The synthesized novel fluorinated compound (LJJ-10) induces death receptor- and mitochondria-dependent apoptotic cell death in the human osteogenic sarcoma U-2 OS cells

Article abstract of DOI:10.1016/j.ejmech.2011.03.059

We designed the 6-fluoro-2-(3-fluorophenyl)-4-substituted anilinoquinazoline derivatives as less toxic anti-cancer candidates. Our result demonstrated that LJJ-10 has greater cytotoxicity than that of the other compounds in human osteogenic sarcoma U-2 OS cells. LJJ-10-induced apoptosis was associated with enhancing ROS generation, DNA damage, and an increase of the protein levels of Fas, FasL, FADD, caspase-8, cytochrome c, Apaf-1, AIF, Endo G, caspase-9 and caspase-3 in U-2 OS cells. LJJ-10-triggered growth inhibition was significantly attenuated by N-acetylcysteine, cyclosporine A, anti-FasL monoclonal antibody, and caspase-8, -9 and -3 specific inhibitors in U-2 OS cells. We suggest that LJJ-10-induced apoptotic cell death in U-2 OS cells through death receptor- and mitochondria-dependent apoptotic signaling pathways.

Full text of DOI:10.1016/j.ejmech.2011.03.059

European Journal of Medicinal Chemistry 46 (2011) 2709e2721
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
The synthesized novel fluorinated compound (LJJ-10) induces death
receptor- and mitochondria-dependent apoptotic cell death in the human
osteogenic sarcoma U-2 OS cells
,
b
c
b
c
d
Mann-Jen Hour a , Jai-Sing Yang , Tai-Lin Chen , Kuan-Tin Chen , Sheng-Chu Kuo , Jing-Gung Chung ,
*
Chi-Cheng Lu e, Chia-Yi Chen b, Yi-Hsuan Chuang b
a School of Pharmacy, China Medical University, Taichung 404, Taiwan
b Department of Pharmacology, China Medical University, Taichung 404, Taiwan
c Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung 404, Taiwan
d Department of Biological Science and Technology, China Medical University, Taichung 404, Taiwan
e Department of Life Sciences, National Chung Hsing University, Taichung 402, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
We designed the 6-fluoro-2-(3-fluorophenyl)-4-substituted anilinoquinazoline derivatives as less toxic
anti-cancer candidates. Our result demonstrated that LJJ-10 has greater cytotoxicity than that of the other
compounds in human osteogenic sarcoma U-2 OS cells. LJJ-10-induced apoptosis was associated with
enhancing ROS generation, DNA damage, and an increase of the protein levels of Fas, FasL, FADD, caspase-
8, cytochrome c, Apaf-1, AIF, Endo G, caspase-9 and caspase-3 in U-2 OS cells. LJJ-10-triggered growth
inhibition was significantly attenuated by N-acetylcysteine, cyclosporine A, anti-FasL monoclonal anti-
body, and caspase-8, -9 and -3 specific inhibitors in U-2 OS cells. We suggest that LJJ-10-induced
apoptotic cell death in U-2 OS cells through death receptor- and mitochondria-dependent apoptotic
signaling pathways.
Received 9 January 2011
Received in revised form
21 March 2011
Accepted 25 March 2011
Available online 3 April 2011
Keywords:
6-Fluoro-2-(3-fluorophenyl)-4-substituted
anilinoquinazoline derivatives
LJJ-10
Ó 2011 Elsevier Masson SAS. All rights reserved.
U-2 OS cells
Death receptor
Mitochondrial apoptotic signaling
1. Introduction
treatment have been focused on novel target therapies including
induction of apoptosis and reduction of cell growth in osteogenic
Osteosarcoma is the most common primary bone malignancy
[1], especially in adolescents and young adults. Contemporary
treatment of osteosarcoma requires therapy incorporating surgery
and systemic chemotherapy [2]. With the advent of chemotherapy,
the prognosis of osteosarcoma patients significantly improved.
Several chemotherapeutic agents, doxorubicin, cisplatin, high-dose
methotrexate with leucovorin rescue and ifosfamide, are consid-
ered the most active agents against osteosarcoma [3]. However,
a plateau in the survival rate seems to have been reached, and high-
grade osteosarcoma predicts a poor outcome with long-term
survival rates between 10 and 40% [4]. In addition, most chemo-
therapeutic agents cause strong normal cells cytotoxicity and side
effects [2]. Recently, studies in bone cancer or osteosarcoma
sarcoma [5e7].
The importance of fluorine in medicinal chemistry is well
recognized [8]. Indeed, an increasing number of drugs on the
market contain fluorine, and the presence of which is often a major
importance to activity. Many fluorinated analogues were recog-
nized by macromolecular recognition sites as the natural substrate
[9]. Fluorinated drugs are used in clinic for treatment of diseases of
the central nervous system [10], various cardiovascular diseases
[11], obesity [12], anti-bacterial agents [13], anti-fungal agents [14]
and anti-cancer therapies [15]. Certain of these agents are well
known, such as the fluoroquinolone, an anti-bacterial agent [16],
fluoroazole, an anti-fungal agent [17] and 5-fluorouracil, an anti-
cancer agent [18]. Herein, the fluorine substitution has played an
important role in the development of more active and more
selective agents. In addition, the advantages to drug design related
to the special properties of fluorine including small size and high
electronegativity, assured that the ability of fluorine-containing
* Corresponding author. Tel.: þ886 4 22053366x5123; fax: þ886 4 2207 8083.
E-mail addresses: mjhour7730@gmail.com, mjhou@mail.cmu.edu.tw (M.-J. Hour).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.059

2710
M.-J. Hour et al. / European Journal of Medicinal Chemistry 46 (2011) 2709e2721
drugs in developing enzyme inhibitors or rendering molecule
resistant to chemical degradation. We designed the novel fluori-
nated compounds with a quinazoline core structure as anti-cancer
candidates in order to increase their metabolic stability and be
recognized by macromolecular recognition sites after entering
human circulation.
Discovery system (Proteineer fc, UltiMate 3000). Elemental anal-
yses (C, H, and N) were performed on a PerkineElmer 2400 Series II
CHNS/O analyzer, and the results were within ꢁ0.4% of the calcu-
lated values.
2.2. General procedure for synthesis of starting 2-aminobenzamides
The analogues of quinazoline nuclei were reported to have
anti-malarial, anti-inflammatory, anti-bacterial and anti-cancer
activities, etc [16e18]. In our study, we design the 6-fluoro-2-(3-
fluorophenyl)-4-substituted anilinoquinazoline derivatives as less
toxic anti-cancer candidates based on the characteristics of fluorine
in medicinal chemistry in our previous study [19]. A series of 6-
fluoro-2-(3-fluorophenyl)-4-substituted anilinoquinazoline deriv-
atives were consequently synthesized and assayed for cytotoxicity
in vitro against seven types of cancer cell lines. Our preliminary
results also showed that these compounds (11e13, 16 and 18) dis-
played anti-proliferate activities on human osteogenic sarcoma
(U-2 OS), leukemia (HL-60), leukemic monocyte lymphoma (U937),
colon adenocarcinoma (HT-29), colon cancer (colo 205), breast
adenocarcinoma (MCF-7) and breast cancer (MDA-MB-231) cell
lines. Compounds 26 and 29 indicated selective effects on HL-60,
U937 and U-2 OS cells, and compounds 25, 26 and 28 showed
only cytotoxicity against the U-2 OS cell line. That is, 25, 26 and 28
showed highly selectivity toward U-2 OS cells. The novel
compound, 6-fluoro-(3-fluorophenyl)-4-(3-methoxyanilino) qui-
nazoline (compound 13, LJJ-10), has greater cytotoxicity than that
of the other compounds in U-2 OS cells with the EC50 (half maximal
(a) Thionyl chloride (1.5 g, 12.6 mmol) was added dropwise to
a suspension of a 5-substituted-2-nitrobenzoic acid (1a: 1.1 g,
6.3 mmol; 1b: 1.2 g, 6.3 mmol) in dichloroethane (30 mL) under
reflux. The resulting mixture was stirred under reflux for 4 h and
dried under vacuum. (b) The residue was dissolved in 200 mL of
dichloroethane and treated with anhydrous ammonia gas at room
temperature. (c) After removing solvent, the intermediate
5-substituted-2-nitrobenzamide (2a,b) was dissolved in MeOH and
hydrogenated over 10% Pd/C for 1e2 h. The catalyst was removed
by filtration, and the solution was dried under vacuum to afford the
5-substituted-2-aminobenzamides as pale yellow powders (3a:
0.7 g, 85%; 3b: 0.8 g, 85%).
2.3. General procedure for synthesis of 6-substituted-2-(3-
substituted phenyl)-4-quinazolinones (4e7)
Sodium hydrogen sulfite (0.8 g, 7.5 mmol) was added to a solu-
tion of 2-aminobenzamide (3a) (1.0 g, 7.3 mmol) and benzaldehyde
(0.8 g, 7.3 mmol) in N,N-dimethylacetamide (DMAC) (20 mL). The
mixture was heated with stirring at 150 ꢀC for 3 h and poured into
ice water (200 mL). The precipitate was collected, washed with
water, and dried in vacuo. After purification by column chroma-
tography (silica gel; chloroform) and followed recrystallization
from EtOH, 2-phenyl-4-quinazolinone (4) was obtained (1.1 g) as
white needles. Yield, melting point, and spectral data of 4 and all
subsequent compounds are summarized in Table 1. The method
used to prepare 4 was used with the indicated substituted benz-
aldehyde and benzamide to afford 5e7.
effective concentration) about 30.26
mM. In this study, we also
investigated the mechanism of apoptotic induction by LJJ-10 in U-2
OS cells. Also, the anti-cancer activity of LJJ-10 is not excellent but
less cytotoxic effects in normal cells (human fetal normal osteo-
blastic cell line, hFOB). Our results suggest that LJJ-10-induced
apoptosis in U-2 OS cells via death receptor- and mitochondria-
mediated apoptotic pathway. LJJ-10 might be selected as the lead
compound of apoptotic anti-cancer agent for further structure
modification in the future.
2.3.1. 2-(3-Fluorophenyl)-4-quinazolinone (5)
1.0 g from 3a and 3-fluorobenzaldehyde (0.9 g, 7.3 mmol), white
needles.
2. Chemistry
The 2-Aminobenzamide (3a) and 5-fluoro-2-aminobenzamide
(3b) were prepared using standard methodology from 2-nitro-
benzoic acid (1a) and 5-fluoro-2-nitrobenzoic acid (1b) respec-
tively, by the reaction with SOCl2, NH3, and H2/Pd/C. The
benzamides (3a, 3b) then were reacted with 3-substituted benz-
aldehydes in N,N-dimethylacetamide (DMAC) in the presence of
NaHSO3 at 150 ꢀC. Thermal cyclodehydration/dehydrogenation
gave the 6-substituted-2-(3-substituted phenyl)-4-quinazolinones
(4e7) as shown in Scheme 1. 6-Substituted-2-(3-substituted
phenyl)-4-quinazolinones (4e7) were chlorinated at the C-4 by
reaction with SOCl2, followed substitution by various substituted
anilines to give the target 6-substituted-2-(3-substituted phenyl)-
4-substituted anilinoquinazolines (8e32) as shown in Scheme 2.
2.3.2. 6-Fluoro-2-phenyl-4-quinazolinone (6)
1.1 g from 3b and benzaldehyde (0.8 g, 7.3 mmol), brown
powders.
2.3.3. 6-Fluoro-2-(3-fluorophenyl)-4-quinazolinone (7)
1.1 g from 3b and 3-fluorobenzaldehyde (0.9 g, 7.3 mmol), white
needles.
2.4. General procedure for synthesis of 6-substituted-2-(3-
substituted phenyl)-4-substituted anilinoquinazolines (8e32)
Thionyl chloride (1.5 g, 12.6 mmol) was added dropwise to
a suspension of 2-phenyl-4-quinazolinone (4) (1.4 g, 6.3 mmol) in
dichloroethane (30 mL) and N,N-dimethylformamide (DMF)
(0.5 mL) at 50 ꢀC. The resulting mixture was stirred at 50 ꢀC for
30 min and dried under vacuum. The residue was dissolved in 20 mL
of DMFand treated with aniline (0.6 g, 6.3 mmol) at 50 ꢀC for 2 h. The
mixture was then poured into ice water (200 mL). The precipitate
was collected, washed with water, and dried in vacuo. After purifi-
cation by column chromatography (silica gel; chloroform) and fol-
lowed recrystallization from the mixed solvent of CHCl3 and EtOH,
4-anilino-2-phenylquinazoline (8) was obtained (1.3 g) as white
needles. The method used to prepare 8 was used with the indicated
substituted aniline and phenylquinazolinone to afford 9e32.
2.1. General experimental procedures
Reagents and solvents were obtained commercially and used
without further purification. Reactions were monitored by thin-
layer chromatography, using Merck plates with fluorescent indi-
cator (TLC Silica Gel 60 F254). Melting points were determined on
a Yanaco MP-500D melting point apparatus and were uncorrected.
1H NMR spectra were obtained on a Bruker Advance DPX-200 FT
NMR spectrometer in DMSO-d6. The following abbreviations are
used: s, singlet; d, doublet; m, multiplet; dd, double doublet. EI-MS
spectra were measured with an HP 5995 GCeMS instrument. ESI-
MS spectra were measured with
a Bruker HCT ultra PTM

M.-J. Hour et al. / European Journal of Medicinal Chemistry 46 (2011) 2709e2721
2711
O
O
R1
R1
NH2
OH
a
b
NO2
NO2
2a-b
1a-b
O
O
N
R1
R1
NH2
NH
c
R2
NH2
3a-b
4-7
a R1=H
b R2=F
4 R1=H, R2=H
5 R1=H, R2=F
6 R1=F, R2=H
7 R1=F, R2=F
Reagents and conditions:
a. (i) SOCl2 reflux; (ii) NH3 (g), dichloroethane, R.T.
b. H2, Pd/C, methanol
c. substituted benzaldehyde, DMAC, NaHSO3, 150
Scheme 1.
Scheme 2.

Table 1
Physical and spectral data of compounds 4e32.
Compounds Yield (%) Mp (ꢀC)
MS (Mþ) m/z 1H NMR (DMSO-d6)
d
13C NMR (DMSO-d6)
d
4
5
6
66
65
69
249e250 222
7.47e7.62 (4H, m, H-6, H-20, H-30, H-50), 7.70e7.87 (2H, m, H-7, H-8), 8.12e8.19
(3H, m, H-5, H-20, H-60), 12.54 (1H, s, NH)
7.37e7.63 (3H, m, H-6, H-40, H-50), 7.70e7.86 (2H, m, H-7, H-8), 7.95e8.05 (2H,
m, H-20, H-60), 8.13 (1H, dd, J ¼ 1.2, 8.0 Hz, H-5), 12.58 (1H, s, NH)
7.48e7.56 (3H, m, H-30, H-40, H-50), 7.61e7.83 (3H, m, H-20, H-60, H-8), 8.12e8.16
(2H, m, H-5, H-7), 12.64 (1H, s, NH)
120.95, 125.84, 126.57, 127.46, 127.75, 128.59, 131.38, 132.70, 134.59, 148.69,
152.33, 162.26
279e280 240
114.29, 114.77, 118.04, 118.46, 121.11, 123.93, 125.87, 126.90, 127.60, 130.65,
130.81, 134.67, 134.92, 135.08, 148.44, 151.01, 159.66, 162.13, 164.51
110.31, 110.77, 114.17, 122.11, 122.28, 122.86, 123.34, 127.78, 128.65, 130.21,
130.37, 131.46, 132.58, 145.58, 151.92, 151.95, 157.57, 161.72, 161.79, 162.25,
162.44, 168.23
110.78, 111.24, 114.72, 115.19, 118.52, 118.94, 122.72, 122.88, 123.34, 123.82,
124.32, 130.75, 130.91, 131.12, 131.29, 135.20, 135.36, 145.73, 151.04, 158.16,
160.09, 162.02, 163.04, 164.93
>280
>300
240
258
7
44
72
79
54
45
7.37e7.46 (1H, m, H-40), 7.52e7.60 (1H, m, H-50), 7.69e7.84 (3H, m, H-8, H-20, H-60),
7.93e8.03 (2H, m, H-5, H-7), 12.72 (1H, s, NH)
8
163ꢂ164 296
152e153 314
177e178 314
169e170 332
7.12e7.19 (1H, m, H-400), 7.42e7.61 (6H, m, H-6, H-30, H-40, H-50, H-300, H-500),
7.79e7.90 (2H, m, H-7, H-8), 7.97e8.01 (2H, m, H-200, H-600), 8.46e8.49 (2H, m,
H-20, H-60), 8.58 (1H, dd, J ¼ 1.2, 8.0 Hz, H-5), 9.88 (1H, s, NH)
114.03, 122.23, 123.02, 123.62, 125.86, 127.91, 128.12, 128.37, 128.47, 130.23,
133.13, 138.36, 139.32, 150.51, 157.92, 159.04
9
7.09e7.29 (2H, m, H-50, H-400), 7.37e7.58 (4H, m, H-6, H-40, H-300, H-500), 7.74e7.81
(2H, m, H-7, H-8), 7.86e7.90(2H, m, H-200, H-600), 8.05 (1H, d, J ¼ 10.6 Hz, H-20),
8.21 (1H, d, J ¼ 7.6 Hz, H-60), 8.50 (1H, dd, J ¼ 1.2, 8.0 Hz, H-5), 9.87 (1H, s, NH)
7.11e7.18 (1H, m, H-400), 7.41e7.48 (5H, m, H-6, H-30, H-50, H-300, H-500), 7.62e7.71
(1H, m, H-7), 7.83e7.98 (3H, m, H-8, H-200, H-600), 8.37e8.42 (3H, m, H-5, H-20, H-60),
9.73 (1H, s, NH)
114.01, 114.17, 114.47, 116.81, 117.23, 122.53, 123.08, 123.91, 126.26, 128.20,
128.52, 130.33, 130.49, 133.31, 139.16, 140.99, 141.14, 150.35, 157.85, 158.03,
160.03, 164.85
107.25, 107.72, 114.34, 114.52, 122.11, 122.60, 123.67, 127.86, 128.34, 128.48,
130.19, 130.69, 130.85, 138.17, 139.18, 147.60, 156.91, 157.54, 157.62,
158.71, 161.77
107.74, 108.21, 114.30, 114.75, 114.91, 115.10, 117.21, 117.64, 122.78, 123.27,
124.15, 124.20, 124.41, 128.94, 130.73, 130.89, 131.22, 131.40, 134.83, 139.35,
141.11, 141.27, 147.81, 147.83, 157.53, 157.81, 157.85, 157.92, 158.05, 158.13,
160.40, 162.40, 165.22
10
11
7.13e7.33 (2H, m, H-40, H-400), 7.41e7.56 (3H, m, H-50, H-300, H-500), 7.68e7.78
(1H, m, H-7), 7.85e7.91 (3H, m, H-8, H-200, H-600), 8.03 (1H, d, J ¼ 10.6 Hz, H-20),
8.20 (1H, d, J ¼ 7.6 Hz, H-60), 8.39 (1H, dd, J ¼ 2.5, 9.8 Hz, H-5), 9.80 (1H, s, NH)
12
13
14
18
12
17
>270
363
3.79 (3H, s, OCH3), 7.02 (2H, d, J ¼ 9.0 Hz, H-300, H-500), 7.27e7.33 (1H, m, H-400),
7.45e7.56 (1H, m, H-50), 7.65e7.71 (1H, m, H-7), 7.73e7.79 (1H, m, H-200, H-600),
7.86e7.90 (1H, m, H-8), 8.02 (1H, d, J ¼ 10.6 Hz, H-20), 8.18 (1H, d, J ¼ 7.6 Hz, H-60),
8.34 (1H, dd, J ¼ 2.5, 9.8 Hz, H-5), 9.72 (1H, s, NH)
55.63, 107.60, 108.08, 114.04, 114.26, 114.72, 114.81, 114.99, 117.05, 117.47,
122.45, 122.94, 124.14, 124.42, 130.58, 130.73, 131.06, 131.23, 132.21, 141.21,
141.36, 147.65, 156.28, 157.43, 157.96, 158.04, 160.34, 162.28, 165.16
144e145 362
166e167 332
3.82 (3H, s, OCH3), 6.76 (1H, dd, J ¼ 2.4, 8.0 Hz, H-400), 7.28e7.39 (2H, m, H-40, H-500),
7.47e7.60 (2H, m, H-200, H-50), 7.70e7.82 (2H, m, H-7, H-600), 7.89e7.97 (1H, m, H-8),
8.11 (1H, d, J ¼ 10.6 Hz, H-20), 8.25 (1H, d, J ¼ 7.6 Hz, H-60), 8.42 (1H, dd, J ¼ 2.5,
9.8 Hz, H-5), 9.80 (1H, s, NH)
55.33, 70.96, 107.53, 107.70, 108.00, 110.12, 114.12, 114.54, 114.75, 114.93,
117.18, 117.60, 122.78, 123.27, 123.97, 129.56, 130.68, 130.83, 131.10, 131.27,
140.39, 140.85, 141.01, 147.58, 157.40, 157.79, 157.87, 159.63, 160.22,
162.27, 165.04
3.80 (3H, s, OCH3), 6.99e7.32 (4H, m, H-40, H-300, H-400, H-500), 7.39e7.50 (1H, m, H-50), 55.99, 107.59, 108.06, 112.11, 114.24, 114.69, 114.75, 114.92, 117.03, 117.46,
7.65e7.76 (2H, m, H-7, H-600), 7.84e7.96 (2H, m, H-8, H-20), 8.10 (1H, d, J ¼ 7.6 Hz,
120.51, 122.63, 123.13, 124.03, 124.07, 126.46, 127.00, 127.08, 127.25, 130.53,
130.69, 131.13, 131.30, 141.19, 141.35, 147.73, 153.55, 157.46, 157.90, 157.95,
158.01, 158.74, 158.82, 162.33, 165.14
109.27, 109.76, 114.58, 114.77, 115.36, 115.64, 115.83, 116.09, 119.33, 119.75,
124.39, 124.89, 125.26, 126.20, 126.36, 126.65, 131.37, 131.53, 134.19, 136.74,
141.56, 156.77, 157.61, 158.06, 158.83, 160.17, 162.45, 162.96, 165.02
107.54, 108.02, 109.25, 110.15, 110.57, 114.23, 114.92, 117.15, 117.57, 117.81,
122.72, 123.22, 124.01, 130.16, 130.35, 130.56, 130.72, 131.17, 131.34, 140.87,
141.03, 141.13, 141.36, 147.67, 157.62, 160.01, 162.35, 162.37, 164.81, 165.17
107.37, 107.83, 113.74, 114.19, 115.77, 116.16, 116.84, 117.25, 122.68, 123.18,
H-60), 8.34 (1H, dd, J ¼ 2.5, 9.8 Hz, H-5), 9.43 (1H, s, NH)
15
16
17
18
19
20
21
22
16
14
25
21
20
22
232e233 351
107e108 351
200e201 351
7.30e7.50 (3H, m, H-40, H-300, H-500), 7.56e7.67 (1H, m, H-50), 7.81e7.97 (3H, m,
H-7, H-200, H-600), 8.08e8.28 (3H, m, H-8, H-20, H-60), 8.71 (1H, dd, J ¼ 2.5,
9.8 Hz, H-5), 11.08 (1H, s, NH)
6.89e6.96 (1H, m, H-400), 7.20e7.27 (1H, m, H-40), 7.33e7.49(2H, m, H-50, H-500),
7.61e7.64 (2H, m, H-200, H-600), 7.73e7.94 (3H, m, H-7, H-8, H-20), 8.08
(1H, d, J ¼ 7.6 Hz, H-60), 8.22 (1H, dd, J ¼ 2.5, 9.8 Hz, H-5), 9.70 (1H, s, NH)
7.22e7.69 (5H, m, H-40, H-50, H-300, H-400, H-500), 7.74e7.98 (4H, m, H-20, H-7, H-8,
H-600), 8.08 (1H, d, J ¼ 7.6 Hz, H-60), 8.35 (1H, dd, J ¼ 2.5, 9.8 Hz, H-5), 9.98 (1H, s, NH) 123.63, 124.41, 125.91, 126.15, 127.58, 127.99, 130.47, 130.86, 131.02, 136.39,
137.62, 140.53, 147.43, 152.49, 154.26, 158.47, 159.17, 161.27, 161.96, 164.70
>300
367.5
7.21e7.28 (1H, m, H-40), 7.40e7.51 (3H, m, H-50, H-300, H-500), 7.62e7.97
(5H, m, H-7, H-8, H-20, H-200, H-600), 8.11 (1H, d, J ¼ 7.6 Hz, H-60), 8.26 (1H, dd,
J ¼ 2.5, 9.8 Hz, H-5), 9.75 (1H, s, NH)
107.22, 107.70, 113.80, 114.27, 114.38, 114.56, 115.28, 116.78, 117.20, 117.62,
122.39, 122.88, 123.62, 127.41, 128.31, 130.26, 130.42, 130.77, 130.94, 137.90,
140.47, 140.62, 147.30, 157.07, 157.33, 157.41, 159.92, 161.94, 164.74
170.10, 107.58, 113.84, 114.30, 114.48, 116.72, 117.14, 119.90, 121.37, 122.29,
122.78, 123.05, 123.60, 129.88, 130.08, 130.23, 130.75, 130.91, 132.76, 140.42,
147.53, 147.26, 157.12, 157.21, 159.93, 161.92, 164.76
107.71, 108.18, 114.22, 114.51, 114.68, 117.19, 117.61, 123.05, 123.55, 124.06,
128.01, 128.33, 129.81, 130.23, 130.64, 130.79, 131.11, 131.30, 131.48, 136.06,
140.99, 141.14, 147.90, 157.52, 157.86, 159.03, 159.11, 160.30, 162.39, 165.12
107.30, 107.78, 113.88, 114.36, 114.57, 115.58, 116.90, 117.32, 122.46, 122.96,
123.81, 124.01, 130.31, 130.47, 130.60, 130.77, 131.25, 138.32, 140.29, 140.43,
147.06, 157.28, 159.94, 161.99, 164.76
223e224 367.5
185e186 367.5
7.12e7.28 (2H, m, H-40, H-400), 7.34e7.49 (2H, m, H-50, H-500), 7.58e7.96 (4H, m,
H-7, H-8, H-200, H-600), 8.08e8.14 (2H, m, H-20, H-60), 8.22 (1H, dd, J ¼ 2.5,
9.8 Hz, H-5), 9.68 (1H, s, NH)
7.19e7.28 (1H, m, H-40), 7.35e7.51 (3H, m, H-300, H-400, H-500), 7.67e7.71 (2H,
m, H-50, H-600), 7.75e7.96 (3H, m, H-20, H-7, H-8), 8.03 (1H, d, J ¼ 7.6 Hz, H-60),
8.35 (1H, dd, J ¼ 2.5, 9.8 Hz, H-5), 9.96 (1H, s, NH)
231
412
7.22e7.31 (1H, m, H-40), 7.41e7.70 (4H, m, H-7, H-50, H-300, H-500), 7.79e7.83
(3H, m, H-8, H-200, H-600), 7.92 (1H, d, J ¼ 10.6 Hz, H-20), 8.09 (1H, d, J ¼ 7.6 Hz, H-60),
8.25 (1H, dd, J ¼ 2.5, 9.8 Hz, H-5), 9.78 (1H, s, NH)

22
23
13
23
224e225 412
199e200 412
7.23e7.51 (4H, m, H-40, H-50, H-400, H-500), 7.61e0.70 (2H, m, H-200, H-600), 7.76e7.81
(2H, m, H-7, H-8), 7.96 (1H, d, J ¼ 10.6 Hz, H-20), 8.13 (1H, d, J ¼ 7.6 Hz, H-60),
8.25 (1H, dd, J ¼ 2.5, 9.8 Hz, H-5), 9.74 (1H, s, NH)
107.21, 107.69, 113.90, 114.37, 114.56, 116.88, 117.30, 120.38, 121.24, 122.47,
122.96, 123.68, 124.35, 126.04, 130.34, 130.86, 131.03, 140.45, 140.69, 147.35,
157.20, 160.01, 162.01, 164.83
107.38, 107.86, 108.14, 110.21, 110.41, 113.99, 114.45, 114.55, 114.73, 116.73,
117.15, 122.21, 122.70, 123.86, 128.74, 130.30, 130.46, 130.73, 130.90, 139.43,
140.80, 140.95, 147.37, 148.98, 157.05, 157.57, 157.71, 157.79, 159.97, 161.91,
164.79
7.15e7.52 (4H, m, H-40, H-50, H-400, H-500), 7.61e7.65 (1H, m, H-300),
7.75e7.92(4H, m, H-7, H-8, H-20, H-600), 7.98 (1H, d, J ¼ 7.6 Hz, H-60), 8.24
(1H, dd, J ¼ 2.5, 9.8 Hz, H-5), 9.92 (1H, s, NH)
24
25
15
17
272
349
6.85 (2H, d, J ¼ 12 Hz, H-300, H-500), 7.24e7.34 (1H, m, H-40), 7.46e7.53
(1H, m, H-50), 7.58e7.76 (3H, m, H-7, H-200, H-600), 7.83e7.90 (1H, m, H-8),
8.02 (1H, d, J ¼ 10.6 Hz, H-20), 8.19 (1H, d, J ¼ 7.6 Hz, H-60), 8.34 (1H, dd,
J ¼ 2.5, 9.8 Hz, H-5), 9.43 (1H, s, OH), 9.71 (1H, s, NH)
107.39, 107.86, 114.09, 114.61, 114.79, 115.25, 116.98, 117.40, 122.39, 122.89,
123.95, 124.83, 130.34, 130.55, 130.70, 130.89, 131.06, 141.01, 141.16, 147.45,
154.38, 157.26, 157.82, 157.99, 158.06, 160.17, 162.12, 164.98
251e252 349
6.76 (1H, dd, J ¼ 1.2, 7.8 Hz, H-400), 7.19e7.42 (4H, m, H-40, H-50, H-200, H-500),
7.49e7.60 (1H, m, H-600), 7.70e7.80 (1H, m, H-7), 7.88e7.95 (1H, m, H-8),
8.10 (1H, d, J ¼ 10.6 Hz, H-20), 8.26 (1H, d, J ¼ 7.6 Hz, H-60), 8.43 (1H, dd,
J ¼ 2.5, 9.8 Hz, H-5), 9.59 (1H, s, OH), 9.75 (1H, s, NH)
107.44, 107.91, 109.39, 111.14, 113.04, 114.00, 114.46, 114.58, 114.76, 116.88,
117.30, 122.43, 122.92, 123.88, 129.17, 130.39, 130.55, 130.84, 131.01, 139.96,
140.71, 140.87, 147.41, 157.13, 157.56, 157.72, 160.00, 161.99, 164.82
26
27
20
20
264
349
6.96e7.30 (4H, m, H-40, H-300, H-400, H-500), 7.42e7.59 (2H, m, H-50, H-600),
7.70e7.80 (1H, m, H-7), 7.87e7.96 (2H, m, H-8, H-20), 8.11 (1H, d, J ¼ 7.6
Hz, H-60), 8.36 (1H, dd, J ¼ 2.5, 9.8 Hz, H-5), 9.52 (2H, s, OH, NH)
5.07 (2H, s, NH2), 6.67 (2H, d, J ¼ 8.2 Hz, H-300, H-500), 7.26e7.30 (1H, m,
H-400), 7.42e0.7.54 (3H, m, H-50, H-200, H-600), 7.63e7.86 (2H, m, H-7, H-8),
8.02 (1H, d, J ¼ 10.6 Hz, H-20), 8.17 (1H, d, J ¼ 7.6 Hz, H-60), 8.30 (1H, dd,
J ¼ 2.5, 9.8 Hz, H-5), 9.59 (1H, s, NH)
107.55, 108.03, 113.90, 114.36, 114.55, 114.73, 116.32, 116.78, 117.20, 118.90,
122.35, 122.84, 123.73, 125.54, 126.89, 127.31, 130.30, 130.46, 130.73, 130.90,
140.81, 140.96, 147.33, 151.74, 157.03, 157.59, 158.69, 159.94, 161.89, 164.75
107.29, 107.77, 113.77, 113.97, 114.43, 114.59, 114.77, 116.78, 117.20, 122.06,
122.55, 123.80, 124.50, 127.50, 130.35, 130.51, 130.71, 130.88, 141.06, 141.21,
145.83, 147.32, 157.08, 157.80, 157.88, 160.05, 161.94, 164.87
235e236 348
183e184 348
213e215 348
197e198 347
28
29
22
33
5.18 (2H, s, NH2), 6.41e7.44 (1H, m, H-200), 7.09e7.11 (3H, m, H-400, H-500,
H-600), 7.26e7.30 (1H, m, H-40), 7.49e7.58(1H, m, H-50), 7.68e7.78 (1H, m,
H-7), 7.85e7.93 (1H, m, H-8), 8.10 (1H, d, J ¼ 10.6 Hz, H-20), 8.25 (1H, d,
J ¼ 7.6 Hz, H-60), 8.42 (1H, dd, J ¼ 2.5, 9.8 Hz, H-5), 9.60 (1H, s, NH)
5.05 (2H, s, NH2), 6.65e6.71 (1H, m, H-300), 6.87e7.28 (4H, m, H-40, H-400, H-500,
H-600), 7.40e4.51 (1H, m, H-50), 7.68e7.77 (1H, m, H-7), 7.85e7.96 (2H,
m, H-8, H-20), 8.11 (1H, d, J ¼ 7.6 Hz, H-60), 8.34 (1H, dd, J ¼ 2.5, 9.8 Hz, H-5),
9.48 (1H, s, NH)
107.37, 107.84, 114.04, 114.22, 114.40, 114.49, 116.98, 117.39, 121.81, 122.82,
123.31, 123.86, 126.89, 130.42, 130.57, 131.04, 131.21, 133.16, 137.30, 140.72,
140.87, 147.62, 151.98, 157.30, 157.68, 158.78, 158.86, 160.06
107.84, 108.30, 113.96, 114.41, 114.67, 114.84, 115.86, 115.97, 116.67, 117.09,
122.08, 122.57, 123.14, 123.75, 127.19, 127.84, 130.18, 130.34, 130.57, 130.74,
140.95, 141.10, 144.46, 147.33, 156.90, 157.70, 158.73, 158.80, 159.92, 161.75
30
31
32
25
22
29
2.31 (3H, s, CH3), 7.19e7.29 (2H, m, H-300, H-500), 7.41e7.85 (6H, m, H-7, H-8,
H-40, H-50, H-200, H-600), 8.01 (1H, d, J ¼ 10.6 Hz, H-20), 8.16 (1H, d, J ¼ 7.6 Hz,
H-60), 8.31 (1H, dd, J ¼ 2.5, 9.8 Hz, H-5), 9.67 (1H, s, NH)
107.24, 107.71, 113.85, 114.31, 114.45, 114.63, 116.64, 117.07, 122.10, 122.25,
122.58, 123.70, 128.86, 130.15, 130.30, 130.68, 130.85, 132.90, 136.35, 140.76,
140.91, 147.29, 157.03, 157.46, 157.54, 159.94, 161.89, 164.76
107.26, 107.73, 113.95, 114.48, 114.67, 116.70, 117.13, 119.26, 122.15, 122.65,
122.83, 123.70, 124.47, 128.28, 130.18, 130.33, 130.75, 130.91, 137.59, 138.96,
140.77, 140.91, 147.36, 157.09, 157.43, 160.01, 161.96, 164.83
107.31, 107.78, 113.70, 114.17, 114.36, 116.61, 117.04, 122.24, 122.72, 123.54,
126.10, 126.39, 127.25, 130.11, 130.26, 130.45, 130.73, 130.90, 134.55, 136.79,
140.73, 140.88, 147.36, 157.01, 157.50, 158.60, 159.86, 161.87, 164.68
173
347
2.49 (3H, s, CH3), 7.07e7.10 (1H, m, H-200), 7.38e7.61 (3H, m, H-40, H-400, H-500),
7.76e7.90 (4H, m, H-7, H-8, H-50, H-600), 8.13 (1H, d, J ¼ 10.6 Hz, H-20), 8.29
(1H, d, J ¼ 7.6 Hz, H-60), 8.43 (1H, dd, J ¼ 2.5, 9.8 Hz, H-5), 9.77 (1H, s, NH)
2.24 (3H, s, CH3), 7.21e7.48 (6H, m, H-40, H-50, H-300, H-400, H-500, H-600),
7.67e7.75 (1H, m, H-7), 7.85e7.89 (2H, m, H-8, H-20), 8.03 (1H, d, J ¼ 7.6 Hz,
H-60), 8.34 (1H, dd, J ¼ 2.5, 9.8 Hz, H-5), 9.71 (1H, s, NH)
194e195 347

2714
M.-J. Hour et al. / European Journal of Medicinal Chemistry 46 (2011) 2709e2721
2.4.1. 4-Anilino-2-(3-fluorophenyl)quinazoline (9)
2.4.17. 6-Fluoro-2-(3-fluorophenyl)-4-(3-hydroxyanilino)
1.5 g from 5 and aniline (0.6 g, 6.3 mmol), pale yellow powders.
quinazoline (25)
1.6 g from 7 and 3-aminophenol (0.7 g, 6.3 mmol), yellow
powders.
2.4.2. 4-Anilino-6-fluoro-2-phenylquiazoline (10)
1.5 g from 6 and aniline (0.6 g, 6.3 mmol), white needles.
2.4.18. 6-Fluoro-2-(3-fluorophenyl)-4-(2-hydroxyanilino)
quinazoline (26)
1.6 g from 7 and 2-aminophenol (0.7 g, 6.3 mmol), yellow
powders.
2.4.3. 4-Anilino-6-fluoro-2-(3-fluorophenyl)quinazoline (11)
1.6 g from 7 and aniline (0.6 g, 6.3 mmol), white needles.
2.4.4. 6-Fluoro-2-(3-fluorophenyl)-4-(4-methoxyanilino)
quinazoline (12)
2.4.19. 6-Fluoro-2-(3-fluorophenyl)-4-(4-aminoanilino)quinazoline
(27)
1.6 g from 7 and 4-methoxyaniline (0.8 g, 6.3 mmol), brown
powders.
1.6 g from 7 and p-phenylenediamine (0.7 g, 6.3 mmol), yellow
needles.
2.4.5. 6-Fluoro-2-(3-fluorophenyl)-4-(3-methoxyanilino)
quinazoline (13)
2.4.20. 6-Fluoro-2-(3-fluorophenyl)-4-(3-aminoanilino)
quinazoline (28)
1.6 g from 7 and m-phenylenediamine (0.7 g, 6.3 mmol), yellow
needles.
1.6 g from 7 and 3-methoxyaniline (0.8 g, 6.3 mmol), yellow
needles.
2.4.6. 6-Fluoro-2-(2-fluorophenyl)-4-(3-methoxyanilino)
quinazoline (14)
1.6 g from 7 and 2-methoxyaniline (0.8 g, 6.3 mmol), yellow
needles.
2.4.21. 6-Fluoro-2-(3-fluorophenyl)-4-(2-aminoanilino)quinazoline
(29)
1.6 g from 7 and o-phenylenediamine (0.7 g, 6.3 mmol), white
needles.
2.4.7. 6-Fluoro-2-(3-fluorophenyl)-4-(4-fluoroanilino)quinazoline (15)
1.6 g from 7 and 4-fluoroaniline (0.7 g, 6.3 mmol), white plate
crystals.
2.4.22. 6-Fluoro-2-(3-fluorophenyl)-4-(4-methylanilino)
quinazoline (30)
2.4.8. 6-Fluoro-2-(3-fluorophenyl)-4-(3-fluoroanilino)quinazoline
(16)
1.6 g from 7 and p-toluidine (0.7 g, 6.3 mmol), yellow needles.
1.6 g from 7 and 3-fluoroaniline (0.7 g, 6.3 mmol), white needles.
2.4.23. 6-Fluoro-2-(3-fluorophenyl)-4-(3-methylanilino)
quinazoline (31)
1.6 g from 7 and m-toluidine (0.7 g, 6.3 mmol), white needles.
2.4.9. 6-Fluoro-2-(3-fluorophenyl)-4-(2-fluoroanilino)quinazoline
(17)
2.4.24. 6-Fluoro-2-(3-fluorophenyl)-4-(2-methylanilino)
quinazoline (32)
1.6 g from 7 and 2-fluoroaniline (0.7 g, 6.3 mmol), white needles.
1.6 g from 7 and o-toluidine (0.7 g, 6.3 mmol), pink plate
crystals.
2.4.10. 6-Fluoro-2-(3-fluorophenyl)-4-(4-chloroanilino)quinazoline
(18)
1.6 g from 7 and 4-chloroaniline (0.8 g, 6.3 mmol), white needles
3. Pharmacology
2.4.11. 6-Fluoro-2-(3-fluorophenyl)-4-(3-chloroanilino)quinazoline
(19)
3.1. Materials and reagents
1.6 g from 7 and 3-chloroaniline (0.8 g, 6.3 mmol), white
needles.
MTT (3-[4,5-dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium
bromide), N-acetylcysteine (NAC), cyclosporine A and dimethyl
sulfoxide (DMSO) were purchased from SigmaeAldrich Corp.
2.4.12. 6-Fluoro-2-(3-fluorophenyl)-4-(2-chloroanilino)quinazoline
(20)
(St. Louis, MO, USA). Fetal bovine serum (FBS),
L-glutamine,
1.6 g from 7 and 2-chloroaniline (0.8 g, 6.3 mmol), pink needles.
penicillin-streptomycin, McCoy’s 5a medium, RPMI 1640 medium,
Dulbecco’s modified eagle medium (DMEM), trypsin-EDTA, 2,7-
dichlorodihydrofluorescein diacetate (H2DCF-DA) and 3,30-dihex-
yloxacarbocyanine iodide (DiOC6) were obtained from Invitrogen
Life Technologies (Carlsbad, CA, USA). Caspase-3 inhibitor (Z-DEVE-
FMK), caspase-8 inhibitor (Z-IETD-FMK) and caspase-9 inhibitor
(Z-LEHD-FMK) (R&D systems, Minneapolis, MN, USA) were dissolved
in DMSO and diluted in cell culture medium before use. Antibodies
against caspase-8, caspase-9 and caspase-3 were purchased from
Cell Signaling Technology (Beverly, MA, USA). Antibodies for Fas/
CD95, FasL, FADD, cytohrome c, Apaf-1, caspase-9, Endo G, AIF, Bcl-2
and Bax were purchased from Santa Cruz Biotechnology, Inc. (Santa
Cruz, CA, USA).
2.4.13. 6-Fluoro-2-(3-fluorophenyl)-4-(4-bromoanilino)
quinazoline (21)
1.6 g from7 and 4-bromoaniline (1.1 g, 6.3 mmol), yellowneedles.
2.4.14. 6-Fluoro-2-(3-fluorophenyl)-4-(3-bromoanilino)
quinazoline (22)
1.6 g from7 and 3-bromoaniline (1.1 g, 6.3 mmol), brownneedles.
2.4.15. 6-Fluoro-2-(3-fluorophenyl)-4-(2-bromoanilino)
quinazoline (23)
1.6 g from 7 and 2-bromoaniline (1.1 g, 6.3 mmol), pink plate
crystals.
2.4.16. 6-Fluoro-2-(3-fluorophenyl)-4-(4-hydroxyanilino)
3.2. Cell culture
quinazoline (24)
1.6 g from 7 and 4-aminophenol (0.7 g, 6.3 mmol), brown
powders.
The human leukemia cell line (HL-60), human monocyte
lymphoma cell line (U937), human colon adenocarcinoma cell line

M.-J. Hour et al. / European Journal of Medicinal Chemistry 46 (2011) 2709e2721
2715
Table 2
In vitro cytotoxicity of 6-substituted-2-(3-substituted phenyl)-4-substituted anilinoquinazolines (8e32).
a
Compounds
IC50
(m
M)
HL-60b
U937b
HT-29b
Colo 205b
U-2 OSb
MCF-7b
MDA-MB-231b
8
>50
>50
>50
>50
>50
>50
>50
9
>50
>50
>50
>50
>50
>50
>50
10
11
12
13 (LJJ-10)
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
>50
>50
>50
>50
>50
>50
>50
48.26 ꢁ 2.01
38.62 ꢁ 4.89
35.49 ꢁ 3.69
>50
45.45 ꢁ 1.06
41.09 ꢁ 3.57
39.62 ꢁ 3.21
>50
48.32 ꢁ 2.07
42.33 ꢁ 3.29
40.35 ꢁ 4.58
>50
>50
>50
49.66 ꢁ 3.04
46.82 ꢁ 3.47
44.68 ꢁ 4.11
>50
>50
>50
47.62 ꢁ 1.09
32.32 ꢁ 3.98
30.26 ꢁ 3.21
>50
49.66 ꢁ 3.47
48.36 ꢁ 4.17
46.33 ꢁ 4.12
>50
47.82 ꢁ 3.25
47.26 ꢁ 4.18
47.05 ꢁ 6.32
>50
>50
>50
>50
>50
>50
43.11 ꢁ 1.29
42.26 ꢁ 3.26
49.62 ꢁ 3.29
48.68 ꢁ 4.47
47.63 ꢁ 2.58
>50
>50
>50
>50
>50
>50
>50
45.69 ꢁ 2.96
>50
>50
>50
>50
>50
>50
>50
32.65 ꢁ 6.24
>50
>50
>50
>50
>50
>50
>50
40.32 ꢁ 3.96
32.64 ꢁ 4.17
38.36 ꢁ 3.08
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
33.32 ꢁ 2.36
41.36 ꢁ 3.69
44.15 ꢁ 6.98
49.65 ꢁ 4.16
37.85 ꢁ 6.96
>50
44.36 ꢁ 2.19
46.25 ꢁ 3.26
>50
>50
>50
>50
41.28 ꢁ 7.45
47.25 ꢁ 5.01
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
a
IC50 was the half maximal inhibitory concentration after 48 h of treatment. The unit is expressed as
m
M.
HL-60 (human leukemia cells), U937 (human leukemic monocyte lymphoma cells), HT-29 (human colon adenocarcinoma cells), Colo 205 (human colon cancer cells), U-2
OS (human osteosarcoma cells), MCF-7 (human breast adenocarcinoma cells) and MDA-MB-231 (human breast cancer cells).
b
(HT-29), human adenocarcinoma cell line (colo 205), human breast
adenocarcinoma cell line (MCF-7), human breast cancer cell line
(MDA-MB-231), human osteosarcoma cell line (U-2 OS) and human
fetal normal osteoblastic cell line (hFOB) were purchased from the
Food Industry Research and Development Institute (Hsinchu,
Taiwan). The HL-60, U937, HT-29 and colo 205 cells were cultured
with RPMI 1640 medium. The MCF-7 and MDA-MB-231 cells were
seeded in DMEM. The U-2 OS cells were cultured with 90% McCoy’s
5a medium. The hFOB cells were cultured with DMEM/Ham’s F12
medium (Invitrogen). All cells were plated onto 75 cm2 tissue
3.4. Cell morphological examination
After U-2 OS cells (2.5 ꢃ 105 cells/well onto 24-well plate) were
treated with 0,15, 30 and 45 mM LJJ-10 for 48 h, the cell morphology
was directly examined and photographed under a contrast-phase
microscope [23].
3.5. Comet assay
After 48 h-treatment with 15, 30 and 45 mM LJJ-10, U-2 OS cells
culture flasks with medium with 2 mM
L-glutamine supplemented
were harvested and mixed with low melting point (LMP,
SigmaeAldrich Corp.) agarose at 37 ꢀC. This mixture was placed on
the top of previous layerof 0.5% agarose normal melting point (NMP)
on the slide, and then covered with a covership at 41 ꢀC until solid.
Subsequently, the covership was removed gently and some agarose
was added onto this slide, and then covered with the covership
again. The slide was placed at 41 ꢀC until the mixture was solid, and
put in chilled alkaline lysis buffer for electrophoresis. Afterward, the
slide was gently washed with neutralized buffer, and stained with
DAPI (Invitrogen) [23]. All results were performed from three
independent experiments. DNA damage was assessed by using free
CometScore software (Tritek Corp., Sumerduck, VA, USA).
with 10% FBS, 100 Units/mL penicillin and 100
m
g/mL streptomycin.
All cells were grown at 37 ꢀC or 34 ꢀC (hFOB cells) in a humidified
atmosphere comprised of 95% air and 5% CO2 [20e22].
3.3. Determination of cell viability by MTT assay
The cell viability was assessed using MTT assay. Cells
(1 ꢃ 104 cells/well) onto 96-well plates were exposed test
compounds, and 0.1% DMSO in media served as a vehicle control.
After a 48 h-incubation,100
ml MTT (0.5 mg/mL) solution was added
to each well, the plate was incubated at 37 ꢀC for 4 h. Then, 100
ml
0.04 N HCl in isopropanol was added and the absorbance at 570 nm
was measured for each well. The cell survival ratio was expressed as
% of control. All results were performed from three independent
experiments [21,22]. Cell viability was also used to examine in U-2
3.6. Assays for reactive oxygen species (ROS) and mitochondrial
membrane potential (DJm)
OS cells after pretreatment with 10 mM NAC, 1 mM cyclosporine A,
FasL mAb, and caspase-3, -8 and -9 inhibitors. U-2 OS cells seeded
at a density of 2.5 ꢃ 105 cells/well into 24-well plates were pre-
treated with NAC and cyclosporine A for 3 h or caspase-8, -9 and -3
The levels of ROS and DJm were determined by flow cytometry
using H2DCF-DA and DiOC6 as fluorescent probes, respectively. U-2
OS cells were seeded at a density of 2.5 ꢃ 105 cells/well into 24-well
inhibitors for 1 h, respectively, followed by treatment with 30
mM
plates and treated with 30
mM LJJ-10 for 0, 2, 6, and 12 h. Then cells
LJJ-10 and 0.1% DMSO as a control. Cells were then harvested after
LJJ-10 for 48 h exposure to determine the percentage of viable cells
as previously described [21,22].
were incubated with 5 mM of H2DCF-DA and 40 nM of DiOC6 at
37 ꢀC for 30 min and then analyzed by flow cytometry. The mean
fluorescence intensity (MFI) was quantities by BD CellQuest Pro

2716
M.-J. Hour et al. / European Journal of Medicinal Chemistry 46 (2011) 2709e2721
A
B
OMe
140
0 µM
10 µM
20 µM
40 µM
80 µM
120
100
80
60
40
20
0
HN
N
F
N
F
U-2 OS
hFOB
LJJ-10
Cell lines
C
LJJ-10 (15 µM)
LJJ-10 (30 µM)
LJJ-10 (45 µM)
Control
D
5
4
3
2
1
0
Control
LJJ-10 (30 µM )
Control 15
30
45
LJJ-10 (µM)
Fig. 1. Effects of LJJ-10 on cell viability, morphological changes, chromatin condensation and DNA damage in human osteosarcoma U-2 OS cells. The chemical structure of LJJ-10 (A).
For cell viability assay, U-2 OS cells were cultured and treated with 0, 10, 20, 40 and 80 M LJJ-10 for 48 h and the percentages of viable cells were detected by MTT assay described in
Materials and Methods (B). After incubation with 0, 15, 30 and 45 M LJJ-10 for 48 h, the cell morphological changes were examined and photographed under a phase-contrast
m
m
microscope (C). DNA damage was determined by Comet assay as described in Materials and Methods (D). Cells were examined and photographed using the fluorescent micros-
copy (ꢃ200). Data from three independent experiments were presented (*p < 0.05 as compared with control treatments).
software [24,25]. All results were performed from three indepen-
dent experiments.
caspase-8 and Z-LEHD-FMK for caspase-9). After treatment with
30 M LJJ-10 for 48 h, activities of caspase-3, -8 and -9 were
m
determined according to the manufacturer’s instructions (Caspase
colorimetric kit, R&D systems). Briefly, cells were harvested and
3.7. Detection of Fas and Fas ligand (FasL) by flow cytometry
U-2 OS cells (2.5 ꢃ 105 cells/well) were seeded into 24-well
lysed in 50
ml lysis buffer containing 2 mM DTT for 10 min. After
centrifugation, the supernatants containing 100
mg proteins were
incubated with caspase-3, -8 and caspase-9 substrate in reaction
buffer (R&D systems). Samples were incubated in a 96-well flat
bottom microplate at 37 ꢀC for 1 h. The levels of released pNA (Z-
DEVE-pNA, Z-IETD-pNA and Z-LEHD-pNA for caspase-3, -8 and -9,
respectively) were measured with an ELISA reader (Anthos Labtec.
Instruments) at a wavelength of 405 nm [26,27].
plates and incubated with 30 mM LJJ-10 for 12 h. Cells were then
harvested and washed twice by PBS. The cells were examined for
Fas and Fas ligand (FasL) by being stained with anti-Fas-fluorescein
isothiocyanate (FITC) and anti-FasL-FITC antibodies (BD Pharmin-
gen Inc. San Diego, CA, USA) and then incubated 1 h and analyzed
by flow cytometry [24].
3.8. Caspase-9, -8 and -3 activities assay
3.9. Western blotting analysis
Approximately 1 ꢃ107 cells of U-2 OS cells onto 75-T flask were
pretreated with/without a pan-caspase inhibitor (Z-VAD-FMK) and
selective inhibitors (Z-DEVE-FMK for caspase-3, Z-IETD-FMK for
Approximately 1 ꢃ107 cells of U-2 OS cells onto 75-T flask were
treated with 30 mM LJJ-10 for 0, 12, 24 and 48 h. Cells were har-
vested and the cytosolic proteins were collected as described

M.-J. Hour et al. / European Journal of Medicinal Chemistry 46 (2011) 2709e2721
2717
Fig. 2. Effects of LJJ-10 on the levels of Fas, FasL, reactive oxygen species (ROS) production and mitochondrial membrane potential (DJm). Cells were pretreated with N-ace-
tylcysteine, cyclosporine A, and anti-FasL monoclonal antibody for 3 h after exposure to LJJ-10, and then incubated for 48 h. Cells were collected to determine the percentage of
viable cells by MTT assay (A). U-2 OS cells were treated with 30
then analyzed by flow cytometry (B). U-2 OS cells were treated with 30
increased (C) and loss of DJm (D). Data from three independent experiments are presented (*p < 0.05 as compared with control treatments).
mM LJJ-10 for 12 h. Subsequently, cells were collected and incubated with Fas-FITC and FasL-FITC mAb for 1 h and
mM LJJ-10 for 0, 2, 6 and 12 h, and the data from flow cytometric analysis was shown that ROS production
elsewhere [28]. The cytosolic proteins (30
m
g) from each sample
-caspase-8, -casepase-9, -AIF, -Endo G, -Bcl-2, -Bax and -Bid (Santa
Cruz Biotechnology, Inc.) at 4 ꢀC overnight. Membranes were
washed three times with TBST for 10 min and incubated with
secondary HRP-conjugated antibody. The blots were developed by
using an ECL kit (Millipore Crop., Billerica, MA, USA) and Kodak Bio-
MAX MR film (Eastman Kodak). All results are from three inde-
pendent experiments. Quantification of each band of protein levels
was appeared by using the NIH ImageJ program [25,29].
were resolved on 12% sodium dodecylsulfate polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred to nitrocellulose
membrane by using the iBot Dry Blotting System (Invitrogen).
Membranes were soaked in blocking buffer (5% non-fat dry milk/
0.05% Tween 20 in 20 mM TBS at pH 7.6) at room temperature for
1 h and then were incubated with primary antibodies (anti-Fas/
CD95, -FasL, -FADD, -cytochrome c, -Apaf-1, -pro-caspase-3,

2718
M.-J. Hour et al. / European Journal of Medicinal Chemistry 46 (2011) 2709e2721
Fig. 3. Effects of LJJ-10 on caspase-8, caspase-9 and caspase-3 activity and apoptosis-associated protein levels in U-2 OS cells. Cells were pre-incubated with or without a pan-caspase
inhibitor(Z-VAD-FMK) for 1 h and then treated with 30 MLJJ-10for 48 h. After incubation, 55 g proteinswere incubatedwith caspase-3-, -8- and -9-specific substrates (Ac-DEVD-pNA,
Ac-IETD-pNA and Ac-LEHD-pNA) with reaction buffer in a 96-well plate at 37 ꢀC for 1 h. The release of pNA was measured at 405 nm by a spectrophotometer (A). U-2 OS cells were pre-
incubated with or without specific caspase inhibitors (Z-DEVE-FMK for caspase-3, Z-IETD-FMK for caspase-8 and Z-LEHD-FMK for caspase-9) for 1 h and then treated with 30 M LJJ-10
for 48 h. Cells were collected to determine the percentage of viable cells by MTT assay. Data from three independent experiments are presented (*p < 0.05 as compared with control
treatments) (B). Cells were treated with 30 M LJJ-10 for 0,12, 24 and 48 h, and cytosolic fractions were prepared and subjected to Western blotting. The resulting blots were probed for
the protein levels of Fas, FasL, FADD, caspase-8, caspase-3, cytochrome c, Apaf-1, AIF, Endo G, caspase-9, Bcl-2, Bax, Bid and Actin (C).
m
m
m
m
3.10. Statistical analysis
However, 1H NMR spectrum of 8e32 displayed signals at
d
¼ 9.27e9.98 ppm, except the compound 15 at 11.08 ppm, char-
Data are presented as the mean ꢁ S.E.M. for the indicated
acteristic for NH of anilinoquinazoline. The physical and spectral
number of separate experiments. Statistical analyses of data were
data of compounds 4e32 were demonstrated in Table 1.
done by one-way ANOVA, and
p < 0.05 was considered
significant.
4.2. 6-Fluoro-2-(3-fluorophenyl)-4-substituted anilinoquinazolines
induced growth inhibition in human cancer cells
4. Results
The anti-proliferation effects of compounds 8e32 on U-2 OS,
HL-60, U937, HT-29, Colo 205, MCF-7, MDA-MB-231 cells were eval-
uated by the MTT assay. The results, IC50 values of compounds 8e32
oncancercelllines,arepresentedinTable 2. Ourresultsdemonstrated
that the compounds 11e13, 16 and 18 had cytotoxic effects toward
a variety of cancer cells, but compound 16 was inactive in HT-29 and
Colo205cells,andcompound18 wasinactiveinMCF-7andMDA-MB-
231cells. Compounds25, 27 and28hadhighlyselectiveeffectsonU-2
OS cells, and compounds 26 and 29 had selective effects on HL-60,
U937 and U-2 OS cells. However, 6-fluoro-(3-fluorophenyl)-4-(3-
4.1. Chemistry
6-Substituted-2-(3-substituted phenyl)-4-quinazolinones (4e7)
and 6-substituted-2-(3-substituted phenyl)-4-substituted anilino-
quinazolines (8e32) were synthesized according to Schemes 1 and
2. Structural elucidation of compounds 4e7 and 8e32 was char-
acterized by correct elemental analysis and careful inspections of
spectral data. 1H NMR spectrum of 4e7 displayed signals at
d
¼ 12.54e12.72 ppm characteristic for NH of quinazolinone ring.

M.-J. Hour et al. / European Journal of Medicinal Chemistry 46 (2011) 2709e2721
2719
methoxyanilino) quinazoline (compound 13, LJJ-10; Fig. 1A) was the
most potent compound against human osteosarcoma U-2 OS cells.
cells (Fig. 3B). Our results suggest that LJJ-10-induced apoptotic cell
death is mediated through the caspase-8, caspase-9 and caspase-3
activations. In order to establish the all possible pathway that
involved in LJJ-10-induced apoptosis in U-2 OS cells, a series of
apoptosis-associated proteins were examined by Western blotting.
As shown in left panel of Fig. 3C, the protein levels of Fas/CD95,
FasL, FADD, and caspase-8 in U-2 OS cells were increased after
treatment with LJJ-10. Observing the protein level of pro-caspase-3
was decreased in LJJ-10-treated U-2 OS cells, we inferred that the
protein level of caspase-3 was increased. Based on these results, LJJ-
10-induced apoptosis in U-2 OS cells via the death receptor-
dependent pathway. In the right panel of Fig. 3C, we also showed
that the protein levels of cytochrome c, Apaf-1, caspase-9, Endo G,
AIF and Bax were increased. However, these of Bcl-2 and Bid were
decreased in LJJ-10-treated U-2 OS cells. The results indicated that
LJJ-10-induced apoptotic cell death in U-2 OS cells through the
mitochondria-dependent pathway. As illustrated above, we suggest
that LJJ-10-induced apoptosis in U-2 OS cells through death
receptor- and mitochondria-dependent signaling pathways.
4.3. LJJ-10-induced growth inhibition, apoptosis and DNA damage
in human osteosarcoma U-2 OS cells
We investigated the growth inhibition effects of LJJ-10 on U-2 OS
cells. As shown in Fig. 1B, exposure to various concentrations of LJJ-
10 (10, 20, 40 and 80 mM) for 48 h resulted in dose-dependent
decreases on cell number of U-2 OS relative to control cultures.
LJJ-10 caused little decrease on cell number of hFOB (Fig. 1B). The
IC50 of LJJ-10-induced U-2 OS cell death was 30.26 ꢁ 3.21
mM. LJJ-
10-induced reduction of cell viability could occur due to DNA
damage or apoptosis. It can be seen in Table 1 and Fig.1C and shown
that LJJ-10 with 30
mM made the U-2 OS cells rounding and
shrinking morphologically after 48 h-treatment. As shown in
Fig. 1D, LJJ-10-induced DNA damage of U-2 OS cells by the notice of
extension of the tails by comet assay (Fig. 1D, left). Furthermore,
analysis of the fluorescent intensity in the comet length of
extending tails revealed that the LJJ-10 worked in
a dose-
dependent manner on U-2 OS cells (Fig. 1D, right). Our data
suggest that LJJ-10-induced apoptotic death in U-2 OS cells.
Moreover, LJJ-10 induced selective cytotoxicity in U-2 OS cells but
had a less cytotoxic effect on human normal hFOB cells (Fig. 1B).
5. Discussion and conclusions
In this study, the newly synthesized compounds 8e18 were
assayed for anti-cancer activity against 7 types of human cancer cell
lines. The IC50 values calculated from the dose-response data
obtained from three independent experiments were listed in
Table 2. The results showed that compounds 11e13, 16 and 18
inhibited cell proliferation in most of the cell lines, but compounds
8e10, 14e15, 17, 19e24 and 30e32 were inactive. In terms of
SAR information, those anilinoquinazoline derivatives without
simultaneously 6- and 2-3-substitutions (8e10) were inactive and
this finding is the same as that of our previous study [33]. Addi-
tionally, compounds bearing F (16), OH (25, 26), OCH3 (12, 13) and
NH2 (27, 28, 29) substituents at 4-anilino moiety that may bind
with some important proteins in cancer cells were active. However,
compounds bearing CH3 (30, 31, 32), Cl (19, 20) and Br (21, 22, 23)
substituents at 4-anilino moiety that may have low potential for
binding with some proteins in cancer cells were inactive. Among
them, LJJ-10 induced significantly dose-dependent growth inhibi-
tion and apoptotic cell death, and the IC50 value of LJJ-10 in human
osteogenic sarcoma U-2 OS cells was much lower than that in
human fetal normal osteoblastic hFOB cells. Therefore, we suggest
that LJJ-10 could be an efficacious and safer anti-cancer agent for
treatment of human osteogenic sarcoma. LJJ-10 was supposed
worthwhile to be the anti-cancer lead and investigate its mecha-
nism. Moreover, further structural modification of compound 13
(LJJ-10) may lead to the discovery of more active anilinoquinazoline
with good selectivity against various cancer types.
4.4. LJJ-10 induced the death receptor- and mitochondria-
dependent apoptotic signaling pathways in U-2 OS cells
Apoptosis is the process of programmed cell death which may
originate in two major signaling pathways [30]. The death receptor
pathway (the extrinsic pathway) activates the death receptor on cell
surface (Fas/FasL) and then promotes caspase-8 activation [31,32]. In
the mitochondrial pathway, some mitochondrial changes are
occurred, including the decrease the DJm, elevated production of
ROS, release of cytochrome c, Apaf-1 and pro-caspase-9 proteins from
mitochondria to cytosol and then activation of caspases. To further
evaluate the role ofdeathreceptorand mitochondrial pathways inLJJ-
10-induced growth inhibition in U-2 OS cells, cells were pretreated
with a ROS scavenger (NAC), mitochondrial membrane potential
resting agent (cyclosporine A), and anti-FasL monoclonal antibody to
block LJJ-10-induced growth inhibition and cell death. As shown in
Fig. 2A, LJJ-10-induced growth inhibition and cell death was signifi-
cantly blocked by NAC, cyclosporine A and anti-FasL monoclonal
antibody. As shown in Fig. 2B, LJJ-10 increased the proportion of Fas
and FasL when comparing with that of the control and the isotype
control experiments. After exposure to 30 mM LJJ-10 for 2, 6 and 12 h,
U-2 OS cells were harvested for determining the ROS and DJm. As
shown in Fig. 2C and D, LJJ-10 promoted ROS production and
decreased the level of DJm in U-2 OS cells in a time-dependent
manner. Our results suggest that the death receptor- and
mitochondria-dependent apoptotic signaling pathways may be
involved in LJJ-10-induced cell death in U-2 OS cells.
Recently, Jantova et al. demonstrated that the use of anilino-
quinazoline derivatives to treat human leukemia cells, and had
revealed some molecular mechanisms include the induction of
caspase-3 activity and cell apoptosis [34]. In our study, we also
demonstrated that 30 mM of LJJ-10 significantly inhibited the cell
4.5. LJJ-10 stimulated caspase-8, caspase-9 and caspase-3 activity
in U-2 OS cells
growth and cell death (Fig. 1B) and triggered apoptosis (Fig. 1C and
D) through the activation of caspase-8, caspase-9 and caspase-3
cascades in U-2 OS cells (Fig. 3). Our results suggest that LJJ-10
induced anti-osteosarcoma effects through the caspase cascade-
dependent apoptotic signaling pathways.
Two apoptotic pathways, the death receptor pathway and the
death receptor pathway, were proved to be associated with the
activation of the caspase family proteins when cells undergo
apoptosis [35]. The death receptor pathway (extrinsic pathway) is
triggered by binding the ligand to its surface receptors such as FasL
and CD95/Fas, and then activating the caspase-8 and caspase-3
[36,37]. The mitochondrial dependent pathway (intrinsic
To investigate the mechanisms of LJJ-10-induced apoptosis, we
determined the caspase-8, caspase-9 and caspase-3 activities in LJJ-
10-treated U-2 OS cells. As shown in Fig. 3A, LJJ-10 caused an
increase of caspase-8, caspase-9 and caspase-3 activities in 48-h
treatment. LJJ-10-induced caspase-8, caspase-9 and caspase-3
activations were also significantly suppressed by a pan-caspase
inhibitor (Z-VAD-FMK). Pre-incubation with caspase-8, caspase-9
and caspase-3 specific inhibitors significantly reduced LJJ-10-
induced apoptotic cell death and growth inhibition in U-2 OS

2720
M.-J. Hour et al. / European Journal of Medicinal Chemistry 46 (2011) 2709e2721
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pathway) that triggered by anti-cancer drugs, DNA damage and ROS
involves the decrease of DJm, and the release of cytochrome c,
Apaf-1, caspase-9, Endo G and AIF. The released cytochrome c and
Apaf-1 form apoptosome and convert pro-caspase-9 into caspase-
9. The pro-caspase-3 is then activated by caspase-9 and finally
leads cells to apoptosis [38e40]. As shown in Fig. 3A, LJJ-10 induced
the cleavage and activation of the caspase-8, caspase-9, and
caspase-3 after 12 h-treatment in U-2 OS cells. From the observa-
tions, we suggest that LJJ-10-induced cell apoptosis through both
the death receptor- and mitochondria-dependent signaling path-
ways. Our results can support the following evidences: (1) after
pretreatment with NAC, cyclosporine A or FasL mAb, respectively,
and then cells were treated with LJJ-10. There is a significant
increase in cell viability (Fig. 2A). (2) LJJ-10 induced a time-
dependent increase protein levels of Fas/CD95 and FasL (Fig. 3C)
and then activation of both caspase-8 and caspase-3 (Fig. 3A). These
results suggest that the involvement of LJJ-10 in the death receptor
apoptotic pathway in U-2 OS cells. (3) The loss of DJm and ROS
production were mediated after LJJ-10 treatment for 2, 6 and 12 h
(Fig. 2C and D) and led to the release of cytochrome c, Apaf-1, pro-
caspase-9, Endo G and AIF from mitochondria into the cytosol
(Fig. 3B) and then activation of caspase-9 cascade (Fig. 3A and C).
Based on these results, we suggest that the mitochondria-
dependent pathway is also involved in LJJ-10-induced apoptosis
in U-2 OS cells. A signaling pathway that is involved in the cross-
talk between both mitochondria-dependent pathway and death
receptor apoptotic pathway has been substantiated by the obser-
vations that the activation of caspase-8 causes the cleavage of Bid
that in turn induces a subsequent activation of caspase-9 [41e43].
LJJ-10 exerts its effects through subsequent caspase-8 activation,
Bid cleavage, caspase-9 activation, and finally caspase-3 activation,
leading to apoptosis in U-2 OS cells.
In conclusion, LJJ-10 displays anti-osteosarcoma effects on U-2
OS cells by inducing cell apoptosis. Our studies have clearly pointed
out that the death receptor protein Fas/CD95 and the mitochondrial
environment are the targets of LJJ-10 and the effects of LJJ-10 on U-2
OS cells are through intrinsic and extrinsic apoptotic signaling
pathways. LJJ-10 might be selected as the lead compound of anti-
osteosarcoma agents for further structure modification in the
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Acknowledgment
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The investigation was supported by research grant from the
National Science Council of the Republic of China (NSC 99-2320-B-
039-013-MY3; NSC 97-2320-B-039-004-MY3) and grant from
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