Novel synthetic 9-benzyloxyacridine analogue as both tyrosine kinase and topoisomerase i inhibitor

Article abstract of DOI:10.1016/j.cclet.2013.05.018

Multi-target agents against tyrosine kinases and topoisomerases are potentially useful for the effective treatment of cancers. Discovery of new multi-target scaffolds are important for developing such agents. A series of five novel acridine analogues, LXL 1-5, were synthesized and their antiproliferative activity against HepG-2 cell lines were evaluated, among which the 9-benzyloxyacridine analogue, LXL-5, showed inhibitory activity against tyrosine kinases, VEGFR-2 and Src. The results of UV-visible absorption spectra and fluorescence emission spectra, as well as DNA topoisomerase I inhibition assay, indicated topoisomerase I inhibitory activity. Our study suggested that acridine scaffold, previously shown to have no multi-target kinase and topoisomerase inhibitory activity, might be potentially developed as a multi-target inhibitor of tyrosine kinases and topoisomerase I.

Full text of DOI:10.1016/j.cclet.2013.05.018

Chinese Chemical Letters 24 (2013) 677–680
Contents lists available at SciVerse ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Novel synthetic 9-benzyloxyacridine analogue as both tyrosine kinase and
topoisomerase I inhibitor
Xu-Liang Lang a,b, Qin-Sheng Sun b, Yu-Zong Chen c, Lu-Lu Li b, Chun-Yan Tan b,d, Hong-Xia Liu b,
b,d,e,
Chun-Mei Gao b,d, , Yu-Yang 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, Shenzhen 518055, China
c Bioinformatics and Drug Design Group, Department of Pharmacy, Centre for Computational Science and Engineering, 117543 Singapore, Singapore
d Shenzhen Anti-Tumor Drug Development Engineering Laboratory, The Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China
e 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:
Multi-target agents against tyrosine kinases and topoisomerases are potentially useful for the effective
treatment of cancers. Discovery of new multi-target scaffolds are important for developing such agents.
A series of five novel acridine analogues, LXL 1–5, were synthesized and their antiproliferative activity
against HepG-2 cell lines were evaluated, among which the 9-benzyloxyacridine analogue, LXL-5,
showed inhibitory activity against tyrosine kinases, VEGFR-2 and Src. The results of UV–visible
absorption spectra and fluorescence emission spectra, as well as DNA topoisomerase I inhibition assay,
indicated topoisomerase I inhibitory activity. Our study suggested that acridine scaffold, previously
shown to have no multi-target kinase and topoisomerase inhibitory activity, might be potentially
developed as a multi-target inhibitor of tyrosine kinases and topoisomerase I.
Received 12 March 2013
Received in revised form 5 May 2013
Accepted 10 May 2013
Available online 10 June 2013
Keywords:
Acridine
Topoisomerases
Tyrosine kinases
Antitumor
ß 2013 Yu-Yang Jiang and Chun-Mei Gao. Published by Elsevier B.V. on behalf of Chinese Chemical
Society. All rights reserved.
DNA-binding
1. Introduction
considerable promise in cancer therapeutics [3]. Several VEGFR-2
inhibitors have been approved in advanced-stage clinical trials as
Cancer has become the main cause of death and the
development of new drugs with promising anticancer efficacies
has attracted great attention [1]. As topoisomerases are highly
expressed in cancer cells, they represent effective targets for cancer
chemotherapy. Topoisomerase I (topo I), one of the topoisomerases
which can break and reseal a single DNA strand, is the target of
several approved anticancer drugs, such as camptosar, topotecan
and their derivatives [2]. The involvement of topo I in various
cancers and the clinical success of topo I inhibitors indicate the
inhibition of topo I is an effective strategy for the treatment of
cancers.
anticancer therapeutics [4].
Acridine and its derivatives have been reported to display
potent antitumor activity primarily due to their inhibition of
topoisomerases [5]. The 9-anilinoacridine derivative, m-amsacrine
(m-AMSA), was the typical analogue approved for clinical use in
1976. It can form DNA-topisomerase complex and the substitution
pattern in the benzyl ring plays an important role in the antitumor
activity. However, few of them have been found to inhibit the
activity of tyrosine kinases [6]. In 2011, our group first reported a
9-benzylaminoacridine derivative which was a dual inhibitor of
the tyrosine kinases, VEGFR-2 and Src, without inhibitory activity
against topoisomerase [7]. In certain cancers, topo I inhibitors
work synergistically with multi-tyrosine kinase inhibitors to kill
cancer cells [8], however no compound with acridine scaffold has
been found as inhibitors of both topoisomerases and tyrosine
kinases. An interesting question is whether compounds of the
acridine scaffold can be potentially developed into novel multi-
target tyrosine kinases and topo I inhibitors.
VEGFR-2 and Src kinases are two types of tyrosine kinases
which play important roles in modulating multiple pathways in
the progression of cancers. Development of novel anticancer
compounds that can inhibit VEGFR-2 and Src kinases may hold
* Corresponding authors at: The Ministry-Province Jointly Constructed Base for
State Key Lab-Shenzhen Key Laboratory of Chemical Biology, Shenzhen 518055,
China.
This study is based on the structures of m-AMSA derivatives and
our earlier work in the discovery of 9-benzylaminoacridine
compound [7] and 9-aminoacridine derivatives with chloro and
methoxy groups substituted at C-2 and C-6 positions of acridine
E-mail addresses: chunmeigao@sz.tsinghua.edu.cn (C.-M. Gao),
jiangyy@sz.tsinghua.edu.cn (Y.-Y. Jiang).
1001-8417/$ – see front matter ß 2013 Yu-Yang Jiang and Chun-Mei Gao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
http://dx.doi.org/10.1016/j.cclet.2013.05.018

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X.-L. Lang et al. / Chinese Chemical Letters 24 (2013) 677–680
ring [7,9]. In this work as part of our continuous efforts for
developing anticancer compounds, we addressed this question by
synthesizing a series of novel 9-benzylaminoacridine and the
bioisostere, 9-benzyloxyacridine, with antitumor activity. Our new
compound LXL-5 showed inhibitory activity against topo I, Src and
VEGFR-2 and showed cytotoxicity against HepG-2 cell lines in vitro,
which represented, for the first time, the success of this scaffold as
a multi-target inhibitor of both topoisomerases and tyrosine
kinases.
J = 7.3 Hz), 7.35 (d, 1H, J = 9.1 Hz), 7.30 (d, 2H, J = 6.1 Hz), 7.24 (dd,
1H, J = 7.8, 1.5 Hz), 7.16 (t, 2H, J = 7.0 Hz), 4.91 (s, 2H), 3.78 (s, 3H);
13C NMR (101 MHz, CDCl3):
d 156.38, 149.70, 147.10, 136.33,
135.37, 133.18, 132.40, 130.89, 129.84, 129.56, 129.37, 128.87,
127.38, 125.26, 124.03, 99.35, 55.34, 52.04; HR-MS(ESI): calcd. for
C
21H16Cl2N2O [M+H]+ 383.0718; found: 383.0717.
6-Chloro-2-methoxy-N-(3-bromobenzyl)acridin-9-amine (LXL-
4): Yield 69%; mp 156–159 8C; 1H NMR (400 MHz, CDCl3):
d
8.08 (d, 1H, J = 1.8 Hz), 7.99 (d, 1H, J = 9.4 Hz), 7.91 (d, 1H,
J = 9.3 Hz), 7.61 (s, 1H), 7.45 (d, 1H, J = 7.8 Hz), 7.39 (dd, 1H, J = 9.4,
2.6 Hz), 7.28 (dd, 2H, J = 6.6, 2.7 Hz), 7.22 (t, 1H, J = 7.7 Hz), 7.09 (d,
1H, J = 2.5 Hz), 4.76 (s, 2H), 3.76 (s, 3H); 13C NMR (101 MHz,
2. Experimental
CDCl3):
d 156.43, 149.09, 148.15, 146.93, 141.72, 134.92, 131.49,
The synthetic methods and the preparation of compounds 3 and
4 can be found in the Supporting information. The synthesis of the
acridine derivatives LXL 1–5 is described in Scheme 1.
130.99, 130.55, 130.38, 128.33, 125.88, 125.25, 124.99, 123.62,
123.13, 118.57, 116.52, 99.04, 55.39, 53.87; HR-MS(ESI): calcd. for
C
21H16BrClN2O [M+H]+ 427.0213; found: 427.0224.
2.1. General procedure for compounds (LXL 1–4)
2.2. 6-Chloro-2-methoxy-9-(benzyloxy)acridine (LXL-5)
Various amines (2.00 mmol) were dissolved in absolute alcohol
(15 mL) and then potassium carbonate (2.00 mmol) was added.
The mixture was stirred for 45 min at room temperature.
Compound 4 (1.00 mmol) and potassium iodide (0.25 mmol) were
added and the mixture stirred and refluxed overnight. Then the
mixture was poured into water (50 mL), extracted with ethyl
acetate to give the crude product. The crude product was purified
by column chromatography using petroleum ether and ethyl
acetate.
Benzyl alcohol (3.00 mmol) was dissolved in dry THF (15 mL)
and then sodium hydride (3.00 mmol) was added. The mixture was
stirred for 45 min at room temperature. Compound 4 (1.00 mmol)
and potassium iodide (0.25 mmol) were added and the mixture
stirred and refluxed overnight. Then the solution was evaporated.
The solid was poured into water (50 mL), and extracted with ethyl
acetate to give the crude product. The crude product was purified
by column chromatography using petroleum ether and ethyl
acetate (20:1, v/v). Yield 17%; mp 128–130 8C; 1H NMR (400 MHz,
6-Chloro-2-methoxy-N-(3-methoxybenzyl)acridin-9-amine (LXL-
CDCl3):
J = 9.2 Hz), 7.53–7.37 (m, 7H), 7.25 (d, 1H, J = 2.8 Hz), 5.32 (s, 2H),
3.83 (s, 3H); 13C NMR (101 MHz, CDCl3):
158.92, 157.20, 148.79,
148.50, 136.56, 135.29, 131.25, 128.88, 128.79, 128.28, 127.23,
126.72, 126.12, 123.69, 121.45, 119.15, 98.08, 55.47; HR-MS(ESI):
calcd. for C21H16ClNO2 [M+H]+ 350.0948; found: 350.0951.
DNA topo I inhibition assay, kinase assays, experiments on
absorption and fluorescence emission; 1H NMR and 13C NMR
spectra, and High resolution mass spectrometry can be found in
Supporting information.
d 8.19 (d, 1H, J = 1.9 Hz), 8.14 (d, 1H, J = 9.2 Hz), 8.07 (d, 1H,
1): Yield 77%; mp 101–104 8C; 1H NMR (400 MHz, CDCl3):
d 8.08 (d,
1H, J = 1.7 Hz), 8.04–7.91 (m, 2H), 7.39 (dd, 1H, J = 9.4, 2.5 Hz),
7.34–7.27 (m, 2H), 7.16 (d, 1H, J = 2.4 Hz), 6.98 (d, 1H, J = 7.5 Hz),
6.93 (s, 1H), 6.90 – 6.79 (m, 1H), 4.84 (s, 2H), 3.78 (s, 3H), 3.76 (s,
d
3H); 13C NMR (101 MHz, CDCl3):
d 160.25, 156.26, 149.66, 148.07,
140.92, 135.02, 131.29, 130.90, 130.17, 128.14, 125.02, 124.91,
123.88, 119.59, 118.23, 116.19, 113.39, 113.05, 99.39, 55.42, 55.30,
54.57; HR-MS(ESI): calcd. for C22H19ClN2O2 [M+H]+ 379.1213;
found: 379.1220.
6-Chloro-2-methoxy-N-(2-methylbenzyl)acridin-9-amine (LXL-
2): Yield 56%; mp 150–151 8C; 1H NMR (400 MHz, CDCl3):
d
8.08 (t, 1H, J = 2.2 Hz), 7.99 (dd, 1H, J = 9.4, 2.4 Hz), 7.92 (dd, 1H,
J = 9.2, 5.3 Hz), 7.54 (dd, 1H, J = 5.9, 2.7 Hz), 7.39 (dt, 1H, J = 9.4,
2.8 Hz), 7.31–7.19 (m, 4H), 7.10 (t, 1H, J = 2.4 Hz), 4.90 (s, 1H), 4.79
3. Results and discussion
Utilizing commercial materials, the Ullmann reaction of 2, 4-
dichloro-benzoic acid 1 with 4-methoxyaniline 2 in DMF using Cu
as the catalyst and under basic condition gave anthranilic acid 3,
which was stirred in POCl3 to afford the 9-chloroacridine
derivative 4. The compounds LXL 1–4 were obtained by the
reaction of substituted benzylamines and compound 4 using KI
and K2CO3 in absolute ethanol. Compound LXL-5 was achieved by
the etherification of compound 4 with benzyl alcohol in the
presence of NaH and catalytic amounts of potassium iodide in THF.
(s, 2H), 3.70 (s, 3H), 2.23 (s, 3H); 13C NMR (101 MHz, CDCl3):
d
156.12, 149.69, 148.34, 147.06, 137.30, 135.79, 134.83, 131.58,
130.81, 128.43, 128.14, 128.07, 126.64, 124.91, 124.74, 123.66,
118.02, 116.03, 99.30, 55.30, 52.65, 19.00; HR-MS(ESI): calcd. for
C
22H19ClN2O [M+H]+ 363.1264; found: 363.1252.
6-Chloro-2-methoxy-N-(2-chlorobenzyl)acridin-9-amine (LXL-3):
Yield 61%; mp 163–165 8C; 1H NMR (400 MHz, CDCl3):
d
8.04 (s,
1
H), 7.99 (d, 1H, J = 9.2 Hz), 7.94 (d, 1H, J = 9.3 Hz), 7.43 (d, 1H,
COOH
COOH
Cl
OCH
3
(i)
OCH
3
+
Cl
N
H
H2N
Cl
2
1
3
R2
R1
Cl
N
LXL-1: R1=H, R2=OCH3, Z=NH
LXL-2: R1=CH3, R2=H, Z=NH
LXL-3: R1=Cl, R2=H, Z=NH
LXL-4: R1=H, R2=Br, Z=NH
LXL-5: R1=H, R2=H, Z=O
(ii)
OCH
(iii)
3
Z
OCH
Cl
3
4
Cl
N
LXL1-5
Scheme 1. Synthesis of LXL 1–5. Reagents and conditions: (i) K2CO3, Cu, DMF, 130 8C; (ii) POCl3, 140 8C; (iii) LXL 1–4: benzylamines, K2CO3, KI, ethanol, reflux; LXL-5: benzyl
alcohol, NaH, KI, THF, reflux.

X.-L. Lang et al. / Chinese Chemical
[
Letters 24 (2013) 677–680
679
6x105
5x105
4x105
3x105
2x105
Table 1
Antiproliferative activity of compounds against HepG-2 cells.
Compound
R1
R2
Z
IC50 (m
mol/L)a
LXL-1
LXL-2
LXL-3
LXL-4
LXL-5
Colchicin
H
OCH3
H
NH
NH
NH
NH
O
13.70 (Æ1.21)
11.43 (Æ0.25)
20.21 (Æ1.64)
9.12 (Æ1.08)
8.78 (Æ0.32)
1.80 (Æ0.23)
CH3
Cl
H
H
Br
H
H
a
All values are means of three experiments (ÆSD).
1x105
0
Table 2
Percent inhibition effect of compounds selected at 20
kinases.a
m
mol/L on the activity of two
VEGFR-2
450
550
600
W5a0v0elength/nm
Compound
Src
Fig. 2. Spectrofluorimetric titration of LXL-5 (10
increasing amounts of ct DNA ([DNA] = 0, 1, 2, 4, 5, 7.5, 10
[F(ig._3)TD$FIG]
m
mol/L) in the presence of
LXL-4
(12.3 Æ 6.1)%
(63.8 Æ 2.4)%
(99.7 Æ 0.1)%
(9.2 Æ 0.9)%
(12.6 Æ 3.7)%
(99.4 Æ 2.5)%
mmol/L).
LXL-5
Staurosporine
a
All values are means of two experiments (ÆSD).
The ability of compounds LXL 1–5 to inhibit cell growth was
evaluated against hepatoma HepG-2 cells by using the MTT assay.
The results are depicted in Table 1. Colchicin was used as the
positive control. Most of the compounds had low micromolar IC50
values, among which compounds LXL-4 containing the bromo-
benzylamino group and LXL-5 comprising the benzyloxy group
showed good activity against HepG-2 cells with IC50 values at 9.12
and 8.78
mmol/L, respectively.
Fig. 3. Effect of the compound LXL-5 on the relaxation of plasmid DNA by human
topoisomerase I (S: superhelix; R: relaxation). Lane 1, DNA pBR322; Lane 2, topo
I + DNA pBR322 + DMSO; Lanes 3–5, DNA pBR322 relaxation by topo I and LXL-5 at
In order to identify whether compounds can inhibit tyrosine
kinases Src and VEGFR-2, we conducted in vitro kinase inhibition
assay studies with compounds LXL-4 and LXL-5 which displayed
good antiproliferative activity. The kinase assay results were
shown in Table 2. Staurosporine was used as a positive reference
compound. It was determined that LXL-4 displayed weak activity
against the two kinases, while LXL-5 exhibited moderate activity
with Src and VEGFR-2 inhibition rates of 63.8% and 12.6% at
concentrations of 1, 10, 100
mmol/L, respectively.
absence and presence of ct DNA. In Fig. 1, the spectrum of LXL-5
presented two signals at 385 nm and 410 nm, while DNA did not
absorb light in this region. With the increase of the concentration
of ct DNA, the intensity of the spectrum of LXL-5 was obviously
decreased, which suggested that the compound LXL-5 might
interact with DNA.
In addition to the electronic absorption spectroscopy, the
binding capacity was also tested by the fluorescence emission
spectroscopy. The compound LXL-5 was gradually mixed with
increasing equivalents of ct DNA in Tris-HCl at pH 7.0. As shown in
Fig. 2, the fluorescence of LXL-5 was gradually quenched with the
increasing amounts of ct DNA, suggesting the interaction between
LXL-5 and ct DNA.
20
mmol/L, respectively. The results suggested that LXL-5 may
inhibit the antiproliferative activity partly by inhibiting the
activity of Src and VEGFR-2, while LXL-4 might inhibit cancer
cell proliferation by another mechanism. Therefore, the 9-
benzyloxyacridine analogue LXL-5 was selected to confirm
whether it could interact with ct DNA and inhibit the activity of
topo I.
Absorption spectroscopy has been widely applied for investi-
gating the interaction between drugs and DNA. To detect the
binding properties of compound LXL-5 with ct DNA, LXL-5 was
investigated using UV-visible spectral absorbance analysis in the
[(Fig._1)TD$FIG]
To test whether the novel tyrosine kinases inhibitor LXL-5 also
possesses activity to inhibit topo I activity, the assay of compound
LXL-5 on the relaxtion of plasmid pBR322 DNA mediated by topo I
was performed. As shown in Fig. 3, the compound LXL-5 displayed
moderate topo I inhibitory activity at 100
mmol/L, whereas there
0.5
0.4
0.3
0.2
0.1
were no detectable activities at low concentration. These data
indicated that LXL-5 could also inhibit topo I, and might be a
potential lead compound for the development of topo I inhibitors.
4. Conclusion
In conclusion, a series of novel 9-benzylaminoacridine deriva-
tives and 9-benzyloxyacridine, LXL 1–5, had been synthesized and
showed good antiproliferative activity against HepG-2 cells in
vitro, among which the 9-benzyloxyacridine analogue, LXL-5,
could inhibit the activity of topoisomerase I, VEGFR-2 and Src. Our
study for the first time suggested that the acridine scaffold, which
had been historically used as topoisomerase inhibitors, had the
potential to be developed as multi-target topoisomerase and
tyrosine kinase inhibitors. Further optimizations of the structure to
improve topo I, VEGFR-2 and Src activity are ongoing.
380
400
420
440
Wavelength/nm
Fig. 1. UV–vis absorption spectra of LXL-5 (50
m
mol/L) in 10 mmol/L Tris-HCl buffer
containing 10 mmol/L NaCl (pH 7.0) by increasing the concentrations of ct DNA
([DNA] = 0, 5, 10, 15, 20, 25, 30 mol/L).
m

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X.-L. Lang et al. / Chinese Chemical Letters 24 (2013) 677–680
[2] Y. Pommier, DNA topoisomerase I inhibitors: chemistry, biology, and interfacial
Acknowledgments
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The authors would like to thank the financial supports from the
Ministry of Science and Technology of China (Nos.
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National Natural Science Foundation of China (Nos. 21272134
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Appendix A. Supplementary data
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