Synthesis and anticancer potential of novel xanthone derivatives with 3,6-substituted chains

Article abstract of DOI:10.1016/j.bmc.2016.07.020

In an effort to develop new drug candidates with enhanced anticancer activity, our team synthesized and assessed the cytotoxicity of a series of novel xanthone derivatives with two longer 3,6-disubstituted amine carbonyl methoxy side chains on either benzene ring in selected human cancer cell lines. An MTT assay revealed that a set of compounds with lower IC₅₀values than the positive control, 5-FU, exhibited greater anticancer effects. The most potent derivative (XD8) exhibited anticancer activity in MDA-MB-231, PC-3, A549, AsPC-1, and HCT116 cells lines with IC₅₀values of 8.06, 6.18, 4.59, 4.76, and 6.09?μM, respectively. Cell cycle analysis and apoptosis activation suggested that the mechanism of action of these derivatives includes cell cycle regulation and apoptosis induction.

Full text of DOI:10.1016/j.bmc.2016.07.020

Accepted Manuscript
Synthesis and anticancer potential of novel xanthone derivatives with 3,6-sub-
stituted chains
Chaomei Liu, Mei Zhang, Zhenhuan Zhang, Steven B. Zhang, Shanmin Yang,
Amy Zhang, Liangjie Yin, Steven Swarts, Sadasivan Vidyasagar, Lurong
Zhang, Paul Okunieff
PII:
S0968-0896(16)30524-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.07.020
BMC 13133
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
12 May 2016
7 July 2016
11 July 2016
Please cite this article as: Liu, C., Zhang, M., Zhang, Z., Zhang, S.B., Yang, S., Zhang, A., Yin, L., Swarts, S.,
Vidyasagar, S., Zhang, L., Okunieff, P., Synthesis and anticancer potential of novel xanthone derivatives with 3,6-
substituted chains, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.07.020
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Synthesis and anticancer potential of novel xanthone derivatives with 3,6-
substituted chains
Chaomei Liu, Mei Zhang, Zhenhuan Zhang, Steven B. Zhang, Shanmin Yang, Amy
Zhang, Liangjie Yin, Steven Swarts, Sadasivan Vidyasagar, Lurong Zhang, and Paul
Okunieff
Department of Radiation Oncology, University of Florida, Cancer/Genetics Research
Complex, 2033 Mowry Road, Suite 145, P.O. Box 103633, Gainesville, FL 32610, USA
Keywords: xanthone derivatives; 3,6-substituted long chains; synthesis; anticancer
potential; IC50; A549; structure-activity relationship; apoptosis; cell cycle arrest; caspase
3/7 activity
Conflicts of interest: None to disclose.
Corresponding Author:
Chaomei Liu, PhD
2000 SW Archer Road
PO Box 100385
Gainesville, FL 32610-0385
Phone: 352-265-0287
FAX: 352-265-8417
liucha@shands.ufl.edu
1 | P a g e

Abstract
In an effort to develop new drug candidates with enhanced anticancer activity, our team
synthesized and assessed the cytotoxicity of a series of novel xanthone derivatives with
two longer 3,6-disubstituted amine carbonyl methoxy side chains on either benzene ring
in selected human cancer cell lines. An MTT assay revealed that a set of compounds with
lower IC50 values than the positive control, 5-FU, exhibited greater anticancer effects.
The most potent derivative (XD8) exhibited anticancer activity in MDA-MB-231, PC-3,
A549, AsPC-1, and HCT116 cells lines with IC50 values of 8.06, 6.18, 4.59, 4.76, and
6.09 µM, respectively. Cell cycle analysis and apoptosis activation suggested that the
mechanism of action of these derivatives includes cell cycle regulation and apoptosis
induction.
2 | P a g e

1. Introduction
With their diverse structures and bioactivity, natural products (NP) have been an
exceptional source for drug discovery. Of the drugs in use today, more than 50% of them
developed over the past 30 years are from NP or their structural scaffolds.1, 2 For
anticancer drugs, 74.8% of these drugs approved worldwide between 1940 and 2010 owe
their origins to NP. Anticancer agents derived from plants and their derivatives have been
proven to be effective for cancer prevention and therapeutics.3 More recently,
considerable attention has been given to NP due to the increased understanding of their
broad spectrum of antitumor activity.1, 4
O
35
34
30
13
29
O
O
28
OH
25
31
17
26
O
19
O
24
40
1
O
20
15
23
22
18
O
O
H3C
2
36
A
12
B
C
D
21
CH3
O
11
39
9
O
6
8
4
10
OH
OH
O
Xanthone skeleton
Gambogic Acid
DMXAA
Fig.1. Structures of xanthone skeleton, DMXAA and gambogic acid
Xanthone (9H-xanthen-9-one or 9-xanthone) has a planar and oxygenated tricyclic
skeleton in which one pyranone ring is fused with two benzene rings on both sides (Fig.
1). Natural xanthones are mainly isolated from higher plants, fungi, and lichens as
secondary metabolites. They have diverse biological profiles, including antioxidative,5, 6
anticancer,7-13 antibacterial, antifungal,14, 15 anti-inflammatory,16 and antihypertensive
activities,17 depending on their diverse structures modified by substituents on the ring
3 | P a g e

system.18 Their interesting structural scaffolds and significant biological activities have
prompted many scientists to isolate and synthesize xanthones and their derivatives for the
development of novel drug candidates, particularly for anticancer drug candidates.19 For
example, one synthetic xanthone compound, 5,6-dimethylxanthone-4-acetic acid
(DMXAA, Fig. 1), has been shown to interact with diverse biological targets providing
its anticancer activity.20, 21 Gambogic acid (Fig.1) is a naturally caged xanthone
compound with potent anticancer activities. Multiple mechanisms, including apoptosis
induction, cell cycle regulation, angiogenesis inhibition, tumor cell adhesion inhibition,
and metastasis prevention, have been proposed as the basis of the ability of gambogic
acid to inhibit the growth of a broad panel of cancer cell lines both in vitro and in vivo.
Gambogic acid has recently passed phase IIa clinical trials in China.22, 23 Although the
primary molecular targets of these xanthone drug candidates are still debatable, their
anticancer activities might be related to their interaction with DNA and can be
significantly altered by ring substituents with different positions because of their planar
pharmacophoric structure involving a three-ring skeleton.24 After a careful analysis of the
structure of many natural and synthetic xanthone compounds, we found that most have
simple small substituents on both benzene rings in different positions, such as the
hydroxyl, methoxyl, or methyl groups. In addition to these common substituents, some
natural xanthones are also substituted with isoprene, which binds to the outer benzene
ring carbon atom, as with mangostins.13 On occasion, the isoprenyl substituent groups
form a bridge-like structure with the benzene ring to form caged xanthone compounds,
such as gambogic acid.8, 22, 24 Of the synthetic xanthone derivatives, studies have shown
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that one longer side-chain substituent on one of the benzene rings has much more potent
anticancer activity.7, 9, 10, 12
In order to search for new xanthone drug candidates with potential antitumor
properties, we synthesized a series of novel xanthone derivatives (XD) with two longer
side chain substituents on either benzene ring (e.g., 3,6-disubstituted carbonyl methoxy
groups) and screened their anticancer activities in vitro. A mechanism of action was
suggested through cell cycle analysis and apoptosis induction.
2. Material and methods
Solvents and reactants were of the highest commercial grade available and were used
without further purification unless otherwise noted. Thin layer chromatography (TLC)
plates (Partisil K6 thin layer chromatography plates, silica gel 60, Whatman, Maidstone,
United Kingdom) and column chromatography (Purasil 60 A Silica gel [230-400 mesh],
Whatman) were used to purify intermediates and final derivatives. 1HNMR spectra were
recorded on a Mercury 300 MHz broadband (mbb) or Mercury 300 MHz 4-nuc (m4n)
spectrometer (Oxford Instruments, Oxfordshire, United Kingdom) in either CDCl3 with
tetramethylsilane (TMS) used as an internal standard or dimethyl sulfoxide (DMSO)-d6
without TMS and trace DMSO used as an internal standard. 13CNMR spectra were
recorded on an Inova 500 MHz 2 RF channels (i2C) spectrometer (Oxford) in DMSO-d6
without TMS and trace DMSO used as an internal standard. Chemical shifts (δ) were in
parts per million (ppm) relative to tetramethylsilane or trace DMSO as an internal
standard, and coupling constants (J values) were in Hertz. Mass spectra were measured
on a DSQ low-resolution mass spectrometer or on a Shimadzu LCMS-2010A liquid
5 | P a g e

chromatography mass spectrometer (Shimadzu Corporation, Kyoto, Japan). Melting
points were determined on an electrothermal digital melting point apparatus and were
uncorrected.
2.1.
Synthesis of 3,6-dihydroxy-4-methylxanthone
2.1.1. Synthesis of 2-hydroxy-3-methyl-2’,4,4’-trimethoxybenzophenone or 2’-
hydroxy-3-methyl-2,4,4’-trimethoxybenzophenone and 3-methyl-2,2’,4,4’-
tetramethoxybenzophenone
2,4-Dimethoxybenzoic acid (18.2 g, 100mmol), benzene (190 mL), and thionyl chloride
(50 mL) were combined in a 500-mL, 3-necked flask. The mixture was refluxed for 3 h.
Then the solvent and excess thionyl chloride were removed by distillation. The residual
was used in the next step without any further purification.
To the above acyl chloride, 2,6-dimethoxytoluene (15.2 g, 100mmol) and dry ethyl
ether (200 mL) were added. The mixture was cooled to 5-10°C with ice. Then AlCl3
(34.2 g, 256mmol) was added in small portions while stirring to keep the temperature of
the reaction mixture below 10°C. After AlCl3 was added, the mixture was left overnight.
An analysis of the post-reaction mixture revealed a combination of 2-hydroxy-3-methyl-
2’,4,4’-trimethoxybenzophenone or 2’-hydroxy-3-methyl-2,4,4’-
trimethoxybenzophenone and 3-methyl-2,2’,4,4’-tetramethoxybenzophenone; it weighed
29.7 g and yielded 98.3%. There were clearly two spots on TLC plate (eluent, ethyl
acetate/petroleum ether, 1/4). The mixture was separated by silica gel column
chromatography (eluent, ethyl acetate/petroleum ether, 1/5) to yield two products:
6 | P a g e

1. 2-hydroxy-3-methyl-2’,4,4’-trimethoxybenzophenone and 2’-hydroxy-3-methyl-
2,4,4’-trimethoxybenzophenone; a light yellow solid; m.p. 113-115°C; 1H NMR
(300 MHz, δ, ppm, CDCl3), 12.76 (s, 1 H, OH), 7.20-7.28 (m, 2 H, aromatic H),
6.60-6.50 (m, 2 H, aromatic H), 6.36 (d, J = 9.0 Hz, 1 H, aromatic H), 3.87 (s,
3H, OCH3), 3.87 (s, 3H, OCH3), 3.76 (s, 3H, OCH3), 2.14 (s, 3H, CH3); 13CNMR
(500 MHz, δ, ppm, DMSO-d6), 200.45, 163.91, 161.91, 160.42, 156.67, 133.79,
127.67, 124.94, 119.48, 114.40, 112.28, 106.31, 103.16, 62.17, 56.47, 56.34, 9.33,
8.51
2. 3-methyl-2,2’,4,4’-tetramethoxybenzophenone; a white solid; m.p. 75-77°C; 1H
NMR (300 MHz, δ, ppm, CDCl3), 7.49 (d, J=8.4 Hz, 1 H, aromatic H), 7.33 (dd,
J= 8.4 Hz, 0.6 Hz, 1 H, aromatic H), 6.63 (d, J= 8.7 Hz, 1 H, aromatic H), 6.51-
6.45 (m, 2 H, aromatic H), 3.87 (s, 3 H, OCH3), 3.85 (s, 3 H, OCH3), 3.69 (s, 3 H,
OCH3), 3.56 (s, 3 H, OCH3), 2.14 (s, 3 H, CH3).
2.1.2. Transformation of 3-methyl-2,2’,4,4’-tetramethoxybenzophenone to 2-
hydroxy-3-methyl-2’,4,4’-trimethoxybenzophenone or 2’-hydroxy-3-methyl-
2,4,4’-trimethoxybenzophenone
A CH2Cl2 (50 ml) solution of 3-methyl-2,2’,4,4’-tetramethoxybenzophenone (3.16 g,
10mmol), and BBr3 (2.51 g, 10mmol) was stirred at -5°C for 2 h. Some cold water (30
mL) was added and the mixture was stirred for further 10 min. The CH2Cl2 layer was
separated and water layer was extracted with CH2Cl2 (30 mL x 2). The combined CH2Cl2
solution was washed with water (20 mL x 2) and brine (30 mL), and then dried over
Na2SO4. After the removal of the solvent, the residual was recrystallized in methanol
7 | P a g e

giving a light yellow solid; weighed 2.7g and yielded 89.4%. The sample was purified by
dry-column flash chromatography to afford a light yellow solid; m.p. 113-114°C.
1HNMR confirmed that this product is the same as 2-hydroxy-3-methyl-2’,4,4’-
trimethoxybenzophenone or 2’-hydroxy-3-methyl-2,4,4’-trimethoxybenzophenone.
2.1.3. Synthesis of 3,6-dimethoxy-4-methylxanthone
2-Hydroxy-3-methyl-2’,4,4’-trimethoxybenzophenone or 2’-hydroxy-3-methyl-2,4,4’-
trimethoxybenzophenone (20.6 g, 68.2mmol), methanol (150 mL), potassium carbonate
(34.5g, 250mmol), and water (300 mL) were combined in a 1000-mL, 3-necked flask.
The mixture was stirred under reflux for 28 h. After cooling to room temperature, the
white solid was filtered out and washed with water until a pH of approximately 7 was
obtained. The solid was recrystallized with methanol to afford 3,6-dimethoxy-4-
methylxanthone; white solid; m.p. 175-177°C; weight of 17.0 g and yield of 92.4%; 1H
NMR (300 MHz, δ, ppm, DMSO-d6), 8.03 (d, J=9.0 Hz, 1 H, aromatic H), 8.01 (d, J=8.7
Hz, 1 H, aromatic H), 7.14 (d, J= 9.3 Hz, 1 H, aromatic H), 7.12 (d, J= 2.4 Hz, 1 H,
aromatic H), 7.00 (dd, J= 9.0 Hz, 2.4 Hz, 1 H, aromatic H), 3.94 (s, 3 H, OCH3), 3.92 (s,
3 H, OCH3), 2.30 (s, 3 H, CH3); 13CNMR (500 MHz, δ, ppm, DMSO-d6), 174.97, 164.98,
162.18, 158.02, 154.86, 127.76, 125.16, 115.52, 114.95, 113.74, 113.01, 108.41, 100.91,
56.68, 56.52, 8.51.
2.1.4. Synthesis of 3,6-dihydroxy-4-methylxanthone
3,6-Dimethoxy-4-methylxanthone (13.5 g, 50mmol), conc. hydrobromic acid (120 mL),
and glacial acetic acid (120 mL) were combined in a 500-mL, 3-necked flask. After the
8 | P a g e

mixture was stirred under reflux for 48 h, the solid was filtered out, washed with water,
dried, and recrystallized in methanol to afford a white solid, m.p. >260°C. It weighed 8.1
g and yielded 66.9%; 1H NMR (300 MHz, δ, ppm, DMSO-d6), 10.89 (s, 1 H, OH), 10.75
(s, 1 H, OH), 8.08 (d, J=9.3 Hz, 1 H, aromatic H), 7.95 (d, J= 8.7 Hz, 1 H, aromatic H),
7.03 (d, J= 8.7 Hz, 1 H, aromatic H), 6.98-6.94 (m, 2 H, aromatic H), 2.37 (s, 3 H, CH3);
13CNMR (500 MHz, δ, ppm, DMSO-d6), 179.59, 168.51, 166.07, 162.73, 160.57, 132.93,
129.49, 119.20, 118.90, 118.84, 117.62, 115.85, 107.35, 13.32.
2.2.
Synthesis of 3,6-dihydroxyxanthone
3,6-Dihydroxyxanthone was synthesized in the same way as 3,6-dihydroxy-4-
methylxanthone. It also produced a white solid, m.p. >260°C; 1H NMR (300 MHz, δ,
ppm, DMSO- d6), 10.91 (s, 2 H, OH x 2), 8.08 (d, J = 8.7 Hz, 2 H, aromatic H), 6.92-
6.98 (m, 4 H, aromatic H); 13CNMR (500 MHz, δ, ppm, DMSO-d6), 179.15, 168.59,
162.69, 133.00, 119.22, 118.88, 107.31.
2.3.
Synthesis of xanthone derivatives (XD) 1-10
K2CO3 (4.14 g, 30mmol) and N-substituted or N,N-disubstituted-α-chloroacetamide
(2mmol) were added to a solution of 3,6-dihydroxy-4-methylxanthone (484 mg, 2mmol)
or 3.6-dihydroxyxanthone (456 mg, 2mmol) in acetone (60 mL). The mixture was stirred
under reflux for 3h, after which more N-substituted or N,N-disubstituted-α-
chloroacetamide (2mmol) were added. Then, the mixture was stirred under reflux
overnight. The solid was filtered out and washed with acetone. The residual left after the
9 | P a g e

removal of the acetone by a rotavapor was purified by dry-column flash chromatography
to afford XD1-10.
2.3.1. 3,6-Di-[(2,6-dimethylpiperidinyl)-carbonylmethoxy]xanthone (XD1).
White solid; yield 68.4%; formed from 3,6-dihydroxyxanthone and α-chloro-(2,6-
dimethylpiperidinyl)acetamide; m.p. 179-180°C; 1H NMR (300 MHz, δ, ppm, DMSO-d6),
8.23 (d, J = 9.0 Hz, 2 H, aromatic H), 7.00-6.94 (m, 4 H, aromatic H), 4.84 (s, 4 H, OCH2
x 2), 1.95-1.20 (m, 28 H, piperidine ring 8H x 2, CH3 x 4); 13CNMR (500 MHz, δ, ppm,
DMSO-d6), 174.47, 165.84, 164.09, 157.76, 127.82, 115.56, 114.11, 102.00, 66.92, 46.49,
43.80, 30.32, 29.91, 21.93, 13.98; ESIMS (M/Z): 535.28 [M+H]+, 557.26 [M+Na]+.
2.3.2. 3,6-Di-(morpholinyl-carbonylmethoxy)xanthone (XD2).
White solid; yield 89.6%; formed from 3,6-dihydroxyxanthone and α-chloro-
morpholinylacetamide; m.p. 251-253°C; 1H NMR (300 MHz, δ, ppm, DMSO-d6), 8.06 (d,
J = 8.7 Hz, 2H, aromatic H), 7.09-7.03 (m, 4 H, aromatic H), 5.08 (s, 4 H, OCH2 x 2),
3.64-3.36 (m, 16 H, morpholine ring CH2 x 8); 13CNMR (500 MHz, δ, ppm, DMSO-d6),
174.47, 165.74, 163.92, 157.75, 127.85, 115.65, 114.10, 102.03, 66.55, 66.44, 45.06,
42.10; ESIMS (M/Z): 483.18 [M+H]+, 505.16 [M+Na]+.
2.3.3. 3,6-Di-(N-tetrahydrofurfurylamino-carbonylmethoxy)xanthone (XD3).
White solid; yield 30.6%; formed from 3,6-dihydroxyxanthone and α-chloro-N-
tetrahydrofurfurylacetamide; m.p. 183-184°C; 1H NMR (300 MHz, δ, ppm, DMSO-d6),
10 | P a g e

8.24 (br. s, 2 H, NH x 2), 8.09 (d, J = 8.4 Hz, 2 H, aromatic H), 7.10-7.07 (m, 4 H,
aromatic H), 4.71 (s, 4 H, OCH2 x 2), 3.88 (m, 2 H, tetrahydrofuran ring OCH x 2), 3.74
(q, J = 6.6 Hz, 2 H, tetrahydrofuran ring OCH2), 3.61 (q, J = 6.9 Hz, 2 H, tetrahydrofuran
ring OCH2), 3.21 (t, J = 5.7 Hz, 4 H, =NCH2- x 2), 1.82-1.70 (m, 6 H, tetrahydrofuran
ring CH x 2 and CH2 x 2), 1.58-1.46 (m, 2 H, tetrahydrofuran ring CH x 2); 13CNMR
(500 MHz, δ, ppm, DMSO-d6), 174.48, 167.43, 163.47, 157.73, 127.99, 115.82, 114.23,
101.98, 77.41, 67.69, 67.62, 42.88, 28.89, 25.59; ESIMS (M/Z): 511.21 [M+H]+, 533.19
[M+Na]+.
2.3.4. 3,6-Di-[(2,6-dimethylpiperidinyl)-carbonylmethoxy]-4-methylxanthone
(XD4).
White solid; yield 87.2%; formed from 3,6-dihydroxy-4-methylxanthone and α-chloro-
(2,6-dimethylpiperidinyl)acetamide; m.p. 245-247°C; 1H NMR (300 MHz, δ, ppm,
DMSO-d6), 8.04 (d, J = 9.0 Hz, 1 H, aromatic H), 7.97 (d, J = 9.0 Hz, 1 H, aromatic H),
7.11 (d, J = 2.1 Hz, 1 H, aromatic H), 7.02 (dd, J = 9.0 Hz, 2.4 Hz, 2 H, aromatic H), 5.18
(br. s, 2 H, OCH2), 4.91 (br. s, 2 H, OCH2), 4.53 (br. s, 2 H, =NCH= x 2), 4.09 (br. s, 2 H,
=NCH= x 2), 2.37 (s, 3 H, CH3), 1.90-1.10 (m, 24 H, piperidine ring H, CH3 x 4);
13CNMR (500 MHz, δ, ppm, DMSO-d6), 174.93, 166.06, 165.84, 164.06, 161.49, 157.87,
155.06, 127.74, 124.80, 115.59, 115.17, 114.17, 113.38, 109.70, 102.02, 67.03, 66.87,
46.45, 43.68, 30.34, 21.96, 20.68, 13.97, 8.82; ESIMS (M/Z): 549.30 [M+H]+, 1097.57
[2M+H]+.
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2.3.5. 3,6- Di-(morpholinyl-carbonylmethoxy)-4-methylxanthone (XD5).
White solid; yield 91.3%; formed from 3,6-dihydroxy-4-methylxanthone and α-chloro-
morpholinylacetamide; m.p. 249-251°C; 1H NMR (300 MHz, δ, ppm, DMSO-d6), 8.13 (d,
J = 9.0 Hz, 1 H, aromatic H), 8.05 (d, J = 9.0 Hz, 1 H, aromatic H), 7.24 (d, J = 2.4 Hz, 1
H, aromatic H), 7.14 (d, J = 9.0 Hz, 1 H, aromatic H), 7.13-7.10 (m, 1 H, aromatic H),
5.17 (s, 2 H, OCH2), 5.15 (s, 2 H, OCH2), 3.71-3.55 (m, 16 H, morpholine ring CH2 x 8),
2.45 (s, 3 H, CH3); 13CNMR (500 MHz, δ, ppm, DMSO-d6), 174.95, 165.99, 165.76,
163.91, 161.26, 157.89, 155.06, 127.77, 124.80, 115.70, 115.26, 114.20, 113.46, 109.72,
102.03, 66.72, 66.50, 45.09, 42.10, 8.79; ESIMS (M/Z): 497.19 [M+H]+, 519.16
[M+Na]+, 1015.41 [2M+Na]+.
2.3.6. 3,6-Di-(N-tetrahydrofurfurylamino-carbonylmethoxy)-4-methylxanthone
(XD6).
White solid; yield 48.3%; formed from 3,6-dihydroxy-4-methylxanthone and α-chloro-N-
tetrahydrofurfurylacetamide; m.p. 202-203°C; 1H NMR (300 MHz, δ, ppm, DMSO-d6),
8.31 (m, 1 H, NH), 8.16 (m, 1 H, NH), 8.15 (d, J = 8.7 Hz, 1 H, aromatic H), 8.07 (d, J =
8.7 Hz, 1 H, aromatic H), 7.22 (d, J = 2.4 Hz, 1 H, aromatic H), 7.17-7.10 (m, 2 H,
aromatic H), 4.80 (s, 4 H, OCH2 x 2), 3.99-3.94 (m, 2 H, tetrahydrofuran ring OCH2),
3.85-3.79 (m, 2 H, tetrahydrofuran ring OCH2), 3.72-3.65 (m, 2 H, tetrahydrofuran ring
OCH x 2), 3.30 (t, J = 5.4Hz, 4H, =NCH2- x 2), 2.47 (s, 3 H, CH3), 1.95-1.82 (m, 6 H,
tetrahydrofuran ring CH x 2, CH2 x 2), 1.63-1.55 (m, 2 H, tetrahydrofuran ring CH x 2);
12 | P a g e

13CNMR (500 MHz, δ, ppm, DMSO-d6), 174.94, 167.65, 167.45, 163.46, 160.90, 157.82,
155.00, 127.89, 125.01, 115.91, 115.39, 114.25, 113.80, 110.00, 102.00, 77.42, 67.66,
67.64, 42.87, 42.81, 28.89, 28.84, 25.63, 25.60, 8.76; ESIMS (M/Z): 525.22 [M+H]+,
547.21 [M+Na]+, 1071.42 [2M+Na]+.
2.3.7. 3,6- Di-[(3,5-dimethylmorpholinyl)-carbonylmethoxy]-4-methylxanthone
(XD7).
White solid; yield 73.7%; formed from 3,6-dihydroxy-4-methylxanthone and α-chloro-
(3,5-dimethylmorpholinyl)acetamide; m.p. 170-171°C; 1H NMR (300 MHz, δ, ppm,
DMSO-d6), 8.13 (d, J = 9.0 Hz, 1H, aromatic H), 8.05 (d, J = 9.0 Hz, 1 H, aromatic H),
7.22 (d, J = 3.6 Hz, 1 H, aromatic H), 7.16-7.10 (m, 2 H, aromatic H), 5.27-5.18 (m, 2 H,
OCH2), 5.12-5.05 (m, 2 H, OCH2), 4.30-3.60 (m, 4 H, morpholine ring =CHO- x 4),
3.60-3.19 (m, 4 H, morpholine ring =NCH= x 4), 2.88-2.35 (m, 4 H, morpholine ring
=NCH= x 4), 2.45 (s, 3 H, CH3), 1.24-1.14 (m, 12 H, CH3 x 4); 13CNMR (500 MHz, δ,
ppm, DMSO-d6), 174.94, 166.26, 166.01, 165.68, 165.45, 163.92, 161.26, 157.89, 155.06,
127.76, 124.80, 115.69, 115.24, 114.21, 113.43, 109.72, 101.99, 71.74, 71.60, 66.79,
66.52, 66.10, 65.32, 49.92, 49.71, 46.99, 46.27, 19.12, 18.86, 17.80, 17.53, 8.81; ESIMS
(M/Z): 553.26 [M+H]+, 575.24 [M+Na]+, 1127.48 [2M+Na]+.
2.3.8. 3,6- Di-[(3,5-dimethylpiperidinyl)-carbonylmethoxy]-4-methylxanthone
(XD8).
White solid; yield 30.1%; formed from 3,6-dihydroxy-4-methylxanthone and α-chloro-
(3,5-dimethylpiperidinyl)acetamide; m.p. 188-190°C; 1H NMR (300 MHz, δ, ppm,
13 | P a g e

DMSO-d6), 8.03 (d, J = 9.0 Hz, 1 H, aromatic H), 7.96 (d, J = 8.7 Hz, 1 H, aromatic H),
7.14 (d, J = 2.4 Hz, 1 H, aromatic H), 7.04 (d, J = 9.0 Hz, 1 H, aromatic H), 7.01 (dd, J =
8.7 Hz, 2.4 Hz, 1 H, aromatic H), 5.16-4.92 (m, 4 H, OCH2 x 2), 4.36-4.23 (m, 2 H,
piperidine ring =NCH= x 2), 3.80-3.70 (m, 2 H, piperidine ring =NCH= x 2), 3.55-3.43
(m, 1 H, piperidine ring =NCH=), 3.06-2.92 (m, 1 H, piperidine ring =NCH=), 2.58-2.42
(m, 2 H, piperidine ring =NCH= x 2), 2.36 (s, 3 H, CH3), 1.98-1.32 (m, 6 H, C3, C4 and
C5 positions of two piperidine rings CH x 6), 0.96—0.68 (m, 12 H, CH3 x 4); 13CNMR
(500 MHz, δ, ppm, DMSO-d6), 174.96, 165.22, 164.98, 163.97, 161.31, 157.87, 155.05,
127.76, 124.83, 115.65, 115.19, 114.19,113.40, 110.00, 109.66, 101.97, 67.03, 66.71,
51.40, 48.67, 42.14, 32.00, 31.96, 31.20, 19.42, 19.21, 14.34, 8.77; ESIMS (M/Z): 549.29
[M+H]+, 571.28 [M+Na]+, 1119.56 [2M+Na]+.
2.3.9. 3,6- Di-[(N-2-methylcyclohexyl)amino-carbonylmethoxy]-4-methylxanthone
(XD9).
White solid; yield 42.4%; formed from 3,6-dihydroxy-4-methylxanthone and N-(α-
chloroacetyl)-2-methylcyclohexylamine; m.p. 242-243°C; 1H NMR (300 MHz, δ, ppm,
DMSO-d6), 8.14 (d, J = 9.0, 1 H, aromatic H), 8.13 (d, J = 9.0 Hz, 1 H, NH), 8.06 (d, J =
9.0 Hz, 1 H, aromatic H), 7.94 (d, J = 9.0 Hz, 1 H, NH), 7.21-7.12 (m, 3 H, aromatic H),
4.86-4.85 (m, 2 H, OCH2), 4.78-4.77 (m, 2 H, OCH2), 3.95-4.04 (m, 2 H, cyclohexane
ring =CHN= x 2), 2.45 (s, 3 H, CH3), 1.81-1.25 (m, 18 H, cyclohexane ring H), 0.92-0.87
(m, 6 H, CH3 x 2); 13CNMR (500 MHz, δ, ppm, DMSO-d6), 174.93, 166.94, 166.75,
166.51, 163.54, 161.08, 157.79, 155.00, 127.85, 124.96, 115.84, 115.32, 114.25, 113.79,
14 | P a g e

109.46, 101.96, 68.19, 67.75, 53.85, 53.80, 37.35, 34.45, 33.24, 25.87, 25.67, 19.57, 8.72;
ESIMS (M/Z): 549.29 [M+H]+, 1135.53 [2M+K]+.
2.3.10. 3,6- Di-[(4-ethoxycarbonylpiperazinyl)-carbonylmethoxy]-4-methylxanthone
(XD10).
White solid; yield 70.4%; formed from 3,6-dihydroxy-4-methylxanthone and ethyl 4-(α-
chloroacetyl)piperazine-1-carboxylate; m.p. 170-171°C; 1H NMR (300 MHz, δ, ppm,
DMSO-d6), 8.08 (d, J = 9.0 Hz, 1 H, aromatic H), 8.01 (dd, J = 9.0 Hz, 0.6 Hz, 1 H,
aromatic H), 7.21 (d, J = 2.1 Hz, 1 H, aromatic H), 7.10 (d, J = 9.3 Hz, 1 H, aromatic H),
7.07 (dd, J = 8.7 Hz, 2.4 Hz, 1 H, aromatic H), 5.14 (s, 2 H, OCH2), 5.13 (s, 2 H, OCH2),
4.14-4.05 (m, 4 H, ethoxy group OCH2 x 2), 3.51-3.42 (m, 16 H, piperazine ring H), 2.41
(s, 3 H, CH3), 1.26-1.18 (m, 6 H, ethoxy group CH3 x 2); 13CNMR (500MHz, δ, ppm,
DMSO-d6), 174.95, 166.02, 165.79, 163.88, 161.24, 157.89, 155.08, 155.06, 127.77,
124.81, 115.71, 115.26, 114.18, 113.46, 102.06, 66.81, 66.60, 61.43, 44.27, 41.49, 15.03,
8.82; ESIMS (M/Z): 639.26 [M+H]+, 1299.50 [2M+Na]+.
2.4.
Synthesis of XD11 and XD12
2,2’-[(xanthone-3,6-diyl)bis(oxy)]bis-acetic acid (344 mg, 1mmol) or 2,2’-[(xanthone-4-
methyl-3,6-diyl)bis(oxy)]bis-acetic acid (358 mg, 1mmol) was dissolved in DMF (20
mL). N,N’-dicyclohexylcarbodiimide (DCC, 247 mg, 1.2mmol) and 4-
dimethylaminopyridine (DMAP, 2.4 mg, 0.02mmol) were added to the solution. After
stirring for 1 h at room temperature, 1-methyl piperazine (300 mg, 3mmol) was added,
and the mixture was stirred for 6 h at 70°C. After the mixture was cooled to room
15 | P a g e

temperature, ice water (40 mL) was added while the mixture was stirred. The solid was
filtered out, washed with water, and dried. XD11 and XD12 were purified by dry-column
flash chromatography.
2.4.1. 3,6- Di-[(4-methylpiperazinyl)-carbonylmethoxy]xanthone (XD11).
White solid; yield 63.6%; formed from 2,2’-[(xanthone-3,6-diyl)bis(oxy)]bis-acetic acid
and 1-methyl piperazine; m.p. 193-195°C; 1H NMR (300 MHz, δ, ppm, DMSO-d6), 8.06
(dd, J = 8.7 Hz, 0.9 Hz, 2 H, aromatic H), 7.07-7.02 (m, 4 H, aromatic H), 5.06 (s, 4 H,
OCH2 x 2), 3.47-3.44 (m, 8 H, piperazine ring CH2 x 4), 2.36-2.27 (m, 8 H, piperazine
ring CH2 x 4), 2.19 (s, 6 H, NCH3 x 2); 13CNMR (500 MHz, δ, ppm, DMSO-d6), 174.47,
165.47, 163.92, 157.73, 135.79, 127.85, 115.62, 114.11, 102.00, 66.66, 55.05, 54.71,
46.15, 44.40, 41.68; ESIMS (M/Z): 509.24 [M+H]+, 1039.45 [2M+Na]+.
2.4.2. 3,6- Di-[(4-methylpiperazinyl)-carbonylmethoxy]-4-methylxanthone (XD12).
White solid; yield 49.1%; formed from 2,2’-[(xanthone-4-methyl-3,6-diyl)bis(oxy)]bis-
acetic acid and 1-methyl piperazine; m.p. 175-176°C; 1H NMR (300 MHz, δ, ppm,
DMSO-d6), 8.13 (d, J = 9.3 Hz, 1 H, aromatic H), 8.05 (d, J = 9.0 Hz, 1 H, aromatic H),
7.24 (d, J = 2.4 Hz, 1 H, aromatic H), 7.14-7.08 (m, 2 H, aromatic H), 5.16 (s, 2 H,
OCH2), 5.14 (s, 2 H, OCH2), 3.50-3.57 (m, 8 H, piperazine ring CH2 x 4), 2.51-2.58 (m, 8
H, piperazine ring CH2 x 4), 2.37 (s, 3 H, CH3), 2.29 (s, 3 H, NCH3), 2.28 (s, 3 H, NCH3);
13CNMR (500 MHz, δ, ppm, DMSO-d6), 174.94, 165.74, 165.50, 163.91, 161.27, 157.87,
155.05, 127.76, 124.80, 115.67, 115.23, 114.20, 113.43, 110.00, 102.00, 66.83, 66.58,
16 | P a g e

55.04, 54.66, 46.09, 44.40, 41.64, 8.79; ESIMS (M/Z): 523.25 [M+H]+, 1045.49
[2M+H]+.
2.5.
Cell culture
Five human cancer cell lines, breast adenocarcinoma (adenocarcinoma MDA-MB-231),
prostate adenocarcinoma (PC-3), lung carcinoma (A549), pancreas adenocarcinoma
(AsPC-1), and colorectal carcinoma (HCT116) were obtained from ATCC (Manassas,
VA). All cell lines were maintained at 37°C in 5% CO2 in culture flasks in RPMI-1640
medium (Sigma-Aldrich, St. Louis, MO), supplemented with 10% fetal calf serum (Life
Technologies, Grand Island, NY), 100-U/ml penicillin, and 0.1-mg/mL streptomycin.
Upon reaching confluence, the cells were trypsinized with 0.25% trypsin containing
0.01% EDTA for 5 minutes at 37°C and then stopped by the addition of complete
medium. About 5 X 105 of the viable cells were then resuspended in complete medium.
2.6.
In vitro cell viability assay
MDA-MB-231 (5 × 103 cells), PC-3 (8 × 103 cells), A549 (5 × 103 cells), AsPC-1 (5 ×
103 cells), and HCT116 (1 × 104 cells) were seeded in 96-well plates and incubated for 24
h to allow the cells to attach. A stock solution of 400 mM of XD was prepared in DMSO
(Sigma, St. Louis, MO, USA) and stored at −20°C. The stock solution was diluted to the
appropriate concentrations with culture medium. The final concentration of DMSO was
less than 0.1% (vol/vol). The same amount of DMSO was used as the vehicle control
throughout this study. Xanthone derivatives were serial diluted (final concentration 0 -
400μM) and added to the microtiter plate. 5-Fluorouracil (5-FU) was added as a positive
17 | P a g e

control. The cells were incubated with compounds for 48 h. MTT (3-(4,5-dimethy-1-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide), a tetrazole assay that uses mitochondrial
metabolic enzyme activity as an indicator of cell viability, was conducted following the
protocol described previously.25 The cell viability was determined at 560-nm absorbance
using a Spectra Max M2 plate reader (Molecular Devices, Sunnyvale, CA). Experiments
were performed in triplicate. Cell viability was calculated as the ratio of the mean of OD
obtained for the treated cell to that of the vehicle control.
2.7.
Cell cycle analysis
After exposure to XD for 48 h, A549 cells were collected and immediately fixed with
70% ethanol overnight. Samples were stained with propidium iodide staining solution
containing 50-µg/mL propidium iodide, 200-µg/ml RNase A, and 0.3% Triton-X100 for
30 min at room temperature. The cells were then measured by Accuri C6 flow cytometry
to measure the fractions of cell populations in the sub-G1 (fractional DNA content), G0/1
(quiescent state/growth phase), S (initiation of DNA replication), and G2/M
(biosynthesis/mitosis) phases. Data were analyzed using the C6 software (BD
Biosciences, San Jose, CA).
2.8.
Cell apoptosis assay
A549 cells (2 × 105) were seeded in 12-well plates and treated with 25 µM of XD. 48 h
after treatment, cells were trypsinized, harvested and washed with ice cold PBS.
Apoptosis was assayed using the Annexin V: FITC Apoptosis Detection Kit (BD
Biosciences) according to the manufacturer's instructions. 1 × 105 cells were incubated
18 | P a g e

with 100 µl of binding buffer containing Annexin V - FITC and propidium iodide at
room temperature for 15 min. After incubation, 400 µl of binding buffer was added to
each sample, and cells were kept on ice. Samples were analyzed with an Accuri C6
cytometer (BD Biosciences).
2.9.
Caspase-3/7 activity assay
To investigate the mechanism underlying the apoptotic activity of the XD, the activation
of caspase-3 and caspase-7 was detected using the Caspase-Glo 3/7 Assay (Promega,
Madison, WI). A549 cells were plated in duplicate in 96-well plates and treated with XD.
48 hours after exposure, the caspase 3/7 activities were measured according to the
manufacturer's instructions. Samples were read after 1 h of incubation with the caspase
substrate. The luminescence was detected using the VICTOR 3V Multilabel Plate Reader
(PerkinElmer, Waltham, MA). The background luminescence associated with the cell
culture and assay reagent (blank reaction) was subtracted from the experimental value.
The relative fold increase of caspase 3/7 was calculated by dividing the luminescence
reading from the compound treatment to that of control.
3. Results and discussion
3.1.
Chemistry
Scheme 1 shows the synthetic route used to produce the lead xanthone compounds. 3,6-
Dihydroxy-4-methylxanthone was synthesized from 2,4-dimethoxybenzoic acid in a
54.3% general yield. The key factor ensuring a high yield of the desired xanthone
derivative was the hydrolysis of 2-hydroxy or 2’-hydroxy during or after the Friedel-
19 | P a g e

Crafts reaction in step “b” in an acidic condition, which yielded 2-hydroxy-3-methyl-
2’,4,4’-trimethoxybenzophenone or 2’-hydroxy-3-methyl-2,4,4’-
trimethoxybenzophenone. If there is no free hydroxyl group in the 2- or 2’-position, there
is no cyclization in the next step to yield 3,6-dimethoxy-4-methylxanthone.26 The last
step “c” yielded 66.9% in the hydrolysis of 3,6-dimethoxy using a mixture of conc.
hydrobromic acid and glacial acetic acid. The yield for this step might have been higher if
a more costly demethylation agent, such as boron tribromide, was used. 3,6-
Dihydroxyxanthone was synthesized in the same way, with a general yield of 58.3%.
O
O
O
H3CO
a
b
H3CO
H3CO
OH
H3CO
OH
Cl
OCH3
OCH3
OCH3
R1
O
O
O
H3CO
d
c
+
OCH3
HO
HO
O
OH
H3CO
O
OCH3
R1
R1
OCH3
R1
3,6-dihydroxy-4-methylxanthone, R1=CH3; 3,6-dihydroxyxanthone, R1=H
Scheme 1. Synthetic route of 3,6-dihydroxylxanthone with or without a 4-methyl group.
Reagents and conditions: (a) Benzene, SOCl2, refluxed for 3h; (b) 2,6-dimethoxytoluene
or 2,6-dimethoxybenzene, AlCl3, diethyl ether, 0-10° C, 10h; (c) CH3OH, K2CO3, H2O,
28h; (d) conc. hydrobromic acid, glacial acetic acid, refluxed for 48 h.
The target xanthone derivative compounds (XD), 3,6-N-substituted or 3,6-N,N-
disubstituted aminocarbonylmethoxy xanthones with or without a 4-methyl group, were
synthesized in two ways. Scheme 2 illustrates the synthesis of XD 1-10, in which single-
20 | P a g e

or double-substituted amines react with alpha-chloroacetic chloride under conditions of
1,2-dichloroethane as a solvent and sodium hydroxide as a catalyst to yield alpha-
chloroacetamide (step “e”). These alpha-chloroacetamides were then introduced into 3,6-
dihydroxy-4-methylxanthone or 3,6-dihydroxyxanthone molecule by etherification with
potassium carbonate as a catalyst and acetone as a solvent (step “f”) to achieve the final
products. The second approach is represented in scheme 3: it illustrates the synthesis of
XD11 and 12 in which 3,6-dihydroxy-4-methylxanthone or 3,6-dihydroxyxanthone were
first etherified with ethyl alpha-bromoacetate in the catalysis of K2CO3 (step “g”) to give
the diethyl carboxylate derivatives. These derivatives were then hydrolyzed with sodium
hydroxide solution in ethanol to yield the dicarboxylic acid product (step “h”). Finally,
this dicarboxylic acid compound was condensed with 1-methylpiperazine under the
catalysis of DCC and DMAP (step “i”), thus yielding the target compounds.
21 | P a g e

O
O
R3
N
R3
R3
N
R3
N
f
e
H
N
R2
R2
Cl
R2
O
O
R2
O
O
R1
O
XD 1-10
H3C
N
R3
R3
XD2: R1=H;
XD1: R1=H;
XD3: R1=H;
N
N
N
=
=
=
N
O
R2
R2
H3C
H3C
N
R3
R3
R2
H
N
XD4: R1=CH3;
XD6: R1=CH3;
N
N
=
R2
O
H3C
R3
R3
R2
H
N
XD5: R1=CH3;
XD7: R1=CH3;
N
N
N
O
=
=
=
R2
R3
R2
O
CH3
CH3
R3
XD8: R1=CH3;
XD10: R1=CH3;
N
=
N
O
N
R2
CH3
CH3
H
CH3
O
R3
R3
N
N
XD9: R1=CH3;
N
N
=
N
=
R2
R2
OC2H5
Scheme 2. Synthetic route of 3,6-N-substituted, or 3,6-N,N-disubstituted
aminocarbonylmethoxyxanthone with or without a 4-methyl group by direct
etherification. Reagents and conditions: (e) ClCH2COCl, ClCH2CH2Cl, 20% NaOH
solution, 0°C; (f) 3,6-dihydroxylxanthone with or without a 4-methyl group, K2CO3,
acetone, refluxed for 8-14 h.
22 | P a g e

O
O
O
O
g
h
C2H5O
OC2H5
O
O
HO
HO
OH
O
R1
O
R1
O
O
O
R3
N
R3
N
i
OH
O
O
R2
O
O
O
R2
O
R1
O
O
R1
O
XD 11,12
R3
R3
XD 11: R1=H;
XD 12: R1=CH3;
=
N
N
N
CH3
N
=
N
N
CH3
R2
R2
Scheme 3. Synthetic route of 3,6- di-(4-methyl-1-piperazinyl-carbonylmethoxy)xanthone
with or without 4-methyl group by amidation of dicarboxylic acid. Reagents and
conditions: (g) ethyl alpha-bromoacetate, K2CO3, acetone, refluxed for 6h; (h) 2N NaOH
solution, ethanol, refluxed for 1h; (i) DCC, DMAP, DMF, 70° C, 6 h.
3.2.
Cytotoxicity on human cancer cell lines
The effect of XD and 5-FU on cell growth in five human cancer cell lines (breast
adenocarcinoma [MDA-MB-231], prostate adenocarcinoma [PC-3], lung carcinoma
[A549], pancreatic adenocarcinoma [AsPC-1], and colorectal carcinoma [HCT116]) was
determined with an MTT assay. The adherent cancer cells were treated with different
concentrations of XD and 5-FU, and each treatment was performed in triplicate. The fifty
percent (IC50) values of the derivatives were calculated by plotting the cell viability
against the concentration of compounds. As Table 1 shows, some synthesized XD
exhibited strong antiproliferative effects on cancer cell viability in vitro. Out of 12
derivatives, compounds XD1, XD4, and XD8-9 were found to be potent growth
23 | P a g e

inhibitors (IC50 ˂ 25 µM), whereas compounds XD6 and XD10 had a more moderate
effect on growth inhibition with IC50 values close to those of 5-FU. The IC50 values
showed that the compounds XD4, XD8, and XD9 exhibited similar anticancer potency
against all cancer cell lines. Compounds XD1 and XD6 were more active against prostate
cancer cells and less active against breast cancer cells. Compound XD10 demonstrated
strong growth inhibition against colorectal cancer cells.
Table 1. Growth inhibition effect (IC50) of xanthone derivatives against human cancer cell
lines.
Breast
Cancer
MDA-MB-
231
Prostate
Cancer
Lung
Pancreatic
Cancer
Colorectal
Cancer
Cancer
Compounds
PC-3
A549
AsPC-1
HCT116
35.91
24.33
>400
72.07
6.68
39.86
17.25
>400
>400
10.58
>400
27.53
295.00
4.59
98.00
13.80
>400
>400
7.10
15.38
15.73
>400
>400
7.63
5-FU
XD1
XD2
XD3
XD4
XD5
XD6
XD7
XD8
>400
249.20
4.27
>400
7.31
>400
>400
34.50
213.50
6.18
>400
78.74
115.54
4.76
>400
33.81
140.88
6.09
98.44
385.23
8.06
24 | P a g e

11.03
64.49
>400
23.30
53.32
>400
89.17
22.32
84.85
>400
15.41
34.13
>400
97.79
18.33
8.16
XD9
XD10
XD11
XD12
>400
119.39
257.10
180.56
The IC50 values are given in µM. 5-FU was used as a positive control.
According to a structure-activity relationship analysis of the XD cytotoxicity results, we
found that most XD with a 4-methyl group exhibited more potent anticancer activities
than their counterparts without a 4-methyl group. Some xanthones with a 2,6-dimethyl
piperidine ring, 3,5-dimethyl piperidine ring, or 2-methyl cyclohexane amine in their side
chains more potently inhibited cancer cell growth than those with a morpholine ring, 3,5-
dimethyl morpholine ring, or 4-methyl and ethoxycarbonyl piperazine rings in their side
chains. These results suggest that electronegative atoms like oxygen, which could form
hydrogen bonds with biological macromolecules in their side chains, reduce the growth
inhibitory properties of the XD. It also appears that steric hindrance around the nitrogen
atom in acetamide side chains decreases the IC50 of the XD (e.g., the compound XD9
with the larger N-2-methylcyclohexyl acetamide side chain has a smaller IC50 than XD6
with its N-tetrahydrofurfuryl acetamide side chain).
3.3.
Cell cycle analysis
To establish whether the tested compounds inhibited cell growth by interrupting cells in
the cell cycle progression, cellular DNA was analyzed and stained with PI and the cells
were analyzed using flow cytometry. The cell cycle profile (Fig. 2) was represented
25 | P a g e

through 3 independent experiments on A549 cell lines. The results in Figure 2 indicate
that a significant increased percentage of cells were in the G0/G1 phase 48 hours
following XD4, XD6, XD8, and 5-FU treatment. XD1 and XD9 exposure induced cell
accumulation in the G2/M phase. Consistent with the MTT results, compounds without
cytotoxic properties did not affect cell cycle distribution. It has been reported that some
XD exhibit cytotoxicity in cancer cell lines through cell cycle arrest and resulting
apoptosis.27-29 The alterations in cell cycle reported here for five of our XD suggest that
cell cycle arrest is one of the primary mechanisms responsible for the anticancer activity
of these derivatives.
26 | P a g e

Figure 2. Effect of XD on cell cycle progression. A549 cells were incubated with XD (25
µM) for 48 hours. Cells were collected, fixed in 70% ethanol, and stained with propidium
iodide solution. M1, M2, and M3 in the figure represent the G0/1 (quiescent state/growth
phase), S (initiation of DNA replication), and G2/M (biosynthesis/mitosis) phases,
respectively.
3.4.
Cell apoptosis analysis
In addition to the cell cycle analysis, we tested the XD for apoptosis activity using
Annexin V/propidium iodide staining. Flow cytometry analysis of stained cells
distinguished between four groups: (1) viable (annexin V- PI-), (2) early apoptosis
(annexin V+ PI-), (3) late apoptosis (annexin V+ PI+), and (4) necrotic (annexin V- PI+)
cells. Apoptosis staining showed that treatment with XD1, XD4, and XD8 increased
27 | P a g e

apoptosis and necrosis in A549 cells. As shown in Figure 3, there was increased late
apoptotic activity in cells treated with XD1 (control 1.2% and XD1 treated 9.1%). XD4
was found to induce apoptotic activity in early (control 0.5% and XD4 7.3%) and late
(control 1.2% and XD4 9.8%) apoptotic populations. XD8 could inhibit the cancer cell
growth at IC50 ˂10 µM with an accompanying increase in early apoptotic cell population
(control 0.5% and XD8 37.5%), followed by a late apoptotic population (control 1.2%
and XD8 12.4%). These results suggest that apoptosis is an anticancer mechanism for
XD1, XD4, and XD8.
Although XD6, XD9, and XD10 exhibited potent anticancer properties in the cytotoxicity
assay, these compounds did not cause significant alterations in cell apoptosis. In contrast,
the control compound, 5-FU, was found to induce an increase in the early apoptosis
population.
28 | P a g e

Figure 3. Cell apoptosis analysis of A549 cell exposed to xanthone derivatives at a
concentration of 25 µM for 48 hours. Cells were treated with tested compounds and
collected for evaluation of apoptosis via Annexin V: FITC Apoptosis Detection Kit per
manufacture’s protocol. The lower left quadrant shows the viable cells, the upper left
shows necrotic cells, the lower right shows the early apoptotic cells while the upper right
shows late apoptotic cells.
3.5.
Caspase 3/7 activity
To further explore the molecular mechanism underlying the apoptotic activity of the XD,
we tested the induction of caspase 3/7 activity. As shown in Figure 4, we observed an
elevation in caspase 3/7 activity after XD1, XD4, and XD8 treatment. Caspase 3/7
activity increased 2.31-fold, 3.45-fold, and 2.04-fold for XD1, XD4, and XD8,
29 | P a g e