hz, 1H), 6.42 ( d, J = 15.0 Hz, 1H), 6.33 (dd, J = 5.0 and
13
15.0 Hz), 4.41 (d, J = 5.0 Hz, 2H), 2.29 (s, 3H); C-NMR (DMSO-d6): δ 154.43, 140.00, 139.50,
136.48, 135.85, 131.24, 130.06, 128.63, 128.28, 127.45, 126.25, 122.07, 119.88, 51.53, 20.66. Anal.
Calcd. for C23H22N2O (342.4337); C, 80.67; H, 6.48; N, 8.18%. Found: C, 80.06; H, 7.04; N, 8.72%.
N-(4-Methoxyphenyl)-N-[(2E)-3-phenylprop-2-en-1-yl]urea (22). Oil; yield 98%; FT-IR (KBr,
υ cm−1): 3421 (N-H), 2956/2929/2839 (C-H), 1672 (C=O), 1597 (C=C); 1247/1031 (O-C-O); 1178
(C-CO-O); 1H-NMR (DMSO-d6): δ 7.69 s, 1H), 7.39 (m, J = 5.0 and 10.0 Hz, 4H), 7.29 (d, J = 5.0 Hz,
2H), 7.25 (d, J = 10.0 Hz, 2H), 7.20 (m, J = 5.0 and 10.0 Hz, 3H), 6.96 (d, J = 10.0 Hz, 2H), 6.92 (t,
J = 5.0 and 10.0 Hz, 1H), 6.41 (d, J = 20.0 Hz, 1H), 6.30 (dd, J = 5.0 and 20.0 Hz), 4.37 (d, J = 5.0 Hz,
13
2H) 3.75 (s, 3H); C-NMR (DMSO-d6): δ 157.94, 154.53, 140.02, 136.51, 134.40, 131.37, 129.38,
128.84, 128.63, 128.24, 127.52, 126.26, 122.05, 119.93, 118.22, 114.78, 55.25, 51.68. Anal. Calcd. for
C23H22N2O (358.4331); C, 77.07; H, 6.19; N, 7.82%. Found: C, 76.83; H, 6.01; N, 8.04%.
N-(4-Chlorophenyl)-N-[(2E)-3-phenylprop-2-en-1-yl]urea (23). White solid; m.p. 98–100 °C; yield
100%; FT-IR (KBr, υ cm−1): 3431 (N-H), 2922/2852 (C-H), 1654 (C=O), 1595/1527 (C=C); 1H-NMR
(DMSO-d6): δ 8.21 (s, 1H), 7.43 (m, J = 5.0 and 10.0 Hz, 4H), 7.38 (d, J = 10.0 Hz, 2H), 7.34 (d,
J = 10.0 Hz, 2H), 7.29 (t, J = 5.0 and 10.0 Hz, 2H), 7.21 (t, J = 10.0 Hz, 3H), 6.44 (d, J = 15.0 Hz,
1H), (dd, J = 5.0 and 15.0 Hz, 1H), 4.44 (d, J = 5.0 Hz, 2H); 13C-NMR (DMSO-d6): δ 154.32, 141.33,
139.95, 136.40, 131.38, 130.47, 129.34, 129.10, 128.65, 128.29, 127.60, 126.30, 126.09, 122.27,
120.13, 51.39. Anal. Calcd. for C22H19ClN2O (362.8518); C, 72.82; H, 5.28; N, 7.72%. Found: C,
73.08; H, 4.98; N, 7.93%.
N-(4-Bromophenyl)-N-[(2E)-3-phenylprop-2-en-1-yl]urea (24). Oil; yield 99%; FT-IR (KBr, υ cm−1):
1
3423 (N-H), 2925/2854 (C-H), 1668 (C=O), 1597/1492 (C=C); H-NMR (DMSO-d6): δ 8.24 (s, 1H),
7.56 (dd, J = 5.0 and 10.0 Hz, 2H), 7.40 (m, J = 5.0 and 10.0 Hz, 4H), 7.28 (d, J = 5.0 Hz, 2H), 7.27
(d, J = 5.0 Hz, 2H), 7.21 (m, J = 5.0 and 10.0 Hz, 4H), 6.94 (t, J = 5.0 and 10 Hz, 1H), 6.44 (d, J = 15.0 Hz,
1H), 6.34 (dd, J = 5.0 and 15.0 Hz), 4.44 (d, J = 6.0 Hz, 2H); 13C-NMR (DMSO-d6): δ 154.29, 141.81,
139.94, 136.40, 132.26, 131.36, 129.37, 128.65, 128.30, 127.60, 126.52, 126.29, 126.08, 122.28,
120.13, 118.82, 118.23. Anal. Calcd. for C22H19BrN2O (407.3031); C, 64.87; H, 4.70; N, 6.88%.
Found: C, 65.02; H, 4.59; N, 7.06%.
N-(4-Fluorphenyl)-N-[(2E)-3-phenylprop-2-en-1-yl]urea (25). White solid; m.p. 75–78 °C; yield 99%;
1
FT-IR (KBr, υ cm−1): 3323 (N-H), 2920/2854 (C-H), 1660 (C=O), 1595/1506 (C=C); H-NMR

Molecules 2012, 17
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(DMSO-d6): δ 7.99 (s, 1H), 7.40 (d, J = 10.0 Hz, 2H), 7.38 (m, J = 5.0 and 10 Hz, 4H), 7.29 (t,
J = 10.0 Hz, 2H), 7.23 (d, J = 10.0 Hz, 2H), 7.21 (m, J = 5.0 and 10.0 Hz, 3H), 6.93 (t, J = 5.0 and
10.0 Hz, 1H), 6.41 (d, J = 15.0 Hz, 1H), 6.33 (dd, J = 5.0 Hz and 15.0 Hz), 4.42 (d, J = 5.0 Hz); 13C-NMR
(DMSO-d6): δ 161.48, 159.55, 154.43, 139.99, 138.38, 136.45, 131.47, 129.96, 129.89, 128.63,
128.25, 127.57, 126.29, 126.13, 122.19, 120.14, 116.29, 116.11, 51.62. Anal. Calcd. for C22H19FN2O
(346.3975); C, 76.28; H, 5.53; N, 8.09%. Found: C, 76.84; H, 5.16; N, 8.23%.
3.5. Materials for DNA-Relaxation Assays
The DNA-topoisomerase II-α (topo II-α) drug screening kits were provided by TopoGEN [27] and
contained supercoiled (form I) plasmid substrate DNA (25 μg in 10 mL TE buffer). TE buffer (10 mM
Tris–HCl at pH 7.5 and 1 mM EDTA) and the assay buffer (50 mM Tris–HCl, pH 8, 120 mM KCl,
10 mM MgCl2, 100 mM EDTA, 3 mg/mL bovine serum albumin, 0.5 mM dithiothreitol, and 0.5 mM
ATP) were used. The supercoiled pBR322 plasmid DNA was purchased from Gibco (Grand Island,
NY, USA). The loading buffer contained 25% bromophenol blue, 50% glycerol and 10% SDS. The
agarose and the substances utilised in these assays were purchased from Sigma® (St. Louis, MO, USA).
3.6. Procedure for the DNA-Topoisomerase Assay
The topo II-α inhibition assay was performed as described in the TopoGEN screening kit. Briefly,
the reaction mixture (10 μL) contained the drug, DNA, assay buffer, 2U of topo II-α, and water. In the
topo II-α assay, two units were utilised to relax 0.125 μg/mL pBR322 (Gibco). The mixture was
incubated at 37 °C for 30 min, and the reaction was terminated by addition of 1 μL of a dye solution
containing 25% bromophenol blue, 50% glycerol and 10% SDS (sodium dodecyl sulfate). The
products were submitted to electrophoresis using 1% agarose gel in TAE buffer (50× stock: 242 g Tris
base, 57.1 mL glacial acetic acid, and 100 mL of 0.5 M EDTA) at 15 V for 3.5 h. The gels were
stained with ethidium bromide (0.5 μg/mL) for 30–45 min, washed, and photographed under UV light.
3.7. Docking Studies
Compounds 20–25 were constructed and energy-minimised with the PM3 semi-empirical method [28]
as implemented in the Spartan’08 program (Wavefunction, Inc., Irvine, CA, USA). The resulting
structures were used for the docking study of the DNA and the ATP binding sites. Due to the
significant structural modifications of the enzyme after ATP binding (and also of its N-substituted
analogue) to its specific allosteric site, it was necessary to use different crystal structures for the study
of these two binding sites. Docking solutions with the DNA binding site were obtained with the human
topo II-α co-crystallised with DNA and etoposide (PDB code 3QX3, resolution 2.16 Å) [29]. For the
ATP binding site, human topo II-α co-crystallised with phosphoaminophosphonic acid-adenylate ester,
an ATP analogue, in the ATPase domain (PDB code 1ZXM, resolution 1.87 Å) was used [30]. For the
docking studies of compounds 20–25, the etoposide and water molecules were deleted from the 3QX3
structure, and the ATP analogue and water molecules were deleted from the 1ZXM crystal structure.
Both TOPO-II structures contain Mg2+ ions, and the 3QX3 structure contains a co-crystallised DNA
segment, which was not removed.

Molecules 2012, 17
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The docking procedure was accomplished with the GOLD 5.1 program (CCDC Software Ltd.), and
the hydrogen atoms were added to the protein structures based on ionisation and tautomeric states
defined by the program. The number of genetic operations (crossover, migration, mutation) for each
run was set to 100,000 in the searching procedure. All of the scoring functions available in the
program, such as GoldScore [31], ChemScore [32], ASP [33] and ChemPLP [34], were evaluated and
compared for the docking calculations.
The internal binding site for docking was defined with a 20 Å radius from residue Asp479 in the
DNA binding site and with a 10 Å radius from the magnesium atom in the ATP binding site. Both are
located at the centre of each site. For the docking evaluation and adequate scoring function selection,
the co-crystallised structures (etoposide and the ATP analogue, in the respective binding site) were
redocked to each original site. The resulting poses of compounds (20–25) were compared and analysed
to identify structural characteristics of the complexes formed by these molecules and topo-II.
4. Conclusions
In summary, a new series of asymmetrically N,N’-substituted ureas was prepared using solvent free
conditions, which is an eco-friendly methodology, for all of the preparation steps involving Schiff
bases and the synthesis of the corresponding secondary amines. The synthesised ureas were obtained
after remarkably short times and in good yields compared to other methodologies.
The evaluation of the topo II-α activity in the relaxation assays showed that all of the compounds
inhibited the enzyme activity at 10 μM, except for urea 24. The docking results of compounds 20–25
into topo II-α indicated that these compounds are able to interact with the same binding site as the
anticancer drug etoposide. These results suggest that the same mechanism of action observed for
etoposide (i.e., prevents re-ligation of the DNA strands) may also be responsible for the observed
enzyme inhibition by the asymmetrically N,N'-substituted ureas.
Acknowledgments
The authors wish to thank CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico),
CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), FAPERJ (Fundação de Apoio
a Pesquisa do Estado do Rio de Janeiro) and INCT-INOFAR for financial support and fellowships.
Conflict of Interest
The authors declare no conflict of interest.
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