Novel synthetic acridine-based derivatives as topoisomerase i inhibitors

Article abstract of DOI:10.1016/j.cclet.2014.03.028

Novel DNA binding agents against topoisomerases are needed for effective treatment of cancers. A series of new acridine-based derivatives 7a-7d were synthesized and their antiproliferative activity against K562 and HepG-2 cell lines were evaluated. Compound 7c with pyridin-2-yl-methanamino group substituted at the C9 position of acridine showed good antitumor activity against both cell lines. The DNA-binding affinity of compound 7c was evaluated by UV-vis absorption spectra and fluorescence emission spectra. DNA topoisomerase I mediated relaxation of plasmid pBR322 DNA was also tested. Our results suggested that compound 7c with good antitumor activity and topoisomerase I inhibition activity can be developed as a prime candidate for further chemical optimization.

Full text of DOI:10.1016/j.cclet.2014.03.028

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CCLET-2914; No. of Pages 4
Chinese Chemical Letters xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Chinese Chemical Letters
journal homepage: www.elsevier.com/locate/cclet
Original article
Novel synthetic acridine-based derivatives as topoisomerase I
inhibitors
b
b
Bin Li a,b, Chun-Mei Gao b,c, , Qin-Sheng Sun , Lu-Lu Li , Chun-Yan Tan b,c, Hong-Xia Liu b,
*
Yu-Yang Jiang b,c,d,
*
a Tsinghua University, Department of Chemistry, Beijing 100084, China
b The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of Chemical Biology, Shenzhen 518055, China
c Shenzhen Anti-Tumor Drug Development Engineering Laboratory, the Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, China
d 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:
Novel DNA binding agents against topoisomerases are needed for effective treatment of cancers. A series
of new acridine-based derivatives 7a–7d were synthesized and their antiproliferative activity against
K562 and HepG-2 cell lines were evaluated. Compound 7c with pyridin-2-yl-methanamino group
substituted at the C9 position of acridine showed good antitumor activity against both cell lines. The
DNA-binding affinity of compound 7c was evaluated by UV–vis absorption spectra and fluorescence
emission spectra. DNA topoisomerase I mediated relaxation of plasmid pBR322 DNA was also tested. Our
results suggested that compound 7c with good antitumor activity and topoisomerase I inhibition activity
can be developed as a prime candidate for further chemical optimization.
Received 25 December 2013
Received in revised form 4 March 2014
Accepted 5 March 2014
Available online xxx
Keywords:
Acridine
DNA binding agent
Topoisomerase
Anticancer
ß 2014 Chun-Mei Gao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights
reserved.
1. Introduction
developed [18,14]. By far, most of the modification of the m-AMSA
was focused on the position and nature of substituents in the 9-
Cancer is one of the most common malignant diseases
obsessing mankind and new effective drugs are needed [1–6].
DNA topoisomerase, either in prokaryotes or eukaryotes, plays a
very important role in cell proliferation, survival and apoptosis,
which has been one of the most potential targets for the
development of new anticancer agents [7–9].
Acridine analogs have been used for treatment of inflammation
and cancer for many years. The unique planar ring structure makes
its strong interaction with DNA base pairs [10,11]. A variety of
acridine derivatives have been designed and synthesized, some of
which have entered clinical studies, such as DACA [12–14], C-1305
[15] and m-AMSA, etc. (Fig. 1) [16]. Among them, m-AMSA was the
first used in clinical treatment for several cancers as topoisomerase
inhibitors and much attention had been paid to the modification of
m-AMSA to improve its activity and bioavailability [17]. A variety
of analogs of m-AMSA, such as AHMA and D3CLP (Fig. 1), have been
aminobenzene moiety and acridine rings. A variety of bis-acridine
derivatives have also been developed to increase the DNA binding
affinity. However, little attention has been paid to the replacement
of benzene ring by naphthalene or heterocycles, such as pyridine.
Previously, we have found that the linker between acridine and
benzene ring played an important role in the antitumor activity
and identified the –NHCH2– or –NHCH2CH2– linker was appropri-
ate [18] as part of our continuous efforts [18,16,19–23] in
developing novel antitumor compounds. In this paper we
synthesized a series of naphthalene, pyridine and indole substi-
tuted-acridine analogs with –NHCH2– or –NHCH2CH2– linker as
potent antitumor agents. Our results indicated that some of these
9-heteroaromatic substituted acridines displayed better antipro-
liferative activity than the corresponding 9-benzene substituted
acridine 5b [18]. The DNA binding capability and topoisomerase I
(topo I) mediated relaxation of plasmid pBR322 DNA were also
evaluated.
*
Corresponding authors at: The Ministry-Province Jointly Constructed Base for
2. Experimental
State Key Lab-Shenzhen Key Laboratory of Chemical Biology, Shenzhen 518055,
China.
The synthetic methods and the preparation of compounds 3 and
4 can be found in the Supporting information.
E-mail addresses: chunmeigao@sz.tsinghua.edu.cn (C.-M. Gao),
jiangyy@sz.tsinghua.edu.cn (Y.-Y. Jiang).
http://dx.doi.org/10.1016/j.cclet.2014.03.028
1001-8417/ß 2014 Chun-Mei Gao. Published by Elsevier B.V. on behalf of Chinese Chemical Society. All rights reserved.
Please cite this article in press as: B. Li, et al., Novel synthetic acridine-based derivatives as topoisomerase I inhibitors, Chin. Chem. Lett.
(2014), http://dx.doi.org/10.1016/j.cclet.2014.03.028

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Fig. 1. The structures of some acridine derivatives.
R
Cl
HN
N
O
N
COOH
Cl
H2N
i
ii
iii
iv
+
N
H
3
COOH
N
1
2
4
7a-7d
5
Scheme 1. Synthesis of acridine derivatives 7a–7d. Reagents and conditions: (i) K2CO3, Cu, DMF, 130 8C; (ii) POCl3, 105 8C; (iii) phenol, 60 8C. (iv) the corresponding amines
7a–7d, 120 8C.
General procedure for compounds (7a-7d): Initially, compound
4 (1.0 mmol) and phenol (10 mmol) were added to a 100 mL dried
round-bottom. The mixture was incubated at 60 8C for 1 h under
argon atmosphere to give the intermediate 5. Then, the corre-
sponding amine 6 (1.1 mmol) was added and the mixture was
heated to 130 8C for 2 h. The mixture was then poured into a
mixture of N-methyl morpholine (1 mL) and ethyl acetate
(100 mL). The precipitation was separated by vacuum filtration
to give the crude products (7a–7d) (Scheme 1).
as the catalyst gave anthranilic acid 3, which was then stirred in
POCl3 to afford the 9-chloroacridine 4. The reaction of 4 with
phenol gave the intermediate 5, which was then reacted with the
corresponding amines to afford the desired acridines 7a–7d.
MTT assay was used to test whether compounds 7a–7d
displayed antiproliferative activity. The cytotoxicity of compounds
7a–7d against K562 and HepG-2 cells was evaluated for
comparison with that of the 9-benzylamino acridine (5b) reported
in our earlier paper [18]. Colchicine and podophyllotoxin were
used as the positive controls. The results can be reported in Table 1.
The newly synthesized four acridines displayed moderate to good
antiproliferative activity against the two tumor cell lines. The 4-
pyridine substituted acridine 7b showed about 3-fold less activity
against K562 cells than 5b. The other three compounds demon-
strated similar, or more cytotoxicity compared to 5b. The low
micromolar IC50 values of the compounds indicate that the
replacement of benzene ring by naphthalene or other heterocycles
may improve the antitumor activity. As the mode of toxic action of
N-(2-(1H-indol-3-yl)ethyl)acridin-9-amine (7a). Compound 7a
was purified by column chromatography (methanol/ethyl ace-
tate = 1/4, v/v). Yield 57%; mp 245–247 8C; 1H NMR (400 MHz,
DMSO-d6): d10.92 (s, 1H), 8.60 (d, 2H, J = 8.3 Hz), 8.00–7.86 (m, 4H),
7.58 (d, 1H, J = 7.8 Hz), 7.50 (t, 2H, J = 6.7 Hz), 7.33 (d, 1H, J = 8.1 Hz),
7.25 (d, 1H, J = 2.2 Hz), 7.06 (t, 1H, J = 7.2 Hz), 6.95 (t, 1H, J = 7.2 Hz),
5.30–5.29 (m, 1H), 4.37 (t, 2H, J = 7.4 Hz), 4.35 (t, 2H, J = 7.4 Hz). HR-
MS (ESI): Calcd. for [M+H]+: m/z 338.1657; found: 338.1670.
N-((Pyridin-4-yl)methyl)acridin-9-amine (7b). Compound 7b
was purified by recrystallization from ethyl acetate. Yield 27%; mp
225–228 8C; 1H NMR (400 MHz, DMSO-d6):
d 8.75 (s, 1H), 8.67–
Table 1
8.48 (m, 3H), 8.03–7.95 (m, 4H), 7.92 (d, 1H, J = 7.8 Hz), 7.51 (s, 2H),
7.43 (dd, 1H, J = 7.7, 4.8 Hz), 5.41 (s, 2H). HR-MS (ESI): Calcd. for
[M+H]+: m/z 286.1344; found: 286.1345.
Antiproliferative activity of compounds against K562 and HepG-2 cells.
Compound
R
IC50 (mmol/L)
K562 IC50
2.92
(m
mol/L)
HepG-2 IC50
6.187
(mmol/L)
N-((Pyridin-2-yl)methyl)acridin-9-amine (7c). Compound 7c
was purified by recrystallization from ethyl acetate. Yield 25%; mp
7a
228–229 8C; 1H NMR (400 MHz, DMSO-d6):
d 8.77 (s, 1H), 8.66–
8.46 (m, 3H), 8.08–7.97 (m, 2H), 7.92–7.74 (m, 3H), 7.52–7.45 (m,
3H), 5.41 (s, 2H). HR-MS (ESI): Calcd. for [M+H]+: m/z 286.1344;
found: 286.1338.
7b
15.163
8.592
N-((Naphthalen-1-yl)methyl)acridin-9-amine (7d). Compound
7d was purified by recrystallization from DMSO/ethyl acetate (1/1,
v/v). Yield 46%; mp 249–252 8C; 1H NMR (400 MHz, DMSO-d6):
d
7c
2.517
4.673
5.24
10.73
7.849
6.07
8.13–8.09 (m, 2H), 8.09–8.04 (m, 1H), 8.01–7.93 (m, 5H), 7.73 (d,
1H, J = 7.0 Hz), 7.65 (dd, 3H, J = 6.3, 3.3 Hz), 7.59–7.51 (m, 1H), 7.43
(s, 2H), 5.77 (s, 2H). HR-MS (ESI): Calcd. for [M+H]+: m/z 335.1548;
found: 335.1533.
The experiments of UV–vis absorption spectroscopy and fluo-
rescence emission; DNA topo I inhibition assay; 1H NMR and high
resolution mass spectra can be found in Supporting information.
7d
5b [18]
3. Results and discussion
Colchicine
Imatinib
NDa
0.47
1.8
NDa
Synthesis of the acridine-based derivatives 7a–7d was accom-
plished as described in Scheme 1. First, an Ullmann coupling
reaction of 2-chloro-benzoic acid 1 with aniline 2 in DMF using Cu
a
Not detected.
Please cite this article in press as: B. Li, et al., Novel synthetic acridine-based derivatives as topoisomerase I inhibitors, Chin. Chem. Lett.
(2014), http://dx.doi.org/10.1016/j.cclet.2014.03.028

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3
7x106
b
0.7
a
6x106
5x106
4x106
3x106
2x106
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1x106
0
400
425
450
475
500
350
375
400
425
450
475
500
Wavelength/nm
Wavelength/nm
Fig. 2. (a) UV–vis absorption spectra of 7c (62.5
mmol/L) in 10 mmol/L Tris–HCl buffer containing 10 mmol/L NaCl (pH 7.0) by increasing the concentrations of ctDNA ([DNA]/
[7c] = 0, 0.125, 0.25, 0.5). The arrow indicates the absorbance changes upon increasing DNA concentrations. (b) Spectrofluorimetric titration of 7c (62.5
mmol/L) in the
presence of increasing amounts of ctDNA ([DNA]/[7c] = 0, 1, 2, 4). The arrow indicates the fluorescence emission changes upon increasing DNA concentrations.
good antiproliferative activity against both K562 and HepG-2 cells.
The identified compound 7c exhibited its antitumor activity
through binding with DNA and inhibiting topo I activity, which can
be developed into novel topoisomerase inhibitors. Further
optimizations of the structure to improve the bioavailability and
solubility are ongoing.
Acknowledgments
Fig. 3. Effect of the compound 7c on the relaxation of plasmid DNA by human topo I
(S: superhelix; R: relaxation).
The authors would like to thank the Ministry of Science and
Technology of the People’s Republic of China (Nos. 2012AA020305
acridines is mainly attributed to DNA and its related enzymes,
compound 7c with the best antiproliferative activity against K562
cells was selected to study the DNA binding property.
and 2011DFA30620), the National Natural Science Foundation of
China (Nos. 21272134 and 21372141), and Shenzhen Sci. & Tech.
Bureau (No. JCYJ20120831165730905) for the financial supports.
UV–vis absorption spectroscopy is extensively utilized to
detecting the interaction between DNA and compounds. The
interaction of compound 7c with ctDNA was evaluated and the
result shown in Fig. 2a. The major peaks in the spectrum of 7c were
observed at about 426 nm and 470 nm, while DNA did not absorb
light in this region. The absorption spectra of 7c decreased
obviously by the addition of increasing amounts of ctDNA. In
addition, a slight bathochromic effect was observed when ctDNA
was added and a clear isosbestic point appeared. All the result
suggested that compound 7c can interact with DNA.
In addition to UV–vis absorption spectroscopy, fluorescence
emission spectroscopy is also widely used to evaluate the binding
abilityofdrugsandtheirtargets.AsshowninFig.2b,thefluorescence
of 7c was gradually decreased when the concentration of ct DNA
increased, which suggested that there are interactions between 7c
and ct DNA.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.cclet.2014.03.028.
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Please cite this article in press as: B. Li, et al., Novel synthetic acridine-based derivatives as topoisomerase I inhibitors, Chin. Chem. Lett.
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Please cite this article in press as: B. Li, et al., Novel synthetic acridine-based derivatives as topoisomerase I inhibitors, Chin. Chem. Lett.
(2014), http://dx.doi.org/10.1016/j.cclet.2014.03.028