Novel synthetic acridine derivatives as potent DNA-binding and apoptosis-inducing antitumor agents

Article abstract of DOI:10.1016/j.bmc.2013.05.008

Acridine derivatives have been explored as DNA-binding anticancer agents. Some derivatives show undesired pharmacokinetic properties and new derivatives need to be explored. In this work, a series of novel acridine analogues were synthesized by modifying previously unexplored linkers between the acridine and benzene groups and their antiproliferative activity and the DNA-binding ability were evaluated. Among these derivatives, compound 5c demonstrated DNA-binding capability and topoisomerase I inhibitory activity. In K562 cell lines, 5c induced apoptosis through mitochondria-dependent intrinsic pathways. These data suggested that compound 5c and other acridine derivatives with modified linkers between the acridine and benzene groups might be potent DNA-binding agents.

Full text of DOI:10.1016/j.bmc.2013.05.008

Bioorganic & Medicinal Chemistry 21 (2013) 4170–4177
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel synthetic acridine derivatives as potent DNA-binding
and apoptosis-inducing antitumor agents
Xuliang Lang a,b, Lulu Li b, Yuzong Chen c, Qinsheng Sun b, Qin Wu a,b, Feng Liu b, Chunyan Tan b,d
,
Hongxia Liu b, Chunmei Gao b,d, , Yuyang Jiang b,d,e,
⇑
⇑
a Tsinghua University, Department of Chemistry, Beijing 100084, PR China
b The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of Chemical Biology, Shenzhen 518055, PR China
c Bioinformatics and Drug Design Group, Department of Pharmacy, Centre for Computational Science and Engineering, Singapore 117543, Singapore
d Shenzhen Anti-Tumor Drug Development Engineering Laboratory, The Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China
e School of Medicine, Tsinghua University, Beijing 100084, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Acridine derivatives have been explored as DNA-binding anticancer agents. Some derivatives show unde-
sired pharmacokinetic properties and new derivatives need to be explored. In this work, a series of novel
acridine analogues were synthesized by modifying previously unexplored linkers between the acridine
and benzene groups and their antiproliferative activity and the DNA-binding ability were evaluated.
Among these derivatives, compound 5c demonstrated DNA-binding capability and topoisomerase I inhib-
itory activity. In K562 cell lines, 5c induced apoptosis through mitochondria-dependent intrinsic path-
ways. These data suggested that compound 5c and other acridine derivatives with modified linkers
between the acridine and benzene groups might be potent DNA-binding agents.
Received 16 February 2013
Revised 28 April 2013
Accepted 7 May 2013
Available online 16 May 2013
Keywords:
Acridine derivatives
Antiproliferative activity
DNA-binding
Ó 2013 Elsevier Ltd. All rights reserved.
Topoisomerase I inhibitor
Apoptosis
1. Introduction
tive m-amsacrine (m-AMSA, Fig. 1) has attracted much attention
for its DNA-binding capability.18 Because of the short half life of
Cancer has become the main cause of mortality and there is an
urgent need for more effective anticancer drugs.1,2 Extensive ef-
forts have been directed at the development of new drugs with
promising anticancer properties. DNA targeted drugs are a class
of clinically highly successful anticancer agents that produce anti-
cancer effects by causing DNA damage and subsequent cell apopto-
sis,3,4 having significantly increased the survival rate of cancer
patients.5 Nonetheless, a main drawback that clinically-used DNA
targeted agents frequently encounter is drug resistance, which
has led to the efforts in discovering new agents that can bind to
DNA and DNA regulatory enzymes, particularly the agents effective
against drug resistant cancers.6–12
m-AMSA in the presence of fresh mouse blood at 37 °C, various
analogues, such as 3-(9-acridinylamino)-5-hydroxymethylaniline
(AHMA, Fig. 1), have been designed and tested for anticancer activ-
ities.19 Thus far, most of m-AMSA derivatives are 9-anilinoacri-
dines.20–22 Little attention has been paid to the derivatives with
modified linkers between the acridine and benzene groups.23
As part of our continuous efforts for developing anticancer com-
pounds,24–33 in this work, we synthesized new m-AMSA deriva-
tives (Table 1; 5a–5f, 6b, 7b) with modified linkers between the
acridine and benzene groups for the discovery of potent DNA-bind-
ing agent and evaluated the structure–activity relationship of these
derivatives. This study is based on our earlier work in the discovery
Previous reports have demonstrated that a planar chromophore,
such as a tri- or tetracyclic ring, is a fundamental structural ele-
ment for DNA intercalation, one of the major interaction modes
by known DNA binding agents.13 Acridine is one of the extensively
explored DNA intercalating scaffolds for developing anticancer,
antiviral, antimalarial, and antibacterial agents, and for biological
fluorescent probes.14–17 In particular, the 9-anilinoacridine deriva-
⇑
Corresponding authors. Tel.: +86 755 2603 6017; fax: +86 755 2603 2094.
E-mail addresses: chunmeigao@sz.tsinghua.edu.cn (C. Gao), jiangyy@sz.tsin-
ghua.edu.cn (Y. Jiang).
Figure 1. Chemical structures of m-AMSA, AHMA, and compound X1.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.008

X. Lang et al. / Bioorg. Med. Chem. 21 (2013) 4170–4177
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Table 1
Antiproliferative activity in K562 and HepG-2 cells
Compound
R1
R2
R3
X
K562 IC50
(
lM)
HepG-2 IC50 (lM)
5a-1
5a-2
5a-3
6b
Cl
Cl
Cl
H
OCH3
OCH3
OCH3
H
H
H
H
H
CH2–NH
(CH2)2–NH
(CH2)3–NH
CH2–O
0.65
0.65
2.20
>50
1.40
2.19
3.26
>50
7b
H
H
H
CO–NH
>50
>50
5b
5c
5d
5e
H
H
Cl
Cl
H
H
OCH3
H
OCH3
H
H
H
H
CH3
CH3
CH2–NH
CH2–NH
CH2–NH
CH2–NH
CH2–NH
5.24
0.54
9.24
3.09
4.86
0.47
NDa
6.07
0.89
11.64
6.05
3.64
NDa
1.80
5f
Imatinib
Colchicin
a
ND: not detected.
of 9-(2-methoxybenzyl) acridine derivative compound X1 (Fig. 1)
as an antiproliferative agent24 and on the synthesis of 9-anilino-
azaacridines derivatives with chloro and methoxy groups substi-
tuted at C-2 and C-6 positions of acridine ring, respectively.29
The topoisomerase I (topo I) inhibitory activity and cytotoxicity
against K562 and HepG-2 cells of new compound 5c represents
the success of this exploration in the linker modifications of acri-
dine derivatives.
of the alkyl chain between the 9-anilinoacridine and benzene
groups has modest influence on the cytotoxicity as indicated by
the measured activities of compounds 5a-1, 5a-2 and 5a-3. The
compound 5a-1 with the shortest chain length showed better cyto-
toxicity than those of 5a-2, 5a-3. It seems that increase in the
length of the alkyl chain leads to modest reduction of cytotoxicity.
Second, a substitution of alternative linkers, that is, compounds 6b
comprising an alkoxy linker and 7b containing an acylamino linker
led to decrease in antiproliferative activity. Finally, we investigated
the modifications in the acridine ring. The cytotoxic activity of
compound 5c with a methoxy group substituted at C-2 position
of acridine ring was almost 10 times higher in potency than that
of 5b with no substituents of the acridine ring. The compounds
5d and 5a-1 showed the similar or slightly reduced activity, com-
paring with 5b and 5c, respectively. These suggest that a chloro
group substituted at C-6 position of acridine ring has no obvious
effect on the activity of this class of derivatives, but a methoxy
group substituted at C-2 position of acridine ring significantly al-
tered their activity. On the other hand, a methyl group on the C-
4 position of acridine ring appeared to be unimportant for the
activity by the fact that 5f was more active than 5b while 5e was
less active than 5a-1.
2. Results and discussion
2.1. Chemistry
Synthesis of the acridine derivatives 5 were accomplished as
described in Scheme 1(a). Compounds 3 (3a–3f) were obtained
from an Ullmann reaction of benzoic acids 1 with the anilines 2
in DMF using Cu as the catalyst under basic condition. Compounds
3 (3a–3f) were stirred in POCl3 giving the 9-chloroacridine deriva-
tives 4 (4a–4f). The compounds 5 (5a–5f) were obtained by the
reaction of substituted benzylamines and compounds 4 using KI
and K2CO3 in absolute ethanol under reflux conditions.
In Scheme 1(b) and (c), the synthesis of compounds 6b, 7b by
the same synthetic method failed due to the low nucleophilic
activity of benzyl alcohol and benzamide. Therefore a stronger base
such as NaH was used. Compound 6b was achieved by compounds
4b with benzyl alcohol in the presence of NaH and catalytic
amounts of potassium iodide in THF under reflux conditions. The
compound 7b was obtained by the reaction of 4b and benzamide
according to the routes described in the references.34
2.3. Fluorescence spectra
Fluorescence spectroscopy has been widely applied for investi-
gating the interaction between drugs and DNA.6 All compounds
were gradually mixed with increasing equivalent of deoxyribonu-
cleic acid from calf thymus (ct DNA) in Tris–HCl at pH 7.0. The typ-
ical spectra were shown in Figure 2(a) and the other spectra can be
seen in Figure 1s (Supporting information). The fluorescence of
2.2. In vitro cytotoxicity
acridine derivatives (50 lM) were gradually quenched with the
increasing amounts of ct DNA, suggesting the interaction between
In vitro cytotoxicity of the 10 acridine derivatives was evaluated
against K562 leukemia cells and hepatoma HepG-2 cells by MTT
assay using imatinib and colchicin as the positive controls. As
shown in Table 1, most of the compounds had low micromolar
IC50 values, among which compound 5c showed the best activity
against both K562 and HepG-2 cells with IC50 values at 0.54 and
acridines and ct DNA.
We used the classical Stern–Volmer Eq. (1)35 to calculate the
quenching constant Kq for the binding of the compound with
DNA, which was usually in the range of 104–106 Mꢀ1
.
F0=F ¼ 1 þ Kq½Qꢁ
ð1Þ
0.89 lM, respectively.
Data in Table 1 indicate that the modification of the linker be-
tween acridine ring and benzene ring and the substituents of the
acridine significantly influenced the cytotoxicity. First, the length
The quenching constant Kq for compound 5c was shown in Fig-
ure 2(b). The Kq of all the synthesized compounds were shown in
Table 2. From the results of fluorescence spectroscopy, most of

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X. Lang et al. / Bioorg. Med. Chem. 21 (2013) 4170–4177
Scheme 1. Reagents and conditions: (a) (i) K2CO3, Cu, DMF, 130 °C; (ii) POCl3, 140 °C; (iii) various benzylamines, K2CO3, KI, ethanol, reflux. (b) (i) Benzylalcohol, NaH, KI, THF,
reflux. (c) (i) Benzamide, NaH, DMF, reflux.
Figure 2. (a) Spectrofluorimetric titration of compound 5c (50 lM) in 10 mM Tris–HCl buffer containing 10 mM NaCl (pH 7.0) by increasing the concentrations of ct DNA
([DNA] = 0, 1, 2, 3, 4, 5, 6 lM). The arrow indicates the fluorescence emission changes upon increasing DNA concentrations. (b) Stern–Volmer quenching plots of the
fluorescence titration, Kq = 3.50 ꢂ 105 Mꢀ1. (c) UV–vis absorption spectra of compound 5c (50
lM) in the presence of increasing amounts of ct DNA; [DNA] = 0, 2, 4, 6, 8,
10 lM. DNA titration of the compound was performed in 10 mM Tris–HCl buffer containing 10 mM NaCl at pH 7.0. The arrow indicates the absorbance changes upon
increasing DNA concentrations. (d) The plot of [DNA]/(eaꢀef) as a function of DNA concentrations as determined from the absorption data.

X. Lang et al. / Bioorg. Med. Chem. 21 (2013) 4170–4177
4173
the compounds can interact with DNA. The compound 5a-1 with
the shortest chain length had better binding capacity than those
of 5a-2, 5a-3. Compounds 6b and 7b with little antiproliferative
activity had low DNA-binding capacity. Compound 5c displayed
the highest DNA binding activity. The results were generally con-
sistent with the results of cell growth inhibition assay.
2.4. UV–visible spectral absorbance
In addition to the electronic fluorescence spectroscopy, the DNA
binding capacity was also detected by the absorption spectroscopy.
Due to the strong stacking interaction between an aromatic chro-
mophore and the base pairs of DNA, compound binding to DNA
can result in hypochromism and red-shifting in the absorption of
the compound. The absorption spectra of 5c in the absence and
presence of ct DNA were presented in Figure 2(c). With the in-
crease of the concentration of ct DNA, the intensity of the spectrum
of 5c was decreased obviously. In addition, we also detected an
obvious bathochromic shift of the chromophore in the absorbance,
which suggested that the compound 5c interact with DNA.
We used formula (2) to calculate the constant Kb for the binding
of the compound with DNA,36 where [DNA] represents the DNA
concentrations in base pair, while ea, ef and eb are the extinction
coefficient of the compound absorption band at a given DNA con-
centration, the complex free in solution and the complex when
fully bound to DNA, respectively.36 When the equilibrium is
reached to an optimum level and the intensity of the spectrum is
not obviously decreased when more DNA were added, which
seems to indicate that the compound is fully bound to DNA. The
Kb was calculated by the gradient ratio to the intercept. The bind-
ing constant Kb of compound 5c was 1.42 ꢂ 105 Mꢀ1, which is gen-
erally consistent with the data of fluorescence spectroscopy.
Figure 3. Circular dichroism spectra of ct DNA (40
concentrations of 5c (0, 40, 80
upon increasing the concentrations of compound 5c.
l
M) by increasing the
l
M).The arrow indicates the CD spectra changes
of the compounds with DNA-binding capacity displayed certain
topo I inhibitory activity at 100
5c and 5f which displayed good cytotoxicity against K562 and
HepG-2 cells, showed good topo I inhibitory activity at 25 M.
lM. In addition, compounds 5b,
l
These data suggest that these compounds may have antiprolifera-
tive activity by interacting with DNA and inhibiting topo I, and
some of their derivatives may thus be developed into potent topo
I inhibitors.
2.7. Apoptosis induced by compound 5c
Our spectroscopic studies consistently suggested that com-
pound 5c may bind with DNA and inhibit topo I activity, which
was expected to subsequently lead to apoptosis in cancer cells.39
To test this hypothesis, an Annexin-V/PI binding assay was con-
ducted in K562 cells. As shown in Figure 5, the upper and lower
right-hand quadrants represented the late stage apoptotic cells
(Annexin-V and PI positive) and the early stage apoptotic (Annex-
in-V positive but PI negative). K562 cells were treated with 5c at
½DNAꢁ=ðea
ꢀ
ef Þ ¼ ½DNAꢁ=ðeb
ꢀ
ef Þ þ 1=Kbðeb
ꢀ
ef
Þ
ð2Þ
2.5. CD spectroscopy
Circular dichroic (CD) spectral technique was used to search for
the conformational changes of ct DNA on addition of compound 5c
(Fig. 3). The CD spectrum of ct DNA produced a classical diagram of
right-handed B-form DNA with positive and negative bands at 275
and 248 nm due to base-stacking and helicity, respectively.31 The
increased intensity of negative bands with slight red shifts were
observed with increasing amount of compound 5c. The positive
band at 275 nm was also increased with no significant red shift
when the concentration of 5c was increased. The enhancement of
the two bands indicated that 5c interacts with DNA. Slight or no
red shifts suggested the binding of 5c did not lead to any signifi-
cant change in conformation of ct-DNA.37,38
the concentrations of 0, 0.5, 1.0 and 2.5 lM for 48 h. As the concen-
tration of 5c increased, the early and the late stage apoptotic cells
increased from 4.32% to 73.15%, indicating that 5c induces apopto-
sis of K562 cells in a dose-dependent manner.
2.6. DNA topoisomerase I inhibition assay
Because of the capabilities of acridine derivatives to interact
with ct DNA, it is of interest to evaluate whether they can also in-
hibit some of the nuclear enzymes involved in DNA processing,
such as topo I. Figure 4 shows the relative affinity of all compounds
on the relaxation of plasmid pBR322 DNA mediated by topo I. Most
Table 2
The quenching constants Kq for the binding of all compounds with DNA
Figure 4. Effect of the compounds on the relaxation of plasmid DNA by human
topoisomerase I. (a) Lane 1, DNA pBR322; Lane 2, topo I + DNA pBR322 + DMSO;
Lanes 3–12, DNA pBR322 relaxation by topo I and 5a-1, 5a-2, 5a-3, 6b, 7b, 5b, 5c,
Compound 5a-1
Kq
Compound 5b
5a-2
5a-3
6b
7b
1.97 ꢂ 105 1.19 ꢂ 105 1.05 ꢂ 105 <1.00 ꢂ 104 <1.00 ꢂ 104
5d, 5e and 5f at concentrations of 100
Lane 2, topo I + DNA pBR322 + DMSO; Lanes 3–8, DNA pBR322 relaxation by topo I
and 5a-2, 5b, 5c, 5d, 5e and 5f at concentrations of 25 M, respectively.
lM, respectively. (b) Lane 1, DNA pBR322;
5c
5d
5e
5f
Kq
3.71 ꢂ 104 3.50 ꢂ 105 1.33 ꢂ 104 1.11 ꢂ 105
6.22 ꢂ 104
l

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X. Lang et al. / Bioorg. Med. Chem. 21 (2013) 4170–4177
Figure 5. Flow cytometric analysis of phosphoatidylserine externalization (Annexin-V binding) and cell membrane integrity (PI staining). K562 cells were treated with
compound 5c at 0, 0.5, 1, 2.5 M, respectively.
l
dose-dependent manner, indicating a mitochondrial-dependent
apoptosis. Based on this finding, we went on to study Bcl-2 family
proteins. The Bcl-2 family proteins play critical roles in controlling
the mitochondrial pathway. Either increase of pro-apoptotic pro-
tein Bax or decrease of anti-apoptotic protein BCL-XL promotes cell
apoptosis. As shown in Figure 6, compound 5c at 2.5 lM effectively
increased Bax and decreased BCL-XL protein, illustrating that com-
pound 5c induced apoptosis may proceed through a caspase-
dependent intrinsic mitochondria pathway.
3. Conclusion
A series of novel acridine analogues were synthesized by mod-
ifying the previously unexplored linkers between the acridine and
the benzene group, and most of them showed promising cytotox-
icity in K562 and HepG-2 cells. In particular, compound 5c dis-
played highest cytotoxicity against both K562 and HepG-2 cells.
DNA-binding and DNA topo I inhibition assay demonstrated that
5c interacted with ct DNA and inhibited topoisomerase I activity,
and induced apoptosis in K562 cells via the caspase-dependent
intrinsic mitochondria pathway. Our studies suggested that com-
pound 5c is a highly promising lead compound for further optimi-
zation into potentially potent DNA binding and apoptotic inducing
agents. Its derivatives may also be developed into DNA topo I
inhibitors. Further modifications of 5c are underway.
Figure 6. K562 cells were treated with different concentrations of compound 5c,
and the levels of C-caspase-7, C-caspase-9, C-caspase-3, BAX, BCL-XL were
determined by Western blot analysis.
We further investigated apoptotic proteins to further confirm
that compound 5c induces apoptosis and to dissect which parts
of the apoptotic pathway are affected by the compound. We first
studied caspases, a family of cystein proteases, can cleave essential
cellular substrates after aspartic residues and are critical initiators
and effectors of apoptosis. Caspase-3 and caspase-7 are effectors
activated by upstream initiators of apoptosis and play an irreplace-
able role in cell apoptosis and caspase-9 is involved in the mito-
chondrial pathway. As shown in Figure 6, compound 5c at
4. Experimental section
4.1. Synthesis and characterization
2.5
lM for 48 h induced significant cleavages of caspase-3 and cas-
See Supporting information for synthetic methods and the
pase-7. Compound 5c also induced the activation of caspase-9 in a
preparation of compounds 3a–3f and 4a–4f.

X. Lang et al. / Bioorg. Med. Chem. 21 (2013) 4170–4177
4175
4.1.1. General procedure for compounds 5a-1–5a-3, 5b–5f
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. The com-
pound 4(a–f) (1.00 mmol) and potassium iodide (0.25 mmol) were
added. The mixture was stirred and refluxed overnight. Then the
mixture was poured into water (50 mL), extracted with ethyl ace-
tate to give the crude product. The crude product was purified by
column chromatography using petroleum ether and ethyl acetate
(5:1 v/v).
115.08, 55.12; HR-MS(ESI): calcd for C20H15ClN2 [M+H]+
319.1002; found: 319.0993.
4.1.1.7.
amine (5e).
6-Chloro-4-methyl-2-methoxy-N-benzylacridin-9-
Yield 37%; mp 107–110 °C; 1H NMR (400 MHz,
CDCl3) d 8.18 (s, 1H), 7.97 (d, J = 9.0 Hz, 1H), 7.44–7.27 (m, 7H),
7.02 (s, 1H), 4.81 (s, 2H), 3.78 (s, 3H), 2.84 (s, 3H); 13C NMR
(101 MHz, CDCl3)
d 155.97, 129.02, 128.82, 127.89, 127.50,
125.08, 124.16, 123.73, 97.22, 55.22, 54.83, 18.68; HR-MS(ESI):
calcd for C22H19ClN2O [M+H]+ 363.1264; found: 363.1265.
4.1.1.1. 6-Chloro-2-methoxy-N-benzylacridin-9-amine (5a-
1).
4.1.1.8. 4-Methyl-N-benzylacridin-9-amine (5f).
Yield 39%;
Yield 82%; mp 158–160 °C; 1H NMR (400 MHz, CDCl3) d
mp 75–77 °C; 1H NMR (400 MHz, CDCl3) d 8.19 (d, J = 8.6 Hz, 1H),
8.15 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.50–7.37 (m, 5H),
7.33 (t, J = 7.0 Hz, 1H), 7.23 (d, J = 6.0 Hz, 1H), 7.16–7.04 (m, 2H),
5.02 (s, 2H), 2.08 (s, 3H). 13C NMR (101 MHz, CDCl3) d 176.95,
137.96, 132.73, 131.99, 130.88, 129.15, 128.86, 127.83, 126.80,
124.42, 123.48, 123.23, 122.71, 122.34, 114.55, 113.51, 52.81,
18.70; HR-MS(ESI): calcd for C21H18N2 [M+H]+ 299.1548; found:
299,1544.
8.10 (d, J = 1.7 Hz, 1H), 8.01 (d, J = 9.4 Hz, 1H), 7.96 (d, J = 9.3 Hz,
1H), 7.46–7.27 (m, 7H), 7.13 (d, J = 2.5 Hz, 1H), 4.92 (s, 1H), 4.84
(s, 2H), 3.77 (s, 3H); 13C NMR (101 MHz, CDCl3) d 156.27, 149.44,
148.36, 147.13, 139.39, 134.84, 131.68, 129.09, 128.51, 128.01,
127.48, 125.08, 124.84, 123.76, 118.42, 116.43, 99.23, 55.39,
54.79; HR-MS(ESI): calcd for
found: 349.1096.
C
21H17ClN2O [M+H]+ 349.1107;
4.1.1.2. 6-Chloro-2-methoxy-N-phenethylacridin-9-amine (5a-
2).
Yield 67%; mp 122–124 °C; 1H NMR (400 MHz, CDCl3) d
4.1.2. Preparation of 9-(benzyloxy)acridine (6b)
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. The compound 4b
(1.00 mmol) and potassium iodide (0.25 mmol) were added. The
mixture was stirred and refluxed overnight. Then the solution
was evaporated. The solid was poured into water (50 mL), and ex-
tracted 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 20%; mp >250 °C; 1H NMR
(400 MHz, Acetone) d 8.29 (d, J = 8.6 Hz, 2H), 8.12 (d, J = 8.8 Hz,
2H), 7.81–7.74 (m, 2H), 7.60 (d, J = 6.6 Hz, 2H), 7.52 (ddd, J = 8.4,
6.6, 0.9 Hz, 2H), 7.45–7.34 (m, 3H), 5.42 (s, 2H). 13C NMR
(101 MHz, Acetone) d 160.32, 150.62, 136.97, 130.19, 129.84,
128.63, 128.51, 128.48, 125.29, 122.70, 120.44, 78.56; HR-MS(ESI):
calcd for C20H15NO [M+H]+ 286.1232; found: 286.1231.
8.06 (d, J = 1.9 Hz, 1H), 7.98 (d, J = 9.4 Hz, 1H), 7.86 (d, J = 9.3 Hz,
1H), 7.44–7.33 (m, 3H), 7.29 (t, J = 6.7 Hz, 3H), 7.25 (d, J = 2.0 Hz,
1H), 4.64 (s, 1H), 3.96 (t, J = 6.4 Hz, 2H), 3.82 (s, 3H), 3.03 (t,
J = 6.6 Hz, 2H); 13C NMR (101 MHz, CDCl3) d 156.26, 149.27,
148.42, 146.84, 138.12, 134.74, 131.64, 129.01, 128.98, 128.38,
127.07, 124.73, 124.70, 124.17, 118.53, 116.46, 98.53, 55.46,
51.10, 37.24; HR-MS(ESI): calcd for
363.1264; found: 363.1263.
C
22H19ClN2O [M+H]+
4.1.1.3. 6-Chloro-2-methoxy-N-phenethylacridin-9-amine (5a-
3).
Yield 78%; mp 79–81 °C; 1H NMR (400 MHz, CDCl3) d 8.04
(s, 1H), 7.96 (d, J = 9.3 Hz, 1H), 7.81 (d, J = 9.2 Hz, 1H), 7.37 (d,
J = 8.8 Hz, 1H), 7.19 (ddd, J = 38.0, 13.5, 6.6 Hz, 7H), 4.67 (s, 1H),
3.87 (s, 3H), 3.66 (t, J = 6.8 Hz, 2H), 2.71 (t, J = 7.2 Hz, 2H), 2.05
(dd, J = 14.0, 6.9 Hz, 2H); 13C NMR (101 MHz, CDCl3) d 156.04,
149.47, 148.34, 146.81, 140.91, 134.72, 131.51, 128.65, 128.35,
128.27, 126.30, 124.52, 123.88, 117.99, 115.92, 99.25, 55.52,
50.03, 33.29, 33.18; HR-MS(ESI): calcd for C23H21ClN2O [M+H]+
377.1420; found: 377.1410.
4.1.3. Preparation of N-(acridin-9-yl)benzamide (7b)
To a suspension of NaH (60% in mineraloil, 80 mg) in 10 mL of
DMF was added benzamide (2.00 mmol) and the mixture was stir-
red at room temperature for 15 min. To the resultant yellow solu-
tion was added 4b (1.00 mmol) and the mixture was heated at
reflux for 2 h. The reaction mixture was cooled and added to a mix-
ture of 1 M HCl and ethyl acetate. The mixture was filtered and the
crude product was purified by column chromatography using
petroleum ether and ethyl acetate (10:1 v/v). Yield 75%; mp 166-
169 °C; 1H NMR (400 MHz, DMSO-d6) d 8.18 (d, J = 20.0 Hz, 2H),
8.05–7.78 (m, 5H), 7.68 (d, J = 42.1 Hz, 1H), 7.57–7.26 (m, 6H).
13C NMR (101 MHz, DMSO-d6) d 167.87, 134.27, 133.37, 132.12,
131.12, 128.60, 128.12, 127.40, 125.98, 120.94, 117.27, 106.57;
4.1.1.4. N-benzylacridin-9-amine (5b).
Yield 76%; mp 137–
140 °C; 1H NMR (400 MHz, CDCl3) d 8.07 (d, J = 8.5 Hz, 2H), 8.02
(d, J = 8.7 Hz, 2H), 7.67–7.57 (m, 2H), 7.38–7.22 (m, 7H), 5.25 (s,
1H), 4.89 (s, 2H); 13C NMR (101 MHz, CDCl3) d 151.14, 149.22,
139.31, 129.87, 129.25, 128.98, 127.89, 127.58, 123.23, 122.84,
117.05, 55.04; HR-MS(ESI): calcd for C20H16N2 [M+H]+ 285.1391;
found: 285.1400.
4.1.1.5. 2-Methoxy-N-benzylacridin-9-amine (5c).
Yield
HR-MS(ESI): calcd for
299.1188.
C
20H14N2O [M+H]+ 299.1184; found:
31%; mp 179–180 °C; 1H NMR (400 MHz, DMSO-d6) d 8.28 (d,
J = 8.7 Hz, 1H), 7.84 (s, 2H), 7.57 (dd, J = 16.7, 9.1 Hz, 2H), 7.46 (d,
J = 7.4 Hz, 2H), 7.42–7.15 (m, 6H), 4.93 (s, 2H), 3.69 (s, 3H); 13C
NMR (101 MHz, CDCl3) d 156.02, 149.25, 148.10, 146.61, 139.70,
131.64, 130.05, 129.01, 128.89, 127.86, 127.51, 124.42, 124.17,
121.94, 118.48, 118.34, 99.42, 55.34, 54.73. HR-MS(ESI): calcd for
4.2. Bioassay
4.2.1. Cell culture
K562 (suspension cells line) was cultured in RPMI-1640, HepG-
2 (adherent cell lines) was cultured in DMEM, with 10% fetal bo-
C
20H18N2O [M+H]+ 315.1497; found: 315.1506.
vine serum (FBS), 100 lg/mL penicillin, and 100 lg/mL streptomy-
4.1.1.6. 6-Chloro-N-benzylacridin-9-amine (5d).
Yield 62%;
cin in humidified air at 37 °C with 5% CO2.
mp 112–116 °C; 1H NMR (400 MHz, CDCl3) d 8.06 (d, J = 8.6 Hz,
2H), 8.03–7.97 (m, 2H), 7.75–7.62 (m, 1H), 7.43–7.30 (m, 6H),
7.24 (d, J = 1.9 Hz, 1H), 4.96 (s, 2H). 13C NMR (101 MHz, CDCl3) d
151.37, 149.66, 138.94, 136.09, 130.91, 130.50, 129.47, 129.19,
128.20, 127.88, 127.66, 124.62, 124.30, 123.79, 122.48, 116.93,
4.2.2. Cell growth inhibition assay
The cells were seeded into 96-well plates at 1.5 ꢂ 105 cells/mL,
treated with the synthesized compounds at various concentra-
tions. After 48 h treatment, the cells were incubated with 15 lL

4176
X. Lang et al. / Bioorg. Med. Chem. 21 (2013) 4170–4177
MTT (3-(4,5-dimethyl-thiazol-2-yl)-2, 5-diphenyl-tetrazolium bro-
mide from Sigma) solution (5 mg/mL) for 4 h at 37 °C, 5% CO2. The
formazan precipitates were dissolved in 100 lL DMSO. At 490 nm,
the absorbance was measured by Multimode Detector DTX 880
sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis
(PAGE), and electrophoretically transferred to PVDF membrane
(amc Biobind NT-200). After blotting, the membrane was blocked
in 5% Bovine serum albumin (BSA) (Trisbuffered saline (TBS),
0.5 mM Na3VO4; 1 mMNaF) for 1 h, and incubated with the specific
primary antibody for 2 h at room temperature. Protein bands were
detected using the Super Signal West Pico Chemiluminescent Sub-
strate (Thermo scientific) after hybridization with the antibody.
(Beckman Coulter).
4.3. Biophysical evaluation
4.3.1. Materials
4.6. Flow cytometry assay
The measurements involving the interaction of the compound
with ct DNA was carried out in Ultra-pure MilliQ water
(18.2 mO) buffer containing 10 mM Tris and 10 mM NaCl, and ad-
justed to pH 7.0 with hydrochloric acid. UV–vis spectrometer was
employed to check a solution of ct DNA (Sigma, purification P90%)
purity (A260:A280 = 1.86) and the concentration of DNA per mole
phosphate was determined optically using a molar extinction coef-
ficient of 6600 Mꢀ1 cmꢀ1 at 260 nm in the buffer.
Phosphatidylserine externalization was measured by Annex-
inV-FITC/PI apoptosis detection kit (Beyotime Company) according
to the manufacturer’s instructions.
Acknowledgments
The authors would like to thank the financial supports from the
Ministry of Science and Technology of China (2012ZX09506001-
010, 2012CB722605, 2013AA092902, 2012AA020305 and
2011DFA30620), the Chinese National Natural Science Foundation
(21272134 and 20902053), and Shenzhen Sci & Tech Bureau
(JCYJ20120831165730905).
4.3.2. Fluorescence emission spectra
All the emission spectra were measured on Fluorolog spectrom-
eter. Xenon arc lamp was used as the excitation light source in the
measurements of emission spectra. 5
pound (the final concentration was 50
Tris–HCl buffer (pH 7.0) solution, and then ct DNA was also added
with the final concentration from 0 to 6
length was set at 420 nm.
l
L of 10 mM tested com-
lM) was incubated in
Supplementary data
lM. The excitation wave-
Supplementary data (general methods and the preparation of
compounds 3a–3f and 4a–4f; fluorescence spectroscopy and
absorption spectroscopy; 1H NMR and 13C NMR spectrum; high
resolution mass spectrometry) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.bmc.2013.05.008.
4.3.3. Absorption spectra
UV–vis absorption spectra were all recorded on a computer-
controlled Beckman Coulter DU 800 spectrophotometer by using
a quartz cell having 1.0 cm pathway. 5 mM tested compounds
DMSO solution (1
50 L above buffer solution, and then a known volume (0–5)
of 0.2 mM ct DNA was also added. The above solutions were mixed
and then diluted to 100 L with the Tris–HCl buffer (pH 7.0) solu-
lL)were transferred to the quartz cell with
l
lL
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