Synthesis and potent antileukemic activities of 10-benzyl-9(10H)-acridinones

Article abstract of DOI:10.1016/j.bmc.2008.07.086

A novel series of 10-benzyl-9(10H)-acridinones and 1-benzyl-4-piperidones were synthesized and tested for their in vitro antitumor activities against CCRF-CEM cells. Assay-based antiproliferative activity study using CCRF-CEM cell lines revealed that the acridone group and the substitution pattern on the benzene unit had significant effect on cytotoxicity of this series of compounds, among which 10-(3,5-dimethoxy)benzyl-9(10H)-acridinone (3b) was found to be the most active compound with IC₅₀ at about 0.7 μM. Compound 3b was also found to have antiproliferative activity against two other human leukemic cell lines K562 and HL60 using the MTT assay. The antitumor effect of 3b is believed to be due to the induction of apoptosis, which is further confirmed by PI (Propidium iodide) staining and Annexin V-FITC/PI staining assay using flow cytometry analysis.

Full text of DOI:10.1016/j.bmc.2008.07.086

Bioorganic & Medicinal Chemistry 16 (2008) 8670–8675
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and potent antileukemic activities of 10-benzyl-9(10H)-acridinones
a
a
a
c,
Chunmei Gao a, Yuyang Jiang a,b, , Chunyan Tan , Xuyu Zu , Huachen Liu , Derong Cao
*
*
a The Key Laboratory of Chemical Biology, Guangdong Province, Graduate School at Shenzhen, Tsinghua University, Lishui Road, Shenzhen 518055, PR China
b School of Medicine, Tsinghua University, Beijing 100084, PR China
c College of Chemistry, South China University of Technology, Guangzhou 510640, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 10-benzyl-9(10H)-acridinones and 1-benzyl-4-piperidones were synthesized and tested
for their in vitro antitumor activities against CCRF-CEM cells. Assay-based antiproliferative activity study
using CCRF-CEM cell lines revealed that the acridone group and the substitution pattern on the benzene
unit had significant effect on cytotoxicity of this series of compounds, among which 10-(3,5-dime-
thoxy)benzyl-9(10H)-acridinone (3b) was found to be the most active compound with IC50 at about
Received 3 July 2008
Revised 29 July 2008
Accepted 30 July 2008
Available online 5 August 2008
0.7 lM. Compound 3b was also found to have antiproliferative activity against two other human leuke-
Keywords:
mic cell lines K562 and HL60 using the MTT assay. The antitumor effect of 3b is believed to be due to the
induction of apoptosis, which is further confirmed by PI (Propidium iodide) staining and Annexin V-FITC/
PI staining assay using flow cytometry analysis.
Acridinone derivatives
Antileukemia agent
Antiproliferative activity
Apoptosis
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
this paper, we describe the synthesis, SAR, and antileukemia effects
of 10-benzyl-9(10H)-acridinone and its derivatives in vitro.
Cancer is now believed to result from unlimited growth of a gi-
ven cell.1,2 Cells die in two ways: necrosis and apoptosis. The reg-
ulation of apoptosis is crucial for development and sustained
health.3–5 The dysregulation of apoptosis will result in a variety
of clinical disorders including cancer. Most chemotherapeutic
agents used in cancer therapy kill particular types of tumor cells
through apoptosis. Because cells killed by apoptosis do not damage
the organism, anticancer drugs, which could kill tumour cells
through apoptosis, have been widely studied in medicinal Chemis-
try.6–9
Acridones are naturally occurring alkaloids, which can be con-
sidered as aza-analogs of anthrones10–12 or xanthones.13 Acridone
derivatives show various interesting biological properties,14–26
which have been studied as antiviral, antiallergy drugs, antitumour
agents, etc. Now, many acridone derivatives with potent antitumor
activity in vitro/vivo against a range of murine and human tumors
have been developed.19,20,24,25 In our search for potent and selec-
tive antitumor agents, a novel series of 10-benzyl-9(10H)-acridi-
none and 1-benzyl-4-piperidone with substituent(s) at the
benzene ring were synthesized for structure-activity relationship
(SAR) study. The 10-(3,5-dimethoxy)benzyl-9(10H)-acridinone
(3b) was found to be the most active compound against human T
cell lymphoblast-like cell (CCRF-CEM) growth. We also proved that
compound 3b treatment increases apoptosis in CCRF-CEM cells. In
2. Results and discussion
2.1. Chemistry
Scheme 1 shows the preparation of the 10-benzyl-9(10H)-
acridinone and its derivatives. Starting from the commercially
available material acridone 1 and NaH, the N-benzylated deriva-
tives of acridone were obtained in high yields by reaction of acri-
done with the appropriate benzyl chlorides in the presence of
catalytic amounts of potassium iodide in N,N-dimethylformamide.
Most benzyl chlorides used in this study were commercially avail-
able, while 2c and 2f were obtained according to the literature,27,28
R1
R2
R1
O
NaH
+
R3
CH2Cl
R2
R3
N
N
H
O
1
2a-l
3a-l
* Corresponding authors. Tel./fax: +86 755 2603 2094 (Y. Jiang).
E-mail addresses: jiangyy@sz.tsinghua.edu.cn (Y. Jiang), drcao@scut.edu.cn
(D. Cao).
Scheme 1. Preparation of the 10-benzyl-9(10H)-acridinones derivatives 3a–l.
Reagents and conditions: NaH, DMF, KI, rt, overnight.
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.07.086

C. Gao et al. / Bioorg. Med. Chem. 16 (2008) 8670–8675
8671
Table 1
and 2g–2i were prepared from benzyl alcohols synthesized by the
reaction of 3,5-dihydroxybenzylalcohol with the corresponding
alkyl bromides.
The synthesis of 1-benzyl-4-piperidone and its derivatives 5a–c
were accomplished via the routes illustrated in Scheme 2. The
N-alkylation was developed by reaction of the hydrochloric salt
of 4-piperidone 4 with substituted benzyl chlorides 2a–c in dichlo-
romethane in the presence of triethylamine under reflux condi-
tions overnight.29,30 after workup, products 5a–c were obtained
in moderate yields.
Chemical data and antiproliferative activity against CCRF-CEM cells of compounds 1
and 3a–l
a
R1
R2
R3
Yield (%)
IC50
(l
M)
1
—
H
OCH3
OCH3
OCH3
H
—
H
H
OCH3
H
OCH3
H
H
H
H
—
H
OCH3
OCH3
H
H
OBn
OCH2 CH3
O(CH2)2CH3
O(CH2)3 CH3
H
H
H
—
>100
52
3a
3b
3c
3d
3e
3f
3g
3h
3i
90
91
89
91
90
87
88
85
95
90
86
85
0.7
1.5
>100
>100
>100
6.7
39.1
>100
8.8
OBn
OCH2 CH3
O(CH2)2CH3
O(CH2)3 CH3
CH3
F
CF3
The structures of all compounds were confirmed by NMR and
mass spectral data. All the structure features and synthetic yields
are listed in Tables 1 and 2.
3j
3k
3l
H
H
H
62
36
2.2. In vitro cell growth inhibition assay
a
IC50 values were determined from MTT proliferation assays after incubation
The compounds 9(10H)-acridinone 1 and 10-benzyl-9(10H)-
acridinone derivatives 3a–l were initially evaluated for
antiproliferative activity against CCRF-CEM leukemia cells. Cell
proliferation was determined using the MTT [3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide] assay after 48 h of
treatment. As shown in Table 1, the compound 1 showed no cyto-
with test compound for 48 h.
Table 2
Chemical data and antiproliferative activity against CCRF-CEM cells of compound
5a–c
a
toxicity (IC50 > 100 lM), while some of the compounds 3 showed
R1
R2
R3
Yield (%)
IC50
(l
M)
minor to good inhibitory effects on CCRF-CEM cell growth. The ob-
served antiproliferative activities were obviously dependent on the
substitution pattern of the benzene part. In this series, compound
3a with no substituents on the benzene ring exhibited moderate
5a
5b
5c
H
OCH3
OCH3
H
H
OCH3
H
OCH3
OCH3
60
65
62
>100
>100
>100
a
IC50 values were determined from MTT proliferation assays after incubation
inhibitory effects (IC50 = 52 lM). Compounds, which only have
with test compound for 48 h.
one methoxy group on either C3 or C4 position (3d and 3e),
showed no activity, while those compounds with two or three
methoxy groups, such as 3b and 3c, showed much stronger inhibi-
tion effect. So the amount and position of substituents –OMe did
greatly affect the antiproliferative potency. By changing the alkyl
chain length of the dialkoxyl substituents on C3 and C5 positions
from methyl to butyl, compounds showed decreasing inhibitory ef-
fect. Specifically, 3g which has diethoxy groups was determined to
be 9-fold less potent than 3b, 3h with dipropoxy substituents was
determined to be 56-fold less potent than 3b, and 3i with dibutoxy
groups was essentially inactive. This comparison clearly demon-
strates that the cytotoxicity of 10-(3,5-dialkyloxy)benzyl-9(10H)-
acridinone decreases with increasing length and size of the alkyl
group.
In contrast to compound 3b, with the introduction of –OBn to
the C3 and C5 positions, 3f did not display any antiproliferative
activity against CCRF-CEM cells. Compounds 3d with electron-
donating methoxy group at 3-position on the benzene ring had
no cytotoxicity against CCRF-CEM cells. When the weaker elec-
tron-donating group methyl was introduced to the benzene ring,
than 3l. The results indicated that electron-negativity and steric ef-
fect in the benzene ring may change the cytotoxic profile.
Among the twelve compounds, compound 3b with dimethoxy
groups at C3 and C5 positions on the benzene ring had the lowest
IC50 at about 0.7
activity.
lM, which displayed the most potent inhibition
Not only the substitution pattern of the benzene part but also
the acridone group played significant influence on the antitumor
activity of these classes of compounds. In order to study the role
of the acridone group on the 10-benzyl-9(10H)-acridinones, com-
pounds 5a-c bearing a 4-piperidone group instead of the acridone
group were also prepared (Scheme 2). The inhibition of CCRF-CEM
leukemia cells growth was also studied (Table 2). Unfortunately, all
these three compounds were essentially inactive, indicating that
the acridone group is a necessary structure unit in affecting the
cytotoxicity of the 10-benzyl-9(10H)-acridinone derivatives. In
spite of these factors, the acridone itself was inactive toward
CCRF-CEM leukemia cells. These findings, combined with the
results above, demonstrated that both the substituted benzyl
group and the acridone group affected the cytotoxicity of 10-ben-
zyl-9(10H)-acridinones.
compound 3j produced high activity (IC50, 8.8 lM). Introduction
of electron-withdrawing group at the benzene ring in the case of
3k (3-fluoro) and 3l (3-trifluoromethyl) resulted in reduced anti-
proliferative activity compared with 3j. Compound 3l was about
4-fold less potent than 3j, while 3k was about 2-fold less potent
Two other leukemia cells K562 (human chronic myelogenous
leukemia cell) and HL60 (human promyelocytic leukemia cell) cells
were chosen to further investigate the antiproliferative potential
effect of the highly active compounds 10-benzyl-9(10H)-acridi-
none derivatives 3b by cellular metabolic activity using the MTT
assay. Compound 3b displayed good cytotoxocities in K562 and
R1
R2
R1
O
K2CO3
+
R3
CH2Cl
R2
R3
HL60 cells with IC50 values 11.4 lM and 0.6 lM respectively, sug-
gesting that 3b has potential antileukemia effect.
N
N
H
2.3. Compound 3b induces apoptosis in CCRF-CEM cells
O
4
2a-c
5a-c
On the basis of the antiproliferative effect study, compound 3b
was selected for further examinations to determine whether the
cytotoxicity is mediated by the induction of apoptosis. Apoptosis
Scheme 2. Preparation of the 1-benzyl-4-piperidone derivatives 5a–c. Reagents
and conditions: K2CO3, CH2Cl2, N(CH2CH3)3, reflux, overnight.

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C. Gao et al. / Bioorg. Med. Chem. 16 (2008) 8670–8675
is characterized by morphological and biochemical changes in the
nucleus, including chromatin condensation and internucleosomal
DNA fragmentation. Flow cytometry is a useful tool to study the
cell apoptosis. One of the simple methods is to observe the sub-
diploid peak due to the loss of DNA small fragments by propidium
iodide (PI) staining. As shown in Figure 1, the sub-Go cell popula-
tion was observed clearly in a dose-dependent manner after the
treatment with 3b for 24 h. Specifically, cells at sub-Go increased
minary in vitro antileukemia activity test showed that 3b had sig-
nificant growth inhibitory effect against CCRF-CEM, K562 and HL60
cells. Structure–activity relationship studies revealed that both the
3,5-dimethoxy substitution and acridone pattern in the 10-benzyl-
9(10H)-acridinones played an important role in cellular growth
inhibition. Such activity was proved to be associated with the
induction of apoptosis by the flow cytometry analysis using PI
staining and annexin-V/PI assays. Our results suggest that 3b
may be an attractive lead compound for further development as
a chemotherapeutic agent for leukemia therapy. Further investiga-
tions of the mechanism of apoptosis induction are under way.
as the concentration of 3b increased from 0.5 lM to 2.5 lM (see
Fig. 1).
In addition, the Annexin-V/PI binding assay was used to further
confirm compound 3b induced apoptosis effect. Annexin-V conju-
gated with the fluorochrome FITC serves as a marker for apoptotic
cells because it has a strong binding affinity to phosphatidylserine
(PS), which re-distributes from the inner to the outer layer of the
plasma membrane in apoptotic cells.31–33 As shown in Figure 2,
the dual parametric dot plots show four quadrants and among
them the lower left quadrant represents the viable cell population
(annexin-V negative and PI negative), the upper right represents
apoptotic cells undergoing secondary necrosis at the last stage or
dead cells (annexin-V and PI double positive), and the lower right
represents the early stage apoptotic cell population (annexin-V po-
sitive and PI negative). As the concentration of 3b increased from
4. Experimental
4.1. Chemistry
General methods: Commercial chemicals were used without
further purification unless otherwise indicated. Melting points
are uncorrected. 1H NMR and 13C NMR spectra were obtained in
CDCl3 at 300 MHz or 500 MHz for 1H NMR and 75 MHz or
125 MHz for 13C NMR with tetramethylsilane as the internal stan-
dard, respectively, unless otherwise indicated. Chemical shifts are
expressed in ppm relative to TMS (1H, 0.00 ppm) or residual CDCl3
(
13C, 77.0 ppm). Splitting patterns are indicated as s, singlet; d,
1 lM to 5 lM, the annexin-V positive/PI negative cells increased
from 9.5% to 27.4%, and then decreased back to 21.7%, whereas
the double positive cells increased from 10.0% to 41.6%, suggesting
that more and more apoptotic cells progressed from the early stage
to the late stage resulting in either death or secondary necrosis un-
der the effect of 3b at higher concentrations. This confirms again
that 3b induced the cell apoptosis of CCRF-CEM cells.
doublet; t, triplet; q, quartet signal; m, multiplet. ESI-MS were ob-
tained with Waters Quattro Premier XE Spectrometer. HRMS were
recorded on a QSTAR XL spectrometer and Waters Q-Tof Premier
spectrometer. Column chromatography was performed on silica
gel under pressure.
4.1.1. General procedure for preparation of 3,5-
dialkyloxybenzyl alcohol
3. Conclusion
3, 5-Dihydroxybenzyl alcohol (700 mg, 5.0 mmol), dried potas-
sium carbonate (1.73 g, 12.5 mmol), 18-crown-6 (264 mg,
1.0 mmol), and the appropriate alkyl bromide (10.0 mmol) in dry
In conclusion, we have designed and synthesized a series of 10-
benzyl-9(10H)-acridinones and 1-benzyl-4-piperidones. The preli-
Figure 1. Flow cytometry analysis of cell membrane integrity (PI staining). CCRF-CEM cells were treated with 3b at different concentrations for 24 h. (a) 0.0
0.5 M; (c) 1.0 M; (d) 2.5 M. Peak A is the sub-diploid peak.
lM, Control; (b)
l
l
l

C. Gao et al. / Bioorg. Med. Chem. 16 (2008) 8670–8675
8673
Figure 2. Flow cytometric analysis of phosphatidylserine externalization (annexin V-binding) and cell membrane integrity (PI staining). CCRF-CEM cells were treated with 3b
at 1.0 M, 2.5 M and 5 M for 48 h.
l
l
l
acetone were heated at reflux and stirred vigorously under nitro-
gen for 48 h. The volatile parts were removed under reduced pres-
sure and the residue was treated with 20 mL CH2Cl2 and 20 mL
H2O. The separated water was extracted three times with 20 mL
CH2Cl2. The combined organic phases were dried with Na2SO4
and the products were purified by column chromatography.
4.1.3.2. 10-(3, 5-Dimethoxybenzyl)-9(10H)-acridinone (3b). Yel-
low crystals (314 mg), yield 91%, mp 229–230 °C; 1H NMR
(300 MHz, CDCl3): d 3.72 (s, 6H), 5.51 (s, 2H), 6.35 (m, 2H), 6.39
(m, 1H), 7.26-7.37 (m, 4H), 7.62 (m, 2H), 8.57 (m, 2H); 13C NMR
(75 MHz, CDCl3): d 51.0, 55.4, 99.0, 103.8, 115.2, 121.7, 122.6,
127.7, 134.1, 138.2, 142.6, 161.6, 178.3; HRMS calcd for C22H20NO3
[M+H]+ 346.1443, found 346.1414.
4.1.1.1. 3,5-Diethoxybenzyl alcohol.34
Colorless oil (837 mg),
yield 85%, ESI-MS: [M+H]+ 197.
4.1.3.3. 10-(3,4,5-Trimethoxybenzyl)acrid in-9-one (3c). Yel-
low crystals (334 mg), yield 89%, mp 211–213 °C. 1H NMR
(300 MHz, CDCl3): d 3.71 (s, 6H), 3.84 (s, 3H), 5.52 (s, 2H), 6.40
(s, 2H), 7.28-7.39 (m, 4H), 7.65 (m, 2H), 8.59 (m, 2H); 13C NMR
(75 MHz, CDCl3): d 51.3, 56.2, 60.9, 102.4, 115.3, 121.7, 122.6,
127.7, 131.3, 134.1, 137.4, 142.7, 154.0, 178.3; HRMS calcd for
4.1.1.2. 3,5-Dipropoxybenzyl alcohol.35 Colorless oil (997 mg),
yield 89%, ESI-MS: [M+H]+ 225.
4.1.1.3. 3,5-Dibutoxybenzyl alcohol.36 Colorless oil (1.0 g), yield
80%, ESI-MS: [M+H]+ 253.
C
23H22NO4 [M+H]+ 376.1549, found 376.1521.
4.1.2. General procedure for preparation of 3,5-
dialkyloxybenzyl chloride
4.1.3.4. 10-(3-Methoxybenzyl)-9(10H)-acridinone (3d). Slight
yellow crystals (287 mg), yield 91%, mp 211–212 °C. 1H NMR
(300 MHz, CDCl3): d 3.72 (s, 3H), 5.53 (s, 2H), 6.73–6.84 (m, 3H),
7.23–7.34 (m, 5H), 7.60 (m, 2H), 8.57 (m, 2H); 13C NMR (75 MHz,
CDCl3): d 50.8, 55.2, 111.8, 112.7, 115.2, 117.9, 121.6, 122.5, 127.7,
130.4, 134.1, 137.3, 142.6, 160.3, 178.2; HRMS calcd for C21H18NO2
[M+H]+ 316.1338, found 316.1324.
To a mixture of the appropriate benzyl alcohol (3 mmol) in car-
bon tetrachloride (10 mL) was added triphenylphosphine
(4.0 mmol), and the reaction mixture was heated at reflux under
nitrogen overnight. The volatile parts were removed under reduced
pressure and the residue was purified by column chromatography.
4.1.2.1. 3,5-Diethoxybenzyl chloride (2g). Colorless crystals
(546 mg), yield 85%, mp 48–49 °C; 1H NMR (500 MHz, CDCl3): d
1.40 (t, 6H, 3J = 7.0 Hz), 4.01 (q, 4H, 3J = 7.0 Hz), 4.50 (s, 2H), 6.40
(m, 1H), 6.51 (m, 2H).
4.1.3.5. 10-(4-Methoxybenzyl)-9(10H)-acridinone (3e). Slight
yellow crystals (284 mg), yield 90%, mp 220–221 °C; 1H NMR
(500 MHz, CDCl3): d 3.77 (s, 3H), 5.53 (s, 2H), 6.87 (d, 3J = 8.7 Hz,
2H), 7.12 (m, 2H), 7.29 (m, 2H), 7.36 (d, 3J = 8.7 Hz, 2H), 7.62 (m,
2H), 8.58 (m, 2H); 13C NMR (125 MHz, CDCl3): d 52.2, 57.2, 116.6,
117.1, 123.5, 124.6, 128.8, 129.2, 129.7, 135.9, 144.5, 161.2,
4.1.2.2.
3,5-Diproxybenzyl chloride (2h). Colorless oil
(640 mg), yield 88%; 1H NMR (500 MHz, CDCl3): d 1.03 (t, 6H,
3J = 7.4 Hz), 1.78 (m, 4H), 3.90 (t, 4H, 3J = 6.6 Hz), 4.49 (s, 2H), 6.40
(m, 1H), 6.51 (m, 2H).
180.1; HRMS calcd for
316.1337.
C
21H18NO2 [M+H]+ 316.1338, found
4.1.3.6. 10-(3, 5-Dibenzyloxybenzyl)-9(10H)-acridinone (3f). Slight
yellow crystals (482 mg), yield 97%, mp 170–171 °C. 1H NMR
(500 MHz, CDCl3): d 4.93 (s, 4H), 5.46 (s, 2H), 6.41(m, 2H), 6.55(m,
1H), 7.25–7.31(m, 14H), 7.60(m, 2H), 8.58(m, 2H); 13C NMR
(125 MHz, CDCl3): d 52.9, 72.1, 103.2, 106.9, 117.1, 123.5, 124.6,
129.3, 129.7, 129.9, 130.5, 135.9, 138.4, 140.1, 144.5, 162.7, 180.1;
HRMS calcd for C34H28NO3 [M+H]+ 498.2069, found 498.2069.
4.1.2.3. 3,5-Dibutoxybenzyl chloride (2i). Colorless oil
(648 mg), yield 80%; 1H NMR (500 MHz, CDCl3): d 0.97 (t, 6H,
3J = 7.4 Hz), 1.48 (m, 4H), 1.75 (m, 4H), 3.94 (t, 4H, 3J = 6.5 Hz), 4.50
(s, 2H), 6.40 (m, 1H), 6.51 (m, 2H).
4.1.3. General procedure for preparation of 10-benzyl-9(10H)-
acridinones
A mixture of 1 (195 mg, 1 mmol), NaH (48 mg, 1.2 mmol) in dry
DMF (10 mL) was stirred vigorously under nitrogen for 1 h at room
temperature. The corresponding benzyl chloride 2 (2 mmol) and KI
(33.2 mg, 0.2 mmol) were added. The mixture was stirred over-
night. Water was slowly added with rapid stirring under ice-water
bath. Yellow crystals were obtained after filtration. The crystals
were purified by recrystallization from trichloromethane/ethanol.
4.1.3.7. 10-(3,5-Diethoxybenzyl)-9(10H)-acridinone (3g). Slight
yellow crystals (329 mg), yield 88%, mp 200–201 °C.
1H NMR (500 MHz, CDCl3): d 1.34 (t, 6H, 3J = 7.0 Hz), 3.92 (q, 4H,
3J = 7.0 Hz), 5.49 (s, 2H), 6.32 (m, 2H), 6.37 (m, 1H), 7.29 (m, 2H),
7.36 (m, 2H), 7.63 (m, 2H), 8.58 (m, 2H); 13C NMR (125 MHz,
CDCl3): d 14.6, 51.1, 63.6, 100.1, 104.3, 115.3, 121.6, 122.6, 127.7,
134.0, 138.1, 142.7, 161.0, 178.3; HRMS calcd for C24H24NO3
[M+H]+ 374.1756, found 374.1747.
4.1.3.1. 10-Benzyl-9(10H)-acridinone (3a). Slight yellow crystals
(257 mg), yield 90%, mp 180–181 °C; 1H NMR (300 MHz, CDCl3): d
5.61 (s, 2H), 7.20-7.37(m, 9H), 7.62(m, 2H), 8.60 (m, 2H).
4.1.3.8. 10-(3,5-Dipropoxybenzyl)-9(10H)-acridinone (3h). Slight
yellow crystals (341 mg), yield 85%, mp 217–218 °C.

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C. Gao et al. / Bioorg. Med. Chem. 16 (2008) 8670–8675
1H NMR (500 MHz, CDCl3): d 0.97 (t, 6H, 3J = 7.4 Hz), 1.73 (4,
55.3, 62.0, 99.0, 106.7, 140.7, 160.8, 209.4; HRMS calcd for
C
4H), 3.81(t, 4H, 3J = 6.6 Hz), 5.49 (s, 2H), 6.32 (m, 2H), 6.38 (m,
1H), 7.28 (m, 2H), 7.36 (m, 2H), 7.62 (m, 2H), 8.57 (m, 2H); 13C
NMR (125 MHz, CDCl3): d 10.4, 22.5, 51.2, 69.6, 100.1, 104.2,
115.3, 121.6, 122.6, 127.7, 134.0, 138.0, 142.7, 161.2, 178.3; HRMS
calcd for C26H28NO3 [M+H]+ 402.2069, found 402.2059.
14H20NO3 [M+H]+ 250.1443, found 250.1437.
4.1.4.3. 1-(3,4,5-Trimethoxybenzyl)-4-piperidone (5c). Colorless
crystals (346 mg), yield 62%, mp 102–103 °C; 1H NMR (300 MHz,
CDCl3): d 2.48 (t, 4H, 3J = 6.1 Hz), 2.76 (t, 4H, 3J = 6.1 Hz), 3.55(s,
2H), 3.85(s, 3H), 3.88(s, 6H), 6.60 (s, 2H); 13C NMR (75 MHz,
CDCl3): d 41.3, 53.0, 56.1, 60.9, 62.2, 105.5, 134.1, 153.2, 209.3;
HRMS calcd for C15H22NO4 [M+H]+ 280.1549, found 280.1549.
4.1.3.9. 10-(3,5-Dibutoxybenzyl)-9(10H)-acridinone (3i). Slight
yellow crystals (408 mg), yield 95%, mp 191–192 °C.
1H NMR (500 MHz, CDCl3): d 0.92 (t, 6H, 3J = 7.4 Hz), 1.42 (m,
4H), 1.69 (m, 4H), 3.86 (t, 4H, 3J = 6.5 Hz), 5.50 (s, 2H), 6.32 (m,
2H), 6.38 (m, 1H), 7.30 (m, 2H), 7.37(m, 2H), 7.64 (m, 2H), 8.59
(m, 2H); 13C NMR (125 MHz, CDCl3): d 13.8, 19.2, 31.2, 51.2, 67.8,
100.1, 104.2, 115.3, 121.6, 122.6, 127.7, 134.0, 138.0, 142.7,
161.2, 178.3; HRMS calcd for C28H32NO3 [M+H]+ 430.2382, found
430.2362.
4.2. Bioassay
4.2.1. Cell culture
CCRF-CEM leukemia cells (Human T cell lymphoblast-like cell
line) and HL60 (human promyelocytic leukemia Cell) were pur-
chased from the Chinese Academy of Sciences Cell Bank. The cells
were cultured in RPIM 1640 medium (Cibco), containing 10% fetal
bovine serum (FBS) (Hyclone Laboratories Inc.), 100 U/mL penicil-
lin, and 100 lg/mL streptomycin in a 5% CO2-humidified atmo-
sphere at 37 °C.
4.1.3.10. 10-(3-Methylbenzyl)-9(10H)-acridinone(3j). Slight
yellow crystals (269 mg), yield 90%, mp 196–198 °C.
1H NMR (500 MHz, CDCl3): d 2.30(s, 3H), 5.56 (s, 2H), 6.99 (m,
1H), 7.02 (br s, 1H), 7.12 (m, 1H), 7.23 (m, 1H), 7.29 (m, 2H),
7.35 (m, 2H), 7.62(m, 2H), 8.60 (m, 2H); 13C NMR (125 MHz,
CDCl3): d 21.5, 50.9, 115.3, 121.6, 122.6, 122.7, 126.2, 127.8,
128.6, 129.1, 134.0, 135.5, 139.1, 142.7, 178.2; HRMS calcd for
4.2.2. Materials
3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium
bro-
mide (MTT) and propidium iodide (PI)were purchased from Sigma
Chemical Co. Annexin V-FITC Apoptosis Detection Kit I and FITC-
conjugated antibody were purchased from Beyotime Institute of
Biotechnology.
C
21H18NO [M+H]+ 300.1388, found 300.1373.
4.1.3.11. 10-(3-Fluorobenzyl)-9(10H)-acridinone (3k). Yellow
crystals (261 mg), yield 86%, mp 197–198 °C.
1H NMR (500 MHz, CDCl3): d 5.58 (s, 2H), 6.92 (m, 1H), 7.01 (m,
2H), 7.30–7.34 (m, 5H), 7.63 (m, 2H), 8.60 (m, 2H); 13C NMR
(125 MHz, CDCl3): d 50.4, 112.8, 113.0, 114.8, 114.9, 115.0, 121.3,
121.3, 121.8, 122.7, 128.0, 130.9, 131.0, 134.1, 138.3, 138.4,
142.5, 162.4, 164.5, 178.1; HRMS calcd for C20H15FNO [M+H]+
304.1138, found 304.1145.
4.2.3. Cell viability
The cells were suspended at a concentration of 2 Â 105 cells/mL
and seeded in 96-well microtiter plates. Various concentrations of
compound were added to each well in quintuplet followed by incu-
bation for the indicated times. After treatment, the cells were incu-
bated with MTT (5 mg /mL) for 4 h. The formazan precipitate was
dissolved in 100 lL DMSO, and the absorbance at 490 nm was
4.1.3.12. 10-(3-Trifluoromethylbenzyl)-9(10H)-acridinone
(3l). Yellow crystals (300 mg), yield 85%, mp 200–201 °C.
1H NMR (500 MHz, CDCl3): d 5.64 (s, 2H), 7.26–7.33 (m, 5H),
7.45 (m, 1H), 7.59 (m, 2H), 7.64 (m, 2H), 8.60 (m, 2H); 13C NMR
(125 MHz, CDCl3): d 50.5, 114.8, 121.9, 122.0, 122.6, 122.6, 122.7,
122.8, 124.8, 124.8, 124.9, 124.9, 125.6, 127.9, 128.9, 129.9,
131.1, 131.5, 131.9, 132.3, 134.2, 136.8, 142.3, 178.1; HRMS calcd
for C21H15F3NO [M+H]+ 354.1106, found 354.1118.
measured by a Benchmark microplate reader (Molecular Devices
Corporation). IC50 values are the concentration at which cell
growth was inhibited by 50%.
4.2.4. Flow cytometric analysis of apoptosis
The CCRF-CEM cells were exposed to different concentrations of
compound for 24 h at 37 °C, and then detached, and collected. The
untreated and treated CCRF-CEM cells were washed twice with
phosphate-buffered saline (PBS), fixed with 0.5 mL ice-cold 70%
ethanol, and stored at 4 °C for 1–2 h. DNA content was then mea-
4.1.4. General procedure for the synthesis of 1-benzyl-4-
piperidone
sured after staining with PI solution (100 lg/mL propidium iodide)
To a suspension of 4-piperidone hydrochloride (2 mmol) in
CH2Cl2 (10 mL) were added the appropriate substituted benzyl
chloride (2.2 mmol) and triethylamine (4.4 mmol). The mixture
was heated at reflux overnight, diluted with CH2Cl2 (20 mL), and
then washed with water and brine. The organic layer was sepa-
rated. The separated water was extracted three times with 20 mL
CH2Cl2. The combined organic phases dried (Na2SO4), and evapo-
rated to give a residue, which was purified by column chromatog-
raphy on silica gel using EtOAc/petroleum ether/triethylamine
(50:50:1, v/v/v) as eluent.
for 30 min. Finally, the cells were analyzed with a flow cytometer
using an Epics system (Coulter Epics XL) equipped with an ar-
gon-ion laser operated at a wavelength of 488 nm. Surface expo-
sure of phosphatidylserine in apoptotic cells was measured by
Annexin V-FITC/PI apoptosis detection kit I.
Acknowledgments
The authors thank the financial supports from the Ministry of
Science and Technology of China (2007AA02Z160), the Chinese Na-
tional Natural Science Foundation (20672068, 20472043), and Chi-
na Postdoctoral Science Foundation (20070410547).
4.1.4.1. 1-Benzyl-4-piperidone (5a). slight yellow oil (227 mg),
yield 60%;1H NMR (500 MHz, CDCl3): d 2.45 (t, 4H, 3J = 6.0 Hz), 2.75
(t, 4H, 3J = 6.0 Hz), 3.62(s, 2H), 7.26–7.36 (m, 5H); HRMS calcd for
References and notes
C
12H16NO [M+H]+ 190.1232, found 190.1230.
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