Synthesis, SAR and pharmacological characterization of novel anthraquinone cation compounds as potential anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2016.10.012

Emodin, a natural anthraquinone derivative isolated from Rheum palmatum L., has been demonstrated to exhibit good anti-cancer effect. In this study, a series of novel quaternary ammonium salts of emodin, anthraquinone and anthrone were synthesized and their anticancer activities were tested in vitro. The effects of emodin quaternary ammonium salts on cell viability, apoptosis, intracellular ROS, and mitochondrial membrane potential were investigated in A375, BGC-823, HepG2 and HELF cells. The results demonstrated that compound 4a induced morphological changes and decreased cell viability. Apoptosis triggered by compound 4a was visualized using DAPI staining and Annexin V-FITC/PI staining. Compound 4a-induced apoptosis of A375 cells were showed to be associated with the dissipation of mitochondrial membrane potential (ΔΨm) as a result of the up-regulation of P53 and Caspase-3. When cancer cells were treated with emodin derivative, their ability to generate reactive oxygen species (ROS) rose significantly and the mitochondrial membrane potential decreased. Additionally, confocal microscopy assay confirmed that compound 4a was primarily located in the mitochondria of A375 cells. These results suggested that compound 4a has the potential for use in cancer therapy.

Full text of DOI:10.1016/j.ejmech.2016.10.012

Accepted Manuscript
Synthesis, SAR and pharmacological characterization of novel anthraquinone cation
compounds as potential anticancer agents
Yanyan Zheng, Li Zhu, Lulu Fan, Wenna Zhao, Jianlong Wang, Xianxiao Hao, Yunhui
Zhu, Xiufang Hu, Yaofeng Yuan, Jingwei Shao, Wenfeng Wang
PII:
S0223-5234(16)30874-1
10.1016/j.ejmech.2016.10.012
EJMECH 8977
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 August 2016
Revised Date: 5 October 2016
Accepted Date: 6 October 2016
Please cite this article as: Y. Zheng, L. Zhu, L. Fan, W. Zhao, J. Wang, X. Hao, Y. Zhu, X. Hu, Y. Yuan,
J. Shao, W. Wang, Synthesis, SAR and pharmacological characterization of novel anthraquinone cation
compounds as potential anticancer agents, European Journal of Medicinal Chemistry (2016), doi:
10.1016/j.ejmech.2016.10.012.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
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ACCEPTED MANUSCRIPT
Graphical abstract
Fifteen new emodin derivatives were synthesized and investigated for their anticancer activities. Compound 4a was shown to induce
apoptosis in cancer cells in vitro.

ACCEPTED MANUSCRIPT
Synthesis, SAR and pharmacological characterization of novel
anthraquinone cation compounds as potential anticancer agents
a
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Authors: Yanyan Zheng , Li Zhu , Lulu Fan , Wenna Zhao , Jianlong Wang ,
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Xianxiao Hao , Yunhui Zhu , Xiufang Hu , Yaofeng Yuan , Jingwei Shao *,
Wenfeng Wang *
Affiliations:
a College of Chemistry, Fuzhou University, 350108, P. R. China
*
Corresponding authors Jingwei Shao and Wenfeng Wang
Address: 523 Industry Road, Science Building, 3FL. Fuzhou, Fujian 350002, China
E-mail: shaojingwei@fzu.edu.cn; wangwf@fzu.edu.cn
Telephone: + 86-13600802402(Shao); +86-13905900679(Wang)
Fax: +86-591-83576912(Shao); +86-591-22866161(Wang)
No potential conflicts of interests were disclosed.

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Abstract
Emodin, a natural anthraquinone derivative isolated from Rheum palmatum L.,
has been demonstrated to exhibit good anti-cancer effect. In this study, a series of
novel quaternary ammonium salts of emodin, anthraquinone and anthrone were
synthesized and their anticancer activities were tested in vitro. The effects of emodin
quaternary ammonium salts on cell viability, apoptosis, intracellular ROS, and
mitochondrial membrane potential were investigated in A375, BGC-823, HepG2 and
HELF cells. The results demonstrated that compound 4a induced morphological
changes and decreased cell viability. Apoptosis triggered by compound 4a was
visualized using DAPI staining and Annexin V-FITC/PI staining. Compound
4a-induced apoptosis of A375 cells were showed to be associated with the dissipation
of mitochondrial membrane potential (∆Ψm) as a result of the up-regulation of P53
and Caspase-3. When cancer cells were treated with emodin derivative, their ability to
generate reactive oxygen species (ROS) rose significantly and the mitochondrial
membrane potential decreased. Additionally, confocal microscopy assay confirmed
that compound 4a was primarily located in the mitochondria of A375 cells. These
results suggested that compound 4a has the potential for use in cancer therapy.
Keyword: Quaternary ammonium salt; Reactive oxygen species; Anticancer
activity; Quinoid structure; Synthesis.
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1. Introduction
Emodin (1,3,8-trihydroxy-6-methyl-9,10-anthraquinone) has been identified as a
natural product from herbs used in traditional medicine for treatment of constipation
jaundice, gastro-intestinal hemorrhage and ulcers in China for more than two
thousand years. It has been shown to display a number of biological activities such as
anti-tumor [1], antimicrobial [2], immunosuppressive [3], and enzyme inhibition [4].
Recently, pharmacological studies showed that emodin is capable of inhibiting
cellular proliferation [5], and activation of caspase cascade and mitochondrial death
pathways [6] in different cancer cells. .
In addition, chemical modifications of anthraquinone have been more commonly
reported than those of emodin due to its accessibility. Shrestha et al [7] synthesized a
series of novel analogs of cationic anthraquinones whose possible modes of
interaction with cancer cells are intercalation of nucleic acids and disruption of
cellular redox processes. Joksovic et al [8] synthesized a series of
anthraquinone-chalcone hybrids which showed promising activity in inhibition of
HeLa cells and low cytotoxicity against healthy MRC-5 cell lines. Lee et al [9] used
ring fusion strategy to synthesize sulfur-substituted anthrax [1,2-c][1,2,5]
thiadiazole-6,11-dione derivatives which not only induced apoptosis of cancer cells
but also attenuated the ERK1/2 and p38 signaling pathways. Zhao et al [10] reported
that an introduction of mustard group into hydrioxyanthraquinone moiety enhanced
drug’s anticancer activities based on rational . In addition, natural anthraquinones,
such as Rubiadin and Soranjidiol [11], are reported to induce apoptosis in cancer cells.

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These works indicate that quinoid structure may play an important role in anticancer
activity. Figure 1 shows some structures of derivatives of anthraquinone mentioned
above.
We previously reported that emodin quaternary ammonium salt derivatives
showed significant anticancer activities against hepatoma cells both in vitro and in
vivo [12,13]. We also reported that lipophilicity and the length of the carbon chains on
the quaternary (4°) nitrogen atom are important factors and chemical moieties
accounting for the anticancer activities of the emodin quaternary ammonium salt
derivatives [14,15]. To fully address the structure-activity relationship (SAR) and
further refine the molecular structure of the emodin derivatives for anticancer
activities, a number of questions should be addressed first. (1) Can other alkyls, such
as nonyl, bring better anticancer activity for emodin quaternary ammonium salts? (2)
If lipophilic long carbon chain is substituted by polyoxyethylene chain to enhance the
water solubility of emodin quaternary ammonium salt, how will the anticancer
activity change? (3) what is the mechanism of action of these emodin quaternary
ammonium salt derivatives? (4) Is quinoid structure necessary for the anticancer
activities of emodin quaternary ammonium salts? In order to answer these questions,
fifteen new quaternary ammonium salts of emodin, aloe emodin, anthraquinone and
anthrone were synthesized and their anticancer activities were tested in vitro. In
addition, the anti-cancer mechanism of compound 4a was also evaluated against A375
cell line.
2. Materials and methods

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2.1. General
The chemical agents and the apparatus to test the structures of compounds
synthesized in this work are the same as those have been reported in our recent work
[12,13,19].
2.2. Synthesis
2.2.1. The preparation of compound 1 (N-methylnonan-1-amine)
1-Bromononane (15 mL, 16.3 g, 79.1 mmol) in 15 mL ethanol was added into 50
mL aqueous methylamine (40%, excessive). The mixture was stirred at room
temperature for 5 h. The mixture was washed by 80 mL distilled water for 3 times to
remove unreacted methylamine and the water phase was extracted by CHCl (30
3
mL×3). The organic phases were combined and dried by Na SO . After the solvent
2
4
had been removed under reduced pressure, the residue (light yellow sticky liquid) was
chromatographed on a silica gel column by using dichloromethane/ethanol (v/v 20:1)
as eluents to give product 1.
1
Yield: 94.0%; Colorless sticky liquid; H NMR (400MHz, CDCl ) δ: 3.69 (s, 1H,
3
-NH), 2.61 (t, J = 6.8Hz, 2H, -NCH -), 2.45 (s, 3H, -NCH ), 1.53 (m, 2H,
2
3
-NCH CH -), 1.26 (m, 12H, 6×-CH -), 0.87 (t, J = 6.8Hz, 3H, -CH ); ESI-MS, m/z:
2
2
2
3
+
158.18[M+H] .
2.2.2. General procedure for preparation of compounds 2a-2c
Compound 1 (0.015 mol), brominated alkanes (0.015 mol) and NaOH (0.6 g,
0.015 mol) were dissolved in 30 mL ethanol. The mixture was stirred and refluxed for
5 h and then washed by 30 mL distilled water for 3 times. The water phase was

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extracted by CHCl (30 mL×3). The organic phases were combined and dried by
3
Na SO . After the solvent had been removed under reduced pressure, the residue
2
4
(light yellow sticky liquid) was chromatographed on a silica gel column by using
dichloromethane as eluents to give product 2.
2.2.2.1. N-methyl-N-octylnonan-1-amine (2a) According to the general procedure,
compound 1 was treated with 1-bromooctane and then purified on silica gel column to
give compound 2a.
1
Yield: 94.1%; Colorless liquid; H NMR (400MHz, CDCl ) δ: 2.33 (m, 4H,
3
-CH NCH -), 2.23 (s, 3H, -NCH ), 1.48 (m, 4H, 2×-NCH CH -), 1.29 (m, 22H,
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11
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11×-CH -), 0.90 (m, 6H, 2×-CH ); ESI-MS, m/z: 270.37[M+H] .
2 3
2.2.2.2. N-methyl-N-nonylnonan-1-amine (2b) According to the general procedure,
compound 1 was treated with 1-bromononane and then purified on silica gel column
to give compound 2b.
1
Yield: 95.1%; Colorless liquid; H NMR (400MHz, CDCl ) δ: 2.33 (t, J = 7.2Hz,
3
4H, -CH NCH -), 2.22 (s, 3H, -NCH ), 1.47 (m, 4H, 2×-NCH CH -), 1.27 (m, 24H,
2
2
3
2
2
+
12×-CH -), 0.88 (t, J = 7.2Hz, 6H, 2×-CH ); ESI-MS, m/z: 284.34[M+H] .
2
3
2.2.2.3. N-methyl-N-nonyldecan-1-amine (2c) According to the general procedure,
compound 1 was treated with 1-bromodecane and then purified on silica gel column
to give compound 2c.
1
Yield: 95.0%; Colorless liquid; H NMR (400MHz, CDCl ) δ: 2.32 (t, J = 7.6Hz,
3
4H, -CH NCH -), 2.22 (s, 3H, -NCH ), 1.47 (m, 4H, 2×-NCH CH -), 1.28 (m, 26H,
2
2
3
2
2
+
13×-CH -), 0.89 (t, J = 6.8Hz, 6H, 2×-CH ); ESI-MS, m/z: 298.39[M+H] .
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2.2.3. The preparation of compound 2d (N,N-dimethylnonan-1-amine)
4 mL aqueous dimethylamine (33%, m/m, excessive) reacted with 5 mmol
1-bromononane in 30 mL ethanol under stirring and reflux for 5 h, then the mixture
was washed by distilled water (3×30 mL) to get rid of unreacted dimethylamine. The
water phase was extracted by CHCl (3×30 mL). The organic phases were combined
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and dried by Na SO . After the solvent had been removed under reduced pressure, the
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crude product (light yellow sticky liquid) was chromatographed on a silica gel column
by using dichloromethane as eluents to give product 2d.
1
Yield: 95.0%; Colorless liquid; H NMR (400MHz, CDCl ) δ: 3.61 (t, J = 8.8Hz,
3
4H, -CH NCH -), 3.51 (s, 6H, -N(CH ) ), 1.73 (m, 2H, -NCH CH -), 1.23 (m, 12H,
2
2
3 2
2
2
+
6×-CH -), 0.84 (t, J = 7.2Hz, 3H, -CH ); ESI-MS, m/z: 172.24[M+H] .
2
3
2.2.4. General procedure for preparation of compound 4a-4d
Compound 3 (0.27 mmol) and equimolar tertiary amine (2a-2d) in 35 mL CHCl3
were stirred and refluxed for 24 h. After removal of the solvent, the crude product was
chromatographed on a silica gel column by gradient elution to give products 4a-4d,
the eluent order were dichloromethane first and then dichloromethane/ethanol (v/v
from 50:1 to 25:1).
2.2.4.1.
N-octyl-N-((4,5-dihydroxy-7-methoxy-9,10-anthraquinone-2-yl)methyl)-N-methylno
nan-1-aminium bromide (4a). According to the general procedure, compound 3 was
treated with N-methyl-N-octylnonan-1-amine and then purified on silica gel column
to give compound 4a.

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Yield: 63.7%; Purple red solid; m.p.135-138˚C; H NMR (400MHz, CDCl ) δ:
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12.07 (s, 1H, OH), 12.04 (s, 1H, OH), 7.84 (s, 1H, Ar-H), 7.77 (s, 1H, Ar-H), 7.31 (d,
+
J = 2.4Hz, 1H, Ar-H), 6.69 (d, J = 2.4Hz, 1H, Ar-H), 5.27 (s, 2H, ArCH N ), 3.97 (s,
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+
3H, OCH ), 3.50 (t, J = 8.0 Hz ,4H, -CH N CH -), 3.35 (s, 3H, -N CH ), 1.84 (m, 4H,
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2
2
3
+
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2×-N CH CH -), 1.42-1.30 (m, 22H, 11×-CH ,), 0.90 (t, J = 6.0Hz, 6H, 2×-CH ); C
2
2
2
3
NMR (400 MHz, CDCl ) δ: 189.9, 180.1, 167.0, 165.5, 162.2, 136.2, 134.3, 133.4,
3
129.7, 122.9, 116.4, 109.7, 108.8, 106.8, 63.8, 61.0, 56.2, 48.2, 31.7, 29.3, 29.1, 26.3,
22.6, 14.0; Anal. Calcd for C H BrNO ·0.6H O: C 63.46, H 8.02, N 2.18; Found: C
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4
50
5
2
+
63.48, H 7.88, N 1.93; ESI-MS, m/z: 552.51 [M-Br] .
2.2.4.2.
N-nonyl-N-((4,5-dihydroxy-7-methoxy-9,10-anthraquinone-2-yl)methyl)-N-methylno
nan-1-aminium bromide (4b). According to the general procedure, compound 3 was
treated with N-methyl-N-nonylnonan-1-amine and then purified on silica gel column
to give compound 4b.
1
Yield: 67.5%; Purple red solid; m.p.110-112˚C; H NMR (400MHz, CDCl ) δ:
3
12.09 (s, 1H, OH), 12.07 (s, 1H, OH), 7.89 (s, 1H, Ar-H), 7.77 (s, 1H, Ar-H), 7.33 (d, J
+
= 2.0Hz, 1H, Ar-H), 6.69 (d, J = 2.0Hz, 1H, Ar-H), 5.23 (s, 2H, ArCH N), 3.95 (s, 3H,
2
+
+
OCH ), 3.47 (m, 4H,-CH N CH -); 3.34 (s, 3H, -N CH ), 1.88-1.74 (m, 4H,
3
2
2
3
+
13
2×-N CH CH -), 1.43-1.26 (m, 24H, 12×-CH -), 0.88 (t, J = 6.8Hz, 6H, 2×-CH ); C
2
2
2
3
NMR (400 MHz, CDCl ) δ: 189.9, 180.1, 167.1, 165.6, 162.1, 136.2, 134.3, 133.4,
3
129.6, 122.9, 116.4, 109.7, 108.8, 106.8, 63.7, 61.0, 56.2, 53.4, 48.2, 31.7, 29.4, 29.2,
29.1, 26.3, 22.7, 22.6, 14.0; Anal. Calcd for C H BrNO ·1.3H O: C 62.73, H 8.21, N
3
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2.09; Found: C 62.78, H 8.00, N 1.87; ESI-MS, m/z: 566.50 [M-Br] .
2.2.4.3.
N-nonyl-N-((4,5-dihydroxy-7-methoxy-9,10-anthraquinone-2-yl)methyl)-N-methylde
can-1-aminium bromide (4c). According to the general procedure, compound 3 was
treated with N-methyl-N-nonyldecan-1-amine and then purified on silica gel column
to give compound 4c.
1
Yield: 63.5%; Purple red solid; m.p.110-112˚C; H NMR (400MHz, CDCl ) δ:
3
12.12 (s, 1H, OH), 12.08 (s, 1H, OH), 7.88 (s, 1H, Ar-H), 7.78 (s, 1H, Ar-H), 7.35 (d,
+
J=2.4Hz, 1H, Ar-H), 6.71 (d, J = 2.0Hz, 1H, Ar-H), 5.25 (s, 2H, ArCH N ), 3.97 (s, 3H,
2
+
+
OCH ), 3.51(s, 4H,-CH N CH -), 3.35 (s, 3H, -N CH ), 1.86-1.78 (m, 4H,
3
2
2
3
+
13
2×-N CH CH -), 1.42-1.29 (m, 26H, 13×-CH ), 0.90 (t, J = 6.8Hz, 6H, 2×-CH ); C
2
2
2
3
NMR (400 MHz, CDCl ) δ: 190.0, 180.3, 167.1, 165.6, 162.2, 136.0, 134.4, 133.5,
3
129.7, 122.8, 116.5, 109.8, 108.9, 106.8, 65.8, 61.0, 56.2, 48.4, 31.8, 31.7, 29.4, 29.3,
29.2, 29.1, 26.3, 22.7, 22.6, 14.0; Anal. Calcd for C H BrNO ·1.4H O: C 63.04, H
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54
5
2
+
8.35, N 2.04; Found: C 63.06, H 7.97, N 2.06; ESI-MS, m/z: 580.38 [M-Br] .
2.2.4.4.
N,N-dimethyl-N-((4,5-dihydroxy-7-methoxy-9,10-anthraquinone-2-yl)methyl)nonan-
1-aminium bromide (4d). According to the general procedure, compound 3 was
treated with N,N-dimethylnonan-1-amine and then purified on silica gel column to
give compound 4d.
1
Yield: 65.5%; Bright yellow solid; m.p.160-162˚C; H NMR (500MHz, CDCl ) δ:
3
12.00 (s, 2H, OH), 7.79 (s, 1H, Ar-H), 7.76 (s, 1H, Ar-H), 7.21 (d, J = 2.5Hz, 1H,

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Ar-H), 6.64 (d, J = 2.5Hz, 1H, Ar-H), 5.29 (s, 2H, ArCH N ), 3.96 (s, 3H, OCH ),
2 3
+
+
3.58 (t, J = 8.0Hz, 2H, -N CH -), 3.41 (s, 6H, 2×-N CH ), 1.90-1.87 (m, 2H,
2
3
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-N CH CH -), 1.40-1.28 (m, 12H, 6×-CH -), 0.89 (t, J = 7.0Hz, 3H, -CH ); C NMR
2
2
2
3
(400 MHz, CDCl ) δ: 190.0, 180.3, 167.1, 165.6, 162.2, 136.4, 134.4, 133.5, 129.5,
3
123.1, 116.6, 109.7, 108.9, 106.8, 66.7, 64.6, 63.8, 56.2, 53.4, 51.4, 50.2, 31.8, 31.6,
29.3, 29.2, 29.1, 26.3, 23.0, 22.8, 22.6, 14.0; Anal. Calcd for C H BrNO ·0.4H O: C
2
7
36
5
2
59.87, H 6.85, N 2.59; Found: C 59.85, H 7.15, N 2.67; ESI-MS, m/z: 454.37
+
(M-Br) .
2.2.5.
The
preparation
of
compound
6a
(N,N-dioctyl-N-((4,5,7-trimethoxy-9,10-anthraquinone-2-yl)methyl)octan-1-aminium
bromide)
Compound 3 (100 mg, 0.260 mmol) and 0.1 mL trioctylamine (excessive) in 20
mL CHCl were stirred and refluxed for 10 h. After removal of the solvent, the crude
3
product was chromatographed on
a
silica gel column by using
dichloromethane/ethanol (v/v from 20:1 to 25:1) as eluent to give 6a (74 mg) as
product.
1
Yield: 46.2%; Yellow solid; m.p.146-148˚C; H NMR (400MHz, CDCl ) δ: 8.43
3
(s, 1H, Ar-H), 7.60 (s, 1H, Ar-H), 7.29 (s, 1H, Ar-H), 6.73 (s, 1H, Ar-H), 5.23 (s, 2H,
+
ArCH N ), 4.16 (s, 3H, -OCH ), 3.97 (s, 3H, -OCH ), 3.95 (s, 3H, -OCH ), 3.35 (t, J
2
3
3
3
+
+
= 8.0 Hz, 6H, 3×-N CH -), 1.84 (m, 6H, 3×-N CH CH -), 1.32 (m, 30H, 15×-CH -),
2
2
2
2
13
0.87 (t, J = 6.8 Hz, 9H, 3×-CH ); C-NMR (400 MHz, CDCl ) δ: 183.0, 180.7, 164.0,
3
3
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18
19
20
21
22
58.9, 58.2, 56.4, 55.9, 53.4, 31.6, 29.0, 26.5, 22.9, 22.6, 14.0; Anal. Calcd for
C H BrNO ·0.6H O: C 66.75, H 8.96, N 1.85; Found: C 66.60, H 8.75, N 1.88;
42
66
5
2
+
ESI-MS, m/z: 664.47[M-Br] .
2.2.6. The preparation
of
compound
6b
(2-(2-methoxyethoxy)-N-(2-(2-methoxyethoxy)ethyl)-N-((4,5,7-trimethoxy-9,10-anth
raquinone-2-yl)methyl)ethanaminium bromide)
Compound 5 (150 mg, 0.384 mmol) and tris(3,6-dioxaheptyl)amine (124 mg,
0.384 mmol) in 20 mL methylglycol were stirred and refluxed for 24 h. After removal
of the solvent, the crude product was chromatographed on a silica gel column by
using dichloromethane/ethanol (v/v from 20:1 to 25:1) as eluent to give 6b (86 mg) as
product.
1
Yield: 31.4%; Yellow viscous liquid; H NMR (400MHz, CDCl ) δ: 8.22 (s, 1H,
3
Ar-H), 8.04 (s, 1H, Ar-H), 7.34 (d, J = 2.8 Hz, 1H, Ar-H), 6.82 (d, J = 2.8 Hz, 1H,
+
Ar-H), 5.23 (s, 2H, ArCH N ), 4.17 (m, 6H, 3×CH OCH ), 4.14 (s, 3H, -OCH ), 4.01
2
2
3
3
+
(s, 3H, -OCH ), 3.98 (s, 3H, -OCH ), 3.98-3.96 (m, 6H, 3×-N CH ), 3.72-3.67 (m,
3
3
2
1
3
12H, 3×CH OCH ), 3.39 (s, 9H, 3×CH OCH ); C-NMR (400 MHz, CDCl ) δ: 183.4,
2
2
2
3
3
181.1, 164.2, 161.8, 160.2, 136.1, 134.7, 133.3, 125.0, 124.8, 123.1, 118.2, 105.4,
102.3, 71.7, 71.5, 70.4, 70.3, 64.8, 58.9, 56.5, 55.9, 54.7; ESI-MS, m/z:
+
+
+
634.37[M-Br]
634.3222.
2.2.7.
;
HRMS (ESI ), calcd for C H NO 634.3227 [M-Br] ; Found,
3
3
48
11
The
preparation
of
compound
(1,3-bis((4-bromomethyl)benzyl)oxy)-8-hydroxy-6-methylanthraquinone (7a) and

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(1,6-bis((4-bromomethyl)benzyl)oxy)-8-hydroxy-3-methylanthraquinone (7b)
Emodin 100mg(0.37 mmol) and K CO 102 mg(0.74 mmol) were dissolved in
2
3
100 mL acetone and the mixture was heat till reflux, then
1,4-bis(bromomethyl)benzene 195 mg (0.74 mmol) was added to react 2 h. When the
mixture cooled to room temperature, dilute hydrochloric acid was added to make the
solution acidic. After an addition of 200 mL water, a lot of precipitates were obtained
as crude product by filtration. The crude was purified by column chromatography
with dichloromethane as eluent to give the mixture of 7a+7b (115 mg, the molar
ration is about 2:1).
1
Yield: 49.1%; Orange solid; m.p.218-221˚C; HNMR(500MH ,CDCl )
δ
: 13.24 (s,
Z
3
0.5H, OH), 13.21 (s, 1H, OH), 7.81 (d, J=1.0Hz, 0.5H, Ar-H), 7.61 (m, 4H,
ArOC H Br), 7.58 (d, J=2.5Hz, 1.0H, Ar-H), 7.50 (t, J=2.5Hz, 2H, ArOC H Br), 7.48
6
4
6
4
(d, J=2.5Hz, 2H, ArOC H Br), 7.47 (m, 4H, ArOC H Br), 7.44 (s, 1H, Ar-H), 7.41 (d,
6
4
6
4
J=2.5Hz, 0.5H, Ar-H), 7.19 (s, 0.5H, Ar-H), 7.13 (d, J=0.5Hz, 1H, Ar-H), 6.90 (d,
J=2.5Hz, 1H, Ar-H), 6.81 (d, J=2.5Hz, 0.5H, Ar-H), 5.30 (s, 2H, ArOCH C H CH Br),
2
6
4
2
5.23 (s, 2H, ArOCH C H CH Br), 5.21 (s, 1.0H, ArOCH C H CH Br), 5.15 (s, 1.0H,
2
6
4
2
2
6
4
2
ArOCH C H CH Br), 4.55 (s, 2H, ArOCH C H CH Br), 4.54 (ArOCH C H CH Br),
2
6
4
2
2
6
4
2
2
6
4
2
1
3
2.50 (s, 1.5H, ArCH ), 2.46 (s, 3H, ArCH ); C-NMR (400 MHz, CDCl ) δ: 187.2,
3
3
3
182.9, 165.2, 164.3, 164.1, 162.7, 161.6, 159.8, 147.0, 146.8, 138.2, 138.0, 137.7, 137.6,
136.5, 136.2, 135.9, 135.7, 135.5, 134.3, 132.3, 129.49, 129.48, 129.45, 128.1, 127.9,
127.0, 124.9, 121.5, 120.6, 120.0, 116.0, 114.8, 111.9, 108.3, 106.9, 106.8, 105.1, 70.8,
+
70.3, 70.0, 33.2, 33.1, 32.9, 32.8, 22.3, 22.0; ESI-MS, m/z: 637.26 [M+H] ; HRMS

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261
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264
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266
(ESI ): calcd for C H Br O [M+H] = 637.0048; Found, 637.0062.
3
1
24
2
5
2.2.8.
The
preparation
of
N,N’-(((((8-hydro-6-methyl-9,10-anthraquinone-1,3-diyl)bis(oxy)bis(methylene))bis(
4,1-phenylene))bis(methylene))bis(N-methyl-N-nonyldecan-1-aminium) bromide (8a)
and
N,N’-(((((8-hydro-3-methyl-9,10-anthraquinone-1,6-diyl)bis(oxy)bis(methylene))bis(
4,1-phenylene))bis(methylene))bis(N-methyl-N-nonyldecan-1-aminium) bromide (8b)
The mixture of 7a+7b 100 mg (0.16 mmol) was dissolved in 20 mL CHCl and
3
the mixture was heated to reflux, then equimolar tertiary amine (2c) was added to
react for 8 h. After removal of the solvent, the residue was chromatographed on a
silica gel column by gradient elution to give the mixture of 8a+8b (the molar ratio is
about 5:1), the eluent order were dichloromethane first and then
dichloromethane/ethanol (v/v from 50:1 to 10:1)
1
Yield:26.1%; Orange solid; mp:128-130˚C; HNMR (400Hz, CDCl ) δ: 13.21 (s,
3
1.0H, OH), 13.13 (s, 0.2H, OH), 7.74 (m, 2.0H, OCH C H CH N), 7.65 (m, 2.4H,
2
6
4
2
OCH C H CH N), 7.61 (m, 2.4H, OCH C H CH N), 7.52 (s, 1.2H, Ar-H), 7.35 (m,
2
6
4
2
2
6
4
2
0.6H, OCH C H CH N), 7.33 (m, 2.0H, OCH C H CH N), 7.19 (m, 0.4H,
2
6
4
2
2
6
4
2
OCH C H CH N), 7.09 (m, 1.2H, Ar-H), 7.02 (s, 1.0H, Ar-H), 6.61 (d, J=2.4Hz,
2
6
4
2
1.0H, Ar-H), 6.44 (d, J=2.4Hz, 0.2H, Ar-H), 5.71 (s, 0.4H, ArOCH C H CH N), 5.37
2
6
4
2
(s, 0.4H, ArOCH C H CH N), 5.29 (s, 2.0H, ArOCH C H CH N), 5.26 (s, 2.0H,
2
6
4
2
2
6
4
2
ArOCH C H CH N), 5.21 (s, 0.4H, ArOCH C H CH N), 5.11 (s, 0.4H,
2
6
4
2
2
6
4
2
ArOCH C H CH N), 5.08 (s, 2.0H, ArOCH C H CH N), 4.94(s, 2.0H,
2
6
4
2
2
6
4
2

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78
79
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83
84
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87
88
ArOCH C H CH N), 3.49-3.34 (m, 9.6H, 2×CH NCH ), 3.18 (s, 6.0H, 2×NCH ),
2
6
4
2
2
2
3
3.14 (s, 1.2H, 2×NCH ), 2.45 (s, 0.6H, ArCH ), 2.39 (s, 3.0H, ArCH ), 1.78 (s, 9.6H,
3
3
3
2×CH CH NCH CH ),
1.39-1.28
(m,
62.4H,
2
2
2
2
13
2×NCH CH (CH ) CH +2×NCH CH (CH ) CH ), 0.89 (m, 14.4H, 4×CH );
C
2
2
2 6
3
2
2
2 7
3
3
NMR (400 MHz, CDCl ) δ: 187.0, 182.4, 165.0, 163.4, 162.6, 161.5, 146.9, 138.4,
3
138.0, 137.3, 134.1, 133.7, 133.6, 132.2, 129.9, 129.8, 127.6, 127.2, 127.1, 126.9,
126.6, 126.3, 120.0, 115.1, 114.8, 104.9, 69.4, 64.4, 63.4, 60.8, 47.8, 35.9, 31.84,
31.80, 31.76, 29.5, 29.4, 29.31, 29.28, 29.23, 29.13, 29.10, 29.06, 26.5, 26.2, 25.8,
22.7, 22.6, 14.1; Anal. Calcd for C H Br N O ·0.5H O: C 68.50, H 9.03, N 2.25;
7
1
110
2
2
5
2
2
+
Found: C 68.83, H 9.31, N 2.05; ESI-MS, m/z: 535.75[M-2Br] .
2.2.9. The preparation of compound 9 (2-bromomethyl-9,10-anthraquinone)
2-methyl-9,10-anthraquinone 444 mg (2.0 mmol) and NBS 356 mg (2 mmol)
were dissolved in 60 mL CCl and the mixture was stirred under reflux for 10
4
minutes, then 48 mg BPO (0.2 mmol) was added as initiator. The mixture was stirred
under reflux for another 24 h. After removal of the solvent under reduced pressure,
the faint yellow crude product was chromatographed on a silica gel column by using
dichloromethane/petroleum ether (v/v = 1:2) as eluent to give compound 9 (530 mg)
as product.
1
Yield: 88.3%; Faint yellow solid; m.p.196-198˚C; H NMR(400 MHz, CDCl )
3
δ: 8.32-8.34 (M, 4H, Ar-H), 7.84-7.86 (m, 2H, Ar-H ), 4.62 (s, 2H, -CH Br). ESI-MS,
2
+
m/z: 301.14 [M+H] .
2.2.10. General procedure for preparation of compounds 10a and 10b

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91
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96
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08
09
10
Compound 9 110 mg (0.50 mmol) was dissolved in 20 mL chloroform and then
0.1 mL tertiary amine was added to the solution. The mixture was stirred under reflux
for 7 h, and then concentrated under reduced pressure. The residue was
chromatographed on a silica gel column by gradient elution to give products 10a and
10b, with dichloromethane first and then chloroform/ethanol (v/v from 40:1 to 10:1,
based on the polarity of final products).
2.2.10.1.
N-octyl-N-((9,10-anthraquinone-2-yl)methyl)-N-methyloctan-1-aminium
bromide (10a). According to the general procedure, compound 9 was treated with
N-methyl-N-octylnoctan-1-amine and then purified on silica gel column to give
compound 10a.
1
Yield: 88.6%; Faint yellow solid; m.p.116-118˚C; H NMR(400 MHz, CDCl ) δ:
3
8.48 (d, J = 8.0Hz, 1H, Ar-H), 8.29-8.25 (m, 2H, Ar-H), 8.21-8.17 (m, 2H, Ar-H), 7.78
+
(t, J = 3.6Hz, 2H, Ar-H), 5.41 (s, 2H, ArCH N ), 3.48 (t, J = 8.4Hz, 4H,
2
+
+
+
2× NCH C H ), 3.32 (s, 3H, NCH ), 1.89-1.83 (m, 4H, 2× NCH CH C H ),
2
7
15
3
2
2
6
13
1
3
1.37-1.25 (m, 20H, 2×-(CH ) CH ), 0.90 (t, J = 7.2Hz, 6H, 2×-CH ); C NMR (400
2
5
3
3
MHz, CDCl ) δ: 181.90, 180.88, 139.5, 134.50, 134.47, 134.3, 133.7, 133.4, 133.04,
3
132.97, 130.9, 128.2, 127.31, 127.29, 64.0, 60.0, 48.0, 31.6, 29.1, 26.3, 22.7, 22.6, 14.0;
+
+
+
ESI-MS, m/z: 476, (M-Br) ; HRMS (ESI ): calcd for C H NO [M-Br] = 476.3523;
3
2
46
2
Found, 476.3519.
2.2.10.2. N-decyl-N-((9,10-anthraquinone-2-yl) methyl)-N-methyldecan-1-aminium
bromide (10b).
According to the general procedure, compound
9
was treated with

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31
32
N-methyl-N-decylndecan-1-amine and then purified on silica gel column to give
compound 10b.
1
Yield: 90.1%; Faint yellow solid; m.p.119-121˚C; H NMR(400 MHz, CDCl ) δ:
3
8.58(d, J = 8.0Hz, 1H, Ar-H), 8.36 (d, J = 8.0Hz, 1H, Ar-H), 8.30-8.28 (m, 3H, Ar-H),
+
7.84 (t, J = 4.0Hz, 2H, Ar-H), 5.40 (s, 2H, ArCH N ), 3.48 (t, J = 7.6Hz, 4H,
2
+
+
+
2× NCH C H ), 3.34 (s, 3H, NCH ), 1.86-1.82 (m, 4H, 2× NCH CH C H ),
2
7
15
3
2
2
8
17
1
3
1.41-1.27 (m, 28H, 2×-(CH ) CH ), 0.90 (t, J = 6.8Hz, 6H, 2×-CH ); C NMR (400
2
7
3
3
MHz, CDCl ) δ: 181.9, 180.9, 139.6, 134.51, 134.47, 134.3, 133.7, 133.4, 133.1, 133.0,
3
130.9, 128.3, 127.34, 127.31, 64.0, 60.9, 48.0, 31.8, 29.4, 29.2, 26.4, 22.7, 22.6, 13.9;
+
+
+
ESI-MS, m/z: 532, (M-Br) ; HRMS (ESI ): calcd for C H NO [M-Br] = 532.4149;
3
6
54
2
Found, 532.4144.
2.2.11. The
preparation
of
compound
12
(3-((dimethylamino)methyl)-1,6,8-trimethoxyl-9,10-anthraquinone)
KOH 358 mg was dissolved in 5 mL water and added into the mixture of
compound 1 200 mg (1.27mmol) and TEBA 120 mg, then a solution of compound 5
(250 mg, 0.64 mmol) in 30 mL chloroform was added into the mixture. The mixture
was refluxed under stirring for 8h and then extracted by CHCl (3×15 mL). The
3
organic phases were combined and dried by Na SO . After the solvent had been
2
4
removed under reduced pressure, the crude product (light yellow solid) was
chromatographed on a silica gel column by gradient elution to give product 12, the
eluent order were dichloromethane first and then dichloromethane/acetone (v/v from
30:1 to 10:1).

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342
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344
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346
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350
351
352
353
354
Yield 36.5%; Yellow solid. m.p.52-54˚C; H NMR (400MHz, CDCl ) δ: 7.74 (s,
3
1H, Ar-H), 7.41 (s, 1H, Ar-H), 7.35 (d, J = 2.0Hz, 1H, Ar-H), 6.79 (d, J = 2.0Hz, 1H,
Ar-H)，4.02 (s, 3H, -OCH ), 3.98 (s, 3H, -OCH ), 3.97 (s, 3H, -OCH ) , 3.59 (s, 2H,
3
3
3
ArCH N) , 2.25 (s, 3H, N-CH ), 2.38 (t, J = 6.8Hz, 2H, -NCH ), 1.53 (m, 2H,
2
3
2
-NCH CH C H ), 1.27-1.44 (m, 12H, -NCH CH C H CH ), 0.89 (t, J = 6.8Hz, 3H,
2
2
7
15
2
2
6
12
3
13
-CH3); C NMR (400 MHz, CDCl ) δ: 184.4, 181.9, 163.8, 161.7, 160.0, 136.6,
3
135.3, 134.4, 125.0, 122.8, 119.2, 118.6, 105.2, 102.0, 61.9, 57.6, 56.5, 55.9, 42.3,
32.2, 31.9, 29.6, 29.2, 27.4, 27.2, 26.4, 23.4, 22.7, 14.1; ESI-MS, m/z: 468.22
+
+
+
[M+H] ; HRMS (ESI ): calcd for C H NO [M+H] = 468.2750; Found, 468.2758.
2
8
37
5
2.2.12.
The
preparation
of
compound
13
(N-2-(2-(2-hydroxyethoxy)ethoxy)ethyl)-N-methyl-N-((4,5,7-trimethoxy-9,10-anthra
quinone-2-yl)methyl)nonan-1 aminium iodide
Compound 11 was prepared according to the document [20] and its structure was
+
+
affirmed by HRMS (ESI ) with the m/z being 260.9973 (calculated (M+H) is
260.9982). A solution of compound 12 (150 mg, 0.32 mmol) and 11 (83.5 mg, 0.32
mmol) in 15 mL methylglycol was refluxed and stirred for 24 h. After the solvent had
been removed under reduced pressure, the crude product (yellow solid) was
chromatographed on a silica gel column by gradient elution to give product 13, the
eluent order were dichloromethane first and then dichloromethane/ethanol (v/v from
25:1 to 4:1).
1
Yield 45.6%; Yellow solid, m. p. 138-140˚C; H NMR(400MHz, CDCl ) δ: 8.38
3
(s, 1H, Ar-H), 7.82 (s, 1H, Ar-H), 7.33 (d, J = 2.0 Hz, 1H, Ar-H), 6.82 (d, J = 2.0 Hz,

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56
57
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1H, Ar-H), 5.32 (s, 2H, NCH Ar), 4.17 (s, 3H, -OCH ), 4.00 (s, 3H, -OCH ) , 3.99 (s,
2
3
3
3H, -OCH ) , 4.14 (m, 2H, -CH OH), 3.93 (m, 2H, -CH CH OH), 3.80 (m, 4H,
3
2
2
2
OC H O), 3.73 (m, 2H, NCH CH O), 3.70 (m, 2H, NCH CH O), 3.66 (m, 2H,
2
4
2
2
2
2
NCH C H ), 3.34 (s, 3H, -NCH ), 1.40-1.29 (m, 14H, NCH C H CH ), 0.90 (t, J =
2
8
17
3
2
7
14
3
1
3
7.2Hz, 3H, NC H CH ); C NMR (400 MHz, CDCl ) δ: 183.3, 180.7, 164.0, 161.9,
8
16
3
3
160.2, 135.9, 134.7, 132.9, 125.1, 124.7, 122.1, 117.9, 105.4, 102.3, 72.5, 70.5, 70.2,
65.2, 64.9, 62.5, 61.5, 61.1, 58.1, 56.4, 55.9, 31.8, 29.4, 29.2, 29.1, 26.3, 23.4, 22.6,
14.0; Anal. Calcd for C H INO : C 56.12, H 6.93, N 1.92; Found: C 56.04, H 7.29,
34
50
8
+
N 1.53; ESI-MS, m/z: 600.16 [M-I] .
2.2.13. The preparation
of
compound
15
(N-decyl-N-methyl-N-((4,5,7-trihydroxy-9-anthrone-2-yl)methyl)decan-1-aminium
bromide)
A solution of compound 14 (323 mg , 0.5 mmol) in 15 mL acetic acid was heated
to 100˚C, then a solution of SnCl ·2H O (226mg, 1 mmol) in 10 mL c-HCl was added
2
2
dropwise. The mixture was stirred and refluxed for another 3 h and then poured into
20 mL water. 15 mL CHCl was used to extract the mixture twice. The organic phases
3
were combined and then product 15 was deposited from CHCl3.
1
Yield 25.4%; Dark-green solid, m. p. 126-128˚C. H NMR(400MHz, DMSO) δ:
12.35 (s, 1H, Ar-OH), 12.23 (s, 1H, Ar-OH), 11.13 (s, 1H, Ar-OH), 7.11 (s, 1H, Ar-H),
7.08 (s, 1H, Ar-H), 6.52 (s, 1H, Ar-H), 6.31 (s, 1H, Ar-H), 4.53 (s, 2H, -CH N), 4.42
2
(s, 2H, ArCH Ar), 3.27-3.17 (m, 4H, 2×NCH ), 2.97 (s, 3H, NCH ), 1.76-1.68 (m, 4H,
2
2
3
+
2×NCH CH ), 1.30-1.26 (m, 20H, 2×N CH CH (CH ) CH ), 0.86 (t, J = 6.8Hz, 6H,
2
2
2
2
2 5
3

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80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
2×-CH ); C NMR (400 MHz, CDCl3) δ: 191.3, 166.0, 165.3, 161.9, 145.3, 142.9,
3
135.5, 123.5, 119.3, 116.6, 108.8, 108.1, 101.6, 64.2, 61.3, 47.8, 31.6, 28.8, 26.2, 22.5,
+
22.0, 14.1; ESI-MS, m/z: 510.35 (M-Br) ; HRMS (ESI+): calcd for C H NO
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[M-Br] = 510.3578; Found, 510.3557.
2.3. Cell lines and culture
A375, BGC-823, HepG2 and HELF cells obtained from our own laboratory were
cultured in RPMI 1640 medium supplemented with 10% FBS, 100 U/mL penicillin,
and 100 mg/mL strepto-mycin in a humidified atmosphere of 5.0% CO at 37˚C.
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2.3.1. MTT assay for cell viability
Cell viability was assessed by the MTT assay described as previously [21]. Briefly,
A375 cells in logarithmic growth phase were seeded in 96-well plates at a density of
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concentrations of emodin derivatives for 24 h. After two times wash of cells by 200
mL PBS, 100 mL of 5 mg/mL MTT were added to each well, and then cells were for
incubated another 4 h. The MTT-formazon formed by metabolically viable cells was
dissolved in 150 mL of DMSO. After shaken for 10 min, the amount of reduced MTT
was measured by an ELISA reader at 492 nm. Results are the means of three
independent experiments performed in triplicate. The 50% inhibitory concentration
(IC 50 ) was determined as the anticancer drug concentration causing 50% reduction
in cell viability and calculated from the cytotoxicity curves.
2.4. Cell apoptosis measurement by DAPI nuclear staining and flow cytometry.
The method was similar to what we used previously [22]. A375 cells were

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incubated with compound 4a (1,2,4 µM) for 24 h, and then washed once with PBS,
fixed with cold methanol: acetic acid (3:1) for 10 min, and washed three times with
PBS. Then, these cells were treated with 1 µg/mL DAPI for at 37˚C 5 min. After
staining, the cells were examined and photographed at 364 nm by a fluorescence
microscope.
Apoptosis was detected using Annexin V-FITC/PI staining and was analyzed by flow
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cytometry (FCM). HeLa cells were seeded in 6-well plates (10 cells per well) for 24
h and incubated with 1 µM compound 4a, Untreated cells were also used as control..
After 24 h incubation, the cells were collected, washed twice with PBS, carefully
trypsinized to avoid mechanical damage ofthe membrane and stained with 5 µM of
the solution containing Annexin V-FITCand PI for 15 min in the dark according to the
manufacture’s instructions. Cells were than analyzed by using flow cytometry (BD
Bioscience, FACS AriaIII).
2.5. Western bloting analysis
A375 cells were treated for 24 h with various concentrations of compound 4a (5
and 10 µM). Cell dishes were washed with cold phosphate-buffered saline and treated
with lysis buffer for several seconds at an ice-cold bath, and then cell lysates were
clarified by centrifugation at 12,000 g for 5 min under 4°C and the supernatants were
recovered. The concentrations of proteins were determined by the BCA method.
Samples with equivalent amounts of proteins were resolved by SDS-PAGE using a
10% gel. Proteins were transferred to a polyvinyldene difluoride (PVDF) membrane.
The membranes were washed for ten minutes with TBS and were blocked by blocking

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solution. The blots were washed three times with TBST, and they were incubated with
specific primary antibodies diluted 1:1000 in TBST solution overnight at 4°C.
Subsequently, the blots were washed three times with TBST, and then the blots were
incubated with secondary antibody diluted 1:5000 in TBST solution for 2 h at 37°C.
The membranes were washed with TBST and TBS. Chemiluminescent signals were
generated using a Super Signal West Pico Chemiluminescent Substrate kit (Pierce),
and detected by using the ChemiDoc XRS system (Bio-Rad). The target proteins
expression was quantified by use of Image Lab analysis software
(Bio-Rad)
2.6. Measurement of intracellular ROS.
2,7-Dichlorodihydrofluorescein diacetate (DCF-DA) (Invitrogen, USA) was
frequently used to monitor intracellular ROS level. DCF-DA was cleaved
intracellularly by non-specific esterases and oxidized by ROS to the highly
fluorescent compound 2,7-dichlorofluorescein (DCF). After A375 cells was exposed
to various drugs for 24 h, washed once with PBS, treated cells were incubated with 20
µM DCF-DA in serum-free DMEM at 37˚C for 30 min. Then, Cells were washed
twice with ice-cold PBS and harvested, and the fluorescence intensity of the cells was
detected by flow cytometry. The average intensity of DCF stands for intracellular
ROS levels.
2.7. Measurement of mitochondrial membrane potential.
Mitochondrial
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described previously [23]. Briefly, the treated A375 cells were incubated with 50 nM
DiOC6 in serum-free DMEM at 37˚C for 30 min and then washed twice with ice-cold
PBS and harvested, and analyzed by flow cytometry.
2.8. Measurement of subcellular localization.
The fluorescent mitochondrial probe, Mito-Tracker Green, was used to measure
the subcellular localization of compound 4a in A375 cells, as described previously
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[24]. A375 cells (10 cells/mL) were plated in petri dish for 24 h, when reached 70%
confluence, they were exposed to compound 4a (2 µM) at 37˚C for 24 h. Then, cells
were washed twice and incubated in the medium containing 100 nM of Mito-Tracker
Green (emitted at 488 nm), for 30 min. After washed twice with PBS, the intracellular
localization of compound 4a (emitted at 568 nm) was detected by a laser confocal
microscope (Olympus FluoView FV1000, Japan).
2.9. Drug treatment of isolated mitochondria
A number of microliters of isolated mitochondria was treated with compound 4a
at indicated concentrations (0、1、10、50、100 µM) for 2 h at 37 ℃. Than the
mitochondria samples were washed once with MT buffer, and the pellets were
resuspended in 200 µL of DiOC6 and incubated for 15 min at 37 ℃. The optimal
DiOC6 staining concentration was indentified to be 500 nM. After washing twice with
MT buffer, the DiOC6(3)–stained mitochondria was resuspended in 50 µL of MT
buffer for HSFCM (a laboratory-built high sensitivity flow cytometer analysis).
2.9. Statistical analysis
All data were presented as mean±S.D of three determinations. Statistical analysis

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was evaluated using Student’s t-test and one-way analysis of variance. Multiple
comparisons of the means were performed by the least significant difference (LSD)
test. Differences were considered statistically significant at P < 0.05. Data variation
analysis was made by using SPSS statistical software (version 16.0).
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3.1. Chemistry
Scheme 1 shows a synthetic route of four new tertiary amines 2a-2d containing
at least one n-nonyl chain. The tertiary amines reacted with emodin derivative 3 to
give corresponding quaternary ammonium salts 4a-4d (scheme 2). Compounds 6a
and 6b were prepared from the reaction between emodin derivative 5 and
corresponding tertiary amines. The synthetic method of compounds 3 and 5 had been
reported in our earlier work [12,13].
Scheme 3 and scheme 4 showed the synthetic routes of emodin bis-quaternary
ammonium salts 8a+8b and anthraquinone quaternary ammonium salts 10a-10b,
respectively.
In scheme 5, iodotriglycol 11 was obtained by a nucleophilic substitution by KI
on chlorotriglycol. Compound 5 reacted with compound 1 to give an emodin tertiary
amine 12 whose alkylation with iodotriglycol gave quaternary ammonium salt 13 as
product. 13 is slightly water soluble. In scheme 6, an emodin quaternary ammonium
salt 14 was transformed into anthrone quaternary ammonium salt 15 by reduction with
SnCl . Along with this reaction, demethylation of three methoxy groups of reactant
2
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took place, which was affirmed by the appearance of three peaks of hydroxyl in H

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NMR of compound 15.
3.2. In vitro anticancer activity screening and structure-activity relationship
The in vitro cytotoxicity activity was evaluated for all of the synthesized
compounds against human malignant melanoma cell line A375, human gastric cancer
cell line BGC-823, human hepatoma cell line HepG2 and human embryonic lung
fibroblast cell line HELF. All compounds were dissolved in DMSO, and then diluted
with culture medium containing 0.1% DMSO for assay. Paclitaxel was used as a
positive control. The results are listed in Table 1.
Compounds 4a-4d all contain at least one long carbon chain with 9 carbon atoms.
Compound 4d showed the lowest anticancer activity due to its insufficient lipophilic
property. Compounds 4a-4c all contain two long carbon chains on the 4° N atom
showed higher anticancer activities than that of paclitaxel, the positive control, on
cancer cells. Moreover, Compound 4a-4c demonstrated anticancer activities that are
slightly higher than those of our previously reported emodin quaternary ammonium
salts [12,13]. We observe here that quaternary ammonium salt with hydrocarbon
chains containing 9 carbons exhibit highest anticancer activity compared to those
containing 8 or 10 carbons. In addition, Emodin bis-quaternary ammonium salts
8a+8b and two anthraquinone quaternary ammonium salts 10a-10b also show high
anticancer activities.
Compound 6a and 6b possess similar lengths of chains in 4° N atom, but they
have big difference of water solubility. Compound 6b is water soluble with a ClogP
value of (2.40), much smaller than that of 6a (9.77). As a result, compound 6a shows

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high anticancer activity and compound 6b hardly possesses anticancer activity.
Compound 13 also possesses similar length of chains in 4° N atom with those of
compounds 4a-4c. Unlike compounds 4a-4c which show high anticancer activities
and big ClogP values (from 6.85 to 7.90), compound 13 is slightly soluble in water
with ClogP of 3.44 and exhibits low anticancer activity. Thus, we conclude that
increasing aqueous solubility of the emodin 4° ammonium salt derivatives negatively
impact the potency of these compounds against cancer cells.
At last, an emodin quaternary ammonium salt 14, whose structure and anticancer
activity have been reported previously by our group [13] was reduced into anthrone
quaternary ammonium salt 15 by using SnCl as reducing reagent. Compound 14 has
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anticancer activity against HepG2 cell line (10.28 µM/24h), but anthrone quaternary
ammonium salt 15 exhibited no inhibitive activity against HepG2 cell line. Our early
work [12] had proved that a transformation of methoxyl into hydroxyl could improve
the anticancer activities of emodin derivatives, so the loss of anticancer activity of
compound 15 is likely due to the lack of quinoid structure. Now we can draw a
conclusion that quinoid structure plays an important role in the anticancer activities of
emodin derivatives.
Since A375 cells exhibit well sensitivity to emodin quaternary ammonium salts,
the growth-inhibitory mechanism of the most active compound 4a in vitro on A375
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3.3. compound 4a induced apoptosis in A375 cells
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were stained with 4’,6-diamidine-2’-phenylindole dihydrochloride (DAPI) after
treatment with compound 4a (1, 2, 4 µM) for 24 h, and detected by fluorescence
microscopy. As shown in Fig. 2A, control cells showed even distribution of the stain
and round homogeneous nuclei feature, while cells exposed to compound 4a
displayed typical changes in a dose-dependent manner including smaller and brighter
stained nuclei with condensed chromatin forming apoptotic bodies.
To evaluate whether compound 4a triggered cell apoptosis, AnnexinV-FITC/PI
double staining kit was used to analyze apoptosis in HeLa cells after the cells were
treated with 1 µM of compound 4a. The results are shown in Fig. 2B. Different states
of cell death were defined as follows: Lower-left quadrant stands for normal cells,
lower-right quadrant represents early apoptosis cells, late apoptosis cells are localized
in the upper-right quadrant, and the necrotic cells are localized in the upper-left
quadrant. As shown in Fig. 2B, exposure to compound 4a for 24 h increased the
proportion of late apoptotic cells compared to negative control, with little increase
in the number of early apoptotic cells, suggesting an increase in the number of
necrotic cells. The results suggested that compound 4a can effectively induce
apoptosis/necrosis in HeLa cells.
3.4. Down –regulation the protein expression of cell apoptosis molecule by compound
4a in A375
To confirm whether the anti-tumor effect of compound 4a in vitro was related to
the apoptosis activity, two proteins related to the apoptosis including caspase-3 and
P53 were traced by western blot technique. As shown in Fig. 2C, the bands for these

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indicator proteins in groups administrated with different concentrations of compound
4a (5 µM and 10 µM) increased in a dose-dependent manner. Using GAPDH as an
internal reference, the related protein expression levels were displayed under the Fig.
2C. Compared with the control group, A375 cells treated with compound 4a
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expressed considerably more caspase-3 and P53 ( P<0.05). Caspase family of
cysteinyl-proteases plays a critical role in the initiation and execution of programmed
cell death. The P53 tumor suppressor gene is an apoptosis regulator, it can induce
tumor cells apoptosis. Thus, the over expression of caspase-3 and P53 in A375 cells as
we observed indicates that compound 4a may possesse the ability to inhibit
proliferation and induce apoptosis of A375 cells via activating caspases and
upregulating P53.
3.5. Measurement of ROS generation
We next assessed the intracellular ROS production in the treated A375 cells using
DCF-DA fluorescence dye. As is shown in Fig. 3A, exposure of cells to 0.5 µM of
compound 4a hardly elicited any elevation of cellular ROS level. However, 2.7 and
5.8-fold increase in the ROS level detected in cells treated with 1 and 2 µM of
compound 4a, respectively, compared to those in untreated cells.
Our recent work [16] showed that there was not a direct correlation between
alkylatiing reactivitiesof emodin derivatives and their anticancer activities. Moreover,
many spectral experiments also confirmed that there was only a weak interaction
between emodin and DNA [17,18]. These results imply that DNA may not be the
main target of emodin derivatives. So in this paper, the abilities of emodin and its

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quaternary ammonium salt derivatives to generate ROS are tested, with the result
listed in Fig. 3B. Compound c whose structure is listed in Fig 3B is an emodin
quaternary ammonium salt which has been reported in our early work [15].
Compound c is water-solubility but does not exhibit any anticancer activity. Fig. 3B
also shows that compound c’s ability to generate ROS is lower than that of emodin,
which implies that the compound’s hydrophilicity is unfavorable in intracellular
generation of ROS. Since water-soluble compounds 6b and 13 exhibit no or low
anticancer activity, a conclusion can be drawn that there is a close relationship
between the ability to generate ROS and anticancer activity. Both compound 4d and
4a are fat soluble and they contain one and two long carbon chains in N cation
respectively. Fig. 3B shows that compound 4a has the highest capability to generate
ROS (4a > 4d > emodin), which correlates to their respective anticancer activities in
vitro. This experiment indicates that there is a positive correlation between
anticancer activity and the ability to generate ROS. Emodin quaternary ammonium
salts possess positive charge and quinoid structure. They are not only accumulated in
the matrix of mitochondria due to relatively high mitochondrial membrane potential
of cancer cells compared to normal cells, but are also capable of capturing electrons
leaking from respiratory chain due to their quinoid structure. But before these
functional moieties induce apoptosis of cancer cells intracellularly, penetration
through various membranes of the cell is required. This rational requires the
hydrophobic interactions between the compound and the fatty acyl domains of the
lipid bilayer. So we conclude that positive charge, lipophilicity and quinoid structure