Antiproliferative effects of novel urea derivatives against human prostate and lung cancer cells; And their inhibition of β-glucuronidase activity

Article abstract of DOI:10.1007/s00044-013-0702-5

Twenty-one novel urea derivatives were synthesized and their structures characterized by mass, NMR, IR, and UV spectroscopy. These compounds were evaluated for their antiproliferative profile against human PC-3 (prostate) and NCI-H460 (lung) cancer cell lines. Among them, compound 21 N-(3-nitrophenyl)- N′-(1-phenylethyl)urea was found to be active against both PC-3 (IC ₅₀ ± SEM: 20.13 ± 0.91 μM) and NCI-H460 (GI ₅₀: 22 ± 2.6 μM) cell lines; hence has the potential to be further studied as anticancer agent. These compounds were also investigated for their ability to inhibit urease, β-glucuronidase, and phosphodiesterase enzymes. N-(2,6-Dimethylphenyl)-N′-(4′-nitrophenyl)urea (1) demonstrated 90 % inhibition of β-glucuronidase enzyme (IC₅₀ ± SEM: 3.38 ± 0.043 μM).

Full text of DOI:10.1007/s00044-013-0702-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1099–1113
DOI 10.1007/s00044-013-0702-5
ORIGINAL RESEARCH
Antiproliferative effects of novel urea derivatives against human
prostate and lung cancer cells; and their inhibition
of b-glucuronidase activity
•
Shahnaz Perveen Sana Mustafa Kehkashan Qamar Ahsana Dar
•
•
•
•
•
•
Khalid M. Khan Muhammad Iqbal Choudhary Ajmal Khan
Wolfgang Voelter
Received: 8 March 2013 / Accepted: 8 August 2013 / Published online: 27 August 2013
Ó Springer Science+Business Media New York 2013
Abstract Twenty-one novel urea derivatives were synthe-
sized and their structures characterized by mass, NMR, IR,
and UV spectroscopy. These compounds were evaluated for
their antiproliferative profile against human PC-3 (prostate)
and NCI-H460 (lung) cancer cell lines. Among them, com-
pound 21 N-(3-nitrophenyl)-N0-(1-phenylethyl)urea was
found to be active against both PC-3 (IC50 SEM:
20.13 0.91 lM) and NCI-H460 (GI50: 22 2.6 lM) cell
lines; hence has the potential to be further studied as anti-
cancer agent. These compounds were also investigated for
their ability to inhibit urease, b-glucuronidase, and phospho-
diesterase enzymes. N-(2,6-Dimethylphenyl)-N0-(40-nitro-
phenyl)urea (1) demonstrated 90 % inhibition of b-
glucuronidase enzyme (IC50 SEM: 3.38 0.043 lM).
Keywords Urea derivatives ꢀ Prostate cancer
(PC-3) cell line ꢀ Lung cancer (NCI-H460) cell line ꢀ
b-Glucuronidase ꢀ Urease ꢀ Phosphodiesterase
Introduction
Cancer is the third leading cause of death in the developing
world after cardiac and infectious diseases (Efferth et al.,
2008). Lung cancer is the most common malignancy
worldwide (Globocan, 2008), whereas cancer of prostate is
the second most frequently diagnosed malignancy in men
(Youlden et al., 2008). Since most of the known anticancer
drugs have serious side effects, or the tumor cells acquire
resistance to these agents, therefore the search for novel
anticancer agents is an ongoing process. In the last decade,
urea derivatives have attained importance as the focus of
research for the rational design of new generation of urea
anticancer drugs (Li et al., 2009; Lam et al., 1994; Castro
et al., 1996). Aromatic urea derivatives, such as N-phenyl-
N0-(2-chloroethyl) ureas and benzoylureas, demonstrated
excellent anticancer activity by acting as tubulin ligands
and inhibiting the polymerization of tubulin (Fortin et al.,
2011). Moreover, a series of 3-haloacylamino benzoyl urea
(HBU) exhibited anticancer activity with IC50 values
between 0.01 and 0.30 lM against nine human tumor cell
lines, including CEM (leukemia), Daudi (lymphoma),
MCF-7 (breast cancer), Bel-7402 (hepatoma), DU-145
(prostate cancer), PC-3 (prostate cancer), DND-1A (mela-
noma), LOVO (colon cancer), and MIA Paca (pancreatic
cancer) (Song et al., 2008). Li et al. (2009) reported that
heterocyclic urea derivatives demonstrated good inhibitory
activity against various molecules involved in tumorigen-
esis, such as receptor tyrosine kinases (RTKs), raf kinases,
S. Perveen (&)
PCSIR Laboratories Complex, Shahrah-e-Dr.
Salimuzzaman Siddiqui, Karachi 75280, Pakistan
e-mail: dr_shahnaz_perveen@yahoo.com
S. Mustafa
Department of Chemistry, University of Karachi, Karachi
75270, Pakistan
K. Qamar ꢀ A. Dar ꢀ K. M. Khan ꢀ M. I. Choudhary
H. E. J. Research Institute of Chemistry, International Center for
Chemical and Biological Sciences, University of Karachi,
Karachi 75270, Pakistan
A. Khan
Department of Chemistry, Faculty of Science, King Saud
University, Riyadh, Saudi Arabia
W. Voelter
¨
¨
¨
¨
Interfakultares Institut fur Biochemie der Universitat Tubingen,
Hoppe-Seyler-Straße 4, 72076, Tu¨bingen, Germany
e-mail: wolfgang.voelter@uni-tuebingen.de
123

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Med Chem Res (2014) 23:1099–1113
protein tyrosine kinases (PTKs), and NADH oxidase, and
hence could be valuable as novel anticancer agents.
dimethylphenyl)-N0-(40-nitrophenyl)urea which showed
90.5 % inhibition of E. coli b-glucuronidase (IC50 SEM:
3.38 0.043 lM) (Table 1). In addition, structure–activ-
ity relationships (SAR), and various other biological
activities of novel urea derivatives were carried out.
Previously, enzyme inhibition studies have led to the
discovery of a wide variety of effective drugs. Specific
inhibitors interact with enzymes like ureases, phosphodi-
esterases, and glucuronidases, and block their activity
toward their corresponding natural substrates. Urease
decomposes urea to ammonia and carbamates, which
spontaneously decomposes to ammonia and carbonic acid.
Bacterial ureases are directly involved in the formation of
stones in the kidney and contribute to the pathogenesis of
pyelonephritis, encephalopathy, hepatic coma, and peptic
ulceration. Therefore, the strategies based on urease inhi-
bition are now considered as the first line of treatment for
infections caused by urease producing bacteria. In agri-
culture too, high urease activities cause significant envi-
ronmental and economic problems by releasing abnormally
large amounts of ammonia into the atmosphere during urea
fertilization. To reduce the problems encountered using
urea fertilizers, several approaches have been suggested
and the most promising one is to apply urease inhibitors
(Perveen, 2008, 2010, 2011; Perveen et al., 2008).
Results and discussion
Chemistry
For the synthesis of compounds 1–3, 5–13, 17–19, and 21,
o-, m-, and p-nitro phenyl isocyanates were treated with
aliphatic/aromatic primary and secondary amines in the
presence of 1,4-dioxane at room temperature (Scheme 1).
On the other hand for the preparation of symmetrical 1,3-
disubstituted urea ‘‘derivatives 14–16’’, o-, m-, and p-
nitrophenyl isocyanates treated with triethylamine in 1,4-
dioxane (Scheme 2), whereas for the synthesis of com-
pounds 4 and 20 (o- and m-nitrophenyl isocyanates,
respectively) were treated with N-phenylpiperazine and
2-pyrrolidone separately in 1,4-dioxane at room tempera-
ture with continuous stirring. Progress of reaction was
monitored by TLC. The reaction was completed in 3 h
(Scheme 3). After completion of the reaction, mixture was
poured into ice-cold water with continuous stirring. The
solids obtained were filtered, and after purification afforded
clear substituted urea ‘‘derivatives 1–13’’ and 17–21, and
symmetrical 1,3-disubstituted urea ‘‘derivatives 14–16’’.
The structures of synthetic compounds are characterized by
mass, NMR, IR, and UV spectroscopy, and melting points.
Finally, different biological and enzyme inhibition activi-
ties were determined.
A phosphodiesterase (PDE) is an enzyme that breaks a
phosphodiester bond and hydrolyzes cyclic nucleotides.
Thus, phosphodiesterases play a key role in regulating
intracellular levels of the second messengers cAMP (cyclic
adenosine monophosphate) and cGMP (cyclic guanosine
monophosphate), and hence cell function. Inhibitors of
PDE can prolong or enhance the effects of physiological
processes mediated by cAMP or cGMP by inhibition of
their degradation by PDE. Non-selective PDE inhibitors
including theophylline and papaverine have been used
therapeutically for a range of diseases for over 70 years.
Sildenafil, a phosphodiesterase inhibitor, used for treating
erectile dysfunction has made worldwide impact. Benzoyl
salireposide and other salireposides are pure non-compet-
itive inhibitors of phosphodiesterase from snake venom,
and are potential candidates for the therapy of arthritis
(Choudhary et al., 2004).
Antiproliferative and enzymes inhibition studies
Antiproliferative effects of compounds 1–21 against PC-3
human prostate and NCI-H460 human non-small cell lung
cancer cell lines
Glucuronidases are enzymes that catalyze the hydrolysis
of glucuronic acid residues from complex carbohydrates.
Human b-glucuronidase is located in the lysosomes of
cells, however, its high levels are present in necrotic areas
of large tumors (De Graaf et al., 2002). Glucuronide pro-
drugs hold promise for the treatment of cancer patients as
enhancement of their therapeutic effects may possibly be
achieved by techniques that target b-glucuronidase spe-
cifically at the site of the tumor. For example, a glucu-
ronidated prodrug of the nitrogen mustard bis-(2-
chloroethyl)amine was 27-fold more cytotoxic to colon
cancer cells than the parent drug (Ray and Chaturvedi,
2004; Shing-Ming et al., 1992). During the present study,
21 urea derivatives were synthesized including N-(2,6-
The use of in vitro human cancer cell lines is commonly
employed for the primary screening of compounds for their
potential cytotoxic/cytostatic activity. Several in vitro
assays have been developed for rapid assessment of cyto-
toxic or growth inhibitory activity, alone and in combina-
tions. The SRB assay is an efficient method for screening a
large number of samples within 7 days, the results are
linear over a 20-fold range of cell concentration and the
sensitivity is comparable to those of MTT colorimetric
method (Keepers et al., 1991). The amount of pink SRB
dye (sulforhodamine B) bound to the cells after staining
gives a measure of cell growth/growth inhibition and/or
cell killing (Boyd and Paull, 1995). Whereas, the MTT
123

Med Chem Res (2014) 23:1099–1113
1101
Table 1 Results of synthesized
symmetrical/unsymmetrical 1,
3-disubstituted ureas 1–21
1
2
3
4
5
6
7
8
9
10
11
12
123

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Med Chem Res (2014) 23:1099–1113
Table 1 continued
13
14
15
16
17
18
19
20
21
assay measures the activity of cellular enzymes that reduce
the yellow tetrazolium dye to its insoluble purple colored
formazan in living cells. This assay measures the cellular
metabolic activity via NAD(P)H-dependent cellular oxi-
doreductase enzymes. It allows assessing the viability and
the proliferation of cells since rapidly dividing cells exhibit
high rates of MTT reduction. It can also be used to measure
cytotoxicity or cytostatic activity of potential medicinal
agents since those agents would result in cell toxicity, and
therefore, metabolic dysfunction and decreased perfor-
mance in the assay (Yung, 1989; Kim et al., 2009).
All synthetic compounds 1–21 were screened for their
cytotoxicity against human prostate and lung cancer cell
lines using MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphe-
nyl-tetrazolium bromide) and sulforhodamine B (SRB)
assays according to literature protocols (Mosmann, 1983;
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Med Chem Res (2014) 23:1099–1113
1103
Scheme 1 Synthetic route for
unsymmetrical ureas
O
H
R
N
N C N
C
O
R'
1,4-Dioxane
r.t., 1 hr
+
R, R'NH
Primary/Sec. amine
O2N
O2N
R, R' = H, Alkyl, Aryl
o-,m-,p-Nitrophenyl isocynate
Unsymmetrical 1,3-disubstituted ureas
1-3, 5-13, 17-19 and 21
Scheme 2 Synthetic route for
symmetrical ureas
O
HN C NH
N
C
O
R3N
1,4-Dioxane
r.t.; 5-30 min
+
Tertiary Amine
R= Alkyl or Aryl
O2N
O2N
O2N
o-,m-,p-Symmetrical 1, 3-disubstituted ureas
o-,m-,p-Nitrophenyl isocynate
14-16
O
N
HN C
N
r
h
e
r
3
n
,
i
.
z
t
.
a
NO2
r
r
,
e
pe
n
e
i
a
p
x
4
l
o
-
y
N
i
C
O
n
D
-
h
4
,
P
1
N
-
P
O
1
,
2
4
D
i
r
o
r
x
a
n
d
HN C
N
e
,
r
-
.
O2N
O2N
t
.
,
y
3
o
h
l
i
o
n
e
o-,m-Nitrophenyl isocynate
O
20
Scheme 3 Synthetic routes for compounds 4 and 20
Monks et al., 1991). The standard drug doxorubicin was
used as positive control against both the cell lines.
Five compounds 8, 12, 15, ‘‘20 and’’ 21 were found to be
cytotoxic against PC-3 cell line (Table 2) having IC50 values
of 66, 78.5, 20, ‘‘55 and’’ 20 lM, respectively, using the
MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazo-
lium bromide) colorimetric assay (Mosmann, 1983). The
potency order regarding IC50 values was 21 = 15 > 20 >
8 > 12.
growth inhibition (62–95 %), and hence were further eval-
uated at five doses to determine their GI50, TGI, and LC50
values (Table 4). This study indicates that the compound 21
(GI50: 22, TGI: 36, and LC50: 66 lM) possesses consider-
able cytotoxic activity (Table 4), whereas compounds 3
(GI50: 12 lM), 12 (GI50: 18 lM), 8 (GI50: 34 lM), and 15
(GI50: 44 lM) exhibit moderate growth inhibitory activity.
The potency order regarding GI50 was 21–3–
12 > 8–15 > 6–9, and for TGI, it was 21 = 3 > 12 > 8. The
compound 21 was the only one for which LC50 could be
calculated, while the compound 12 exhibited 48 % cell
killing at 100 lM, hence its LC40 was found to be 91 lM.
Although, these compounds exhibited \20-fold activity
against both cell lines in comparison to doxorubicin which
is a highly effective anthracycline antibiotic used to treat a
All 21 synthesized urea derivatives were initially evalu-
ated against NCI-H460 (human non-small cell lung cancer)
cell lines at the dose of 100 lM (Table 3) using the sulfo-
rhodamine B (SRB) assay (Monks et al., 1991). The com-
pounds 3, 8, 12, and 21 exhibited 14–58 % cell killing at this
dose while compounds 6, 9, and 15 demonstrated substantial
123

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Med Chem Res (2014) 23:1099–1113
Table 2 Antiproliferative
effects of compounds 1–21
against PC-3 human prostate
cancer cell line
Compound no.
Compounds
IC50 (lM)
1
N-(2,6-Dimethylphenyl)-N0-(40-nitrophenyl)urea
N-(2,6-Dimethylphenyl)-N0-(2-nitrophenyl)urea
N-(2-Nitrophenyl)-N0-(10-phenylethyl)urea
N-(2-Nitrophenyl)-N0-(N-phenylpiprazinyl)urea
N-Butyl-N0-(20-nitrophenyl)urea
[100
2
[100
3
[100
4
[100
5
[100
6
N-(4-Chlorophenyl)-N0-(20-nitrophenyl)urea
N-(2,6-Dimethylphenyl)-N0-(30-nitrophenyl)urea
N-Butyl-N0-(30-nitrophenyl)urea
[100
7
[100
66.31 0.53a
8
9
N-(4-Chlorophenyl)-N0-(30-nitrophenyl)urea
N-(4-Nitrophenyl)-N0-(10-phenylethyl)urea
N-Isopropyl-N0-(20-nitrophenyl)urea
[100
10
11
12
13
14
15
16
17
18
19
20
21
[100
[100
78.49 1.2b
N-Butyl-N0-(40-nitrophenyl)urea
Data are mean SEM of three
independent experiments
analyzed by ANOVA
N-(4-Chlorophenyl)-N0-(40-nitrophenyl)urea
N-(2-Nitrophenyl)-N0-(20-nitrophenyl)urea
N-(3-Nitrophenyl)-N0-(30-nitrophenyl)urea
N-(4-Nitrophenyl)-N0-(40-nitrophenyl)urea
N-Isopropyl-N0-(30-nitrophenyl)urea
[100
[100
20.0 0.91c
For comparison, least significant
difference and Duncan’s
multiple range tests were used
[100
[100
In columns statistically different
values are represented by
dissimilar alphabetical
superscripts
N-Isopropyl-N0-(40-nitrophenyl)urea
[100
N-(2-Methylphenyl)-N0-(40-nitrophenyl)urea
N-(3-Nitrophenyl)-2-one-1-pyrrolidine carboxamide
N-(3-Nitrophenyl)-N0-(1-phenylethyl)urea
Doxorubicin
[100
55.02 1.74d
20.13 0.91c
0.912 0.12e
IC50 concentration of the
compound causing 50 % growth
inhibition of cells
wide array of cancers for over 30 years, but its use is
limited owing to its toxicity to most major organs, espe-
cially life-threatening cardiotoxicity, which forces the
treatment to become dose-limiting (Gibson et al., 2013).
Moreover, doxorubicin also induces apoptosis and necrosis
in other healthy tissues causing toxicity in the brain, liver,
and kidney (Tacar et al., 2013). On the other hand, these
compounds might lead to the development of effective and
less toxic novel types of anticancer agents/therapies.
demonstrate that in this series of compounds, the position of
nitro group at the phenyl ring and the substituent group at
the nitrogen of urea bridge induces a pronounced effect on
the antiproliferative activities of these compounds. Fur-
thermore, since the compound 21 demonstrated maximum
cytotoxicity against both cell lines (Tables 2, 4), therefore
has the potential to be developed as an anticancer agent.
Enzyme inhibition assays
Structure–activity relationship
Herein, all the 21 synthetic compounds were screened
against three clinically important enzymes i.e., b-glucu-
ronidase, urease, and phosphodiesterase.
It was remarkable that compounds 8, 12, 15, and 21
exhibited significant antiproliferative against both the cell
lines even when two different anticancer assays were
employed. It is suggested that the presence of a nitro group
at meta position in compounds 8 and 21, a nitro group at
para position in compound ‘‘12, and’’ a nitro group at meta
position at both the phenyl rings in the compound 15, which
is a symmetrical 1,3-disubstituted urea, could be responsi-
ble for their greater activity. The presences of nitro group at
meta and para positions causes less steric hindrance as
compared to its ortho analogs and another important thing
in all these compounds except 21 have an aliphatic alkyl
chain and alkyl benzylic group directly attached to nitrogen
of the urea bridge enhancing the activity. These results
b-Glucuronidase inhibition activity
Against b-glucuronidase (Table 5), N-(2,6-dimethyl-
phenyl)-N0-(40-nitrophenyl)urea (1) showed a 90 % inhibi-
tion with an IC50 = 3.38 lM, 14-fold more active than the
standard, D-saccharic acid 1,4-lactone (IC50 = 48.7
1.25 lM). The nitro group in para position of compound 1,
and an aryl group (2,6-dimethy phenyl) is directly attached
to the nitrogen of the urea bridge, and we assume that both
the structural properties are responsible for the b-glucu-
ronidase activity. Two isomers, N-(2,6-dimethylphenyl)-N0-
(20-nitrophenyl)urea (2), and N-(2,6-dimethylphenyl)-N0-
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Med Chem Res (2014) 23:1099–1113
1105
Table 3 Antiproliferative
effects of compounds 1–21 at
Compound no. Compounds
% Growth inhibition/cytotoxicity
the dose of 100 lM against
NCI-H460 human non-small
cell lung cancer cell line
1
N-(2,6-Dimethylphenyl)-N0-(40-nitrophenyl)urea
?28
?47
-23
?22
?10
?95
?08
-14
?62
?02
?04
-48
?06
?02
?68
?01
?03
?01
?05
2
N-(2,6-Dimethylphenyl)-N0-(2-nitrophenyl)urea
N-(2-Nitrophenyl)-N0-(10-phenylethyl)urea
N-(2-Nitrophenyl)-N0-(N-phenylpiprazinyl)urea
N-Butyl-N0-(20-nitrophenyl)urea
N-(4-Chlorophenyl)-N0-(20-nitrophenyl)urea
N-(2,6-Dimethylphenyl)-N0-(30-nitrophenyl)urea
N-Butyl-N0-(30-nitrophenyl)urea
N-(4-Chlorophenyl)-N0-(30-nitrophenyl)urea
N-(4-Nitrophenyl)-N0-(10-phenylethyl)urea
N-Isopropyl-N0-(20-nitrophenyl)urea
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
N-Butyl-N0-(40-nitrophenyl)urea
N-(4-Chlorophenyl)-N0-(40-nitrophenyl)urea
N-(2-Nitrophenyl)-N0-(20-nitrophenyl)urea
N-(3-Nitrophenyl)-N0-(30-nitrophenyl)urea
N-(4-Nitrophenyl)-N0-(40-nitrophenyl)urea
N-Isopropyl-N0-(30-nitrophenyl)urea
N-Isopropyl-N0-(40-nitrophenyl)urea
N-(2-Methylphenyl)-N0-(40-nitrophenyl)urea
N-(3-Nitrophenyl)-2-one-1-pyrrolidine carboxamide ?02
N-(3-Nitrophenyl)-N0-(1-phenylethyl)urea
-58
? Growth inhibition, -
cytotoxicity
Table 4 Antiproliferative effects of compounds 3, 6, 8, 9, 12, 15, and 21 against NCI-H460 human non-small cell lung cancer cell line
Compound no.
Compounds
GI50
12 1.7a
59 8.5b
34 7c
TGI (lM)
LC50
3
N-(2-Nitrophenyl)-N0-(10-phenylethyl)urea
N-(4-Chlorophenyl)-N0-(20-nitrophenyl)urea
N-Butyl-N0-(30-nitrophenyl)urea
35 1a
[100
87 2.4b
[100
6
[100
8
[100
9
N-(4-Chlorophenyl)-N0-(30-nitrophenyl)urea
N-Butyl-N0-(40-nitrophenyl)urea
N-(3-Nitrophenyl)-N0-(30-nitrophenyl)urea
N-(3-Nitrophenyl)-N0-(1-phenylethyl)urea
Doxorubicin
68 10.2b
18 8.8a
44 6.7c
22 2.6a
0.05 0.01d
[100
[100
12
15
21
65 4.0c
[100
91 2.6a,*
[100
66 3.0b
0.8 0.1c
36 2.6a
0.25 0.01d
Data are mean SEM of three independent experiments analyzed by ANOVA
For comparison, least significant difference and Duncan’s multiple range tests were used in columns statistically different values are represented
by dissimilar alphabetical superscripts
GI50 concentration of the compound causing 50 % growth inhibition of cells
TGI concentration of the compound causing total growth inhibition of cells
LC50 lethal concentration of the compound that killed 50 % of cells
LC40 lethal concentration of the compound that killed 40 % of cells
* 91 ? 2.6
(30-nitrophenyl)urea (7), in which a nitro group is attached at
ortho and meta positions, respectively, were inactive. The
difference in inhibitory potential of these compounds clearly
indicates that the presences of a para nitro group is a pre-
requisite for inhibition of the b-glucuronidase enzyme,
along with a 2,6-dimethyl group on the other phenyl residue.
Conclusively, compound 1 seems to be a promising com-
pound. The rest of the compounds showed less than 50 %
inhibition, and therefore were not evaluated for their IC50
values.
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Table 5 b-Glucuronidase, urease, and phosphodiesterase activities of unsymmetrical/symmetrical 1,3-disubstituted urea derivatives 1–21
Compound no.
Compounds
b-Glucuronidase (%)
Urease (%)
16.3
Phosphodiesterase (%)
NA
1
N-(2,6-Dimethylphenyl)-N0-(40-nitrophenyl) 90.5
urea
2
N-(2,6-Dimethylphenyl)-N0-(20-nitrophenyl) NA
urea
45.9
NA
3
4
N-(2-Nitrophenyl)-N0-(10-phenylethyl)urea
N-(2-Nitrophenyl)-N0-(N-phenylpiprazinyl)
urea
NA
10.9
29.1
NA
N.A
15.6
5
6
7
N-Butyl-N0-(20-nitrophenyl)urea
N-(4-Chlorophenyl)-N0-(20-nitrophenyl)urea NA
N-(2,6-Dimethylphenyl)-N0-(30-nitrophenyl) NA
urea
24.25
38.2
7.0
18.0
23.8
NA
35.1
8
N-Butyl-N0-(30-nitrophenyl)urea
N-(4-Chlorophenyl)-N0-(30-nitrophenyl)urea NA
28.4
7.9
NA
NA
24.4
N.A
26.1
16.5
NA
NA
48.9
NA
26.0
NA
NA
9
NA
10
11
12
13
14
15
16
17
18
19
20
N-(4-Nitrophenyl)-N0-(10-phenylethyl)urea
N-Isopropyl-N0-(20-nitrophenyl)urea
N-Butyl-N0-(40-nitrophenyl)urea
NA
NA
NA
43.7
5.0
42.1
N-(4-Chlorophenyl)-N0-(40-nitrophenyl)urea NA
38.2
N-(2-Nitrophenyl)-N0-(20-nitrophenyl)urea
N-(3-Nitrophenyl)-N0-(30-nitrophenyl)urea
N-(4-Nitrophenyl)-N0-(40-nitrophenyl)urea
N-Isopropyl-N0-(30-nitrophenyl)urea
NA
NA
N.A
NA
35.9
NA
30.35
IC50 = 1.25 lM
NA
0.2
N-Isopropyl-N0-(40-nitrophenyl)urea
N-(2-Methylphenyl)-N0-(40-nitrophenyl)urea NA
NA
NA
N-(3-Nitrophenyl)-2-one-1-pyrrolidine
carboxamide
NA
21
N-(3-Nitrophenyl)-N0-(1-phenylethyl)urea
D-Sacchuric acid,1,4-lactonea
NT
IC50 SEM:d
6.4
NT
48.7 1.25 lM
Thioureab
EDTAc
IC50 SEM:
21.0 0.01 lM
IC50 SEM:
274.0 0.007 lM
NA not active, NT not tested
a
Standard inhibitor for b-glucuronidase
b
Standard inhibitor for urease
c
Standard inhibitor for phosphodiesterase
d
Standard error mean
Urease inhibition activity
bridge the most active compound is 2 (46 %) with nitro
group at ortho position of phenyl ring. When an alkyl
group (butyl) is present at the nitrogen of urea bridge in
compounds 5, 8, and 12, the most active compound is 12
(42 %) having a nitro group at para position. Compounds
6, 9, and 13 with 4-chlorophenyl group at nitrogen of urea
bridge; among them, compound 13 with nitro group at para
position is active (38 %). A number of urea derivatives
were reported to test for urease and a-chymotrypsin
inhibitory properties; it was found that the symmetrical N-
In assays of urease, we found that compounds 2, 5, 7, 10,
12, 13, and 15 showed % inhibition of 46, 38, 35, 44, 42,
38, and 30, respectively, (Table 5). Compounds 3, 10, and
21 which are o, p, and m derivatives, respectively, having
1-phenylethyl group at the nitrogen of urea bridge, among
them the most active compound is 10 with nitro group at
para position (44 %). In compounds 1, 2, and 7, the 2,6-
dimethyl phenyl group is present at the nitrogen of urea
123

Med Chem Res (2014) 23:1099–1113
1107
(4-nitrophenyl)-N0-(40-nitrophenyl)urea 16 is a potent ure-
ase inhibitor with IC50 value of 1.25 lM (Perveen, 2008,
2010, 2011; Perveen et al., 2008).
The extent of MTT reduction to formazan within cells was
calculated by measuring the absorbance at 570 nm, using a
microplate ELISA reader (Spectra Max plus, Molecular
Devices, CA, USA). The cytotoxicity was recorded as
concentration causing 50 % growth inhibition for 3T3 cells
(Mosmann 1983).
These results demonstrate that the position (para) and
number of nitro group play vital role for urease inhibition.
Phosphodiesterase inhibition activity
Sulforhodamine B (SRB) assay
In case of phosphodiesterase enzymes inhibition, the same
compound N-(4-nitrophenyl)-N0-(40-nitrophenyl)urea (16)
which is symmetrical 1,3-disubstituted urea having nitro
group at para position in the phenyl ring is most active
showed 49 % inhibition while compounds 6, 10, 12, and
18 showed percentage inhibitions 24, 24, 26, and 26
(Table 5) less than 30 %, and are therefore not evaluated
for their IC50 values and considered to be inactive. Fig-
ure 1 demonstrates the activity of compounds 1–21
against b-glucuronidase, urease, and phosphodiesterase
enzymes.
The sulforhodamine B (SRB) protein staining assay was
employed for the evaluation of in vitro growth inhibition and
cytotoxicity (Monks et al., 1991). About 1 9 104 NCI-H460
cells were added in each well (100 lL) of 96-well plates, and
then incubated for 24 h leading to the formation of a
monolayer. Subsequently, the test compounds were added in
five tenfold dilutions and the plates were incubated for an
additional 48 h. This was followed by fixation of cells with
ice-cold trichloroacetic acid (50 lL). The plates were then
stained using SRB dye which was solubilized in Tris-base
solution, and then the optical density measurements were
measured at 545 nm. Initially, all the compounds were tested
at a single dose (100 lM). The compounds displaying more
than 50 % growth inhibition were further tested at five doses
(1–100 lM) to obtain their GI50, TGI, and LC50 values. Drug
concentrations causing a growth inhibition of 50 % (GI50),
total growth inhibition (TGI), and killing 50 % cells (LC50)
were calculated from dose response curves. Doxorubicin
(anthracycline anticancer drug) was used as positive control
(GI50 = 0.05, TGI = 0.25, LC50 = 0.8 lM).
Cytotoxicity assays
MTT assay
Cytotoxic activity of compounds was evaluated in 96-well
flat-bottomed microplates by using the standard MTT (3-
[4,5-dimethylthiazole-2-yl]-2,5-diphenyl-tetrazolium bro-
mide) colorimetric assay. For this purpose, PC-3 prostate
cancer cells were cultured in Dulbecco’s Modified Eagle’s
Medium, supplemented with 5 % of fetal bovine serum
(FBS), 100 IU/mL of penicillin, and 100 lg/mL of strep-
tomycin in 25 cm3 flasks, and kept in 5 % CO2 incubator at
37 °C. Exponentially growing cells were harvested, counted
with haemocytometer, and diluted with a particular medium.
Cell culture with the concentration of 1 9 105 cells/mL was
prepared and introduced (100 lL/well) into 96-well plates.
After overnight incubation, medium was removed and
200 lL of fresh medium was added with different concen-
trations of compounds (1–100 lM). After 72 h, 50 lL MTT
(2 mg/mL) was added to each well and incubated further for
4 h subsequently, 100 lL of DMSO was added to each well.
Statistical analysis
The data are expressed as the mean SEM for three
experiments. Statistical comparisons between the various
treatments were performed using SPSS 17 statistical soft-
ware, and the data were evaluated by Analysis of Variance
(ANOVA). Comparisons of data were conducted by least
significant difference (LSD) and Duncan’s multiple range
tests. P values of \0.05 were considered statistically
significant.
Fig. 1 Effect of synthetic
compounds 1–21 against b-
glucuronidase, urease, and
β-Glucuronidase
Urease
Phosphodiesterase
100
phosphodiesterase enzymes
50
0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21
Compound
EDTA
D-SaT.h..iourea
123

1108
Med Chem Res (2014) 23:1099–1113
Enzyme inhibition assays
Urease inhibition assay
After 30 min pre-incubation of the enzyme with the test
samples, the enzyme activity was monitored spectropho-
tometrically at 37 °C on a microtitre plate reader (Spectra
Max, Molecular Devices, CA, USA) by following the rate
(change in OD/min) of release of p-nitrophenol from p-
nitrophenyl phosphate at 410 nm. All assays were con-
ducted in triplicate (Mamillapalli et al., 1998).
Reaction mixtures comprising one unit of urease solution
in water (Bacillus pasteurii or Jack bean), and 55 lL of
buffers (0.01 M K2HPO4ꢀ3H2O, 1 mM EDTA, 0.01 M
LiCl; pH 8.2) containing 100 mM urea were incubated
with 5 lL of test compounds (1 mM) at 30 °C for 15 min
in 96-well plates.
Urease inhibitory activity was determined by measur-
ing ammonia production using the indophenol method.
Briefly, 45 lL of phenol reagent (1 % w/v phenol and
0.005 % w/v sodium nitroprusside) and 70 lL of alkali
reagent (0.5 % w/v NaOH and 0.1 % w/v NaOCl) were
added to each well. The increasing absorbance at 630 nm
was measured after 50 min, using a microplate reader
(Molecular Devices, USA). All reactions were performed
in triplicate in a final volume of 200 lL. The results
(change in absorbance per min) were processed by using
Soft-Max Pro software (Molecular Devices, CA, USA)
(Khan et al., 2004).
Experimental
Melting points were taken on a Gallenkamp melting point
apparatus. Thin layer chromatography was performed on
pre-coated silica gel plates (Kieselgel 60 F254, E. Merk,
Germany) and spots were visualized under UV (Dual
range, UK-Germany) at 254 and 365 nm and/or by spray-
ing cerium sulfate (heat). EI MS were performed on MAT-
312 and on JEOL JMS-HX 110 instruments. 1H NMR
spectra were recorded on a Bruker AVANCE instruments
at 400 or 500 MHz, d is given in ppm related to SiMe4
(0 ppm) as internal standard. All the solvents used were of
reagent grade. Bioscreenings for urease, b-glucuronidase,
phosphodiesterase inhibition, and cytotoxicity were carried
in various laboratories of the H. E. J. Research Institute of
Chemistry, International Center for Chemical and Biolog-
ical Sciences, University of Karachi, Pakistan. However,
the compounds were synthesized in Pharmaceutical
Research Centre, PCSIR Laboratories Complex, Karachi.
b-Glucuronidase inhibition assay
b-Glucuronidase activity was determined by measuring the
absorbance at 405 nm of p-nitrophenol formed from the
substrate (50 lL of 0.4 mM p-nitrophenyl-b-D-glucuro-
nide) by the spectrophotometric method. 185 lL of 0.1 M
acetate buffer (13.608 g/L of sodium acetate, pH adjusted
to 5.0 with 0.1 M acetic acid), 5 lL (0.5 mM) of test
compound solution, and 10 lL of enzyme solution (30 U)
were incubated at 37 °C for 30 min. The plates were read
on a multiplate reader (Spectra Max plus 384) at 405 nm
after the addition of 50 lL of 0.4 mM p-nitrophenyl-b-D-
glucuronide counting for a total reaction volume of
250 lL. All the assays were performed in triplicate (Riaz
et al., 2003).
Chemistry
General procedure for the synthesis of symmetrical
(14–16) and unsymmetrical 1,3-disubstituted ureas (1–13
and 17–21)
Unsymmetrical 1,3-disubstituted ureas 1–3, 5–13, 17–19,
and 21 were synthesized (Table 1) by weighing 1.4 mol of
primary/secondary amine in a round-bottom flask con-
taining 20–25 mL of 1,4-dioxane. 0.2 Mol of ortho-, meta-,
and para-nitrophenyl isocyanates, respectively, were added
dropwise at room temperature separately with continuous
stirring. The reaction was completed in 1 h (Scheme 1).
For the preparation of the symmetrical 1,3-disubstituted
ureas 14–16, 0.3 mol of ortho-, meta-, and para-nitro-
phenyl isocyanates, respectively, in 20–25 mL of 1,4-
dioxane, 2 mol of triethyl amine were added at room
temperature (Scheme 2) (Hai et al., 2003), and the progress
of reaction was monitored via TLC. For the preparation of
compounds 4 and 20, 1.4 mol of N-phenylpiperazine and
2-pyrrolidone, respectively, were taken in a round-bottom
flask containing 20-25 mL of 1,4-dioxane. Then, 0.2 mol
of ortho- and meta-nitrophenyl isocyanates were added
Phosphodiesterase inhibition assay
Activity against snake venom phosphodiesterase (PDE)
was assayed by a modified method of (Mamillapalli et al.,
1998) by mixing 97 lL (33 mM) Tris–HCl buffer pH 8.8,
20 lL (30 mM) Mg-acetate with 0.000742 U/well of final
enzyme concentration. A microtiter plate assay was applied
and 60 lL (0.33 mM) of bis-(p-nitrophenyl) phosphate
(Sigma N-3002) was used as substrate and 8 lL (0.5 mM)
as test compound. 8 lL (0.5 mM) of EDTA (Merck,
Darmstadt) were used as positive controls (1C50 = 748
lM 0.015, 274 lM 0.007, respectively).
123

Med Chem Res (2014) 23:1099–1113
1109
dropwise at room temperature with continuous stirring.
After completion of the reaction in 3 h (Scheme 3), the
mixture was poured into ice-cold water with continuous
stirring, the solid was filtered and crystallized from an
appropriate solvent to obtain the desired product.
(38)—13C NMR (125 MHz, CDCl3) d; 155.50 (C=O),
139.27 (C-10), 134.29 (C-2), 133.33 (C-5), 131.95 (C-1),
127.62 (C-40), 126.64 (C-30, 50), 126.63 (C-20, 60), 121.83
(C-3), 119.82 (C-4), 119.35 (C-6), 52.38 (CH), 20.95
(CH3)—Anal. Calcd for C15H15N3O3: (285): C, 63.15; H,
5.30; N, 14.73 %. Found: C, 63.19; H, 5.28; N, 14.75 %.
N-(2,6-Dimethylphenyl)-N0-(40-nitrophenyl)urea (1) Rf =
0.65 (CH2Cl2/C6H14, 9:1). IR (KBr):mmax: 3,326, 1,663,
1,540, 1,499, 1,332, 1,230, 1,103, 837, 751 cm-1. 1H NMR
N-(2-Nitrophenyl)-N0-(1-phenylpiprazinyl)urea (4) Rf =
0.76 (CH2Cl2/C6H14, 7:3). IR (KBr): mmax: 3,366, 2,835,
1,666, 1,599, 1,589, 1,499, 1,450, 1,344, 1,270, 1,229,
1,139 cm-1. 1H NMR (400 MHz, DMSO) d; 9.3 (1H, br.s,
0
0
0
0
(4000 MHz, CDCl3) d; 8.13 (2H, d, J3 ,2 =5 ,6 = 9.0 Hz,
0
0
0
H3 ,5 ), 7.49 (2H, d, J2 ,3 =6 ,5 = 9.0 Hz, H2 ,6 ), 7.22 (2H, d,
J3,4=5,4 = 9.0 Hz, H3,5), 7.19 (1H, t, J4,(3,5) = 9.0 Hz, H4),
6.41 (1H, br.s, NH), 5.94 (1H, br.s, NH), 2.32 (6H, s,
2CH3)—EI MS: m/z (rel. abund. %), 285 (M?, 89), 270 (3),
255 (4), 164 (5), 147 (100), 138 (88), 132 (19), 121 (72),
108 (56), 92 (25)—13C NMR (125 MHz, CDCl3) d; 159.46
(C=O), 144.34 (C-10), 140.80 (C-40), 138.96 (C-1), 131.16
(C-2,6), 128.45 (C-3,5), 125.10 (C-30,50), 123.19 (C-4),
116.92 (C-20,60), 18.18 (2CH3)—Anal. Calcd for
C15H15N3O3: (285): C, 63.15; H, 5.30; N, 14.73 %. Found:
C, 63.18; H, 5.28; N, 14.75 %.
0
0
0
0
NH), 7.92 (2H, d, J3,4=6,5 = 8.0 Hz, H3,60), 7.66 (2H, dd,
0
J3 ,2 =5 6 = 8.1, J3 ,4 =5 4 = 7.0 Hz, H3 ,5 ), 7.23 (2H, t,
0
0
0
0
0
0
0 0
J4,(3,5)=5,(4,6) = 08.0 Hz, H4,5), 6.97 (2H, d, J2 ,3 =6 ,5
0
0
0
0
0
0
= 8.1 Hz, H2 ,6 ), 6.81 (1H, t, J4 ,(3 ,5 ) = 7.0 Hz, H4 ), 3.60
0
0
0
00 00
(4H, t, J1 ,2 = 4.9 Hz, 2CH2), 3.17 (4H, t,
00 00
J2 ,1 = 4.9 Hz, 2CH2)—EI MS: m/z (rel. abund. %), 326
(M?, 1), 162 (27), 147 (7), 132 (35), 119 (28), 105 (76),
104 (86), 91 (100)—13C NMR (125 MHz, CDCl3) d;
153.32 (C=O), 151.33 (C-10 phenyl), 148.04 (C-3), 137.74
(C-1), 129.84 (C-5), 129.10 (C-30, 50 phenyl), 123.82 (C-6),
120.30 (C-40 phenyl), 115.93 (C-20, 60 phenyl), 112.43 (C-
2), 50.66 (2CH2-300, 500 piprazine), 45.22 (2CH2-200, 600
piprazine)—Anal. Calcd for C17H18N4O3: (326): C, 62.57;
H, 5.56; N, 17.17 %. Found: C, 62.54; H, 5.58; N,
17.19 %.
N-(2,6-Dimethylphenyl)-N0-(20-nitrophenyl)urea (2) Rf =
0.60 (CH2Cl2/C6H14, 6:4). IR (KBr): mmax: 3,322, 3,256,
1,646, 1,585, 1,548, 1,499, 1,336, 1,283, 1,225, 1,152,
1
1,090, 858, 764 cm1. H NMR (500 MHz, CD0Cl3) d; 9.63
(1H, br.s, NH), 8.75 (1H, d, J3 ,4 = 8.5 Hz, H3 ), 8.10 (1H,
0
0
0
d, J6 ,5 = 8.2 Hz,0 H6 ), 7.60 (1H, dt, J5 ,(4 ,6 ) = 7.3,
N-Butyl-N0-(20-nitrophenyl)urea (5) Rf = 0.71 (CH2Cl2/
CH3OH, 9:1). IR (KBr): mmax: 3,342, 2,970, 1,665, 1,507,
1,465, 1,340, 1,262, 1,148, 743 cm-1. 1H NMR (400 MHz,
0
0
0
0
0
J5 ,3 = 0.8 Hz, H5 ), 7.26 (1H, m, H4), 7.19 (2H, br.d,
0
0
J3,4=5,4 = 7.3 Hz, H3,5), 7.03 (1H, dt, J4 ,(3 ,5 ) = 7.3,
0
0
0
0
J4 ,6 = 0.9 Hz, H4 ), 6.03 (1H, br.s, NH), 2.33 (3H, s,
CH3), 1.50 (3H, s, CH3)—EI MS: m/z (rel. abund. %), 285
(M?, 45), 255 (2), 239 (5), 222 (2), 164 (5), 147 (89), 138
(100), 121 (70), 106 (26), 92 (38)—13C NMR (125 MHz,
CDCl3) d; 156.16 (C=O), 136.33 (C-1), 134.68 (C-20),
133.74 (C-50), 133.25 (C-10), 131.16 (C-2, 6), 128.45 (C-
3,5), 123.19 (C-4), 122.23 (C-30), 120.07 (C-40), 119.75 (C-
60), 18.18 (2CH3)—Anal. Calcd for C15H15N3O3: (285): C,
63.15; H, 5.30; N, 14.73 %. Found: C, 63.12; H, 5.32; N,
14.71 %.
CDCl3) d; 9.76 (1H, br.s, NH), 8.64 (1H, d, J3 ,4 = 8.5 Hz,
0
0
0
0
0
0
H3 ), 8.16 (1H, dd, J6 ,5 = 8.5, J6 ,4 = 1.1 Hz, H6 ), 7.57
(1H, dt, J4 ,(3 ,5 ) = 8.5, J4 ,6 = 1.1 Hz, H4 ), 7.02 (1H, dt,
0
0
0
0
0
0
0
0
0
0
0
J5 ,(6 ,4 ) = 8.5, J5 ,3 = 0.6 Hz, H5 ), 4.76 (1H, s, NH), 3.29
(2H, q, J1,(2,NH) = 7.3 Hz, CH2), 1.56 (2H, qin,
J2,(1,3) = 7.3 Hz, CH2), 1.39 (2H, sex, J3,(2,4) = 7.3 Hz,
CH2), 0.95 (3H, t, J4,3 = 7.3 Hz, CH3)—EI MS: m/z (rel.
abund. %), 237 (M?, 3), 148 (4), 139 (7), 138 (100), 122
(3), 108 (19), 92 (29)—13C NMR (125 MHz, CDCl3) d;
151.49 (C=O), 134.42 (C-20), 133.33 (C-50), 131.64 (C-10),
121.83 (C-30), 119.82 (C-40), 119.49 (C-60), 42.47 (CH2-1),
32.00 (CH2-2), 20.00 (CH2-3), 13.70 (CH3)—Anal. Calcd
for C11H15N3O3: (237): C, 55.69; H, 6.37; N, 17.71 %.
Found: C, 55.66; H, 6.40; N, 17.73 %.
0
0
0
0
0
N-(2-Nitrophenyl)-N0-(10-phenylethyl)urea (3) Rf = 0.81
(CH2Cl2/C6H14, 9:1). IR (KBr): mmax: 3,374, 3,305, 1,658,
1,552, 1,503, 1,315, 1,225, 1,111, 849, 747 cm-1. 1H NMR
(400 MHz, CDCl3) d; 9.72 (2H, br.s, NH), 8.62 (1H, d,
J3,4 = 8.4 Hz, H3), 8.14 (1H, d, J6,5 = 7.3 Hz, H6), 7.53
(1H, t, J5,(4,6) = 7.3 Hz, H5), 7.36 0 (4H, t,
N-(4-Chlorophenyl)-N0-(20-nitrophenyl)urea
(6) Rf =
0.68 (CH2Cl2/CH3OH, 9:1). IR (KBr): mmax: 3,334, 16,667,
1,618, 1,585, 1,540, 1,491, 1,334, 1,344, 1,274, 1,205,
0
0
0
J2 ,(3 ,1 )=3 ,(2 ,4 )=4 ,(3 ,5 ),5 ,(4 ,6 ) = 6.1 Hz, H2 ,3 ,4 ,5 ), 7.29
0
0
00
0
0
0
0
0
0
0
0
0
0
(1H, dd, J6 ,5 = 6.1, J6 ,4 = 2.4 Hz, H6 ), 7.01 (1H, t,
1,095, 1,017 cm-1. H NMR (400 MHz, CDCl3) d; 9.97
1
0
0
0
0
0
J4(3,5) = 7.3 Hz, H4), 5.00 (1H, sex, J1 (2 ,2 ,6 ) = 6.7 Hz,
(1H, br.s, NH), 8.62 (1H, d, J3 ,4, = 8.5 Hz, H3 ), 8.18 (1H,
00 00
0
0
0
0
CH), 1.52 (3H, q, J2 ,(1 ,2 ,6 ) = 6.7 Hz, CH3)—EI MS: m/
z (rel. abund. %), 285 (M?, 6), 268 (2), 164 (57), 147 (31),
138 (89), 132 (86), 120 (10), 106 (100), 92 (33), 90
d, J6 ,5 = 7.5 Hz, H6 ), 7.61 (1H, t, J4 ,(3 ,5 ) = 7.5 Hz,
00
00
0
0
0
0
0
0
0
0
H4 ), 7.37 (2H, d, J3,4=5,6 = 8.7 Hz, H3,5), 7.32 (2H, d,
J2,3=6,5 = 8.7 Hz, H2,6), 7.09 (1H, t, J5 ,(4 ,6 ) = 7.5 Hz,
0
0
0
123

1110
Med Chem Res (2014) 23:1099–1113
0
H5 ), 6.76 (1H, br.s, NH)—EI MS: m/z (rel. abund. %), 293
NH), 6.45 (1H, br.s, NH)—EI MS: m/z (rel. abund. %), 293
(M?2, 4), 291 (M?, 16), 165 (5), 155 (3), 153 (12), 138
(31), 127 (100), 111 (13), 99 (21), 92 (40)—13C NMR
(125 MHz, CDCl3) d; 156.55 (C=O), 148.10 (C-30), 138.74
(C-10), 138.03 (C-1), 129.90 (C-50), 129.33 (C-3, 5), 128.52
(C-4), 123.14 (C-60), 121.49 (C-2, 6), 115.95 (C-40), 111.75
(C-20)—Anal. Calcd for C13H10N3O3Cl: (293): C, 53.63;
H, 3.46; N, 14.43 %. Found: C, 53.60; H, 3.48; N,
14.41 %.
(M2?, 4), 291 (M?, 11), 155 (6), 153 (21), 148 (4), 138
(100), 127 (90), 125 (15), 111 (9), 108 (14), 92 (39), 90
(52)—13C NMR (125 MHz, CDCl3) d; 153.26 (C=O),
135.41 (C-1), 134.68 (C-20), 133.74 (C-50), 133.24 (C-10),
129.33 (C-3, 5), 128.52 (C-4), 122.23 (C-30), 121.49 (C-20,
60), 120.07 (C-40), 119.75 (C-60)—Anal. Calcd for
C13H10N3O3Cl: (293) C, 53.63; H, 3.46; N, 14.43 %.
Found: C, 53.60; H, 3.44; N, 14.40 %.
N-(2,6-Dimethylphenyl)-N0-(30-nitrophenyl)urea (7) Rf =
0.74 (CH2Cl2/C6H14, 6:4). IR (KBr): mmax: 3,326, 1,638,
N-(4-Nitrophenyl)-N0-(10-phenylethyl)urea
(10) Rf =
0.74 (CH2Cl2/CH3OH, 9:1). IR (KBr): mmax: 3,350, 3,277,
2,978, 1,659, 1,565, 1,503, 1,450, 1,344, 1,283,
1,524, 1,340, 1,217, 1,074, 764, 730 cm-1
.
1H NMR
0
(400 MHz, CDCl3) d; 8.06 (1H, br.s, H2 ),0 7.84 (2H, dt,
1,156 cm-1. H NMR (400 MHz, CDCl3) d; 8.11(2H, d,
1
0
0
0
0
0
0
0
0
0
J
= 7.9, J6 ,(2 ,4 ) = 1.6 Hz, H5 ,6 ), 7.41 (1H,0 t,
J3,2=5,6 = 9.1 Hz, H3,5), 7.41 (2H, d, J2,3=6,5 = 9.1 Hz,
5 ,(4 ,6 )=6 ,5
0
0
J4,(3,5) = 8.2 Hz, H4), 7.20 (1H, d, J4 ,5 = 7.9 Hz, H4 ),
7.19 (2H, m, H3,5), 6.27 (1H, br.s, NH), 5.87 (1H, br.s,
NH), 2.34 (6H, s, 2CH3)—EI MS: m/z (rel. abund. %), 285
(M?, 92), 270 (2), 255 (2), 238 (3), 164 (4), 147 (86), 138
(97), 121 (100), 106 (49), 92 (41)—13C NMR (125 MHz,
CDCl3) d; 159.46 (C=O), 148.10 (C-30), 138.96 (C-1),
138.75 (C-10), 131.16 (C-2,6), 129.90 (C-50), 128.45 (C-3,
5), 123.19 (C-4), 123.14 (C-60), 115.95 (C-40), 111.75 (C-
20), 18.18 (2 CH3)—Anal. Calcd for C15H15N3O3: (285): C,
63.15; H, 5.30; N, 14.73 %. Found: C, 63.17; H, 5.35; N,
14.70 %.
H2,6), 7.35 (2H, d, J2 ,3 =6 ,5 = 5.9 Hz, H2 ,6 ), 7.34 (1H, m,
0
0
0
0
0
0
0
H4 ),0 7.29 (2H, dd, J3 ,2 =5 ,6 = 5.9, J3 ,5 =5 ,3 = 2.4 Hz,
0
0
0
0
0
0
0
0
0
H3 ,5 ), 6.57 (1H, br.s, NH), 4.93 (1H, quin,
00
J1 ,(2 ,NH) = 6.6 Hz, CH), 3.68 (1H, s, NH), 1.52 (3H, t,
00
00
00
0
J2 ,(1 ,2 ) = 3.2 Hz, CH3)—EI MS: m/z (rel. abund. %), 285
(M?, 2), 270 (4), 164 (5), 148 (53), 138 (98), 132 (17), 120
(11), 105 (100), 92 (17)—13C NMR (125 MHz, CDCl3) d;
158.79 (C=O), 143.04 (C-1), 140.55 (C-4), 139.27 (C-10),
127.62 (C-40), 126.64 (C-30, 50), 126.36 (C-20, 60), 124.70
(C-3, 5), 116.52 (C-2, 6), 52.38 (CH), 20.95 (CH3)—Anal.
Calcd for C15H15N3O3: (285): C, 63.15; H, 5.30; N,
14.73 %. Found: C, 63.21; H, 5.33; N, 14.69 %.
N-Butyl-N0-(30-nitrophenyl)urea (8) Rf = 0.72 (CH2Cl2/
C6H14, 6:4). IR (KBr): mmax: 3,342, 2,942, 1,712, 1,638,
N-Isopropyl-N0-(20-nitrophenyl)urea
(11) Rf = 0.65
(CH2Cl2/C6H14, 7:3). IR (KBr): mmax: 3,396, 2,960, 1,641,
1,524, 1,422, 809, 735 cm-1. 1H NMR (400 MHz, CDCl3)
0
d; 8.14 (1H, t, J2 ,(4 ,6 ) = 2.0 Hz, H2 ), 7.85 (1H, dd,
1,557, 1,503, 1,348, 1,285, 734 cm-1. 1H NMR (400 MHz,
0
0
0
0
J6 ,5 = 8.1, J6 ,4 = 1.4 Hz, H6 ), 7.78 (1H, dd, J4 ,5 = 8.01,
CDCl3) d; 9.71 (1H, br.s, NH), 8.63 (1H, d, J3 ,4 = 8.4 Hz,
0
0
0
0
0
0
0
0
0
0
0
J4 ,6 = 1.4 Hz, H4 ), 7.41 (1H, t, J5 ,(4 ,6 ) = 8.1 Hz, H5 ),
6.50 (1H, br.s, NH), 4.66 (1H, br.s, NH), 3.27 (2H, q,
J1,(2,NH) = 6.9 Hz, CH2), 1.53 (2H, qin, J2,(1,3) = 6.9 Hz,
CH2), 1.37 (2H, sex, J3,(2,4) = 7.2 Hz, CH2), 0.93 (3H, t,
J4,3 = 7.2 Hz, CH3)—EI MS: m/z (rel. abund. %), 237
(M?, 3), 270 (3), 165 (1), 138 (100), 108 (7), 92 (59), 65
(24)—13C NMR (125 MHz, CDCl3) d; 154.79 (C=O),
147.69 (C-30), 137.14 (C-10), 129.50 (C-50), 122.88 (C-60),
115.70 (C-40), 111.49 (C-20), 42.47 (CH2-1), 32.00 (CH2-
2), 20.00 (CH2-3), 13.70 (CH3)—Anal. Calcd for
C11H15N3O3: (237): C, 55.69; H, 6.37; N, 17.71 %. Found:
C, 55.71; H, 6.34; N, 17.68 %.
H3 ), 8.16 (1H, dd, J6 ,5 = 8.4, J6 ,4 = 0.9 Hz, H6 ), 7.56
0
0
0
0
0
0
0
0
0
0
(1H, t, J5 ,(4 ,6 ) = 8.4 Hz, H5 ), 7.01 (1H, t,
0
0
0
0
J4 ,(3 ,5 ) = 8.4 Hz, H4 ), 4.70 (1H, d, JNH,1 = 5.4 Hz, NH),
3.98 (1H, sept, J1,(2a,2b) = 6.5 Hz, CH), 1.22 (6H, d,
J2a,1=2b,1 = 6.5 Hz, 2CH3)—EI MS: m/z (rel. abund. %),
223 (M?, 23), 167 (4), 164 (6), 149 (19), 138 (100), 92
(68), 85 (15), 83 (16), 69 (22), 57 (37)—13C NMR
(125 MHz, CDCl3) d; 155.86 (C=O), 134.53 (C-20), 133.33
(C-50), 131.48 (C-10), 121.83 (C-30), 119.82 (C-40), 119.59
(C-60), 42.26 (CH-1), 23.73 (2CH3)—Anal. Calcd for
C10H13N3O3: (223): C, 53.78; H, 5.87; N, 18.83 %. Found:
C, 53.71; H, 5.82; N, 18.88 %.
0
0
0
N-(4-Chlorophenyl)-N0-(30-nitrophenyl)urea
(9) Rf =
0.88 (CH2Cl2/C6H14, 6:4). IR (KBr): mmax: 3,363, 1,687,
N-Butyl-N0-(40-nitrophenyl)urea (12) Rf = 0.67 (CH2Cl2/
C6H14, 7:3). IR (KBr): mmax: 3,379, 3,305, 2,845, 1,650,
1,593, 1,540, 1,487, 1,344, 1,230, 1,095, 878 cm-1. H
1,556, 1,503, 1,319, 1,225, 1,111, 849, 747, 678 cm-1. H
1
1
0
0
0
NMR (500 MHz, CDCl3) d; 8.21 (1H, t, J2 ,(4 ,6 ) = 1.9 Hz,
NMR (400 MHz, CDCl3) d; 9.35 (1H, br.s, NH), 8.15 (2H,
0
0
0
H2 ), 7.91 (2H, dd, J2,3=6,5 = 8.4, J2,6=6,2 = 1.4 Hz, H2,6),
d, J3 ,2 =5 ,6 = 8.7 Hz, H3 ,5 ), 7.50 (2H, d, J2 ,3
0
0
0
0
0
0
0
0
7.82 (2H, dd, J3,2=5,6 = 8.4, J3,5=5,3 = 1.3 Hz, H3,5), 7.4
= 8.7 Hz, H2 ,6 ), 6.68 (1H, br.s, NH), 3.27 (2H, t,
=60,50
0
(1H, d, J4 ,5 = 8.1 Hz, H4 ), 7.44 (1H, dd, J6 ,5 = 8.5,
J1,(2,NH) = 7.2 Hz, CH2), 1.52 (2H, qin, J2,(1,3) = 7.2 Hz,
CH2), 1.36 (2H, sex, J3,(2,4) = 7.2 Hz, CH2), 0.92 (3H, t,
0
0
0
0
0
0
J6 ,4 = 1.7 Hz, H6 ), 7.32 (1H, m, H5 ), 6.66 (1H, br.s,
0
0
123

Med Chem Res (2014) 23:1099–1113
1111
J4,3 = 7.2 Hz, CH3)—EI MS: m/z (rel. abund. %), 237
(M?, 17), 219 (2), 219 (2), 207 (2), 164 (6), 138 (100), 122
(7), 108 (30), 92 (17), 78 (21)—13C NMR (125 MHz,
CDCl3) d; 154.79 (C=O), 142.73 (C-10), 140.55 (C-40),
124.70 (C-30, 50), 116.66 (C-20, 60), 42.47 (CH2-1), 32.00
(CH2-2), 20.00 (CH2-3), 13.70 (CH3)—Anal. Calcd for
C11H15N3O3: (237): C, 55.69; H, 6.37; N, 17.71 %. Found:
C, 55.71; H, 6.39; N, 17.68 %.
302 (M?, 3), 164 (23), 138 (87), 118 (15), 108 (6), 106
(12), 92 (73), 90 (97), 65 (100)—13C NMR (125 MHz,
CDCl3) d; 156.55 (C=O), 148.10 (C-3, 30), 138.74 (C-1, 10),
129.90 (C-5, 50), 123.14 (C-6, 60), 115.95 (C-4, 40), 111.75
(C-2, 20)—Anal. Calcd for C13H10N4O5: (302): C, 51.66;
H, 3.33; N, 18.54 %. Found: C, 51.64; H, 3.30; N,
18.51 %.
N-(4-Nitrophenyl)-N0-(40-nitrophenyl)urea (16) IR (KBr):
N-(4-Chlorophenyl)-N0-(40-nitrophenyl)urea (13) Rf =
0.70 (CH2Cl2/C6H14, 6:4). IR (KBr): mmax: 3,366, 1,723,
1
mmax: 3,286, 3,190, 1,649, 1,600, 1,556, 1,312 cm-1. H
NMR (400 MHz, CD3OD) d; 9.62 (2H, br.s, NH), 8.22
1,595, 1,539, 1,492, 1,345, 1270, 1173, 1113, 835 cm-1
.
0
0
0
0
0
0
0
0
(4H, dd, J3,2=5,6=3 ,2 =5 ,6 = 8.1, J3,6=5,2=3 ,6 =5 ,2 = 0.2 Hz,
1H NMR (400 MHz, DMSO) d; 9.45 (1H, br.s, NH), 9.04
0
0
H3,5
,
H3 ,5 ), 8.13 (4H, dd, J2,3=6,5=2 ,3 =6 ,5 = 8.1,
0 0 0 0
(1H, br.s, NH), 8.19 (2H, d, J3,2=5,6 = 9.3 Hz, H3,5), 7.67
0
0
J2,5=6,3=2 ,5 =6 ,3 = 0.2 Hz, H2,6, H2 ,6 )—EI MS: m/z (rel.
abund. %), 302 (M?, 6), 257 (15), 181 (20), 162 (100), 123
(55)—13C NMR (125 MHz, CDCl3) d; 156.55 (C=O),
144.33 (C-1, 10), 140.80 (C-4, 40), 125.10 (C-3, 5, 30, 50),
116.92 (C-2, 6, 20, 60)—Anal. Calcd for C13H10N4O5:
(302): C, 51.66; H, 3.34; N, 18.54 %. Found: C, 51.59; H,
3.28; N, 18.47 %.
0
0
0
0
(2H, dd, J2,3=6,5 = 9.3, J2,6=6,2 = 01.4 Hz, H2,6), 7.49 (2H,
0
d,
J2 ,3 =6 ,5 = 8.8 Hz,
H2 ,6 ),
7.35
(2H,
d,
0
0
0
0
0
0
J3 ,2 =5 ,6 = 8.8 Hz, H3 ,5 )—EI MS: m/z (rel. abund. %),
293 (M?2, 4), 291 (M?, 11), 164 (21), 155 (48), 153 (100),
138 (81), 129 (18), 127 (72), 125 (69), 108 (54), 92 (28), 90
(52), 65 (33)—13C NMR (125 MHz, CDCl3) d;156.55
(C=O), 144.33 (C-10), 140.80 (C-40), 138.03 (C-1), 129.33
(C-3, 5), 128.52 (C-4), 125.10 (C-30,50), 121.49 (C-1, 6),
116.92 C-10, 60)—Anal. Calcd for C13H10N3O3Cl: (293): C,
53.63; H, 3.46; N, 14.43 %. Found: C, 53.68; H, 3.39; N,
14.46 %.
0
0
0
0
N-Isopropyl-N0-(30-nitrophenyl)urea (17) Rf = 0.72
(CH2Cl2/C6H14, 7:3). IR (KBr): mmax: 3,326, 1,638, 1,565,
1
1,527, 1,345, 728 cm-1. H NMR (400 MHz, CDCl3) d;
0
8.16 (1H, t, J2 ,(4 ,6 ) = 1.7 Hz, H2 ), 7.84 (1H, dd,
0
0
0
0
J4 ,5 = 8.1, J4 ,6 = 1.2 Hz, H4 ), 7.76 (1H, dd, J6 ,5 = 8.01,
N-(2-Nitrophenyl)-N0-(20-nitrophenyl)urea (14) Rf = 0.70
(CH2Cl2/C6H14, 6:4). IR (KBr): mmax: 3,366, 1,659, 1,589,
1,556, 1,499, 1,344, 1,344, 1,288, 1,209, 1,160, 849, 739,
727, 682 cm-1. 1H NMR (400 MHz, CDCl3) d; 10.13 (1H,
br.s, NH), 8.55 (10H, d, J3,4 = 7.5 Hz, H3), 8.23 (1H, d,
0
0
0
0
0
0
0
J6 ,4 = 1.2 Hz, H6 ), 7.41 (1H, t, J5 ,(4 ,6 ) = 8.1 Hz, H5 ),
6.30 (1H, br.s, NH), 4.39 (1H, br.s, NH), 3.98 (1H, sept,
J1,(2a,2b) = 6.6 Hz, CH), 1.21 (6H, d, J2a,1=2b,1 = 6.6 Hz,
2CH3)—EI MS: m/z (rel. abund. %), 223 (M?, 51), 164 (8),
139 (84), 138 (100), 92 (100), 80 (35), 58 (22)—13C NMR
(125 MHz, CDCl3) d; 159.15 (C=O), 147.69 (C-30), 136.98
(C-10), 129.50 (C-50), 122.98 (C-60), 115.70 (C-40), 111.59
(C-20), 42.26 (CH-1), 23.73 (2CH3)—Anal. Calcd for
C10H13N3O3: (223): C, 53.78; H, 5.87; N, 18.83 %. Found:
C, 53.81; H, 5.91; N, 18.86 %.
0
0
0
0
0
J3 ,4 = 7.6 Hz, H3 ), 8.10 (1H, d, J6,5 = 8.5 Hz, H6), 7.65
0
0
(1H, t, J4,(3,5) = 7.5 Hz, H4), 7.34 (1H, t, J5,(4,6) = 7.5 Hz,
0
H5), 7.16 (1H, t, 0 J4 ,(3 ,5 ) = 7.6 Hz, H4 ), 6.78 (1H, d,
0
0
0
0
J6 ,5 = 8.3 Hz, H6 ), 6.69 (1H, t, J5 ,(4 ,6 ) = 7.6 Hz, H5 ),
6.01 (1H, br.s, NH)—EI MS: m/z (rel. abund. %), 302 (M?,
2), 256 (1), 164 (5), 138 (80), 121 (7), 92 (63), 90 (59), 80
(34), 78 (12), 77 (10), 76 (6), 66 (19), 65 (100), 52
(13)—13C NMR (125 MHz, CDCl3) d; 149.96 (C=O),
134.68 (C-2, 20), 133.74 (C-5, 50), 130.62 (C-1, 10), 122.23
(C-3, 30), 120.07 (C-4, 40), 119.75 (C-6, 60)—Anal. Calcd
for C13H10N4O5: (302): C, 51.66; H, 3.33; N, 18.54 %.
Found: C, 51.68; H, 3.31; N, 18.52 %.
0
0
0
0
0
N-Isopropyl-N0-(40-nitrophenyl)urea (18) Rf = 0.75
(CH2Cl2/C6H14, 7:3). IR (KBr): mmax: 3,395, 3,302, 2,926,
1,662, 1,551, 1,507, 1,324, 1,236, 855 cm-1
.
1H NMR
0
0
0
0
(4000 MHz, CDCl3) d; 8.14 (1H, d, J3 ,2 =5 ,6 = 9.1 Hz,
0
0
0
H3 ,5 ), 7.49 (2H, d, J2 ,3 =6 ,5 = 9.0 Hz, H2 ,6 ), 6.56 (1H,
br.s, NH), 4.54 (1H, d, JNH,1 = 6.5 Hz, NH), 3.99 (1H,
sept, J1,(2a,2b) = 6.5 Hz, CH), 1.20 (6H, d, J2a,1=2b,1
= 6.5 Hz, 2CH3)—EI MS: m/z (rel. abund. %), 223 (M?,
52), 165 (184), 138 (100), 108 (36), 92 (98)—13C NMR
(125 MHz, CDCl3) d; 159.15 (C=O), 142.57 (C-10), 140.55
(C-40), 124.70 (C-30, 50), 116.76 (C-20, 60), 42.26 (CH-1),
23.73 (2CH3)—Anal. Calcd for C10H13N3O3: (223): C,
53.78; H, 5.87; N, 18.83 %. Found: C, 53.83; H, 5.82; N,
18.89 %.
0
0
0
0
N-(3-Nitrophenyl)-N0-(30-nitrophenyl)urea (15) Rf = 0.77
(CH2Cl2/C6H14, 7:3). IR (KBr): mmax: 3,346, 1,720, 1,691,
1,524, 1,417, 1,352, 809, 756, 682 cm-1 1H NMR
.
0
0
0
(400 MHz, CDCl3) d; 8.02 (2H, t, J2,(4,6)=2 ,(4 ,6 ) = 1.9 Hz,
0
H2,2 ), 7.78 (2H, dd, J4,5=4 ,5 = 8.2, J4,6=4 ,6 = 1.6 Hz,
0
0
0
0
0
H4,4 ), 7.80 (2H, dd, J6,5=6 ,5 = 8.2, J6,4=6 ,4 = 01.6 Hz,
0
0
0
0
0
H6,6 ), 7.38 (2H, t, J5,(4,6)=5 ,(4 ,6 ) = 8.2 Hz, H5,5 ), 7.23
0
0
0
(1H, s, NH), 3.08 (1H, s, NH)—EI MS: m/z (rel. abund. %),
123

1112
Med Chem Res (2014) 23:1099–1113
N-(2-Methylphenyl)-N0-(40-nitrophenyl)urea (19) Rf = 0.73
(CH2Cl2/C6H14, 7:3). IR (KBr): mmax: 3,300, 1,692, 1,496,
21 with IC50 value of 20 lM against PC-3 and GI50 of
22 lM against NCI-H460 was most active against both the
cell lines. These results revealed that the position, number
of nitro groups, and presence of an aryl group play a vital
role for the antiproliferative activities of these compounds.
While for the b-glucuronidase, the position of nitro
group at para is a precondition for inhibition of b-glucu-
ronidase enzyme along with aryl group on the second
phenyl ring of the urea bridge.
1,298, 1,178, 1,114, 846 cm-1 1H NMR (500 MHz,
.
0
0
0
0
CDCl3 & CD3OD) d; 8.06 (2H, d, J3 ,2 =5 ,6 = 9.2 Hz,
0
0
H3 ,5 ), 7.63 (1H, d, 0 J3,4 = 7.5 Hz, H3), 7.51 (2H, d,
0
J2 ,3=6 ,5 = 9.2 Hz, H2 ,6 ), 7.10 (1H, d, J6,5 = 7.5 Hz, H6),
7.08 (1H, t, J5,(4,6) = 7.5 Hz, H5), 6.93 (1H, t,
J4,(3,5) = 7.5 Hz, H4)—EI MS: m/z (rel. abund. %), 271
(M?, 41), 164 (1), 138 (40), 107 (100), 91 (42), 65
(47)—13C NMR (125 MHz, CDCl3) d; 158.00 (C=O),
144.33 (C-10), 140.80 (C-40), 137.93 (C-1), 132.58 (C-3),
129.88 (C-2), 125.94 (C-5), 125.10 (C-30, 50), 122.67 (C-6),
122.88 (C-4), 116.92 (C-20, 60), 17.49 (CH3)—Anal. Calcd
for C14H13N3O3: (271): C, 61.99; H, 4.83; N, 15.50 %.
Found: C, 61.86; H, 4.80; N, 15.47 %.
0
0
0
Acknowledgments Authors gratefully acknowledge the Higher
Education Commission (HEC) for granting fellowship to Ph. D. stu-
dent Ms. Sana Mustafa. Dr. Shahnaz Perveen, and Prof. Dr. Khalid M.
Khan are thankful to Deutscher Akademischer Austauschdienst
(DAAD, Bonn, Germany) for a short term scholarship.
N-(3-Nitrophenyl)-20-one-1-pyrrolidine
carboxamide
References
(20) Rf = 0.56 (CH2Cl2/C6H14, 6:4). IR (KBr): mmax
:
3,300, 1,527, 1,343, 1,229, 1,172, 878, 735, 698 cm-1. H
NMR (400 MHz, CDCl3) d; 8.25 (1H, br.s, H2), 7.95 (1H, d,
J4,5 = 7.9 Hz, H4), 7.84 (1H, d, J6,5 = 7.9 Hz, H6), 7.50
(1H, t, J5,(4,6) = 7.9 Hz, H5), 6.77 (1H, br.s, NH), 3.47 (2H,
1
Boyd MR, Paull KD (1995) Some practical considerations and
applications of the NCI in vitro anticancer drug discovery screen.
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0
0
d, J5 ,4 = 5.6 Hz, H5 ), 1.24 (2H, br.s, H4 ), 0.85 (2H, m,
0
0
0
H3 )—EI MS: m/z (rel. abund. %), 249 (M?, 30), 165 (10),
164 (83), 138 (100), 136 (40), 112 (82), 106 (18), 92
(82)—13C NMR (125 MHz, CDCl3) d; 171.93 (C=O),
154.03 (C=O), 147.61 (C-3), 137.25 (C-1), 129.41 (C-5),
122.10 (C-6), 116.40 (C-4), 110.72 (C-2), 47.99 (CH2-5,
pyrrolidine), 30.68 (CH2-4, pyrrolidine), 16.59 (CH2-3, pyr-
rolidine)—Anal. Calcd for C11H11N3O4: (249): C, 53.01; H,
4.45; N, 42.02 %. Found: C, 52.99; H, 4.42; N, 42.00 %.
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Choudhary MI, Fatima N, Muhammad AA, Jalil S, Ahmed VU, Atta-
ur-Rahman (2004) Phenolic glycosides, a new class of human
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Efferth T, Kahl S, Paulus K, Adams M, Rauh R, Boechzelt H, Hao X,
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ics of natural products derived from traditional Chinese medicine
and Chinese materia medica with activity against tumor cells.
Mol Cancer Ther 7:152–161
N-(3-Nitrophenyl)-N0-(1-phenylethyl)urea (21) Rf = 0.62
(CH2Cl2/CH3OH, 9:1). IR (Solid): mmax: 3,366, 3,313, 2,361,
1,654, 1,556, 1,352, 1,233, 1,021 cm-1
.
1H NMR
(400 MHz, CDCl3) d 8.08 (1H, t, J2,(4,6) = 1.4 Hz, H2), 7.82
(1H, dd, J4,5 = 8.3, J4,2 = 1.4 Hz, H4), 7.68 (1H, dd,
J6,5 = 8.3, J6,2 = 1.2 0H0z,0 H6), 7.39 (1H, t, J5,(4,6) = 8.3 Hz,
Fortin S, Bouchon B, Chambon C, Lacroix J, Moreau E, Chezal JM,
Degoul F, C-Gaudreault R (2011) Characterization of the
covalent binding of N-phenyl-N0-(2-chloroethyl)ureas to b-
Tubulin: importance of Glu 198 in microtubule stability.
J Pharmacol Exp Ther 336(2):460–467
0
0
H5), 7.36 (4H, m, H2 ,3 ,5 ,6 ), 7.28 (1H, m, H4 ), 6.38 (2H,
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cin-induced vascular dysfunction and its attenuation by exercise
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amines promoted synthesis of symmetrical 1,3-disubstituted
ureas. Nat Prod Res 17:351–354
Keepers YP, Pizao PE, Peters GJ, van Ark-Otte J, Winograd B, Pindo
HM (1991) Comparison of sulforhodamine B protein and
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00 00
br.s, NH), 4.92 (1H, m, CH), 1.52 (3H, d, J2 ,1 = 7.4 Hz,
CH3))—EI MS: m/z (rel. abund. %), 285 (M?, 25), 270 (3),
227 (1), 181 (1), 164 (5), 138 (100), 120 (5), 105 (92), 92
(17)—13C NMR (125 MHz, CDCl3) d; 158.79 (C=O),
147.69 (C-3), 139.27 (C-10), 137.45 (C-1), 129.50 (C-5),
127.62 (C-40), 126.64 (C-30,50), 126.63 (C-20, 60), 122.74 (C-
6), 115.70 (C-4), 111.35 (C-2), 52.38 (CH), 20.95 (CH3)—
Anal. Calcd for C15H15N3O3: (285): C, 63.15; H, 5.30; N,
14.73 %. Found: C, 63.20; H, 5.31; N, 14.68 %.
Conclusion
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