Design, synthesis and evaluation of acridine derivatives as multi-target Src and MEK kinase inhibitors for anti-tumor treatment

Article abstract of DOI:10.1016/j.bmc.2015.12.011

Clinical studies have shown enhanced anticancer effects of combined inhibition of Src and MEK kinases. Development of multi-target drugs against Src and MEK is of potential therapeutic advantage against cancers. As a follow-up of our previous studies, and by using molecular docking method, we designed and synthesized a new series of 9-anilinoacridines containing phenyl-urea moieties as potential novel dual Src and MEK inhibitors. The anti-proliferative assays against K562 and HepG-2 tumor cells showed that most of the derivatives displayed good cytotoxicity in vitro. In particular, kinase inhibition assays showed that compound 8m inhibited Src (59.67%) and MEK (43.23%) at 10 μM, and displayed moderate inhibitory activity against ERK and AKT, the downstream effectors of both Src and MEK. Moreover, compound 8m was found to induce K562 cells apoptosis. Structure-activity relationships of these derivatives were analyzed. Our study suggested that acridine scaffold, particularly compound 8m, is of potential interest for developing novel multi-target Src and MEK kinase inhibitors.

Full text of DOI:10.1016/j.bmc.2015.12.011

Accepted Manuscript
Design, Synthesis and Evaluation of Acridine Derivatives as Multi-Target Src
and MEK Kinase Inhibitors for Anti-tumor Treatment
Zhishan Cui, Xi Li, Lulu Li, Bin Zhang, Chunmei Gao, Yuzong Chen, Chunyan
Tan, Hongxia Liu, Weiyi Xie, Ti Yang, Yuyang Jiang
PII:
S0968-0896(15)30179-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.12.011
BMC 12704
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 October 2015
1 December 2015
7 December 2015
Please cite this article as: Cui, Z., Li, X., Li, L., Zhang, B., Gao, C., Chen, Y., Tan, C., Liu, H., Xie, W., Yang, T.,
Jiang, Y., Design, Synthesis and Evaluation of Acridine Derivatives as Multi-Target Src and MEK Kinase Inhibitors
for Anti-tumor Treatment, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.
2015.12.011
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Design, Synthesis and Evaluation of Acridine Derivatives as Multi-Target Src and MEK Kinase
Inhibitors for Anti-tumor Treatment
Zhishan Cuia,b,†, Xi Lib,†, Lulu Lia, b, Bin Zhangb*, Chunmei Gaoa,b*,Yuzong Chenc, Chunyan Tana,b,
Hongxia Liua,b, Weiyi Xieb, TiYangb, Yuyang Jianga, b, d
aThe Guangdong Province Key Laboratory of Chemical Biology, the Graduate School at Shenzhen,
Tsinghua University, Shenzhen 518055, PR China
bNational & Local United Engineering Lab for Personalized anti-tumor drugs, the Graduate School
at Shenzhen, Tsinghua University, Shenzhen 518055, P. R. China
cBioinformatics and Drug Design Group, Department of Pharmacy, Centre for Computational
Science and Engineering, 117543, Singapore
dSchool of Medicine, Tsinghua University, Beijing 100084, PR China
* Corresponding author. Tel.: +86 755 2603 6017; fax: +86 755 2603 2094;
Email address: binzhang86@126.com; chunmeigao@sz.tsinghua.edu.cn
†Authors with equal contributions.

Abstract
Clinical studies have shown enhanced anticancer effects of combined inhibition of Src and MEK
kinases. Development of multi-target drugs against Src and MEK is of potential therapeutic
advantage against cancers. As a follow-up of our previous studies, and by using molecular docking
method, we designed and synthesized a new series of 9-anilinoacridines containing phenyl-urea
moieties as potential novel dual Src and MEK inhibitors. The anti-proliferative assays against K562
and HepG-2 tumor cells showed that most of the derivatives displayed good cytotoxicy in vitro. In
particular, kinase inhibition assays showed that compound 8m inhibited Src (59.67%) and MEK
(43.23%) at 10 μM, and displayed moderate inhibitory activity against ERK and AKT, the
downstream effectors of both Src and MEK. Moreover, compound 8m was found to induce K562
cells apoptosis. Structure−activity relationships of these derivatives were analyzed. Our study
suggested that acridine scaffold, particularly compound 8m, is of potential interest for developing
novel multi-target Src and MEK kinase inhibitors.
Key words: Src; MEK; kinase inhibitor; acridine; antitumor; apoptosis

1. Introduction
Src is one of non-receptor type tyrosine kinases, which was first identified as a protein encoded
by sarcoma sirus,1 and subsequently found to be a critical regulator of a large number of
intracellular signaling pathways.2 Recent studies have shown that dysregulation of Src is strongly
associated with malignant progression of human tumors.3 As Src was wildly expressed in tumor
cells, and controlled a myriad of cellular processes including proliferation, migration, invasion,
gene transcription, differentiation, angiogenesis, and survival,4 it has been pursued as a potential
drug target for cancer and related bone disease.5 Development of Src inhibitors has become a main
focus in antitumor drug campaigns.2c, 5b, 6 At present, a series of Src inhibitors have been developed,
some of which have entered into clinical research phases, such as Dasatinib,7 Bosutinib,8
Ponatinib,9 and Saracatinib, et al.2d, 10
Although Src inhibitor drugs have exhibited highly promising anticancer effects, much work
needs to be done since most of the drugs suffered from low tumor response or resistance.11 The
resistance to Src inhibitors may partly arise from the crosstalk between Src-pathway and other
pathways.12 Therefore, the exploration of multi-target drugs or drug combinations to simultaneously
inhibit the drug target and resistant pathways can provide a more effective approach for cancer.13
Recent study indicated that combined inhibition of Src and MEK was a promising approach to
15
improve the efficacy of cancer therapy.14 MEK kinase itself is an important anticancer target
because of its key roles in regulating mitogen-activated protein kinase (MAPK) signaling pathway
in apoptosis and oncogenic transformation.16 However, only 22% patients with BRAF mutations are
sensitive to MEK inhibitor (Trametinib) in a clinical trial for melanoma.17 Combinatorial
approaches are necessary to improve the clinical response.18 As Src and MEK inhibitors displayed
synergistic effects14a, development of multi-target drugs targeted both Src and MEK may therefore

offer a better therapeutic advantage.
Acridine derivatives have been explored as potential therapeutic agents for cancer treatment19
particularly for targeting DNA and DNA related enzymes, such as topoisomerases, telomerase, et
al.20 However, some acridine derivatives have been found to inhibit the activity of tyrosine
kinases.21 In our pursuit of novel acridine analogues with antitumor activity,22 through using
molecular docking and SVM high throughput virtual screening methods, we found that the acridine
derivative OA (Figure 1) inhibited Src activity.23 However, the inhibitory activity is very weak
likely due in part to only one hydrogen bond formed between the acridine derivative and the hinge
of Src protein. Shokat et al. had found that small molecules bearing a moity such as urea bonds with
the ability to bind to the DFG-out conformation of c-Src could improve the kinase inhibition
affinity greatly.24 In addition, some kinase drugs approved or in clinical trails, such as Imatinib,25
Ponatinib,26 and Regorafenib,27 all contain amido or urea moieties, which contribute much to the
kinase binding affinity due to that they can form hydrogen bonds with the amino-acid residues of
the DFG-out pocket of kinases.28
Based on the above considerations and the characteristics of protein kinase inhibitors with the
current state of Src and MEK kinase inhibitors, we introduced the urea moities to the
9-anilinoacridine substituents (Table 1) such as compound 8a (Figure 2a) to improve the bioactivity.
In order to further understand the interaction between compound 8a and kinases, molecular docking
studies were performed using the Discovery Studio 3.1. Figure 2b and 2c indicated that compound
8a could dock into ATP binding sites of both Src and MEK. For Src, nitro group of compound 8a
could form a hydrogen bond with Met341 in the hinge region. Meanwhile, as we expected, -NH of
the urea moiety formed a hydrogen bond with Glu310 in the DFG-out pocket. In addition, π-π
interaction was formed between the acridine ring with Phe405. Cation–π interaction was also

occurred between the phenyl group and Tyr340. For MEK, the nitro group of compound 8a enabled
the formation of hydrogen bonds with Asn221 and Arg234 in the hinge region of MEK. -NH of the
urea linkage formed two hydrogen bonds with Phe209 in the DFG-out region. All the results
revealed that compound 8a could be a potential dual inhibitor of Src and MEK kinases.
In this article, a series of compound 8a derivatives with different substitents at acridine and
phenyl rings were designed and synthesized. Preliminary in vitro anti-proliferative ability of
synthetic compounds against related human cancer cell-lines (K562 and HepG-2 cells) were studied,
and structure–activity relationship was disclosed. Inhibition rates of the representative synthetic
compounds against Src and MEK kinase were also evaluated.
2. Results and discussions
2.1 Chemistry
The synthetic pathway was illustrated in Scheme 1. The reaction of 2-chlorobenzoic acid
derivatives 1 with the anilines 2 in the presence of Cu afforded the corresponding anthranilic acids 3
(Ullmann conditions).29 Cyclization of compound 3 using POCl3 gave the 9-chloroacridine
derivatives 4 (4a-4g) by using column chromatography (petroleum ether: ethyl acetate=20:1).
Compound 5 was reacted with anilines bearing different substituents to give compound 6 (6a-6l).
Subsequent reduction of 6 (6a-6l) in the presence of Fe and NH4Cl led to the expected phenyl-urea
derivatives 7 (7a-7l).30 Compound 7 (7a-7l) (0.22 mmol) in THF (10 mL) which then underwent
nucleophilic substitution reaction with compound 4 (4a-4g) (0.20 mmol) in the presence of three
drops of concentrated hydrogen chloride.The mixture was stirred for two days at room temperature.
The raw products were filtered off, washed with diethyl ether. Finally, the raw products were
filtered off, washed with diethyl ether to obtain the desired compound 8 (8a-8r) in moderate to

good yields.
2.2 In vitro cell growth inhibition assay
The antiproliferative activity of the desired acridine derivatives bearing phenyl-urea group at
9-position was tested against K562 and HepG-2 cells by MTT assay, and Imatinib was used as the
positive control. As shown in Table 2, most of the acridine derivatives displayed good
antiproliferative activity with low micromolar IC50 values, and both the substituents in the acridine
ring (A) and phenyl ring (B) affected the antiproliferative activity significantly. As synthesized
compounds were more sensitive to K562 cells than HepG-2 cells, K562 cells were selected to study
the structure-activity relationship.
2.2.1. Substitution Effects of the benzene ring A (R5, R6, R7)
As literature reported, the substituents in phenyl ring A played an important role in the
bioactivity.31 A series of compounds 8 (8a−8l) bearing the same acridine core but with different
substituents atR5, R6, R7 were synthesized (Table 2). For R5, the introduction of methoxyl group had
almostly no effect on the cytotoxicity (8a vs. 8b). For R6, compound 8c with methoxyl group
dispalyed similar IC50 value compared with 8a. By changing methoxyl (8c) to methyl (8h) or ethyl
(8i) group, compounds showed decreasing inhibitory effect. However, introduction of
tri-fluoromethyl substituent showed much stronger inhibition effect, approximately 3-fold more
potent than 8a. For R7, a different trend could be seen compared to that for R6. For example, when
methxoyl group was introduced, compound 8d displayed an apparent reduced activity. However,
introduction of alkyl group (8j:ethyl; 8k:propyl; 8l:butyl) led to an increased antiproliferative
activity compared to 8a. These results indicated that the position, electron-negativity and steric
effect in the benzene ring may change the cytotoxic profile.
2.2.2. Substitution Effects of the ring of acridine (ring B)
Molecular modeling results (Figure 2, Figure 1s and Table 1s) indicated that nitro or methoxyl
group at R1 position could form hydrogen bonds to the key residues of Src and MEK. Therefore,
most of the acridine compounds were modified with these moieties, such as 8a-8m, 8o-8p. As seen

from Table 1, the antiproliferative activity of compound 8m with methoxyl group was comparable
to that of 8a with nitro group. The results indicated that the R1 position might tolerate both nitro and
methoxyl group, which was in agreement with molecular docking results. When methoxyl group
was introduced to the R3 or R4 position, almost no distinct effect on the bioactivity was observed,
such as 8o-8q. To our surprise, compound 8n with no substitents at R1 position and methoxyl group
at R2 position displayed the best antiproliferative activity with an IC50 value at 0.49 μM. As for B
ring, the introduction of tri-fluoromethyl group at R6 position (8g) could improve the cytotoxity,
compound 8r containing both substituents of 8n and 8g was synthesized to increase the
antiproliferative activity. To our disappointment, this modification led to approximately 4-fold less
activity than that of compound 8n.
2.3 In vitro kinase inhibition assay
As compounds 8m, 8n and 8q had good bioactivities against both K562 and HepG-2 cells with
IC50 values all below10 μM, they were firstly selected to screen in vitro kinase inhibition assay to
evaluate whether they can truly inhibit Src activity. Unfortunately, only compound 8m potently
inhibited 59.67% of Src activity at 10 μM. At the same condition, compound 8m exhibited
moderate activity against MEK and PI3K with inhibition rates of 43.23% and 44.41%, respectively.
Although the kinases activities of compound 8m were weaker than the antiproliferative activity, the
synergistic effects of multi-target agents of moderate activities might contribute to the
cytotoxicity.32
Furthermore, the influences of compound 8m on the protein expression levels of ERK and AKT,
the downstream kinases of Src, MEK and PI3K were evaluated. K562 cells were treated with
compound 8m at different concentrations (0, 0.5, 2.5, 5, and 10 µM) for 48 h. The results (Figure 3)
indicated that 8m reduced the expression of ERK and AKT in a dose-dependent manner. In addition,
DNA binding ability of 8m was evaluated by UV-vis spectrophotometric titration test. The binding
constant was 0.083 M-1, which was much lower than DNA binding agents (Figure 2s, supporting
data). Therefore, DNA binding ability was not contributed to the antiproliferative activity of

compound 8m. All the results suggested that the inhibition of Src, MEK and PI3K might mainly
contribute to the good activities of compound 8m against K562 and HepG-2 cell lines.
2.4 Effects of Compound 8m on Cell Apoptosis
Literature reported that inhibition Src activity can induce cancer cell apoptosis, such as the
preclinical drugs AZM475271,33 SU6656,34 etc.35 Compound 8m with the best kinases inhibition
rates was selected to test its impact on apoptosis. As shown in Figure 4, compound 8m induced
apoptosis in K562 cells in a concentration-dependent manner. At the concentration increased from 0
μM to 10 μM, the early apoptosis ratio increased from 3.14% to 54.98%, when K562 cells were
treated with compound 8m for 48 h, which demonstrated that compound 8m could effectively
induce K562 cells apoptosis.
In addition, the expression of cleaved-caspase-3 and cleaved-caspase-7 were evaluated to further
evaluate the apoptosis induced. As we know, caspases play an important role in apoptosis, among
which caspase-3 and caspase-7 are executioner caspases. They will be cleaved by an initiator
caspase after apoptotic signaling events occurred. Figure 5 showed that compound 8m could induce
significant activation of cleaved-caspase-3 and cleaved-caspase-7 at 10 µM for 48 h, suggesting the
capability of 8m to induce apoptosis. Moreover, PARP (poly ADP-ribose polymerase) can be
activated in cells experiencing DNA damage to repair them, which become inactivated by caspase
cleavage. Caspase-3 and caspase-7 are responsible for PARP cleavage. Therefore, we tested the
induction of PARP cleavage by compound 8m. The results in Figure 5 revealed the generation of
the cleaved PARP. The results above suggested that compound 8m could induce K562 cells
apoptosis.
3. Conclusion
In this study, a series of novel multi-target inhibitors of Src and MEK bearing acridine and
phenyl-urea scaffolds were designed and synthesized based on rational design and molecular
docking methods. Most of these compounds displayed low micromolar IC50 values against K562

cells in vitro. Structure−activity relationship indicated that substitutions of the benzene ring (A) and
acridine ring (B) play important roles in the antiproliferative activities of the tested compounds.
Kinase assay study confirmed that compound 8m is a novel multi-target Src and MEK kinases
inhibitor. Moreover, compound 8m was found to induce cancer cell apoptosis. Further optimization
of this structure for improved anti-proliferative activity is ongoing. Our study suggested the
potential use of the acridine compounds as multi-target kinase inhibitors, and indicated new
possibilities for expanding the application of acridine compounds beyond their conventional DNA
and DNA related enzyme targets.
4. Experimental section
4.1. Synthesis and characterization
See supporting information for synthetic methods and the preparation of compounds 4a-4g and
7a-7l.
4.1.1. General procedure for compounds 8 (8a-8r)
Compound 7 (7a-7l) (0.22 mmol) in THF (10 mL) was added three drops HCl (6 mM). The
mixture was stirred for 0.5 h and then compound 4 (4a-4g) was added respectively (0.20 mmol).The
mixture was stirred for two days at room temperature under nitrogen until TLC indicated the
completion of reaction. The products were filtered off, washed with diethyl ether, and the pure
target compounds 8 (8a-8q) were obtained.
4.1.1.1. 1-(4-((3-Nitroacridin-9-yl)methyl)phenyl)-3-phenyl)urea (8a). Yield 73%; red solid, mp
268.2-267.9 °C；1H NMR (400 MHz, DMSO-d6) δ 9.34 (s, 1H), 9.12 (s, 1H), 8.68 (s, 1H), 8.43 (s,
1H), 8.42-8.41(m, 1H), 8.12 (s, 1H), 8.04 (d, J = 8.8 Hz, 1H), 7.94-7.90 (m, 2H), 7.60 (d, J = 8.8 Hz,
2H), 7.47 (d, J = 7.6 Hz, 2H), 7.42 (d, J = 8.8 Hz, 1H), 7.29 (dd, J = 8.0 Hz, 7.6 Hz, 4H), 6.98 (dd,
J = 8.0Hz, 7.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ：156.29, 153.37, 149.68, 145.80, 144.90,
141.60, 133.33, 130.48, 130.29, 130.16, 129.85, 129.01, 128.62, 126.04, 123.43, 123.28, 122.01,

121.65, 121.54, 119.85, 118.05, 118.01, 117.03, 114.63, 114.39, 114.34.HRMS (ESI) m/z calculated
for [M+H]+ 450.1566, found 450.1544.
4.1.1.2. 1-(2-Methoxyphenyl)-3-(4-((3-nitroacridin-9-yl)amino)phenyl)urea (8b). Yield 75%；red
solid, mp 232.8-233.9 °C；1H NMR (400 MHz, DMSO-d6) δ 11.74(s, 1H), 9.73 (s, 1H), 9.58, (s,
1H), 8.96 (s, 1H), 8.38 (s, 1H), 7.92 (s, 1H), 7.53-7.47 (m, 5H), 7.32 (s, 1H), 7.28 (m, 3H), 7.16 (s,
13
1H), 7.03 (s, 1H), 6.95 (s, 1H), 4.07 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 156.29, 153.28,
153.02, 149.68, 148.15, 145.78, 144.45, 133.32, 129.35, 129.0, 126.05, 123.28, 122.12, 121.75,
121.55, 121.20, 121.07, 120.95, 119.50, 119.27, 118.79, 118.63, 117.03, 114.70, 111.28. HRMS
(ESI) m/z calculated for [M+H]+ 480.1672, found 480.1667.
4.1.1.3. 1-(3-Methoxyphenyl)-3-(4-((3-nitroacridin-9-yl)amino)phenyl)urea (8c). Yield 80%; red
1
solid, mp 274.1-275.8 °C；H NMR (400 MHz, DMSO-d6) δ 9.32 (s, 1H), 9.13 (s, 1H), 8.66 (s, 1H),
8.42 (s, 1H), 8.41 (s, 1H), 8.10 (s, 1H), 8.02 (d, J = 8.8 Hz, 1H), 7.94-7.88 (m, 2H), 7.59 (d, J = 8.8
Hz, 2H), 7.40(s, 1H), 7.26 (d, J = 7.6 Hz, 2H), 7.21 (s, 1H), 7.17 (d, J = 8.0 Hz, 1H), 6.94 (dd, J =
8.0 Hz, 8.0 Hz, 1H), 6.56 (dd, J = 8.0 Hz, 8.0 Hz, 1H), 3.73 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 156.61, 153.75, 153.15, 149.86, 140.42, 140.24, 140.18, 139.05, 137.02, 134.12,
129.39, 129.29, 128.77, 126.10, 125.19, 124.27, 122.35, 121.64, 119.28, 119.14, 118.65, 118.56,
116.55, 115.95, 115.59, 104.10, 56.40. HRMS (ESI) m/z calculated for [M+H]+ 480.1672, found
480.1661.
4.1.1.4. 1-(4-Methoxyphenyl)-3-(4-((3-nitroacridin-9-yl)amino)phenyl)urea (8d). Yield 53%;
1
red solid, mp 283.1-284.3 °C； H NMR (400 MHz, DMSO-d6) δ 9.32 (s, 1H), 8.95, (s, 1H), 8.76 (s,
1H), 8.45 (d, J = 4.8 Hz, 1H), 8.19 (s, 1H), 8.08 (d, J = 9.2 Hz, 1H), 7.98 (s, 2H), 7.62 (d, J = 8.8
13
Hz, 2H), 7.46 (s, 1H), 7.38-7.33 (m, 3H), 6.88 (d, J = 9.2 Hz, 2H), 3.72 (s, 3H); C NMR (100
MHz, DMSO-d6) δ 155.07, 153.25, 150.41, 141.52, 140.19, 136.18, 133.13, 128.97, 126.52, 125.25,

124.65, 120.49, 119.74, 119.29, 116.47, 115.14, 114.54, 55.70. HRMS (ESI) m/z calculated for
[M+H]+ 480.1672, found 480.1661.
4.1.1.5. 1-(3-Fluorophenyl)-3-(4-((3-nitroacridin-9-yl)amino)phenyl)urea (8e). Yield 71%; red
1
solid, mp >300 °C；H NMR (400 MHz, DMSO-d6) δ 9.49 (s, 2H), 8.68, (s, 1H), 8.41 (d, J = 8.0 Hz,
1H), 8.11 (s, 1H), 8.02 (d, J = 9.2 Hz, 1H), 7.95-7.88 (m, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.50 (d, J =
13
11.6 Hz, 1H), 7.38 (s, 1H), 7.34-7.28 (m, 3H), 7.12 (d, J = 8.0 Hz, 1H), 6.81-6.76 (m, 1H); C
NMR (100 MHz, DMSO-d6) δ 164.09, 161.70, 154.61, 152.95, 150.20, 142.10, 141.99, 141.61,
140.38, 139.06, 135.72, 130.84, 130.75, 128.94, 126.61, 124.53, 124.32, 119.71, 118.16, 116.22,
115.02, 114.31, 114.29, 108.73, 108.52, 105.34, 105.08. HRMS (ESI) m/z calculated for [M+H]+
468.1472, found 468.1490.
4.1.1.6. 1-(3-Chlorophenyl)-3-(4-((3-nitroacridin-9-yl)amino)phenyl)urea (8f). Yield 59%; red
solid, mp >300 °C；1H NMR (400 MHz, DMSO-d6) δ 9.52 (s, 2H), 8.67, (s, 1H), 8.39 (s, 1H), 8.10
(s, 1H), 8.01 (d, J = 9.2 Hz, 1H), 7.91 (d, J = 10.8 Hz, 2H), 7.69 (s, 1H), 7.60 (d, J = 8.8 Hz, 2H),
7.38 (s, 1H), 7.30-7.28 (m, 4H), 7.01 (d, J = 5.2 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 154.62,
152.94, 150.20, 141.73, 141.58, 140.34, 139.13, 135.75, 133.70, 128.93, 126.59, 124.60, 124.35,
121.92, 119.70, 117.87, 116.94, 116.23, 115.02.HRMS (ESI) m/z calculated for [M+H]+ 484.1176,
found 484.1145.
4.1.1.7.1-(4-((3-Nitroacridin-9-yl)amino)phenyl)-3-(3(trifluoromethyl)phenyl)urea (8g). Yield
81%; red solid, mp 268.7-270.9 °C；1H NMR (400 MHz, DMSO-d6) δ 11.95(s, 1H),9.75 (s, 1H),
9.68 (s, 1H), 8.80 (s, 1H), 8.47 (d, J = 8.4 Hz, 1H), 8.22 (s, 1H), 8.10 (d, J = 9.6 Hz, 1H), 8.01 (s,
3H), 7.65 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 1H), 7.54-7.49 (m, 2H), 7.41 (d, J = 8.4 Hz, 2H),
7.32 (d, J = 7.6 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ 155.59, 153.08, 150.50, 141.46, 141.02,
140.18, 139.87, 136.43, 130.45, 130.23, 129.91, 128.96, 126.44, 126.05, 125.66, 124.86, 123.34,

122.15, 119.73, 118.63, 117.06, 116.62, 115.19, 114.47, 114.43, 65.38. HRMS (ESI) m/z calculated
for [M+H]+ 518.1440, found 518.1450.
4.1.1.8. 1-(4-((3-Nitroacridin-9-yl)amino)phenyl)-3-(m-tolyl)urea (8h). Yield 63%; red solid, mp
277.8-278.9 °C；1H NMR (400 MHz, DMSO-d6) δ 9.47 (s, 1H), 9.16, (s, 1H), 8.70 (s, 1H), 8.41 (s,
1H), 8.11 (s, 1H), 8.03 (d, J = 6.8 Hz, 1H), 7.91 (s, 2H), 7.60 (d, J = 7.6 Hz, 2H), 7.38 (s, 1H),
7.34-7.28 (m, 4H), 7.16 (d, J = 6.8 Hz, 1H), 6.78 (d, J = 6.4 Hz, 1H), 2.27 (s, 3H); 13C NMR (100
MHz, DMSO-d6) δ 155.57, 153.00, 150.47, 141.49, 140.19, 140.14, 137.86, 136.40, 129.03, 126.47,
125.79, 124.87, 124.07, 120.75, 119.72, 119.40, 119.28, 116.94, 116.58, 115.18, 114.47, 25.64.
HRMS (ESI) m/z calculated for [M+H]+464.1723, found464.1729.
4.1.1.9. 1-(3-Ethylphenyl)-3-(4-((3-nitroacridin-9-yl)amino)phenyl)urea (8i). Yield 47%; red
1
solid, mp 275.4-275.8 °C；H NMR (400 MHz, DMSO-d6) δ 9.37 (s, 1H), 9.10 (s, 1H), 8.65 (s, 1H),
8.39 (d, J = 8.8 Hz, 1H), 8.2 (s, 1H), 8.00 (d, J = 9.2 Hz, 1H), 7.91-7.86 (m, 2H), 7.59 (d, J = 8.8 Hz,
2H), 7.35 (s, 1H), 7.31 (s, 1H), 7.28 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 7.18 (m, 1H), 6.82
(d, J = 7.2 Hz, 1H), 2.57 (q, J = 7.6 Hz, 2H), 1.18 (t, J = 7.6 Hz, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 154.27, 153.11, 150.13, 144.82, 141.69, 140.49, 140.16, 139.16, 135.46, 129.16,
128.95, 126.68, 124.14, 123.81, 121.87, 119.70, 119.58, 118.16, 117.98, 116.08, 115.29, 114.98,
28.78, 16.00. HRMS (ESI) m/z calculated for [M+H]+ 478.1879, found 478.1873.
4.1.1.10. 1-(4-Ethylphenyl)-3-(4-((3-nitroacridin-9-yl)amino)phenyl)urea (8j). Yield 43%; red
1
solid, mp 253.2-254.7 °C；H NMR (400 MHz, DMSO-d6) δ 9.50 (s, 1H), 9.14 (s, 1H), 8.82 (s, 1H),
8.47 (s, 1H), 8.22 (s, 1H), 8.10 (d, J = 9.6 Hz, 1H), 8.02 (d, J = 7.6 Hz, 2H), 7.63 (d, J = 8.8 Hz,
2H), 7.55 (d, J = 8.8 Hz, 1H), 7.40-7.36 (m, 4H), 7.12 (d, J = 8.4 Hz, 2H), 2.52 (m, 2H), 1.16 (t, J =
13
7.6 Hz, 3H); C NMR (100 MHz, DMSO-d6) δ 155.60, 153.12, 150.49, 141.44, 140.44, 140.18,
137.81, 137.72, 136.44, 128.97, 128.47, 126.43, 125.71, 124.86, 123.96, 119.73, 119.35, 118.82,

116.60, 115.18, 28.00, 16.22. HRMS (ESI) m/z calculated for [M+H]+ 478.1879, found 478.1895.
4.1.1.11. 1-(4-((3-Nitroacridin-9-yl)amino)phenyl)-3-(4-propylphenyl)urea (8k). Yield 80%; red
1
solid, mp 271.0-272.3 °C；H NMR (400 MHz, DMSO-d6) δ 9.48 (s, 1H), 9.13 (s, 1H), 8.81 (s, 1H),
8.48(s, 1H), 8.21 (s, 1H), 8.10 (d, J = 1.2 Hz, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.53 (d, J =8.4 Hz, 1H),
7.45 (s, 1H), 7.37(d, J = 8.4 Hz,2H), 7.25 (s, 1H), 7.22 (d, J = 9.6 Hz, 1H),7.10 (d, J = 8.4 Hz, 1H),
7.08 (s, 1H), 1.58-1.54(q, J = 7.6 Hz, 2H), 0.89(t, J = 7.6 Hz, 3H) ; 13C NMR (100 MHz, DMSO-d6)
δ153.25, 137.44, 135.11, 129.12, 129.05, 126.57, 123.36, 122.94, 119.75, 119.11, 118.20, 114.94,
113.80, 37.03, 24.60,14.01.HRMS (ESI) m/z calculated for [M+H]+ 492.2036, found492.2032
4.1.1.12. 1-(4-Butylphenyl)-3-(4-((3-nitroacridin-9-yl)amino)phenyl)urea (8l). Yield 44%; red
1
solid, mp 279.4-281.2 °C；H NMR (400 MHz, DMSO-d6) δ 9.43 (s, 1H), 9.08 (s, 1H), 8.79 (s, 1H),
8.46 (s, 1H), 8.22 (s, 1H), 8.10 (d, J = 8.8 Hz, 1H), 8.04-8.01 (m, 2H), 7.64 (m, 2H), 7.49 (s, 1H),
7.40-7.35 (m, 4H), 7.10 (d, J = 8.0 Hz, 2H), 1.54-1.51 (m, 2H), 1.33-1.27 (m, 2H), 0.90 (t, J = 7.8
Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 153.10, 150.50, 141.45, 140.20, 137.67, 136.38, 129.01,
126.44, 125.64, 124.84, 123.84, 119.74, 119.38, 118.81, 116.60, 115.19, 114.51, 34.64, 33.74, 22.18,
14.26. HRMS (ESI) m/z calculated for [M+H]+ 506.2192, found 506.2177.
4.1.1.13. 1-(4-((3-Methoxyacridin-9-yl)amino)phenyl)-3-phenylurea (8m). Yield 79%；yellow
1
solid, mp 233.4-234.8 °C; H NMR (400 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.71 (s, 1H), 9.63 (s,
1H), 8.09 (d, J = 8.0 Hz, 1H), 8.02 (m 1H), 7.93 (dd, J =7.6 Hz, 7.6 Hz, 1H), 7.84 (d, J = 8.0
Hz,1H), 7.71 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.4 Hz, 2H), 7.55 (m, 2H), 7.37 (d, J = 8.40 Hz, 2H),
7.31 (dd, J = 8.0 Hz, 7.6 Hz, 2H), 7.25 (d, J = 8.6 Hz, 1H), 7.13 (t, J = 8.0 Hz, 1H), 3.73 (s, 3H);
13C NMR (100 MHz, DMSO-d6) δ 155.89, 153.89, 153.12, 140.16, 139.77, 136.06, 134.88, 134.73,
129.27, 128.51, 126.15, 125.93, 124.09, 122.36, 121.52, 119.79, 119.34, 118.61, 115.05, 113.44,
103.92, 56.17. HRMS (ESI) m/z calculated for [M+H]+ 435.1821, found 435.1822.

4.1.1.14. 1-(4-((4-Methoxyacridin-9-yl)amino)phenyl)-3-phenylurea (8n). Yield 75%; yellow
1
solid, mp 227.1-228.3 °C；H NMR (400 MHz, DMSO-d6) δ11.20 (s, 1H), 9.50 (s, 1H), 9.26 (s, 1H),
8.09 (d, J = 8.8 Hz, 1H), 8.03-8.00 (m, 1H), 7.92 (m, J = 7.4 Hz, 1H), 7.72-7.70 (m, 2H), 7.63 (d, J
= 8.8 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 8.0 Hz, 1H), 7.38-7.35 (m, 2H), 7.29 (dd, J =
7.6 Hz, 7.2 Hz, 2H), 6.98 (dd, J =7.6 Hz, 7.6 Hz, 1H), 3.74 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ 155.85, 153.71, 153.11, 140.15, 139.79, 139.64, 134.88, 134.75, 129.25, 128.38, 125.98, 125.90,
124.04, 122.34, 121.61, 119.88, 119.34, 118.60, 115.13, 113.54, 103.92, 56.15. HRMS (ESI) m/z
calculated for [M+H]+ 435.1821, found 435.1840.
4.1.1.15.1-(4-((2-Methoxy-6-nitroacridin-9-yl)amino)phenyl)-3-phenylurea (8o). Yield 62%；red
solid, mp >300°C；1H NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H), 9.73 (s, 1H), 9.58 (s, 1H), 8.96
(s, 1H), 8.38 (s, 1H), 7.92 (s, 1H), 7.53-7.47 (m, 5H), 7.32 (s, 1H), 7.27 (m, 3H), 7.15 (s, 1H), 7.03
(s, 1H), 6.95 (m, 1H), 4.07 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ: 165.75, 158.62, 157.48,
152.58, 140.27, 137.35, 129.80, 129.44, 128.88, 128.06, 123.31, 123.01, 121.65, 120.60, 119.22,
115.76, 113.03, 109.88, 46.14.HRMS (ESI) m/z calculated for [M+H]+ 480.1672, found 480.1695.
4.1.1.16. 1-(4-((2-Methoxy-6-nitroacridin-9-yl)amino)phenyl)-3-phenylurea (8p). Yield 77%;
1
yellow solid, mp >300 °C；H NMR (400 MHz, DMSO-d6) δ 11.68 (s, 1H), 9.69 (d, J = 2.0 Hz, 1H),
9.40 (d, J = 1.6.Hz, 1H), 8.89 (s, 1H), 8.29 (d, J = 9.2 Hz, 1H), 8.10-8.04 (m, 2H), 7.77 (d, J = 7.6
Hz, 2H), 7.64 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 7.6 Hz, 2H), 7.29 (dd, J=
7.6 Hz, 8.0 Hz, 2H), 6.97 (dd, J = 7.2 Hz, 7.2 Hz, 1H), 3.77 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 156.56, 153.73, 153.04, 149.83, 140.17, 140.07, 138.99, 136.97, 139.38, 129.25,
128.67, 126.05, 122.36, 121.68, 119.33, 118.57, 116.56, 115.87, 115.60, 103.97, 56.30. HRMS (ESI)
m/z calculated for [M+H]+ 480.1672, found 480.1692.
4.1.1.17. 1-(4-((6-Chloro-2-methoxyacridin-9-yl)amino)phenyl)-3-phenylurea (8q). Yield 80%;

yellow solid, mp 297.0-298.2 °C；1H NMR (400 MHz, DMSO-d6) δ 11.34 (s, 1H), 9.68 (s, 1H),
9.40 (s, 1H), 8.05 (d, J = 1.6 Hz, 1H), 7.99 (d, J = 9.2 Hz,2H), 7.98 (s, 1H), 7.71(s, 1H), 7.84 (d, J =
9.2 Hz, 2H), 7.64 (t, J = 8.0 Hz, 2H), 7.47(t, J = 8.0 Hz, 1H), 7.38(t, J = 8.0 Hz, 2H),7.29 (t, J = 8.0
Hz, 2H), 7.15(s, 1H), 6.97 (dd, J = 7.6 Hz, 7.2 Hz, 1H), 3.75 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 156.16, 155.66, 153.88, 140.69, 140.34, 139.30, 133.72, 128.76, 128.58, 128.69,
127.60, 125.98, 124.26, 121.42, 118.93, 118.31, 115.28, 111.80, 104.03, 56.27. HRMS (ESI) m/z
calculated for [M+H]+ 469.1431, found 469.1433.
4.1.1.18.1-(4-((2-Methoxyacridin-9-yl)amino)phenyl)-3-(3(trifluoromethyl)phenyl)urea(8r).
Yield 78%; yellow solid, mp 233.4-234.8 °C；1H NMR (400 MHz, DMSO-d6) δ 11.46 (s, 1H), 9.86
(s, 1H), 9.76 (s, 1H), 8.38 (d, J =8.4 Hz, 1H),8.23 (d, J = 5.6 Hz, 1H), 8.02 (s, 1H), 7.97 (s, 1H),
7.79 (d, J = 6.4 Hz, 1H), 7.62 (d, J = 4.8 Hz, 2H), 7.53 (s, 1H),7.46 (s, 1H), 7.43 (s, 2H), 7.39 (m,
2H), 7.33(s, 1H), 4.16(s, 3H); 13C NMR (100 MHz, DMSO-d6) δ155.81, 153.15, 148.88, 141.08,
140.35, 139.68, 135.50, 134.82, 132.32, 130.49, 130.27, 126.11, 125.94, 124.48, 124.11, 123.39,
122.21, 120.46, 119.63, 118.67, 117.15, 114.61, 114.09, 113.59, 57.39. HRMS (ESI) m/z calculated
for [M+H]+ 503.1695, found 503.1694.
4.2. Molecular docking
The molecular modeling of compound 8a was performed with Discovery Studio.3.1/CDOCKER
protocol (Accelrys Software Inc.) according to the reported process.23, 36 The dimensional structure
of Src (PDB ID: 3G6H) was downloaded from Protein Data Bank (PDB). The following process
was used to carry out molecular docking: (1) deleting the water crystallization involved 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.

4.3. Biological assays
4.3.1. Cell culture
K562 and HepG-2 cells were obtained from Cell Resources Center of Shanghai Institutes for
Biological Science, Chinese Academy of Science. They were maintained in Dulbecco’s modified
Eagle’s medium (DMEM) with 10% fetal bovine serum, 100 μgml-1 penicillin and 100 μgmL-1
streptomycin in humidified air at 37 °C with 5% CO2.
4.3.2. Cell Viability Assays
4.3.2.1. K562 cells
The synthesized compounds were dissolved in DMSO, and then diluted with culture medium to
the final concentrations ranging from 0.01 to 50 μM (the final DMSO concentration was less than
1%) for tumor cells assay. 100 μL of cell solution with the concentration of 7×105 cells mL-1 was
seeded to each well of a 96-well plate and incubated for 12 h at 37 °C in a 5% CO2 incubator. The
test compound solution was added to each well for the treatment of maintained cells in triplicate per
concentration, and incubated at 37 °C in a 5% CO2 incubator for 48 h. After this treatment, 10 μL
MTT solution (5 mgmL-1) was then added to each well and incubated for 4 h at 37 °C. The
formazan precipitate was dissolved in 100 μL DMSO and the absorbance at 495 nm was determined
using Multimode Detector DTX880 (Beckman Coulter).
4.3.2.2. HepG-2 cells
The synthesized compounds were dissolved in DMSO, and then diluted with culture medium to
the final concentrations ranging from 0.01 to 50 μM (the final DMSO concentration was less than
1%) for tumor cells assay. 100 μL of cell solution with the concentration of 5 ×105 cells mL-1 was
seeded to each well of a 96-well plate and incubated for 12 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 concentration, and incubated at 37 °C in a
5% CO2 incubator for 48 h. After this treatment, 10 μL MTT solution (5 mgmL-1) was then added to
each well and incubated for 4 h at 37 °C. The formazan precipitate was dissolved in 100 μL DMSO

and the absorbance at 495 nm was determined using Multimode Detector DTX880 (Beckman
Coulter).
4.3.3. Kinase Inhibition Assays
In vitro kinase assays were carried out by Medicilon Co., Ltd in Shanghai, China. Kinases
inhibitory activities of synthetic compounds against Src and MEK. The general procedures were as
follows: mix enzyme, substrate, ATP and compounds in a buffer solution (pH 7.0) of 50 mM
HEPES/NaOH, 0.02% NaN3, 0.01% BSA, 0.1m Mortho-vanadate, 5 mM MgCl2, 1 mM DTT in an
OptiPlate-384. The assay plate was incubated at room temperature for 15 min and compounds in 10
µM dissolved in 2.5% DMSO. Then the mixture was added by Streptavidin-XL665 and TK
antibody europium cryptate (1:100) solution 10 μL (50 mM HEPES/NaOH pH 7.0, 0.1% BSA, 0.8
M KF, 20 mM EDTA). The Instrument (Perkinelmer) to detect the signal at room temperature. The
luminescence was read at envision. The signal was correlated with the amount of ATP remaining in
the reaction and was inversely correlated with the kinase activity.
4.3.4. Cell Apoptosis Assays
A total of 1 × 105cells mL-1 K562 cells were plated in a six-well plate and treated with 8m for 48
h at 37 °C. After incubation, the cells were harvested and washed with ice-cold PBS. The cell cycle
progression was analyzed using a cell cycle and apoptosis analysis kit (Beyotime), and the
apoptosis ratio was performed with an annexin V-FITC Apoptosis Detection Kit (keygentec).
4.3.5. Western Blot Analysis
After treatment with a series of concentrations of 8m for 48 h at 37 °C, K562 cells were
harvested, a centrifuge separates the cells from substratum at the speed of 800 rap for 5min.
Removed supernate, lysed (10 mM HEPES, pH 7.9; 10 mM KCl; 1 mM EDTA; 0.1% NP-40; 0.5
mM Na3VO4; 1 mM NaF; and protease inhibitor cocktail (Thermo scientific)) on ice for 30 min.
Whole-cell protein lysates were prepared and centrifuged for 10 min at 12000 g and 4 °C to remove
any insoluble material. The total proteins were determined using the Bradford method, and an
equivalent quantity of protein was combined with an SDS−PAGE loading buffer in boiled water for

5 min. Cell lysates were separated by SDS−PAGE and electro-transferred onto PVDF membranes
(amcBiobind NT-200). The PVDF membranes were incubated with each antibody and detected
according to the immunoblot analysis principle. The antibodies were purchased from Cell Signaling
Technology, and the dilutions of the antibodies were according to the instruction from Cell
Signaling Technology.
Acknowledgements
The authors would like to thank the financial supports from the Ministry of Science and Technology
of China (2012AA020305), the Chinese National Natural Science Foundation (21272134 and 2137
2141), and Shenzhen Sci & Tech Bureau (JCYJ20130402164027386, JCYJ20150331151358131,
CXZZ20150529165045064, CXB201104210013A and CXB201104210014A).
Supplementary data
Supplementary data (Molecular modeling of compound 8m with Src and MEK proteins,
Synthesis of compounds 4a-4g and 7a-7l, the UV-vis absorption spectra of compound 8m binding
1
with ctDNA, H NMR and 13C NMR spectra and high resolution mass spectrometry of 8a-8r)
associated with this article can be found, in the online version.
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Legends
Figure 1.the structure of compound OA.
Figure 2. (a) the structure of compound 8a; (b) Molecular modeling of compound 8a binding to Src
protein (PDB ID: 2OIQ); (c) Molecular modeling of compound 8a binding to MEK protein (PDB
ID: 4LMN).
Figure 3. 8m affected the expression of ERK and AKT in cell cultures. K562 cells were treated
with compound 8m for 48 h. Cells were lysed, and the proteins were analyzed by Western blot
analysis.
Figure 4. 8m induced apoptosis of K562 cells. K562 cells were harvested after treatment with
various concentrations of compound 8m for 48 h. Cells were stained with an annexin V-FITC
apoptosis detection kit.
Figure 5. 8m affected the expression of C-Caspase-3, C-caspase-7, C-PARP in cell cultures. K562
cells were treated with compound 8m for 48 h. Cells were lysed, and the proteins were analyzed by
Western blot analysis.
Scheme 1. Reagents and conditions: a. (i) Cu, K2CO3, DMF, 130 ℃, overnight; (ii) POCl3, reflux,
3 h; b. (i) different substituent of aniline, Et3N, THF, reflux, 3-4 h; (ii) Fe, NH4Cl, EtOH, 2 h; c. (i)
HCl, THF, r.t., 2 days.

Figure 1

(a)
(b)
(c)
Figure 2