Synthesis and antiproliferative activity of 2,7-diamino l0-(3,5-dimethoxy)benzyl-9(10H)-acridone derivatives as potent telomeric G-quadruplex DNA ligands

Article abstract of DOI:10.1016/j.bioorg.2015.04.002

A novel series of l0-(3,5-dimethoxy)benzyl-9(10H)-acridone derivatives with terminal ammonium substituents at C2 and C7 positions on the acridone ring were successfully synthesized as antiproliferation agents. The biologic activity of the acridone compoun

Full text of DOI:10.1016/j.bioorg.2015.04.002

Bioorganic Chemistry 60 (2015) 30–36
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and antiproliferative activity of 2,7-diamino
l0-(3,5-dimethoxy)benzyl-9(10H)-acridone derivatives
as potent telomeric G-quadruplex DNA ligands
Chunmei Gao a,b, , Wei Zhang a,b,c, Shengnan He a,b,c, Shangfu Li , Feng Liu
⇑
a
a,b,
⇑
, Yuyang Jiang a,b,d
a
The Ministry-Province Jointly Constructed Base for State Key Lab-Shenzhen Key Laboratory of Chemical Biology, The Graduate School at Shenzhen, Tsinghua University,
Shenzhen 518-55, PR China
National & Local United Engineering Lab for Personalized Anti-Tumor Drugs, The Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, PR China
b
c
Department of Chemistry, Tsinghua University, Beijing 100084, PR China
d
Department of Pharmacology and Pharmaceutical Sciences, 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:
Received 26 December 2014
Available online 17 April 2015
A novel series of l0-(3,5-dimethoxy)benzyl-9(10H)-acridone derivatives with terminal ammonium sub-
stituents at C2 and C7 positions on the acridone ring were successfully synthesized as antiproliferation
agents. The biologic activity of the acridone compounds against leukemia CCRF-CEM cells demonstrated
that some of the compounds displayed good antiproliferative activity, among which compound 6a con-
taining dimethylamine substituents at the terminal C2 and C7 positions exhibited the highest cytotoxi-
Keywords:
Acridone derivatives
Synthesis
Antiproliferative activity
G-quadruplex DNA
city with IC50 at 0.3
lM. In addition compound 6a showed little toxicity against normal 293T cells
proliferation with IC50 more than 100
l
M. Further study indicated that compound 6a had strong binding
activity to human telomeric G-quadruplex DNA, as detected by mass spectrometry, CD spectroscopy, UV
absorption, FRET and fluorescence quenching assays. Our data suggested that the activity of 6a might be
associated with its stabilization of G-quadruplex DNA, which can be developed as potent antitumor
agent.
Ó 2015 Elsevier Inc. All rights reserved.
1
. Introduction
Cancer has become one of the most serious diseases in the pre-
their telomeres due to the end replication problem, until they enter
replicative senescence. As 85–90% cancer cells display telomerase
activity, which renders tumor cells with a capacity to continue cel-
lular proliferative and to be immortal, telomerase can be regarded
as an important target for cancer chemotherapy [6–9]. In addition,
telomerase needs a linear, non-folded telomere DNA substrate to
extend telomeres. If the telomere overhang DNA forms higher
order structures, such as G-quadruplex, telomerase cannot recog-
nize them and the activity of telomerase would be inhibited.
Thus the design and synthesis of efficient compounds with the
ability to stabilize G-quadruplex DNA is a potent strategy for antic-
ancer chemotherapy [10–13].
sent society. One of the well-know hallmarks of cancer cells is their
ability to sustain chronic proliferation [1,2]. Therefore great efforts
have been made to identify new entities with good antiprolifera-
tive activity. As most of antitumor agents have various adverse
effects, such as toxicity against normal cells, design and develop-
ment of new compounds with good antiproliferative activity and
low toxic effects on normal cells has attracted great attention
[
3–5].
Telomerase is a cellular reverse transcriptase which can add
telomere repeats to telomere DNA to maintain chromosome integ-
rity and stability. Most normal somatic cells lack telomerase activ-
ity, and after each cell division they would continually shorten
Acridine and acridone derivatives show potent antitumor activ-
ity in vitro/vivo, some of which have been developed as good
G-quadruplex stabilizers and antitumor agents, such as BRACO-
1
9 and AS1410 [14–26]. Previously, we have reported that
compounds based on planar tricyclic chromophore scaffolds
exhibited antiproliferative activity in vitro, among which 10-(3,5-
dimethoxy)benzyl-9(10H)-acridone with terminal amino sub-
stituents at C2 position on the acridone ring had strong binding
activity to calf thymus DNA [27]. In order to further improve the
⇑
Corresponding authors at: The Ministry-Province Jointly Constructed Base for
State Key Lab-Shenzhen Key Laboratory of Chemical Biology, The Graduate School
at Shenzhen, Tsinghua University, Shenzhen 518-55, PR China. Fax: +86 (755)
2
6032094.
E-mail addresses: chunmeigao@sz.tsinghua.edu.cn (C. Gao), liu.feng@sz.
tsinghua.edu.cn (F. Liu).
http://dx.doi.org/10.1016/j.bioorg.2015.04.002
0
045-2068/Ó 2015 Elsevier Inc. All rights reserved.

C. Gao et al. / Bioorganic Chemistry 60 (2015) 30–36
31
anticancer activity and reduce the cytotoxicity to normal cells, two
0-(3,5-dimethoxy)benzyl-9(10H)-acridone with two terminal
2.2.2.2. 2,7-Bis[3-(diethylamino)propionamido]-10-(3,5-dimethoxy-
1
benzyl)-9,10-di-hydroacridone (6b). Yield 107 mg, 85%; m.p. 235–
1
) d: 0.98 (t, 12H, 3J = 7.2 Hz),
amino substituents at C2 and C7 positions on the acridone ring
were developed as selective G-quardruplex stabilizers [28].
However, the two compounds displayed moderate antiprolifera-
tive activity. In continuation of our interest on the development
of new acridones with potent antiproliferative activity, this study,
we report the synthesis and biochemistry of new acridone deriva-
tives with two terminal amino substituents at C2 and C7 positions
to discover new antiproliferative entities.
236 °C; H NMR (400 MHz, DMSO-d
6
3
2.42–2.53 (m, 12H), 2.76 (t, 4H, J = 7.2 Hz), 3.66 (s, 6H), 5.69 (s,
2H), 6.26 (m, 2H), 6.41 (m, 1H), 7.31 (m, 1H), 7.60 (d, 2H,
3
5
3
5
J = 9.6 Hz), 7.93 (dd, 2H, J = 9.6 Hz, J = 2.4 Hz), 8.59 (d, 2H,
13
6
J = 2.4 Hz), 10.31 (s, 2H); C NMR (100 MHz, DMSO-d ) d: 12.1,
34.5, 46.4, 48.6, 55.4, 98.6, 104.3, 115.4, 116.9, 121.5, 126.7,
133.7, 138.1, 139.2, 161.2, 170.7, 176.3; HRMS calcd for
+
C
36
48
H N
5
O
5
[M + H] 630.3655, found 630.3651.
2
.2.2.3.
2,7-Bis[3-(diethanolamino)propionamido]-10-(3,5-
2
. Experimental
dimethoxybenzyl)-9,10-di-hydroacridone (6c). Yield 76 mg, 55%;
1
6
m.p. 205–206 °C; H NMR (400 MHz, DMSO-d ) d: 2.46 (t, 4H,
2.1. Materials and methods
3
3
3
J = 6.6 Hz), 2.59 (t, 8H, J = 6.2 Hz), 2.85 (t, 4H, J = 6.6 Hz), 3.47
3
(
(
(
m, 8H), 3.66 (s, 6H), 4.40 (t, 4H, J = 5.3 Hz), 5.69 (s, 2H), 6.26
m, 2H), 6.41 (m, 1H), 7.31 (m, 1H), 7.60 (d, 2H, J = 9.4 Hz), 7.92
dd, 2H, J = 9.4 Hz, J = 2.5 Hz), 8.63 (d, 2H, J = 2.5 Hz), 10.36 (s,
Melting points (mp) were recorded on a SGW X-4 melting point
3
1
13
apparatus and were uncorrected. H NMR and C NMR spectra
3
5
5
1
13
were obtained at 400 MHz for H NMR and 100 MHz for C NMR
in DMSO-d6 solution with tetramethylsilane as the internal stan-
dard, respectively. Splitting patterns are indicated as s, singlet; d,
doublet; t, triplet; m, multiplet; brs, broad singlet. HRMS were
recorded on a QSTAR XL spectrometer and Waters Q-Tof Premier
spectrometer.
13
2
5
1
6
6
H); C NMR (100 MHz, DMSO-d ) d: 35.0, 49.4, 51.2, 55.6, 56.7,
9.7, 98.8, 104.6, 115.8, 117.0, 121.7, 127.1, 133.9, 138.4, 139.5,
61.4, 171.0, 176.5; HRMS calcd for
94.3452, found 694.3452.
[M + H]+
36 48 5 9
C H N O
2.2.2.4. 2,7-Bis(3-pyrrolidinopropionamido)-10-(3,5-dimethoxyben-
CCRF-CEM leukemia cells, human lung adenocarcinoma cells
zyl)-9,10-di-hydroacridone (6d). Yield 100 mg, 80%; m.p. 245–
2
(
A549), human breast adenocarcinoma cells (MCF7) and human
1
6
46 °C; H NMR (400 MHz, DMSO-d ) d: 1.69 (m, 8H), 2.48–2.52
embryonic kidney cells (293T), human leukemia cells (K562), and
Human colon carcinoma cells (HCT-116) were purchased from
the Cell Bank of Chinese Academy of Sciences.
3
(
m, 12H), 2.74 (t, 4H, J = 6.8 Hz), 3.66 (s, 6H), 5.69 (s, 2H), 6.26
3
(
m, 2H), 6.41 (m, 1H), 7.61 (d, 2H, J = 9.6 Hz), 7.92 (dd, 2H,
J = 9.6 Hz, J = 2.4 Hz), 8.61 (d, 2H, J = 2.4 Hz), 10.27 (s, 2H);
3
5
5
13
C
4
Single-stranded oligonucleotides (TTAGGG) were purchased
NMR (100 MHz, DMSO-d
6
) d: 23.4, 36.4, 49.6, 51.8, 53.7, 55.4,
from invitrogen (Guangdong, China), which were converted to
the G-quadruplex according to our previous paper and references
9
1
6
8.6, 104.3, 115.5, 116.9, 121.5, 126.7, 133.7, 138.1, 139.2, 161.2,
70.4, 176.3; HRMS calcd for C36
26.3344.
+
44 5 5
H N O [M + H] 626.3342, found
[
28,29]. The concentration of quadruplex was determined
spectroscopically.
2.2.2.5.
2,7-Bis(3-piperidinopropionamido)-10-(3,5-dimethoxyben-
2
2
.2. Chemistry
zyl)-9,10-dihydro-acridone (6e). Yield 111 mg, 85%; m.p. 229–
1
2
6
30 °C; H NMR (400 MHz, DMSO-d ) d: 1.39 (m, 2H), 1.51 (m,
3
3
.2.1. Synthesis of 2,7-bis(3-chloropropionamido)-10-(3,5-
8H), 2.39 (m, 8H), 2.48 (t, 4H, J = 6.8 Hz), 2.62 (t, 4H, J = 6.8 Hz),
dimethoxybenzyl)9,10-dihydro-acrid-inone (5)
A suspension of 4 (375 mg, 1 mmol), 3-chloropropanoyl chlo-
ride (382 mg, 3 mmol) and N-methyl morpholine (303 mg,
3.65 (s, 6H), 5.68 (s, 2H), 6.26 (m, 2H), 6.41 (m, 1H), 7.60 (d, 2H,
3
1
3
J = 9.2 Hz), 7.92 (d, 2H, J = 9.2 Hz), 8.59 (brs, 1H), 10.33 (s, 2H);
3
6
C NMR (100 MHz, DMSO-d ) d: 24.3, 25.9, 34.3, 49.2, 53.9, 54.7,
3
mmol) was refluxed in dry tetrahydrofuran until TLC indicated
55.4, 98.6, 104.3, 115.4, 116.9, 121.5, 126.7, 133.6, 138.2, 139.2,
161.2, 170.6, 176.3; HRMS calcd for
654.3655, found 654.3653.
38 48 5 5
C H N O
[M + H]+
completion of reaction and then water was added with rapid stir-
ring. Yellow solids were obtained after filtration. The solids were
washed with ethanol to afford 5 (501 mg, 90%) as yellow solids,
which were used without further purification.
2.2.2.6. 2,7-Bis(3-morpholinopropionamido)-10-(3,5-dimethoxyben-
zyl)-9,10-di-hydro-acridone (6f). Yield 113 mg, 86%; m.p. 265–
1
2
66 °C; H NMR (400 MHz, DMSO-d
6
) d: 2.42 (m, 8H), 2.51 (t, 4H,
2.2.2. General aminolysis procedure of compound (6a–6g)
3
3
J = 6.8 Hz), 2.65 (t, 4H, J = 6.8 Hz), 3.58 (m, 8H), 3.66 (s, 6H),
.69 (s, 2H), 6.26 (m, 2H), 6.41 (m, 1H), 7.61 (d, 2H, J = 9.2 Hz),
.93 (dd, 2H, J = 9.6 Hz, J = 2.4 Hz), 8.62 (d, 2H, J = 2.4 Hz),
6
0.24 (s, 2H); C NMR (100 MHz, DMSO-d ) d: 34.1, 49.2, 53.3,
4.4, 55.4, 66.4, 98.6, 104.3, 115.5, 116.9, 121.5, 126.7, 133.6,
38.2, 139.2, 161.1, 170.3, 176.3; HRMS calcd for
To a stirred refluxing suspension of 5 (111 mg, 0.2 mmol) and KI
3
5
(
66 mg, 0.4 mmol) in EtOH (2 mL) was added to the corresponding
3
5
5
7
1
5
1
C
secondary amines (5 mmol). The mixture was stirred at reflux until
TLC indicated completion of reaction. After cooling to room tem-
perature, water was added with rapid stirring under ice-water
bath. Yellow solids were obtained after filtration. The products
were purified by recrystallization from cold ethanol.
13
+
36
H
44
N
5
O
7
[M + H] 658.3241, found 658.3239.
2.2.2.7.
2,7-Bis[3-(4-hydroxyl-piperidino)propionamido]-10-(3,5-
2
.2.2.1. 2,7-Bis[3-(dimethylamino)propionamido]-10-(3,5-dimethoxy-
dimethoxybenzyl)-9, 10-dihydro-acridone (6g). Yield 114 mg, 83%;
m.p. 140–142 °C; H NMR (400 MHz, DMSO-d ) d: 1.41 (m, 4H),
6
1.72 (m, 4H), 2.08 (m, 4H), 2.48 (m, 4H), 2.63 (m, 4H), 2.75 (m,
4H), 3.44 (m, 2H), 3.66 (s, 6H), 4.54 (m, 2H), 5.69 (s, 2H), 6.26
1
benzyl)-9,10-di-hydroacridone (6a). Yield 94 mg, 82%; m.p. 246–
2
4
1
47 °C; H NMR (400 MHz, DMSO-d
6
) d: 2.18 (s, 12H), 2.47 (t,
H, J = 7.2 Hz), 2.58 (t, 4H, 3J = 7.2 Hz), 3.66 (s, 6H), 5.69 (s, 2H),
.26 (m, 2H), 6.41 (m, 1H), 7.61 (d, 2H, J = 9.6 Hz), 7.93 (dd, 2H,
3
3
6
(m, 2H), 6.41 (m, 1H), 7.60 (m, 2H), 7.91 (m, 2H), 8.60 (m, 2H),
3
J = 9.6 Hz, J = 2.4 Hz), 8.61 (d, 2H, 5J = 2.4 Hz), 10.31 (s, 2H);
5
13
13
C
6
10.33 (s, 2H); C NMR (100 MHz, DMSO-d ) d: 34.6, 34.7, 49.2,
NMR (100 MHz, DMSO-d
6
) d: 35.0, 45.2, 49.1, 55.4, 98.6, 104.3,
50.9, 54.0, 55.4, 66.5, 98.6, 104.3, 115.4, 116.9, 121.5, 126.6,
1
C
15.5, 116.9, 121.5, 126.7, 133.6, 138.1, 139.2; HRMS calcd for
133.6, 138.1, 139.2, 161.1, 170.5, 176.3; HRMS calcd for
+
+
32
H
40
N
5
5
O [M + H] 574.3029, found 574.3025.
C
38
48
H N
5
O
7
[M + H] 686.3554, found 686.3553.

3
2
C. Gao et al. / Bioorganic Chemistry 60 (2015) 30–36
2
.2.3. General preparation of compound 8
To a solution of Boc-amino acid (1.2 mmol) in dry THF (10 mL)
was added HOBt (162 mg, 1.2 mmol), DIC (152 mg, 1.2 mmol) and
,7-diamino-10-(3,5-dimethoxybenzyl)-9,10-dihydro-acridone
187 mg, 0.5 mmol). The reaction suspension was stirred under
2H), 6.27 (m, 2H), 6.43 (m, 1H), 7.69 (m, 2H), 7.99 (m, 2H), 8.41
(brs, 6H), 8.71 (m, 2H), 11.20 (s, 2H); C NMR (100 MHz, DMSO-d )
6
d: 18.2, 18.7, 30.2, 49.2, 55.4, 59.2, 98.5, 104.3, 116.2, 117.3, 121.5,
13
2
(
4
126.9, 132.5, 138.8, 139.0, 161.2, 167.0. 176.2; HRMS calcd for
+
C
32
H
40
N
5
O
5
[M-HCl
2
] 574.3029, found 574.3026.
nitrogen overnight at room temperature. The volatile parts were
removed under reduced pressure and compound 7 was obtained
by column chromatography. Compound 7 was suspended and stir-
red in hydrochloride 1,4-dioxane solution (15 ml), The suspension
was stirred at room temperature until TLC indicated completion of
reaction. Yellow solids were obtained after filtration.
2.3. Measurement of G-quardruplex binding ability
2.3.1. Circular dichroism spectra
The CD spectra of DNA oligonucleotides 10
4
lM (TTAGGG) with
0, 20 and 40 M 6a in NH OAc buffer were carried out at room
l
4
temperature by using a J-815 spectropolarimeter (JASCO) with a
0.1 cm path-length quartz cell. The CD spectrum was scanned
three times and obtained by taking the average of them. The scan
for buffer was subtracted from the average scan each time.
2
.2.3.1. 2,7-Bis-(2-ammonium-methyl-carbamoyl)-10-(3,5-dimethoxy-
benzyl)-9,10-di-hydro-acridoneBis-hydrochlorate (8a). Yield 140 mg,
1
5
3
7
2
1
6
7%; yellow solids; m.p. > 300 °C; H NMR (400 MHz, DMSO-d ) d:
.66 (s, 6H), 3.84 (s, 4H), 5.72 (s, 2H), 6.28 (m, 2H), 6.42 (m, 1H),
.68 (m, 2H), 7.95 (m, 2H), 8.31 (brs, 6H), 8.68 (m, 2H), 11.02 (s,
2
.3.2. Absorption spectra
M compound 6a were incubated in NH
in the presence or absence of 7 or 14 M of the G-quadruplex DNA.
1
3
H); C NMR (100 MHz, DMSO-d
6
) d: 41.2, 49.3, 55.4, 98.6, 104.3,
2
0
l
4
OAc buffer (pH 7.4)
15.8, 117.3, 121.5, 126.7, 132.7, 138.7, 139.1, 161.2, 165.0, 176.2;
l
+
28 5 5 2
HRMS calcd for C26H N O [M-HCl ] 490.2090, found 490.2087.
UV–Vis absorption spectra were all recorded using DU 800 spec-
trophotometer (BeckmanCoulter, Atlanta, GA, USA) with a quartz
cell having 1.0 cm pathway.
2
.2.3.2. 2,7-Bis-(3-ammonium-ethyl-carbamoyl)-10-(3,5-dimethoxy-
benzyl)-9,10-di-Hydro-acridoneBis-hydrochlorate (8b). Yield
55 mg, 60%; yellow solids; m.p. 262–264 °C; H NMR (400 MHz,
1
1
2.3.3. Mass spectra
DMSO-d
5
6
) d: 2.78 (t, 4H, 3J = 6.8 Hz), 3.12 (m, 4H), 3.66 (s, 6H),
The mass spectra were acquired using a Waters Q-Tof Premier
.70 (s, 2H), 6.26 (m, 2H), 6.41 (m, 1H), 7.63 (m, 2H), 7.90–7.94
mass spectrometer equipped with an electrospray ionization
ESI) source. The instrument was operated in the negative-ion
mode. Compound 6a and DNA solution was diluted with 20:80
v/v) methanol/100 mm ammonium acetate. The binding assays
were performed at 25 M DNA and compound 6a (25 M).
Methanol was added to obtain a good spray [29]. The direct infu-
sion flow rate was 10 l/min. The electrospray source conditions
were spray voltage of 2.4 kV and capillary temperature of 120 °C
30].
13
(
d
1
C
m, 8H), 8.70 (m, 2H), 10.49 (s, 2H); C NMR (100 MHz, DMSO-
) d: 33.4, 35.2, 49.1, 55.4, 98.6, 104.3, 115.8, 117.0, 121.5,
26.8, 133.3, 138.3, 139.1, 161.1, 168.6, 176.3; HRMS calcd for
(
6
(
+
28
32 5 5 2
H N O [M-HCl ] 518.2403, found 518.2393.
l
l
2
.2.3.3. 2,7-Bis-(4-ammonium-propyl-carbamoyl)-10-(3,5-dimethoxy-
l
benzyl)-9,10-dihydro-acridoneBis-hydrochlorate (8c). Yield 166 mg,
1
6
1%; yellow solids; m.p. 267–269 °C; H NMR (400 MHz, DMSO-
) d: 1.92 (m, 4H), 2.50 (m, 4H), 2.87 (m, 4H), 3.66 (s, 6H), 5.71
s, 2H), 6.26 (m, 2H), 6.42 (m, 1H), 7.62 (m, 2H), 7.96 (m, 2H),
.05 (brs, 6H), 8.67 (m, 2H), 10.43 (s, 2H); 13C NMR (100 MHz,
[
d
6
(
8
2.3.4. FRET assay
0
The labeled oligonucleotides F21T [5 -FAM-d(GGG[TTAGGG]3)-
DMSO-d
1
forC30
6
) d: 22.8, 32.7, 38.8, 48.7, 54.9, 98.1, 103.9, 115.2, 116.4,
0
TAMRA-3 ] were purchased from invitrogen (Guangdong, China)
21.0, 126.3, 133.1, 137.8, 138.7, 160.7, 170.1, 175.9; HRMS calcd
+
and the FRET assay was carried out on a real-time PCR apparatus
36 5 5 2
H N O [M-HCl ] 546.2716, found 546.2725.
(
Roche LightCycler 2). The FRET probes were diluted to the correct
concentration (200 nM) in Tris–HCl buffer (10 mM, pH 7.4) con-
taining 60 mM KCl. Compound 6a was diluted to concentrations
of 1.0, 1.4, and 2.0 lM, respectively. Samples were incubated for
1 h. and then put into LightCycler capillaries. Measurements were
made in triplicate on a RT-PCR with excitation at 470 nm and
detection at 530 nm. Fluorescence readings were taken at intervals
of 1 °C over the range 37–99 °C, with a constant temperature being
maintained for 30 sprior to each reading to ensure a stable
value.
2
.2.3.4. 2,7-Bis-(5-ammonium-butyl-carbamoyl)-10-(3,5-dimethoxy-
benzyl)-9,10-dihydro-acridoneBis-hydrochlorate (8d). Yield 172 mg,
6
1
0%; yellow solids; m.p. 199–201 °C; H NMR (400 MHz, DMSO-
) d: 1.62–1.68 (m, 8H), 2.40 (m, 4H, 3J = 6.8 Hz), 2.81 (m, 4H),
d
3
2
6
.66 (s, 6H), 5.70 (s, 2H), 6.25 (m, 2H), 6.41 (m, 1H), 7.61 (m,
H), 7.93–7.95 (m, 8H), 8.67 (m, 2H), 10.32 (s, 2H); 13C NMR
(
125 MHz, DMSO-d
6
) d: 22.6, 27.0, 36.1, 39.0, 49.3, 55.6, 98.8,
1
1
04.6, 115.8, 117.1, 121.7, 127.0, 133.9, 138.4, 139.5, 161.4,
+
71.4, 176.6; HRMS calcd for
32 40 5 5 2
C H N O [M-HCl ] 574.3029,
found 574.3028.
2.4. In-vitro cytotoxic activity evaluation by MTT assay
2
.2.3.5. 2,7-Bis-(2-ammonium-ethyl-carbamoyl)-10-(3,5-dimethoxy-
MTT assays were performed according to the methods reported
by our previous papers [27,28,31–33].
benzyl)-9,10-dihydro-acridoneBis-hydrochlorate (8e). Yield 137 mg,
5
1
3%; yellow solids; m.p. 252–253 °C; H NMR (400 MHz, DMSO-
d
6
) d: 1.52 (d, 6H, 3J = 6.8 Hz), 3.66 (s, 6H), 4.14 (m, 2H), 5.73 (s,
2
H), 6.27 (m, 2H), 6.42 (m, 1H), 7.68 (m, 2H), 7.99 (m, 2H), 8.43
3. Results and discussion
13
(
brs, 6H), 8.71 (m, 2H), 11.11 (s, 2H); C NMR (100 MHz, DMSO-
) d: 17.7, 49.4, 49.5, 55.6, 98.9, 104.6, 116.4, 117.5, 121.7,
27.1, 133.0, 138.9, 139.3, 161.4, 168.7, 176.4; HRMS calcd for
d
6
3.1. Chemistry
1
C
+
28
H
32
N
5
O [M-HCl
5
2
] 518.2403, found 518.2402.
The synthetic route of desired compound is similar to our
previous papers [27]. As shown in Scheme 1, starting from acridone,
2
.2.3.6. 2,7-Bis-(2-ammonium-3-methyl-propyl-carbamoyl)-10-(3,5-
dimethoxybenzyl)-9,10-dihydro-acridoneBis-hydrochlorate (8f). Yield
72 mg, 60%; yellow solids; m.p. 242–244 °C; H NMR (400 MHz,
DMSO-d ) d: 1.02–1.04 (m, 12H), 3.66 (s, 6H), 3.90 (m, 1H), 5.73 (s,
2,7-diamino-l0-(3,5-dimethoxy)benzyl-9(10H)-acridone
4
was
obtained by three-stage reaction. Acylation reaction of compound
4 with 3-chloropropionyl chloride yielded the 2,7-bis(3-Chloro-
propionamido)-10-(3,5-dimethoxybenzyl) 9,10-dihydro-acridone
1
1
6

C. Gao et al. / Bioorganic Chemistry 60 (2015) 30–36
33
5
in high yields. The followed nucleophilic displacement of the chlo-
amine substituents, compounds 6b and 6c showed decreasing inhi-
bitory effect. Compound 6b was determined to be 15-fold less
potent than 6a. 6c was determined to be 8-fold less potent than
6b. In addition, compounds bearing terminal cyclic amino moiety
(6d–6) were also evaluated, among which compound 6e with
piperidine groups displayed the highest antiproliferative activity
ride by the appropriate amine in the presence of KI gave the desired
compounds 6(a–g) in good yields. Compounds 8(a–f) were synthe-
sized according to the reference [28], as seen in Scheme 2 and the
above Experimental section. All synthesized new compounds were
characterized pure by H NMR, C NMR and high resolution mass
spectral data.
1
13
with an IC50 at 2.3 lM. The replacement of piperidine to 4-hy-
droxyl piperidine reduced about 10-fold cytotoxicity. Derivatives
with pyrrolidine (6d) and morpholine (6f) groups were essentially
inactive. The data indicated that the pattern of tertiary amines
played important roles on the cytotoxicity.
Compared with the antiproliferative activity of 6a–6g, ana-
logues 8 displayed lower activity. Compounds 8a–8d with different
length of the alkyl chain on the amino acid moiety were inactive.
By changing the methylene group (8a) to methyl methylene (8e)
or isopropyl methylene (8f), compounds showed moderate inhibi-
tion effect against CCRF-CEM cells.
As compound 6a displayed the highest activity against CCRF-
CEM cells, to further investigate its antiproliferative potential and
toxicity against different cancer cells and normal cells, the cyto-
toxic activity against A549, MCF7, 293T, K562, and HCT-116 cells
was evaluated. As shown in Table 2, compound 6a possessed little
activities against the growth of the three solid tumor cell lines. The
results indicated that 6a had high selectivity to leukemia cells with
3
.2. Cytotoxic activity
Two series of synthesized new l0-(3,5-dimethoxy)benzyl-
(10H)-acridone derivatives with a range of side chain substituents
9
at C2 and C7 position on the acridone ring were tested for their
antiproliferative activity against leukemia CCRF-CEM using 3-
(
(
4,5-dimethylthiazol-2-yl)-2,5-di-phenyltetrazolium
MTT) assay after 48 h of treatment with increasing concentrations
bromide
of the tested compounds. Etoposide was used as the positive con-
trol. The results were shown in Table 1.
Compound 6(a–g) with tertiary amines at the termini of the
chains displayed moderate to good antiproliferation activity, some
of which had IC50 values at a low micromolar range. Compound 6a
containing dimethylamine substituents was considered as the
most potent antiproliferation agent with an IC50 at 0.3 lM. By
changing dimethylamine group to diethylamine and diethanol
O
O
H
H
O
N
iv
O
N
iii
i
N
O
N
ii
NO2
O N
2
O N
NO2
2
H N
NH2
O
2
2
O
O
4
1
3
O
O
O
O
N
O
O
N
v
v
i
O
O
H N
Cl
NH
Cl
H N
NH
NR
2
R N
2
O
5
O
6(a-g)
Scheme 1. Reagents and conditions (i) HNO
NH, EtOH, KI, reflux.
3
, HOAC (ii) HNO
3
, H
2
SO
4
(iii) 3,5-dimethoxybenzyl chloride, NaH (iv) Na
2
Sꢀ9H
2 2 5
O, C H OH (v) chloropropionyl chloride, THF (vi)
R
2
Scheme 2. Reagents and conditions (i) HOBt, DIC (ii) HCl in 1,4-dioxane.

3
4
C. Gao et al. / Bioorganic Chemistry 60 (2015) 30–36
Table 1
Chemical data and antiproliferative activity against CCRF-CEM cells of compound 6a–6g and 8a–f.
Compound
R
IC50
(
l
M)
Compound
8a
Z
IC50
>50
(lM)
6
a
0.3 ± 0.1
CH
2
6
b
4.4 ± 0.5
8b
8c
(CH
(CH
2
)
2
3
>50
>50
6
c
35.5 ± 3.2
2
)
6
6
d
e
>50
8d
8e
(CH
)
2 4
>50
2.3 ± 0.2
30.9 ± 0.8
9.7 ± 0.8
6
6
f
>50
8f
g
26.1 ± 2.2
Etoposide
0.14 ± 0.03
no toxic effects on normal 293T cells, which made it a good candi-
date for further development.
3.3. G-quadruplex DNA binding ability
3.3.1. UV–Vis absorption spectra
Generally, when acridine or acridone derivatives intercalated
into DNA, the stacking interaction between aromatic chromophore
and the base pairs of DNA will lead to bathochromic shifts and
hypochromicity on the UV–Vis curve of compound-DNA complex
Table 2
IC50 values (
lines.
lM) ofcompound 6a against A549, MCF-7, 293T, HCT-116 and K562 cell
[
34–36]. To evaluate the G-quadruplex DNA binding capacity of
A549
>50
MCF-7
>50
293T
>100
HCT-116
21.3 ± 2.6
K562
our compounds, the representative spectra of 6a in the absence
and presence of G-quadruplex DNA were investigated. As shown
6
a
4.7 ± 0.2
Fig. 1. (a) UV–Vis absorption spectra of 20
lM 6a with 0, 7, 14, 21
lM G-quadruplex DNA in NH
4
OAc buffer. DNA Vertical arrows indicate the increase of the absorbance
change upon increasing DNA concentration. (b) The plot of [DNA]/(e
a
f
–e ) as a function of DNA concentration determined from the absorption spectral data.

C. Gao et al. / Bioorganic Chemistry 60 (2015) 30–36
35
in Fig. 1a, compound 6a showed the maximal absorption around
14 nm with an absorption bands in the region of 360–460 nm,
which was shown in Fig. 1s (Supplementary material). The results
indicated that 6a displayed moderate binding selectivity toward
G-quadruplex DNA.
4
while the maximal absorption is about 420 nm in the presence of
G-quadruplex DNA. The absorption spectra also demonstrated sig-
nificant hypochromicities. Both the bathochromic shifts and
hypochromicity indicated that compound 6a had the ability to
bind G-quadruplex DNA.
3.3.2. Fluorescence emission spectra
The fluorescence of acridine or acridone derivatives always
quenched severely after forming compound-DNA complex. In
order to gain further insight into the binding properties of the
human telomeric G-quadruplex DNA with 6a, the fluorescence
In order to further elucidate the G-quadruplex DNA binding
ability of 6a, the binding constant K
ing the changes in absorbance with increasing concentrations of
G-quadruplex DNA. The binding constant K was calculated by
the ratio of slope to the intercept [37] (Fig. 1b), which was derived
b
was determined by monitor-
spectra in NH OAc aqueous solution had been recorded in the
4
b
absence and presence of G-quadruplex DNA. About 50% decrease
of the peak intensity in the presence of DNA was observed
(Fig. 2). This result can be served as another proof of the interaction
between compound 6a and G-quadruplex DNA.
5
ꢂ1
to be 6.7 ꢁ 10 M . In addition, the binding ability of compound
5
ꢂ1
6
a to duplex ctDNA was also investigated (K
b
= 1.3 ꢁ 10 M ),
3
.3.3. Circular dichroism spectroscopy
Circular dichroism (CD) has been used as a powerful tool to
3
2
2
1
1
000000
500000
000000
500000
000000
monitor the change of biomacromolecules for years due to its out-
standing sensitivity. To further confirm the interaction mode
between 6a and G-quadruplex DNA, CD spectra of free G-quadru-
plex in the absence and presence of 6a were studied (Fig. 3). The
CD spectrum of free G-quadruplex DNA had a negative peak near
a
b
c
2
35 nm, a small positive peak at about 250 nm and a positive peak
near 295 nm associated with a 274 nm positive shoulder in
NH OAc buffer, which indicated that the coexistence of antiparal-
5
00000
0
4
lel, parallel G-quadruplex structure and other hybrid forms. After
the addition of 6a, a strong enhancement in the CD intensity at
both 265 and 295 nm, which suggested that 6a could increase both
the parallel and antiparallel structures.
450
500
550
600
Wavelength (nm)
Fig. 2. Fluorescence emission spectra of 20
concentrations of G-quadruplex DNA in NH
kex = 380 nm. (a) 0 M G-quadruplex DNA; (b) 10
M G-quadruplex DNA.
l
M acridone derivatives 6a with various
OAc buffer after excitation at
M G-quadruplex DNA; (c)
4
3.3.4. Mass spectrometry
l
l
Electrospray ionization mass spectrometry (ESI–MS) is a highly
sensitive method to investigate the binding ability of compounds
and DNA [38,39]. Therefore, the binding of compounds to human
20 l
G-quadruplex DNA was analyzed by ESI–MS. In the NH
the ESI–MS spectrum of the DNA revealed two main ions at m/z
262 and 1514 confirming the formation of the quadruplex
Fig. 4). When compound 6a was added to the G-quadruplex
DNA solution, a mixture of 1:1 and 2:1 drug-DNA complexes were
identified. These indicated that the compound 6a was a suitable
binder for human telomeric quadruplex DNA.
4
OAc buffer,
3
3
2
2
1
1
0
0
.5
.0
.5
.0
.5
.0
.5
.0
1
(
c
b
3.3.5. FRET assays of quadruplexbinding
a
The ability of compound 6a to stabilize human telomeric
quadruplex DNA was also evaluated by fluorescence resonance
-
-
0.5
1.0
Table 3
2
40
260
280
300
320
340
Thermal stabilization of G-quadruplex DNA for compound 6a at different
concentrations.
Wavelength (nm)
6
D
a (
l
(°C)
M)
1.0
10
1.4
14
1.6
23
2
28
Fig. 3. CD spectra of 10
l
M (TTAGGG)
4
with various concentrations of 6a in
M 6a.
T
m
NH OAc buffer: (a) 0 M 6a; (b) 20
4
l
lM 6a; (c) 40 l
Fig. 4. (a) ESI MS spectra of 25
lM G-quadruplex DNA and (b) compounds 6a with G-quadruplex DNA in a 1:1 M ratio in NH
4
OAc buffer.

3
6
C. Gao et al. / Bioorganic Chemistry 60 (2015) 30–36
energy transfer (FRET) methods. The data in Table 3 showed that
compound 6a had great ability to stabilize G-quadruplex DNA. As
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a had the ability to be developed as potent G-quadruplex DNA
stabilizers.
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antiproliferation activity and little toxic activity against normal
cells, which might be associated with its stabilization of G-quadru-
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The authors would like to thank the financial supports from the
Ministry of Science and Technology of China (2012AA020305,
[
[
[
2
013AA092902), the Chinese National Natural Science Foundation
(
(
21272134 and 21372141), and Shenzhen Sci& Tech Bureau
ZDSY20120619141412872 and JCYJ20120831165730905).
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Appendix A. Supplementary material
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8
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