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
Reference: BMC 13133

To appear in: Bioorganic & Medicinal Chemistry

Received Date: 12 May 2016

Revised Date: 7 July 2016
Accepted Date: 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

[email protected]

1|P a ge
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 ge
 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

35
O
34 30
O 29
28 OH
25
O 31 26
19 O 24
40 20 1 17 15
13 23
O 18 O 22
O
H3C O 2
36 A B C 21 D 12
CH3 O 11
39 9
O 4 6 8 10
OH OH O
Xanthone skeleton DMXAA Gambogic Acid
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 ge
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

4|P a ge
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 ge
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 ge
 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 CH 2Cl2 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 ge
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.

1
HNMR 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 ge
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);
13
CNMR (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 ge
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);
13
CNMR (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]+.

11 | P a g e
 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
13
CNMR (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);
13
CNMR (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 Cl OH
H3CO
OCH3 OCH3 OCH3
R1

O
O O
H3CO
+ c d
OCH3
HO H3CO O OCH3 HO O OH
OCH3 R1 R1
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
Scheme 1. Synthetic
conditions: route
(a) Benzene, of2,3,6-dihydroxylxanthone
SOCl with or without
refluxed for 3h; (b) 2,6-dimethoxytoluene, AlCla3,4-methyl group.
diethyl ether, 0-10°
C, 10h; (c) CH3OH, K2CO3, H2O, 48h; (d) concn, hydrobromic acid, HAc, refluxed for 24h.
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
R3 R3
e f R3 R3
H N N
R2 R2 N N
Cl R2 O O O R2
O O R1 O
XD 1-10
H3C
R3 R3
XD1: R1=H; N = N XD2: R1=H; N = N O
R2 R2

H3C
H3C
R3 R3
H
XD3: R1=H; N = N XD4: R1=CH3; N = N
R2 O R2
H3C
R3 R3
XD5: R1=CH3; H
N = N O XD6: R1=CH3; N = N
R2 R2 O
CH3 CH3
R3 R3
XD7: R1=CH3; N = N O XD8: R1=CH3; N = N
R2 R2

CH3 CH3
H CH3 R3 O
R3
XD9: R1=CH3; N N XD10: 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
Scheme a 4-methyl
2. Synthetic group
route ofby direct etherification.or
3,6-N-substituted, Reagents and conditions: (e) ClCH2COCl,
3,6-N,N-disubstituted
ClCH2CH2Cl, 20% NaOH solution, 0°C; (f) 3,6-dihydroxylxanthone with or without a 4-methyl group,
K2CO3, acetone, refluxed for 8-14h.
aminocarbonylmethoxyxanthone with or without a 4-methyl group by direct

etherification. Reagents and conditions: (e) ClCH2 COCl, 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

g h
C2H5O OC2H5
O O O
HO O OH
O R1 O
R1
O O
R3 R3
i
HO OH N N
O O O R2 O O O R2
O R1 O O R1 O

XD 11,12
R3 R3
XD 11: R1=H; N = N N CH3 XD 12: R1=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 Prostate Lung Pancreatic Colorectal

Cancer Cancer Cancer Cancer Cancer

Compounds
MDA-MB-
PC-3 A549 AsPC-1 HCT116
231

5-FU 35.91 72.07 39.86 98.00 15.38

XD1 24.33 6.68 17.25 13.80 15.73

XD2 >400 >400 >400 >400 >400

XD3 >400 249.20 >400 >400 >400

XD4 7.31 4.27 10.58 7.10 7.63

XD5 >400 >400 >400 >400 >400

XD6 98.44 34.50 27.53 78.74 33.81

XD7 385.23 213.50 295.00 115.54 140.88

XD8 8.06 6.18 4.59 4.76 6.09

24 | P a g e
XD9 11.03 23.30 22.32 15.41 18.33

XD10 64.49 53.32 84.85 34.13 8.16

XD11 >400 >400 >400 >400 >400

XD12 257.10 89.17 180.56 97.79 119.39

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
respectively, compared to the untreated control. 5-FU treatment induced a 4.2-fold

increase in caspase 3/7 activity. Although apoptosis is a complex process that includes

caspase-dependent and caspase-independent mechanisms,30 the results shown in Figure 4

suggest that XD1, XD4, and XD8 induce apoptosis through activation of a caspase-

dependent pathway. No activation of caspase 3/7 was observed for the compounds XD6,

XD9, and XD10, suggesting that they induce growth inhibition through a different

pathway.

30 | P a g e
Figure 4. Effect of xanthone derivatives on caspase 3/7 activation. A549 cells (8 x 103)

were plated in duplicate in 96-well plates and treated with xanthone derivatives (25 µM).

48 hours after exposure, caspase 3/7 activities were measured using the Caspase-Glo 3/7

Assay. Data are represented as the mean (± 1 standard deviation) in the bar graph.

Analysis of variance with Dunnett’s post hoc testing was used to analyze any potential

difference between the treated groups and the control group. A P value <0.05 (labeled

with asterisk) was regarded as statistically significant.

4. Conclusions

In summary, a series of novel 3,6-N-substituted or 3,6-N,N-disubstituted

aminocarbonylmethoxyxanthones with or without a 4-methyl group have been

synthesized and characterized by 1HNMR, 13CNMR, and MS. Most of these XD had very

good anticancer activities in vitro and obvious structure-activity relationships. Further

studies aiming at clarifying the mechanisms underlying the anticancer activities of these

XD are continuing in our laboratories, which will be reported in due course. The

compounds XD1, XD4, XD6, and XD8-10 inhibited the growth of human breast cancer

(MDA-MB-231), prostate cancer (PC-3), lung cancer (A549), pancreatic cancer (AsPC-

1), and colorectal cancer (HCT116) cells. Treatment of cancer cells with XD1 and XD4

resulted in cell accumulation in G2/M phase, while XD6 and XD8 led to G0/G1

accumulation. Furthermore, the results from the cell apoptosis staining and caspase 3/7

activation testing suggested that the anticancer mechanism of compounds XD1, XD4, and

XD8 are mediated through apoptosis via a caspase-dependent pathway.

31 | P a g e
Acknowledgments

This project is supported by UF Health Cancer Center startup funds. We thank Dr. Ion

Ghiviriga and Robert Harker in the Department of Chemistry, University of Florida, for

helping us obtain the NMR data. We thank Dr. Kari B. Basso in the Department of

Chemistry, University of Florida, for supplying us with the MS data. We also thank Kate

Casey-Sawicki, MA, for editing and preparing this manuscript for publication.

32 | P a g e
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Graphical Abstract