Design, synthesis and biological evaluation of novel 1-hydroxyl-3- aminoalkoxy xanthone derivatives as potent anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2014.07.076

A series of novel 1-hydroxyl-3-aminoalkoxy xanthone derivatives were designed, synthesized and evaluated for in vitro anticancer activity against four selected human cancer cell lines (nasopharyngeal neoplasm CNE, liver cancer BEL-7402, gastric cancer MGC-803, lung adenocarcinoma A549). Most of the synthesized compounds exhibit effective cytotoxic activity against the four tested cancer cell lines with the IC₅₀ values at micromolar concentration level. Some preliminary structure-activity relationships were also discussed. In this series of derivatives, compound 3g shows excellent broad spectrum anticancer activity with IC₅₀ values ranging from 3.57 to 20.07 μM. The in vitro anticancer activity effect and action mechanism of compound 3g on human gastric carcinoma MGC-803 cell were further investigated. The results showed that compound 3g exhibits dose- and time-dependent anticancer effects on MGC-803 cells through apoptosis, which might be associated with its decreasing intracellular calcium and the mitochondrial membrane potential.

Full text of DOI:10.1016/j.ejmech.2014.07.076

European Journal of Medicinal Chemistry 85 (2014) 487e497
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of novel 1-hydroxyl-3-
aminoalkoxy xanthone derivatives as potent anticancer agents
Zheng-Min Yang , Jun Huang , Jiang-Ke Qin , Zhi-Kai Dai b , Wen-Li Lan , Gui-Fa Su ,
a
a
a
,
*
,
*
a
a
a
a
Huang Tang , Feng Yang
Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmaceutical Sciences,
Guangxi Normal University, Guilin 541004, PR China
a
b
Department of Pharmacology, Guilin Medical University, Guilin 541004, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 November 2013
Received in revised form
A series of novel 1-hydroxyl-3-aminoalkoxy xanthone derivatives were designed, synthesized and
evaluated for in vitro anticancer activity against four selected human cancer cell lines (nasopharyngeal
neoplasm CNE, liver cancer BEL-7402, gastric cancer MGC-803, lung adenocarcinoma A549). Most of the
synthesized compounds exhibit effective cytotoxic activity against the four tested cancer cell lines with
the IC50 values at micromolar concentration level. Some preliminary structureeactivity relationships
were also discussed. In this series of derivatives, compound 3g shows excellent broad spectrum anti-
21 July 2014
Accepted 21 July 2014
Available online 22 July 2014
cancer activity with IC50 values ranging from 3.57 to 20.07 mM. The in vitro anticancer activity effect and
Keywords:
Xanthone
Synthesis
Anticancer activity
Structureeactivity relationships
Apoptosis
Intracellular calcium
Mitochondrial membrane potential
action mechanism of compound 3g on human gastric carcinoma MGC-803 cell were further investigated.
The results showed that compound 3g exhibits dose- and time-dependent anticancer effects on MGC-
803 cells through apoptosis, which might be associated with its decreasing intracellular calcium and the
mitochondrial membrane potential.
© 2014 Published by Elsevier Masson SAS.
1. Introduction
possessing excellent antiproliferative activities were obtained
[4,7,8]. Younghwa Na and his co-workers reported a series of
Xanthone, also named 9H-xanthene-9-one, is a fundamentally
structural framework of active ingredients isolated from many
medicinal plants and microorganism. It has a planar tricyclic skel-
eton, in which one pyran ring is fused with two phenyl rings on
both sides (Fig. 1). Owing to this kind of simple and interesting
structural scaffolds and variable substituents on the rings,
xanthone derivatives show broad pharmacological activities such
epoxypropoxy-substituted xanthone derivatives which show sig-
nificant cytotoxic, topoisomerase inhibitory and DNA cross-linking
activities [9e11]. Due to the planar pharmacophore structure of the
xanthone, its anticancer activity was related with its interaction
with DNA and can be dramatically altered by the ring substituents
and their positions [3]. Though the biological mechanism of action
has not been thoroughly investigated, some xanthone derivatives
are currently undergoing clinical trials for cancer treatment. Among
them, an impressive natural xanthone analogue is psorospermin
(Fig. 1), the xanthone group can interact with DNA base pairs, while
the epoxide group can alkylate N7-guanine of DNA in the presence
of topoisomerase II [12,13]. Another synthetic xanthone known as
antitumor drug candidates is 5,6-dimethylxanthone-4-acetic acid
(DMXAA) (Fig. 1). This compound could interact with diverse bio-
logical targets via various actions and it had entered phase II clinical
trials and shown promising activity against malignant tumors
as antioxidant activity [1,2], anticancer activity [3,4], a-glucosidase
inhibitory activity [5], antibacterial and antifungal activity [6]. Their
interesting structural scaffold and pharmacological importance
have attracted many scientists to isolate or synthesize these com-
pounds as novel drug candidates [3].
In recent years, the development of xanthone derivatives as
effective anticancer drug candidates has attracted considerable
attention, lots of natural or synthetic xanthone compounds
[14e16].
Modifying natural compounds with efficient synthetic methods
and calibrating their pharmacologically active structure were
*
Corresponding authors.
E-mail addresses: jiangkeq@sina.com, jiangkeq@163.com (J.-K. Qin), dzhk110@
126.com (Z.-K. Dai).
regarded to be a key method to find new anticancer drug. In
http://dx.doi.org/10.1016/j.ejmech.2014.07.076
223-5234/© 2014 Published by Elsevier Masson SAS.
0

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Z.-M. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 487e497
O
O
OCH3
O
9
O
O
8
5
1
8
4
a
b
8b
4a
7
6
2
3
O
H
OH
O
H C
3
4
CH3
CH COOH
2
O
Xanthone
Psorospermin
DMXAA
Fig. 1. Chemical structures of some reported xanthone derivatives.
previous works of our groups [17,18] and others [9], 1,3-
dihydroxylxanthone could be used as an important lead com-
pound for chemical modification to obtain potent anticancer can-
2. Results and discussion
2.1. Chemistry
didates. The physicochemical and biological activities of
a
pharmacophore could usually be improved by the introduction of a
nitrogen-containing side chain. Considering that aminoalkoxy
groups are conveniently introduced onto 1,3-dihydroxyxanthone's
pharmacophore via simple etherification and amination reactions,
a series of novel 1,3-dihydroxyxanthone derivatives with their
terminal amines linked by different carbon spacers were designed,
synthesized and evaluated for their anticancer activity in vitro. The
primary aim of this work was to gain some insight into the effect of
terminal amines and linker length on the anticancer activities of
these compounds, and further indicate an interesting structur-
eeactivity relationship for the search of potential anticancer drugs.
In addition, cell and molecular biological experiments, such as cell
growth curves, morphological examination and colonial measure-
ment, etc., were carried out to further investigate the anticancer
effects of the most potent compound 3g on human gastric carci-
noma MGC-803 cell and the preliminary mechanism was also
discussed.
The synthetic route of 1-hydroxyl-3-aminoalkoxy xanthone
derivatives is shown in Scheme 1. 1,3-dihydroxylxanthone was
obtained in a yield of 52% from the condensation reaction of sali-
cylic acid with phloroglucinol in the presence of anhydrous zinc
chloride and phosphorus oxychloride [17e19]. Later, the ether-
ification of the hydroxyl group in the position
dihydroxyxanthone with 1, -dibromoalkane was carried out in
CO /acetone to offer the key intermediates 2ae2e, which
3 of 1,3-
u
K
2
3
possessed various side chains with different length. Then, com-
pounds 2ae2e were treated with appropriate alkyl amines in
ethanol under reflux to yield the target compounds 3ae3i, which
were finally quaternized with methyl iodide in chloroform to
obtain the corresponding quaternary ammonium salts 4ae4i with
improved water solubility. The twenty-three new compound
2ae2e, 3ae3i, 4ae4i were characterized by elemental analysis, IR,
NMR and MS. The NMR assignments were provided explicitly in the
section of spectral data.
OH
O
OH
COOH
i
ii
+
OH
O
HO
OH
O
1
OH
OH
O
O
OH
(
)n
iii
iv
O
O
Br
( )n
R1
O
N
R2
2a~2e, n=2~6 respectively
3a~3i
O
OH
(
)n
N
R1
R2
O
O
I
4a~4i
a
b
c
d
e
f
g
i
h
n
2
3
4
5
6
4
N
4
4
4
R1
O .
N
N
N
N
N
N
N
N
N
R2
ꢀ
Scheme 1. The synthetic route of 1-hydroxyl-3-aminoalkoxy xanthone derivatives. Reagents and conditions: (i) ZnCl
appropriate amine, EtOH, reflux 2 h; (iv) CH I, CHCl , r. t., 24 h.
2
, POCl
3
, 60 C, 2 h; (ii) Br(CH
2
)
n
Br, K
2
CO
3
reflux 24 h; (iii)
3
3

Z.-M. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 487e497
489
2
2
.2. Biological activity
tetrahydropyrrol ring via different length of linker, were synthe-
sized to investigate the influence of the length of side chains on the
anticancer activities. The results showed that the length of side
chains had no obviously regular influence on their anticancer ac-
.2.1. MTT assay and structureeactivity relationships
Except for the insoluble lead compound 1, the antiproliferative
activities of all of the other synthesized compounds were tested
in vitro on four human tumor cell lines, including CNE (nasopha-
ryngeal neoplasm), BEL-7402 (liver cancer), MGC-803 (gastric
cancer) and A549 (lung adenocarcinoma). 5-flurouracil (5-Fu) was
used as the reference compound. The results were shown as IC50
value in Table 1.
2 4
tivities. Compound 3c, bearing a linker of e(CH ) e between
xanthone pharmacophore and terminal amino group, showed
potent inhibitory activities on the four tested cancer cell lines.
Based on the potent inhibitory activities of compound 3c and our
previous experience [18], the length of the linker between
xanthone pharmacophore and terminal amino group was fixed as
The results in Table 1 indicated that most of the tested com-
pounds exhibit effective cytotoxic activities against the four tested
cancer cell lines with IC50 values at micromole concentration level.
In the case of bromoalkoxyl substituted xanthone derivatives
2 4
e(CH ) e in the next step. That is, compounds 3c and 3fe3i con-
taining four carbon spacers were tested on cancer cell lines for
investigating the influence of different amino substituents on the
antiproliferative activity. The IC50 values were shown in Fig. 2, it
was obviously that compound 3g with diethylamine substituent
exhibit the best inhibitory activity in most cases, with IC50 value in
(2ae2e), no obvious cytotoxic activities were observed. It suggested
that the introduction of bromoalkoxyl group to xanthone ring core
was not helpful to improve its anticancer activity. In contrast,
aminoalkoxy xanthone derivatives 3ae3i show excellent anti-
the range of 3.57e20.07
activities were observed for the morpholinyl substituted compound
3i, with IC50 higher than 100 M for all the tested cancer cell line,
mM. Interestingly, no notable inhibitory
cancer activities with IC50 values in the range of 1.24e88.42
mM
m
(
3
except for compound 3i). Comparing the IC50 values of compounds
ae3i with those of compounds 2ae2e, it was obviously that the
similar to our previous reports of other xanthones [17,18]. From
the IC50 values of this series, it strongly suggested that the
different terminal amino groups at the side chains had obvious
impact on their anticancer activities, showing an order of
diethylaminyl > dimethylaminyl > pyrrolidinyl > piperidinyl >
morpholinyl.
introduction of terminal amino groups could greatly improve their
anticancer activities, which confirmed our designing idea. But for
the case of corresponding quaternary ammonium derivatives
4
ae4i, their anticancer activities became weaker relative to com-
pounds 3ae3i. It might be due to that good water solubility of
compounds 4ae4i led to poor cytomembrane penetration.
To optimize the structure favorable for anticancer activities, two
important structural factors were explored, including the types of
terminal amino groups and the length of methylene linkers. Firstly,
for compounds 3ae3e, the xanthone ring core linked with terminal
Although further studies are necessary, a structureeactivity
relationship could be roughly established based on the discussion
mentioned above. (1) The introduction of a N-containing side chain
such as aminoalkoxy group to xanthone pharmacophore can
greatly increase its anticancer activity, but further quaternarization
of the amino group might decline its anticancer capability. (2) The
Table 1
Cytotoxic activity of the compounds 2ae4i against four tested cancer cells.
Compounds
IC50
CNE
(mM)
BEL-7402
MGC-803
A549
2
2
2
2
2
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
5
a
b
c
d
e
a
b
c
d
e
f
g
h
i
a
b
c
d
e
f
g
h
i
>100
>100
>100
>100
67.9
21.5
28.9
22.7
24.4
>100
5.63
6.66
4.50
3.81
5.32
5.99
3.57
28.18
>100
83.34
9.66
8.04
12.73
14.81
38.09
4.61
5.13
17.61
7.76
>100
>100
>100
>100
>100
12.85
11.64
18.51
5.99
85.61
>100
>100
11.32
6.25
5.97
12.11
1.24
77.2
>100
14.61
13.03
20.83
18.16
17.94
20.32
20.07
54.43
>100
>100
>100
>100
>100
23.89
>100
>100
>100
>100
19.84
8.94
8.83
7.57
3.81
3.63
16.20
>100
65.88
>100
>100
3.88
88.42
>100
>100
>100
19.49
10.71
9.95
83.88
12.99
19.21
>100
7.62
4.82
38.48
>100
26.51
92.94
15.07
-Fu

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Z.-M. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 487e497
compound 3g at different concentrations for 24, 48, and 72 h,
respectively, and then the cell viability was measured by MTT assay.
Cell viability was calculated as a percentage of untreated cells
(
100%). Human MGC-803 cells treated with compound 3g showed a
time- and dose-dependent proliferation inhibition in vitro (Fig. 3A).
At concentrations higher than1 M, cell viability was statistically
m
significantly decreased (all P < 0.05). The half maximal inhibitory
concentration (IC50) of compound 3g on MGC-803 cells for 24, 48
and 72 h were 11.89, 3.57 and 1.82
mM, respectively. Moreover,
inverted phase contrast microscopy was used to evaluate the cell
survival and apoptotic changes of MGC-803 cells treated with
compound 3g for 72 h (Fig. 3B). The results showed that when
MGC-803 cells were exposed to compound 3g at 100 mM, the cells
showed shrinkage and deformation while distribution of the cells
was markedly sparseness as compared with the control group. To
observe the delayed cytotoxic effects of this agent on MGC-
803 cells, clonogenic survival assay was carried out. Colony for-
mation was calculated as a percentage of untreated cells (100%).
Experimental results revealed long-term proliferation inhibition
effect of compound 3g against MGC-803 cells by compound 3g in a
dose-dependent manner (Fig. 3C). Cell viability decreased signifi-
Fig. 2. Effects of terminal amino groups on cells proliferation.
cantly when the concentration of compound 3g was above 1 mM (all
P < 0.001). These results indicated that compound 3g might be a
potential anticancer agent.
length of the linker between xanthone pharmacophore and the
terminal amino group has no obviously regular influence on its
anticancer activity. (3) The anticancer activity of aminoalkoxy
xanthone derivatives were greatly influenced by the structure of
the terminal amino group.
Because compound 3g was the most potent derivative pos-
sessing broad anticancer activities among all of the synthesized
compounds, so its anticancer effect and preliminary anticancer
mechanism on MGC-803 cell lines were further discussed in the
following paragraphs.
2.2.3. Compound 3g induced apoptosis against MGC-803 cells
Apoptosis is the process of programmed cell death that may
occur in multicellular organisms. Biochemical events lead to char-
acteristic cell changes (morphology) and death. These changes
include blebbing, cell shrinkage, nuclear fragmentation, chromatin
condensation, chromosomal DNA fragmentation and so on. Previ-
ous studies have showed that inducing apoptosis is one of the
anticancer mechanisms of nature xanthone products [4]. In this
study, apoptosis analysis of MGC-803 cells treated with compound
3g were observed under a fluorescence microscope after being
stained by Hochest 33258/Propidium iodide (PI). Hoechst 33258
limitedly passes the plasma membrane with intact membranes and
2.2.2. In vitro anticancer effect of compound 3g on human gastric
carcinoma MGC-803 cell
The anticancer effect of compound 3g on human gastric carci-
noma MGC-803 cell was determined by MTT, morphological ex-
amination and clonogenic assay. MGC-803 cells were treated with
Fig. 3. Effects of compound 3g on MGC-803 cell proliferation. A. Cell viability of MGC-803 cells treated with 3g for 24 h, 48 h, and 72 h. Values were mean ± SD (n ¼ 5); results are
representative of three independent experiments. A two-tailed t-test was carried out. B. Images of MGC-803 cells with 3g treated for 72 h (a) MGC-803 cells untreated as control, (b)
MGC-803 cells treated with 3g at 100
Values were mean ± SD (n ¼ 3); results are representative of three independent experiments.
m
M, (c) MGC-803 cells treated with 5-Fu at 100
m
M. Original magnification ꢁ100. C. 3g inhibited the long-term proliferation of MGC-803 cells.

Z.-M. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 487e497
491
readily enters cells with damaged membranes and stains DNA blue;
whereas PI, a highly polar dye which is impermeable to cells with
preserved membranes, stains DNA red. The nuclei of the living cells
were shown light blue. Apoptotic cells were identified by nuclear
condensation and/or fragmentation depending on the stage in the
process (bright blue fluorescence indicates an early phase in
apoptosis where the cell membrane is still intact, while a red
fluorescence is an index for cells in the late phase of apoptosis,
where the cell membrane integrity is lost). Necrotic cells were
characterized by homogeneous red nucleus. After MGC-803 cells
were exposed to compound 3g, cell shrinkage was occurred in
morphology as described above. With the incubation time pro-
longing to 24 h, nuclear condensation was observed. These char-
acteristic cell changes indicated that compound 3g induced
apoptosis of MGC-803 cells. More than 1000 cells were counted,
and apoptosis percentage was calculated according to it. Treatment
with compound 3g for 24 h induced MGC-803 cell apoptosis in a
dose-dependent manner (Fig. 4). It appears that apoptosis associ-
ated cell death might be a main mechanism for compound 3g to
exhibiting proliferative inhibition to cancer cells.
2
þ
2.2.4. Compound 3g affected [Ca ]i of MGC-803 cells
2þ
Intracellular Ca homeostasis is required for maintenance of
2þ
cell survival. Apoptosis can be brought about by a loss of Ca ho-
meostatic control, but can also be finely tuned, positively or
2
þ
negatively, by more subtle changes in Ca
distribution within
2
þ
intracellular compartments. In its dual role, Ca can either protect
cells or cause their demise [20]. A biphasic mechanism of increased
[Ca ] has been reported in many studies, where moderate [Ca ]i
2þ
2þ
i
2
þ
elevations are antiapoptotic [21] but excessive [Ca ] elevations
i
2
þ
are proapoptotic [22]. Diminutions in the concentrations of Ca in
cytosol, endoplasmic reticulum, or other intracellular compart-
ments either directly or indirectly regulate apoptosis [23]. In this
2
þ
work, we measured [Ca ]i using the calcium indicator fura-2 AM,
Fig. 4. Effect of compound 3g on apoptosis in MGC-803 cells. (A) Morphological features of apoptosis and necrosis were examined after MGC-803 cells were exposed to 3g for 24 h.
B) Apoptosis percentage. Data are means ± SD. *P < 0.05; **P < 0.01.
(

4
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Z.-M. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 487e497
2þ
Ca2 -sensitive fluorescent indicator, to examine whether [Ca
þ
]
i
change were involved in the compound 3g induced cancer cell
apoptosis. F340,510/F380,510, which represents relative changes in
intracellular [Ca2 ], was visualized under a fluorospectropho-
þ
tometer. After MGC-803 cells were treated with compound 3g at 0,
2
þ
]
i
1
0, 50 and 100
ined. Distinct decrease of F340, 510/F380, 510 with compound 3g
treated at 10, 50 and 100 M was showed in a dose-dependent
manner when comparing with that in control (Fig. 5). The [Ca
m
M, respectively, altered [Ca
levels were exam-
m
2
þ
i
]
changes induced by compound 3g might be associated with one or
more mechanisms including inhibiting calcium efflux from intra-
cellular calcium pond, inducing calcium uptaking of endoplasmic
reticulum (ER) or/and mitochondria, chelating intracellular calcium
and other mechanisms. Further studies are needed to elucidate the
potential mechanism.
2
.2.5. Compound 3g changed Djm of MGC-803 cells
Mitochondrial dysfunction has been shown to participate in the
Fig. 6. Effect of compound 3g on Dj of MGC-803 cells.
induction of apoptosis and has even been suggested to be central to
the apoptotic pathway. Opening of the mitochondrial permeability
transition pore has been demonstrated to induce depolarization of
the transmembrane potential (Djm), release of apoptogenic factors
and loss of oxidative phosphorylation. Loss of Djm may be an early
event or a consequence of the apoptotic-signaling pathway [24]. In
this study, we measured mitochondrial membrane potential using
the cationic fluorophore R123 to examine whether mitochondrial
membrane potential change was involved in the compound 3g-
induced cancer cell apoptosis. For quantitation of R123 fluores-
cence as a semiquantitative assay of Dj, fluorescent intensity of
cells with compound 3g addition was normalized to baseline values
of control. Dj decrease of MGC-803 cells treated with compound
anticancer activity shown by compound 3g prompts our further
investigation of the in vitro anticancer effect on human gastric
carcinoma MGC-803 cell as well as its action mechanism. Com-
pound 3g exhibited dose- and time-dependent anticancer effects
on MGC-803 cells through apoptosis, which might be associated
with its decreasing intracellular calcium and the mitochondrial
membrane potential. The 1-hydroxyl-3-aminoalkoxy xanthone
derivatives have simple structure and are easy to be synthesized.
These finding encouraged us to further optimize their structure and
investigate their in vivo efficacy in animal model as well as the
detail pharmacological mechanism.
4. Experiment section
3
g at 10, 50 and 100
mM was showed in a dose-dependent manner
when compared with the control (Fig. 6).
4.1. General
3
. Conclusion
All the solvents and reactants were of analytical grade and were
used without further purification unless noted. All reactions were
detected by thin layer chromatography (TLC) and spots were
visualized by UV lamp or in iodine chamber. Melting points were
measured in X-4 micro-melting point instrument and were un-
corrected. IR spectra were taken on Nicolet ESP 360 FI-IR using KBr
pellets. Direct MS spectra were performed on ESQUIRE HTC in-
In summary,
a novel series of 1-hydroxyl-3-aminoalkoxy
xanthone derivatives were designed, synthesized and evaluated
for anticancer activities against four human cancer cell lines. The
results showed that most of the compounds exhibited effective
cytotoxic activities against the four tested cancer cell lines with the
IC50 values at micromolar concentration level. The dimethylamine
substituted compound 3g demonstrated potent inhibitory activity
with IC50 values of 3.63, 3.57, 7.57 and 20.07 against CNE, MGC-803,
A549 and BEL-7402 respectively. The broad and significant
1
13
strument. H NMR and C NMR spectra were recorded in CDCl
DMSO-d on Bruker AVANCE AV 500/125 MHz instruments.
Chemical shifts were reported as ppm using tetramethylsilane
3
or
6
d
(TMS) as the internal standard and couplings expressed in Hertz.
Spin multiplicities were given as follows: s (singlet), d (doublet), t
(triplet), m (multiplet), or br (broad). Column chromatography was
accomplished on Qingdao silica gel (100e200 or 200e300 mesh).
4.2. Procedures for the synthesis of 1, 3-dihydroxyxanthone (1)
1
, 3-dihydroxyxanthone (1) was prepared according to the
ꢀ
literature [17,19]. Yield: 52%; m.p. 260e262 C; IR (KBr),
654, 1610, 1570, 1491, 1470, 1445, 1222, 1163, 1078, 827, 762 cm
H NMR (500 MHz,
H, 3-OH), 8.10 (dd, J ¼ 1.5, 8.0 Hz, 1H, H-8), 7.80e7.83 (m, 1H, H-6),
.55 (d, J ¼ 8.0 Hz, 1H, H-5), 7.42e7.45 (m, 1H, H-7), 6.37 (d,
J ¼ 2.1 Hz, 1H, H-4), 6.20 (d, J ¼ 2.1 Hz, 1H, H-2).
n
: 3327,
ꢂ1
1
;
1
6
d, ppm, DMSO-d ): 12.80 (s, 1H, OH-1), 11.07 (s,
1
7
4
.3. General procedure for the synthesis of -bromoalkoxy
u
substituted xanthones (2ae2e)
To a mixture of 1, 3-dihydroxyxanthone (1.01 g, 4.4 mmol) and
CO (1.2 g, 8.8 mmol) in anhydrous acetone (15 mL), 1, 2-
Fig. 5. Effect of compound 3g on [Ca2þ
]
level of MGC-803 cells.
K
2
i
3

Z.-M. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 487e497
493
dibromoethane or 1, 3-dibromopropane or 1, 4-dibromobutane or
, 5-dibromopentane or 1, 6-dibromohexane (each 6.2 mmol) was
(OCH
IR (KBr)
2 2 2 2 2 2 2
), 33.4 (CH Br), 32.4 (OCH CH ), 28.2(CH CH Br), 24.7 (CH );
1
n
ꢂ1
: 3461, 2943, 1662, 1608, 1572, 1481, 1299, 1166, 1038,
þ
added by a syringe. The reaction mixture was stirred under reflux
for about 24 h until the starting material disappeared by TLC. After
the reaction was completed, the reaction mixture was filtered,
washed with acetone (30 mL) and then the filtrate was concen-
trated to afford the crude product, which could be recrystallized in
acetone to give compound 2ae2e respectively.
822 cm ; APCI-MS m/z: 377 [MþH] . Anal. Calcd for C18
4
H17BrO : C,
57.31; H, 4.54. Found: C, 57.29; H, 4.56.
4.3.5. 3-(6-Bromohexyloxy)-1-hydroxy-9H-xanthen-9-one (2e)
Yield: 51% from compound 1 and 1, 6-dibromohexane; a yellow
solid; m.p.: 139e140 C; H NMR (500 MHz, d, ppm, CDCl ): 12.85
3
ꢀ
1
(
s, 1H, OH), 8.24 (dd, J ¼ 1.7, 8.0 Hz,1H, H-8), 7.71 (ddd, J ¼ 1.7, 7.2,
4.3.1. 3-(2-Bromoethoxy)-1-hydroxy-9H-xanthen-9-one (2a)
8.5 Hz, 1H, H-6), 7.42 (d, J ¼ 8.5 Hz, 1H, H-5), 7.35e7.39 (m, 1H, H-7),
Yield: 51% from compound 1 and 1, 2-dibromoethane; a yellow
6.42 (d, J ¼ 2.2 Hz, 1H, H-4), 6.33 (d, J ¼ 2.2 Hz, 1H, H-2), 4.05 (t,
ꢀ
1
solid; m.p.: 184e186 C; H NMR (500 MHz,
d, ppm, CDCl
3
): 12.87
J ¼ 6.4 Hz, 2H, OCH
2H, OCH CH
OCH CH CH
2
), 3.43 (t, J ¼ 6.7 Hz, 2H, CH
2
Br), 1.88e1.94 (m,
N), 1.50e1.56 (m, 4H,
N); C NMR (125 MHz, , ppm, CDCl ):
(
8
6
s, 1H, OH), 8.25 (dd, J ¼ 1.7, 7.9 Hz, 1H, H-8), 7.72 (ddd, J ¼ 1.7, 7.2,
2
2 2 2
), 1.81e1.87 (m, 2H, CH CH
1
3
.6 Hz, 1H, H-6), 7.43 (d, J ¼ 8.6 Hz, 1H, H-5), 7.40e7.36 (m, 1H, H-7),
2
2
2
CH
2
CH
2
CH
2
d
3
.45 (d, J ¼ 2.3 Hz, 1H, H-4), 6.35 (d, J ¼ 2.3 Hz, 1H, H-2), 4.38 (t,
180.8 (C-9), 166.3 (C-3), 163.6 (C-1), 157.8 (C-4a), 156.1 (C-4b), 134.9
(C-6), 125.9 (C-8), 124.0 (C-7), 120.7 (C-8a), 117.6 (C-5), 103.4 (C-8b),
1
3
J ¼ 6.3 Hz, 2H, OCH
2
), 3.67 (t, J ¼ 6.3 Hz, 2H, BrCH
2
); C NMR
(
125 MHz,
d
, ppm, CDCl
3
): 180.9 (C-9), 165.0 (C-3), 163.7 (C-1), 157.8
97.4 (C-2), 93.3 (C-4), 68.5(OCH
28.9 (CH CH Br), 27.9 (OCH CH
(KBr) : 3427, 2944, 1668, 1602, 1570, 1466, 1296, 1178, 1076,
2
), 33.6 (CH
2
Br), 32.7 (OCH
2 2
CH ),
(
8
C-4a), 156.0 (C-4b), 135.1 (C-6), 125.9 (C-8), 124.1 (C-7), 120.7 (C-
a),117.6 (C-5), 104.2 (C-8b), 97.4 (C-2), 93.4 (C-4), 68.1 (CH O), 28.2
: 3438, 2940, 1656, 1608, 1570, 1464, 1287, 1161,
2
2
2
2
CH ), 25.2 (CH
2
2
CH CH Br); IR
2
2
2
n
ꢂ1
þ
(
CH
2
Br); IR (KBr)
n
823 cm ; APCI-MS m/z: 391 [MþH] . Anal. Calcd for C19
4
H19BrO :
ꢂ1
þ
1
C
081, 824 cm ; ESI-MS m/z: 335 [MþH] . Anal. Calcd for
C, 58.33; H, 4.89. Found: C, 58.27; H, 4.93.
15 4
H11BrO : C, 53.76; H, 3.31. Found: C, 53.59; H, 3.33.
4.4. General procedure for the synthesis of 1-hydroxyl-3-
4.3.2. 3-(3-Bromopropoxy)-1-hydroxy-9H-xanthen-9-one (2b)
aminoalkoxy xanthone derivatives (3ae3i)
Yield: 58% from compound 1 and 1, 3-dibromopropane; a pale
ꢀ
1
yellow solid; m.p.: 119e121 C; H NMR (500 MHz,
2.85 (s, 1H, OH), 8.24 (dd, J ¼ 1.7, 7.9 Hz, 1H, H-8), 7.71 (ddd, J ¼ 1.7,
.3, 8.6 Hz, 1H, H-6), 7.42 (d, J ¼ 8.6 Hz, 1H, H-5), 7.37 (dd, J ¼ 7.3,
.9 Hz, 1H, H-7), 6.43 (d, J ¼ 2.2 Hz, 1H, H-4), 6.34 (d, J ¼ 2.2 Hz, 1H,
H-2), 4.20 (t, J ¼ 5.9 Hz, 2H, OCH ), 3.61 (t, J ¼ 6.4 Hz, 2H, BrCH ),
.32e2.41 (m, 2H, OCH CH CH , ppm,
CDCl ): 180.8 (C-9), 165.7 (C-3), 163.6 C-1), 157.7 (C-4a), 156.0 (C-
b), 135.0 (C-6), 125.8 (C-8), 124.0 (C-7), 120.6 (C-8a), 117.5 (C-5),
04.1 (C-8b), 97.5 (C-2), 93.2 (C-4), 66.0 (CH O), 31.9 (CH Br), 29.6
CH ); IR (KBr) : 3445, 2966, 1661, 1610, 1573, 1466, 1299, 1158,
d
, ppm, CDCl
3
):
To a solution of 2ae2e (0.7 mmol) in refluxed ethanol (15 mL),
corresponding secondary amines (dimethylamine or diethylamine
or pyrrolidine or piperidine or morpholine, 2.5 mmol) in ethanol
(8 mL) was added dropwise by a syringe. The reaction mixture was
stirred under reflux for about 0.5 h until the starting material dis-
appeared monitored by TLC. On completion, the reaction mixture
was cooled, concentrated under vacuum to afford the crude prod-
uct, which could be recrystallized in acetone to give compound
3ae3e respectively.
1
7
7
2
2
13
2
2
2
2
Br); C NMR (125 MHz, d
3
4
1
(
1
2
2
2
n
ꢂ1
þ
038, 825 cm ; APCI-MS m/z: 269 [MþH] . Anal. Calcd for
4.4.1. 1-Hydroxy-3-(2-(pyrrolidin-1-yl)ethoxy)-9H-xanthen-9-one
(3a)
C
16 4
H13BrO : C, 55.04; H, 3.75. Found: C, 55.07; H, 3.71.
Yield: 88% from compound 2a and pyrrolidine; a yellow solid;
ꢀ
1
4.3.3. 3-(4-Bromobutoxy)-1-hydroxy-9H-xanthen-9-one (2c)
m.p.: 121e122 C; H NMR (500 MHz, d, ppm, CDCl ): 12.84 (s, 1H,
3
Yield: 47% from compound 1 and 1, 4-dibromobutane; a yellow
OH), 8.24 (dd, J ¼ 1.7, 8.0 Hz, 1H, H-8), 7.70 (ddd, J ¼ 1.7, 7.3, 8.6 Hz,
1H, H-6), 7.42 (dd, J ¼ 0.8, 8.6 Hz, 1H, H-5), 7.36 (ddd, J ¼ 0.8, 7.3,
8.0 Hz, 1H, H-7), 6.45 (d, J ¼ 2.3 Hz, 1H, H-4), 6.36 (d, J ¼ 2.3 Hz, 1H,
ꢀ
1
solid; m.p.: 133e135 C; H NMR (500 MHz,
2.88 (s, 1H, OH), 8.23 (dd, J ¼ 1.5, 7.9 Hz, 1H, H-8), 7.87 (ddd, J ¼ 1.5,
.3, 8.5 Hz, 1H, H-6), 7.56 (d, J ¼ 8.5 Hz, 1H, H-5), 7.49 (m, 1H, H-7),
.59 (d, J ¼ 2.0 Hz, 1H, H-4), 6.38 (d, J ¼ 2.0 Hz, 1H, H-2), 4.25 (t,
J ¼ 6.2 Hz, 2H, OCH ), 3.63 (t, J ¼ 6.6 Hz, 2H, CH Br), 2.76e2.78 (m,
); C NMR (125 MHz, , ppm, CDCl ): 180.8 (C-9), 166.0
C-3), 163.5 (C-1), 157.7 (C-4a), 156.0 (C-4b), 135.0 (C-6), 125.8 (C-8),
24.0 (C-7), 120.6 (C-8a), 117.6 (C-5), 103.9 (C-8b), 97.4 (C-2), 93.2
3 2 6
d, ppm, (CD ) CO-d ):
1
7
6
H-2), 4.21 (t, J ¼ 5.9 Hz, 2H, OCH
2
), 2.95 (t, J ¼ 5.8 Hz, 2H,
in pyrrole ring),1.81e1.85 (m,
in pyrrole ring); C NMR (125 MHz, , ppm, CDCl ): 180.8
OCH
4H,
2
b
CH
-CH
2
N), 2.64e2.68 (m, 4H,
a
-CH
2
13
2
2
2
d
3
13
4
(
1
H, CH
2
CH
2
d
3
(C-9), 166.0 (C-3), 163.5 (C-1), 157.7 (C-4a), 156.0 (C-4b), 135.0 (C-6),
125.9 (C-8), 124.0 (C-7), 120.6 (C-8a), 117.6 (C-5), 104.0 (C-8b), 97.5
2 2
(C-2), 93.4 (C-4), 67.8 (OCH ), 54.8 (2a-CH in pyrrole ring), 54.7
(
3
C-4), 67.6 (CH
2
O), 33.2 (CH
2
Br), 29.3 (CH
2
), 27.6 (CH
2
); IR (KBr)
n
:
;
(NCH ), 23.5 (2 -CH in pyrrole ring); IR (KBr)
2
b
2
n
: 3418, 2957, 2780,
ꢂ1
ꢂ1
546, 2959, 1663, 1609, 1572, 1469, 1299, 1158, 1081, 824 cm
1656, 1609, 1570, 1467, 1364, 1296, 1161, 1076, 823, 759 cm ; APCI-
þ
þ
APCI-MS m/z: 363 [MþH] . Anal. Calcd for C17
H15BrO
4
: C, 56.22; H,
MS m/z: 326 [MþH] . Anal. Calcd for C19
4
H19NO : C, 70.14; H, 5.89;
4
.16. Found: C, 56.25; H, 4.13.
N, 4.31. Found: C, 70.11; H, 5.91; N, 4.33.
4
.3.4. 3-(5-Bromopentyloxy)-1-hydroxy-9H-xanthen-9-one (2d)
4.4.2. 1-Hydroxy-3-(3-(pyrrolidin-1-yl)propoxy)-9H-xanthen-9-
one (3b)
Yield: 55% from compound 1 and 1, 5-dibromopentane; a yellow
ꢀ
1
solid; m.p.: 129e131 C; H NMR (500 MHz,
d
, ppm, CDCl
3
): 12.85
Yield: 87% from compound 2b and pyrrolidine; a yellow solid;
ꢀ
1
(
8
s, 1H, OH), 8.24 (dd, J ¼ 1.7, 7.9 Hz, 1H, H-8), 7.70 (ddd, J ¼ 1.7, 7.3,
m.p.: 107e109 C; H NMR (500 MHz, d, ppm, CDCl ): 12.81 (s, 1H,
3
.5 Hz, 1H, H-6), 7.42 (d, J ¼ 8.5 Hz, 1H, H-5), 7.34e7.40 (m, 1H, H-7),
.41 (d, J ¼ 2.2 Hz, 1H, H-4), 6.33 (d, J ¼ 2.2 Hz, 1H, H-2), 4.06 (t,
OH), 8.23 (dd, J ¼ 1.7, 7.9 Hz, 1H, H-8), 7.69 (ddd, J ¼ 1.7, 7.3, 8.5 Hz,
1H, H-6), 7.40 (d, J ¼ 8.5 Hz, 1H, H-5), 7.35 (m, 1H, H-7), 6.42 (d,
J ¼ 2.2 Hz, 1H, H-4), 6.33 (d, J ¼ 2.2 Hz, 1H, H-2), 4.12 (t, J ¼ 6.4 Hz,
6
J ¼ 6.4 Hz, 2H, OCH
H, OCH CH CH
OCH CH CH CH CH
C NMR (125 MHz,
C-1), 157.7 (C-4a), 156.1 (C-4b), 135.0 (C-6), 125.9 (C-8), 124.0 (C-7),
20.7 (C-8a), 117.6 (C-5), 103.9 (C-8b), 97.4 (C-2), 93.2 (C-4), 68.3
2
), 3.45 (t, J ¼ 6.7 Hz, 2H, CH
2
Br), 1.92e2.00 (m,
(m, 2H,
CH N);
): 180.8 (C-9), 166.2 (C-3), 163.6
2
2
2
2
CH
N), 1.62e1.68 (m, 2H, OCH
, ppm, CDCl
2
CH
2
N),
1.83e1.89
2H, OCH
-CH
(m, 4H,
2
), 2.63 (t, J ¼ 7.4 Hz, 2H, OCH
2
CH
2
CH
2
N), 2.50e2.58 (m, 4H,
CH CH N),1.75e1.84
in pyrrole ring); C NMR (125 MHz, , ppm, CDCl ):
2
2
2
2
2
2
CH
2
CH
2
2
CH
2
a
2
in pyrrole ring), 2.01e2.06 (m, 2H, OCH
2
2
2
13
13
d
3
b
-CH
2
d
3
(
1
180.8 (C-9), 166.3 (C-3), 163.5 (C-1), 157.8 (C-4a), 156.1 (C-4b), 134.9
(C-6), 125.9 (C-8), 124.0 (C-7), 120.7 (C-8a), 117.6 (C-5), 103.9 (C-8b),

4
94
Z.-M. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 487e497
ꢂ1
þ
9
5
3
8
C
4
7.6 (C-2), 93.3 (C-4), 67.1 (OCH
2.8 (NCH ), 28.6 (CH ), 23.5 (2
414, 2960, 2804, 1666, 1606, 1571, 1469, 1324, 1299, 1172, 1078,
2
b
), 54.3 (2
a
-CH
2
in pyrrole ring),
759 cm ; ESI-MS m/z: 382 [MþH] . Anal. Calcd for C23
4
H27NO : C,
2
2
-CH
2
in pyrrole ring); IR (KBr)
n
:
72.42; H, 7.13; N, 3.67. Found: C, 72.39; H, 7.15; N, 3.69.
ꢂ1
þ
23, 793 cm ; APCI-MS m/z: 340 [MþH] . Anal. Calcd for
4.4.6. 3-(4-(Dimethylamino)butoxy)-1-hydroxy-9H-xanthen-9-one
(3f)
20 4
H21NO
.11.
: C, 70.78; H, 6.24; N, 4.13. Found: C, 70.75; H, 6.27; N,
Yield: 85% from compound 2c and dimethylamine; a yellow
ꢀ
1
3
solid; m.p.: 121e122 C; H NMR (500 MHz, d, ppm, CDCl ): 12.96
(
1
s, 1H, OH), 8.36 (d, J ¼ 7.0 Hz, 1H, H-8), 7.81 (ddd, J ¼ 1.7, 7.2, 8.4 Hz,
4
(
.4.3. 1-Hydroxy-3-(4-(pyrrolidin-1-yl)butoxy)-9H-xanthen-9-one
3c)
Yield: 85% from compound 2c and pyrrolidine; a yellow solid;
H, H-6), 7.54 (d, J ¼ 8.4 Hz, 1H, H-5), 7.48 (m, 1H, H-7), 6.53 (s, 1H,
), 2.47 (t, J ¼ 7.1
), 1.95e1.98 (m, 2H,
H-4), 6.44 (s, 1H, H-2), 4.18 (t, J ¼ 5.8 Hz, 2H, OCH
Hz, 2H, NCH ), 2.33e2.37(m, 6H, N(CH
OCH CH CH
NMR (125 MHz,
2
ꢀ
1
2
CH
2 3
)
6
m.p.: 123e124 C; H NMR (500 MHz, d, ppm, DMSO-d ): 12.82 (s,
13
2
2
2
2
N), 1.77e1.79 (m, 2H, OCH
2
CH
2
CH
2
CH
2
N);
C
1
8
6
H, OH), 8.22 (dd, J ¼ 1.7, 7.9 Hz, 1H, H-8), 7.69 (ddd, J ¼ 1.7, 7.2,
d
, ppm, CDCl
3
): 180.7 (C-9), 166.2 (C-3), 163.5 (C-1),
.6 Hz, 1H, H-6), 7.40 (d, J ¼ 8.6 Hz, 1H, H-5), 7.33e7.37 (m, 1H, H-7),
.40 (d, J ¼ 2.2 Hz, 1H, H-4), 6.31 (d, J ¼ 2.2 Hz, 1H, H-2), 4.06 (t,
1
57.7 (C-4a), 156.0 (C-4b), 134.8 (C-6), 125.8 (C-8), 123.8 (C-7), 120.6
C-8a), 117.5 (C-5), 103.8 (C-8b), 97.4 (C-2), 93.2 (C-4), 68.4 (OCH ),
9.2 (NCH ), 45.4 (2NCH ), 26.8 (OCH CH ), 24.0 (NCH CH ); IR
(KBr) : 3649, 2935, 2757, 1661, 1608, 1569, 1467, 1316, 1296, 1163,
(
5
2
J ¼ 6.5 Hz, 2H, OCH
OCH CH CH CH
.66e1.73 (m, 2H, OCH
CDCl ): 180.7 (C-9), 166.3 (C-3), 163.5 (C-1), 157.7 (C-4a), 156.0 (C-
b), 134.9 (C-6), 125.9 (C-8), 123.9 (C-7), 120.7 (C-8a), 117.5 (C-5),
03.8 (C-8b), 97.5 (C-2), 93.2 (C-4), 68.5 (OCH ), 56.0 (NCH ), 54.2
-CH in pyrrole ring), 27.1 (OCH CH ), 25.5 (NCH CH ), 23.4 (2
in pyrrole ring); IR (KBr)
2
), 2.48e2.54 (m, 6H, N(CH
2
)
3
),1.83e1.89 (m, 2H,
in pyrrole ring),
N); C NMR (125 MHz, , ppm,
2
3
2
2
2
2
2
2
2
2 2
N), 1.76e1.81 (m, 4H, b-CH
13
n
1
2
CH
2
CH
2
CH
2
d
ꢂ1
þ
1
C
4
079, 823, 758 cm . APCI-MS m/z: 328 [MþH] . Anal. Calcd for
3
19 4
H21NO : C, 69.71; H, 6.47; N, 4.28. Found: C, 69.69; H, 6.50; N,
4
1
.27.
2
2
(
2
a
2
2
2
2
2
b-
4
.4.7. 3-(4-(Diethylamino)butoxy)-1-hydroxy-9H-xanthen-9-one
(3g)
Yield: 86% from compound 2c and diethylamine; a yellow solid;
CH
2
n
: 3447, 2959, 2790, 1663, 1607, 1569,
ꢂ1
1468, 1318, 1296, 1165, 1080, 823, 756 cm . ESI-MS m/z: 354
þ
[
MþH] . Anal. Calcd for C21
4
H23NO : C, 71.37; H, 6.56; N, 3.96.
ꢀ
1
m.p.: 184e186 C; H NMR (500 MHz,
1
8
d
, ppm, DMSO-d
H, OH), 8.17 (dd, J ¼ 1.5, 8.0 Hz, 1H, H-8), 7.90 (ddd, J ¼ 1.5, 7.2,
.5 Hz, 1H, H-6), 7.63 (d, J ¼ 8.5 Hz, 1H, H-5), 7.51 (m, 1H, H-7), 6.67
6
): 12.81 (s,
Found: C, 71.40; H, 6.55; N, 3.97.
4
.4.4. 1-Hydroxy-3-(5-(pyrrolidin-1-yl)pentyloxy)-9H-xanthen-9-
one (3d)
Yield: 92% from compound 2d and pyrrolidine; a pale yellow
(
2
d, J ¼ 2.1 Hz,1H, H-4), 6.43 (d, J ¼ 2.1 Hz,1H, H-2), 4.18 (t, J ¼ 6.1 Hz,
H, OCH ), 2.91e3.02(m, 6H, N(CH ), 1.77e1.84 (m, 2H,
OCH CH CH CH N), 1.70e1.75 (m, 2H, OCH CH CH CH N), 1.14 (t,
J ¼ 7.1 Hz, 6H, N(CH ); C NMR (125 MHz, , ppm, CDCl ):
80.7 (C-9), 166.3 (C-3), 163.5 (C-1), 157.7 (C-4a), 156.0 (C-4b), 134.9
C-6), 125.9 (C-8), 123.9 (C-7), 120.7 (C-8a), 117.6 (C-5), 103.8 (C-8b),
2
2 3
)
2
2
2
2
2
2
2
d
2
ꢀ
1
solid; m.p.: 107e108 C; H NMR (500 MHz,
d
, ppm, CDCl
3
): 12.82
13
2
CH
3
)
2
3
(
8
6
s, 1H, OH), 8.22 (dd, J ¼ 1.7, 7.9 Hz, 1H, H-8), 7.69 (ddd, J ¼ 1.7, 7.2,
1
(
.5 Hz, 1H, H-6), 7.40 (d, J ¼ 8.5 Hz, 1H, H-5), 7.33e7.38 (m, 1H, H-7),
.39 (d, J ¼ 2.2 Hz, 1H, H-4), 6.31 (d, J ¼ 2.2 Hz, 1H, H-2), 4.03 (t,
9
2
3
7
7
7.5 (C-2), 93.2 (C-4), 68.6 (OCH
7.1 (OCH CH ), 23.6 (NCH CH
671, 2964, 2790, 1669, 1603,1571,1472,1329,1298,1179, 1079, 821,
2
), 52.5 (NCH
2
2 3 2
), 46.9 (N(CH CH ) ),
J ¼ 6.5 Hz, 2H, OCH
2
), 2.44e2.52 (m, 6H, N(CH
2
)
3
), 1.81e1.87 (m, 2H,
-CH in pyrrole ring),
N), 1.47e1.54 (m, 2H,
N); C NMR (125 MHz, , ppm, CDCl ): 180.8
2
2
2
2
), 11.7 (N(CH
2
CH ); IR (KBr)
3
)
2
n:
OCH
2
CH
2
CH
2
CH
2
CH
2
N), 1.74e1.80 (m, 4H,
CH CH CH CH
b
2
1.56e1.63 (m, 2H, OCH
2
2
2
2
2
ꢂ1
þ
58 cm . APCI-MS m/z: 356 [MþH] . Anal. Calcd for C21
4
H25NO : C,
1
3
OCH
2
CH
2
CH
2
CH
2
CH
2
d
3
0.96; H, 7.09; N, 3.94. Found: C, 70.91; H, 7.11; N, 3.92.
(
1
C-9), 166.3 (C-3), 163.6 (C-1), 157.8 (C-4a), 156.1 (C-4b), 134.9 (C-6),
25.9 (C-8), 123.9 (C-7), 120.7 (C-8a), 117.6 (C-5), 103.9 (C-8b), 97.5
4
.4.8. 1-Hydroxy-3-(4-(piperidin-1-yl)butoxy)-9H-xanthen-9-one
(
C-2), 93.2 (C-4), 68.5 (OCH
ring), 28.9 (OCH CH ), 28.4 (NCH
-CH in pyrrole ring); IR (KBr)
2
), 56.3 (NCH
2
), 54.2 (2
a
-CH
CH
2
in pyrrole
CH ), 23.4
(3h)
2
2
2
CH
2
), 24.0 (NCH
2
2
2
Yield: 83% from compound 2c and piperidine; a yellow solid;
(
2
b
2
n
: 3430, 2949, 2793, 1662, 1608,
ꢀ
1
6
m.p.: 114e115 C; H NMR (500 MHz, d, ppm, DMSO-d ): 12.79 (s,
ꢂ1
1571,1468,1316,1299,1166,1082, 824, 763 cm ; APCI-MS m/z: 368
1
1
7
H, OH), 8.15 (d, J ¼ 7.8 Hz, 1H, H-8), 7.88 (ddd, J ¼ 1.6, 7.3, 8.5 Hz,
þ
[
MþH] . Anal. Calcd for C22
4
H25NO : C, 71.91; H, 6.86; N, 3.81.
H, H-6), 7.61 (d, J ¼ 8.5 Hz, 1H, H-5), 7.50 (dd, J ¼ 7.3, 7.8 Hz, 1H, H-
), 6.65 (d, J ¼ 1.7 Hz, 1H, H-4), 6.40 (d, J ¼ 1.7 Hz, 1H, H-2), 4.14 (t,
Found: C, 71.85; H, 6.89; N, 3.83.
J ¼ 6.6 Hz, 2H, OCH
OCH CH CH CH
1.47e1.52 (m, 4H,
6
CH in piperidine ring); C NMR (125 MHz, d, ppm, DMSO-d ):
180.6 (C-9), 166.7 (C-3), 163.6 (C-1), 157.9 (C-4a), 156.1 (C-4b), 135.5
(C-6), 125.5 (C-8), 124.2 (C-7), 120.5 (C-8a), 117.6 (C-5), 103.4 (C-8b),
97.4 (C-2), 93.1 (C-4), 68.7 (OCH
piperidine ring), 26.7 (OCH CH ), 25.9 (2
24.5 (NCH CH ), 23.0 ( -CH in piperidine ring); IR (KBr)
2959, 2790, 1662, 1607, 1569, 1468, 1319, 1296, 1165, 1080, 823,
2
), 2.27e2.50 (m, 6H, N(CH
2
)
3
), 1.71e1.77 (m, 2H,
CH CH CH N),
in piperidine ring), 1.37e1.39 (m, 2H, g-
4
.4.5. 1-Hydroxy-3-(6-(pyrrolidin-1-yl)hexyloxy)-9H-xanthen-9-
one (3e)
Yield: 87% from compound 2e and pyrrolidine; a pale yellow
2
2
2
2
N), 1.55e1.59 (m, 2H, OCH
-CH
2
2
2
2
b
2
13
2
ꢀ
1
solid; m.p.: 104e105 C; H NMR (500 MHz,
d
, ppm, CDCl
3
): 12.86
(
8
s, 1H, OH), 8.26 (dd, J ¼ 1.6, 7.8 Hz, 1H, H-8), 7.71 (ddd, J ¼ 1.6, 7.2,
.4 Hz, 1H, H-6), 7.43 (d, J ¼ 8.4 Hz, 1H, H-5), 7.39 (m, 1H, H-7), 6.42
2
), 58.3 (NCH
2
), 54.4 (2
in piperidine ring),
: 3447,
2
a-CH in
(
d, J ¼ 2.0 Hz, 1H, H-4), 6.34 (d, J ¼ 2.0 Hz, 1H, H-2), 4.06 (t,
J ¼ 6.0 Hz, 2H, OCH ), 2.54e2.64 (m, 4H, -CH in pyrrole ring),
.46e2.49 (m, 2H, OCH CH CH CH CH CH N), 1.79e1.88 (m, 6H,
OCH CH CH CH CH CH N), 1.61e1.62 (m, 2H, -CH in pyrrole
ring), 1.50-1.53 (m, 2H, -CH in pyrrole ring), 1.41-1.43 (m, 2H,
OCH CH CH CH CH CH
80.7 (C-9), 166.3 (C-3), 163.5 (C-1), 157.7 (C-4a), 156.0 (C-4b), 134.9
C-6), 125.8 (C-8), 123.9 (C-7), 120.6 (C-8a), 117.5 (C-5), 103.8 (C-8b),
2
2
b
-CH
2
2
a
2
2
2
g
2
n
2
2
2
2
2
2
2
ꢂ1
þ
2
2
2
2
2
2
b
2
756 cm ; APCI-MS m/z: 368 [MþH] . Anal. Calcd for C22
4
H25NO : C,
b
2
71.91; H, 6.86; N, 3.81. Found: C, 71.94; H, 6.87; N, 3.80.
1
3
2
2
2
2
2
2 3
N); C NMR (125 MHz, d, ppm, CDCl ):
1
(
4.4.9. 1-Hydroxy-3-(4-morpholinobutoxy)-9H-xanthen-9-one (3i)
Yield: 84% from compound 2c and morpholine; a yellow solid;
m.p.: 143e144 C; H NMR (500 MHz, d, ppm, CDCl ): 12.86 (s, 1H,
OH), 8.26 (dd, J ¼ 1.6, 7.9 Hz, 1H, H-8), 7.73 (ddd, J ¼ 1.6, 7.2, 8.5 Hz,
1H, H-6), 7.44 (dd, J ¼ 0.4, 8.5 Hz,1H, H-5), 7.40 (m,1H, H-7), 6.43 (d,
J ¼ 2.2 Hz, 1H, H-4), 6.34 (d, J ¼ 2.2 Hz, 1H, H-2), 4.10 (t, J ¼ 6.4 Hz,
ꢀ
1
9
7.4 (C-2), 93.2 (C-4), 68.6 (OCH
pyrrole ring), 28.9 (OCH CH ), 28.8 (NCH
), 23.4 (2 -CH in pyrrole ring); IR (KBr)
946, 2785, 1659, 1607, 1570, 1466, 1315, 1296, 1164, 1078, 823,
2
), 56.5 (NCH
2
), 54.2 (2
a
-CH
2
in
3
2
2
2
CH ), 27.4 (OCH
2
2
CH
2
CH
2
),
2
2
5.9 (NCH
2
CH
2
CH
2
b
2
n: 3407,

Z.-M. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 487e497
495
2
2
H, OCH
.37e2.55 (m, 6H, NCH
CH
): 180.7 (C-9), 166.2 (C-3), 163.6 (C-1), 157.8 (C-4a), 156.1 (C-
2
), 3.75 (t, J ¼ 4.6 Hz, 4H,
b
-CH
2
in morphoing ring),
CH CH CH N),
N); C NMR (125 MHz, , ppm,
7.9 Hz, 1H, H-7), 6.70 (d, J ¼ 2.1 Hz, 1H, H-4), 6.45 (d, J ¼ 2.1 Hz, 1H,
H-2), 4.20 (t, J ¼ 6.1 Hz, 2H, OCH ), 3.39e3.52 (m, 6H, N(CH ), 3.02
(s, 3H, NCH ), 2.06e2.13 (m, 4H, -CH in pyrrole ring), 1.86e1.94
(m, 2H, OCH CH CH CH N), 1.78e1.85 (m, 2H, OCH CH CH CH N);
C NMR (125 MHz, , ppm, DMSO-d ): 180.6 (C-9), 166.3 (C-3),
2
), 1.85e1.92 (m, 2H, OCH
2
2
2
d
2
2
2 3
)
1
3
1.69e1.75 (m, 2H, OCH
2
2
CH
2
CH
2
3
b
2
CDCl
3
2
2
2
2
2
2
2
2
13
4
b), 134.9 (C-6), 125.9 (C-8), 124.7 (C-7), 120.8 (C-8a), 117.5 (C-5),
), 67.0 (2 -CH in
in morphoing ring),
d
6
103.9 (C-8b), 97.4 (C-2), 93.2 (C-4), 68.4 (OCH
2
a
2
163.1 (C-1), 157.8 (C-4a), 156.0 (C-4b), 136.5 (C-6), 125.8 (C-8), 125.1
(C-7), 120.3 (C-8a), 118.2 (C-5), 103.6 (C-8b), 98.0 (C-2), 93.8 (C-4),
morphoing ring), 58.4 (NCH
2
), 53.8 (2
b-CH
2
2
6.9 (OCH
2
CH
2
), 23.0 (NCH
2
CH ); IR (KBr)
2
n
: 3441, 2944, 2773,1664,
68.3 (OCH
2
), 64.0 (2
), 25.9 (OCH CH
CH ); IR (KBr) : 3425, 2954, 1665, 1607, 1569, 1483, 1325,
a
-CH
2
in pyrrole ring), 63.2 (NCH
2
), 48.1
ꢂ1
1605, 1569, 1467, 1317, 1297, 1167, 1077, 826, 765 cm . ESI-MS m/z:
(NCH
(NCH
3
2
2
), 21.6 (2
b
-CH in pyrrole ring), 20.4
2
þ
3
70 [MþH] . Anal. Calcd for C21
H23NO
5
: C, 68.28; H, 6.28; N, 3.79.
2
2
n
ꢂ1
þ
Found: C, 68.26; H, 6.31; N, 3.81.
1294, 1165, 1081, 828, 780 cm ; ESI-MS m/z: 368 [MþH] . Anal.
Calcd for C22 26INO : C, 53.34; H, 5.29; N, 2.83. Found: C, 53.30; H,
5.32; N, 2.85.
H
4
4.5. General procedure for the preparation of the corresponding
quaternary ammonium derivatives (4ae4i)
4.5.4. 1-(5-(1-Hydroxy-9-oxo-9H-xanthen-3-yloxy)pentyl)-1-
To a solution of 3ae3i (0.2 g) in chloroform (10 mL), iodo-
methane (1 mL) was added and then it was stirring at room tem-
perature for about 3 days until the starting material disappeared by
TLC. After the reaction was completed, the precipitate was filtrated,
washed by chloroform (3 ꢁ 10 mL) and dried to give corresponding
quaternary ammonium derivatives 4ae4i respectively.
methylpyrrolidinium iodide (4d)
Yield: 90% from compound 3d and iodomethane; pale yellow
ꢀ
1
6
solid; m.p.: 242e243 C; H NMR (500 MHz, d, ppm, DMSO-d ):
12.79 (s, 1H, OH), 8.16 (dd, J ¼ 1.7, 7.9 Hz, 1H, H-8), 7.89 (ddd, J ¼ 1.7,
7.2, 8.5 Hz, 1H, H-6), 7.61 (d, J ¼ 8.5 Hz, 1H, H-5), 7.48e7.52 (m, 1H,
H-7), 6.65 (d, J ¼ 2.0 Hz,1H, H-4), 6.41 (d, J ¼ 2.0 Hz,1H, H-2), 4.16 (t,
J ¼ 6.4 Hz, 2H, OCH
2
), 3.41e3.51 (m, 6H, N(CH
-CH
N), 1.42e1.49
N); C NMR (125 MHz, , ppm, DMSO-d
6
2
)
3
), 3.00 (s, 3H,
in pyrrole ring), 1.76e1.84 (m, 4H,
(m, 2H, OCH CH
): 180.6 (C-9),
4.5.1. 1-(2-(1-Hydroxy-9-oxo-9H-xanthen-3-yloxy)ethyl)-1-methyl
NCH
OCH
3
), 2.05e2.12 (m, 4H,
CH CH CH CH
CH
b
2
pyrrolidinium iodide (4a)
2
CH
2
2
2
2
2
2
13
Yield: 93% from compound 3a and iodomethane; white solid;
CH
2
2
2
d
ꢀ
1
m.p.: 233e234 C; H NMR (500 MHz,
1
8
d
, ppm, DMSO-d
H, OH), 8.16 (dd, J ¼ 1.6, 7.9 Hz, 1H, H-8), 7.90 (ddd, J ¼ 1.6, 7.3,
.5 Hz, 1H, H-6), 7.63 (d, J ¼ 8.5 Hz, 1H, H-5), 7.51 (m, 1H, H-7), 6.76
6
): 12.80 (s,
166.5 (C-3), 163.1 (C-1), 157.8 (C-4a), 156.0 (C-4b), 136.5 (C-6), 125.8
(C-8), 125.1 (C-7), 120.3 (C-8a), 118.2 (C-5), 103.6 (C-8b), 98.0 (C-2),
93.8 (C-4), 68.8 (OCH
48.2 (NCH ), 28.3 (OCH
21.6 (2 -CH in pyrrole ring); IR (KBr)
2
), 64.0 (2
a
-CH
2
in pyrrole ring), 63.5 (NCH
2
),
),
(
d, J ¼ 2.1 Hz,1H, H-4), 6.51 (d, J ¼ 2.1 Hz,1H, H-2), 4.64 (t, J ¼ 4.8 Hz,
H, OCH CH N), 3.55e3.65 (m, 4H,
), 3.88 (t, J ¼ 4.8 Hz, 2H, OCH
CH in pyrrole ring), 3.12 (s, 3H, NCH ), 2.10e2.16 (m, 4H, -CH
pyrrole ring); C NMR (125 MHz, , ppm, DMSO-d ): 181.4 (C-9),
65.4 (C-3), 163.4 (C-1), 158.2 (C-4a), 156.4 (C-4b), 137.3 (C-6), 126.2
C-8), 125.8 (C-7), 120.6 (C-8a), 118.8 (C-5), 104.5 (C-8b), 98.7 (C-2),
3
2
CH ), 23.1 (NCH
2
2
CH ), 22.9 (NCH CH CH
: 3431, 2946, 1668, 1605,
2
2
2
2
2
2
2
2
a
-
b
2
n
ꢂ1
2
3
b
2
in
1569, 1470, 1330, 1293, 1168, 1081, 829, 787 cm ; ESI-MS m/z: 382
1
3
þ
d
6
[MþH] . Anal. Calcd for C23
4
H28INO : C, 54.23; H, 5.54; N, 2.75.
1
(
Found: C, 54.29; H, 5.51; N, 2.73.
9
4
4.6 (C-4), 65.8 (2
a
-CH
2
in pyrrole ring), 63.9 (OCH ), 62.7(NCH
2
2
),
4.5.5. 1-(6-(1-Hydroxy-9-oxo-9H-xanthen-3-yloxy)hexyl)-1-
methylpyrrolidinium iodide (4e)
9.3 (NCH ), 22.0 (2
3
b
-CH
2
in pyrrole ring); IR (KBr) n: 3467, 2959,
ꢂ1
1656, 1606, 1573, 1470, 1323, 1294, 1169, 1081, 828, 787 cm ; ESI-
Yield: 89% from compound 3e and iodomethane; white solid;
þ
ꢀ
1
MS m/z: 340 [MþH] . Anal. Calcd for C20
H22INO
4
: C, 51.40; H, 4.75;
m.p.: 225e226 C; H NMR (500 MHz,
1H, OH), 8.11 (dd, J ¼ 1.3, 7.9 Hz, 1H, H-8), 7.85 (ddd, J ¼ 1.3, 7.2,
.3 Hz, 1H, H-6), 7.57 (d, J ¼ 8.3 Hz, 1H, H-5), 7.46 (dd, J ¼ 7.2, 7.9 Hz,
6
d, ppm, DMSO-d ): 12.74 (s,
N, 3.00. Found: C, 51.37; H, 4.77; N, 3.02.
8
4.5.2. 1-(3-(1-Hydroxy-9-oxo-9H-xanthen-3-yloxy)propyl)-1-
1H, H-7), 6.59 (d, J ¼ 2.1 Hz, 1H, H-4), 6.35 (d, J ¼ 2.1 Hz, 1H, H-2),
methylpyrrolidinium iodide (4b)
4.10 (t, J ¼ 6.4 Hz, 2H, OCH
2
), 3.34e3.44 (m, 6H, N(CH
2
)
3
), 2.94 (s,
Yield: 94% from compound 3b and iodomethane; white solid;
3H, NCH
3
), 2.04 (s, 4H,
b
-CH
CH
2
in pyrrole ring), 1.67e1.75 (m, 4H,
ꢀ
1
m.p.: 231e232 C; H NMR (500 MHz,
d
, ppm, DMSO-d
H, OH), 8.13 (dd, J ¼ 1.6, 8.0 Hz, 1H, H-8), 7.88 (ddd, J ¼ 1.6, 7.3,
.6 Hz, 1H, H-6), 7.60 (dd, J ¼ 0.8, 8.6 Hz, 1H, H-5), 7.49 (ddd, J ¼ 0.8,
6
): 12.77 (s,
OCH
OCH
2
CH
CH
2
CH
CH
2
CH
CH
2
CH
CH
2
2
N),
1.41e1.48
(m, 2H,
1
8
2
2
2
2
2
CH
2
N), 1.29e1.36 (m, 2H, OCH
2 2 2
CH CH
1
3
CH
2
CH
2
CH
2
N); C NMR (125 MHz,
d
, ppm, DMSO-d ): 181.3 (C-9),
6
7.3, 8.0 Hz, 1H, H-7), 6.65 (d, J ¼ 2.0 Hz, 1H, H-4), 6.40 (d, J ¼ 2.0 Hz,
167.2 (C-3), 163.8 (C-1), 158.5 (C-4a), 156.7 (C-4b), 137.1 (C-6), 126.5
(C-8), 125.8 (C-7), 121.1 (C-8a), 118.9 (C-5), 104.3 (C-8b), 98.7 (C-2),
1
H, H-2), 4.22 (t, J ¼ 6.1 Hz, 2H, OCH
.06 (s, 3H, NCH ), 2.20e2.30 (m, 2H, OCH
-CH in pyrrole ring); C NMR (125 MHz,
): 181.3 (C-9), 166.6 (C-3), 163.7 (C-1), 158.4 (C-4a), 156.6 (C-4b),
2
), 3.48e3.59 (m, 6H, N(CH
CH CH N), 2.08e2.16
, ppm, DMSO-
2 3
) ),
3
3
2
2
2
d
94.4 (C-4), 69.6 (OCH
48.9 (NCH ), 29.3 (OCH
24.0 (NCH CH CH ), 22.3 (2
2
), 64.7 (2
CH ), 26.7 (NCH
-CH in pyrrole ring); IR (KBr)
a
-CH
2
in pyrrole ring), 64.3 (NCH
2
),
),
13
(
m, 4H,
b
2
3
2
2
2
CH ), 26.1 (OCH CH CH
2
2
2
2
d
6
2
2
2
b
2
n
: 3464,
ꢂ1
1
37.1 (C-6), 126.4 (C-8), 125.7 (C-7), 121.0 (C-8a), 118.9 (C-5), 104.4
C-8b), 98.7 (C-2), 94.4 (C-4), 66.9 (OCH ), 64.8 (2 -CH in pyrrole
ring), 61.4 (NCH ), 48.8 (NCH ), 24.3 (NCH CH ), 22.2 (2 -CH in
pyrrole ring); IR (KBr) : 3432, 2960, 1657, 1604, 1571, 1467, 1324,
295, 1172, 1076, 825, 782 cm ; ESI-MS m/z: 354 [MþH] . Anal.
Calcd for C21 24INO : C, 52.40; H, 5.03; N, 2.91. Found: C, 52.37; H,
2943, 1662, 1603, 1568, 1469, 1328, 1294, 1168, 1078, 833, 783 cm
;
þ
(
2
a
2
ESI-MS m/z: 396 [MþH] . Anal. Calcd for C24
4
H30INO : C, 55.07; H,
2
3
2
2
b
2
5.78; N, 2.68. Found: C, 55.04; H, 5.71; N, 2.65.
n
ꢂ1
þ
1
4.5.6. 4-(1-Hydroxy-9-oxo-9H-xanthen-3-yloxy)-N,N,N-
trimethylbutan-1-aminiu-m iodide (4f)
H
4
5
.06; N, 2.94.
Yield: 90% from compound 3f and iodomethane; white solid;
m.p.: 232e233 C; H NMR (500 MHz, d, ppm, DMSO-d ): 12.77 (s,
6
ꢀ
1
4
.5.3. 1-(4-(1-Hydroxy-9-oxo-9H-xanthen-3-yloxy)butyl)-1-
1H, OH), 8.16 (dd, J ¼ 1.6, 7.9 Hz, 1H, H-8), 7.90 (ddd, J ¼ 1.6, 7.2,
8.6 Hz, 1H, H-6), 7.61 (d, J ¼ 8.6 Hz, 1H, H-5), 7.52 (dd, J ¼ 7.2, 7.9 Hz,
1H, H-7), 6.66 (d, J ¼ 1.8 Hz, 1H, H-4), 6.42 (d, J ¼ 1.8 Hz, 1H, H-2),
methylpyrrolidinium iodide (4c)
Yield: 89% from compound 3c and iodomethane; pale yellow
ꢀ
1
solid; m.p.: 253e254 C; H NMR (500 MHz,
2.81 (s, 1H, OH), 8.18 (dd, J ¼ 1.5, 7.9 Hz, 1H, H-8), 7.91 (ddd, J ¼ 1.5,
.3, 8.5 Hz, 1H, H-6), 7.63 (d, J ¼ 8.5 Hz, 1H, H-5), 7.52 (dd, J ¼ 7.3,
d
, ppm, DMSO-d
6
):
4.21 (t, J ¼ 6.1 Hz, 2H, OCH
N(CH ), 1.85e1.92 (m, 2H, OCH
OCH
2
), 3.38e3.44 (m, 2H, NCH
CH CH CH
N); C NMR (125 MHz, , ppm, DMSO-d
6
2
), 3.09 (s, 9H,
N), 1.77e1.84 (m, 2H,
): 180.6
1
7
3
)
CH
3
2
2
2
2
13
2
2
CH
2
CH
2
d

496
Z.-M. Yang et al. / European Journal of Medicinal Chemistry 85 (2014) 487e497
(
1
(
(
1
C-9), 166.3 (C-3), 163.2 (C-1), 157.8 (C-4a), 156.0 (C-4b), 136.4 (C-6),
25.8 (C-8), 125.1 (C-7), 120.4 (C-8a), 118.2 (C-5), 103.7 (C-8b), 98.1
C-2), 93.9 (C-4), 68.3 (OCH ), 65.6 (NCH ), 52.8 (3NCH ), 25.8
OCH CH ), 19.6 (NCH CH ); IR (KBr)
4.6. Cell culture
2
2
3
Cell lines were obtained from Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences (Shanghai, China) and
cultured as a monolayer in RPMI 1640 medium (Gibco BRL, Grand
Island, NY, USA.) supplemented with 10% FBS (Gibco BRL, Grand
2
2
2
2
n
: 3473, 2952, 1654, 1606,
ꢂ1
570, 1469, 1324, 1293, 1166, 1081, 830, 782 cm ; ESI-MS m/z: 342
þ
[
MþH] . Anal. Calcd for C20
4
H24INO : C, 51.18; H, 5.15; N, 2.98.
Found: C, 51.22; H, 5.17; N, 2.95.
Island, NY, USA.), penicillin (100 units/mL) and streptomycin
ꢀ
(100
mg/mL), in a humid atmosphere at 37 C with 95% air and 5%
4.5.7. N,N-diethyl-4-(1-hydroxy-9-oxo-9H-xanthen-3-yloxy)-N-
CO
2
.
methylbutan-1-a-minium iodide (4g)
Yield: 93% from compound 3g and iodomethane; white solid;
4.7. Cell viability assay
ꢀ
1
m.p.: 236e237 C; H NMR (500 MHz,
d
, ppm, DMSO-d
H, OH), 8.19 (dd, J ¼ 1.5, 7.9 Hz, 1H, H-8), 7.91 (ddd, J ¼ 1.5, 7.3,
.4 Hz, 1H, H-6), 7.64 (d, J ¼ 8.4 Hz, 1H, H-5), 7.52 (m, 1H, H-7), 6.71
6
): 12.82 (s,
1
8
(
Inhibition of cell proliferation of xanthone derivatives (2ae4i)
was measured by the MTT assay in vitro [25]. Briefly, cells were
plated in 96-well culture plates at the density of 5000 cells per well
in RPMI 1640 medium with 200
incubation, cells were treated with various concentrations of tested
compounds for 48 h, 20
and the cells were further incubated at 37 C for another 4 h. The
supernatant was discarded, and 150 mL DMSO was added into per
well and then the absorbance (A) was measured at 490 nm. The cell
viability ratio was calculated by the following equation: Inhibitory
ratio (%) ¼ [(Acontrol ꢂ Atreated)/(Acontrol)] ꢁ 100%. IC50 value was
taken as the concentration that caused 50% inhibition of cell via-
bilities and calculated by the Logit method [26].
d, J ¼ 2.0 Hz, 1H, H-4), 6.46 (d, J ¼ 2.1 Hz, 1H, H-2), 4.21 (s, 2H,
OCH ), 3.28e3.33 (m, 6H, N(CH ), 2.93 (s, 3H, NCH ), 1.80e1.81
m, 4H, OCH CH CH CH
N), 1.23 (t, J ¼ 6.8 Hz, 6H, N(CH
NMR (125 MHz, , ppm, DMSO-d ): 180.6 (C-9), 166.3 (C-3), 163.1
C-1), 157.8 (C-4a), 156.1 (C-4b), 136.4 (C-6), 125.8 (C-8), 125.1 (C-7),
20.3 (C-8a), 118.2 (C-5), 103.7 (C-8b), 98.0 (C-2), 93.8 (C-4), 68.3
OCH ), 59.6 (NCH ), 56.2 (2NCH ), 47.1(NCH ), 25.8 (OCH CH ),
8.8 (NCH CH ), 8.0 (2CH ); IR (KBr) : 3436, 2954, 1663, 1607, 1571,
2
2
)
3
3
mL aliquots separately. After 24 h
13
(
2
2
2
2
2
CH
3
)
2
);
C
d
6
m
L 5 mg/mL MTT was added to each well,
ꢀ
(
1
(
1
1
[
2
2
2
3
2
2
2
2
3
n
ꢂ1
469, 1320, 1296, 1166, 1082, 820, 781 cm ; ESI-MS m/z: 370
þ
MþH] . Anal. Calcd for C22
4
H28INO : C, 53.13; H, 5.67; N, 2.82.
Found: C, 53.17; H, 5.64; N, 2.80.
For anticancer effects of compound 3g on MGC-803 cells, the
compound was exposed to the cells for 24, 48 and 72 h respectively.
Cell survival rates were calculated according to the following
equation: Survival rate ¼ (experimental absorbance value/control
absorbance value) ꢁ 100%.
4
.5.8. 1-(4-(1-Hydroxy-9-oxo-9H-xanthen-3-yloxy)butyl)-1-
methylpiperidinium iodide (4h)
Yield: 89% from compound 3h and iodomethane; pale yellow
ꢀ
1
solid; m.p.: 235e236 C; H NMR (500 MHz,
2.75 (s, 1H, OH), 8.12 (dd, J ¼ 1.4, 7.8 Hz, 1H, H-8), 7.86 (ddd, J ¼ 1.4,
.4, 8.5 Hz, 1H, H-6), 7.58 (d, J ¼ 8.5 Hz, 1H, H-5), 7.47 (dd, J ¼ 7.4,
.8 Hz, 1H, H-7), 6.63 (d, J ¼ 1.8 Hz, 1H, H-4), 6.39 (d, J ¼ 1.9 Hz, 1H,
), 3.34e3.38 (m, 6H, N(CH ), 2.97
-CH in
6
d, ppm, DMSO-d ):
1
7
7
4.8. Colony formation assay
H-2), 4.14 (t, J ¼ 6.0 Hz, 2H, OCH
s, 3H, NCH ), 1.75e1.81 (m, 8H, OCH
piperidine ring), 1.47e1.52 (m, 2H, -CH
NMR (125 MHz, , ppm, DMSO-d
C-1), 157.8 (C-4a), 155.9 (C-4b), 136.5 (C-6), 125.8 (C-8), 125.1 (C-7),
20.3 (C-8a), 118.2 (C-5), 103.6 (C-8b), 98.0 (C-2), 93.8 (C-4), 68.3
OCH ), 62.3 (NCH ), 60.5 (2 -CH in piperidine ring), 47.6 (NCH ),
5.8 (OCH CH ), 21.1 (NCH CH ), 19.7 (2 in piperidine ring),
-CH in piperidine ring); IR (KBr)
2
2
)
3
Delayed cytotoxic effects of agents on MGC-803 cells were
determined using the clonogenic assay [27]. Briefly, after MGC-
803 cells seeded in 6-well tissue culture plates (200 cells per well)
(
3
2
CH
2
CH
2
CH
2
N,
b
2
1
3
g
2
in piperidine ring);
C
d
6
): 180.6 (C-9), 166.3 (C-3), 163.1
were treated with compound 3g for 24 h, all the cells were then
ꢀ
(
1
(
2
incubated in humidified CO
2
atmosphere at 37 C for 10 d. Colonies
(more than >50 cells/colony) were counted and colony survival in
percentage was calculated relative to the control (100%) group.
2
2
a
2
3
2
2
2
2
b-CH
2
18.4 (
g
2
n
: 3423, 2948, 1657, 1613,
4.9. Fluorescence microscopy
ꢂ1
1570, 1469, 1326, 1293, 1166, 1081, 828, 778 cm ; ESI-MS m/z: 382
þ
[
MþH] . Anal. Calcd for C23
4
H28INO : C, 54.23; H, 5.54; N, 2.75.
MGC-803 cells were seeded in 24-well tissue culture plates and
incubated for 24 h before treatment. Cells were exposed to com-
Found: C, 54.19; H, 5.57; N, 2.71.
pound 3g for 24 h before stain with PI (20
mg/mL) and Hoechst
4.5.9. 4-(4-(1-Hydroxy-9-oxo-9H-xanthen-3-yloxy)butyl)-4-
33258 (10 g/mL, Sigma, St.Louis, MI) for 30 min. Images indicative
m
methylmorpholin-4-ium iodide (4i)
of apoptosis were automatically captured with a fluorescence mi-
croscope (Leica DM4000B, Leica Microsystems, Nussloch, Ger-
many). In each condition and experiment, minimally 1000 cells
were counted for apoptosis percentage calculation.
Yield: 94% from compound 3i and iodomethane; pale yellow
ꢀ
1
solid; m.p.: 224e226 C; H NMR (500 MHz,
2.78 (s, 1H, OH), 8.16 (dd, J ¼ 1.7, 7.8 Hz, 1H, H-8), 7.90 (ddd, J ¼ 1.7,
.2, 8.6 Hz, 1H, H-6), 7.61 (d, J ¼ 8.6 Hz, 1H, H-5), 7.52 (m, 1H, H-7),
.67 (d, J ¼ 2.0 Hz, 1H, H-4), 6.43 (d, J ¼ 2.0 Hz, 1H, H-2), 4.21 (t,
J ¼ 5.8 Hz, 2H, OCH ), 3.92e3.96 (m, 4H, -CH in morpholine ring),
.55e3.56 (m, 2H, NCH ), 3.44e3.45 (m, 4H, -CH in morpholine
ring), 3.17 (s, 3H, NCH ), 1.86e1.90 (m, 2H, OCH CH CH CH N),
.79e1.84 (m, 2H, OCH CH N); C NMR (125 MHz, , ppm,
DMSO-d ): 180.7 (C-9), 166.3 (C-3), 163.1 (C-1), 157.8 (C-4a), 156.0
C-4b), 136.5 (C-6), 125.8 (C-8), 125.1 (C-7), 120.4 (C-8a), 118.2 (C-5),
03.7 (C-8b), 98.1 (C-2), 93.9 (C-4), 68.3 (OCH ), 63.8 (NCH ), 60.4
in morpholine ring),
6
d, ppm, DMSO-d ):
1
7
6
4.10. Intracellular calcium ([Ca2
þ
]
) measurement
i
2
b
2
a
3
2
2
Changes in [Ca2
þ
] were measured by the ratiometric method in
i
6
3
2
2
2
2
fura-2 AM-loaded cells [28]. Briefly, MGC-803 cells (1 ꢁ 10 /mL)
13
ꢀ
1
2
2
CH
2
CH
2
d
were dyed with 5
m
M fura-2 AM (Sigma, St.Louis, MI) at 37 C for
6
30 min in dark. The fluorescence intensity ratio of the Fluo-2 AM
loaded cells was monitored using a dual-excitation fluorospec-
trophotometer (EL05113056, Cary Eclipse, Australia) at 340 and
380 nm for excitation and 510 nm for emission (F340, 510/F380,
(
1
2
2
(
2
a-CH
2
in morpholine ring), 59.6 (2
b
-CH
2
4
2
6.8 (NCH
3
), 25.8 (OCH CH ), 18.3 (NCH
2
2
2
CH
2
); IR (KBr)
n
: 3439,
510) after a pharmacological agent was administered and used as
ꢂ1
2þ
959, 1665, 1607, 1570, 1469, 1326, 1293, 1167, 1081, 820, 786 cm
;
an index of [Ca
i
] . The results were normalized to baseline pre-
þ
ESI-MS m/z: 384 [MþH] . Anal. Calcd for C22
H
26INO
4
: C, 51.67; H,
treatment values by dividing the average ratio and expressed as
means ± SD.
5
.12; N, 2.74. Found: C, 51.61; H, 5.15; N, 2.71.

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This work was financially supported by grants from the
National Natural Science Foundation of China (21002015),
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