Synthesis and evaluation of N-Benzyl-acridinone derivatives induced apoptosis in human liver cancer cell-lines

Article abstract of DOI:10.2174/157018011796235176

A series of N-benzyl-9(10H)-acridinones were synthesized and tested for their antitumor activities in vitro against HepG2 cells Assay-based antiproliferative activity study using HepG2 cell lines revealed that several compounds had significant effects on cytotoxicity, among which compound 5h was found to be the most active compound with IC50 at about 1.33 μM using the MTT assay. The antitumor effect of compound 5h is believed to be due to the induction of apoptosis, which was further confirmed by Hoechst 33258 fluorescence staining, agarose gel electrophoresis and Annexin VFITC/ PI staining assay using flow cytometry analysis. Above all, compound 5h would be a potential anticancer agent which deserves further research.

Full text of DOI:10.2174/157018011796235176

606
Letters in Drug Design & Discovery, 2011, 8, 606-611
Synthesis and Evaluation of N-Benzyl-Acridinone Derivatives Induced
Apoptosis in Human Liver Cancer Cell-Lines
Xian-Feng Huang*,a, Yulan-Zhua and Hai-Liang Zhu*,b
aSchool of Pharmaceutical Engineering & Life Science, Changzhou University, Changzhou, 213164, P.R. China
bState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, P.R. China
Received February 14, 2011: Revised May 13, 2011: Accepted May 16, 2011
Abstract: A series of N-benzyl-9(10H)-acridinones were synthesized and tested for their antitumor activities in vitro
against HepG2 cellsꢀꢁAssay-based antiproliferative activity study using HepG2 cell lines revealed that several compounds
had significant effects on cytotoxicity,ꢁamong which compound 5h was found to be the most active compound with IC50 at
about 1.33 μM using the MTT assay. The antitumor effect of compound 5h is believed to be due to the induction of apop-
tosis, which was further confirmed by Hoechst 33258 fluorescence staining, agarose gel electrophoresis and Annexin V-
FITC/PI staining assay using flow cytometry analysis. Above all, compound 5h would be a potential anticancer agent
which deserves further research.
Keywords: Acridinone, Antiproliferative activity, Apoptosis, HepG2 cell.
1. INTRODUCTION
In this paper, we describe the synthesis, SAR, and anti-
tumor effects of a series of 10-benzyl-9(10H)-acridinone
derivatives including some new compounds and reported
ones on one human liver carcinoma cell line (Hep G2) in
vitro. The activity has also been confirmed by Hoechst
33258 fluorescence staining, agarose gel electrophoresis and
Annexin V-FITC/PI staining assay using flow cytometry
analysis.
Hepatocellular carcinoma (HCC) is one of the most
common cancers worldwide, with a particularly high preva-
lence in Asian countries, due to endemic hepatitis B virus
(HBV) infection [1-4]. HCC is a rapidly fatal disease and
HCC patients have caused cirrhosis and impaired liver func-
tion, making treatment of HCC more difficult than many
other cancers [5, 6]. The survival of cancer cells depends on
their ability to adapt changes in their microenvironment and
to escape from the growth-inhibitory effects of neighboring
normal cells and to resist apoptosis and growth-inhibitory
signals, leading to tissue invasion and metastasis. The regu-
lation of apoptosis is crucial for development and sustained
health [7-9]. The dysregulation of apoptosis will result in a
variety of clinical disorders including cancer. Most che-
motherapeutic agents used in cancer therapy kill particular
types of tumor cells through apoptosis and do not damage
organism. Therefore, anticancer drugs killing tumor cells
through apoptosis have been widely studied in medicinal
chemistry [10-13].
2. CHEMISTRY
Acridone derivatives 5a-5j variously functionalized at
the C-ring were synthesized as reported [25] in Scheme 1.
We first synthesized N-(carboxyphenyl) anthranilic acid (3)
on a preparative scale, using the method of Jourdan–Ulmann
copper-catalyzed condensation of o-chlorobenzoic acid and
4-chloroaniline. The reactants were heated preferably at re-
flux in the presence of copper powder and K2CO3 in DMF
medium. The N-(carboxyphenyl) anthranilic acids were cy-
o
clized with polyphosphoric acid at 120 C to form variously
functionalized acridones (4). Following this methodology,
we prepared a series of N-benzyl-9(10H)-acridinones. Then
target compounds were obtained by N10-alkylation by corre-
sponding benzyl chloride and K2CO3 in DMF. All com-
Acridone derivatives have been known for many years as
antimicrobial agents [14]. The planar, heterocyclic and con-
siderably hydrophobic nature of acridone, making it to inter-
act with several biomolecular targets, led to the investiga-
tions of a number of acridone derivatives for their anti-tumor
[15-18], anti-protozoan [19-21] and anti-viral [22] proper-
ties. Recently, several research groups have reported a series
of 10-benzyl-9(10H)-acridinones and their in vitro antitumor
activities against various cell lines [23, 24].
1
pounds were fully characterized by H NMR, ESI MS and
elemental analysis. The structure of compound 5d was also
established by X-ray crystallographic analysis (Fig. 1). Of all
the compounds, 5d, 5f, 5h and 5j were new compounds and
others have been reported [26, 27].
3. CYTOTOXICITY MEASUREMENT
In vitro cytotoxic activities of these N-Substituted acri-
dones were studied on a panel of one human liver carcinoma
cell line (Hep G2). All of the compounds displayed potent
cytotoxic activities against Hep G2. The cytotoxic activity of
the 10 compounds in Hep G2 was closely associated with
their structures, as shown in Table 1 and expressed as the
half maximal inhibitory concentration (IC50).
*Address correspondence to these authors at the School of Pharmaceutical
Engineering & Life Science, Changzhou University, Changzhou, 213164, P.
R. China; Tel: +86-519-8633 0167; Fax: +86-25-8633 0167;
E-mail: xianfenghh@163.com
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, P. R. China; Tel: +86-25-8359 2572; Fax: +86-25-8359
2672; E-mail: zhuhl@nju.edu.cn
1570-1808/11 $58.00+.00
© 2011 Bentham Science Publishers Ltd.

Synthesis and Evaluation of N-Benzyl-Acridinone Derivatives
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 7 607
O
COOH
COOH
b
a
+
R1
R1
R1
Cl H2N
N
N
H3
H4
2
1
O
N
5
c
R1
Cl
R2
R2
Scheme 1. Synthesis of compounds 5a–5j. Reagents and conditions: (a) K2CO3, Cu, DMF, rt, 6 h; (b) PPA, 120 oC, 3 h; (c) K2CO3, DMF,
90-100 oC, 3-6 h.
Out of the 10 compounds, 5 compounds exhibited good
activity with IC50 values of <6.0 μM, among which 3 com-
pounds (5d, 5g, 5h) demonstrated IC50 values of 1.0-3.0 μM,
compound 5h exhibited the most potential inhibitory activity
in tumor growth inhibition (IC50=1.33 μM), being better than
that of 5-Fu.
acridone ring demonstrated that a 4-substituted group (5c,
5d, 5g, 5h) may have more significant improved antiprolif-
erative activity than a 2-substituted group (5e, 5f, 5i, 5j), and
it showed the most potent inhibitory activity when the 4 po-
sition on the acridone was substituted by methoxy group.
This result may be explained because of the dihedral angle
between acridone ring and N-phenyl ring resulting from
steric hindrance of 4-substituted group, and the different
electronic effect. In addition, this trend was also observed by
comparison of the two most potent compounds 5g and 5h;
the latter with a 3ꢀ-mothoxy group showed a better activity.
However, it remains to be further investigated how the
change of the substitute on the N-phenyl ring influences the
cytotoxic activity. In summary, the 4-substituted was found
to play a very important role in the cytotoxic potency of
these acridone derivatives.
Table 1. Structures and Cytotoxicity of Compounds 5a-5j
Against Hep-G2
R1
R2
IC50 (μM)
5a
5b
5c
H
H
OCH3
H
19.15±1.13
6.01±0.35
5.92±0.21
2.85±0.11
21.71±1.24
11.19±0.98
2.58±0.15
1.33±0.13
17.20±1.7
12.15±0.61
1.52±0.14
H
4-CH3
4-CH3
2-CH3
2-CH3
4-OCH3
4-OCH3
2-OCH3
2-OCH3
5d
5e
OCH3
H
5f
OCH3
H
5g
5h
5i
Fig. (1). Crystal structure of the compound 5d.
OCH3
H
Structure-activity relationships of these acridones dem-
onstrated that the electronic effect on the N-phenyl ring has
some influence on the cytotoxic potency. For example, these
derivatives (5b, 5d, 5f, 5h, 5j) with methoxy group on the
ring have lower IC50 values than those corresponding deriva-
tives (5a, 5c, 5e, 5g, 5i). These results are according to that
reported by Gao and co-workers, whose studies revealed that
compounds, which have more methoxy group on N-phenyl
ring showed better activity against CCRF-CEM cells, and
the compound with dimethoxy groups at C3 and C5 posi-
tions on the ring had the lowest IC50 at about 0.71 μM. On
the other hand, a comparison of the substitute position on
5j
OCH3
5-Fu
IC50 = 50% inhibitory concentration represents the mean ± S. D. from dose-response
curves of at least three experiments.
4. THE INDUCTION OF APOPTOSIS IN HEP G2
CELLS BY COMPOUND 5H
In order to identify whether these acridone derivatives
induced cell death in apoptotic mode, we observed com-

608 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 7
Huang et al.
a
b
Fig. (2). Fluorescent microscopic analysis of apoptotic cells. Cells stained with DAPI after treated with compound 5h for 24 h. (a) control,
(b) 2.50 μM.
pound 5h-treated Hep G2 cells by staining with DAPI. The
apoptotic nuclei were filled with condensed or fragmented
chromatin, which showed enhanced fluorescence with DAPI
staining. Distinguished apoptosis was seen in the cells
treated with compound 5hcompared with the control group
(Fig. 2).
shifts (ꢀ) for 1H NMR spectra were reported in parts per
million to residual solvent protons. Melting points were
measured on a Boetius micro melting point apparatus. The
ESI-MS spectra were recorded on a Mariner System 5304
Mass spectrometer. All chemicals and reagents used in cur-
rent study were of analytical grade. TLC was running on the
silica gel coated aluminum sheets (Silica Gel 60 GF254, E.
Merk, Germany) and visualized in UV light (254 nm).
To further confirm that compound 5h leads to apoptosis,
Hep G2 cells were stained with annexin V-FITC and propid-
ium iodide, and subsequently analyzed by flow cytometry.
As shown in Fig. (3), a significant induction of apoptosis
was shown by compound 5h treatment. Compared to the
control, cells treated with different concentration of com-
pound 5h for 24 h had an increase in the percentage of early
apoptotic cells (Annexin V positive but PI negative) from
0.01% to 27.38%, and of late apoptotic cells (Annexin V and
PI double-positive cells) from 0.20% to 16.88%. It is obvi-
ous, that as the concentration increased, the apoptosis rate
gradient grew.
6.2. General Procedure for Compounds 5a-5j Prepara-
tions
To a mixture of o-chlorobenzoic acid (0.064 mol), corre-
sponding aniline (0.064 mol), and copper powder (0.2 g) in
60 mL of DMF, dry K2CO3 (10 g) was slowly added and the
contents were refluxed for 5 h. The mixture was poured into
1 L of hot water, and acidified. Precipitate formed was fil-
tered, washed with hot water and collected. The crude acid
was dissolved in aqueous sodium hydroxide solution, boiled
in the presence of activated charcoal, and filtered. On acidi-
fication, light yellowish precipitate was obtained which was
washed with hot water and recrystallized from aqueous
methanol to give a light yellow solid 3 (yield 72-86 %).
5. CONCLUSION
A series of acridones were synthesized and their antipro-
liferative activities against the human liver cancer cell line
Hep G2 were evaluated. Some compounds displayed good
cytotoxic activities and the SARs have also been studied.
The consequences demonstrated that substitution at the 4
position of the acridone ring plays a very important role in
the activity against Hep G2 cells. Among the compounds
tested, compound 5h exhibited the most potent activity in
tumor growth inhibition (IC50=1.5 μM). Through DAPI
staining and Annexin V-FITC and PI staining for apoptosis,
we have found the compound 6 induced cell death in apop-
totic mode. Above all, compound 5h would be a potential
anticancer agent which deserves further research. More in-
formation regarding SAR and the mechanism of apoptosis
induction are currently under way in our laboratory.
3 (5 g) was taken in a flask to which was added 52 g of
polyphosphoric acid. The reaction mixture was heated on
oil-bath at 120 oC for 3 h with stirring. Appearance of yellow
color indicated the completion of the reaction. Then, it was
poured into 1 L of hot water, and made alkaline by liquor
ammonia. The yellow precipitate formed was filtered,
washed with hot water and collected. The sample of 4 was
recrystallized from acetic acid (yield 71-85%).
Compound 4 (1 mmol), proportional benzyl chloride, and
K2CO3 in 50 ml of dry DMF were stirred at 90–100 oC for 3–
6 h. After the completion of reaction, the solution was added
to a mixture of ice and H2O, and the yellow solution was
extracted with three 10-ml portions of AcOEt. The combined
organic layers were washed with H2O, dried (Na2SO4), fil-
tered, evaporated, and recrystallized from AcOEt.
6. EXPERIMENTAL PROTOCOLS
6.1. Chemistry
6.2.1. 10-benzylacridin-9(10H)-one (5a)
o
1
All the NMR spectra were recorded on a Bruker DRX
500 or DPX 300 model Spectrometer in CDCl3. Chemical
Yellow powder, yield: 71%. Mp: 180-181 C. H NMR
(500 MHz, CDCl3, ꢀ ppm): 4.50 (s, 2H), 7.17 (d, 1H, J = 7.5

Synthesis and Evaluation of N-Benzyl-Acridinone Derivatives
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 7 609
Fig. (3). Induction of apoptosis by compound 5h. Flow cytometric analysis of annexin V-FITC and propidium iodide double stained cells.
Cells were untreated or treated with different concentration of compound 5h for 24 h. Data represent the percentage of aopototic cells. (a)
control, (b) 1.0 μM , (c) 2.0 μM , (d) 4.0 μM.
Hz), 7.23 (m, 1H), 7.31-7.44 (m, 8H), 7.65 (d, 1H, J = 8.5
Hz), 7.76 (t, 1H, J = 7.5 Hz), 8.41 (m, 1H). MS (ESI): 286.9
([M+H]+). Anal. Calcd for C20H15NO: C, 84.19; H, 5.30; N,
4.91. Found: C, 84.56; H, 5.31; N, 4.93.
1H, J = 8.8 Hz), 7.64 (m, 2H), 8.20 (m, 2H). MS (ESI):
300.3 ([M+H]+). Anal. Calcd for C21H17NO: C, 84.25; H,
5.72; N, 4.68. Found: C, 84.58; H, 5.71; N, 4.63.
6.2.4. 10-(3-methoxybenzyl)-4-methylacridin-9(10H)-one
(5d)
6.2.2. 10-(3-methoxybenzyl)acridin-9(10H)-one (5b)
o
1
Yellow powder, yield: 73%. Mp: 211-212 C. H NMR
(300 MHz, CDCl3, ꢀ ppm): 3.70 (s, 3H), 5.78 (s, 2H), 6.64
(d, 1H, J = 7.5 Hz), 6.81 (s, 1H), 6.85 (d, 1H, J = 8.0 Hz),
7.22 (t, 1H, J = 8.0 Hz), 7.35 (t, 2H, J = 7.5 Hz), 7.64 (d, 2H,
J = 8.5 Hz), 7.77 (m, 2H), 8.38 (m, 2H). MS (ESI): 316.1
([M+H]+). Anal. Calcd for C21H17NO2: C, 79.98; H, 5.43; N,
4.44. Found: C, 79.46; H, 5.41; N, 4.40.
Colorless crystal (petroleum ether/EtOAc, CCDC num-
o
1
ber: 804502), yield: 64%. Mp: 119-120 C. H NMR (300
MHz, CDCl3, ꢀ ppm): 2.61 (s, 3H), 3.74 (s, 2H), 5.61 (s,
2H), 6.59 (d, 1H, J = 8.0 Hz), 6.62 (s, 1H), 6.78 (m, 1H),
7.17 (t, 1H, J = 7.5 Hz), 7.21-7.29 (m, 2H),7.58 (d, 1H, J =
8.8 Hz), 7.64 (m, 2H), 8.20 (m, 2H). MS (ESI): 330.8
([M+H]+). Anal. Calcd for C22H19NO2: C, 80.22; H, 5.81; N,
4.25. Found: C, 80.53; H, 5.85; N, 4.27.
6.2.3. 10-benzyl-4-methylacridin-9(10H)-one (5c)
6.2.5. 10-benzyl-2-methylacridin-9(10H)-one (5e)
o
1
Yellow powder, yield: 61%. Mp: 125-126 C. H NMR
(500 MHz, CDCl3, ꢀ ppm): 2.61 (s, 3H), 5.65 (s, 2H), 7.03
(d, 2H, J = 7.5 Hz), 7.19 (m, 1H), 7.22-7.31 (m, 4H), 7.57 (d,
o
1
Yellow powder, yield: 85%. Mp: 163-164 C. H NMR
(500 MHz, CDCl3, ꢀ ppm): 2.46 (s, 3H), 5.59 (s, 2H), 7.20

610 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 7
Huang et al.
(d, 2H, J = 7.0 Hz), 7.25-7.37 (m, 6H), 7.45 (dd, 1H, J = 2.0
Hz, 8.8 Hz), 7.62 (m, 1H), 8.39 (d, 1H, J = 2.0 Hz), 8.61 (m,
1H). MS (ESI): 300.1 ([M+H]+). Anal. Calcd for C21H17NO:
C, 84.25; H, 5.72; N, 4.68. Found: C, 84.71; H, 5.76; N,
4.71.
h, cell survival was determined by the addition of MTT solu-
tion (10μL of 5 mg/mL MTT in PBS). After 4 h, 100 μL of
10% SDS (Sigma) in 0.01 N HCl was added, and the plates
o
were incubated at 37 C for a further 18 h; optical absor-
bance was measured at 570 nm on LX300 Epson Diagnostic
microplate reader. Survival ratios are expressed in percent-
ages with respect to untreated cells. IC50 values were deter-
mined from replicates of 6 wells from at least two independ-
ent experiments.
6.2.6. 10-(3-methoxybenzyl)-2-methylacridin-9(10H)-one
(5f)
o
1
Yellow powder, yield: 80%. Mp: 156-158 C. H NMR
(300 MHz, CDCl3, ꢀ ppm): 2.45 (s, 3H), 3.72 (s, 3H), 5.52
(s, 2H), 6.73–6.88 (m, 4H), 7.23–7.35 (m, 5H), 7.61 (m, 1H),
8.32 (m, 1H). MS (ESI): 330.0 ([M+H]+). Anal. Calcd for
C22H19NO2: C, 80.22; H, 5.81; N, 4.25. Found: C, 80.46; H,
5.85; N, 4.20.
6.4. DAPI Staining Assay
Hep G2 cells were seeded in 6-well plates at a seeding
density of 105 cells/ml. When all the cells were adhered,
compound 5h was added. After 24 h incubation, the cells
were fixed with 4% paraformaldehyde for 20 minutes and
permeabilized by incubation in 0.1% sodium citrate contain-
ing 0.1% Triton® X-100 (Sigma) for 2 minutes at 4˚C, then
labeled with DAPI (Sigma, 300 nM). After labeling, the
apoptotic cells were visualized using a Nikon E800 (Nikon,
Japan) microscope. The cells were considered to be apop-
totic when they showed either fragmented or condensed nu-
clei. The apoptotic cells were defined morphologically by
cytoplasmic and nuclear shrinkage and chromatin condensa-
tion or fragmentation [28].
6.2.7. 10-benzyl-4-methoxyacridin-9(10H)-one (5g)
o
1
Amorphous crystal, yield: 63%. Mp: 145-146 C. H
NMR (300 MHz, CDCl3, ꢀ ppm): 3.63 (s, 3H), 5.76 (s, 2H),
7.16 (m, 2H), 7.17 (m, 1H), 7.20-7.29 (m, 4H), 7.32 (m, 1H),
7.51 (d, 1H, J = 8.0 Hz), 7.68 (m, 1H), 7.96 (m, 1H), 8.28
(m, 1H). MS (ESI): 316.1 ([M+H]+). Anal. Calcd for
C21H17NO2: C, 79.98; H, 5.43; N, 4.44. Found: 79.68; H,
5.45; N, 4.49.
6.2.8. 10-(3-methoxybenzyl)-4-methoxyacridin-9(10H)-one
(5h)
o
1
Amorphous crystal, yield: 61%. Mp: 139-141 C. H
NMR (300 MHz, CDCl3, ꢀ ppm): 3.59 (s, 3H), 3.81 (s, 3H),
5.61 (s, 2H), 6.80-6.95 (m, 3H), 7.17 (m, 1H), 7.21-7.29 (m,
3H), 7.37 (m, 1H), 7.57 (m, 1H), 8.19 (m, 1H), 8.50 (m, 1H).
MS (ESI): 346.5 ([M+H]+). Anal. Calcd for C22H19NO3: C,
76.50; H, 5.54; N, 4.06. Found: C, 76.18; H, 5.53; N, 4.10.
6.5. Annexin-V/PI Double-Staining Assay
The Annexin V- FITC binding assay was performed by
an Annexin V-FITC apoptosis detection kit (Nanjing Key-
Gen Biotech. Co. Ltd.) according to the manufacturer’s pro-
tocol. The cells were seeded and treated with compound 5h
as for the DAPI staining assay. After 24 h incubation, the
cells were trypsinized, washed in PBS and resuspended in
binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM
NaCl, 2.5 mM CaCl2). The cells were incubated with An-
nexin V-FITC (100 ng/ml) in the dark for 10 min and 10 ml
propidium iodide (PI) was added to each group before flow
cytometric analysis. Positive Annexin V staining indicated
apoptosis, while positive PI indicated necrosis. For each
group, a minimum of 10,000 cells were counted. Data analy-
sis was performed with standard Cell Quest software [29].
(FACSCA2BUR, Becton Dickinson, USA)
6.2.9. 10-benzyl-2-methoxyacridin-9(10H)-one (5i)
o
1
Yellow powder, yield: 83%. Mp: 210-212 C. H NMR
(300 MHz, CDCl3, ꢀ ppm): 3.90 (s, 3H), 5.52 (s, 2H), 7.20-
7.29 (m, 4H), 7.32-7.59 (m, 6H), 7.96 (m, 1H), 8.49 (m, 1H).
MS (ESI): 316.0 ([M+H]+). Anal. Calcd for C21H17NO2: C,
79.98; H, 5.43; N, 4.44. Found: C, 79.63; H, 5.49; N, 4.41.
6.2.10.
10-(3-methoxybenzyl)-2-methoxyacridin-9(10H)-
one (5j)
o
1
Yellow powder, yield: 74%. Mp: 234-255 C. H NMR
(300 MHz, CDCl3, ꢀ ppm): 3.75 (s, 3H), 3.92 (s, 3H), 5.51
(s, 2H), 6.73-6.89 (m, 4H), 7.17–7.26 (m, 4H), 7.62 (m, 1H),
7.96 (m, 1H), 8.49 (m, 1H). MS (ESI): 346.3 ([M+H]+).
Anal. Calcd for C22H19NO3: C, 76.50; H, 5.54; N, 4.06.
Found: C, 76.14; H, 5.58; N, 4.09.
ACKNOWLEDGEMENTS
The work was supported by the Doctoral Fund (Projectꢀ
ZMF 08020066) of Changzhou University.
6.3. Cytotoxicity Study on Hep G2 Cells
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