Synthesis and biological evaluation of novel N, N′-disubstituted urea and thiourea derivatives as potential anti-melanoma agents

Article abstract of DOI:10.3109/14756366.2011.608665

Two series of urea and thiourea derivatives (1a11a, 1b11b) have been synthesized; all the 22 compounds were reported for the first time. Their anti-proliferative activities against the melanoma cell line B16-F10 were evaluated. Among the compounds tested,

Full text of DOI:10.3109/14756366.2011.608665

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(5): 708–714
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.608665
research artIcle
Synthesis and biological evaluation of novel N,
N′-disubstituted urea and thiourea derivatives as potential
anti-melanoma agents
Qing-Shan Li, Peng-Cheng Lv, Huan-Qiu Li, Xiang Lu, Zi-Lin Li, Ban-Feng Ruan, and
Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, P. R. China
abstract
Two series of urea and thiourea derivatives (1a–11a, 1b–11b) have been synthesized; all the 22 compounds were
reported for the first time. Their anti-proliferative activities against the melanoma cell line B16-F10 were evaluated.
Among the compounds tested, compound 6b exhibited the most potent activity in melanoma cells growth inhibition
(IC50 = 0.33 μM). The bioassay tests showed that anti-proliferative activities of these novel compounds were possibly
caused by inhibition of ERK1/2 phosphorylation level. Therefore, compound 6b can be a potential anti-melanoma
agent and an inhibitor of ERK1/2 phosphorylation deserving further research.
Keywords: Melanoma, ERK, phosphorylation, urea, thiourea
Introduction
inhibition of these proteins is particularly desirable7.
e ERK proteins consist of two isoforms (the 44 kDa
Skin cancer is one of the most common human malig-
nancies and its global incidence is rising at an alarming
rate, with basal cell carcinoma, squamous cell carcinoma
and melanoma being the most common forms1. Due to
the rising incidence and lack of effective treatments for
advanced disease, melanoma is the most dangerous
form, accounting for most skin cancer deaths, so new
treatment strategies are urgently needed2,3. e growth
of melanoma depends on stimulatory effects of various
growth factors, which bind to tyrosine kinase receptors
ERK1 and the 42 kDa ERK2; referred to as ERK1/2)8,9
.
e activities of ERK1/2 are tightly controlled by dual-
phosphorylation at special r 183/202 and Tyr 185/204
residues10,11. e dual-phosphorylated (full-active) forms
have determined the structural features required for full-
activation11–13. Phosphorylated ERK (pERK, active ERK)
1/2 protein transactivates numerous growth-related
genes contribute to tumour cell proliferation, tumour
cell survival, migration and metastasis14,15. is pathway
has emerged recently as the central growth-stimulatory
pathway in melanoma, with ERK being hyperactivated in
to activate various intracellular signalling pathways1,4
.
Recent studies have provided some new therapeutic
approaches to this disease. Extracellular signal-regulated
kinase (ERK) is one of the most important members
of mitogen-activated protein kinase (MAPK) family,
which is activated by tyrosine kinase receptors and other
upstream signal molecules, such as RAS (RAS-GTP),
RAF-1 and MEK1/25,6. e pathway offers several junc-
tures for signal-transduction blockade; due to the
converging functions of MEK1/2 and ERK1/2, specific
up to 90% of human melanomas1,16,17
.
e urea derivatives such as N-nitrosoureas, ben-
zoylureas, thioureas generally represent one of the
most useful classes of anticancer agents, with a wide
range of activities against various leukemias and solid
tumours18–21. Our interest in this area is to design and
synthesize diverse active urea and thiourea derivatives
for anti-tumour agents22,23. In continuation of our earlier
Address for Correspondence: Hai-Liang Zhu, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, P.
R. China. Tel: +86-25-83592672. Fax: +86-25-83592572. E-mail: zhuhl@nju.edu.cn
(Received 02 June 2011; revised 23 July 2011; accepted 25 July 2011)
708

Evaluation of novel N, N′-disubstituted urea and thiourea derivatives as anti-melanoma agents 709
studies, two novel series of N-benzyl-N-(4-hydroxy-3-
1H); 6.96–7.31 (m, 9H). MS (ESI): 363.1 ([M+H]+). Anal.
Calcd for C22H22N2O3: C, 72.91; H, 6.12; N, 7.73. Found: C,
72.78; H, 6.05; N, 7.87.
methoxybenzyl)-N′-phenyl-urea and thiourea (1a–11a,
1b–11b) derivatives were synthesized in this paper, then
we found that some of these compounds could inhibit
the proliferation of melanoma cell line B16-F10 via block
RAS–MEK–ERK signalling pathway.
1-(4-Hydroxy-3-methoxybenzyl)-1-(4-methoxyphenyl)-3-
phenylurea (3a)
1
Needle crystals, yield: 60%. Mp: 139°C–142°C. H NMR
(500 MHz, CDCl3, δ ppm): 3.80 (s, 3H); 3.85 (s, 3H); 4.81
(s, 2H); 5.56 (s, 1H); 6.62–6.65 (dd, 1H); 6.75–6.78 (d, J= 8.0
Hz, 1H); 6.89–7.23 (m, 8H); 7.27–7.32 (m, 2H). MS (ESI):
379.1 ([M+H]+). Anal. Calcd for C22H22N2O4: C, 69.83; H,
5.86; N, 7.40. Found: C, 69.79; H, 5.87; N, 7.23.
experimental procedure chemistry
All the NMR spectra were recorded on a Bruker DRX
500 or DPX 300 model Spectrometer in CDCl3. Chemical
1
shifts (δ) for H NMR spectra were reported in parts per
million to residual solvent protons. Melting points were
measured on a Boetius micro melting point apparatus.
e ESI-MS spectra were recorded on a Mariner System
5304 Mass spectrometer. All chemicals and reagents used
in current study were of analytical grade. e TLC was
run on the silica gel-coated aluminium sheets (Silica Gel
60 GF254, E. Merck, Germany) and visualized in UV light
(254 nm).
1-(4-Fluorophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylurea (4a)
Needle crystals, yield: 74%. Mp: 126°C–128°C. H NMR
(300 MHz, CDCl3, δ ppm): 3.84 (s, 3H); 4.80 (s, 2H); 5.57
(s, 1H); 6.60–6.63(dd, J1 = 2.01, J2 = 8.04 Hz, 1H); 6.77–6.80
(d, J= 8.04 Hz, 1H); 6.86–6.89 (d, J= 1.83 Hz, 1H); 6.98–7.03
(m, 1H); 7.08–7.24 (m, 6H); 7.27–7.30 (m, 2H). MS (ESI):
367.1 ([M+H]+). Anal. Calcd for C21H19FN2O3: C, 68.84; H,
5.23; N, 7.65. Found: C, 68.90; H, 5.43; N, 7.52.
1
General procedure for N-benzyl-N-(4-hydroxy-3-
methoxy-benzyl)-N′-phenyl-urea (1a–11a) and
thiourea (1b–11b) preparations
1-(4-Chlorophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylurea (5a)
e primary substituted amines (12 mmol) was added
to a solution of 3-methoxy-4-hydroxybenzaldehyde (10
mmol), in ethanol (30 mL). e mixture was stirred at
room temperature, and the reaction was monitored
by TLC. e products were filtered and recrystallised
in ethanol. Without further purification, to an etha-
nolic solution of synthesized Schiff bases, NaBH4 (10
mmol) was slowly added in an ice bath with stirring.
e mixture was refluxed for 2 h, then the solvent was
evaporated and water (20 mL) was added. e product
extraction was carried out with CH2Cl2 (3 × 20 mL). e
organic layer was dried over Na2SO4, and after solvent
evaporation affording the crude product as yellow oil
(1′–11′). To chloroform solution of amines (1′–11′),
phenylisocyanate or phenylisothiocyanate (1:1) was
slowly added with stirring. e mixture was refluxed
overnight. e completion of reaction was checked
by TLC. e CHCl3 was removed under reduced pres-
sure and the crude product was purified by column
chromatography.
1
Needle crystals, yield: 67%. Mp: 143°C–145°C. H NMR
(500 MHz, CDCl3, δ ppm): 3.84 (s, 3H); 4.81 (s, 2H); 5.58(s,
1H); 6.60–6.62 (dd, 1H); 6.77–6.79 (d, J= 8.0 Hz, 1H); 6.86
(d, J= 1.0 Hz, 1H); 6.98–7.08 (m, 3H); 7.23–7.30 (m, 4H);
7.36–7.38 (d, J= 8.5 Hz, 2H). MS (ESI): 383.1 ([M+H]+).
Anal. Calcd for C21H19ClN2O3: C, 65.88; H, 5.00; N, 7.32.
Found: C, 65.91; H, 4.97; N, 7.26.
1-(4-Bromophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylurea (6a)
Crystals, yield: 80%. Mp: 150°C–152°C. 1H NMR (300 MHz,
CDCl3, δ ppm): 3.84 (s, 3H); 4.81 (s, 2H); 5.58 (s, 1H);
6.60–6.64 (dd, J1 = 1.83, J2 = 8.03 Hz, 1H); 6.77–6.80 (d,
J= 7.8 Hz, 1H); 6.85–6.86 (d, J= 1.83 Hz, 1H); 6.98–7.03 (m,
3H); 7.22–7.31 (m, 4H); 7.51–7.54 (m, 2H). MS (ESI): 427.0
([M+H]+). Anal. Calcd for C21H19BrN2O3: C, 59.03; H, 4.48;
N, 6.56. Found: C, 59.16; H, 4.45; N, 6.62.
1-(2-Fluorophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylurea (7a)
1
Spectral properties of compounds 1a–11a and 1b–11b
White powder, yield: 43%. Mp: 126°C–129°C. H NMR
1-(4-Hydroxy-3-methoxybenzyl)-1, 3-diphenylurea (1a)
White powder, yield: 75%. Mp: 124°C–126°C. H NMR
(500 MHz, CDCl3, δ ppm): 3.83 (s, 3H); 4.80 (s, 2H); 5.57
(s, 1H); 6.61–6.64 (m, 1H); 6.74–7.32 (m, 12H). MS (ESI):
349.1 ([M+H]+). Anal. Calcd for C21H20N2O3: C, 72.40; H,
5.79; N, 8.04. Found: C, 72.51; H, 5.97; N, 8.19.
(300 MHz, CDCl3, δ ppm): 3.85 (s, 3H); 4.76 (s, 2H); 5.65
(s, 1H); 6.57–7.19 (m, 8H); 7.27–7.43 (m, 4H). MS (ESI):
367.1 ([M+H]+). Anal. Calcd for C21H19FN2O3: C, 68.84; H,
5.23; N, 7.65. Found: C, 68.63; H, 5.09; N, 7.75.
1
1-(2-Chlorophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylurea (8a)
1
1-(4-Hydroxy-3-methoxybenzyl)-3-phenyl-1-p-tolylurea (2a)
Needle crystals, yield: 56%. Mp: 117°C–119°C. H NMR
1
Needle crystals, yield: 79%. Mp: 146°C–148°C. H NMR
(300 MHz, CDCl3, δ ppm): 3.84 (s, 3H); 4.79 (s, 2H); 5.57
(s, 1H); 6.58–7.03 (m, 5H); 7.17–7.34 (m, 7H). MS (ESI):
383.1 ([M+H]+). Anal. Calcd for C21H19ClN2O3: C, 65.88; H,
5.00; N, 7.32. Found: C, 65.71; H, 5.07; N, 7.51.
(300 MHz, CDCl3, δ ppm): 2.37 (s, 3H); 3.84 (s, 3H); 4.81
(s, 2H); 5.54 (s, 1H); 6.61–6.65 (dd, J1 = 1.83, J2 = 8.04 Hz,
1H); 6.76–6.78 (d, J= 8.04 Hz, 1H); 6.88–6.89 (d, J= 1.83 Hz,
© 2012 Informa UK, Ltd.

710 Q.-S. Li et al.
(s, 1H); 6.62–6.65 (dd, 1H); 6.76–6.78 (d, J= 8.04 Hz, 1H);
6.89 (s, 1H); 7.06–7.32 (m, 9H). MS (ESI): 383.1 ([M+H]+).
Anal. Calcd for C21H19FN2O2S: C, 65.95; H, 5.01; N, 7.32.
Found: C, 66.02; H, 5.03; N, 7.51.
1-(2-Bromophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylurea (9a)
White powder, yield: 80%. Mp: 134°C–137°C. H NMR
(300 MHz, CDCl3, δ ppm): 3.84 (s, 3H); 4.81 (s, 2H); 5.69
(s, 1H); 6.59–7.51 (m, 12H). MS (ESI): 427.0 ([M+H]+).
Anal. Calcd for C21H19BrN2O3: C, 59.03; H, 4.48; N, 6.56.
Found: C, 60.07; H, 4.59; N, 6.46.
1
1-(4-Chlorophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylthiourea (5b)
1
Needle crystals, yield: 65%. Mp: 138°C–140°C. H NMR
(500 MHz, CDCl3, δ ppm): 3.85 (s, 3H); 5.45 (s, 2H);
5.58(s, 1H); 6.63–6.65 (dd, J1 = 1.5 Hz, J2 = 8.0 Hz, 1H);
6.77–6.78 (d, J= 8.0 Hz, 1H); 6.88 (s, 1H); 7.02–7.04 (d,
J= 8.5 Hz, 2H); 7.10–7.11 (d, J= 1.5 Hz, 1H); 7.18–7.21 (m,
1H); 7.31–7.40(m, 5H). MS (ESI): 399.1 ([M+H]+). Anal.
Calcd for C21H19ClN2O2S: C, 63.23; H, 4.80; N, 7.02. Found:
C, 63.14; H, 4.86; N, 6.88.
1-(2, 4-Difluorophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylurea (10a)
White powder, yield: 53%. Mp: 142°C–145°C. H NMR
(300 MHz, CDCl3, δ ppm): 3.83 (s, 3H); 4.76 (d, 2H); 5.62
(s, 1H); 6.60–6.64 (m, 1H); 6.73–7.30 (m, 10H). MS (ESI):
385.1 ([M+H]+). Anal. Calcd for C21H18F2N2O3: C, 65.62; H,
4.72; N, 7.29. Found: C, 65.81; H, 4.75; N, 7.40.
1
1-(4-Bromophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylthiourea (6b)
1-(2, 4-Dichlorophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylurea (11a)
White powder, yield: 46%. Mp: 144°C–145°C. H NMR
(300 MHz, CDCl3, δ ppm): 3.83 (s, 3H); 4.82 (d, 2H); 5.61
(s, 1H); 6.59–6.62 (dd, 1H); 6.77–7.11 (m, 5H); 7.23–7.36
(m, 5H). MS (ESI): 417.1 ([M+H] +). Anal. Calcd for
C21H18Cl2N2O3: C, 60.44; H, 4.35; N, 6.71. Found: C, 60.52;
H, 4.33; N, 6.58.
Crystals, yield: 73%. Mp: 150°C–152°C. 1H NMR (300 MHz,
CDCl3, δ ppm): 3.85 (s, 3H); 5.44 (s, 2H); 5.58 (s, 1H); 6.63–
6.66 (dd, 1H); 6.76–6.79 (d, J= 7.86 Hz, 1H); 6.88 (s, 1H);
6.96–6.99 (m, 2H); 7.10–7.11 (d, J= 1.83 Hz, 1H); 7.17–7.21
(m, 1H); 7.30–7.56 (m, 5H). MS (ESI): 443.0 ([M+H]+).
Anal. Calcd for C21H19BrN2O2S: C, 56.89; H, 4.32; N, 6.32.
Found: C, 56.81; H, 4.29; N, 6.24.
1
1-(4-Hydroxy-3-methoxybenzyl)-1, 3-diphenylthiourea (1b)
1
1-(2-Fluorophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylthiourea (7b)
White powder, yield: 38%. Mp: 133°C–135°C. H NMR
(300 MHz, CDCl3, δ ppm): 3.85 (s, 3H); 5.39 (s, 2H); 5.64
(s, 1H); 6.65–7.05 (m, 4H); 7.16–7.26 (m, 5H); 7.30–7.37
(m, 3H). MS (ESI): 383.1 ([M+H]+). Anal. Calcd for
C21H19FN2O2S: C, 65.95; H, 5.01; N, 7.32. Found: C, 65.91;
H, 5.06; N, 7.26.
Needle crystals, yield: 71%. Mp: 136°C–138°C. H NMR
(500 MHz, CDCl3, δ ppm): 3.84 (s, 3H); 5.48 (s, 2H);
5.57(s, 1H); 6.64–6.66 (dd, 1H); 6.75–6.77 (d, J= 8.0 Hz,
1H); 6.93 (s, 1H); 7.09–7.10 (m, 2H); 7.16–7.19 (m, 1H);
7.30–7.43 (m, 6H). MS (ESI): 365.4([M+H]+). Anal. Calcd
for C21H20N2O2S: C, 69.20; H, 5.53; N, 7.69. Found: C, 69.13;
H, 5.46; N, 7.56.
1
1-(4-Hydroxy-3-methoxybenzyl)-3-phenyl-1-p-tolylthiourea
(2b)
Needle crystals, yield: 65%. Mp: 130°C–132°C. H NMR
(300 MHz, CDCl3, δ ppm):2.36 (s, 3H); 3.85 (s, 3H); 5.46(s,
2H); 5.56 (s, 1H); 6.63–6.66(dd, J1 = 1.83, J2 = 8.07 Hz, 1H);
6.75–6.78 (d, J= 8.07 Hz, 1H); 6.95–6.98 (m, 3H); 7.13–
7.34 (m, 7H). MS (ESI): 379.1 ([M+H]+). Anal. Calcd for
C22H22N2O2S: C, 69.81; H, 5.86; N, 7.40. Found: C, 69.85;
H, 6.00; N, 7.47.
1-(2-Chlorophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylthiourea (8b)
Needle crystals, yield: 34%. Mp: 146°C–149°C. H NMR
(300 MHz, CDCl3, δ ppm): 3.85 (s, 3H); 5.43 (s, 2H); 5.58(s,
1H); 6.59–7.38 (m, 12H). MS (ESI): 399.1 ([M+H]+). Anal.
Calcd for C21H19ClN2O2S: C, 63.23; H, 4.80; N, 7.02. Found:
C, 63.30; H, 4.72; N, 7.16.
1
1
1-(2-Bromophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylthiourea (9b)
White powder, yield: 63%. Mp: 143°C–145°C. H NMR
(300 MHz, CDCl3, δ ppm): 3.85 (s, 3H); 5.31 (s, 2H); 5.49
(s, 1H); 6.57–7.17 (m, 9H); 7.19–7.23 (m, 1H); 7.29–7.32
(m, 2H). MS (ESI): 443.0 ([M+H]+). Anal. Calcd for
C21H19BrN2O2S: C, 56.89; H, 4.32; N, 6.32. Found: C, 56.94;
H, 4.22; N, 6.39.
1-(4-Hydroxy-3-methoxybenzyl)-1-(4-methoxyphenyl)-3-
phenylthiourea (3b)
Needle crystals, yield: 67%. Mp: 146°C–148°C. H NMR
(300 MHz, CDCl3, δ ppm): 3.81 (s, 3H); 3.85 (s, 3H); 5.44
(s, 2H); 5.55 (s, 1H); 6.63–6.66 (dd, J1 = 1.83 Hz, J1 = 8.04
Hz, 1H); 6.75–6.78 (d, J= 8.04 Hz, 1H); 6.89–7.00 (m, 5H);
7.12–7.20 (m, 1H); 7.27–7.35 (m, 4H). MS (ESI): 395.1
([M+H]+). Anal. Calcd for C22H22N2O3S: C, 66.98; H, 5.62;
N, 7.10. Found: C, 66.85; H, 5.70; N, 7.26.
1
1
1-(2, 4-Difluorophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylthiourea (10b)
1
White powder, yield: 45%. Mp: 135°C–137°C. H NMR
1-(4-Fluorophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylthiourea (4b)
(300 MHz, CDCl3, δ ppm): 3.85 (s, 3H); 5.44 (s, 2H);
5.57 (s, 1H); 6.59–6.63 (dd, J1 = 1.8 Hz, J2 = 7.86 Hz, 1H);
6.74–6.77 (d, J= 7.86 Hz, 1H); 6.86–7.05 (m, 5H); 7.18–7.23
1
Needle crystals, yield: 59%. Mp: 136°C–139°C. H NMR
(300 MHz, CDCl3, δ ppm): 3.85 (s, 3H); 5.45 (s, 2H); 5.57
Journal of Enzyme Inhibition and Medicinal Chemistry

Evaluation of novel N, N′-disubstituted urea and thiourea derivatives as anti-melanoma agents 711
(m, 1H); 7.29–7.36 (m, 3H). MS (ESI): 401.1 ([M+H]+).
Anal. Calcd for C21H18F2N2O2S: C, 62.99; H, 4.53; N, 7.00.
Found: C, 62.95; H, 4.48; N, 6.85.
anti-pERK1/2 (Boster, 1:100). e cells were incubated
with secondary antibodies: FITC-labelled goat anti-
rabbit IgG (Boster) at dilution 1:100 for 1h at 37°C in the
dark. Slides were analysed under Nikon E800 (Nikon,
Japan) microscope.Results and discussion
1-(2, 4-Dichlorophenyl)-1-(4-hydroxy-3-methoxybenzyl)-3-
phenylthiourea (11b)
White needle crystals yield: 38%. Mp: 125°C–127°C. H
1
Chemistry
NMR (300 MHz, CDCl3, δ ppm): 3.83 (s, 3H); 5.39 (d,
2H); 5.60 (s, 1H); 6.60–6.62 (dd, 1H); 6.77–7.21 (m, 7H);
7.32–7.41 (m, 3H). MS (ESI): 433.1 ([M+H]+). Anal. Calcd
for C21H18Cl2N2O2S: C, 58.20; H, 4.19; N, 6.46. Found: C,
58.07; H, 4.23; N, 6.53.
e synthesis of compounds 1a–11a and 1b–11b fol-
lowed the general pathway outlined in Scheme 1. e
two series of urea and thiourea derivatives were synthe-
sized in three steps. First, we chose 11 different substi-
tuted anilines to prepare the corresponding Schiff bases.
e secondary amines (1′-11′) were obtained from the
corresponding Schiff bases after the reduction reactions
with sodium borohydride. In the next step, the secondary
amines were condensed with phenylisocyanate or phe-
nylisothiocyanate in chloroform as the solvent, affording
the target compounds 1a-11a and 1b-11b. e reactions
were monitored by thin layer chromatography (TLC) and
the products were purified by column chromatography.
All the urea and thiourea 1a–11a and 1b–11b were syn-
thesized for the first time and all the compounds were
Cell culture
B16-F10 cells obtained from American Type Culture
Collection (ATCC) were cultured in RPMI-1640 medium
(GibcoBRL) with 10% (v/v) fetal bovine serum (FBS),
100 U/mL penicillin G and 100 μg/mL streptomycin.
Cells were incubated at 37°C in a humidified atmosphere
of air/CO2 (95%: 5%).
Cell proliferation assay
1
e anti-proliferative activity was determined using
a standard (MTT)-based colorimetric assay (Sigma).
Briefly, cell lines were seeded at a density of 7 × 103 cells/
well in 96-well microtiter plates (Costar). After 24 h,
exponentially growing cells were exposed to the indi-
cated compounds at final concentrations ranging from
0.1 to 100 μg/mL. After 48 h, cell survival was determined
by the addition of an MTT solution (10 μL of 5mg/mL
MTT in PBS). After 4 h, 100 μL of 10% SDS (Sigma) in 0.01
N HCl was added, and the plates were incubated at 37°C
for a further 18 h; optical absorbance was measured at
570 nm on an LX300 Epson Diagnostic microplate reader.
Survival ratios are expressed in percentages with respect
to untreated cells. e IC50 values were determined
from replicates of 6 wells from at least two independent
experiments.
fully characterized by H NMR, ESI MS and elemental
analysis. Furthermore, the crystal data, data collection
and refinement parameter for compound 6b are listed
in supplementary information. e structure was solved
by direct methods and refined on F2 by full-matrix least-
squares methods using SHELX-9724. Biological activity
study
In vitro cytotoxic activities of these N-benzyl-N-(4-
hydroxy-3-methoxybenzyl)-N′-phenylurea and thiourea
derivatives were studied in B16-F10 melanoma cell line.
A number of synthesized compounds displayed potent
cytotoxic activities against B16-F10. e cytotoxic activity
of the 22 compounds in B16-F10 was closely associated
with their structures, as shown in Table 1 and expressed
as the half maximal inhibitory concentration (IC50). All
22 compounds exhibited good activity with IC50 values
of <10.0 μM, among which 7 compounds demonstrated
IC50 values of 5.0–10.0 μM, 15 compounds showed IC50
less than 5.0 μM, compound 6b exhibited the most
potent inhibitory activity in melanoma cells growth
inhibition (IC50 = 0.33 μM), which was more potent than
positive control cisplatin (IC50 =4.24 μM) and carboplatin
(IC50 = 7.35 μM).
Structure-activity relationships (SARs) of these urea
and thiourea derivatives demonstrated that phenyl
thiourea derivatives have less IC50 values than those
corresponding phenyl urea derivatives. e result also
indicated that compounds with substitution at the para
(1a-6a, 1b-6b) position showed more potent activities
than those with substitution on the ortho position (7a-
9a, 7b-9b). A comparison of the para position substitu-
tion on benzene ring demonstrated that a para halogen
group (4a-6c, 4b-6b) may have slightly more improved
anti-proliferative activity than a methyl or a methoxy
group, and it showed the most potent inhibitory activity
when the para position was substituted by bromine. A
Cell-based enzyme inhibition measurement by ELISA
Detection of the effect of compounds on p-MEK1/2,
t-ERK1/2, pERK1/2, p-Rsk1 and p-Elk-1 activity in B16-
F10 cells was performed using ELISA kits (Invitrogen)
and strictly according to the manufacturer’s instructions.
Cells were pre-treated with 30 nM PMA (Sigma) in the
absence or presence of these compounds.
Immunofluorescence
e B16-F10 cells were seeded in 6-well plates at a
seeding density of 105 cells/mL. When all the cells
were adhered, various concentrations of compound
6b added. For immunofluorescence analysis, B16-F10
cells were treated with PMA (30 nM) and two different
concentrations (0 and 0.25 μM) of compound 6b. After
24 h incubation, cells washed with PBS, fixed with 2%
paraformaldehyde for 2h and permeabilized with 0.2%
Triton X-100. After blocking with 1% BSA, the cells were
incubated for 3h at room temperature with primary
© 2012 Informa UK, Ltd.

712 Q.-S. Li et al.
Scheme 1. Synthesis route of compounds 1a–11a and 1b–11b. Reagents and conditions: (i) ethanol, rt, 4 h; (ii) ethanol, NaBH4, reflux, 2h;
(iii) phenylisocyanate, chloroform, reflux, overnight; (iiii) phenylisothiocyanate, chloroform, reflux, overnight.
Table 1. Cytotoxicity of compounds 1a–11a and 1b–11b against B16-F10 cells.
B16-F10
B16-F10
Compound
R1
H
H
H
H
H
H
F
R2
H
(IC50 SD, μM) Compound
R1
H
H
H
H
H
H
F
R2
H
(IC50 SD, μM)
1a
2.07 0.25
4.54 0.97
5.31 0.61
1.57 0.54
1.49 0.42
0.99 0.25
5.98 1.31
8.96 0.72
7.95 0.47
4.03 1.07
3.33 0.63
4.24 0.67
7.35 0.89
1b
2b
1.23 0.25
3.39 0.83
2.47 0.32
1.35 0.24
1.27 0.36
0.33 0.10
5.55 0.76
6.09 0.36
5.38 0.62
3.56 0.94
2.57 0.91
2a
Me
OMe
F
Me
OMe
F
3a
3b
4a
4b
5a
Cl
Br
H
5b
Cl
Br
H
6a
6b
7a
7b
8a
Cl
Br
F
H
8b
Cl
Br
F
H
9a
H
9b
H
10a
F
10b
11b
F
11a
Cl
Cl
Cl
Cl
Cisplatin
Carboplatin
Journal of Enzyme Inhibition and Medicinal Chemistry

Evaluation of novel N, N′-disubstituted urea and thiourea derivatives as anti-melanoma agents 713
Table 2. Inhibition (IC50) of pERK1/2 in B16-F10.
Compound
pERK (μM)
6.77
Compound pERK (μM)
1a
2a
3a
4a
5a
6a
7a
8a
9a
10a
11a
1b
2b
3.35
5.47
3.21
3.12
3.28
1.27
7.22
6.99
6.14
6.92
4.13
6.12
5.76
3b
4.19
4b
4.07
5b
2.14
6b
9.15
7b
10.08
10.17
6.47
8b
9b
10b
11b
5.22
Cells were pre-treated with 30nM PMA in the absence or
presence of these compounds.
Figure 1. Immunofluorescence staining of pERK1/2 in B16-F10
cells after 24h incubation. Cells were treated in the absence or
presence of 6b. Green staining indicates the immunofluorescence
of human pERK1/2 (FITC-labelled) in B16-F10 cells (a) untreated
(b) or treated with 0.25 μM compound 6b.
significant loss of activity was observed when the halo-
gen substituent was moved to the ortho (7a-9a, 7b-9b)
position. is trend was observed in all compounds
whether they were urea or thiourea. e compounds
(10a, 11a, 10b, 11b) substituted by halogen groups both
on para position and ortho position showed more potent
activities than compounds with ortho halogen substitu-
ents, but less than compounds with para halogen sub-
stituents. So we deduced that substitution at the para
position of the N-phenyl ring plays an important role in
the anti-proliferative activity.
Table 3. Inhibition of p-MEK1/2, t-ERK1/2, p-Rsk1 and p-Elk1
in B16-F10 cell line by tested compounds.
IC50 (μM)
Compounds
p-MEK
> 25
t-ERK
> 25
> 25
> 25
> 25
22.7
p-Rsk1
1.93
p-Elk1
2.42
3b
4b
5b
6a
6b
> 25
2.71
1.95
19.6
2.76
2.47
> 25
1.38
1.24
As described above, the ERK signalling pathway plays
a central role in several steps of melanoma development,
including cancer cell proliferation and the development
of resistance to apoptosis. e ERK1/2 is therefore con-
sidered a prominent therapeutic target for the develop-
ment of chemotherapeutic drugs. e synthesized urea
and thiourea were evaluated for their ability to inhibit
phosphorylation (activation) of ERK in B16-F10 cells.
As illustrated in Table 2, all the compounds displayed
potent activities to induce the reduction in the amounts
of pERK1/2. ese results suggested that compounds
with substitution at the para (1a-6a, 1b-6b) position
showed more potent activities than those with substi-
tution at the ortho position, and compound 6b, which
displayed the most potent activity in tumour growth
inhibition, also showed the lowest IC50 value (IC50 = 1.27
μM). It is indicated that there is a reasonable correla-
tion between the pERK1/2 inhibitory activities and the
cytotoxicities against B16-F10 cells of these compounds.
Next, compound 6b was evaluated for its ability to inhibit
pERK1/2 level by using an immunofluorescence analy-
sis25. Samples were immunocytochemically labelled with
fluorescein isothiocyanate (FITC) for phosphorylated
ERK1/2 in green. e results were summarized in Figure
1. Compared to controls, the cells treated with compound
6b displayed a significant reduction of fluorescence,
which verified that this thiourea derivative could inhibit
pERK1/2 level in B16-F10 cell line.
17.8
0.97
1.53
Cells were pre-treated with 30nM PMA in the absence or
presence of these compounds.
of pERK), phosphorylated Rsk1 and phosphorylated
Elk-1 (downstream factors of pERK). ese compounds
decreased p-Rsk1 and p-Elk-1 amounts (the downstream
substrates of ERK1/2) in B16-F10 cells with good effects.
However, these compounds did not show obvious inhibi-
tory ability against p-MEK1/2, t-ERK1/2. Combining
these enzyme assay results, we deduced that these com-
pounds could specially inhibit the phosphorylation of
ERK1/2 and then block the RAS–MEK–ERK signalling
pathway in B16-F10 cells.
conclusion
In summary, two series of novel N-benzyl-N-(4-
hydroxy-3-methoxy-benzyl)-N′-phenylurea and thio-
urea derivatives (1a–11a, 1b–11b) were synthesized.
eir anti-proliferative activities against the melanoma
cell line B16-F10 were evaluated. Some compounds
displayed good inhibitory activities and the SARs have
also been studied. e consequences demonstrated
that substitution at the para position of the N-phenyl
ring played an important role in the anticancer activity.
Among the compounds tested, we found compound
6b had demonstrated significant ERK phosphorylation
inhibitory activity (IC50 = 1.27 μM against pERK1/2) and
anti-proliferative activity in melanoma cells growth inhi-
bition (IC50 = 0.33 μM). In enzyme assays, 6b did not show
As shown in Table 3, we tested the inhibitory activities
of selected compounds (3b, 4b, 5b, 6a, 6b) against phos-
phorylated MEK1/2 and total ERK1/2 (upstream factors
© 2012 Informa UK, Ltd.

714 Q.-S. Li et al.
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acknowledgement
Qing-Shan Li and Peng-Cheng Lv have contributed
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Declaration of interest
is work was supported by Jiangsu National Science
Foundation (No. BK2009239).
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