Exploration of 1-(3-chloro-4-(4-oxo-4H-chromen-2-yl)phenyl)-3-phenylurea derivatives as selective dual inhibitors of Raf1 and JNK1 kinases for anti-tumor treatment

Article abstract of DOI:10.1016/j.bmc.2012.04.006

Based on the roles of Raf1 and JNK1 in hepatocarcinoma development, scaffold-based drug design was employed to produce a series of compounds, which subsequently were synthesized and explored as potential dual inhibitors Raf1 and JNK1 kinases for anti-tumor treatment. The compound 1-(3-chloro-4-(6-ethyl-4- oxo-4H-chromen-2-yl)phenyl)-3-(4-chloro-phenyl)urea (3d) showed 66%, 67% and 13% inhibition rate at 50 μM against Raf1, JNK1 and p38-alpha, respectively, but no effect on ERK1 and ERK2, and inhibited the expression of pERK1/2 markedly and HepG2 cells proliferation with IC₅₀ at 8.3 μM. Furthermore, 3d showed lower toxicity against normal liver cell-lines QSG7701 and HL7702. Molecular docking study further showed that 3d can fit into binding domain of JNK1 and Raf1. Our data suggested the activities of 3d were associated with dual inhibition of JNK1 and Raf1 kinases.

Full text of DOI:10.1016/j.bmc.2012.04.006

Bioorganic & Medicinal Chemistry 21 (2013) 824–831
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Exploration of 1-(3-chloro-4-(4-oxo-4H-chromen-2-yl)phenyl)-3-phenylurea
derivatives as selective dual inhibitors of Raf1 and JNK1 kinases for anti-tumor
treatment
Feng Jin a,b, Dan Gao b, Cunlong Zhang b, Feng Liu b, Bizhu Chu a,b, Yan Chen b, Yu Zong Chen c,
b,d,
Chunyan Tan b, , Yuyang Jiang
⇑
⇑
a Department of Chemistry, Tsinghua University, Beijing 100084, China
b The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of Chemical Biology, the Graduate School at Shenzhen, Tsinghua University,
Shenzhen 518055, China
c Bioinformatics and Drug Design Group, Department of Pharmacy, Centre for Computational Science and Engineering, National University of Singapore, Singapore 117543, Singapore
d Department of Pharmacology and Pharmaceutical Sciences, School of Medicine, Tsinghua University, Beijing, 100084, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Based on the roles of Raf1 and JNK1 in hepatocarcinoma development, scaffold-based drug design was
employed to produce a series of compounds, which subsequently were synthesized and explored as
potential dual inhibitors Raf1 and JNK1 kinases for anti-tumor treatment. The compound 1-(3-chloro-
4-(6-ethyl-4-oxo-4H-chromen-2-yl)phenyl)-3-(4-chloro-phenyl)urea (3d) showed 66%, 67% and 13%
Received 1 February 2012
Revised 31 March 2012
Accepted 4 April 2012
Available online 11 April 2012
inhibition rate at 50
ERK2, and inhibited the expression of pERK1/2 markedly and HepG2 cells proliferation with IC50 at
8.3 M. Furthermore, 3d showed lower toxicity against normal liver cell-lines QSG7701 and HL7702.
lM against Raf1, JNK1 and p38-alpha, respectively, but no effect on ERK1 and
Keywords:
Flavones
Aromatic urea
Raf
JNK
l
Molecular docking study further showed that 3d can fit into binding domain of JNK1 and Raf1. Our data
suggested the activities of 3d were associated with dual inhibition of JNK1 and Raf1 kinases.
Ó 2012 Elsevier Ltd. All rights reserved.
Kinases inhibitors
Hepatocellular carcinoma
1. Introduction
more effective anti-cancer effects,6–8 in particular, dual-targeting
of Ras–Raf–ERK and JNK pathways by drug combinations has
The Raf protein is a key component of the Ras–Raf–ERK mito-
gen-activated protein kinases (MAPKs) pathway, which regulates
cellular processes, for example cell proliferation, migration and
apoptosis.1 c-Jun-N-terminal Kinase (JNK), another MAPK member,
is activated in some cancers by Ras to collectively promote cancer
together with the Ras–Raf–ERK signaling.2 Dysregulation of both
Raf-MAPKs and JNK pathways had been well elucidated in various
human cancers, for example, the mutant of Ras proteins often re-
sult in carcinogenesis by continuously activating downstream
effectors, and it is well known that Raf-MAPKs are not the only
downstream targets of mutant Ras, another an important target
is the JNK pathway.3 Some compounds targeting either Raf or
JNK had been approved or in preclinical trails for anti-cancer treat-
ment.4,5 However, because of the heterogenetic nature of cancers,
multi-target drugs or drug combinations directed at multiple com-
ponents of cancer proliferation and survival pathways produce
shown promising potential in inducing anti-cancer effect.9 There-
fore, development of multi-target inhibitors of Raf and JNK may
be a promising strategy for specific cancer therapy.
Hepatocellular carcinoma (HCC) accounts for up to 70% of can-
cer deaths in Eastern Asia and Central Africa, in HCC as well as
other cancers, pathways of mitogen-activated protein kinases,
including extracellular signal-regulated kinase (ERK), JNK and
p38 kinase, had been implicated,10,11 and ERK regulators such as
Raf have been explored as target against HCC.4 High JNK activation
has been found in various cancer cell lines and is linked to cancer
development,12,13 JNK1 is over-expressed in 55% Chinese HCC pa-
tients,12,14 and JNK inhibitor has been found to facilitate the inhibi-
tion of HCC growth.15 In addition to their individual roles in HCC,
Raf1 and JNK1 synergistically regulate MAPK signaling via a posi-
tive feedback loop established by functional interaction between
Raf1 and JNK1, which enables both JNK1 activation of Raf1 (and
thus subsequent activation of ERK)16 and Raf1 activation of JNK1.
On the other hand, p38-alpha antagonized JNK pathway in liver
cancer development.17 Thus, MAPKs map had been identified as
promising target against HCC,18,19 multi-target agents that
⇑
Corresponding authors. Tel./fax: +86 755 26036017 (Y.J.).
E-mail addresses: tancy@sz.tsinghua.edu.cn (C. Tan), jiangyy@sz.tsinghua.edu.cn
(Y. Jiang).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.006

F. Jin et al. / Bioorg. Med. Chem. 21 (2013) 824–831
825
simultaneously inhibit Raf1 and JNK1 with no or minimal inhibi-
tion of p38-alpha may produce enhanced anti-HCC effect than
agents targeting either Raf1 or JNK1 alone.
dried in vacuo to obtain 3a–3g. A solution of 1d, phenylacetic acid
derivatives (2d–2e), N,N-diisopropylcarbodiimide (DIC), N,N-diiso-
propylethylamine (DIEA) and N-hydroxy-benzotriazole (HOBt) in
dry THF was stirred at room temperature for about 18 h. Evapora-
tion of the solvent and column chromatography on silica (CH2Cl2/
CH3OH = 40:1, v/v) gel afforded 3h and 3i.
A key step towards the design of multi-target Raf1 and JNK1
inhibitors is to select relevant appropriate scaffolds from chemical
libraries. There are accumulating evidences suggesting the poten-
tial of flavones scaffold in driving protein kinases inhibitors due
to their suitable molecular skeleton.20,21 Many flavonoids exert
extensive biological benefits including inhibition of MAPKs.22–24
For example, quercetin shows potent anti-tumor activities partly
due to inhibition of Raf1 and ERK1.20,25 Some naturally occurring
flavonoids with various substituted groups exhibit moderate inhib-
itory activity against p38-alpha and JNK3, 6-substituted flavones
displayed selectivity for JNK3 over p38-alpha.26 On the other hand,
it has been reported that aromatic urea moiety has been identified
as a promising fragment to produce novel inhibitors of Raf kinase.
One urea derivative sorafenib had been approved as Raf1 inhibitors
for treatments of advanced HCC,27 two others urea derivatives, CP-
547623 and KRN951, were in clinical trials. Aromatic urea deriva-
tives of benzothiazole was also explored as Raf1 inhibitors.28 Also,
certain compounds containing aminopyrimidines and urea moiety
have been designed as JNK kinase inhibitors for anti-inflammation
therapeutics.29
To the best of our knowledge, flavones scaffold has not been
specifically explored for developing multi-target Raf and JNK inhib-
itors. Thus, novel multi-target Raf1 and JNK1 inhibitors may be
potentially derived from this scaffold. It was well noted that 40-
amino or 20-chloro substituent of flavones was interesting for
kinases inhibition activity.30,31 In this study, a 6-ethyl-20-chloro-
40 -aminoflavones compound (1d) was found to have moderate
kinases inhibition activity against Raf1 and p38-alpha, further
structural optimization was conducted by scaffold-based drug de-
signed and thereby produced a series of 1-(3-chloro-4-(4-oxo-4H-
chromen-2-yl)phenyl)-3-phenylurea derivatives, then evaluated as
multi-target Raf1 and JNK1 inhibitors, which were tested by
in vitro Raf1, JNK1 and p38-alpha kinase inhibition assays, anti-
proliferative activities against hepatocellular carcinoma cell-line
HepG2, lung adenocarcinoma epithelial cell-line A549, and colon
cancer cell-line HCT116, and toxicity activities against normal hu-
man liver cells QSG7701 and HL7702.
2.2. In vitro kinase inhibition assay
In vitro kinases inhibition activities of 1b, 1d and 3d at the spe-
cific concentration against Raf1, JNK1, ERK1, ERK2 and p38-alpha
were tested. As shown in Table 1, 3d inhibited 66% and 67% of
Raf 1 and JNK1 activity at 50 lM, respectively. Notably, only 13%
of p38-alpha activity was inhibited, ERK1 and ERK2 activities were
not influenced at the same concentration, which indicated the
selectivity of 3d towards the desired targets Raf1 and JNK1. While
individual target agents such as SP600125 (a widely used selective
JNK inhibitor)34 and FR180204 (a widely used selective ERK inhib-
itor)35 are highly potent (with low nM IC50) against their respective
target, multi-target drug may produce good therapeutic effect via
balanced activity across targets.32 For example, some (S)-3-amino-
pyrrolidine derivatives with similar activity against multiple tar-
gets were reported to be potent anti-proliferation agents against
tumor cell-lines.8 In a similar manner, our compound 3d may be
balanced dual inhibitor with similar activity between Raf1 and
JNK1, and essential selectivity against p38-alpha. Besides, its pre-
cursor compound 1d inhibited 27% Raf1 activity at the same con-
centration. Our newly designed compound 3d showed improved
activity against Raf1 over its precursor because of structural opti-
mization. Based on comparative analysis of compounds 1b and
1d, structural arrangement and physicochemical properties of the
substituent at A-ring of flavones seem to significantly influence
their inhibitory activity towards their kinase targets. 6-substituted
flavone derivatives may be better inhibitor against Raf1 than 7-
substituted analogs, and ethyl-substituted at A-ring was more ben-
eficial for inhibition of Raf1 than hydroxyl-substituted analogs.
2.3. Western blot analysis
ERK1/2 plays a pivotal role in regulating cell proliferation,36 and
it has specific roles in live tumorigenesis.37 As the key downstream
effector of Raf1, high phosphorylation levels of ERK1/2 have been
observed in most human HCC samples.38 Inhibition of Raf often
led to dephosphorylation of ERK1/2.39,40 Thus, we investigated
2. Results and discussions
2.1. Scaffold-based drug design and synthesis of compounds
the effect of 10 lM 3d on the phosphorylation of ERK1/2 by wes-
tern blot analysis. As shown in Figure 1(A), 3d markedly inhibited
the phosphorylation of ERK1/2 in HepG2 cells. After a 4 h drug
exposure, high activation of pERK1/2 was restrained, but not disap-
peared, which may contributed to relative low toxicity of 3d be-
cause activation of ERK was critical for a large number of normal
cellular response.41
Furthermore, the influences of JNK inhibition upon the phos-
phorylation levels of c-Jun were studied. Treatment with 10 lM
3d had no obvious effect on the phosphorylation of c-Jun, a down-
stream protein of JNK, which might due to low baseline phosphor-
ylation.42,43 When cells were stimulated by 1 mM MMS (methyl
methane sulfonate), the phosphorylation level of c-Jun was
strongly enhanced, a detectable inhibition effect was observed
A general strategy for designing multi-target agents is to conju-
gate or combine pharmacophoric elements for each target by con-
necting them with a metabolically stable linker,32 which is highly
effective if certain degrees of commonalities exist in the pharmaco-
phoric elements of each target. Scaffold-based design strategy was
especially suitable for kinase inhibitors discovery because of the
conserved construction of kinase domain.33 Firstly, a 6-ethyl-20-
chloro-40-aminoflavone compound (1d) was tested as low-affinity
scaffold, which inhibits 14% p38-alpha activity at 100
lM and
27% raf1 activity at 50 M, based on this scaffold novel compound
l
3d was generated by merging 6-ethyl-20-chloro-40-aminoflavones
and diphenylurea (Scheme 1). In order to clarify structure–activity
relationship, some analogs also were designed and synthesized. As
shown in Scheme 2, the designed compounds were synthesized by
a one-step routine. 20-chloro-40-aminoflavones derivatives (1a–1e)
and phenylisocyanate derivatives (2a–2c) were refluxed in dry tet-
rahydrofuran (THF), 4-dimethylamiopryidine (DMAP) as catalyst,
until raw materials disappeared in solution (monitored by thin-
layer chromatography, TLC). After evaporation of the solvent, the
residue was washed sequentially with deionizer water and ether,
after co-treatment with 100 lM 3d and 1 mM MMS for 2 h, shown
in Figure 1(B), which further supported the biological activities of
3d derived from its dual inhibition against Raf1 and JNK1.
2.4. In vitro cell cytotoxicity assay
In vitro cell cytotoxicity assay of compounds 3a–3i was evalu-
ated against HepG2 cells by MTT methods. As shown in Table 2,

826
F. Jin et al. / Bioorg. Med. Chem. 21 (2013) 824–831
NH2
O
O
Cl
H
N
H
N
6-ethyl-2'-chloro-4'-aminoflavone
O
O
O
merging
Cl
Cl
3d
H
N
H
N
CF3
Cl
N
H
N
O
O
O
Sorafenib
Scheme 1. Scaffold-based drug design to produce 1-(3-chloro-4-(6-ethyl-4-oxo-4H-chromen- 2-yl)phenyl)-3-(4-chlorophenyl)urea (3d).
H
N
H
N
NH2
NCO
R2
O
O
O
O
i
R2
+
R1
R1
Cl
Cl
O
1a:
2a
3a
: R1 = H, R2 = 3-Cl
R1 = H
: R2 = 2-Cl
1b: R1 = 7-OH
2b: R2 = 3-Cl
3b: R1 = 6-CH2CH3, R2 = 3-Cl
3c: R1 = 6-OCH3, R2 = 3-Cl
1c: R1 = 6-OCH3
2c: R2 = 3,4-Cl
1d
3d
: R1 = 6-CH2CH3, R2 = 4-Cl
: R1 = 6-CH2CH3
1e: R1 = F
3e: R1 = 6-OCH3, R2 = 3,4-Cl
3f: R1 = 6-F, R2 = 3-Cl
3g
: R1 = 7-OH, R2 = 3-Cl
H
N
NH2
R3
O
O
O
O
R3
ii
+
Cl
Cl
HOOC
2d
O
1d
3h
: R3 = 3-F
3i: R3 = 4-F
: R3 = 3-F
2e: R3 = 4-F
Scheme 2. Synthesis of compounds 3a–3i. Reagents and conditions: (i) dry THF, DMAP, reflux; (ii) dry THF, DIC, DIPEA, HoBt, rt for 48 h.
Table 1
Inhibitory activity of 1b, 1d and 3d at 50 lM against different kinases (inhibitory rate, %)
Kinases
1b
1d
3d
Staurosporine
Sorafenib
SB202190
Raf1
JNK1
p38-alpha
ERK2
ERK1
naa
nt
na
nt
27.04 3.74
65.57 4.65
67.40 2.36
13.51 4.55
na
ntb
95.99 1.22
nt
100.30 9.53
99.37 9.35
101.36 2.09
nt
nt
nt
nt
nt
nt
nt
14.47 0.2c
105.85 3.21
nt
nt
nt
nt
nt
na
a
b
c
na: not activity.
nt: not tested.
Inhibitory rate at 100
l
M.
compounds 3a, 3d, and 3e exhibited good anti-proliferative activ-
ity with <10 M IC50 values. 3d had an IC50 of 8.3 M, which was
comparative with approved inhibitor sorafenib (5.0 M, a reported
IC50 was 6.0
M44). By comparing the data, we obtained some pre-
liminary structure–activity relationships (SAR) as follows: (1)
when R2 = 3-Cl, compounds 3b, 3c and 3f with 6-substituted at
A-ring of flavones had similar anti-proliferation ability, all three
and electron-withdrawing substituents at A-ring of flavones de-
creased the activity, for example 3a, an unsubstituted compound
at A-ring, had stronger activity than substituted analogs 3b, 3c,
3f and 3g; (4) compounds with the substitution of R2 = 4-Cl had
better anti-tumor activity than compounds with the substitution
of R2 = 3-Cl, as demonstrated by the results of 3b and 3d; Also,
R2 = 3,4-Cl substituted compounds showed stronger inhibitory
activity than R2 = 3-Cl substituted compounds. For example, 3c
l
l
l
l
compounds had an IC50 at about 30 lM; (2) when R2 = 3-Cl, 7-hy-
droxyl substituted 3g had better activity than 6-substituted ana-
with IC50 at 26.3
lM, whereas 3e containing chlorination at
logs 3b, 3c and 3f; (3) when R2 = 3-Cl, both electron-donating
3,4-positions displayed a better effect with IC50 at 9.9
l
M; (5)

F. Jin et al. / Bioorg. Med. Chem. 21 (2013) 824–831
827
Table 3
Toxicity evaluation of synthetic compounds against QSG7701 cells and HL7702 cells
(inhibitory rate%)
Compd
Inhibition rate* (50
lM)
Inhibition rate* (100
lM)
QSG7701
HL7702
QSG7701
HL7702
3a
3b
3c
3d
3e
3f
3g
3h
48.9
12.6
15.9
23.7
13.2
26.8
34.6
22.4
16.2
4.2
26.8
9.4
62.4
18.0
30.1
31.8
42.5
57.7
43.2
27.4
36.9
23.2
47.1
37.2
40.6
28.3
77.9
54.2
83.8
44.6
45.2
20.2
35.7
18.7
66.4
20.1
80.2
20.4
29.1
5.3
3i
Sorafenib
Figure 1. The effect of 3d on the levels of phosphorylated ERK1/2 and c-Jun
proteins. HepG2 cells were treated with indicated compound for specific times,
then cell extracts were subjected to western blot analysis using specific antibodies.
*
Inhibition rate were determined from MTT assays after incubation with test
compounds for 48 h.
Table 2
3d not only substantially enhanced its Raf1 inhibitory activities
but also significantly reduced its toxicity towards novel liver cells.
A number of studies have suggested that multi-target drugs are
often low-affinity binders against each individual targets, but low-
affinity against individual targets not necessarily translate into low
efficacy against disease models like cell-lines, and in some cases
significantly higher efficacy than that of individual targets can be
achieve by multi-target drugs due to synergistic actions against
multiple components of relevant pathways.6 Our compound 3d
Anti-proliferative ability of synthetic compounds against HepG2, A549 and HCT116
cells
*
Compd
IC50
(l
M)
Compd
IC50
(lM)
HepG2 A549 HCT116
HepG2 A549 HCT116
3a
3b
3c
3d
9.7
30.7
26.3
8.3
14.0 25.4
3e
3f
3g
3h
3i
9.9
31.7
12.2
19.8
15.6
>50
16.8
>50
>50
8.6
24.2
25.9
>50
>50
>50
11.0
>50
4.5
27.9
9.6
28.7
2.3
Sorafenib
5.0
>50
also showed this behavior, its 8.3
significantly better than its 65.57% and 67.40% inhibition rate at
50 M against Raf1 and JNK1. Moreover, compounds of low po-
lM IC50 value against HepG2 is
*
IC50 values were determined from MTT assays after incubation with test com-
pounds for 48 h.
l
tency against individual targets might lead to lower toxicity than
compounds of higher potency, which may partly explain the rela-
tively lower toxicity of our synthesized compounds.
substitution of urea bond with amide bond had a negative effect on
cytotoxicity, for example, IC50 of 3i was found to be greater than
50 lM; (6) after substitution of urea bond with amide bond, 3-F
substituted compound 3h had better anti-tumor activity than 4-F
2.5. Impact of 3d on apoptosis and cell cycle in HepG2 cells
substituted compound 3i.
Furthermore, different activation status of JNK1 and Raf1 in dif-
ferent cell lines might result in different responses. Based on spe-
cific role of Raf and JNK in lung cancer and colon cancer,45 we
evaluated anti-proliferative ability of our synthesized compounds
against A549 cells and HCT116 cells (Table 2). Excessive activity
of Raf1 and JNK1 in HepG2 cells had been well studied, in which
3d demonstrated the most effective inhibition with the IC50 value
Some widely used JNK1 or Raf1 kinase inhibitor, for example,
SP600125 had minimal effect on apoptosis alone,42 and sorafenib
was a poor apoptotic inducer in HCC cells.50 Some other kinases
inhibitors, for example, only highly potent Abl inhibitors were
capable of inducing apoptosis, and selective PI3K inhibitors in-
duced apoptosis only at high concentration and prolonged expo-
sure time.8 In order to further explore similar biological function
of 3d with classical inhibitors of JNK1 or Raf1 kinases, flow cytom-
etry analysis were conducted to identify the effects of 3d on apop-
tosis and cell cycle in HepG2 cells. As shown in Figure S1, a
at 8.3 lM. Moreover, both ERK and JNK signal pathways were acti-
vated in about 50% human colon cancer,46 which may be a major
reason why most synthetic compounds had a similar IC50 between
HCT116 cells and HepG2 cells; On the other hand, it had been re-
ported that activation of JNK pathway was important for cell apop-
tosis and inhibition of cancer cell growth induced by synthetic
compounds or some drugs in clinical trials in A549 cells.47,48 Our
synthetic compound 3d or some other compounds had little effect
minimal apoptotic effect was detected after 10 lM 3d treatment
for varied time (12 h, 24 h, 36 h and 48 h, respectively) in HepG2
cells, which was consistent with classical Raf1 and JNK1 kinases
inhibitors. Cell cycle arrest was observed at S phase with the pro-
portion of about 6%, shown in Figure S2.
on A549 cells (IC50 >50
against JNK.
lM), which may derived from its inhibition
2.6. Molecular docking
Drugs targeting cancer-specific pathways need to have minimal
adverse effects against normal cells so as to achieve the desired
efficacy and safety.49 Herein, our synthesized compounds were
tested against two normal human liver cell lines, QSG7701 and
HL7702 cells, at two concentrations of 100 lM and 50 lM, respec-
tively (Table 3). There were no over-expression of Raf1 and JNK1 in
both normal cell lines, therefore, most of the compounds have a
lower inhibition rate of <45% at 100 lM. In particular, compound
3d had very weak effect on the two normal liver cell lines, with
32% and 28% inhibition rate for QSG7701 and HL7702 cells, respec-
tively. In contrast, its precursor 1d had relatively stronger cytotox-
icity against both normal liver cell lines (>50% inhibition rate at
To better understand the interaction of compound 3d with its
target kinases, molecular docking studies were conducted using
the Discovery Studio 3.0/CDOCKER protocol. As shown in Figure 2A,
specific interactions of 3d in the Raf1 (PDB ID: 3OMV) model
included: (1) two hydrogen bonds, one formed between C@O at
B-ring of flavones and NH2 of Lys431 (C@Oꢀ ꢀ ꢀH–NH), which was
considered to be an important site of action,51 with the distance
of 2.34 Å; the other formed between the same C@O and OH of
Ser427 (C@Oꢀ ꢀ ꢀH–O), with the distance of 2.11 Å; (2) three
interactions forming between the three rings at flavones backbone
(A-ring, B-ring and C-ring) and Trp423; (3) a cation– interaction
existing between 4-chlorination benzene ring and an ammonium
p–p
p
50 lM), which indicates that structural optimization of 1d into

828
F. Jin et al. / Bioorg. Med. Chem. 21 (2013) 824–831
Figure 2. Molecular modeling of 3d binding to target proteins. Molecules are colored by atom type and hydrogen bonds are represented by green dotted lines. White:
hydrogen atom; red: oxygen atom; blue: nitrogen atom; green: chlorine atom; cyan: the backbone and carbon atom of 3d. (A: Raf1, PDB ID: 3OMV; B: JNK1, PDB ID: 3ELJ).
cation of Lys375. Figure 2B shows two hydrogen bonds (NHꢀ ꢀ ꢀO)
between 3d and Met111 of JNK1 (PDB ID: 3ELJ) with the distance
of 1.98 and 2.29 Å, respectively, which contains one of key action
the 20-chloro-40-aminoflavones scaffold may be potentially ex-
plored for deriving multi-target Raf1 and JNK1 inhibitors with sub-
stantial selectivity against p38-alpha for the treatment of HCC at
lower toxicity against normal liver cells.
model.52,53 One
p–sigma interaction was also observed between
A-ring of flavones and Lys55. Molecular docking analysis identified
that 3d could fit into the binding domain of Raf1 and JNK1, thus
further supported that anti-tumor activity of 3d derived from its
dual inhibition of Raf1 and JNK1.
4. Experimental
4.1. Chemistry
A strategy for enhancing the selectivity of kinase inhibitors
against selected kinases is to explore distinguished features at
binding sites adjacent to the ATP-binding site (non-ATP-competi-
tive sites) where more diverse structural features can be explored,
for example an approved inhibitor imatinib largely explores these
sites for achieving selectivity against Abl.54 In our study, we
particularly explored some primary difference between JNK1 and
p38-alpha MAPKs for preferential selectivity against JNK while
maintaining sufficient interactions in Raf1. From the structural
analysis of the binding of a highly selective JNK1 inhibitor
SP600125 to JNK1, one finds that its selectivity is partly derived
from the variations of the crucial hydrophobic residue by analysis
of co-crystal structure of JNK1 and SP600125.55 Our compound 3d
has a common hydrogen bond with SP600125 in MET111. In addi-
All starting materials 1a–1e were from laboratory-made and
stored in dryer. Tetrahydrofuran (THF) was dried according to
the standard process. All other regents and analytical grade sol-
vents commercial available were directly used without further
purification. Thin Layer Chromatography (TLC) was used to moni-
tor the progress of reactions. Melting points were measured with
a SGW X-4 electrothermal melting apparatus, and data were
uncorrected. 1H NMR and 13C NMR spectra were recorded with a
Bruker spectrometer at 400 MHz for 1H NMR and at 101 MHz for
13C NMR, using TMS as an internal standard in CDCl3 or in
DMSO-d6. Abbreviations: singlet (s), doublet (d), triplet (t), broad
(br), quadruplet (q), multiplet (m). High-resolution mass spectra
were recorded with Waters Q-Tof Premier mass spectrometer.
tion, 3d had three
p–p interactions with Raf1, similar three p–p
interactions also could be observed in co-crystal of Raf1 and its
4.1.1. General procedure for synthesis of compounds 3a–3g
0.10 g 20-chloro-40-aminoflavones derivatives (1a–1e), 1.2 equiv
4-dimethylamio-pryidine (DMAP) and 1.1 equiv (2a–2c) was
added to a 100 mL round-bottomed flask, the resulting mixture
was refluxed in 25 mL dry THF. The reaction was monitored with
TLC until raw materials were completely consumed. After that,
THF was removed by rotating distillation. The crude solids were
washed with deionized water and subsequently with ether to ob-
tain pure products.
inhibitor.51
3. Conclusion
Because of the promising potential of the scaffold of flavones to
develop inhibitors of MAPKs and essential role of Raf1 and JNK1 in
HCC development, a 6-ethyl-20-chloro-40-aminoflavone compound
(1d) was tested to be low affinity inhibitors of Raf1 and p38-alfa.
Based on this scaffold, a series of compounds merging 20-chloro-
40-aminoflavones with diphenylurea were designed, synthesized
and evaluated as Raf1, JNK1 and p38-alfa kinases inhibitors for
anti-hepatocellular carcinoma treatment. One compound 3d was
found to be a dual inhibitor of Raf1 and JNK1 kinases with substan-
tial selectivity against p38-alpha kinase, good anti-proliferative
activity against HepG2 cell-line and inhibition rate against
pERK1/2, and relatively lower toxicity against normal liver cell-
lines QSG7701 and HL7702. Molecular docking studies showed
its binding modes at target binding sites in Raf1 and JNK1, and
structure–activity relationship further revealed the structural fea-
tures in the substructures of flavones and phenylurea on anti-pro-
liferative activity against HepG2 cell-line. Our study suggested that
4.1.1.1. 1-(3-Chloro-4-(4-oxo-4H-chromen-2-yl)phenyl)-3-(3-ch
lorophenyl)urea (3a).
Yield 70%, yellow solid, mp: 290–292
°C, HRMS (ESI) m/z calculated for [M+H]+ 425.0460, found
425.0457. 1H NMR (400 MHz, DMSO-d6) d 9.33 (s, 1H), 9.13
(s, 1H), 8.08 (dd, J = 7.9, 1.5 Hz, 1H), 7.91 (d, J = 2.0 Hz, 1H), 7.84
(ddd, J = 8.7, 7.2, 1.6 Hz, 1H), 7.76 (d, J = 8.6 Hz, 1H), 7.71 (dd,
J = 6.8, 4.9 Hz, 2H), 7.57–7.46 (m, 2H), 7.38–7.26 (m, 2H), 7.11–
7.01 (m, 1H), 6.64 (s, 1H). 13C NMR (101 MHz, DMSO-d6) d 177.23,
162.74, 156.49, 152.61, 143.60, 141.25, 134.99, 133.73, 132.58,
132.22, 130.95, 126.19, 125.39, 124.62, 123.68, 122.54, 119.51,
118.99, 118.54, 117.60, 117.42, 112.28.

F. Jin et al. / Bioorg. Med. Chem. 21 (2013) 824–831
829
4.1.1.2. 1-(3-Chloro-4-(6-ethyl-4-oxo-4H-chromen-2-yl)phenyl)-
3-(3-chlorophenyl)urea (3b). Yield 67%, yellow solid, mp:
13C NMR (101 MHz, DMSO-d6) d 175.90, 162.83, 161.40, 157.67,
152.14, 142.90, 140.77, 133.13, 131.90, 131.54, 130.36, 126.51,
124.04, 121.77, 118.61, 117.62, 116.69, 116.55, 115.84, 115.15,
111.32, 102.33.
229–230 °C, HRMS (ESI) m/z calculated for [M+H]+ 453.0773, found
453.0772. 1H NMR (400 MHz, DMSO-d6) d 9.36 (s, 1H), 9.16 (s, 1H),
7.91 (d, J = 10.4 Hz, 2H), 7.74 (dt, J = 17.9, 8.8 Hz, 3H), 7.62 (d,
J = 8.6 Hz, 1H), 7.52 (t, J = 12.1 Hz, 1H), 7.40–7.24 (m, 2H), 7.06
(d, J = 3.4 Hz, 1H), 6.63 (d, J = 12.9 Hz, 1H), 2.76 (q, J = 7.4 Hz, 2H),
1.24 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) d 177.24,
162.58, 154.93, 152.62, 143.56, 141.96, 141.27, 134.96, 133.73,
132.55, 132.16, 130.94, 129.39, 124.69, 123.39, 122.51, 119.49,
118.90, 118.52, 117.58, 117.40, 112.10, 28.07, 15.98.
4.1.3. Synthesis of compounds 3h and 3i
0.33 mmol (0.1 g) 6-ethyl-20-chloro-40-aminoflavone 1d, equi-
molar (0.051 g) 3-fluoro-phenylacetic acid 2d, 63
pylcarbodiimide (DIC), 146 di-isopropylethylamine (DIEA),
l
L N,N0-diisopro-
lL
0.40 mmol (0.054 g) 1-hydroxybenzotriazole (HoBt) were added to
a 50 mL round-bottomed flask. The resulting mixture was stirred
in 15 mL dry THF at room temperature for 48 h. The solvents were
removed in vacuo, flash chromatography of the residue on silica
gel (CH2Cl2/CH3OH = 40:1, v/v) gave N-(3-chloro-4-(6-ethyl-4-
oxo-4H-chromen-2-yl)phenyl)-2-(3-fluorophenyl)acetamide (3h)
was obtained as a yellow solid, yield 45%, mp: 181–182 °C, HRMS
(ESI) m/z calculated for [M+H]+ 436.1116, found 436.1125. 1H NMR
(400 MHz, CDCl3) d 8.35 (s, 1H), 8.04 (d, J = 2.0 Hz, 1H), 7.81 (d,
J = 1.2 Hz, 1H), 7.62–7.51 (m, 3H), 7.44 (d, J = 8.6 Hz, 1H), 7.33 (td,
J = 7.9, 6.1 Hz, 1H), 7.13 (d, J = 7.7 Hz, 1H), 7.08 (dd, J = 9.5, 2.0 Hz,
1H), 7.01 (td, J = 8.4, 2.1 Hz, 1H), 6.65 (d, J = 6.2 Hz, 1H), 3.78 (s,
2H), 2.77 (q, J = 7.6 Hz, 2H), 1.29 (t, J = 7.6 Hz, 3H). 13C NMR
(101 MHz, CDCl3) d 178.60, 168.96, 163.09 (J = 248.5 Hz), 162.34,
155.08, 141.84, 140.94, 136.40 (J = 7.07 Hz), 134.31, 133.50,
131.10, 130.66 (J = 9. Hz), 127.24, 125.06 (J = 2.0 Hz), 123.77,
123.49, 121.42, 118.14, 117.95, 116.46 (J = 22.2 Hz), 114.72
(J = 21.2 Hz), 112.42, 44.30, 28.41, 15.47.
N-(3-Chloro-4-(6-ethyl-4-oxo-4H-chromen-2-yl)phenyl)-2-(4-
fluorophenyl)acetamide 3i, as a light yellow solid from the same
synthetic process with 3h, yield 65%, mp: 237–238 °C, HRMS
(ESI) m/z calculated for [M+H]+ 436.1116, found 436.1116. 1H
NMR (400 MHz, DMSO-d6) d 10.64 (s, 1H), 8.01 (d, J = 1.9 Hz, 1H),
7.88 (d, J = 1.8 Hz, 1H), 7.77 (d, J = 8.5 Hz, 1H), 7.73–7.55 (m, 3H),
7.37 (dd, J = 8.5, 5.7 Hz, 2H), 7.16 (t, J = 8.9 Hz, 2H), 6.60 (s, 1H),
3.71 (s, 2H), 2.75 (q, J = 7.5 Hz, 2H), 1.23 (t, J = 7.6 Hz, 3H). 13C
4.1.1.3. 1-(3-Chloro-4-(6-methoxy-4-oxo-4H-chromen-2-yl)phe-
nyl)-3-(3-chlorophenyl)urea (3c).
Yield 67%, yellow solid,
mp: 240–243 °C, HRMS (ESI) m/z calculated for [M+H]+ 455.0565,
found 455.0562. 1H NMR (400 MHz, DMSO-d6) d 9.33 (s, 1H),
9.13 (s, 1H), 7.91 (d, J = 1.8 Hz, 1H), 7.81–7.69 (m, 2H), 7.67 (d,
J = 8.8 Hz, 1H), 7.56–7.40 (m, 3H), 7.40–7.26 (m, 2H), 7.12–7.00
(m, 1H), 6.62 (s, 1H), 3.88 (s, 3H). 13C NMR (101 MHz, DMSO-d6)
d 176.34, 161.76, 156.61, 151.97, 150.58, 142.90, 140.64, 133.13,
131.93, 131.49, 130.29, 124.01, 123.76, 123.29, 121.88, 119.93,
118.88, 117.89, 116.93, 116.75, 110.79, 104.69, 55.66.
4.1.1.4. 1-(3-Chloro-4-(6-ethyl-4-oxo-4H-chromen-2-yl)phenyl)-
3-(4-chlorophenyl)urea (3d).
Yield 75%, yellow solid, mp:
258–259 °C, HRMS (ESI) m/z calculated for [M+H]+ 453.0773, found
453.0767. 1H NMR (400 MHz, DMSO-d6) d 9.29 (s, 1H), 9.06 (s, 1H),
7.90 (dd, J = 6.2, 1.8 Hz, 2H), 7.78–7.68 (m, 2H), 7.62 (d, J = 8.6 Hz,
1H), 7.50 (dd, J = 12.4, 5.4 Hz, 3H), 7.36 (d, J = 8.8 Hz, 2H), 6.61 (s,
1H), 2.76 (q, J = 7.5 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H). 13C NMR
(101 MHz, DMSO-d6) d 177.25, 162.60, 154.93, 152.63, 143.65,
141.97, 138.68, 134.97, 132.54, 132.17, 129.18, 126.48, 124.59,
123.46, 123.39, 120.69, 119.40, 118.91, 117.32, 112.09, 28.06,
15.98.
4.1.1.5. 1-(3-Chloro-4-(6-methoxy-4-oxo-4H-chromen-2-yl)phe-
NMR (101 MHz, DMSO-d6) d 177.21, 170.38, 162.37, 161.72
nyl)-3-(3,4-dichlorophenyl)urea (3e).
Yield 54%, yellow so-
(J = 243.4 Hz), 154.91, 142.88, 141.99, 134.99, 132.42, 132.21,
132.00 (J = 3.0 Hz), 131.61 (J = 8.1 Hz), 126.02, 123.44, 123.38,
120.43, 118.90, 118.14, 115.55 (J = 21.2 Hz), 112.25, 42.77, 28.05,
15.96.
lid, mp: 256–258 °C, HRMS (ESI) m/z calculated for [M+H]+
489.0176, found 489.0190. 1H NMR (400 MHz, DMSO-d6) d 9.37
(s, 1H), 9.23 (s, 1H), 7.89 (s, 2H), 7.75 (d, J = 8.8 Hz, 1H), 7.66 (d,
J = 8.9 Hz, 1H), 7.53 (dd, J = 17.5, 8.9 Hz, 2H), 7.44 (d, J = 9.5 Hz,
2H), 7.38 (d, J = 8.9 Hz, 1H), 6.62 (s, 1H), 3.88 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) d 176.96, 162.41, 157.26, 152.55, 151.21,
143.39, 139.95, 132.53, 132.17, 131.59, 131.13, 124.82, 124.39,
124.26, 123.96, 120.59, 120.28, 119.61, 119.29, 117.50, 111.45,
105.33, 56.31.
4.2. Molecular docking
The molecular modeling of 3d was performed with Discovery
Studio.3.0/CDOCK protocol ( Accelrys Software Inc.) according to
the reported process.56,57 Three dimensional structures of Raf-1
(PDB ID: 3OMV) and JNK1 (PDB ID: 3ELJ) were downloaded from
Protein Data Bank (PDB). The following process was used to carry
out molecular docking: (1) deleting the water crystallization in-
volved in protein kinase structure; (2) optimizing protein structure
and ligands; (3) defining receptor and ligand, finding the candidate
binding site; (4) deleting small molecular docking in candidate
binding site; (5) docking designed compounds into the candidate
binding site on the target protein kinase; (6) molecular modeling
based on the above docking data.
4.1.1.6. 1-(3-Chloro-4-(6-fluoro-4-oxo-4H-chromen-2-yl)pheny
l)-3-(3-chlorophenyl)urea (3f).
Yield 40%, yellow solid,
mp >300 °C, HRMS (ESI) m/z calculated for [MꢁH]ꢁ 441.0208,
found 441.0217. 1H NMR (400 MHz, DMSO-d6) d 9.34 (s, 1H),
9.14 (s, 1H), 7.92 (d, J = 2.0 Hz, 1H), 7.86–7.81 (m, 1H), 7.79–7.70
(m, 4H), 7.51 (dd, J = 8.6, 2.0 Hz, 1H), 7.37–7.30 (m, 2H), 7.07 (dt,
J = 4.9, 2.2 Hz, 1H), 6.69 (s, 1H). 13C NMR (101 MHz, DMSO-d6) d
176.00, 162.40, 152.32, 151.99, 143.10, 140.62, 133.13, 132.00,
131.68, 130.36, 124.25 (J = 7.1 Hz), 123.78, 122.60, 122.35,
121.96, 121.25 (J = 9.1 Hz), 118.90, 117.93, 117.00, 116.80,
110.96, 109.43 (J = 24.2 Hz).
4.3. Biological assays
4.3.1. Cell culture
4.1.1.7. 1-(3-Chloro-4-(7-hydroxy-4-oxo-4H-chromen-2-yl)phe-
All human cell lines, including HepG2, HCT116, A549, QSG7701
and HL7702 cells were obtained from Cell Resources Center of
Shanghai Institutes for Biological Science, Chinese Academy of Sci-
ence. They were maintained in Dulbecco’s modified Eagle’s med-
nyl)-3-(3-chlorophenyl)urea (3g).
Yields 45%, yellow solid,
mp >300 °C, HRMS (ESI) m/z calculated for [M+H]+ 441.0409, found
441.0406. 1H NMR (400 MHz, DMSO-d6) d 9.87 (s, 1H), 9.64 (s, 1H),
7.92 (dd, J = 17.9, 9.1 Hz, 2H), 7.73 (d, J = 8.6 Hz, 2H), 7.48 (dd,
J = 8.6, 2.0 Hz, 2H), 7.32 (d, J = 4.9 Hz, 2H), 7.09–7.01 (m, 1H),
6.96 (dd, J = 8.7, 2.2 Hz, 1H), 6.91 (d, J = 2.1 Hz, 1H), 6.49 (s, 1H).
ium (DMEM) with 10% fetal bovine serum, 100
and 100
g mlꢁ1 streptomycin at 37 °C in a humidified atmosphere
of 5% CO2.
l
g mlꢁ1 penicillin
l

830
F. Jin et al. / Bioorg. Med. Chem. 21 (2013) 824–831
4.3.2. Cell growth inhibition assay
The synthesized compounds were dissolved in DMSO, and then
diluted with culture medium to the final concentrations ranging
from 0.5 to 50 lM (the final DMSO concentration was less than
1%) for tumor cells assay, 50 and 100 lM for normal liver cells assay.
against HepG2 and HCT116 cells, and the effect of 3d on the phos-
phorylation level of ATF-2. 1H NMR and 13C NMR spectra and high
resolution mass spectrometry) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.bmc.
2012.04.006.
100
l
L of cell solution with the concentration of 5 ꢂ 105 cells mLꢁ1
was seeded to each well of a 96-well plate and incubated for 24 h at
37 °C in a 5% CO2 incubator. The medium was then removed from
the 96-well plate. The test compound solution was added to each
well for the treatment of maintained cells in triplicate per concen-
tration, and incubated at 37 °C in a 5% CO2 incubator for 48 h. After
this treatment, 10
each well and incubated for 4 h at 37 °C. The formazan precipitate
was dissolved in 100 L DMSO and the absorbance at 495 nm was
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After treatment with indicated compound for specific time,
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Acknowledgements
The authors would like to thank the financial support provided by
the Ministry of Science and Technology of China (2012ZX09506001-
010, 2012CB722605, 2012AA020305 and 2011DFA30620) and the
Science Industry Trade and Information Technology Commission
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Supplementary data
Supplementary data (cell apoptosis and cell cycle arrest in
HepG2 cells induced by 10 lM 3d, anti-proliferative curve of 3d

F. Jin et al. / Bioorg. Med. Chem. 21 (2013) 824–831
831
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