Synthesis, cytotoxicity, and DNA-topoisomerase inhibitory activity of new asymmetric ureas and thioureas

Article abstract of DOI:10.1016/j.bmc.2005.08.031

A new series of N-3,3-diphenylpropyl-N-(p-X-benzyl)-N′-phenylureas (5a-g) and thioureas (6a-g) were synthesized by the reaction of secondary amines and phenyl isocyanate or isothiocyanate. The cytotoxic effects of the urea and thiourea derivatives were ev

Full text of DOI:10.1016/j.bmc.2005.08.031

Bioorganic & Medicinal Chemistry 14 (2006) 492–499
Synthesis, cytotoxicity, and DNA-topoisomerase inhibitory
activity of new asymmetric ureas and thioureas
Andressa Esteves-Souza,a Kenia Pissinate,a Maria da Grac¸a Nascimento,b
Noema Faiga Grynberga and Aurea Echevarriaa,*
a
´
Universidade Federal Rural do Rio de Janeiro, Seropedica, 23851-970, RJ, Brazil
Universidade Federal de Santa Catarina, Florianopolis, 88040-900, SC, Brazil
b
´
Received 18 June 2005; revised 15 August 2005; accepted 16 August 2005
Available online 23 September 2005
Abstract—A new series of N-3,3-diphenylpropyl-N-(p-X-benzyl)-N0-phenylureas (5a–g) and thioureas (6a–g) were synthesized by
the reaction of secondary amines and phenyl isocyanate or isothiocyanate. The cytotoxic effects of the urea and thiourea derivatives
were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay against Ehrlich carcinoma and K562
human leukemia cells. Moreover, the activity of compounds in the inhibition of DNA topoisomerases I and II-a was tested. The
results indicated that the compounds presented important and promising antiproliferative action.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
Diarylsulfonylurea derivatives have been reported to
possess a broad spectrum of antineoplasic activity in
several tumor models. A series of compounds were ini-
tially discovered and assayed against CCRF-CEM,
HT-29, K562, and HTB-54 tumor cell lines, showing
good growth inhibition.8
The DNA-topoisomerases are ubiquitous nuclear en-
zymes that play crucial roles in DNA metabolism events
such as replication, transcription, recombination, repair,
chromatin assembly, and chromosome segregation.1–3
The type I enzymes (TOPO I) are monomeric and cata-
lyze an ATP-independent relaxation of DNA supercoils
by transiently breaking and religating single-stranded
DNA, and the type II topoisomerases (TOPO II) are
dimeric and relax supercoiled DNA through catalysis
of a transient breakage of double-stranded DNA in an
ATP-dependent manner.1,4 Because of the crucial role
of topoisomerases in the maintenance and replication
of DNA during proliferation, cells become highly vul-
nerable when these functions are lost.5 Consequently,
topoisomerase-targeting agents that stabilize the clev-
able complex formed between the enzyme and DNA
have proved to be effective in the treatment of cancer.
The medical importance of these enzymes is highlighted
by the fact that they are the specific targets for many
promising anticancer drugs.6,7
In the literature, the pyrazolyl-ureas are described as
P38 kinase inhibitors and the benzoylureas as new drugs
with strong cancericidal activity.9 Recently, hydroxy-
urea has been described as a clinically useful drug for
the treatment of a wide range of solid tumors as well
as acute and chronic leukemia.10
The urea and the thiourea derivatives have also been
used for brain cancer treatment and as potent inhibitors
of human DNA-topoisomerase II.11 Moreover, other
properties were attributed to urea derivatives such as
HIV-1 protease and cholesterol acyltransferase (ACAT)
inhibitory activities. They are also promising therapeutic
agents for hypercholesteromia and atherosclerosis.12,13
Thus, due to the important properties presented by ure-
as and thiourea derivatives involving particularly
antineoplasic activity, two new series were synthesized
and evaluated for their cytotoxic activity.
Thus, there is now good evidence showing that
topoisomerases are the principal intracellular targets
for a number of clinically important antitumor drugs.7
In this work, we report the synthesis, cytotoxicity evalu-
ation, and human DNA-topoisomerases I and II-a
Keywords: Ureas and thioureas; Cytotoxicity; DNA-topoisomerase.
*
Corresponding author. Tel./fax: +55 21 26 82 28 07; e-mail:
echevarr@ufrrj.br
inhibitory activity of
a series of N-[3,3-diphenyl
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.031

A. Esteves-Souza et al. / Bioorg. Med. Chem. 14 (2006) 492–499
493
propyl]-N-[p-X-benzyl]-N0-phenylureas (5a–g) and thiou-
reas (6a–g), in order to identify the essential structural
elements required for the antiproliferative activity and
to optimize the therapeutic index of these compounds.
the products were purified by recrystallization with eth-
anol. The yields were in the range of 52–98% for ureas
and 45–98% for the thioureas. The best yields observed
for ureas were obtained due to the higher reactivity of
phenylisocyanate. The electrophilic character of the car-
bonylic carbon is stronger than that of the thiocarbony-
lic carbon, thus facilitating the nucleophilic attack of the
secondary amines.
2. Results and discussion
2.1. Synthetic chemistry
The infra-red spectra for the ureas and thioureas showed
the absorption stretching band values in the range of
1647–1623 cmꢀ1 and 1350–1341 cmꢀ1 for C@O and
C@S groups, respectively. Higher values of stretching
absorption bands were observed for the compounds
with electron-withdrawing substituents, both for urea
and thiourea derivatives.
The route adapted for the synthesis of asymmetric ureas
and thioureas is outlined in Scheme 1. The secondary
amines (4a–g) were obtained from the corresponding
Schiff bases (3a–g) after the reduction reactions with
sodium borohydride in 55 to 98% yield.14 In the next
step, the secondary amines were condensed with phenyl-
isocyanate or phenylisothiocyanate in chloroform as the
solvent, affording the target compounds after 2 h for
unsubstituted derivatives (5a and 6a) and derivatives
with electron-donating substituent groups (5b–d and
6b–d), and after 4h for the electron-withdrawing substi-
tuted derivatives (5e–g and 6e–g). The reactions were
monitored by thin layer chromatography (TLC) and
The values obtained in the 1H NMR chemical shifts per-
mitted the characterization of the hydrogens showing
similar values for both ureas and thioureas with the
same substituents. However, deshield effects on H-1
and H-a NMR chemical shift values of thioureas were
observed (c.a. d 0.5) due to the heavy atom effect of
O
H
H
a
N
+
NH2
X
X
3a-g
2a-g
1
H
2''
1
2'
1
α
2''
2'
b
1'
1''
1'
1''
3'
4'
N
α
3
3''
4''
3''
4''
3
N
H
3'
4'
2
2
X
X
4a-g
3a-g
c
d
4'"
3'"
O
4'"
3'"
S
2'
1
2'
1
3
3
1'
X
1'
3'
4'
N
N
1'"
3'
4'
N
N
H
1'"
2
2"'
2
2"'
H
α
α
1"
2"
1"
2"
4"
X
4"
3"
3"
5a-g
6a-g
a: X= H
b: X= CH3
c: X= N(CH3)2
d: X= OCH2O
e: X= Br
f: X= CN
g: X= NO2
Scheme 1. Reagents and conditions: (a) EtOH, reflux; (b) NaBH4, EtOH, reflux; (c) PhNCO, CHCl3, reflux; (d) PhNCS, CHCl3, reflux.

494
A. Esteves-Souza et al. / Bioorg. Med. Chem. 14 (2006) 492–499
sulfur. The signals of aromatic hydrogens occurred as
multiplets in the characteristic region. The 1H NMR
chemical shift of the N–H in the ureas and thioureas
presented a strong deshielding when compared with
the N–H values of the amines, for example, d 1.55 (4a)
and d 5.99 (5a), where X = H.
The cytotoxicity of the ureas and thioureas against K562
leukemia cells showed a dose-dependent effect and the
thiourea derivatives presented the highest antiprolifera-
tive effect (Table 1). The thioureas with N,N-dimethyl
(6c) and methylenedioxyl (6d) groups were the most ac-
tive derivatives with IC50 of 14( 1) and 16( 1) lM,
respectively. Moreover, no significant correlation was
obtained for IC50 values of thioureas against K562 cells
in relation to electronic parameters.
13C NMR spectral analysis of the ureas and thioureas
showed the typical absorptions for aliphatic carbons in
the expected region, such as the signals for the carbony-
lic and thiocarbonylic carbons, in the range of d 155.12–
156.34 and d 181.01–182.17, respectively.
2.3. DNA-Topoisomerase inhibitory activity
The conversion of supercoiled plasmid DNA to relaxed
DNA by human topoisomerases I and II-a was examined
in the presence of urea (5a–g) and thiourea (6a–f)
derivatives. The activity of the ureas and thioureas on
the DNA-topoisomerase I enzyme was observed through
relaxation assays of the supercoiled / · 174 plasmid
DNA. The campothecin, a well-known topoisomerase I
enzyme inhibitor, was used as a positive control.17,18
2.2. Cytotoxic activity
The cytotoxic activity of the N-3,3-diphenylpropyl-N-(p-
X-benzyl)-N0-phenylureas and thioureas on ascitic
Ehrlich carcinoma and K562 human leukemia cells was
measured by MTT assay15 and the IC50 values were
determined in lM and lg/mL, at least in three indepen-
dent experiments.
The results were observed by the alteration of the
electrophoretic mobility of / · 174 plasmid DNA by
the combined action of topoisomerase I and the drugs.
The results were analyzed after development with
ethidium bromide in UV light, and the record was
photographed with a digital camera. As shown in Figure
1, the mobility of the supercoiled closed circular double-
stranded plasmid DNA increased upon topoisomerase I
mediated relaxation, when subjected to electrophoresis
with ethidium bromide, line 3. In the presence of
100 lM campothecin (positive control), the relaxation
effect was not observed, line 1. It can also be seen that
in the presence of topoisomerase I with 6a and 6d (lines
5 and 9), as in the presence of campothecin a significant
relaxation inhibitory effect was observed. The 5a deriva-
tive (line 4) showed a lower inhibitory effect, and 5b, 5e,
5f, and 6b a very weak effect, but with nicked DNA
formation.
The results of the cytotoxic activities for ureas and thi-
oureas against Ehrlich carcinoma cells for 48 h cultures
indicated that the ureas had a higher activity (Table 1).
The ureas with electron-withdrawing groups were more
cytotoxic with IC50 values of 12( 1) and 11( 1) lM for
5e and 5f, respectively. However, the thioureas with
electron acceptors of groups did not present significant
cytotoxicity until a dose of 50 lM.
The preliminary quantitative relationship analysis be-
tween –log (1/IC50) of ureas and the electronic substituent
parameter rp (Hammett substituent constant)16 showed a
significant correlation [correlation equation: ꢀlog
(1/IC50) = (1.21 0.05) ꢀ (0.34 0.11) rp, sd = 0.12,
r = 0.88, n = 5] indicating a linear relationship between
the cytotoxic activity against Ehrlich cells and the substi-
tuent electronic effect. However, other parameters could
have a significant importance, such as, the insolubility
indicating an apparent inactivity, as, for example, the 5g
derivative.
The effects of the ureas (5a–g) and thioureas (6a–f) on
the human DNA topoisomerase II-a enzyme were
observed in the relaxation assays using supercoiled
pBR322 plasmid DNA in the presence of ATP. The
reaction products were analyzed by electrophoretic
mobility and developed in ethidium bromide in the pres-
ence of UV light. As shown in Figure 2, 5g and 5f
(100 lM) presented a significant inhibition of topo II-a
Table 1. IC50 (lM) values of the ureas and thioureas against Ehrlich
carcinoma and K562 human leukemia cells, and substituent constants
Urea/thiourea
IC50 (lM sda)
Substituent
constant
r1p6
Ehrlich carcinoma
K562
5a
5b
5c
5d
5e
5f
20
13
36
nab
12
11
na
33
16
na
na
na
na
3
3
2
42
41
25
na
22
30
na
20
26
14
16
na
na
1
0
6
2
ꢀ0.17
ꢀ0.83
ꢀ0.16
0.23
1
2
3
4
5
6
7
8 9
Linear DNA
1
1
2
2
0.66
0.78
5g
6a
6b
6c
6d
6e
6f
Supercoilled DNA
Nicked DNA
2
2
4
6
1
1
0
ꢀ0.17
ꢀ0.83
ꢀ0.16
0.23
Figure 1. Effect of ureas and thioureas on DNA-topo I. Line 1: topo
I + campothecin 100 lM; line 2: / · 174 (0.125 lg) only; line 3: /
· 174 + topo I; line 4: topo I + 5a 100 lM; line 5: topo I + 6a 100 lM;
line 6: topo I + 5b 100 lM; line 7: topo I + 6b 100 lM; line 8: topo
I + 5e 100 lM; line 9: topo I + 6d 100 lM.
0.66
a sd: standard deviation.
b na: no activity until 50 lM.

A. Esteves-Souza et al. / Bioorg. Med. Chem. 14 (2006) 492–499
495
added, in ethanol. The mixture was refluxed, with stir-
ring and the water was collected in a Dean–Stark appa-
ratus. The reaction was monitored by TLC. The
products were filtered and recrystallized in ethanol.
1
2
3
4
5
6
7
8 9 10
3.1.3. N-Benzyliden-N-3,3-diphenylpropylamine (3a).
85% yield; mp 108–110 ꢁC; IR (KBr): mmax 3026, 2921–
Linear DNA
1
2853, 1641, 1538; H NMR d 2.45 (q, J = 7 Hz, 2H),
Supercoilled DNA
3.53 (t, J = 7 Hz, 2H), 4.03 (t, J = 7.9 Hz, 1H), 7.12–
7.26 (m, 10H), 7.37–7.40 (m, 4H), 7.66–7.70 (m, 1H),
8.09 (s, 1H); 13C NMR d 36.32 (C-2), 48.61 (C-1),
59.60 (C-3), 126.15 (C-40), 127.92 (C-300, C-500), 128.43
(C-30,C-50), 128.54 (C-200, C-600), 129.43 (C-20, C-60),
130.18 (C-400), 136.18 (C-100), 144.50 (C-10), 161.36 (C-a).
Figure 2. Effect of ureas and thioureas on DNA-topo II-a. Line 1:
pBR322 (0.125 lg) only; line 2: pBR322 + topo II-a; line 3: topo II-
a + 5g 100 lM; line 4: topo II-a + 5f 100 lM; line 5: topo II-a + 5e
100 lM; line 6: topo II-a + 6e 100 lM; line 7: topo II-a + 6d 100 lM;
line 8: topo II-a + 6b 100 lM; line 9: topo II-a + 5b 100 lM; line 10:
topo II-a + 6d 100 lM.
3.1.4. N-p-Methyl-benzyliden-N-3,3-diphenylpropylamine
(3b). 79% yield; mp 163–165 ꢁC; IR (KBr): mmax 3023,
2928–2832, 1643, 1493, 859, 740; 1H NMR d 2.36 (s, 3H),
2.45 (q, J = 7 Hz, 2H), 3.51 (t, J = 7 Hz, 2H), 4.02 (t,
J = 7.2 Hz, 1H), 7.12–7.28 (m, 13H), 7.57 (d, J = 8.3 Hz,
1H), 8.05 (s, 1H); 13C NMR d 21.45 (CH3), 36.36 (C-2),
48.60 (C-1), 59.59 (C-3), 126.12 (C-40), 127.92 (C-300, C-
500), 128.42 (C-30-C-50), 129.27 (C-20, C-60), 133.60 (C-100),
140.74 (C-400), 144.53 (C-10), 161.27 (C-a).
1
2
3
4
5
6
7
8
9
10 11
Linear DNA
Supercoilled DNA
3.1.5. N-p-Dimethylamino-benzyliden-N-3,3-diphenylpro-
pylamine (3c). 87% yield; mp 108–110 ꢁC; IR (KBr): mmax
Figure 3. Effect of ureas and thioureas on DNA-topo II-a. Line 1:
etoposide 100 lM + topo II-a; line 2: pBR322 (0.125 lg) only; line 3:
pBR322 + topo II-a; line 4: topo II-a + 5 g 100 lM; line 5: topo II-
a + 5g 50 lM; line 6: topo II-a + 5g 10 lM; line 7: topo II-a + 5f
100 lM; line 8: topo II-a + 5f 50 lM; line 9: topo II-a + 5f 10 lM; line
10: topo II-a + 5a 100 lM; line 11: topo II-a + 6a 100 lM.
1
3023, 2929–2856, 1603, 1531, 813, 749; H NMR d 2.50
(q, J = 6.6 Hz, 2H), 3.00 (s, 6H), 3.58 (t, J = 6.6 Hz, 2H),
4.10 (t, J = 7.6 Hz, 1H), 6.70 (dd, Jo = 9.4; Jp = 1.9 Hz,
2H), 7.30 (m, 10H), 7.62 (dd, Jo = 9.4; Jp = 1.9 Hz,
2H), 8.02 (s, 1H); 13C NMR d 36.52 (C-2), 40.13
(N(CH3)2), 48.56 (C-1), 59.42 (C-3), 111.51 (C-300, C-
500), 124.24 (C-100), 126.00 (C-40), 127.64 (C-200, C-600),
128.33 (C-30, C-50), 129.37 (C-20, C-60), 144.62 (C-10),
151.89 (C-400), 161.33 (C-a).
catalytic activity. In order to investigate the dose-depen-
dent inhibitory effects of 5g and 5f, the reaction products
of the relaxation assays were analyzed using 100, 50, and
10 lM drug concentrations. Figure 3 indicates the dose-
dependent inhibition of topo II catalytic activity by 5g
and 5f. The etoposide, a known topo II inhibitor, was
used as a positive control at 100 lM.
3.1.6. N-3,4-Methylenedioxy-benzyliden-N-3,3-diphenyl-
propylamine (3d). 72% yield; mp 105–107 ꢁC; IR (KBr):
mmax 3065, 2924–2820, 1649, 1495, 1195–1125, 819, 742;
1H NMR d 2.43 (q, J = 7 Hz, 2H), 3.48 (t, J = 7 Hz,
2H), 4.02 (t, J = 7.9 Hz, 1H), 5.98 (s, 2H), 6.80 (d,
J = 7.9 Hz, 1H), 7.01 (d, J = 7.9 Hz, 1H), 7.24 (m,
10H), 7.32 (sl, 1H), 7.97 (s, 1H); 13C NMR d 36.78 (C-
2), 48.59 (C-1), 59.32 (C-3), 101.38 (OCH2O), 106.47
(C-200), 108.02 (C-500), 124.16 (C-600), 126.15 (C-40),
127.92 (C-30, C-50), 128.43 (C-20, C-60), 131.03 (C-100),
144.52 (C-10), 148.19 (C-300), 153.28 (C-400), 160.51 (C-a).
3. Experimental
3.1. Chemistry
3.1.1. Reagents and apparatus. Melting points were
determined with a MELTEMP II apparatus and are
uncorrected. The microanalyses were carried out with
elemental apparatus CHNS-O EA-1110, of Carlo Erba
Instruments. Infra-red spectra (KBr pellets or as films
on a NaCl disk) were recorded on a Perkin-Elmer
1605 FT-IR spectrophotometer. 1H and 13C NMR spec-
tra were obtained on Varian Unity-plus (299.95 MHz
and 75.42 MHz) and Bruker AC200 (200 MHz and
50.3 MHz) spectrometers, with TMS as the internal ref-
erence, and CDCl3 as the solvent. TLC analyses were
performed on silica gel 60 F254 plates. All the reagents
were purchased from Merck or Aldrich.
3.1.7. N-p-Bromo-benzyliden-N-3,3-diphenylpropylamine
(3e). 65% yield; mp 99–102 ꢁC; IR (KBr): mmax 3021,
1
2929–2835, 1639, 1586, 819, 743; H NMR d 2.43 (q,
J = 7 Hz, 2H), 3.48 (t, J = 7 Hz, 2H), 4.02 (t,
J = 7.9 Hz, 1H), 7.14–7.31 (m, 12H), 7.53 (m, 2H),
8.04 (s, 1H); 13C NMR d 36.23 (C-2), 48.66 (C-1),
59.62 (C-3), 124.89 (C-400), 126.21 (C-40), 127.90 (C-30,
C-50), 128.46 (C-200, C-600), 129.41 (C-20, C-60), 131.79
(C-300, C-500), 135.07 (C-100), 144.43 (C-10), 160.09 (C-a).
3.1.8. N-p-Cyano-benzyliden-N-3,3-diphenylpropylamine
(3f). 80% yield; mp 78–80 ꢁC; IR (KBr): mmax 3056,
2934–2844, 2228, 1644, 1550, 836, 750; 1H NMR d
2.45 (q, J = 7 Hz, 2H), 3.57 (t, J = 7 Hz, 2H), 4.01 (t,
3.1.2. General procedure for Schiff base preparations (3a–
g). To a solution of 3,3-diphenyl-propylamine 1
(2 mmol) the p-X-benzaldehyde (2a–g) (2 mmol) was

496
A. Esteves-Souza et al. / Bioorg. Med. Chem. 14 (2006) 492–499
J = 7.8 Hz, 1H), 7.10–7.26 (m, 10H), 7.66 (d, J = 6.7 Hz,
2H), 7.77 (d, J = 6.7 Hz, 2H), 8.11 (s, 1H); 13C NMR d
36.13 (C-2), 48.68 (C-1), 59.76 (C-3), 118.53 (CN),
126.27 (C-400), 127.47 (C-300, C-500), 128.40 (C-30, C-50),
128.50 (C-2 , C-60), 127.86 (C-200, C-600), 129.85 (C-400),
139.98 (C-100), 144.29 (C-10), 159.41 (C-a).
1107, 809, 750; 1H NMR d 1.72 (s, 1H), 2.26 (q,
J = 7.5 Hz, 2H), 2.58 (t, J = 7.5 Hz, 2H), 3.62 (s, 2H),
4.02 (t, J = 7.5 Hz, 1H), 5.92 (s, 2H), 6.77 (1H), 6.71
(2H), 7.13–7.31 (m, 10H); 13C NMR d 35.52 (C–2),
47.29 (C-1), 48.86 (C-a), 53.42 (C-3), 107.92 (C-500),
108.58 (C-200), 110.74 (OCH2O), 121.12 (C-600), 126.07
(C-40), 127.70 (C-30, C-50), 133.86 (C-100), 144.62 (C-20,
C-60), 146.38 (C-400), 147.53 (C-300).
3.1.9. N-p-Nitro-benzyliden-N-3,3-diphenylpropylamine
(3g). 92% yield; mp 96–98 ꢁC; IR (KBr): mmax 3058,
2933–2835, 1645, 1598, 832, 747; 1H NMR 2.47 (q,
J = 7.9 Hz, 2H), 3.59 (t, J = 7 Hz, 2H), 4.03 (t,
J = 7.9 Hz, 1H), 7.14–7.27 (m, 10H), 7.83 (d,
J = 10.4 Hz, 2H), 8.19 (d, J = 10.4 Hz, 2H), d 8.26 (s,
1H); 13C NMR d 36.09 (C-2, C-3), 48.68 (C-1), 59.080 4
(C-4), 123.80 (C-400), 126.25 (C-40), 127.82 (C-200, C-6 ),
128.49 (C-30, C-50), 128.61 (C-20, C-60), 141.61 (C-100),
144.25 (C-10), 148.84 (C-300, C-500), 158.97 (C-a).
3.1.15. N-[3,3-Diphenylpropyl]-N-[p-bromo-benzyl]-amine
(4e). 62% yield; refraction index: volatile; IR (KBr):
mmax 3322, 3059–3026, 2927–2846, 1489, 1116, 819,
749; 1H NMR d 1.55 (s, 1H), 2.23 (q, J = 7.7 Hz,
2H), 2.55 (t, J = 6.3 Hz, 2H), 3.62 (s, 2H), 4.00 (t,
J = 7.7 Hz, 1H), 7.06–7.41 (m, 14H); 13C NMR d
35.60 (C-2), 47.37 (C-1), 48.74 (C-a), 52.90 (C-3),
120.40 (C-400), 126.02 (C-40), 127.61 (C-30, C-50),
128.29 (C-20, C-60), 129.57 (C-200, C-600), 131.16 (C-300,
C-500), 139.19 (C-100), 144.53 (C-10).
3.1.10. General procedure for amine preparations (4a–g).
To an ethanolic solution of Schiff bases (1a–g), (2 mmol)
NaBH4 (2 mmol) was slowly added in an ice bath with
stirring. The mixture was refluxed for 3–6 h. After this
time, the solvent was evaporated and water (2 mL)
was added. The product extraction was carried out with
CHCl3 (3· 2 mL). The organic layer was dried over
Na2SO4, and after solvent evaporation the residue was
purified by flash column chromatography (silica gel 60,
35–70 mesh) using CHCl3 as the eluent.
3.1.16. N-[3,3-Diphenylpropyl]-N-[p-cyano-benzyl]-amine
(4f). 72% yield; refraction index: volatile; IR (KBr): mmax
3314, 3058–3026, 2929–2854, 2226, 1494, 1113, 818, 747;
1H NMR d 2.28 (q, J = 7.5 Hz, 2H), 2.65 (t, J = 6.9 Hz,
2H), 3.78 (s, 2H), 4.03 (t, J = 7.5 Hz, 1H), 7.14–7.37 (m,
14H); 13C NMR d 35.65 (C-2), 47.57 (C-1), 48.89 (C-a),
53.22 (C-3), 126.24 (C-40), 127.71 (C-30, C-50), 128.48
(C-20, C-60, C-300, C-500), 132.12 (C-200, C-600), 144.43
(C-10), 145.80 (C-100).
3.1.11. N-[3,3-Diphenylpropyl]-N-benzylamine (4a). 94%
yield; refraction index: 1.444; IR (KBr): mmax 3408,
3025, 2923–2851, 1492, 1115, 744; H NMR d 1.55 (s,
1H), 2.26 (q, J = 7 Hz, 2H), 2.61 (t, J = 7 Hz, 2H),
3.71 (s, 2H), 4.02 (t, J = 7.8 Hz, 1H), 7.15–7.30 (m,
15H); 13C NMR d 35.82 (C-2), 47.71 (C-1), 49.01
(C-a), 53.89 (C-3), 126.15 (C-40), 126.88 (C-400), 127.82
(C-30, C-50), 128.06 (C-300, C-500), 128.36 (C-20, C-60),
128.43 (C-200, C-600), 140.31 (C-100), 144.77 (C-10).
3.1.17. N-[3,3-Diphenylpropyl]-N-[p-nitro-benzyl]-amine
(4g). 55% yield; refraction: 1.456; IR (KBr): mmax 3332,
3059–3026, 2929–2850, 1495, 845, 748; 1H NMR d
2.21 (s, 1H), 2.30 (q, J = 7 Hz, 2H), 2.59 (t, J = 7 Hz,
2H), 3.81 (s, 2H), 4.06 (t, J = 7.8 Hz, 1H), 7.16–7.29
(m, 10H), 7.76 (dd, J = 8.9 Hz, 2H), 8.05 (dd,
J = 8.9 Hz, 2H); 13C NMR d 36.06 (C-2), 48.65 (C-1),
48.80 (C-a), 52.95 (C-3), 123.76 (C-300, C-500), 126.22
(C-40), 127.79 (C-30,C-50), 128.46 (C-20, C-60), 128.58
(C-200, C-600), 141,56 (C-10), 144.23 (C-100,C-400).
1
3.1.12. N-[3,3-Diphenylpropyl]-N-[p-methyl-benzyl]-amine
(4b). 98% yield; refraction index: volatile; IR (KBr): mmax
3322, 2925–2822, 1493, 804, 749; 1H NMR d 1.58 (s, 1H),
2.26 (q, J = 7 Hz, 2H), 2.33 (s, 3H), 2.61 (t, J = 7 Hz,
2H), 3.68 (s, 2H), 4.03 (t, J = 7.8 Hz, 1H), 7.13–7.31 (m,
14H); 13C NMR d 21.01 (CH3) 35.73 (C-2), 47.59 (C-1),
48.92 (C-a), 53.51 (C-3), 126.06 (C-40), 127.73 (C-300, C-
500), 127.94 (C-200, C-600), 128.36 (C-30, C-50), 128.95 (C-20,
C-60), 136.32 (C-400), 137.19 (C-100), 144.72 (C-10).
3.2. General procedure for urea (5a–g) and thiourea (6a–f)
preparations
To chloroform solution of amines (4a–g) (1.5 mmol),
phenylisocyanate or phenylisothiocyanate (1.5 mmol)
was slowly added with stirring. The mixture was re-
fluxed for 2–4 hours. After this time, the product
was filtered and purified by recrystallization from
ethanol.
3.1.13. N-[3,3-Diphenylpropyl]-N-[p-dimethylamino-ben-
zyl]-amine (4c). 65% yield; refraction index: volatile; IR
(KBr): mmax 3306, 3058–3024, 2928–2854, 1491, 1125, 806,
750; 1H NMR d 2.33 (q, J = 6.5 Hz, 2H), 2.62 (t,
J = 7 Hz, 2H), 2.91 (s, 3H), 3.65 (s, 2H), 4.01 (t,
J = 6.5 Hz, 1H), 6.67 (d, J = 8.7 Hz, 2H), 7.13–7.26 (m,
12H); 13C NMR d 35.38 (C-2), 40.68 (N(CH3)2), 47.29
(C-1), 48.95 (C-a), 53.06 (C-3), 112.62 (C-300, C-500), 126.09
(C-40), 127.42 (C-100), 127.77 (C-30, C-50), 128.39 (C-20,
C-60), 129.15 (C-200, C-600), 144.65 (C-10), 149.82 (C-400).
3.2.1. N-[3,3-Diphenylpropyl]-N-benzyl-N0-phenylurea (5a).
98% yield; mp 123–125 ꢁC; IR (KBr): mmax 3284, 3057,
2930–2863, 1623, 1530, 750; 1H NMR d 2.38 (q,
J = 8 Hz, 2H), 3.28 (t, J = 8 Hz, 2H), 3.89 (t, J = 8 Hz,
1H), 4.51 (s, 2H), 5.99 (s, 1H), 6.96–7.32 (m, 20H);
13C NMR d 33.81 (C-2), 46.43 (C-1), 48.62 (C-a),
50.53 (C-3), 119.69 (C-2000, C-6000), 122.90 (C-4000), 126.58
(C-40), 127.30 (C-400), 127.61 (C-30, C-50, C-200, C-300, C-
500, C-600), 128.46 (C-3000, C-5000) 128.78 (C-20, C-60),
137.56 (C-1000), 138.89 (C-100), 143.91 (C-10), 155.26
(C@O); Found: C = 83.29, H = 6.77, N = 7.09.
C29H28N2O; Requires: C = 82.81, H = 6.72, N = 6.66.
3.1.14. N-[3,3-Diphenylpropyl]-N-[3,4-methylenedioxy-
benzyl]-amine (4d). 75% yield; refraction index: 1.450;
IR (KBr): mmax 3643, 3059–3026, 2926, 1493, 1189,

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497
3.2.2. N-[3,3-Diphenylpropyl]-N-[p-methyl-benzyl]-N0-phe-
nylurea (5b). 84% yield; mp 118–119 ꢁC; IR (KBr): mmax
3412, 3053–3027, 2925, 1632, 1530, 1244, 827, 752; H
(C-2000, C-6000), 121.15 (C-200, C-600), 124.28 (C-4000),
126.91 (C-40), 127.55 (C-30, C-50), 128.86 (C-3000, C-
5000), 129.04 (C-20, C-60), 132.53 (C-300, C-500), 143.53
(C-10), 143.70 (C-100); Found: C = 80.73, H = 6.07,
N = 9.22. C30H27N3O; Requires: C = 80.86, H = 6.12,
N = 9.43.
1
NMR d 2.31 (s, 3H), 2.38 (q, J = 7.4 Hz, 2H), 3.28 (t,
J = 7.4 Hz, 2H), 3.89 (t, J = 8 Hz, 1H), 4.47 (s, 2H), 6.01
(s, 1H), 6.96–7.31 (m, 19H); 13C NMR d 20.99 (CH3),
33.70 (C-2), 46.29 (C-1), 48.54 (C-a), 50.21 (C-3), 119.61
(C-2000, C-6000), 122.70 (C-4000), 126.42 (C-40), 127.22 (C-400),
127.54 (C-200, C-300, C-500,C-600), 128.63 (C-30, C-50),
129.41 (C-20, C-60), 134.37 (C-3000, C-5000), 137.19 (C-1000),
138.94 (C-100), 143.89 (C-10), 155.20 (C@O); Found:
C = 83.07, H = 6.63, N = 6.74. C30H30N2O; Requires:
C = 82.90, H = 6.97, N = 6.45.
3.2.7. N-[3,3-Diphenylpropyl]-N-[p-nitro-benzyl]-N0-phe-
nylurea (5g). 52% yield; mp 110–112 ꢁC; IR (KBr): mmax
3418, 3058, 2940-2876, 1642, 1535, 1241, 841, 753; H
1
NMR d 2.85 (q, J = 6.6 Hz, 2H), 3.62 (t, J = 6.6 Hz,
2H), 4.20 (t, J = 6.9 Hz, 1H), 4.85 (s, 2H), 6.13 (s, 1H),
7.03–7.34 (m, 19H); 13C NMR d 33.79 (C-2), 46.09 (C-
1), 48.55 (C-a), 50.20 (C-3), 119.75 (C-2000, C-6000), 122.73
(C-200, C-600), 123.16 (C-300, C-500), 126.05 (C-4000), 126.68
(C-40), 127.57 (C-30, C-50), 128.85 (C-3000, C-5000), 128.90
(C-20, C-60), 139.49 (C-1000), 142.01 (C-10), 143.01 (C-400),
143.65 (C-100), 155.19 (C@O); Found: C = 74.78,
H = 6.02, N = 9.08. C29H27N3O3; Requires: C = 74.81,
H = 5.86, N = 9.03.
3.2.3. N-[3,3-Diphenylpropyl]-N-[p-dimethylamino-benzyl]-
N0-phenylurea (5c). 75% yield; mp 128–131 ꢁC; IR (KBr):
mmax 3442, 3023, 2934-2859, 1635, 1535, 1243, 922, 750;
1H NMR d 2.06 (s, 6H), 2.25 (q, J = 6.4 Hz, 2H), 3.21 (t,
J = 7.8 Hz, 2H), 3.94 (t, J = 6.4 Hz, 1H), 6.12 (s, 1H),
7.09–7.33 (m, 19H); 13C NMR d 35.66 (C-2), 38.94 (C-1),
39.17 (N(CH3)2), 48.53 (C-a), 48.78 (C-3), 120.62 (C-2000,
C-6000), 123.37 (C-4000), 126.43 (C-100), 126.67 (C-40), 127.67
(C-30, C-50), 128.49 (C-3000, C-5000), 128.57 (C-20, C-60),
138.74 (C-1000), 143.78 (C-10), 144.16 (C-400), 156.34
(C@O); Found: C = 79.98, H = 7.04, N = 9.06.
C31H33N3O; Requires: C = 80.30, H = 7.19, N = 9.06.
3.2.8. N-[3,3-Diphenylpropyl]-N-benzyl-N0-phenylthiourea
(6a). 70% yield; mp 135–137 ꢁC; IR (KBr): mmax 3387, 3085,
2915, 1344, 1517, 1205, 741;1H NMR d 2.50(q, J = 7.6 Hz,
2H), 3.68 (t, J = 8 Hz, 2H), 3.90 (t, J = 7.6 Hz, 1H), 4.94 (s,
2H),5.99(s, 1H),7.11–7.31(m, 20H);13CNMRd32.70(C-
20)0, 48.68 (C-1), 50.22 (C-a), 54.70 (C-3), 125.49 (C-200, C-
6 ), 125.69 (C-400), 126.52 (C-40), 127.52/127.05 (C-200, C-
300, C-500, C-600), 127.91 (C-400), 128.45 (C-30, C-50), 128.66
(C-300, C-500), 128.92 (C-20, C-30), 135.76 (C-100), 139.46
(C-100), 143.62 (C-10), 181.94 (C@S), Found: C = 79.53,
H = 6.51, N = 6.55, S = 1.29. C29H28N2S; Requires:
C = 79.64, H = 6.48, N = 6.42, S = 1.35.
3.2.4.
N-[3,3-Diphenylpropyl]-N-[3,4-methylenodioxy-
benzyl]-N0-phenylurea (5d). 83% yield; mp 83–85 ꢁC; IR
(KBr): mm1ax 3419, 3058, 2932-2896, 1628, 1529, 1242,
809, 751; H NMR d 2.37 (q, J = 7.5 Hz, 2H), 3.25 (t,
J = 7.9 Hz, 2H), 3.90 (t, J = 7.5 Hz, 1H), 4.42 (s, 2H),
5.92 (s, 2H), 6.00 (s, 1H), 6.64–7.33 (m, 19H); 13C
NMR d 33.76 (C-2), 46.15 (C-1), 48.59 (C-a), 50.22
(C-3), 101.10 (OCH2O), 107.96 (C-200), 108.28 (C-600),
119.58 (C-2000, C-6000), 120.64 (C-500), 122.87 (C-4000),
126.63 (C-40), 127.61 (C-30, C-50), 128.82 (C-20, C-60,
C-3000, C-5000), 131.50 (C-100), 138.88 (C-1000), 143.88 (C-
10), 147.00 (C-400), 148.00 (C-300), 155.15 (C@O); Found:
C = 77.34, H = 6.06, N = 6.32. C30H29N2O3; Requires:
C = 77.55, H = 6.07, N = 6.03.
3.2.9. N-[3,3-Diphenylpropyl]-N-[p-methyl-benzyl]-N0-phen-
ylthiourea (6b). 98% yield; mp 150–151 ꢁC; IR (KBr):
1
mmax 3440, 3023, 2915, 1519, 1348, 1205, 836, 759; H
NMR d 2.35 (s, 3H), 2.52 (q, J = 7.9 Hz, 2H), 3.71 (t,
J = 8,1 Hz, 2H), 3.72 (t, J = 7.9 Hz, 1H), 4.89 (s, 2H),
6.80 (s, 1H), 7.15–7.29 (m, 19H); 13C NMR d 21.03
(CH3), 32.66 (C-2), 48.68 (C-1), 50.43 (C-a), 54.86 (C-
3), 125.39 (C-200, C-600), 125.58 (C-400), 126.46 (C-40),
126.91 (C-200, C-600), 127.50 (C-300, C-500), 128.40 (C-300,
C-500), 128.60 (C-30, C-50), 129.58 (C-20, C-30), 132.50
(C-100), 135.66 (C-100), 139.47 (C-400), 143.65 (C-10),
181.82 (C@S); Found: C = 79.85, H = 6.69, N = 6.36,
S = 7.14. C30H30N2S; Requires: C = 79.95, H = 6.72,
N = 6.22, S = 7.12.
3.2.5.
N-[3,3-Diphenylpropyl]-N-[p-bromo-benzyl]-N0-
phenylurea (5e). 55% yield; mp 242–244 ꢁC; IR (KBr):
mmax 3256, 3055, 2972-2874, 1640, 1536, 1238, 830,
753; 1H NMR d 2.37 (q, J = 7.5 Hz, 2H), 3.23 (t,
J = 8.3 Hz, 2H), 3.87 (t, J = 7.5 Hz, 1H), 4.48 (s, 2H),
5.94 (s, 1H), 6.69–7.44 (m, 19H); 13C NMR d 33.74
(C-2), 46.02 (C-1), 48.44 (C-a), 49.69 (C-3), 119.66 (C-
2000, C-6000), 120.95 (C-400), 123.07 (C-600, C-200), 124.10
(C-4000), 126.73 (C-40), 127.57 (C-30, C-50), 128.79 (C-
3000, C-5000),128.89 (C-20, C-60), 129.31 (C-300, C-500),
136.83 (C-100), 138.68 (C-1000), 143.71 (C-10), 155.12
(C@O); Found: C = 69.57, H = 5.76, N = 5.42.
C29H27N2OBr; Requires: C = 69.73, H = 5.46, N = 5.61.
3.2.10.
N-[3,3-diphenylpropyl]-N-[3,4-methylenodioxy-
benzyl]-N0-phenylthiourea (6c). 80% yield; mp 109–
112 ꢁC; IR (KBr): mmax 3409, 3055, 2918-2889, 1345,
1
1519, 1203, 806; H NMR: d 2.46 (q, J = 7.8 Hz, 2H),
3.60 (t, J = 8.2 Hz, 2H), 3.88 (t, J = 7.8 Hz, 1H), 4.85 (s,
2H), 6.61–6.83 (m, 3H), 7.11–7.31 (m, 10H); 13C NMR
d 32.66 (C-2), 48.67 (C-1), 49.64 (C-a), 54.58 (C-3),
101.17 (OCH2O), 107.66 (C-500), 108.39 (C-200), 120.50
(C-600), 125.52 (C-200, C-600), 1250.078 (C-400), 126.62 (C-40),
127.52 (C-30, C-50), 128.52 (C-3 , C-500), 128.73 (C-20, C-
30), 129.64 (C-100), 139.43 (C-100), 143.59 (C-10), 147.33
(C-300), 148.20 (C-400), 181.82 (C@S); Found: C = 74.78,
H = 6.08, N = 5.82. C30H29N2O2S; Requires: C = 74.80,
H = 6.08, N = 5.82.
3.2.6. N-[3,3-Diphenylpropyl]-N-[p-cyano-benzyl]-N0-phe-
nylurea (5f). 74% yield; mp 243–246 ꢁC; IR (KBr): mmax
3324, 3063, 2924-2824, 1647, 1554, 1233, 895, 753; H
1
NMR d 2.40 (q, J = 7 Hz, 2H), 3.21 (t, J = 7 Hz, 2H),
3.90 (t, J = 6.9 Hz,1H), 4.59 (s, 2H), 5.94 (s, 1H),
7.13–7.60 (m, 19H); 13C NMR d 33.81 (C-2), 46.14
(C-1), 48.36 (C-a), 49.99 (C-3), 119.72 (CN), 120.67

498
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3.2.11. N-[3,3-Diphenylpropyl]-N-[p-bromo-benzyl]-N0-
phenylthiourea (6d). 61% yield; mp 129–130 ꢁC; IR
(KBr): mmax 3389, 3054, 2914, 1519, 1350, 1204, 820,
762; 1H NMR d 2.43 (q, J = 7.8 Hz, 2H), 3.50 (t,
J = 8.3 Hz, 2H), 3.84 (t, J = 7.8 Hz, 1H), 4.95 (s, 2H),
6.80 (s, 1H), 7.10–7.27 (m, 18H), 7.41 (s, 1H); 13C NMR
d 32.66 (C-2), 48.53 (C-1), 48.95 (C-a), 54.35 (C-3),
121.69 (C-400), 125.61 (C-200, C-600), 125.93 (C-400), 126.70
(C-40), 128.53 (C-30, C-50), 128.79 (C-300, C-500), 129.10
(C-20, C-30, C-200, C-600), 131.86 (C-300, C-500), 135.18 (C-
100), 139.28 (C-100), 143.38 (C-10), 181.01 (C@S); Found:
C = 67.79, H = 5.19, N = 5.58, S = 6.04. C29H27N2SBr;
Requires: C = 67.56, H = 5.29, N = 5.44, S = 6.23.
Aliquots of 5 · 105 cells/mL (Ehrlich) and 2 · 104 cells/
mL (K562) were seeded onto 96-microtiter flat well
plates in RPMI 1640 medium supplemented with 10%
fetal bovine serum, 2 mM glutamine, 100 lg/mL strepto-
mycin, and 100 U/mL penicillin, containing the indicat-
ed concentration of the drug. All determinations were
carried out in triplicate. After 48 h (Ehrlich) or 96 h
(K562), at 37 ꢁC under 5% of CO2, the cultures were
incubated with MTT (5 mg/mL) for 3 h. The formazan
produced by live cells was solubilized with acidic isopro-
panol, and the absorbance was read at 570 nm. IC50 val-
ues were obtained by a linear regression analysis of the
absorbance percent versus the log of the drug
concentration.
3.2.12. N-[3,3-Diphenylpropyl]-N-[p-cyano-benzyl]-N0-phen-
ylthiourea (6e). 60% yield; mp 138–140 ꢁC; IR (KBr): mmax
3356, 3028, 2927, 2228, 1525, 1348, 1254, 815, 746; H
4.3. Materials for DNA-relaxation
1
NMR d 2.48 (q, J = 6.8 Hz 2H), 3.47 (t, J = 6.6 Hz, 2H),
3.85 (t, J = 6.8 Hz 1H), 4.17 (s, 2H), 6.85 (s, 1H), 7.20–
7.36 (m, 19H); 13C NMR d 32.63 (C-2), 48.34 (C-1, C-a),
54.86 (C-3), 110.00 (C-400), 125.82 (C-200, C-600), 126.10 (C-
400), 126.82 (C-40), 127.37 (C-30, C-50), 128.10 (C-300, C-500),
128.55 (C-20, C-30), 128.86 (C-200, C-600), 132.37 (C-300, C-
500), 139.00 (C-100), 142.14 (C-100), 143.17 (C-10), 182.17
(C@S); Found: C = 77.96, H = 5.88, N = 9.27.
C30H27N3S; Requires: C = 78.04, H = 5.91, N = 9.10.
The DNA-topoisomerase I (topo I) and II-a (topo II-
a) drug screening kits were from TopoGEN contain-
ing supercoiled (form I) plasmid substrate DNA
(25 lg in 10 mL TE buffer). TE buffer 10 mM Tris–
HCl at pH 7.5 and 1 mM EDTA), and the assay buf-
fers (10 mM Tris–HCl pH 7.9, 1 mM EDTA, 0.15 M
NaCl, 0.1% BSA, 0.1 mM spermidine, and 5% glycer-
ol (for topo I), and 50 mM Tris–HCl, pH 8, 120 mM
KCl, 10 mM MgCl2, 100 mM EDTA, 3 mg/mL bo-
vine serum albumin, 0.5 mM dithiothreitol, and
0.5 mM ATP (for topo II-a). The DNAs utilized
were supercoiled / · 174 and pBR322 plasmid pur-
chased from Sigma and Gibco, respectively. The
loading buffer contained 25% bromophenol blue,
50% glycerol, and 10% SDS. The agarose and the
substances utilized in these assays were purchased
from Sigma.
3.2.13. N-[3,3-Diphenylpropyl]-N-[p-nitro-benzyl]-N0-phen-
ylthiourea (6f). 45% yield; mp 180–182 ꢁC; IR (KBr): mmax
3382, 3025, 2925-2858, 1517, 1341, 1196, 840, 748; H
1
NMR d 2.49 (q, J = 6.6 Hz, 2H), 3.58 (t, J = 6.6 Hz, 2H),
3.89 (t, J = 6.8 Hz, 1H), 5.13 (s, 2H), 6.13 (s, 1H), 7.03–
7.34 (m, 2H), 7.17–7.41 (m, 10H), 8.14 (d, J = 8.4 Hz,
1H), 8.24 (d, J = 8.4 Hz, 1H); 13C NMR d 32.67 (C-2),
48.56 (C-1), 49.41 (C-a), 54.60 (C-3), 122.71 (C-300, C-500),
125.53 (C-400), 126.03 (C-200, C-600), 126.75 (C-40), 127.77
(C-30, C-50), 128.54 (C-300, C-500), 128.73 (C-20, C-30),
128.79 (C-200, C-600), 137.70 (C-100), 139.25 (C-100), 143.38
(C-10), 182.08 (C@S); C29H27N3O2S.
4.4. Topo I assay
The topo I inhibition was determined by relaxation as-
say and was carried out as described in the TopoGEN
screening kit. For topo I, one unit of the enzyme was uti-
lized to relax 0.125 lg of the supercoiled / · 174 plas-
mid DNA. The reaction mixture (10 lL) contained the
drug, DNA, assay buffer, 1U of topo I, and water.
The mixture was incubated at 37 ꢁC for 30 min, and
the reaction was finalized by the addition of 1 lL of
dye solution containing 25% bromophenol blue, 50%
glycerol, and 10% SDS.
4. Biological assays
4.1. Cell culture
Ehrlich carcinoma cells were maintained for 12–14 days
in Swiss mice. The tumor cell cultures were started from
mouse Ehrlich ascites with at least one passage in vitro
prior to use. The K562 cells were maintained in RPMI
1640 medium supplemented with fetal calf serum,
50 lM of 2-mercaptoethanol, 100 IU/mL penicillin,
and 100 lg/mL streptomycin. The cultures were incubat-
ed at 37 ꢁC in humidified atmosphere with 5% CO2, and
the culture medium was changed twice a week.
Reaction products were loaded onto a 1% agarose gel
containing ethidium bromide. Electrophoresis was car-
ried out in Tris-acetate–EDTA, pH 8.5, at 15 V for
3.5 h and then photographed with a digital camera by
illumination.
4.5. Topo II-a assay
4.2. Drugs and MTT assay
The topo II-a inhibition was carried out as described in
the TopoGEN screening kit. The reaction mixture
(10 lL) contained the drug, DNA, assay buffer, 2U of
topo II-a, and water. In the topo II-a assay, two units
were utilized to relax 0.125 lg/mL pBR322 (Gibco).
The mixture was incubated at 37 ꢁC for 30 min, and
the reaction was ended by the addition of 1 lL of dye
The stock solutions of the ureas and thioureas were pre-
pared in DMSO. The percentage of DMSO in assays
was in maximum of 0.5% (v/v) in saline solution. The drug
cytotoxicity assays were performed as previously de-
scribed by Mosmann15 using MTT for viable cell
measurements.

A. Esteves-Souza et al. / Bioorg. Med. Chem. 14 (2006) 492–499
499
3. Redinbo, M. R.; Stewart, L.; Kuhn, P.; Champoux, J. J.;
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Acknowledgment
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