Synthesis and evaluation of 1-(substituted)-3-prop-2-ynylureas as antiangiogenic agents

Article abstract of DOI:10.1016/j.bmcl.2012.02.029

Novel urea derivatives of alkynes have been designed, synthesized, and evaluated as potential cancer therapeutics leads. The most active 1-((3-chloromethyl)phenyl)-3-prop-2-ynylurea (1) exhibited cytotoxic effect against HELA and MCF-7 cell lines with IC₅₀ values of 1.55 μM and 1.48 μM, respectively. Further investigation on tube formation assay in human vein umbilical cells (HUVEC) demonstrated that 1 and methyl 4-(3-(3-ethynylureido)benzyloxy) benzoate (6) possess antiangiogenic activity, with minimum effective dose of 25 nM (for 1) and 6.25 μM (for 6). The ED ₅₀ of 1 and 6 were found to be 0.26 μM and 17.52 μM, respectively. The results from in vitro tyrosine kinase assay indicated the EGFR inhibition of 1 over other kinases (VEGFR2, FGFR1 and PDGFRβ). The cytotoxicity of 1 against EGFR overexpressing cell line A431 (IC₅₀ 36 nM) was comparable to that of erlotinib. The binding mode of 1 from docking simulation in the EGFR active site revealed that the urea motif formed hydrogen bonding with Lys745, Thr854 and Asp855 in hydrophobic pocket of EGFR. Compound 1 is considered as a potential lead for further optimization.

Full text of DOI:10.1016/j.bmcl.2012.02.029

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3001–3005
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 1-(substituted)-3-prop-2-ynylureas
as antiangiogenic agents
Kingkan Sanphanya a, Suvara K. Wattanapitayakul b, Orawin Prangsaengtong c, Michiko Jo c,
Keiichi Koizumi c, Naotoshi Shibahara c, Aroonsri Priprem d, Valery V. Fokin e, Opa Vajragupta a,
⇑
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudya Rd, Bangkok 10400, Thailand
b Department of Pharmacology, Faculty of Medicine, Srinakharinwirot University, 114 Sukhumvit 23, Bangkok 10110, Thailand
c Department of Kampo Diagnostics, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama-shi, Toyama 930-0194, Japan
d Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kae 40002, Thailand
e Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Rd, La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel urea derivatives of alkynes have been designed, synthesized, and evaluated as potential cancer
Received 12 September 2011
Revised 20 January 2012
Accepted 10 February 2012
Available online 22 February 2012
therapeutics leads. The most active 1-((3-chloromethyl)phenyl)-3-prop-2-ynylurea (1) exhibited cyto-
toxic effect against HELA and MCF-7 cell lines with IC50 values of 1.55 lM and 1.48 lM, respectively. Fur-
ther investigation on tube formation assay in human vein umbilical cells (HUVEC) demonstrated that 1
and methyl 4-(3-(3-ethynylureido)benzyloxy) benzoate (6) possess antiangiogenic activity, with mini-
mum effective dose of 25 nM (for 1) and 6.25
lM (for 6). The ED50 of 1 and 6 were found to be
Keywords:
1-(Substituted)-3-prop-2-ynylurea
Cytotoxic agents
Tyrosine kinase inhibitors
EGFR kinase inhibitor
Antiangiogenic agent
0.26 M and 17.52 M, respectively. The results from in vitro tyrosine kinase assay indicated the EGFR
l
l
inhibition of 1 over other kinases (VEGFR2, FGFR1 and PDGFRb). The cytotoxicity of 1 against EGFR over-
expressing cell line A431 (IC50 36 nM) was comparable to that of erlotinib. The binding mode of 1 from
docking simulation in the EGFR active site revealed that the urea motif formed hydrogen bonding with
Lys745, Thr854 and Asp855 in hydrophobic pocket of EGFR. Compound 1 is considered as a potential lead
for further optimization.
Ó 2012 Elsevier Ltd. All rights reserved.
Protein tyrosine kinases (TKs), a family of cell signaling proteins
that regulate inter- and intracellular communications, have been
implicated in cancer development. Two classes of TKs, categorized
by structures, functions and locations, are receptor tyrosine
kinases (RTKs) and non-receptor tyrosine kinases (NRTKs). These
TKs play an important role in regulation of cell growth, prolifera-
tion, differentiation, survival and metabolism.1,2 RTKs such as vas-
cular endothelial growth factor receptor-2 (VEGFR2), epidermal
growth factor receptor (EGFR), fibroblast growth factor receptor-
1 (FGFR1), and platelet-derived growth factor receptor (PDGFR)
play a key role in angiogenesis, the sprouting process of capillaries
from pre-existing vessels. These RTKs are important targets in anti-
angiogenic drug development. Inhibition of these enzymes can
lead to suppression of both cell proliferation and angiogenesis
and may become a viable strategy in cancer treatment.3
(Fig. 1).7–9 It is a tyrosine kinase inhibitor (VEGFR and PDGFR) which
was approved by the US FDA for advanced renal cancer.10
The 1-(substituted)-3-prop-2-ynylureas, terminal alkynes bear-
ing urea motif, were firstly designed in our lab as an in-house library
for the preparation of 1,4-disubsituted-1,2,3-triazole derivatives by
copper-catalyzed azide alkyne cycloaddition (CuAAC) reaction.11
Cl
F3C
N
N
O
NO2
O
S
H
N
HN
HN
O
O
N
N
O
N
N
N
N
OCH3
Urea derivatives demonstrate diverse array of biological and
pharmacological activities, such as antibacterial, antifungal, anti-
inflammatory, antiangiogenic and antiproliferative properties.4–6
Sorafenib is an example of an anti-proliferative urea agent
O
3
Sorafenib
A [Ref. 8]
B [Ref. O9C] H
IC50(MDA-MB-231) = 2.5 µM IC50 (KB) = 2.5 µM IC50 (K562) = 3 µM
IC50 (K562) = 0.4 µM
Figure 1. Substituted ureas acting as antiproliferative agents. MDA-MB-231, KB
and K562 are human breast cancer cell line, human carcinoma of the nosopharynx
and human erythroleukemia cell line, respectively.
⇑
Corresponding author. Tel.: +66 26 448 677; fax: +66 26 448 695.
E-mail address: pyovj@mahidol.ac.th (O. Vajragupta).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.02.029

3002
K. Sanphanya et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3001–3005
O
O
a
100
N3
a
OH
R
R
90
1
µM in Hela
100 µM in Hela
80
b
70
60
50
H
N
H
N
N
C
c
R
O
R
O
40
30
20
10
0
Scheme 1. General synthesis of urea compounds 1–5. Reagents and conditions: (a)
DPPA, TEA, toluene or dioxane, rt, 30 min; (b) toluene or dioxane, 100 °C, 1 h; (c)
propargylamine, rt, 30 min.
ref.
1
2
3
4
5
6
7
8
9
10
Compound
b
O
O
H2N
R
HO
R
100
90
80
70
60
50
40
30
20
10
0
/
Cl
X
N
H
N
H
K2CO3, 60°C
overnight
N
H
N
H
R
1
µM in MCF-7
1
6-10
100 µM in MCF-7
Scheme 2. General synthesis of urea compounds 6–10.
Table 1
Yields and melting points of the synthesized 1-(substituted)-3-prop-2ynylureas
O
O
X
R1
R2
N
H
N
H
N
H
N
H
ref.
1
2
3
4
5
6
7
8
9
10
6-10
1-5
Compound
Compoundd R1
X
R2
%
Yield
Mp (°C)
Figure 2. In vitro cytotoxicity effect of 1-(substituted)-3-prop-2-ynylureas at 1
and 100 M against HELA (a) and MCF-7 (b). Doxorubicin at 1 M was used as
reference compound, n = 3.
lM
l
l
1
—
—
—
—
21.48
10.01
158–160
132–134
Cl
H
N
H
N
Cl
2
Cl
O
1
N
3
4
—
—
—
—
50.27
72.48
178–180
122–124
A family of 26 of 1-(substituted)-3-prop-2-ynylureas were next
designed by molecular modeling to increase the potency. Flexible
ligands were docked to a grid representation of VEGFR2 model de-
rived from crystal structure of the VEGFR2 tyrosine kinase domain
in complex with a pyridyl-pyrimidine benzimidazole inhibitor
(PDB: 3EWH)12 using AutoDock4.213 (docking experiment and data
of all 26 compounds are shown in Supplementary data). Ten com-
pounds were selected based on free energy of binding, hydrogen
bond interaction and visual inspection and synthesized and preli-
minary screened for their cytotoxic effects toward two types of hu-
man cancer cell lines, HELA and MCF-7.
Compounds 1–5 were prepared by the reaction between various
phenyl isocyanates and propargylamine. The synthetic route is
illustrated in Scheme 1.14,15 Various phenyl isocyanates were pre-
pared in situ via Curtius rearrangement of corresponding acyl
azides before reacting with propargylamine to give 1-(substi-
tuted)-3-prop-2-ynylureas 1–5. Briefly, acyl azides were prepared
from the corresponding benzoic acids with diphenylphosphoryl
azide (DPPA) in the presence of triethylamine in an appropriate
solvent. Then, acyl azides were heated at 100 °C to achieve the cor-
responding isocyanates that were subsequently reacted with prop-
argylamine yielding the desired compounds 1–5, unoptimized
yield: 10–72% (Supplementary data). Compounds 6–10 were syn-
thesized by displacement of chloride of compound 1 with phenols
or benzylamines in the presence of K2CO3 in one step fashion16 as
illustrated in Scheme 2 (unoptimized yield: 17–80%, synthesis
N
5
6
—
O
—
10.53
38.12
246–248
179–181
HO
O
O
—
7
8
—
—
NH
NH
12.23
25.53
68–72
119–120
9
—
—
NH
NH
15.34
34.87
112–114
99–101
F3C
O
O
10
The synthesized triazole-based ureas, as well as the azide and alkyne
precursors, were screened for their cytotoxic effects against HELA
and MCF-7 cell lines. 1-(3-Chloromethyl)-3-prop-2-ynylurea 1
showed cytotoxic effect against HELA and MCF-7.

K. Sanphanya et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3001–3005
3003
Table 2
MCF-7 were observed. Although all compounds demonstrated poor
cytotoxic activities comparing to doxorubicin, compound 1 exhib-
ited substantial cytotoxic effect at 100 lM against both HELA and
IC50 values of the cytotoxicity against HELA, MCF-7
a
Compound
IC50 (lM)
MCF-7 (growth inhibitions of 86.86% and 85.16% for HELA and
MCF-7, respectively). Data from cytotoxicity assay of compounds
1–5 suggested that both size and position of substituents on 1-
(substituted)-3-prop-2-ynylurea greatly affected on cytotoxic
properties of the compounds in this series. Substituents on para-
position of 1-(phenyl)-3-prop-2-ynylurea decreased cytotoxic
effect regardless of the size of substituents as observed in com-
pounds 2 and 3. Bulky substituent that is, fused ring at position 1
of urea motif (compound 5) also suppressed cytotoxic potency.
The extended structure of compound 1 by displacement of chloride
moiety with various phenyl or benzyl via ether- or amine-linker
(compounds 6–10) did not enhance anticancer activity as antici-
pated, even deteriorated. These emphasized the influence of size
of the substituent on phenyl ring at position 1 of urea motif on
the cytotoxicity.
HELA
MCF-7
1
6
7
8
9
10
1.55
1.48
74.87
343.48
49.89
282.90
194.90
0.06
12.26
NAb
329.10
339.00
391.50
0.19
Doxorubicin
a
Mean values of three independent experiments are reported.
No activity.
b
Table 3
IC50 against the HUVEC
a
Compound
IC50 (lM)
Compounds 1, 6 and 8–10 showed % growth inhibition over 30% at
100 lM against either or both HELA and MCF-7 were selected for the
determination for IC50. Among the compounds in this series, compound
1 is most potent compound showing cytotoxic effect on both HELA and
Primary
EA.hy926
1
6
7
20
100
50
10.54
78.23
191.72
MCF-7 with IC50 of 1.55 lM and 1.48 lM, respectively (Table 2).
a
Mean values of three independent experiments are reported.
Compounds 1 and 6 showing the best activity against two cancer
cell lines were further evaluated for their antiangiogenic effects by
tube formation assay, regardless of the considerably high cytotoxic
details are shown in Supplementary data). All synthesized com-
pounds were displayed in Table 1.
IC50 in
lM level. Compound 7 with poor activity was included in
The synthesized 1-(substituted)-3-prop-2-ynylureas 1–10 were
the assay for comparison. Tube formation assays were performed
at non-cytotoxic doses of each compound against the human vein
umbilical cell line (HUVEC) (Supplementary data). To identify non-
cytotoxic doses for tube formation assay, the in vitro cytotoxicity
of compounds 1, 6 and 7 against HUVEC were performed by MTT
assay in the similar fashion as of HELA and MCF-7. IC50 values of
selected compounds against HUVEC were listed in Table 3.
then tested for their cytotoxic effects at 1 and 100 lM against
HELA and MCF-7 using MTT assay17 (Supplementary data). Doxo-
rubicin was used as reference. The compounds that showed growth
inhibition over 30% at 100 lM against either cancer cell line were
selected for IC50 determination.
The initial screening results at two-point concentration of com-
pounds 1–10 were shown in Figure 2. Concentration dependent
cytotoxic effects of the compounds in this series against HELA and
Tube formation assays of compounds 1, 6 and 7 were per-
formed. Comparing with control, both compounds
1 and 6
120
100
80
60
40
20
0
140
120
100
80
Cont
60
40
20
1
0
Cont
25nM
Cpd 1
Cont
6.25uM
Cpd 6
180
160
140
120
100
80
7
6
60
40
20
0
Cont
12.5nM
Cpd 7
Figure 3. Effect of compounds 1, 6 and 7 on tube formation comparing with control, n = 3, ⁄⁄p <0.01.

3004
K. Sanphanya et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3001–3005
30
25
20
15
10
5
while that of compound 6 was only 6 times. Apparently, compound
1 did not only possess more potent antiangiogenic activity but also
showed safer profile than compound 6. The minimum effective
doses were also determined at the non-cytotoxic dose, compounds
1 and 6 significantly suppressed tube formation of HUVEC at 25 nM
EFGR
FGFR1
KDR
and 6.25
lM with 21.18% and 46.47% inhibition, respectively
(Fig. 3). Interestingly, compound
7
at non-cytotoxic dose
PDGFRb
(12.5 nM) showed the increase in tube formation, this compound
is currently under investigation for its molecular mechanism.
Compounds 1 and 6 exhibiting significant antiangiogenic activ-
ity were evaluated for their effects on tyrosine kinases involving in
angiogenesis. In vitro kinase assay of compounds 1 and 6 at 1 lM
against VEGFR2, FGFR1, EGFR and PDGFRb were performed. The
0
1
6
7
Compound
levels of phosphorylation of the tyrosine kinase-specific ligand pep-
tides18–21 at 1
lM of test compounds were measured (Supplemen-
Figure 4. Kinase inhibitory profile at 1
PDGFRb tyrosine kinases, % inhibition of erlotinip at 1
l
M against EGFR, FGFR1, VEGFR2 and
M against EGFR was 98%.
tary data). The kinase inhibition profile was displayed in Figure 4.
l
The tested compounds predominantly inhibited EGFR tyrosine
kinase more than other kinases. At 1 lM, compound 1 exhibited
significant activity against EGFR and PDGFRb while compounds 6
and 7 inhibited EGFR tyrosine kinase only. The better cytotoxicity
of compound 1 may be due to its ability to inhibit two kinases,
EGFR and PDGFRb.
significantly suppressed tube formation of HUVEC. The half maxi-
mal effective concentration (EC50) of compounds 1 and 6 against
tube formation were found to be 0.26 lM and 17.52 lM, respec-
tively. The observed results from tube formation assay indicated
that compounds 1 and 6 possessed antiangiogenic properties
against HUVEC. The IC50:ED50 ratios for HUVEC of compounds 1
and 6 were 77 and 6, respectively. The antiangiogenic ED50 dose
of compound 1 was 77 times lower than its cytotoxic IC50 dose
Since compounds 1 and 6 inhibited EGFR more than the in-
tended VEGFR2, docking of these compounds into ATP binding site
of EGFR was performed using AutoDock4.2 to simulate a binding
model. The EGFR model was derived from crystal structure of erl-
otinib bound EGFR obtained from RCSB (PDB: 1M17).22 Dock poses
(a)
(b)
T766
T766
hydrophobic
pocket (P1)
adenine binding
region
M769
D831
D831
(c)
(d)
L694
V702
K721
VEGFR2
A719
EGFR
L768
F699
L764
T766
T766
T916
M769
D831
D1046
D831
T830
L820
Figure 5. (a) Overlay of binding models of 1-(substituted)-3-prop-2-ylnylurea 1 (cyan), 6 (magenta) and erlotinib (wheat) in kinase domain of EGFR, (b) Overlay of 1 and 6
and their H-bond interactions in the EGFR active site (green dot line), 1 occupied back cavity of ATP binding site of EGFR (P1), (c) binding modes of 1 and 6 displaying
interacted amino acid residues, (d) overlay binding model of compound 1 in kinase domain of EGFR (blue) and VEGFR2 (pink), key amino acid residues for H-bond interactions
(observed in compounds 1 and 6) in EGFR (T766 and D831) and in VEGFR2 (T916 and D1046) were illustrated.

K. Sanphanya et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3001–3005
3005
Table 4
IC50 against A431 cell lines
effect in vitro. In EGFR overexpressing cell line (A431), the cytotox-
icity of compound 1 was in nM level comparable to that of
erlotinip. The binding mode of 1 from EGFR docking simulation
demonstrated that the smaller in size of 1 facilitates and accom-
modates the hydrogen bond formation in the active sites of the
receptors as evidenced by the inhibition of two types of tyrosine
kinases (EGFR and PDGFRb). The urea motif in the compound plays
an important role in hydrogen bond interaction with amino acid
residues in the active binding site. Terminal alkyne containing urea
motif can be considered as new scaffold for further optimization of
a potential cancer therapeutic lead.
Compound
IC50 (nM)
1
6
7
36.00
215.50
318.00
55.50
Erlotinib
of these compounds were compared with crystal pose of erlotinib
as shown in Figure 5a.
EGFR docking results showed that 1-(substituted)-3-prop-2-
ynylureas 1 and 6 partially occupied the ATP binding pocket of EGFR
whereas erlotinib extensively occupied EGFR kinase domain
(Fig. 5a). The partial occupancy may explain the observed low po-
tency in kinase inhibition of these compounds. The 3-chloromethyl-
phenyl substituent of 1 penetrated deeply in the back cavity of the
ATP which was a poorly conserved hydrophobic area of the ATP
binding site of EGFR.23 This part of structure was surrounded by
the hydrophobic side chains of Phe699, Val702, Ala719, Leu764,
Leu820 and the hydrophobic parts of Lys721, Thr766 and Thr830.
The urea motif established two hydrogen bonds with the carboxyl-
ate of conserved Asp831, a component of DFG motif in the beginning
of the activation loop of EGFR which involved in Mg-ATP binding24
(Fig. 5b). The occupied location of compound 6 was similar to those
of 1, two hydrogen bonds between both HN of urea and COOH of
Asp831, and an extra hydrogen bond between oxygen atom of ether
and OH of Thr766, gatekeeper residue of EGFR (Fig. 5b). In addition
to Thr766 and Asp831, amino acid residues in hydrophobic pocket
that involved hydrophobic interaction included Leu694, Phe699,
Val702, Ala719, Lys721, Leu764, Leu768, Met769, Leu820 and
Thr830 (Fig. 5c). The binding mode cannot explain the higher po-
tency of 1 over 6 since 6 showed more interacted hydrogen bonds
over 1. The overlay binding mode of 1 in kinase domain of EGFR
and VEGFR2 was displayed in Figure 5d. Compound 1 also occupied
back cavity of VEGFR2 and was surrounded by the hydrophobic side
chains of Val848, Phe1047, Leu1049 and the hydrophobic part of
Arg842, Lys868, Asp1046 and Asp1052 (data not shown). The ob-
served hydrogen bond interactions cannot explain the selectivity
of 1 against EGFR over VEGFR2 since 1 located in the similar region
and bound to DFG motif of both kinases (Asp831 of EGFR and
Asp1046 of VEGFR2). However, sequence alignment between EGFR
(PDB: 1M17) and VEGFR2 (PDB: 3EWH) by Needle (EMBOSS)25
showed 28.6% identity and 44.4% similarity suggested that the dif-
ference in the hydrophobic component of back cavity observed be-
tween EGFR and VEGFR2 might be the key factor controlling the
selectivity of 1 and 6 against EGFR over VEGFR2; which appeared
to be consistent with the reported notion.23
As compounds 1 and 6 were found to inhibit different kinases,
especially EGFR, the effect on human epidermoid carcinoma cells
A431, EGFR overexpressing cell lines26–29 was performed (Supple-
mentary data) and the IC50 values were reported in Table 4. The
IC50 values in nM level for EGFR overexpressing A431 cells demon-
strated the significance of EGFR kinase inhibition as those for HUVEC
were in lM level. The cellular cytotoxicity of 1 was comparable to
erlotinip despite of moderate EGFR kinase inhibition, it was possibly
due to its ability to inhibit two kinases, EGFR and PDGFRb.
In summary, a series of novel 1-(substituted)-3-prop-2-ynylu-
reas based on structural modification of compound 1 were pre-
pared to increase the binding capability. The structural
modifications of 1 with extended aromatic side chains to increase
the binding capability did not enhance tyrosine kinase inhibition
nor antiproliferative as expected. Compounds 1 and 6 were cyto-
toxic against human cancer cells and demonstrated antiangiogenic
Acknowledgments
This work was supported by the Royal Golden Jubilee Ph.D. Pro-
gram (RGJ Grant No. PHD/0229/2547) funded by Thailand Research
Fund (TRF) and the commission of Higher Education Thailand
(CHE-RG 2551). The authors thank Dr. Benjamin Fraser and Dr. Neil
P. Grimster for their valuable advice in some synthesis reactions.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.02.029.
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