Discovery of nitroaryl urea derivatives with antiproliferative properties

Article abstract of DOI:10.3109/14756366.2015.1057716

A series of urea derivatives bearing nitroaryl moiety has been synthesized and assayed for their potential antiproliferative activities. Some of the tested compounds displayed activity in RK33 laryngeal cancer cells and TE671 rhabdomyosarcoma cells while being generally less toxic to healthy HSF human fibroblasts cells. One compound was demonstrated to be a moderate CDK2 inhibitor with IC₅₀ = 14.3 μM. Its structure was solved by an X-ray crystallography and molecular modelling was performed to determine structure-activity relationship. Obtained compounds constitute novel structures and generally demonstrated greater cytotoxicity in comparison to cisplatin. This study offers new structural motifs with potential for further development.

Full text of DOI:10.3109/14756366.2015.1057716

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Discovery of nitroaryl urea derivatives with
antiproliferative properties
Tomasz M. Wróbel, Michał Kiełbus, Agnieszka A. Kaczor, Vladimír Kryštof,
Zbigniew Karczmarzyk, Waldemar Wysocki, Andrzej Fruziński, Sylwia K. Król,
Aneta Grabarska, Andrzej Stepulak & Dariusz Matosiuk
To cite this article: Tomasz M. Wróbel, Michał Kiełbus, Agnieszka A. Kaczor, Vladimír Kryštof,
Zbigniew Karczmarzyk, Waldemar Wysocki, Andrzej Fruziński, Sylwia K. Król, Aneta Grabarska,
Andrzej Stepulak & Dariusz Matosiuk (2015): Discovery of nitroaryl urea derivatives with
antiproliferative properties, Journal of Enzyme Inhibition and Medicinal Chemistry
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1057716
Published online: 26 Jun 2015.
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–11
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1057716
!
RESEARCH ARTICLE
Discovery of nitroaryl urea derivatives with antiproliferative properties
4
1
2
1,3
5
Tomasz M. Wr o´ bel , Michał Kiełbus , Agnieszka A. Kaczor , Vladim ´ı r Kry sˇ tof , Zbigniew Karczmarzyk ,
5
6
2
2
2
1
Waldemar Wysocki , Andrzej Fruzi n´ ski , Sylwia K. Kr o´ l , Aneta Grabarska , Andrzej Stepulak , and Dariusz Matosiuk
1
2
Department of Synthesis and Chemical Technology of Pharmaceutical Substances and Department of Biochemistry and Molecular Biology,
3
4
Medical University of Lublin, Lublin, Poland, School of Pharmacy, University of Eastern Finland, Kuopio, Finland, Laboratory of Growth Regulators,
Centre of the Region Han a´ for Biotechnological and Agricultural Research, Institute of Experimental Botany ASCR & Palack y´ University, Olomouc,
5
6
Czech Republic, Department of Chemistry, Siedlce University of Natural Sciences and Humanities, Siedlce, Poland, and Institute of General and
Ecological Chemistry, Technical University of Lodz, Lodz, Poland
Abstract
A series of urea derivatives bearing nitroaryl moiety has been synthesized and assayed for their Antiproliferative, cancer, CDK inhibitor,
Keywords
potential antiproliferative activities. Some of the tested compounds displayed activity in RK33
laryngeal cancer cells and TE671 rhabdomyosarcoma cells while being generally less toxic to
healthy HSF human fibroblasts cells. One compound was demonstrated to be a moderate CDK2
inhibitor with IC50 ¼ 14.3 mM. Its structure was solved by an X-ray crystallography and molecular
modelling was performed to determine structure-activity relationship. Obtained compounds
constitute novel structures and generally demonstrated greater cytotoxicity in comparison to
cisplatin. This study offers new structural motifs with potential for further development.
molecular modelling, synthesis
History
Received 17 February 2015
Revised 22 April 2015
Accepted 19 May 2015
Published online 26 June 2015
Introduction
regulation of transcription in response to a mitogenic stimulation.
This mainly occurs due to phosphorylation and subsequent inacti-
vation of retinoblastoma protein (Rb) resulting in de-repression of
Cancer is the leading cause of death worldwide accounting for 8.2
million deaths in 2012 and it is expected that 14.1 million of all
cancer cases in 2012 will rise to 22 million in the next two
14
multiple genes involved in DNA synthesis and mitosis . There are
15
many compounds spanning from broad-range inhibitors such
1
decades . Currently available options for treatments of cancer
16 17
as flavopidirol and roscovitine to selective inhibitors such as
involve surgery, radiotherapy and pharmacotherapy, but the
general efficacy is not satisfying. Therefore there is an ongoing
need for development of new and more effective drugs.
18 19
palbociclib (PD-033299) or PHA-767491 .
Although 20 CDK inhibitors have entered clinical trials, none
has been approved for therapy yet. This prompted us to look for a
scaffold which would combine structural features of known CDK
Protein kinases are well-established targets for anticancer drug
2
,3
discovery . They regulate diverse cellular functions by phos-
phorylating threonine, serine and tyrosine residues at specific
20
inhibitors together with urea moiety . Our intention was further
supported with findings of urea derivatives active towards
4
positions in proteins . Protein kinases can be altered in many
disorders such as cancer and therefore are attractive molecular
21
CDKs (Scheme 2). The aim of our work was synthesis,
5
targets in drug discovery programmes . These programmes
in vitro studies and molecular modelling of novel nitroaryl urea
derivatives (NUD) which displayed promising antiproliferative
properties. We chose compounds with different substituents to be
able to investigate possible structure-activity relationship.
yielded so far several small molecule inhibitors of protein kinases
that have been approved for clinical applications in oncology
6
sunitinib , imatinib , gefitinib , dasatinib – Scheme 1).
7
8
9
(
There is an ever increasing body of evidence linking CDK-
1
0,11
Experimental
related malfunction with cancer development
. CDKs (cyclin
dependent kinases) are enzymes that have been identified origin- Synthesis
1
ally to play a key role in regulating the cell cycle machinery . They
2
1
3
All reagents were obtained from Sigma-Aldrich (St. Louis, MO)
and used without further purification unless stated otherwise.
NMR spectra were aqcuired on Bruker AVANCE III 600 MHz
and Bruker Fourier 300 MHz spectrometers (Fallanden,
Switzerland). Solvents for NMR spectroscopy were purchased
from Armar Chemicals (Dottingen, Switzerland). MS spectra
were recorded on Bruker micrOTOF-Q II spectrometer using
direct probe method. TLC analysis was carried out on Merck
HPTLC Silica gel 60 F254 plates (Darmstadt, Germany) pre-
washed with MeOH. Merck Silica gel 60 15–40 mm particle size
constitute a family of proteins assigned Cdk1–Cdk20 . Similar
other protein kinases, CDKs also have two-lobed structure with
the active site sandwiched between an amino-terminal lobe
consisting of beta-sheets and a carboxy-terminal lobe based on
1
2
alpha-helices . Activities of CDKs are dependent on regulatory
subunits – proteins called cyclins. The function of CDKs lies in
Address for correspondence: Tomasz M. Wr o´ bel, Department of
Synthesis and Chemical Technology of Pharmaceutical Substances,
Medical University of Lublin, Chodzki 4a, 20-093 Lublin, Poland. Tel:
2
2
+
48-81-448-72-73. E-mail: tomasz.wrobel@umlub.pl
was used for DCVC chromatography .

2
T. M. Wr o´ bel et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
N
N
N
HO
N
O
N
NH
N
N
H
OH
F
N
HN
Sunitinib
N
O
Imatinib
O
N
N
O
N
N
OH
N
HN
Cl
N
N
N
S
Cl
NH
N
F
OH
Gefitinib
Dasatinib
Scheme 1. Structures of sunitinib, imatinib, gefitinib and dasatinib.
OH
NH NH
O
Cl
NH NH
O
N
N
Cl
44 µM
36 µM
OH
NH NH
N
O
7
.6 µM
2
1
Scheme 2. Urea compounds active in CDK assay with their IC50 values .
N-(4-nitrophenyl)ethane-1,2-diamine (3)
N-(5-nitropyridin-2-yl)ethane-1,2-diamine (4)
1
0 g of 1-chloro-4-nitrobenzene (1) (0.06 mol) and 40 g of 4.8 g of 2-chloro-5-nitropyridine (2) (0.03 mol) was carefully
ethylenediamine (0.6 mol) were refluxed together for 90 minutes. added portionwise to a chilled ethylenediamine 40 g (0.6 mol).
Excess of ethylenediamine was distilled and the remaining residue The mixture was stirred for 30 minutes and excess of
was dissolved in hot water, filtered while hot and allowed to cool. ethylenediamine was removed by distillation. The residue was
Crystals formed very quickly. They were filtered, washed with chromatographed over Silica gel with MeOH–DCM–25%
water and air dried. Thus 9.6 g (83%) of orange solid was NH aq 10–90–1 to afford 3.8 g of orange solid (69%) with
3
ꢀ
24
obtained. All analytical data corresponds to that found in the m.p. 124–126 C in accordance to the literature data .
2
literature m.p. 148–150 C. H NMR (300 MHz, DMSO-d ) ꢀ
3
ꢀ
1
1
H NMR (600 MHz, METHANOL-d ) ꢀ 8.93 (d, J ¼ 2.75 Hz,
6
4
7
2
.89–8.07 (m, 2H), 7.30 (br t, J ¼ 4.66 Hz, 1H), 6.56–6.72 (m, 1H), 8.14 (br d, J ¼ 8.36 Hz, 1H), 6.55 (d, J ¼ 9.35 Hz,
1
3
H), 3.13 (q, J ¼ 6.08 Hz, 2H), 2.73 (t, J ¼ 6.29 Hz, 2H), 1.60 (br 1H), 3.53 (br s, 2H), 2.87 (t, J ¼ 6.33 Hz, 2H); C NMR
1
3
s, 2H); C NMR (75 MHz, DMSO-d ) ꢀ 155.2, 135.9, 126.7, (151 MHz, METHANOL-d ) ꢀ 161.7, 146.2, 135.1, 131.7,
6
4
1
11.1, 46.5, 41.1.
107.9, 43.7, 40.4.

DOI: 10.3109/14756366.2015.1057716
Nitroaryl urea derivatives
3
General procedure to obtain compounds 5a–5d and 6a–6f
DMSO-d ) ꢀ 158.7, 158.5, 155.2, 136.1, 133.2, 128.8, 126.7,
6
1
14.1, 111.2, 55.5, 43.5, 42.9, 39.1.
1
mmol of N-(4-nitrophenyl)ethane-1,2-diamine (3) or N-(5-nitro-
pyridin-2-yl)ethane-1,2-diamine (4) was dissolved in a suitable
solvent and solution of 1 mmol of isocyanate in the same solvent
was added dropwise at room temperature. The mixture was stirred
1-Naphthalen-1-yl-3-{2-[(5-nitropyridin-2-yl)amino]ethyl}
urea (6a)
for 30 minutes at RT. Formed precipitate was collected and Compound 6a was obtained using 1-naphthyl isocyanate and ACN
purified as indicated for each individual compound.
-Naphthalen-1-yl-3-{2-[(4-nitrophenyl)amino]ethyl}urea (5a)
as solvent. Product was recrystallized from aqueous MeOH. TLC
on Silica gel MeOH–CHCl –NH 25%aq 10–90–1 R ¼ 0.39.
3
3
f
1
Elemental analysis C H N O Calcd. C(61.53%) H(4.88%)
18 17 5 3
N(19.93%) exp. C(61.15%) H(4.87%) N(19.62%). HRMS
[
Compound 5a was obtained using 1-naphthyl isocyanate and ACN
as solvent. Product was recrystallized from AcOH. TLC on Silica
gel EtOAc–hexane 8–2 R ¼ 0.41. Elemental analysis C H N O
+
M + H] Calcd. 352.1404; exp. 352.1403. H NMR (600 MHz,
1
DMSO-d ) ꢀ 8.92 (d, J ¼ 2.68 Hz, 1H), 8.56 (br. s., 1H), 8.23–
6
f
19 18 4 3
8
.34 (m, 1H), 8.12 (br. s., 1H), 8.03–8.06 (m, 1H), 7.93–7.97 (m,
1H), 7.87–7.92 (m, 1H), 7.57 (d, J ¼ 8.13 Hz, 1H), 7.49–7.55 (m,
H), 7.43 (t, J ¼ 7.85 Hz, 1H), 6.68–6.73 (m, 1H), 6.61 (d,
Calcd. C(65.13%) H(5.18%) N(15.99%) exp. C(64.86%) H(5.11%)
+
1
N(15.91%). HRMS [M + H] Calcd. 351.1452; exp. 351.1453. H
2
NMR (600 MHz, DMSO-d ) ꢀ 8.63 (s, 1H), 8.05–8.08 (m, 1H),
6
13
C
J ¼ 8.59 Hz, 1H), 3.57 (br. s., 2H), 3.38 (q, J ¼ 6.07 Hz, 2H);
8
.00–8.04 (m, 2H), 7.96 (dd, J ¼ 0.80, 7.60 Hz, 1H), 7.88–7.92
NMR (151 MHz, DMSO-d ) ꢀ 162.1, 156.3, 147.3, 135.5, 134.8,
6
(
(
(
m, 1H), 7.58 (d, J ¼ 8.20 Hz, 1H), 7.50–7.56 (m, 2H), 7.42–7.46
1
m, 2H), 6.71–6.76 (m, 3H), 3.32–3.40 (m, 4H); C NMR
3
134.2, 132.2, 128.8, 126.4, 126.2, 125.9, 122.8, 121.9, 117.4,
1
09.2, 41.6, 39.3.
151 MHz, DMSO-d ) ꢀ 156.4, 155.1, 136.2, 135.4, 134.2, 128.8,
6
1
4
26.7, 126.4, 126.3, 126.2, 125.9, 122.8, 122.0, 117.5, 111.2,
3.2, 39.0.
1-(4-Acetylphenyl)-3-{2-[(5-nitropyridin-2-yl)amino]ethyl}
urea (6b)
1
-(4-Acetylphenyl)-3-{2-[(4-nitrophenyl)amino]ethyl}urea (5b)
Compound 6b was obtained using freshly purified 4-acetylphenyl
isocyanate and ACN as solvent. Product was recrystallized from
Compound 5b was obtained using freshly purified 4-acetylphenyl
isocyanate (commercial reagent was heated with carbon tetra-
chloride, filtered and the solvent evaporated under vacuo leaving
pure isocyanate) and THF as solvent. Product was washed with
hot AcOH and subsequently with MeOH. TLC on Silica gel
EtOAc–hexane 8–2 R ¼ 0.24. Elemental analysis C H N O
acetone/cyclohexane. TLC on Silica gel MeOH–CHCl –NH3
3
2
C(55.97%) H(4.99%) N(20.40%) exp. C(55.91%) H(4.93%)
5%aq 10–90–1 R ¼ 0.25. Elemental analysis C H N O Calcd.
f
16 17 5 4
+
N(20.32%). HRMS [M + H] Calcd. 344.1353; exp. 344.1354.
1
H NMR (600 MHz, DMSO-d ) ꢀ 9.00 (br. s., 1H), 8.92 (d,
6
f
17 18 4 4
J ¼ 2.42 Hz, 1H), 8.24 (br. s., 1H), 8.11 (br. s., 1H), 7.83–7.87 (m,
13
Calcd. C(59.64%) H(5.30%) N(16.37%) exp. C(59.54%) H(5.32%)
+
1
N(16.13%). HRMS [M + H] Calcd. 343.1401; exp. 343.1402. H
2H), 7.50–7.54 (m, 2H), 6.59 (d, J ¼ 8.56 Hz, 1H), 6.40–6.49 (m,
1
(
H), 3.53 (br. s., 2H), 3.30–3.35 (m, 2H), 2.49 (s, 3H); C NMR
151 MHz, DMSO-d ) ꢀ 196.7, 162.0, 155.4, 147.3, 145.6, 134.9,
NMR (300 MHz, DMSO-d ) ꢀ 9.10 (s, 1H), 8.03 (d, J ¼ 9.22 Hz,
6
6
2
H), 7.88 (d, J ¼ 8.85 Hz, 2H), 7.56 (d, J ¼ 8.85 Hz, 2H), 7.42 (br
1
32.2, 130.3, 130.1, 117.1, 109.2, 41.4, 39.2, 26.8.
s, 1H), 6.73 (d, J ¼ 9.31 Hz, 2H), 6.47 (s, 1H), 3.28–3.36 (m, 4H),
1
.52 (s, 3H); C NMR (75 MHz, DMSO-d ) ꢀ 196.7, 155.5,
3
2
1
2
6
1-(4-Chlorobenzyl)-3-{2-[(5-nitropyridin-2-yl)amino]ethyl}
urea (6c)
55.1, 145.6, 136.3, 130.4, 130.1, 126.8, 117.2, 111.3, 42.9, 31.2,
6.8.
Compound 6c was obtained using 4-chlorobenzyl isocyanate and
ACN as solvent. Product was recrystallized from EtOAc. TLC on
1
-(4-Chlorobenzyl)-3-{2-[(4-nitrophenyl)amino]ethyl}urea (5c)
Silica gel acetone–hexane 1–1 R ¼ 0.31. Elemental analysis
f
Compound 5c was obtained using 4-chlorobenzyl isocyanate and
THF as solvent. Product was chromatographed over Silica gel
with AcOEt–hexane 95–5. TLC on Silica gel EtOAc–hexane 8–2
R ¼ 0.19. Elemental analysis C H ClN O Calcd. C(55.10%)
C H ClN O
5 3
Calcd. C(51.51%) H(4.61%) Cl(10.14%)
N(20.02%) exp. C(51.36%) H(4.56%) Cl(10.17%) N(19.99%).
1
5
16
+
1
HRMS [M + H] Calcd. 350.1014; exp. 350.1012. H NMR
600 MHz, DMSO-d ) ꢀ 8.91 (d, J ¼ 2.10 Hz, 1H), 8.15–8.24 (m,
f
16 17
4 3
(
6
H(4.91%) Cl(10.16%) N(16.06%) exp. C(55.06%) H(4.92%)
1
H), 8.06–8.14 (m, 1H), 7.35 (d, J ¼ 7.82 Hz, 2H), 7.25 (d,
J ¼ 8.38 Hz, 2H), 6.56 (d, J ¼ 9.21 Hz, 1H), 6.50 (br. s., 1H), 6.14
br. s., 1H), 4.19 (d, J ¼ 6.08 Hz, 2H), 3.39–3.53 (m, 2H), 3.23 (q,
+
Cl(10.18%) N(15.69%). HRMS [M + H] Calcd. 349.1062; exp.
1
3
2
(
4
49.1065. H NMR (600 MHz, DMSO-d ) ꢀ 7.99 (d, J ¼ 9.17 Hz,
6
(
H), 7.34–7.37 (m, 2H), 7.26 (d, J ¼ 8.38 Hz, 2H), 6.67
13
J ¼ 6.20 Hz, 2H); C NMR (151 MHz, DMSO-d ) ꢀ 162.0, 158.6,
6
d, J ¼ 9.27 Hz, 2H), 6.56 (t, J ¼ 6.06 Hz, 1H), 6.15 (s, 1H),
1
4
47.3, 140.5, 134.8, 132.1, 131.5, 129.3, 128.6, 109.1, 42.7,
1.8, 39.4.
1
3
.20 (d, J ¼ 6.01 Hz, 2H), 3.20–3.24 (m, 4H);
C NMR
151 MHz, DMSO-d ) ꢀ 158.7, 155.1, 140.5, 136.1, 131.5,
(
6
1
29.3, 128.6, 126.7, 111.3, 43.4, 42.7, 39.1.
1-(4-Methoxybenzyl)-3-{2-[(5-nitropyridin-2-yl)amino]ethyl}
urea (6d)
1
-(4-Methoxybenzyl)-3-{2-[(4-nitrophenyl)amino]ethyl}urea (5d)
Compound 5d was obtained using 4-methoxybenzyl isocyanate Compound 6d was obtained using 4-methoxybenzyl isocyanate
and ACN as solvent. Product was recrystallized from MeOH. TLC
on Silica gel acetone–hexane 6–4 R ¼ 0.33. Elemental analysis
and THF as solvent. Product was washed with THF and
subsequently with MeOH. TLC on Silica gel EtOAc–hexane 8–
f
2
R ¼ 0.43. Elemental analysis C H N O Calcd. C(59.29%)
C H
16
N
19
5
O
4
Calcd. C(55.64%) H(5.55%) N(20.28%) exp.
+
f
17 20 4 4
H(5.85%) N(16.27%) exp. C(59.27%) H(5.86%) N(16.22%). C(55.44%) H(5.49%) N(20.32%). HRMS [M + H] Calcd.
1
46.1510; exp. 346.1513. H NMR (600 MHz, DMSO-d ) ꢀ
+
1
HRMS [M + H] Calcd. 345.1557; exp. 345.1555. H NMR
3
6
(
600 MHz, DMSO-d ) ꢀ 7.97–8.01 (m, 2H), 7.33–7.38 (m, 1H), 8.90 (d, J ¼ 2.49 Hz, 1H), 8.20 (br. s., 1H), 8.10 (br. s., 1H), 7.15
6
7
6
2
.14–7.19 (m, 2H), 6.84–6.88 (m, 2H), 6.67 (d, J ¼ 9.35 Hz, 2H), (d, J ¼ 8.06 Hz, 2H), 6.85 (d, J ¼ 8.02 Hz, 2H), 6.56
d, J ¼ 9.46 Hz, 1H), 6.35 (br. s., 1H), 6.04 (br. s., 1H), 4.13 (d,
H), 3.73 (s, 3H), 3.19–3.24 (m, 4H); C NMR (151 MHz, J ¼ 5.95 Hz, 2H), 3.72 (s, 3H), 3.45 (br. s., 2H), 3.23
(
.42 (t, J ¼ 5.93 Hz, 1H), 6.03–6.10 (m, 1H), 4.14 (d, J ¼ 5.93 Hz,
1
3

4
T. M. Wr o´ bel et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
1
3
(
1
6
q, J ¼ 6.25 Hz, 2H); C NMR (151 MHz, DMSO-d ) ꢀ 171.1, 2-Imino-3-(4-nitrophenyl)-N-phenylimidazolidine-1-
6
67.9, 167.8, 157.0, 144.9, 143.4, 142.4, 139.2, 125.0, 120.3, carboxamide (8)
8.6, 56.4, 55.5, 53.1.
To a stirred mixture of 1.19 g (10 mmol) phenyl isocyanate in
0 ml of THF there was added 2.06 g (10 mmol) 1-(4-nitrophe-
5
1-(3,4-Dichlorophenyl)-3-{2-[(5-nitropyridin-2-yl)amino]
ethyl}urea (6e)
nyl)imidazolidin-2-imine freebase. The mixture was allowed to
stir for 75 minutes after which time it was filtered (filtrate was
saved) and washed with THF and hot MeOH resulting in 2.35 g
Compound 6e was obtained using 3,4-dichlorophenyl isocyanate
and ACN as solvent. Precipitated product was washed with ACN.
(72%) of yellow solid. TLC on Silica gel EtOAc–hexane–AcOH
1
C(59.07%) H(4.65%) N(21.53%) exp. C(58.93%) H(4.71%)
0–90–1 R ¼ 0.27. Elemental analysis C H N O Calcd.
f
16 15 5 3
TLC on Silica gel acetone–hexane 4–6 R ¼ 0.30. Elemental
f
analysis C H Cl N O Calcd. C(45.42%) H(3.54%) Cl(19.15%)
3
1
4
13
2
5
+
N(21.71%). HRMS [M + H] Calcd. 326.1248; exp. 326.1246.
N(18.92%) exp. C(45.41%) H(3.50%) Cl(19.17%) N(18.97%).
1
+
HRMS [M + H] Calcd. 370.0468; exp. 370.0469. H NMR
1
H NMR (600 MHz, CHLOROFORM-d) ꢀ 12.11 (br. s., 1H),
8
7
4
.33–8.42 (m, 2H), 7.53–7.61 (m, 2H), 7.36–7.41 (m, 2H), 7.30–
.36 (m, 2H), 7.09 (tt, J ¼ 1.06, 7.41 Hz, 1H), 6.45 (br. s., 1H),
(
(
7
6
1
600 MHz, DMSO-d ) ꢀ 8.83–8.98 (m, 2H), 8.22 (br. s., 1H), 8.10
6
br. s., 1H), 7.84 (d, J ¼ 2.54 Hz, 1H), 7.44 (d, J ¼ 8.91 Hz, 1H),
1
.10–4.17 (m, 2H), 3.90–3.97 (m, 2H); C NMR (151 MHz,
3
.25 (dd, J ¼ 2.49, 8.83 Hz, 1H), 6.58 (d, J ¼ 8.78 Hz, 1H), 6.39–
.46 (m, 1H), 3.42–3.60 (m, 2H), 3.31 (d, J ¼ 5.98 Hz, 2H);
CHLOROFORM-d) ꢀ 152.5, 151.4, 145.1, 144.1, 138.4, 129.1,
129.0, 125.8, 123.5, 120.7, 119.7, 44.8, 40.7.
3
C NMR (151 MHz, DMSO-d ) ꢀ 162.0, 155.5, 147.3, 141.2,
6
1
34.9, 132.2, 131.4, 130.9, 122.7, 119.2, 118.2, 109.2, 41.4, 39.2.
1-[1-(4-Nitrophenyl)-4,5-dihydro-1H-imidazol-2-yl]-3-
phenylurea (9)
1-(4-Methoxyphenyl)-3-{2-[(5-nitropyridin-2-yl)amino]ethyl}
urea (6f)
The filtrate from the previous experiment was allowed to
evaporate overnight. The residue was repeatedly recrystallized
from isopropanol/NH . 127 mg (4 %) of yellow solid was
Compound 6f was obtained using 4-methoxyphenyl isocyanate and
ACN as solvent. Product was recrystallized from MeOH/DCM.
3
obtained. TLC on Silica gel EtOAc – hexane – AcOH 10–90–1
R ¼ 0.44. Elemental analysis C H N O Calcd. C(59.07%)
TLC on Silica gel acetone–hexane 8–2 R ¼ 0.48. Elemental
f
f
16 15 5 3
analysis C H N O Calcd. C(54.38%) H(5.17%) N(21.14%) exp.
5 17 5 4
1
H(4.65%) N(21.53%) exp. C(58.43%) H(4.72%) N(21.07%).
+
+
C(54.25%) H(5.09%) N(21.07%). HRMS [M + H] Calcd.
1
HRMS [M + H] Calcd. 326.1248; exp. 326.1243. H NMR
600 MHz, CHLOROFORM-d) ꢀ 9.03 (br s, 1H), 8.28 (br d,
1
3
32.1353; exp. 332.1355. H NMR (600 MHz, DMSO-d ) ꢀ 8.92
6
(
(
7
br. s., 1H), 8.32 (br. s., 1H), 8.23 (br. s., 1H), 8.11 (br. s., 1H), 7.24–
.31 (m, 2H), 6.78-6.85 (m, 2H), 6.58 (d, J ¼ 8.51 Hz, 1H), 6.16
J ¼ 7.91 Hz, 2H), 7.90 (br d, J ¼ 8.47 Hz, 2H), 7.52 (br d,
13
J ¼ 6.78 Hz, 2H), 7.33 (t, J ¼ 7.91 Hz, 2H), 7.13 (br s, 1H), 7.07
1
3
(br. s., 1H), 3.69 (s, 3H), 3.42-3.58 (m, 2H), 3.27–3.32 (m, 2H);
NMR (151 MHz, DMSO-d ) ꢀ 162.0, 156.1, 154.5, 147.3, 134.8,
C
(
br t, J ¼ 6.40 Hz, 1H), 4.01–4.07 (m, 2H), 3.80–3.86 (m, 2H);
C
NMR (151 MHz, CDCl ) ꢀ 159.7, 145.5, 142.6, 139.1, 128.9,
6
3
1
34.0, 132.2, 120.1, 114.3, 109.1, 55.6, 41.6, 39.2.
1
24.8, 124.6, 123.0, 119.1, 118.9, 45.7, 39.9.
1
-Methyl-3-{2-[(5-nitropyridin-2-yl)amino]ethyl}urea (6g)
Cell lines and cell culture
1
82 mg (1 mmol) of N-(5-nitropyridin-2-yl)ethane-1,2-diamine
RK33 laryngeal cancer cells were derived from a patient with
diagnosed laryngeal squamous cell carcinoma, as previously
was added to a solution of 125 mg (1 mmol) N-methyl-1H-
imidazole-1-carboxamide in 10 ml of DCM. This was followed by
addition of 152 ml (1.1 mmol) of TEA. The mixture was stirred
under argon for 24 hours. Solvent was removed on a rotavap and
the residue was chromatographed over Silica gel with EtOAc/
hexane gradient. Elemental analysis C H N O Calcd.
25
described . Human rhabdomyosarcoma TE671 cell line (origin-
ally identified as medulloblastoma) was obtained from ATCC.
HSF (human skin fibroblasts) were obtained as a laboratory strain
from a healthy volunteer by using standard outgrowth technique.
Cell lines were maintained in Dulbecco’s Modified Eagle
Medium (DMEM) culture medium (Sigma), and 1:1 mixture of
DMEM and Nutrient mixture F-12 Ham (Sigma), for RK33 and
TE671 cells respectively. Human skin fibroblasts were cultured in
DMEM. Culture media were supplemented with 10% Fetal
Bovine Serum (FBS) (Sigma) and antibiotics: penicillin
9
13 5 3
C(45.18%) H(5.48%) N(29.27%) exp. C(45.13%) H(5.44%)
+
N(29.10%). HRMS [M + H] Calcd. 240.1091; exp. 240.1092.
1
H NMR (600 MHz, DMSO-d ) ꢀ 8.91 (d, J ¼ 1.16 Hz, 1H), 8.20
6
(
br. s., 1H), 8.03–8.15 (m, 1H), 6.56 (d, J ¼ 8.59 Hz, 1H), 6.04 (br.
s., 1H), 5.81 (br. s., 1H), 3.43 (br. s., 2H), 3.19 (q, J ¼ 6.33 Hz,
1
3
2
1
H), 2.54 (d, J ¼ 4.62 Hz, 3H); C NMR (151 MHz, DMSO-d ) ꢀ
6
(
100 U/ml) and streptomycin (100 mg/ml) (both from Sigma).
61.9, 159.3, 147.3, 134.8, 132.1, 109.1, 41.9, 39.3, 26.9.
ꢀ
Cultures were kept at 37 C in a humidified atmosphere of 95% air
and 5% CO2.
1
-(4-Nitrophenyl)imidazolidin-2-imine hydrobromide (7)
Cell viability assessment
3
.18g (30 mmol) of cyanogen bromide dissolved in 50 ml
of MeOH was added dropwise to 5.44 g (30 mmol) of Cancer cells were seeded on 96-well microplates (Nunc,
4
N-(4-nitrophenyl)ethane-1,2-diamine (3) suspended in 50 ml of Langenselbold, Germany) at a density of 1 ꢁ 10 cells/ml. The
MeOH. During addition the amine went into solution and colour following day, the culture medium was replaced by serial dilutions
turned orange-brown. The reaction was heated under reflux for 90 of examined compounds in fresh culture medium with 10% FBS.
minutes and then solvent removed on a rotavap. Yellow residue was Analyzed compounds were dissolved in DMSO as 25 mM stock
recrystallized from EtOH to afford 7.3 g (85%) of yellow solid. The solution, the final concentration of solvent did not exceed 0.1%
hydrobromide salt was converted to a freebase form by dissolving it v/v. Cell viability was assessed after 72 h by means of MTT
in warm water, adding to 5% NaOH (2.5 eq) and filtering resulting method in which the yellow tetrazolium salt (MTT,
1
precipitate. NMR of the freebase H NMR (300 MHz, DMSO-d ) ꢀ Methylthiazolyldiphenyl-tetrazolium bromide) is metabolized by
6
8
.09–8.18 (m, 2H), 7.96–8.07 (m, 2H), 6.06 (br s, 2H), 3.81–3.94 viable cells to purple formazan crystals. The cells were incubated
13
(m, 2H), 3.35–3.43 (m, 2H); C NMR (75 MHz, DMSO-d ) ꢀ for 3 h in culture medium with MTT solution (5 mg/ml; Sigma).
6
1
58.5, 148.2, 140.0, 124.9, 117.1, 47.5, 40.0.
Formazan crystals were solubilized overnight in sodium dodecyl

DOI: 10.3109/14756366.2015.1057716
Nitroaryl urea derivatives
5
3
sulfate (SDS) buffer (10% SDS in 0.01 N HCl) and the product was three-parameter hybrid functional (B3) with correlation func-
1
3
2
quantified spectrophotometrically by measuring the optical density tional such as the one proposed by Lee, Yang, and Parr (LYP)
(OD) at 570 nm wavelength using an Infinite M200 Pro microplate using 6–31 G(d,p) basis set as included in Gaussian09 .
33
reader (Tecan, M a¨ nnedorf, Switzerland). The viability of the cells Furthermore, electron density and frontal molecular orbital
treated with solvent only (0.1% v/v DMSO) was set as 100%, and (FMO) analysis was performed with Gaussian09 on the B3LYP/
the viability in the other groups was calculated by comparing the 6–31 G(d,p) level of theory. Molecular descriptors were calculated
3
4
OD reading with the control. The GI50 values were calculated using with VegaZZ (molecular surface area, polar surface area,
3
nonlinear regression analysis on GraphPad Prism 5.04 platform. molecular volume and ovality), Discovery Studio v.3.5 (lipo-
5
36
The selectivity index (SI) was calculated as GI50 for normal cell philicity, molecular weight, polarizability) or ACDLabs soft-
line/GI50 for cancer cell line ratio. The data were collected from ware (number of hydrogen bond donors and acceptors).
three independent experiments, each in 8 wells (n ¼ 24).
In order to determine the binding mode of 9 with the active site
of CDK2, molecular docking followed by molecular dynamics
simulations were performed. Crystallographic structure of 9 was
used as a starting conformation for molecular docking to a
Kinase inhibition assays
CDK2/Cyclin E and c-Abl kinases were produced in Sf9 insect
cells via baculoviral infection and purified on a NiNTA column
20
selected crystal structure of CDK2 (PDB ID: 3QXP) . Molecular
docking was performed using the induced-fit approach of
Schr o¨ dinger software. The binding pose of 9 was selected from
most frequent binding poses obtained from docking which
corresponded to the binding pose of the reference inhibitor from
the X-ray structure. The inhibitor-enzyme complex was subjected
(Qiagen, Valencia, CA). The kinases were assayed with 1 mg/mL
histone H1 (CDK2) or with 500 mM GGEAIYAAPFKK peptide
3
3
(
c-Abl) in the presence of 15 mM ATP, 0.05 mCi [g- P]ATP and
of the test compound in a final volume of 10 mL, all in a reaction
buffer (60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl , 3 mM
MnCl , 3 mM Na-orthovanadate, 1.2 mM DTT, 2.5 mg/50 ml
37
2
to molecular dynamics with Desmond . The complex was first
2
hydrated and NaCl was added to the concentration of 0.15 M. The
inhibitor-enzyme complex was minimized and subjected to MD
first in the NVT ensemble for 1 ns and then in NPT ensemble for
PEG20.000). The reactions were stopped by adding 5 mL of 3 %
aq. H PO . Aliquots were spotted onto P-81 phosphocellulose
3
4
(
Whatman, Little Chalfont, UK), washed 3ꢁ with 0.5 % aq.
38
100 ns. Visualization of results was carried out with ArgusLab
39
and PyMol v. 0.99 .
H PO and finally air-dried. Kinase inhibition was quantified
3
4
using a FLA-7000 digital image analyzer (Fujifilm, Tokyo,
Japan). The concentration of the test compounds required to
decrease the CDK activity by 50% was determined from dose-
Results and discussion
response curves and designated as IC50(37)
.
Synthesis
The synthesis of novel nitroaryl urea derivatives was performed
as outlined in Scheme 3. The starting 1-chloro-4-nitrobenzene
X-ray data of 9 were collected on the Bruker SMART APEX II 1 or 2-chloro-5-nitropyridine 2 was reacted with ethylenediamine
Crystallography
CCD diffractometer; crystal sizes 0.50 ꢁ 0.50 ꢁ 0.01 mm, CuKꢁ to afford N-nitroarylethylenediamines 3 and 4. These were
˚
(
l ¼ 1.54178 A) radiation, ! scans, absorption correction: multi- then subjected to reaction with variety of isocyanates a–f or
2
6
scan SADABS , T /T ¼ 0.8308/1.0000. The structure was with N-methyl carbamoylimidazole g due to toxicity of methyl
min max
4
solved by direct methods using SHELXS97 and refined by full– isocyanate .
0
Thus,
1-substituted-3-{2-[(4-nitrophenyl)
2
7
matrix least–squares with SHELXL97 . The N-bound H atoms amino]ethyl}urea 5a–d and 1-substituted-3-{2-[(5-nitropyridin-
were located by difference Fourier synthesis and refined freely. 2-yl)amino]ethyl}urea 6a–g were obtained. 2-Imino-3-(4-nitro-
The remaining H atoms were positioned geometrically and treated phenyl)-N-phenylimidazolidine-1-carboxamide 8 and 1-[1-(4-
˚
as riding on their parent C atoms with C–H distances of 0.93 A nitrophenyl)-4,5-dihydro-1H-imidazol-2-yl]-3-phenylurea 9 were
˚
aromatic) and 0.97 A (CH ). All H atoms were refined with synthesized in one-step reaction from 1-(4-nitrophenyl)-4,5-
(
isotropic displacement parameters taken as 1.5 times those of the dihydro-1H-imidazol-2-amine hydrobromide 7 which had been
2
respective parent atoms. All calculations were performed using freebased and then reacted with phenyl isocyanate. Formation of
2
8
WINGX version 1.64.05 package CCDC-1036098 contains the two regioisomers is possible due to presence of guanidine moiety
supplementary crystallographic data for this paper. These data can which renders bonds between nitrogens equivalent as demon-
4
be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrie- strated by equal bond distances in compound 5. However, there
1
ving.html [or from the Cambridge Crystallographic Data Centre is a preference for addition to a nitrogen contained within the ring
(
1
CCDC), 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0) probably because of a lesser steric hindrance resulting in
223 336 033; email: deposit@ccdc.cam.ac.uk]. compound 8 being the major product. Compound 7 was obtained
Crystal data of 9: C H N O , M ¼ 325.33, monoclinic, from cyclisation of 3 with cyanogen bromide. Compounds 5, 6, 8
1
6 15 5 3
˚
space group P2 , a ¼ 5.2582(1), b ¼ 9.7217(1), c ¼ 14.7605(1) A, and 9 have been obtained in good to excellent yields in a general
1
ꢀ
˚
3
ꢂ3
ꢂ ¼ 91.229(1) , V ¼ 754.363(17) A , Z ¼ 2, d ¼ 1.432 Mg m
,
reaction of amine and isocyanate and constitute novel compounds.
calc
ꢂ1
F(000) ¼ 340, ꢃ(Cu Kꢁ) ¼ 0.855 mm , T ¼ 293 K, 8874 mea- This approach allows for a rapid generation of a library of urea
ꢀ
sured reflections (ꢄ range 2.99–69.62 ), 2649 unique reflections moiety compounds with simple chemistry.
(
Rint ¼ 0.028), extinction coefficient: 0.0139(16), final R ¼ 0.029,
wR ¼ 0.090, S ¼ 1.115 for 2626 reflections with I42ꢅ(I).
Crystallography
The X-ray analysis of compound 9 was performed in order to
confirm the synthesis pathway, identify tautomeric form in the
The investigated compounds were modeled using the LigPrep solid state and provide structural information for docking studies.
Molecular modelling
2
9
protocol from the Schr o¨ dinger Suite . In order to sample The structure and conformation of the molecule 9 in the crystal is
different protonation states of ligands in physiological pH, Epik shown in Figure 1. In the crystalline state, the molecule exists in
3
module was used . The molecular structures of 5a–5d, 6a–6g, 8 N3-amino/N6-imino/O7-keto/N8-amino tautomeric form, as evi-
0
˚
and 9 in the ground state (in vacuo) were further optimized denced by the C7–O7 bond length of 1.2275(17) A typical for the
with the B3LYP DFT (the variant of DFT method using Becke’s carbonyl group and the positions of the H atoms in the vicinity of

6
T. M. Wr o´ bel et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
R
NO2
a
NO2
X
b
NO2
O
NH
NH
(i)
(ii)
X
c
X
Cl
NH
R
HN
Cl
NH2
d
O
H3CO
1
2
X=C
X=N
3 X=C
4 X=N
5a-d X=C
6a-g X=N
Cl
e
Cl
(iii)
f
H
CO
3
H3C
g
O
NO2
NO2
N
N
NH
(
iv)
NH
N
N
N
NH
NH
HBr
HBr
+
O N
2
N
N
H
N
NH
NH2
O
O N
2
7
8
9
Scheme 3. Synthesis of 1-substituted-3-{2-[(4-nitrophenyl)amino]ethyl}urea (5a–d), 1-substituted-3-{2-[(5-nitropyridin-2-yl)amino]ethyl}urea
(
(
6a–g), 2-imino-3-(4-nitrophenyl)-N-phenylimidazolidine-1-carboxamide (8) and 1-[1-(4-nitrophenyl)-4,5-dihydro-1H-imidazol-2-yl]-3-phenylurea
9). (i) NH CH CH NH , reflux. (ii) RNCO or RNHCOIm, room temperature. (iii) BrCN, MeOH, reflux. (iv) 1. NaOH 2.PhNCO, DCM.
2
2
2
2
Figure 1. A view of the X-ray molecular
structure of 9 with the atomic labelling
scheme. Non H-atoms are represented by
displacement ellipsoids of 30% probability.
N3 and N8 in the difference electron-density map. The bond The tautomeric form and the nearly planar conformation of the
4
distances and angles are in normal ranges and are comparable molecule as a whole with the torsion angles C12–C11–N1–C2,
2
to the corresponding values observed in closely related structure N1–C2–N6–C7 and C22–C21–N8–C7 of ꢂ23.21(19),
4
3
ꢀ
of 1-(3-chlorophenyl)-3-(1-p-tolylimidazolidin-2-ylidene)urea . ꢂ178.06(12) and ꢂ175.04(15) , respectively, are stabilized by

DOI: 10.3109/14756366.2015.1057716
Nitroaryl urea derivatives
7
Table 1. Effects of the synthesized compounds on cancer and normal cells in vitro.
HSF
RK33
TE671
GI50 ± SD
GI50 ± SD
GI50 ± SD
SI
SI
5
5
5
5
6
6
6
6
6
6
6
8
9
a
b
c
d
a
b
c
d
e
f
14.73 ± 2.86
41.40 ± 2.73
77.75 ± 2.57
21.52 ± 1.90
56.80 ± 2.63
58.05 ± 1.57
129.08 ± 1.57
24.37 ± 2.09
54.79 ± 3.64
23.24 ± 1.27
4100
16.08 ± 1.10
62.70 ± 1.07
42.33 ± 1.06
6.67 ± 1.05
42.89 ± 1.05
168.20 ± 1.16
105.90 ± 1.18
21.25 ± 1.05
13.71 ± 1.10
20.89 ± 1.09
4100
0.92
0.66
1.84
3.23
1.32
0.35
1.22
1.15
4.00
1.11
12.22 ± 1.07
20.74 ± 1.09
23.01 ± 1.08
11.79 ± 1.07
24.25 ± 1.08
35.47 ± 1.12
37.75 ± 1.10
15.76 ± 1.05
6.46 ± 1.09
30.16 ± 1.10
4100
1.21
2.00
3.38
1.83
2.34
1.64
3.44
1.55
8.48
0.77
g
53.08 ± 1.78
38.01 ± 1.91
146.66 ± 1.1
122.90 ± 1.15
117.10 ± 1.13
83.33 ± 1.1
0.43
0.32
1.8
31.22 ± 1.06
26.44 ± 1.09
56.66 ± 1.2
1.70
1.44
2.6
CDDP
Shown GI50 values are the concentrations [mM] for half-maximal inhibition of cell viability and were calculated
from data obtained during three independent experiments. The selectivity index (SI) represents GI50 for normal
cell line/GI50 for cancer cell line after 72 h treatment with NUD.
with the lack of DMSO, and that difference is statistically
significant (p ¼ 0.006, Mann–Whitney test). For that reason the
cells treated with DMSO in 0.1% (v/v) were used as a control
group. Novel derivatives 5a, 5d, 6d, 6e and 6f showed the
strongest inhibitory influence on the cell viability of laryngeal
cancer cells RK33. Compounds 5a, 5b, 5d, 6d and 6e were the
most potent against rhabdomyosarcoma cells TE671. The highest
inhibitory effect on the cell viability was observed for compounds
Table 2. Effect of the synthesized compounds
on inhibition of protein kinases CDK2/cyclin E
and c-Abl.
IC50 (mM)
Compound
CDK2/cyclin E
Abl
5
5
5
5
6
6
6
6
6
6
6
8
9
a
b
c
d
a
b
c
d
e
f
450
412.5
425
412.5
450
450
450
425
412.5
412.5
4100
450
450
412.5
425
412.5
450
450
450
425
412.5
412.5
4100
450
5
5
a, 5d and 6e against both RK33 and TE671 cells. Compounds
a, 5d, 6d and also 6f produced the highest inhibitory effect on
the HSF cells viability.
Next, the selectivity index (SI) was calculated (as was
described in Experimental section) to determine the selective
activity of investigated compounds against cancer cells over
normal HSF. Two compounds, 5c and 6e, showed the highest
selectivity against both cancer cell lines in comparison to
antiproliferative effects exerted on normal cells. The GI50 value
of the compound 6e for HSF cells was 4 and at least 8 times
higher than observed for laryngeal cancer cells and rhabdomyo-
sarcoma, respectively. Therefore compound 6e shows the highest
SI value against both cancer cell types.
The considerable anticancer properties were shown for
compounds 5c, 5d, 6e and 5c, 6c, 6e against RK33 and
TE671 cancer cells respectively. Four urea derivatives 6b, 6c, 8
and 9 were active against TE671 cell line, nonetheless not
affected on RK33 cells viability. The less active against both
cancer cell lines used in this study were compounds 6g, anywise
no significant influence on HSF cells viability was observed.
Anti-cancer drug cisplatin (CDDP) was used as a positive
control of the cell viability assessment. TE671 cancer cell line
was the most sensitive against CDDP. Calculated GI50 ± SD
values for TE671 and RK33 cells were 56.66 ± 1.2 mM and
g
14.3
4100
the strong N3–H3 . . . O7 [N3–H3 ¼ 0.84(3), H3 . . . O7 ¼ 2.03(3),
˚
ꢀ
N3 . . . O7 ¼ 2.608(2) A and N3–H3 . . . O7 ¼ 125(2) ], C12–
H12 . . . N6 [C12–H12 ¼ 0.93, H23 . . . N6 ¼ 2.31, C12 . . . N6 ¼
˚
ꢀ
2
[
.8863(18) A and C12–H12 . . . N6 ¼ 120 ] and C26–H26 . . . O7
˚
C26–H26 ¼ 0.93, H26 . . . O7 ¼ 2.25, C26 . . . O7 ¼ 2.848(2) A
ꢀ
and C26–H26 . . . O7 ¼ 122 ] intramolecular hydrogen bonds.
Biological activity
Cell viability assessment
Newly synthesized compounds were subjected to MTT bioassay
to determine whether cancer and normal cells responded with
growth inhibition. Cancer and normal cells were treated with
increasing concentrations of indicated nitroaryl urea derivatives
over a period of 72 h. In general, all of the synthesized compounds
significantly reduced cell viability of cancer as well as normal
cells compared with non-exposed cells in a concentration-
dependent manner (Table 1). DMSO used in 0.1% (v/v) concen-
tration as a solvent did not influence viability of analysed cancer
cells, notwithstanding the inhibitory effect of the solvent was
8
3.33 ± 1.1 mM respectively demonstrating greater potency of
some newly synthesized compounds on tested cell lines. Healthy
human cells showed the greatest resistance against CDDP
in MTT assay, the calculated GI50 value was 146.66 ± 1.1 mM.
Estimated SI values for both cancer cell lines (TE671 and
RK33) were 2.6 and 1.8 respectively.
CDK inhibition
observed in the human skin fibroblasts HSF culture. The mean Because of the antiproliferative properties of newly prepared
HSF cell viability for the solvent only was 94.26% ± 1.42 compounds, we sought to find the molecular mechanism of this
(
standard error) in comparison to the viability of cells cultured activity. Aryl substituted urea derivatives have been discovered

8
T. M. Wr o´ bel et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
Number of Number of
Table 3. Molecular descriptors of the investigated compounds.
Surface Polar surface Volume,
˚
HOMO, LUMO,
eV
Molecular
weight
H bond
donors
H bond
acceptors
2
2
3
˚
Compound area, A
˚
area, A
A
Ovality
eV
Polarizability clogP
5
5
5
5
6
6
6
6
6
6
6
8
9
a
b
c
d
a
b
c
d
e
f
620.2
605.9
600.1
620.9
594.2
595.5
598.2
620.4
585.1
585.1
455.5
567.3
548.2
145.7
187.3
152.6
166.4
190.1
227.3
194.3
207.8
196.7
204.7
212.1
178.5
151.8
304.2
297.3
293.7
300.8
301.1
295.4
291.2
301.8
285.5
284.0
205.5
277.9
265.6
1.6601
1.6655
1.6786
1.7046
1.6603
1.6755
1.6762
1.7104
1.6652
1.6556
1.5843
1.6392
1.5764
ꢂ8.83
ꢂ8.92
ꢂ8.82
ꢂ8.74
ꢂ9.25
ꢂ9.39
ꢂ9.32
ꢂ9.20
ꢂ9.34
ꢂ8.76
ꢂ9.61
ꢂ9.10
ꢂ9.03
ꢂ0.72
ꢂ0.79
ꢂ0.71
ꢂ0.66
ꢂ0.93
ꢂ1.07
ꢂ0.97
ꢂ0.93
ꢂ0.10
ꢂ0.99
ꢂ1.21
ꢂ1.30
ꢂ1.35
38.11
35.22
35.05
35.66
36.96
35.03
34.60
35.22
34.78
33.83
23.45
34.05
33.34
3.206
2.037
2.969
2.288
2.594
1.426
2.357
1.676
3.015
1.670
0.109
2.482
2.482
350.371
342.349
348.784
344.365
351.359
343.337
349.772
345.353
370.191
331.327
239.231
325.322
325.322
3
3
3
3
3
3
3
3
3
3
3
2
2
7
8
7
8
8
9
8
9
8
9
8
8
8
g
Figure 2. HOMO (A) and LUMO (B) orbitals for 9. (C) The map of the electrostatic potential onto a surface of the electron density for 9.
4
4–47
51–54
as potent inhibitors of various kinases
and sorafenib is a well- CDKs and antiproliferative effect has been well documented
.
4
8
known example of a drug used for treatment of some cancers . CDK2 is often deregulated in cancer cells usually by cyclin
We decided to pursue CDK inhibition because of the similarity of overexpression or loss of natural inhibitors and provides cells with
our compounds and those described in the literature taking urea growth advantage. We therefore assayed all prepared compounds
2
1,49,50
moiety into account
. Moreover a link between inhibition of with purified recombinant protein kinases CDK2/cyclin E and

DOI: 10.3109/14756366.2015.1057716
Nitroaryl urea derivatives
9
Molecular modelling
In order to determine the structure-activity relationship regarding
the effect of the synthesized compounds on inhibition of protein
kinase CDK2/cyclin E, selected molecular descriptors were
calculated (Table 3). It can be concluded that the active
compound 9 has smaller molecular surface area, polar surface
area, volume, ovality, polarizability, molecular weight, LUMO
energy and number of hydrogen bond donors than most of the
inactive compounds. No trend was found for the number
of hydrogen bond acceptors, HOMO energy and lipophilicity.
Figure 2 presents the HOMO and LUMO orbitals as well as the
map of the electrostatic potential onto a surface of the electron
density for 9.
Next we determined the binding mode of 9 in the active site of
CDK2 using induced-fit molecular docking followed by molecu-
lar dynamics. From many available X-ray structures of CDK2 in
2
0
complex with inhibitors, 3QXP was selected due to structural
similarity of 9 to the complexed inhibitor. Figure 3 depicts the
final binding pose of 9 (A) and this pose aligned to the reference
ligand (B). The binding pose of 9 was selected from the most
frequent binding poses which corresponded to the binding pose of
the reference inhibitor from the X-ray structure.
It was found that the nitro group of 9 forms water molecule
mediated hydrogen bond with the side chain of Gln131.
Moreover, the urea moiety of 9 forms two hydrogen bond with
the main chain of Leu83. The compound 9 fits well to the CDK2
binding pocket and is additionally involved in a set of non-polar
interactions with the hydrophobic residues of the active site
(Ile10, Val18, Ala31, Val64, Phe80, Phe82, Leu134, Ala144). The
urea moiety seems to be a key element of the system. The docking
shows that a polar group substituted at N1 phenyl ring is favorable
for activity. The nitro group may be exchanged into carboxylic,
amide or hydroxyl group. Moreover, as there is some space near
phenyl moiety substituted at urea system, some non-polar
substituents, such as 2-fluoro, 2-chloro or 2-methyl groups
could be favorable. These modifications will be a subject of our
future studies. This inhibitor-enzyme complex was found to be
stable in molecular dynamics simulations which confirm the
probability of rationally selected binding mode.
Conclusion
The novel series of nitroaryl urea derivatives have been
synthesized and tested for biological activity. They displayed an
antiproliferative effect in the lower micromolar range. Two
compounds 5d nitrophenyl (6.7 mM in RK33) and 6e nitropyridyl
(6.5 mM in TE671) were the most active from the series. They
contain 4-methoxybenzyl and 3,4-dichlorophenyl substituents
respectively. Compound 6e proved to be the most selective
towards cancer cells. All compounds have also been tested
towards CDK. However, all compounds except 9 have not
displayed significant activity, suggesting a different mechanism
of action which will be further investigated. Compound 9 being
active in a CDK2 assay has a rigid structure with urea moiety
closer to nitroaryl fragment. The rest of the compounds have
greater conformational flexibility together with a more distanced
urea fragment. In the case of compound 8 which is similarly rigid
Figure 3. Compound 9 in stick representation with magenta carbon atoms
in the binding pocket of CDK2 (protein colored with green carbon atoms)
(
(
A), 2D interaction map (B) and 9 superimposed to the reference ligand
stick representation, cyan carbon atoms, hydrogen atoms not shown in
ligands for clarity; protein shown as green cartoon) (C). Hydrogen bonds
shown as red dashed lines (color version available online).
5
5
c-Abl as described previously . The compounds were tested in a as active compound 9, the urea feature is also moved further away
dose-dependent manner starting from 0.1 mM up to their respect- from the nitroaryl fragment. This feature renders the compounds
ive solubility limits. Results summarized in Table 2 illustrate that significantly less active towards CDK2. Moreover, compound 9 is
the majority of tested compounds did not display significant characterized by decreased values of molecular descriptors which
activity against the two kinases, with the exception of 9 that make this molecule more compact. This corresponds well with
showed modest inhibitory activity towards CDK2 with IC50 of docking studies which also revealed the importance the nitro
1
4.3 mM, indicating some sort of selectivity over tyrosine kinase group. From the above results, it can be concluded that this
c-Abl. This finding however suggests a different mechanism of scaffold may be developed into more active CDK inhibitors
action and prompts for investigation of another targets.
the future.

1
0
T. M. Wr o´ bel et al.
J Enzyme Inhib Med Chem, Early Online: 1–11
Declaration of interest
23. Allali M, Benoist E, Habbadi N, et al. Design and synthesis of new
ethylenediamine or propylenediamine diacetic acid derivatives for
The authors report no declarations of interest.
Re(I) organometallic chemistry. Tetrahedron 2004;60:1167–74.
¨
This paper was developed using the equipment purchased within the
Project ‘‘The equipment of innovative laboratories doing research on new
medicines used in the therapy of civilization and neoplastic diseases’’
within the Operational Program Development of Eastern Poland 2007–
2
2
4. Bremer O. Uber die Synthese nitrierter 2,3-dihydro-pyrimidazole.
Justus Liebigs Annalen der Chemie 1936;521:286–97.
5. Rzeski W, Paduch R, Klatka J, et al. Establishment and preliminary
characterization of two cell lines derived from larynx carcinoma.
Folia Histochem Cytobiol 2002;40:195–6.
2
013. Priority Axis I Modern Economy, Operations I.3 Innovation
Promotion. Support of Grant for Young Scientists MNmb 44 is also
greatly appreciated. V.K. acknowledges financial support from the Czech
Science Foundation (project 15-15264S).
2
2
6. SADABS. 2004/1 ed. Madison (WI): Bruker AXS Inc.; 2005.
7. Sheldrick G. A short history of SHELX. Acta Crystallogr Sect A
2
008;64:112–22.
2
2
8. Farrugia L. WinGX and ORTEP for Windows: an update. J Appl
Crystallogr 2012;45:849–54.
9. LigPrep. Schr o¨ dinger. 2.4 ed. New York: LLC; 2010.
References
1.
2.
3.
4.
5.
6.
WHO. 2014. Available from: http://globocan.iarc.fr/Pages/fact_- 30. Epik. Schr o¨ dinger. 2.1 ed. New York: LLC; 2010.
31. Becke AD. Density–functional thermochemistry. III: the role of
exact exchange. J Chem Phys 1993;98:5648–52.
sheets_cancer.aspx [last accessed Nov 2014].
Cohen P. Protein kinases – the major drug targets of the twenty-first
century? Nat Rev Drug Discov 2002;1:309–15.
Melnikova I, Golden J. Targeting protein kinases. Nat Rev Drug
Discov 2004;3:993–4.
32. Lee C, Yang W, Parr R. Development of the Colle–Salvetti
correlation-energy formula into a functional of the electron density.
Phys Rev B 1988;37:785–9.
Adams JA. Kinetic and catalytic mechanisms of protein kinases. 33. Frisch MJ, Trucks GW, Schlegel HB, et al. Gaussian 09. Wallingford
Chem Rev 2001;101:2271–90.
(CT): Gaussian, Inc.; 2009.
Giamas G, Stebbing J, Vorgias CE, Knippschild U. Protein kinases 34. Pedretti A, Villa L, Vistoli G. VEGA – an open platform to develop
as targets for cancer treatment. Pharmacogenomics 2007;8:1005–16.
Mendel DB, Laird AD, Xin X, et al. In vivo antitumor activity of
chemo-bio-informatics applications, using plug-in architecture and
script programming. J Comput-Aided Mol Des 2004;18:167–73.
SU11248, a novel tyrosine kinase inhibitor targeting vascular 35. Discovery Studio. 3.5 ed: Biovia. Available from: http://accelrys.
endothelial growth factor and platelet-derived growth factor recep-
com/products/discovery-studio/ [last accessed Nov 2014].
tors: determination of a pharmacokinetic/pharmacodynamic rela- 36. ACDLabs. Available from: http://www.acdlabs.com/ [last accessed
tionship. Clin Cancer Res 2003;9:327–37.
Nov 2014].
Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective 37. Bowers KJ, Chow E, Xu H, et al. Scalable algorithms for molecular
7
8
.
.
inhibitor of the Abl tyrosine kinase on the growth of Bcr–Abl
positive cells. Nat Med 1996;2:561–6.
Barker AJ, Gibson KH, Grundy W, et al. Studies leading to the
dynamics simulations on commodity clusters. Proceedings of the
2006 ACM/IEEE conference on Supercomputing. Tampa (FL):
ACM; 2006. p. 84.
identification of ZD1839 (iressaÔ): an orally active, selective 38. ArgusLab. Available from: http://www.arguslab.com/arguslab.com/
epidermal growth factor receptor tyrosine kinase inhibitor targeted
ArgusLab.html [last accessed Nov 2014].
to the treatment of cancer. Bioorg Med Chem Lett 2001;11:1911–14. 39. The PyMOL Molecular Graphics System. Schr o¨ dinger. 0.99 ed:
9
.
Das J, Chen P, Norris D, et al. 2-Aminothiazole as a novel
LLC. Available from: https://www.pymol.org/pymol [last accessed
kinase inhibitor template. Structure–activity relationship studies
Nov 2014].
toward the discovery of N-(2-Chloro-6-methylphenyl)-2-[[6-[4-(2- 40. Duspara PA, Islam MS, Lough AJ, Batey RA. Synthesis and
hydroxyethyl)-1-piperazinyl)]-2-methyl-4-pyrimidinyl]amino)]-1,3-
thiazole-5-carboxamide (Dasatinib, BMS-354825) as a potent pan-
Src kinase inhibitor. J Med Chem 2006;49:6819–32.
reactivity of N-alkyl carbamoylimidazoles: development of
N-methyl carbamoylimidazole as a methyl isocyanate equivalent.
J Org Chem 2012;77:10362–8.
1
1
1
1
1
0. Hunter T, Pines J. Cyclins and cancer II: cyclin D and CDK 41. Dobrowolski MA, Cyra n´ ski MK, Pisklak M, Wawer I, Matosiuk D.
inhibitors come of age. Cell 1994;79:573–82.
1. Krystof V, Uldrijan S. Cyclin-dependent kinase inhibitors as
anticancer drugs. Curr Drug Targets 2010;11:291–302.
Structural studies of 1-aryl-2-aminoimidazolinium bromides: focus
on tautomer preference of the 2-aminoimidazoline moiety in the
solid state. Pol J Chem 2007;81:1037–48.
2. Malumbres M. Cyclin-dependent kinases. Genome Biol 2014; 42. Allen FH, Kennard O, Watson DG, et al. Tables of bond
1
5:122.
lengths determined by X-ray and neutron diffraction. Part 1. Bond
lengths in organic compounds. J Chem Soc Perkin Trans 2 1987;12:
S1–19.
3. Malumbres M, Harlow E, Hunt T, et al. Cyclin-dependent kinases: a
family portrait. Nat Cell Biol 2009;11:1275–6.
4. Malumbres M, Barbacid M. Milestones in cell division: to cycle or 43. Wysocki W, Matosiuk D, Karczmarzyk Z, et al. 1-(3-Chlorophenyl)-
not to cycle: a critical decision in cancer. Nat Rev Cancer 2001;1:
22–31.
3-(1-p-tolylimidazolidin-2-ylidene)urea. Acta Crystallogr Sect E
2009;65:o40.
5. Cicenas J, Valius M. The CDK inhibitors in cancer research and 44. Wang GT, Li G, Mantei RA, et al. 1-(5-Chloro-2-alkoxyphenyl)-3-
2
1
1
1
therapy. J Cancer Res Clin Oncol 2011;137:1409–18.
6. Blachly JS, Byrd JC. Emerging drug profile: cyclin-dependent
kinase inhibitors. Leukemia Lymphoma 2013;54:2133–43.
(5-cyano-pyrazi-2-yl)ureas as potent and selective inhibitors of
Chk1 kinase: synthesis, preliminary SAR, and biological activities.
J Med Chem 2005;48:3118–21.
7. Havl ´ı cˇ ek L, Hanu sˇ J, Vesel y´ J, et al. Cytokinin-derived cyclin- 45. Mitchell SA, Danca MD, Blomgren PA, et al. Imidazo[1,2-
a]pyrazine diaryl ureas: inhibitors of the receptor tyrosine kinase
EphB4. Bioorg Med Chem Lett 2009;19:6991–5.
dependent kinase inhibitors: synthesis and cdc2 inhibitory activity
of olomoucine and related compounds. J Med Chem 1997;40:
4
08–12.
46. Chen Z, Venkatesan AM, Dos Santos O, et al. Stereoselective
synthesis of an active metabolite of the potent PI3 kinase inhibitor
PKI-179. J Org Chem 2010;75:1643–51.
1
8. Fry DW, Harvey PJ, Keller PR, et al. Specific inhibition of cyclin-
dependent kinase 4/6 by PD 0332991 and associated antitumor
activity in human tumor xenografts. Mol Cancer Therap 2004;3: 47. Yin Y, Lin L, Ruiz C, et al. Synthesis and biological evaluation of
1
427–38.
urea derivatives as highly potent and selective rho kinase inhibitors.
J Med Chem 2013;56:3568–81.
restricts initiation of DNA replication and has antitumor activity. Nat 48. Hotte SJ, Hirte HW. BAY 43-9006: early clinical data in patients
1
2
2
2
9. Montagnoli A, Valsasina B, Croci V, et al. A Cdc7 kinase inhibitor
Chem Biol 2008;4:357–65.
with advanced solid malignancies. Curr Pharm Design 2002;8:
2249–53.
and selective diaminothiazole inhibitors of cyclin-dependent kin- 49. Yue EW, DiMeo SV, Higley CA, et al. Synthesis and evaluation of
0. Schonbrunn E, Betzi S, Alam R, et al. Development of highly potent
ases. J Med Chem 2013;56:3768–82.
indenopyrazoles as cyclin-dependent kinase inhibitors. Part 4:
heterocycles at C3. Bioorg Med Chem Lett 2004;14:343–6.
of a new class of potent Cdk4 inhibitors: new de novo design 50. Honma T, Yoshizumi T, Hashimoto N, et al. A novel approach for
1. Honma T, Hayashi K, Aoyama T, et al. Structure-based generation
strategy and library design. J Med Chem 2001;44:4615–27.
2. Pedersen DS, Rosenbohm C. Dry column vacuum chromatography.
Synthesis 2001;16:2431–4.
the development of selective Cdk4 inhibitors: Library design based
on locations of Cdk4 specific amino acid residues. J Med Chem
2001;44:4628–40.

DOI: 10.3109/14756366.2015.1057716
Nitroaryl urea derivatives 11
5
5
5
1. Vermeulen K, Strnad M, Kry sˇ tof V, et al. Antiproliferative effect of 54. Chu X-J, DePinto W, Bartkovitz D, et al. Discovery of [4-
plant cytokinin analogues with an inhibitory activity on cyclin-
dependent kinases. Leukemia 2002;16:299–305.
2. Gray NS, Wodicka L, Thunnissen A-MWH, et al. Exploiting
chemical libraries, structure, and genomics in the search for kinase
inhibitors. Science 1998;281:533–8.
Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimidin-5-yl](2,
3-difluoro-6-methoxyphenyl)methanone (R547), a potent and select-
ive cyclin-dependent kinase inhibitor with significant in vivo
antitumor activity. J Med Chem 2006;49:6549–60.
55. Kry sˇ tof V, Canka rˇ P, Fry sˇ ov a´ I, et al. 4-Arylazo-3,5-diamino-1H-
pyrazole CDK inhibitors: SAR study, crystal structure in complex
with CDK2, selectivity, and cellular effects. J Med Chem 2006;49:
6500–9.
3. Wang S, Meades C, Wood G, et al. 2-Anilino-4-(thiazol-5-yl)pyr-
imidine CDK inhibitors: synthesis, SAR analysis, X-ray crystallog-
raphy, and biological activity. J Med Chem 2004;47:1662–75.