Antioxidant xanthone derivatives induce cell cycle arrest and apoptosis and enhance cell death induced by cisplatin in NTUB1 cells associated with ROS

Article abstract of DOI:10.1016/j.ejmech.2011.01.043

In an effort to develop novel antioxidant as anticancer agents, a series of xanthones were prepared. In vitro screening, the synthetic xanthones revealed significant inhibitory effects on xanthine oxidase and ABTS radical-cation scavenging activity. The selective compounds 2 and 8 induced an accumulation of NTUB1 cells in the G₁ phase arrest and cellular apoptosis by the increase of ROS level. The combination of cisplatin and 2 significantly enhanced the cell death in NTUB1 cells. Compounds 2 and 8 did not show cytotoxic activity in selected concentrations against SV-HUC1 cells. The present results suggested that antioxidants 2 and 8 may be used as anticancer agent for enhancing the therapeutic efficacy of anticancer agents and to reduce their side effect.

Full text of DOI:10.1016/j.ejmech.2011.01.043

European Journal of Medicinal Chemistry 46 (2011) 1222e1231
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antioxidant xanthone derivatives induce cell cycle arrest and apoptosis and
enhance cell death induced by cisplatin in NTUB1 cells associated with ROS
,
,
,
Jen-Hao Cheng a 1, A-Mei Huang b 1, Tzyh-Chyuan Hour b, Shyh-Chyun Yang a
**
,
Yeong-Shiau Pu c, Chun-Nan Lin d
,
*
a School of Pharmacy, Kaohsiung Medical University, Kaohsiung 807, Taiwan
b Institute of Biochemistry, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan
c Department of Urology, College of Medicine, National Taiwan University, Taipei 100, Taiwan
d Faculty of Fragrance and Cosmetics, College of Pharmacy, 100 Shih-Chuan 1st Road, Kaohsiung Medical University, Kaohsiung 807, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
In an effort to develop novel antioxidant as anticancer agents, a series of xanthones were prepared. In
vitro screening, the synthetic xanthones revealed significant inhibitory effects on xanthine oxidase and
ABTS radical-cation scavenging activity. The selective compounds 2 and 8 induced an accumulation of
NTUB1 cells in the G1 phase arrest and cellular apoptosis by the increase of ROS level. The combination of
cisplatin and 2 significantly enhanced the cell death in NTUB1 cells. Compounds 2 and 8 did not show
cytotoxic activity in selected concentrations against SV-HUC1 cells. The present results suggested that
antioxidants 2 and 8 may be used as anticancer agent for enhancing the therapeutic efficacy of anticancer
agents and to reduce their side effect.
Received 9 September 2010
Received in revised form
19 January 2011
Accepted 25 January 2011
Available online 4 February 2011
Keywords:
Xanthone
Antioxidation
Cytotoxicity
Ó 2011 Elsevier Masson SAS. All rights reserved.
Reactive oxygen species
1. Introduction
Xanthine oxidase (XO) is a key enzyme that catalyzes the
oxidation of xanthine and hypoxanthine into uric acid and
Xanthones are found in natural products and synthetic chem-
istry. Xanthone derivatives are characterized with diverse biological
activities, including anti-inflammatory, anti-bacteria, anti-platelet,
anti-hypertensive, vasorelaxing and cytotoxic effects, reported in
the past decade [1e4]. In our previous papers, we have demon-
strated that various synthetic xanthones revealed potential phar-
macological activities [3e5].
Reactive oxygen species (ROS) are resulting in oxidation of
various cell constituents as DNA, lipid, and proteins and conse-
quently cause oxidative damage to cellular substance leading to cell
death [6]. The oxidative damage of DNA induced by ROS lead to
certain cancers, and ROS may also play a role in cell cycle
progression. ROS is implicated in numerous pathological events
including metabolic disorders, cellular aging, reperfusion damage
of DNA, inflammation, atherosclerosis and carcinogenesis [7].
induces hyperuricemia and gout [8]. Antioxidant therapies have
been recognized as a potential strategy for preventing acute CNS
injury, cardiovascular diseases and asthma [8]. In the investiga-
tion on antioxidant agent, we found that synthetic xanthones
significantly inhibited oxidative damage of DNA, XO activity, and
ABTS radical-cation scavenging activity. For continual discovery
of antioxidant agent as anticancer agent, we reported the
synthesis, antioxidant activity, cytotoxicity against NTUB1 cells
(human bladder cancer cell line) and SV-HUC1 cells (a SV-40
immortalized human uroepithelial cell line), and the structur-
eeactivity relationship of various synthetic xanthones in the
present paper.
2. Chemistry
As shown in Schemes 1 and 2, compounds 1 and 7 were
synthesized with appropriated benzoic acids, methoxybenzenes
and related reagents. A mixture of 1 or 7 and 1,3-dibromopropane
was reacted in appropriate solvents and then aminated with
appropriate amines respectively, to give the final products, 2e6 or
8e14 (Schemes 1 and 2) [5].
* Corresponding author. Tel.: þ886 7 3121101; fax: þ886 7 5562365.
** Corresponding author. Tel.: þ886 7 3121101; fax: þ886 7 3210638.
E-mail addresses: scyang@kmu.edu.tw (S.-C. Yang), lincna@cc.kmu.edu.tw
(C.-N. Lin).
1
These authors contributed equally to this work.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.01.043

J.-H. Cheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1222e1231
1223
OCH3
OCH3
H3CO
OCH3
COOH
COCl
a
b
O
O
O
O
c
OCH3
OCH3
O
OCH3
OH OCH3
H3CO HO
O
O
e
d
O
Br
O
OH
1
O
8
5
1
8a
9a
2
f
9
7
6
10
4a
10a
3
O
O
NR1
4
2-6
Scheme 1. Synthesis of aminated derivatives of 3-hydroxyxanthone. Reagents:(a) oxalyl chloride, anhydrous benzene, rt, 2 h; (b) AlCl3, anhydrous ether, rt, 8 h; (c) pyridine, 10%
tetramethylammonium hydroxide, reflux, 36 h; (d) phenol, HI, 160 ꢀC, 8 h; (e) NaOH, H2O, n-BuOH, 3-hydroxyxanthone, 1,3-dibromopropane, reflux, 5 h; (f) absolute EtOH,
appropriate amine, stirred, 60e70 ꢀC, 5 h. Substituents were described in Table 1.
3. Pharmacology
ABTS radical-cation scavenging activity. The selected compounds
2 and 8 with potent antioxidant activity were further tested for
cytotoxicity against NTUB1 and SV-HUC1 cells by MTT assay. The
cytotoxic activity of 2 and 8 combined with cisplatin was also
examined by MTT assay. For further evaluation of cytotoxic effect
All synthesized compounds were tested for their antioxidant
activity, including inhibition of oxidative DNA damage by agarose
gel electrophoresis, inhibitory effect on xanthine oxidase, and
OCH3
COOH
OCH3
H3CO
OCH3
COCl
a
b
H3CO
H3CO
H3CO
O
O
O
O
c
OCH3
H3CO
OCH3
H3CO
OCH3
OH OCH3
H3CO HO
O
O
e
d
HO
O
O
Br
HO
O
OH
7
O
f
HO
O
O
NR2
8-14
Scheme 2. Synthesis of aminated derivatives of 3,6-dihydroxyxanthone. Reagents:(a) oxalyl chloride, anhydrous benzene, rt, 2 h; (b) AlCl3, anhydrous ether, rt, 8 h; (c) pyridine, 10%
tetramethylammonium hydroxide, reflux, 36 h; (d) phenol, HI, 160 ꢀC, 8 h; (e) NaOH, H2O, n-BuOH, 3,6-dihydroxyxanthone, 1,3-dibromopropane, reflux, 5 h; (f) absolute EtOH,
appropriate amine, stirred, 60e70 ꢀC, 5 h. Substituents were described in Table 1.

1224
J.-H. Cheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1222e1231
and mechanisms of 2 and 8, and the combination of 2 and 8 with
cisplatin induced cell death in vitro, we first examined the effects
of 2 and 8 on the intracellular ROS amount in NTUB1 cells and cell
cycle progression determined by using fluorescence-activated cell
sorting (FACS).
4. Results and discussion
The inhibition of oxidative DNA damage by agarose gel elec-
trophoresis, the inhibitory effect on xanthine oxidase, and ABTS
radical-cation scavenging activity were used to evaluate the series
of synthesized xanthones as antioxidants. As shown in Fig. 1,
selected compounds, 2e6 and 8e11 showed protective effects of
oxidative DNA damage caused by OL2 generated by xanthine (XA)
ꢁ
and xanthine oxidase (XO). Compounds 2e6 and 8e11, each
Table 1
showed protective effects on oxidative DNA damage caused by O2L
ꢁ
Structure and basic data of xanthones.
at 300 mM.
O
O
O
For determination of the antioxidant activity of the series of
synthesized xanthones, ABTS radical-cation scavenging activity and
XO inhibitory activity of 2e6 and 8e14 also were analyzed. As
shown in Fig. 2, compound 2 with a piperidine ring substituted at
C-3 of propoxy side chain and allopurinol (positive control)
significantly inhibited the XO activity in a concentration-dependent
O
O
R1
HO
O
R2
8-14
2-6
manner with IC50 values of 37.8 ꢂ 3.9 and 1.9 ꢂ 0.7
mM respectively.
Compounds
R1
R2
Formula
Yield %
30
Compounds 3e6 also showed concentration-dependent manner
but possessed relatively weaker activities than that of 2 (Table 2). As
shown in Fig. 3, compounds 8e14 and allopurinol all inhibit the XO
activity in a concentration-dependent manner. Compound 14 with
a methylpiperazine ring substituted at C-3 of propoxy side chain
N
N
2
C21H23NO3
C20H21NO3
C21H24N2O3
showed significant activity with an IC50 value of 44.4 ꢂ 3.9
mM
3
4
40
35
(Table 2). According to Figs. 2 and 3, a OH (8e14) or without a OH
group substituted (2e6) at C-6 of xanthone moiety revealed no
significant difference in the inhibitory effects on XO activity, but
a piperidine or a piperazine ring substituted at C-3 of propoxy side
chain of xanthone moiety enhanced the inhibitory effect on XO
activity.
N
N
N
We also evaluated the ABTS radical-cation scavenging activities
of 2e6 and 8e14. As shown in Table 3, all synthesized xanthones
showed ABTS radical-cation scavenging activities except for 3.
Among them 8 and 13 possessed stronger ABTS radical-cation
NH
5
6
C20H22N2O3
38
43
scavenging activities with IC50 values of 69.3
65.0 ꢂ 4.1 M respectively than that of the positive control, vitamin
E (IC50 ¼ 77.2 ꢂ 3.5 M). In turn, we found that compounds 5, 8, 13,
ꢂ
2.6 and
N
C20H23NO3
m
m
and 14 with a piperidine or piperazine ring substituted at C-3 of
propoxy side chain showed significant ABTS radical-cation scav-
enging activities. As mentioned above, the synthesized xanthones
with a piperidine or a piperazine ring substituted at C-3 of propoxy
side chain enhanced the antioxidant activity.
N
N
8
9
C21H23NO4
33
39
The MTT microassay was used for the evaluation of cytotoxic
properties of selective compounds, 2 and 8, against NTUB1 cells.
Fig. 4 showed the cell viability of 2 and 8 assessed by the MTT assay
after treating with different concentrations of 2 and 8 for 72 h. The
C20H21NO4
N
H
IC50 values of 2 and 8 were 31.2 ꢂ 2.9 and 35.1 ꢂ 4.0
mM respec-
10
11
C19H19NO4
35
32
tively. The cell cycle progression of 2 and 8 was determined by
using fluorescence-activated cell sorting (FACS) analysis in propi-
dium iodide-stained NTUB1 cells. As shown in Figs. 5 and 6, NTUB1
N
H
cells were exposed to 40 and 60 mM of 2 and 8 respectively for 24 h.
C22H25NO4
Obviously, G1 arrest was induced, accompanied by an increased cell
apoptosis. It also suggested that 2 and 8 revealed same cell cycle
arrest in NTUB1 cells.
It was proven that ROS induced programmed cell death or
necrosis, grand effect on gene expression and activated cell
signaling cascades [9]. As shown in Fig. 7, NTUB1 cells were exposed
N
12
13
14
C20H23NO4
C20H22N2O4
C21H24N2O4
40
42
39
to 20 and 40
mM of 2 and 8 respectively for 24 h, leading to
NH
N
significant enhancement of intracellular ROS levels. ROS cause
adaptive cellular response to apoptosis or necrosis, depending on
the level of ROS. It was well known that cellular ROS were essential
to cell survival, but the effect of ROS on cells is complicated [10].
Experimentally, low concentrations of H2O2 caused a moderate
enhancement in proliferation of many tumor cell lines, while
higher levels resulted in slowed cell growth, cell cycle arrest,
N
N

J.-H. Cheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1222e1231
1225
Fig. 1. Inhibition of DNA strand breaks induced by Oꢃ2 (generated by XA/XO) in the presence of 2e6 and 8e11 studied by gel electrophoresis. Supercoiled plasmid pBR322 DNA
(500 ng) in phosphate buffer (pH 7.4) solution was incubated for 20 min with XA/XO acting as the control. Lane 1, DNA (without XA/XO); lane 2, control; lane 3, control þ SOD
ꢁ
(300
control þ 5 (300
m
M); lane 4, control þ quercetin (300
m
M) serving as positive control; lane 5, control þ 2 (300
m
M); lane 6, control þ 3 (300
m
M); lane 7, control þ 4 (300
m
M); lane 8,
m
M); lane 9, control þ 6 (300
m
M); lane 10, control þ 8 (300 M); lane 11, control þ 9 (300
m
m
M); lane 12, control þ 10 (300
m
M); lane 13, control þ 11 (300
mM).
apoptosis even necrosis [10]. Treatments of NTUB1 cells of 2 and 8
for 24 h exhibited G1 arrest and increased the ROS levels about
250% compared with the control (Fig. 8). It indicated that 2 and 8
mediated through increased ROS in NTUB1 cells induced G1 cell
cycle arrest and apoptosis.
To enhance the therapeutic efficacy of anticancer agents and to
reduce their side effect, antioxidants, such as pyrrolidine dithio-
carbamate, epigallocatechin gallate, genistein, and vitamin E could
enhance the cell death of various types of cancer cells [11].
Cisplatin has been successfully used as a chemotherapeutic
agent against malignant solid tumors in the head and neck region.
However, these have been barriers to the use of cisplatin in the
clinical setting of head and neck cancer including nephrotoxicity
and cisplatin resistance [12]. We hypothesized that antioxidants, 2
and 8 in combination with cisplatin may enhance tumor cell killing.
Thus we examined the effects of combination use of cisplatin
with 2 and 8 respectively by MTT assay. As shown in Fig. 9, cisplatin
(0.3e50
concentration of 50
may not be toxic for normal cells of urinary systems in selected
levels except for 2 in higher levels (>30 M). These results sug-
gested that antioxidant 2 combined with low concentration of
cisplatin may enhance the therapeutic efficacy of cisplatin and to
reduce the side effect induced by cisplatin.
m
M) while
2
showed non-cytotoxic except for the
m
M. It indicated that the clinic use of 2 and 8
m
5. Conclusion
In this study, we have synthesized a series of new compounds of
xanthone derivatives and studied on their biological activities of
antioxidation, xanthine oxidase inhibitory effect, ABTS radical
scavenging, and cytotoxicities. This series of compounds revealed
antioxidanteffects, among them, compounds 2 and 8 showed potent
antioxidant activities, and were selected for the determination of
cytotoxicities against both NTUB1 and SV-HUC1 cells, cell cycle
arrest, ROS level and combined use with cisplatin. The present
results suggested that antioxidant 2 combined with low concen-
tration of cisplatin may enhance the cell death of NTUB1 cells
induced bycisplatin and reduced the side effect induced bycisplatin.
in different concentrations (0.3, 1.0 and 3.0
mM) was cotreated with
15 and 30 M 2 respectively. It clearly indicated that 30
m
mM 2
combined with cisplatin in all selected concentrations revealed
synergistically significant cytotoxicities against NTUB1, while 8 did
not show the same effects (Fig. 10). It indicated that the use of
30 mM 2 combined cisplatin of low level led to more significant
cytotoxicity than that of cisplatin or 2 individually examined. The
reduced dose of cisplatin may contribute to the decrease of side
effects in clinic uses.
6. Experimental protocols
The MTT assay was also used for the evaluation of cytotoxicities
of 2 and 8 against SV-HUC1 cells. Fig. 11 showed the cell viability of
2 and 8 assessed by the MTT assay after treating with different
concentrations of 2 and 8 for 72 h, respectively. Compound 8
showed almost non-cytotoxic in all selected concentrations
6.1. Chemistry, general information
IR spectra were determined with a PerkineElmer system 2000
FTIR spectrophotometer. 1H (400 MHz) and 13C (400 MHz) NMR
spectra were recorded on a Varian UNITY-400 spectrometer, and
mass spectra were obtained on a JMX-HX 100 mass spectrometer.
Mass analyses were within ꢂ0.4% of the theoretical values, unless
otherwise noted. Chromatography was performed using a flash-
column technique on silica gel 60 supplied by E. Merck.
Table 2
Xanthine oxidase inhibitory activities of 2e6 and 8e14.a
Compounds
IC50 (mM)
2
3
4
5
6
8
9
10
11
12
13
14
37.8 ꢂ 3.9
65.5 ꢂ 2.6
47.3 ꢂ 3.3
108.7 ꢂ 6.3
56.5 ꢂ 2.1
82.3 ꢂ 2.8
68.8 ꢂ 2.4
63.0 ꢂ 1.4
72.9 ꢂ 5.4
53.5 ꢂ 2.8
91.9 ꢂ 3.7
44.3 ꢂ 3.9
1.9 ꢂ 0.7
Allopurinol
Fig. 2. Dose-dependent inhibition of XO by 2e6 and allopurinol. Data were presented
as means ꢂ s.e.m., n ¼ 3e6. ap < 0.05, bp < 0.01, and cp < 0.001 compared to the
control value, respectively.
a
Data were presented as means ꢂ s.e.m. (n ¼ 3).
Allopurinol was used as a positive control.

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J.-H. Cheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1222e1231
column chromatography (CHCl3:CH3OH) and recrystallized from
CH3OH to yield yellow needle crystals, 3-hydroxyxanthone (1.40 g,
6.0 mmol). 3-Hydroxyxanthone was reacted with dibromopropane
(1.21 g, 6.0 mmol) to give 1, 3-(3-bromopropoxy)xanthone.
Compound 1 was then refluxed with ethanol and corresponding
amines to yield 2e6. 2,4-Dimethoxybenzoic acid was treated as the
procedure mentioned above to yield 3-(3-bromopropoxy)-6-
hydroxyxanthone (7) and 3,6-di(3-bromopropoxy)xanthone.
Compound 7 was then refluxed with ethanol and corresponding
amines to yield 8e14.
6.2.1. 3-[3-(Piperidin-1-yl)-propoxy]xanthone (2)
Compound 1 (0.2 g, 0.60 mmol) in ethanol (30 mL) was added
piperidine (0.51 g, 5.99 mmol) and then refluxed for 6 h. The
mixture was purified by column chromatography (silica gel and
methanol/n-hexane, 9:1) to afford 2 (0.061 g, 0.18 mmol, 30%) as
yellow powder. IR (KBr) 1675, 1585 cmꢃ1; 1H NMR (CD3OD):
d 1.49
(2H, m, eN(CH2CH2)2CH2), 1.63 (4H, m, eN(CH2CH2)2e), 2.04 (2H,
m, eOCH2CH2e), 2.48 (4H, br.s, eN(CH2CH2)2e), 2.54 (2H, t,
J ¼ 6.0 Hz, eOCH2CH2CH2e), 4.14 (2H, t, J ¼ 6.0 Hz, eOCH2e), 6.96
(1H, dd, J ¼ 8.8, 2.0 Hz, H-2), 6.99 (1H, d, J ¼ 2.0 Hz, H-4), 7.41 (1H,
td, J ¼ 8.0, 1.2 Hz, H-7), 7.52 (1H, dd, J ¼ 8.8, 0.8 Hz, H-5), 7.77 (1H,
td, J ¼ 8.8, 1.6 Hz, H-6), 8.11 (1H, d, J ¼ 8.8 Hz, H-1), 8.20 (1H, dd,
Fig. 3. Dose-dependent inhibition of XO by 8e14 and allopurinol. Data were presented
as means ꢂ s.e.m., n ¼ 3e6. ap < 0.05, bp < 0.01, and cp < 0.001 compared to the
control value, respectively.
J ¼ 7.6, 1.2 Hz, H-8). 13C NMR (CD3OD):
d 25.2 (eN(CH2CH2)2CH2),
6.2. General procedure for the synthesis of xanthone derivatives
26.5 (eN(CH2CH2)2e), 27.2 (eOCH2CH2e), 55.5 (eN(CH2CH2)2e),
56.9 (eOCH2CH2CH2e), 68.3 (eOCH2e), 101.8 (C-4), 115.1 (C-2),
116.3 (C-9a), 119.0 (C-5), 122.7 (C-8a), 125.3 (C-7), 127.2 (C-8), 128.8
(C-1), 136.1 (C-6), 157.7 (C-10a), 159.6 (C-4a), 166.4 (C-3), 178.0 (C]
O). ESIMS m/z [M þ 1]þ 338. HRESIMS m/z [M þ 1]þ 338.1759 (calcd
for C21H24NO3, 338.1756).
2-Methoxybenzoic acid (2.0 g, 13.14 mmol) in anhydrous
benzene (60 mL) was treated with 5.0 mL of oxalyl chloride and
thoroughly stirred at room temperature. After 2 h, the solvent and
the excess reagent were removed under reduced pressure. The
residue, 2-methoxybenzoyl chloride was dissolved in anhydrous
ether (80 mL), 1,3-dimethoxybenzene (1.8 g, 13.03 mmol) and AlCl3
(5.0 g) were added. After stirring for 8 h at room temperature, the
mixture was hydrolyzed with ice-cold water (500 mL) containing
concentrated HCl (45 mL) and extracted with CHCl3. Solvent
removal and purification with gel-column chromatography (CHCl3)
gave yellow oil, 2-hydroxy-4-methoxy-20-methoxybenzophenone
and 2,4-dimethoxy-20-hydroxybenzophenone (2.20 g, 8.53 mmol,
65%). The yellow oil (2.20 g, 8.53 mmol) was treated with pyridine
(100 mL), H2O (50 mL) and aqueous 10% tetramethylammonium
hydroxide (45 mL). The mixture was refluxed for 36 h, poured into
ice, acidified with HCl, and extracted with ether. Purification with
gel-chromatography (CHCl3) and recrystallization (CHCl3) gave
colorless needle crystals, 3-methoxyxanthone (1.60 g, 7.08 mmol,
83%). A mixture of 3-methoxyxanthone (1.60 g, 7.08 mmol), phenol
(42 mL) and HI (35 mL) was refluxed at 160 ꢀC for 8 h, and the
reaction mixture was poured into aqueous NaHSO3 solution. The
resulting yellow precipitate was collected, purified by silica gel-
6.2.2. 3-[3-(Pyrrolidin-1-yl)-propoxy]xanthone (3)
Compound 1 (0.2 g, 0.60 mmol) in ethanol (30 mL) was added
pyrrolidine (0.43 g, 6.05 mmol) and then refluxed for 6 h. The
mixture was purified by column chromatography (silica gel and
methanol/n-hexane, 9:1) to afford 3 (0.078 g, 0.24 mmol, 40%) as
pale-brown powder. IR (KBr) 1675, 1581 cmꢃ1 1H NMR (CD3OD):
;
d
2.13 (4H, m, eN(CH2CH2)2), 2.32 (2H, m, eOCH2CH2e), 3.44 (4H,
br.s, eN(CH2CH2)2e), 3.46 (2H, t, J ¼ 7.6 Hz, eOCH2CH2CH2e), 4.29
(2H, t, J ¼ 6.0 Hz, eOCH2e), 7.05 (1H, dd, J ¼ 9.2, 2.4 Hz, H-2), 7.10
(1H, d, J ¼ 2.4 Hz, H-4), 7.45 (1H, td, J ¼ 8.0, 0.8 Hz, H-7), 7.57 (1H,
Table 3
Free radical scavenging activities of 2e6 and 8e14 of
ABTS.a
Compounds
IC50 (mM)
2
3
90.0 ꢂ 4.9
Inactive
4
5
6
8
9
10
11
12
151.9 ꢂ 1.6
75.1 ꢂ 2.5
84.9 ꢂ 2.1
69.3 ꢂ 2.6
88.8 ꢂ 1.8
107.9 ꢂ 2.7
105.8 ꢂ 1.6
80.5 ꢂ 2.4
65.0 ꢂ 4.1
76.5 ꢂ 1.8
77.2 ꢂ 3.5
13
14
Vitamin E
Fig. 4. Cytotoxicities of 2 and 8 against NTUB1 cells. Cell viability was assessed by the
MTT assay after treating with different concentrations for 72 h. The data were pre-
sented as means ꢂ S.D. (n ¼ 3). ap < 0.05, bp < 0.01, and cp < 0.001 compared to the
control value, respectively.
a
Data were presented as means ꢂ s.e.m. (n ¼ 3).
Vitamin E was used as a positive control.

J.-H. Cheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1222e1231
1227
Fig. 5. Flow cytometry analysis of 2- and 8-treated NTUB1 cells. NTUB1 cells (8 ꢄ 105 cells/10 cm dish) were treated with absence of compounds (control, A), 40
mM 2 (B), 60 mM 2
(C), 40 mM 8 (D) and 60 mM 8 (E), respectively, for 24 h. At the time indicated, cells were stained with propidium iodide (PI), and DNA contents were analyzed by flow cytometry,
apoptosis was measured by the accumulation of sub-G1 DNA contents in cells. Results were representative of three independent experiments.
dd, J ¼ 8.4, 0.8 Hz, H-5), 7.82 (1H, td, J ¼ 8.8, 1.6 Hz, H-6), 8.18 (1H, d,
J ¼ 8.8 Hz, H-1), 8.24 (1H, dd, J ¼ 8.0, 1.6 Hz, H-8). 13C NMR (CD3OD):
(C-9a), 119.1 (C-5), 122.7 (C-8a), 125.4 (C-7), 127.2 (C-8), 129.0 (C-1),
136.3 (C-6), 157.7 (C-10a), 159.6 (C-4a), 165.8 (C-3), 178.0 (C]O).
ESIMS m/z [M þ 1]þ 324. HRESIMS m/z [M þ 1]þ 324.1599 (calcd for
C20H22NO3, 324.1600).
d
24.0 (eN(CH2CH2)2), 26.9 (eOCH2CH2e), 53.6 (eOCH2CH2CH2e),
55.4 (eN(CH2CH2)2), 66.8 (eOCH2e), 102.2 (C-4), 115.0 (C-2), 116.7
6.2.3. 3-[3-(4-Methylpiperazino)-propoxy]xanthone (4)
Compound 1 (0.2 g, 0.60 mmol) in ethanol (30 mL) was added 1-
methylpiperazine (0.61 g, 6.09 mmol) and then refluxed for 6 h. The
mixture was purified by column chromatography (silica gel and
methanol/n-hexane, 9:1) to afford 4 (0.074 g, 0.21 mmol, 35%) as
pale-yellow powder. IR (KBr) 1675, 1585 cmꢃ1 1H NMR (CD3OD):
;
d
2.02 (2H, m, eOCH2CH2e), 2.29 (3H, s, eNCH3), 2.57 (2H, t,
J ¼ 7.6 Hz, eOCH2CH2CH2e), 2.57 (4H, br.s, eN(CH2CH2)2NCH3),
2.57 (4H, br.s, eN(CH2CH2)2NCH3), 4.13 (2H, t, J ¼ 6.4 Hz, eOCH2e),
6.90 (1H, d, J ¼ 9.6 Hz, H-2), 6.93 (1H, s, H-4), 7.39 (1H, td, J ¼ 8.4,
1.2 Hz, H-7), 7.48 (1H, dd, J ¼ 8.4, 0.4 Hz, H-5), 7.75 (1H, td, J ¼ 8.8,
1.6 Hz, H-6), 8.06 (1H, d, J ¼ 9.2 Hz, H-1), 8.18 (1H, dd, J ¼ 7.6, 1.2 Hz,
H-8). 13C NMR (CD3OD):
d 27.2 (eOCH2CH2e), 45.9 (eNCH3),
53.7 (eN(CH2CH2)2NCH3), 55.6 (eN(CH2CH2)2NCH3), 55.9
(eOCH2CH2CH2e), 68.1 (eOCH2e), 101.8 (C-4), 115.1 (C-2), 116.3 (C-
9a), 119.0 (C-5), 122.6 (C-8a), 125.2 (C-7), 127.1 (C-8), 128.8 (C-1),
136.1 (C-6), 157.6 (C-10a), 159.5 (C-4a), 166.4 (C-3), 177.9 (C]O).
ESIMS m/z [M þ 1]þ 353. HRESIMS m/z [M þ 1]þ 353.1867 (calcd for
C21H25N2O3, 353.1865).
Fig. 6. Cell cycle distribution of NTUB1 cells treated with 2 and 8 for 24 h in different
concentrations. Percentages of sub-G1 cells and cells in G0/G1, S, and G2/M phase were
shown. Data refer to a representative experiment one of three. CT (control). ap < 0.05,
and bp < 0.01 compared to the control value, respectively.
6.2.4. 3-[3-(Piperazino)-propoxy]xanthone (5)
Compound 1 (0.2 g, 0.60 mmol) in ethanol (30 mL) was added
piperazine (0.52 g, 6.04 mmol) and then refluxed for 6 h. The mixture

1228
J.-H. Cheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1222e1231
Fig. 7. The effect of 2 and 8 on the production of ROS in NTUB1 cells. (A) control, (B) 1 mM NAC, (C) 10
mM cisplatin, (D) 20 mM 2, (E) 40 mM 2, (F) 20 mM 8, (G) 40 mM 8. Cells were
treated with 1 mM NAC, 10 M cisplatin and different concentrations of 2 and 8 for 24 h, respectively, and the amount of ROS was assayed by H2DCFDA staining. Results were
m
repeated by three independent experiments.
was purified by column chromatography (silica gel and methanol/n-
hexane, 9:1) to afford 5 (0.077 g, 0.23 mmol, 38%) as pale-yellow
(C-4), 115.1 (C-2), 116.3 (C-9a), 119.0 (C-5), 122.7 (C-8a), 125.3 (C-7),
127.2 (C-8),128.8 (C-1),136.1 (C-6),157.7 (C-10a),159.6 (C-4a),166.5
(C-3), 178.0 (C]O). ESIMS m/z [M þ 1]þ 339. HRESIMS m/z [M þ 1]þ
339.1708 (calcd for C20H23N2O3, 339.1709).
powder. IR (KBr) 1684, 1587 cmꢃ1; 1H NMR (CD3OD):
d 2.04 (2H, m,
eOCH2CH2e), 2.51 (1H, br.s, eNH), 2.51 (4H, br.s, eN(CH2CH2)2NH),
2.57 (2H, t, J ¼ 7.6 Hz, eOCH2CH2CH2e), 2.87 (4H, t, J ¼ 4.8 Hz, eN
(CH2CH2)2NH), 4.16 (2H, t, J ¼ 6.0 Hz, eOCH2e), 6.96 (1H, dd, J ¼ 8.8,
2.4 Hz, H-2), 6.99 (1H, d, J ¼ 2.0 Hz, H-4), 7.41 (1H, td, J ¼ 8.0, 0.8 Hz,
H-7), 7.52 (1H, dd, J ¼ 8.4, 0.8 Hz, H-5), 7.77 (1H, td, J ¼ 8.4,1.6 Hz, H-
6), 8.11 (1H, d, J ¼ 9.2 Hz, H-1), 8.20 (1H, dd, J ¼ 7.6, 1.6 Hz, H-8). 13C
6.2.5. 3-[3-(Diethylamino)-propoxy]xanthone (6)
Compound 1 (0.2 g, 0.60 mmol) in ethanol (30 mL) was added
diethylamine (0.44 g, 6.02 mmol) and then refluxed for 6 h. The
mixture was purified by column chromatography (silica gel and
methanol/n-hexane, 9:1) to afford 6 (0.084 g, 0.26 mmol, 43%) as
NMR (CD3OD):
d 27.0 (eOCH2CH2e), 46.1 (eN(CH2CH2)2NH), 54.9
(eN(CH2CH2)2NH), 56.7 (eOCH2CH2CH2e), 68.2 (eOCH2e), 101.8
brown powder. IR (KBr) 1653, 1559 cmꢃ1; 1H NMR (CD3OD):
d 1.07

J.-H. Cheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1222e1231
1229
Fig. 10. Cytotoxicity of 8 combined with cisplatin against NTUB1 cells. Cell viability
was assessed by the MTT assay after treating with different concentrations of 8 and
Fig. 8. The enhancement of 2 and 8 on the production of ROS in NTUB1 cells assayed
by H2DCFDA staining under the treatments of 1 mM NAC, 10 M cisplatin and different
concentrations of 2 and 8 for 24 h respectively. CT (control). Data were presented as
means ꢂ S.D. (n ¼ 3). bp < 0.01, and cp < 0.001 compared to the control value,
respectively.
cisplatin respectively, and 15, 30 mM 8 cotreated with cisplatin of different concen-
m
trations for 72 h. The data were presented as means ꢂ S.D. (n ¼ 3). bp < 0.01, and
cp < 0.001 compared to the control value, respectively.
(6H, t, J ¼ 7.2 Hz, eN(CH2CH3)2), 1.98 (2H, m, eOCH2CH2e), 2.61
(4H, q, J ¼ 6.8 Hz, eN(CH2CH3)2), 2.67 (2H, t, J ¼ 6.0 Hz,
eOCH2CH2CH2e), 4.11 (2H, t, J ¼ 6.0 Hz, eOCH2e), 6.91 (1H, dd,
J ¼ 8.0, 1.6 Hz, H-2), 6.92 (1H, d, J ¼ 1.6 Hz, H-4), 7.38 (1H, td, J ¼ 8.0,
1.2 Hz, H-7), 7.48 (1H, dd, J ¼ 8.4, 0.8 Hz, H-5), 7.75 (1H, td, J ¼ 8.8,
2.0 Hz, H-6), 8.07 (1H, d, J ¼ 8.0 Hz, H-1), 8.17 (1H, dd, J ¼ 8.4, 1.6 Hz,
was purified bycolumn chromatography (silica gel and methanol) to
afford 8 (0.067 g, 0.19 mmol, 33%) as yellow powder. IR (KBr) 3446,
1653, 1560 cmꢃ1
; d 1.54 (2H, m, eN
1H NMR (CD3OD):
(CH2CH2)2CH2), 1.69 (4H, m, eN(CH2CH2)2CH2), 2.08 (2H, m,
eOCH2CH2e), 2.64 (4H, br.s, eN(CH2CH2)2CH2), 2.69 (2H, t,
J ¼ 7.6 Hz, eOCH2CH2CH2e), 4.13 (2H, t, J ¼ 6.0 Hz, eOCH2e), 6.71
(1H, d, J ¼ 2.4 Hz, H-5), 6.80 (1H, dd, J ¼ 9.2, 2.4 Hz, H-7), 6.90 (1H, d,
J ¼ 2.4 Hz, H-4), 6.92 (1H, dd, J ¼ 7.6, 2.4 Hz, H-2), 8.01 (1H, d,
H-8). 13C NMR (CD3OD):
d 11.4 (eN(CH2CH3)2), 26.9 (eOCH2CH2e),
47.8 (eN(CH2CH3)2), 50.2 (eOCH2CH2CH2e), 68.3 (eOCH2e), 101.8
(C-4), 115.0 (C-2), 116.3 (C-9a), 119.0 (C-5), 122.6 (C-8a), 125.2 (C-7),
127.1 (C-8), 128.8 (C-1), 136.0 (C-6), 157.6 (C-10a), 159.6 (C-4a),
166.4 (C-3), 177.9 (C]O). ESIMS m/z [M þ 1]þ 326. HRESIMS m/z
[M þ 1]þ 326.1753 (calcd for C20H24NO3, 326.1756).
J ¼ 8.8 Hz, H-8), 8.07 (1H, d, J ¼ 8.4 Hz, H-1).13C NMR (CD3OD):
d 24.7
(eN(CH2CH2)2CH2), 26.1 (eN(CH2CH2)2CH2), 26.8 (eOCH2CH2e),
55.3 (eN(CH2CH2)2CH2), 56.7 (eOCH2CH2CH2e), 67.9 (eOCH2e),
101.8 (C-5), 103.6 (C-4), 114.2 (C-9a), 114.3 (C-7), 116.4 (C-8a), 116.5
(C-2),128.6 (C-8),128.8 (C-1),159.5 (C-10a),160.2 (C-4a),165.6 (C-6),
168.8 (C-3), 177.4 (C]O). ESIMS m/z [M þ 1]þ 354. HRESIMS m/z
[M þ 1]þ 354.1702 (calcd for C21H24NO4, 354.1705).
6.2.6. 3-Hydroxy-6-[3-(piperidin-1-yl)-propoxy]xanthone (8)
Compound 7 (0.2 g, 0.57 mmol) in ethanol (30 mL) was added
piperidine (0.49 g, 5.76 mmol) and then refluxed for 6 h. The mixture
Fig. 9. Cytotoxicity of 2 combined with cisplatin against NTUB1 cells. Cell viability was
assessed by the MTT assay after treating with different concentrations of
2
and
Fig. 11. Cytotoxicities of 2 and 8 against SV-HUC1 cells. Cell viability was assessed by
the MTT assay after treating with different concentrations of 2 and 8 for 72 h. The data
were presented as means ꢂ S.D. (n ¼ 3). bp < 0.01 compared to the control value,
respectively.
cisplatin respectively, and 15, 30 mM 2 cotreated with cisplatin of different concen-
trations for 72 h. The data were presented as means ꢂ S.D. (n ¼ 3). bp < 0.01, and
cp < 0.001 compared to the control value, respectively.

1230
J.-H. Cheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1222e1231
6.2.7. 3-Hydroxy-6-[3-(pyrrolidin-1-yl)-propoxy]xanthone (9)
Compound 7 (0.2 g, 0.57 mmol) in ethanol (30 mL) was added
pyrrolidine (0.41 g, 5.76 mmol) and then refluxed for 6 h. The
mixture was purified by column chromatography (silica gel and
methanol) to afford 9 (0.075 g, 0.22 mmol, 39%) as brown powder.
(4H, q, J ¼ 7.2 Hz, eN(CH2CH3)2), 2.81 (2H, t, J ¼ 6.0 Hz,
eOCH2CH2CH2e), 4.14 (2H, t, J ¼ 6.0 Hz, eOCH2e), 6.65 (1H, d,
J ¼ 2.4 Hz, H-4), 6.76 (1H, dd, J ¼ 8.8, 2.0 Hz, H-2), 6.92 (1H, dd,
J ¼ 8.8, 2.4 Hz, H-7), 6.94 (1H, d, J ¼ 2.4 Hz, H-5), 7.98 (1H, d,
J ¼ 8.8 Hz, H-1), 8.07 (1H, d, J ¼ 8.8 Hz, H-8). 13C NMR (CD3OD):
d 9.8
IR (KBr) 3461, 1653, 1569 cmꢃ1; 1H NMR (CD3OD):
d
1.84 (4H, m, eN
(eN(CH2CH3)2), 25.4 (eOCH2CH2e), 46.7 (eN(CH2CH3)2), 49.0
(eOCH2CH2CH2e), 66.7 (eOCH2e), 100.6 (C-4), 102.6 (C-5), 112.0
(C-9a), 113.0 (C-2), 115.2 (C-8a), 116.5 (C-7), 127.3 (C-1), 127.4 (C-8),
158.3 (C-4a), 159.3 (C-10a), 164.3 (C-3), 170.2 (C-6), 176.2 (C]O).
ESIMS m/z [M þ 1]þ 342. HRESIMS m/z [M þ 1]þ 342.1708 (calcd for
C20H24NO4, 342.1705).
(CH2CH2)2), 2.02 (2H, m, eOCH2CH2e), 2.63 (4H, m, eN(CH2CH2)2),
2.71 (2H, t, J ¼ 7.6 Hz, eOCH2CH2CH2e), 4.06 (2H, t, J ¼ 6.0 Hz,
eOCH2e), 6.54 (1H, d, J ¼ 2.4 Hz, H-5), 6.69 (1H, dd, J ¼ 8.8, 2.4 Hz,
H-7), 6.82 (1H, d, J ¼ 2.4 Hz, H-4), 6.84 (1H, dd, J ¼ 8.0, 2.4 Hz, H-2),
7.92 (1H, d, J ¼ 8.8 Hz, H-8), 8.01 (1H, d, J ¼ 8.0 Hz, H-1). 13C NMR
(CD3OD):
d
24.1 (eN(CH2CH2)2), 29.0 (eOCH2CH2e), 54.0
(eOCH2CH2CH2e), 55.0 (eN(CH2CH2)2), 67.7 (eOCH2e), 101.6 (C-5),
104.2 (C-4), 111.9 (C-9a), 113.8 (C-7), 116.3 (C-8a), 119.0 (C-2), 128.3
(C-8), 128.3 (C-1), 159.3 (C-10a), 160.8 (C-4a), 165.2 (C-6), 174.5 (C-
3), 177.2 (C]O). ESIMS m/z [M þ 1]þ 340. HRESIMS m/z [M þ 1]þ
340.1551 (calcd for C20H22NO4, 340.1549).
6.2.11. 3-Hydroxy-6-[3-(piperazino)-propoxy]xanthone (13)
Compound 7 (0.2 g, 0.57 mmol) in ethanol (30 mL) was added
piperazine (0.50 g, 5.81 mmol) and then refluxed for 6 h. The
mixture was purified by column chromatography (silica gel and
methanol) to afford 13 (0.085 g, 0.24 mmol, 42%) as pale-yellow
powder. IR (KBr) 3446, 1684, 1569 cmꢃ1 1H NMR (CD3OD):
; d 2.03
6.2.8. 3-[3-(Cyclopropylamino)-propoxy]-6-hydroxyxanthone (10)
Compound 7 (0.2 g, 0.57 mmol) in ethanol (30 mL) was added
cyclopropylamine (0.33 g, 5.78 mmol) and then refluxed for 6 h. The
mixture was purified by column chromatography (silica gel and
methanol) to afford 10 (0.065 g, 0.20 mmol, 35%) as brown powder.
(2H, m, eOCH2CH2e), 2.50 (4H, br.s, eN(CH2CH2)2NH), 2.56 (2H, t,
J ¼ 7.6 Hz, eOCH2CH2CH2e), 2.87 (4H, t, J ¼ 4.8 Hz, eN
(CH2CH2)2NH), 4.14 (2H, t, J ¼ 6.0 Hz, eOCH2e), 6.49 (1H, d,
J ¼ 2.4 Hz, H-5), 6.64 (1H, dd, J ¼ 8.8, 2.0 Hz, H-7), 6.90 (1H, dd,
J ¼ 8.8, 2.4 Hz, H-2), 6.94 (1H, d, J ¼ 2.4 Hz, H-4), 7.91 (1H, d,
J ¼ 8.8 Hz, H-8), 8.07 (1H, d, J ¼ 8.8 Hz, H-1). 13C NMR (CD3OD):
IR (KBr) 3420, 1684, 1569 cmꢃ1 1H NMR (CD3OD):
; d 0.40 (2H, m,
eNHCH(CHaHb)2), 0.51 (2H, m, eNHCH(CHaHb)2), 2.04 (2H, m,
eOCH2CH2e), 2.21 (1H, m, eNHCHe), 2.89 (2H, t, J ¼ 7.2 Hz,
eOCH2CH2CH2e), 4.15 (2H, t, J ¼ 6.4 Hz, eOCH2e), 6.77 (1H, dd,
J ¼ 8.8, 2.4 Hz, H-2), 6.91 (1H, dd, J ¼ 8.4, 2.4 Hz, H-7), 6.92 (1H, d,
J ¼ 2.0 Hz, H-5), 6.97 (1H, d, J ¼ 2.4 Hz, H-4), 7.99 (1H, d, J ¼ 8.8 Hz,
d
27.0 (eOCH2CH2e), 46.0 (eN(CH2CH2)2NH), 54.8 (eN
(CH2CH2)2NH), 56.7 (eOCH2CH2CH2e), 67.9 (eOCH2e), 101.7 (C-5),
104.4 (C-4), 110.9 (C-9a), 113.7 (C-7), 116.4 (C-8a), 120.2 (C-2), 128.1
(C-8), 128.3 (C-1), 159.4 (C-10a), 161.3 (C-4a), 165.2 (C-6), 177.2 (C-
3), 177.2 (C]O). ESIMS m/z [M þ 1]þ 355. HRESIMS m/z [M þ 1]þ
355.1660 (calcd for C20H23N2O4, 355.1658).
H-1), 8.06 (1H, d, J ¼ 8.4 Hz, H-8). 13C NMR (CD3OD):
d
5.9 (eNHCH
(CHaHb)2),
29.8
(eOCH2CH2e),
31.3
(eNHCHe), 47.3
(eOCH2CH2CH2e), 68.1 (eOCH2e), 101.7 (C-4), 103.7 (C-5), 113.7 (C-
9a), 114.3 (C-2), 116.3 (C-8a), 117.1 (C-7), 128.5 (C-1), 128.6 (C-8),
159.5 (C-4a), 160.3 (C-10a), 165.6 (C-3), 170.2 (C-6), 177.4 (C]O).
ESIMS m/z [M þ 1]þ 326. HRESIMS m/z [M þ 1]þ 326.1395 (calcd for
C19H20NO4, 326.1392).
6.2.12. 3-Hydroxy-6-[3-(methylpiperazylamino)-propoxy]xanthone
(14)
Compound 7 (0.2 g, 0.57 mmol) in ethanol (30 mL) was added
methylpiperazine (0.58 g, 5.79 mmol) and then refluxed for 6 h.
The mixture was purified by column chromatography (silica gel
and methanol) to afford 14 (0.082 g, 0.22 mmol, 39%) as yellow
6.2.9. 3-[3-(Cyclohexylamino)-propoxy]-6-hydroxyxanthone (11)
Compound 7 (0.2 g, 0.57 mmol) in ethanol (30 mL) was added
cyclohexylamine (0.57 g, 5.75 mmol) and then refluxed for 6 h. The
mixture was purified by column chromatography (silica gel and
methanol) to afford 11 (0.067 g, 0.18 mmol, 32%) as brown powder.
powder. IR (KBr) 3380, 1675, 1583 cmꢃ1 1H NMR (CD3OD):
; d 2.04
(2H, m, eOCH2CH2e), 2.32 (3H, s, eNCH3), 2.55 (4H, br.s, eN
(CH2CH2)2NCH3), 2.55 (4H, br.s, eN(CH2CH2)2NCH3), 2.60 (2H, t,
J ¼ 7.6 Hz, eOCH2CH2CH2e), 4.17 (2H, t, J ¼ 6.4 Hz, eOCH2e), 6.79
(1H, d, J ¼ 2.4 Hz, H-5), 6.86 (1H, dd, J ¼ 8.8, 2.4 Hz, H-7), 6.96
(1H, dd, J ¼ 8.8, 2.4 Hz, H-2), 6.99 (1H, d, J ¼ 2.4 Hz, H-4),
8.06 (1H, d, J ¼ 8.8 Hz, H-8), 8.10 (1H, d, J ¼ 8.8 Hz, H-1). 13C NMR
IR (KBr) 3420, 1684, 1571 cmꢃ1 1H NMR (CD3OD):
; d 1.17 (2H, m,
eNHCH(CH2CH2)2CH2), 1.30 (4H, m, eNHCH(CH2CH2)2CH2), 1.67
(1H, m, eNHe), 1.81 (2H, m, eOCH2CH2e), 2.04 (4H, m, eNHCH
(CH2CH2)2CH2), 2.72 (1H, m, eNHCHe), 2.96 (2H, t, J ¼ 7.2 Hz,
eOCH2CH2CH2e), 4.05 (2H, t, J ¼ 6.0 Hz, eOCH2e), 6.51 (1H, d,
J ¼ 2.4 Hz, H-4), 6.67 (1H, dd, J ¼ 8.8, 2.4 Hz, H-2), 6.76 (1H, d,
J ¼ 2.4 Hz, H-5), 6.78 (1H, dd, J ¼ 8.8, 2.4 Hz, H-7), 7.91 (1H, d,
J ¼ 8.8 Hz, H-1), 7.97 (1H, d, J ¼ 8.8 Hz, H-8). 13C NMR (CD3OD):
(CD3OD):
(CH2CH2)2NCH3),
d
27.3 (eOCH2CH2e), 45.9 (eNCH3), 53.6 (eN
55.6 (eN(CH2CH2)2NCH3), 55.9
(eOCH2CH2CH2e), 68.0 (eOCH2e), 101.8 (C-5), 103.3 (C-4), 114.6
(C-7), 115.1 (C-9a), 115.4 (C-2), 116.3 (C-8a), 128.7 (C-8), 128.9 (C-
1), 159.6 (C-10a), 159.9 (C-4a), 165.9 (C-6), 166.6 (C-3), 177.5 (C]
O). ESIMS m/z [M þ 1]þ 369. HRESIMS m/z 369.1815 (calcd for
C21H25N2O4, 369.1814).
d
25.9 (eNHCH(CH2CH2)2CH2), 26.7 (eNHCH(CH2CH2)2CH2), 28.8
(eOCH2CH2e), 32.2 (eNHCH(CH2CH2)2CH2), 43.8 (eNHCHe), 58.2
(eOCH2CH2CH2e), 67.4 (eOCH2e), 101.6 (C-4), 104.2 (C-5), 111.9 (C-
9a), 113.7 (C-2), 116.4 (C-8a), 119.2 (C-7), 128.3 (C-1), 128.3 (C-8),
159.2 (C-4a), 160.9 (C-10a), 164.9 (C-3), 174.7 (C-6), 177.1 (C]O).
ESIMS m/z [M þ 1]þ 368. HRESIMS m/z [M þ 1]þ 368.1861 (calcd for
C22H26NO4, 368.1862).
6.3. Assay of XO inhibitory activity
The XO activity with xanthine as the substrate was measured at
25 ꢀC, according to the protocol of Kong and others [13] with
modification. The assay mixture consisting of 50
mL of test solution,
6.2.10. 3-[3-(Diethylamino)-propoxy]-6-hydroxyxanthone (12)
Compound 7 (0.2 g, 0.57 mmol) in ethanol (30 mL) was added
diethylamine (0.42 g, 5.74 mmol) and then refluxed for 6 h. The
mixture was purified by column chromatography (silica gel and
methanol) to afford 12 (0.078 g, 0.23 mmol, 40%) as pale-brown
60 L of 70 mM phosphate buffer (pH 7.5), and 30 m
m
L of enzyme
solution (0.1 unit/mL in 70 mM phosphate buffer (pH 7.5)) was
prepared immediately before use. After preincubation at 25 ꢀC for
15 min, the reaction was initiated by addition of 60
mL of substrate
solution (150 M xanthine in the same buffer). The reaction was
m
powder. IR (KBr) 3446, 1675, 1569 cmꢃ1
;
1H NMR (CD3OD):
d
1.13
monitored for 5 min at 295 nm. The XO activity was expressed as
micromoles of uric acid per minute.
(6H, t, J ¼ 7.2 Hz, eN(CH2CH3)2), 2.03 (2H, m, eOCH2CH2e), 2.73

J.-H. Cheng et al. / European Journal of Medicinal Chemistry 46 (2011) 1222e1231
1231
6.4. ABTS radical scavenging assay
USA) in PBS at room temperature for 30 min. The fractions of cells in
each phase of cell cycle were analyzed using FACScan flow
cytometer and Cell Quest software (Becton Dickinson).
The scavenging activity of ABTS was measured according to the
method described by Ng et al. [14] with some modifications. Briefly,
ABTS was dissolved in deionized water to 7 mM in concentration,
which was then mixed with 2.45 mM potassium persulfate.
6.8. Quantitative analysis of intracellular reactive oxygen species
(ROS)
The scavenging activity was determined by mixing with 180
mL
of ABTS and 40 L of selected compounds and positive control
(vitamin E), followed by measuring absorbance at 630 nm.
m
Production of ROS was analyzed by flow cytometry as described
previously (Pu at al., [18]). Briefly, cells were plated in 6-well plates.
10 mM dichlorofluorescein diacetate (DCFH-DA; Molecular Probes,
6.5. Cell culture
Eugene, OR, USA) was added to the treated cells 30 min prior
harvest. The cells were collected by trypsinization and washed with
PBS. The green fluorescence of intracellular DCF (20,70-dichloro-
fluorescein) was then analyzed by FACScan flow cytometer with
a 525-nm band pass filter (Becton Dickinson).
NTUB1, a human bladder carcinoma cell line, was established
from a high-grade bladder cancer by Yu et al. [15]. NTUB1 and
stable transfected cells were maintained in RPMI 1640 medium
(Invitrogen, USA) supplemented with 10% fetal bovine serum,
100 unit/mL penicillin-G, 100
mg/mL streptomycin and 2 mM L-
6.9. Statistical analysis
glutamine. SV-HUC1 cells (a SV-40 immortalized human uroepi-
thelial cell line) were obtained from ATCC and maintained in F12
medium supplemented with 10% fetal bovine serum, 100 unit/mL
Data were expressed as the means ꢂ S. D. Statistical analysis was
performed using student’s t-test method for two group compar-
ison. P < 0.05 was considered to be statistically significant.
penicillin-G, 100 mg/mL streptomycin and 2 mM L-glutamine. The
cells were cultured at 37 ꢀC in a humidified atmosphere containing
5% CO2.
Acknowledgments
6.6. Cytotoxicity analysis by MTT assay
This work was partially supported by a grant from the National
Science Council of Republic of China (NSC 98e2320eBe037e018).
Cellular cytotoxicity of tested compounds was performed by
using
a modified 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte-
trazolium bromide (MTT, Sigma Chemical Co., USA) (Hour et al.
[16]). Briefly, the cells were plated at a density of 1 ꢄ 103 cells/well
in 96-well plates and incubated at 37 ꢀC overnight before drug
exposure. Cells were then cultured in the presence of graded
concentrations of compounds at 37 ꢀC for 72 h. At the end of the
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