Synthesis of damnacanthal, a naturally occurring 9,10-anthraquinone and its analogues, and its biological evaluation against five cancer cell lines

Article abstract of DOI:10.1007/s00044-012-0197-5

Damnacanthal and nordamnacanthal, two naturally occurring 9,10-anthraquinones, and their analogues were synthesized. Cytotoxic activity against five cancer cell lines was evaluated using MTT assay. 2-Bromomethyl-1,3-dimethoxyanthraquinone was found to display the highest activity against all cell lines with IC₅₀ range of 2-8 μM. Structure-activity relationship (SAR) assessment was considered to rationalise the cytotoxic effect. Bromomethyl group at position C-2 of the anthraquinone was found to be important in exerting cytotoxic activity of this class of compounds. The presence of the flanking methoxyl or hydroxyl groups at C-1 and C-3 also contributes to this activity. Finally, the antioxidant effect of these compounds was evaluated. MTT assay was used to measure the cytotoxicity against different cancer cell lines. Antioxidant activity was measured by FTC and TBA methods. Only two anthraquinones, damnacanthal and nordamnacanthal, were found to be antioxidative.

Full text of DOI:10.1007/s00044-012-0197-5

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2093–2104
DOI 10.1007/s00044-012-0197-5
ORIGINAL RESEARCH
Synthesis of damnacanthal, a naturally occurring 9,10-
anthraquinone and its analogues, and its biological evaluation
against five cancer cell lines
•
Koushik Saha Kok Wai Lam Faridah Abas
•
•
•
A. Sazali Hamzah Johnson Stanslas
•
•
Lim Siang Hui Nordin H. Lajis
Received: 2 February 2012 / Accepted: 8 August 2012 / Published online: 13 September 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Damnacanthal and nordamnacanthal, two natu-
rallyoccurring 9,10-anthraquinones, andtheiranalogueswere
synthesized. Cytotoxic activity against five cancer cell lines
was evaluated using MTT assay. 2-Bromomethyl-1,3-dim-
ethoxyanthraquinone was found to display the highest activity
against all cell lines with IC50 range of 2–8 lM. Structure–
activity relationship (SAR) assessment was considered to
rationalise the cytotoxic effect. Bromomethyl group at posi-
tion C-2 of the anthraquinone was found to be important in
exerting cytotoxic activity of this class of compounds. The
presence of the flanking methoxyl or hydroxyl groups at C-1
and C-3 also contributes to this activity. Finally, the antioxi-
dant effect of these compounds was evaluated. MTT assay
was used to measure the cytotoxicity against different cancer
cell lines. Antioxidant activity was measured by FTC and
TBA methods. Only two anthraquinones, damnacanthal and
nordamnacanthal, were found to be antioxidative.
Keywords Anthraquinones ꢀ Synthesis ꢀ Cytotoxicity ꢀ
Antioxidant activity
Introduction
Anthraquinones continue to attract interest among
researchers due to its diverse potential pharmacological
uses. 9,10-Anthraquinone derivatives are known to
exhibit a quite potent anticancer activity (Jin et al., 2001;
K. Saha ꢀ K. W. Lam ꢀ F. Abas ꢀ N. H. Lajis (&)
Laboratory of Natural Products, Institute of Bioscience,
University Putra Malaysia (UPM), 43400 Serdang,
Selangor Darul Ehsan, Malaysia
F. Abas
Department of Food Science, Faculty of Food Science and
Technology, University Putra Malaysia (UPM), 43400 Serdang,
Selangor Darul Ehsan, Malaysia
e-mail: nordinlajis@gmail.com
e-mail: faridah@food.upm.edu.my
Present Address:
N. H. Lajis
Scientific Chairs Unit, Taibah University,
P.O. Box 30001, Madinah al-Munawarah 41311,
Saudi Arabia
A. Sazali Hamzah
Department of Chemistry, Faculty of Applied Science,
University Technology MARA, 40450 Shah Alam,
Selangor Darul Ehsan, Malaysia
e-mail: asazali@salam.uitm.edu.my
K. Saha
J. Stanslas
Department of Chemistry, Jahangirnagar University, Savar,
Dhaka 1342, Bangladesh
e-mail: ksaha_ju@hotmail.com
Department of Medicine, Faculty of Medicine and Health
Science, University Putra Malaysia (UPM), 43400 Serdang,
Selangor Darul Ehsan, Malaysia
e-mail: rcxjs@medic.upm.edu.my
K. W. Lam
Drug and Herbal Research Centre Faculty of Pharmacy,
Universiti Kebangsaan Malaysia, Jalan Raja Muda Abd. Aziz,
50300 Kuala Lumpur, Malaysia
L. S. Hui
Department of Biomedical Science, Faculty of Medicine and
Health Science, University Putra Malaysia (UPM),
43400 Serdang, Selangor Darul Ehsan, Malaysia
e-mail: david_lam_98@yahoo.com
123

2094
Med Chem Res (2013) 22:2093–2104
O
R1
Zagotto et al., 2000). Anthraquinones are well known for
their antioxidant property (Huang et al., 1995; Yen et al.,
2000), and have also been reported to exhibit diverse
bioactivities including hypotensive, antimicrobial, anti-
plasmodial, antitumor and cytotoxic effects (Chang et al.,
1982; Johnson et al., 1997; Mishra and Gupta, 1982).
Unfortunately, anthraquinones are also reported to display
undesirable side effects, such as cardiotoxicity (Cardia
et al., 2001).
R2
R3
O
R4
Compound
R1
R2
R3
Br
H
R4
H
1
OH
H
2
OH
H
Br
H
In our previous efforts to discover new bioactive con-
stituents from our plant resources, we isolated a new
2-formyl-1-hydroxyanthraquinone along with ten other
known 9,10-anthraquinones, including damnacanthal and
nordamnacanthal (Ismail et al., 1997). Both damnacanthal
and nordamnacanthal exhibited strong and selective cyto-
toxic activity against CEM-SS and MCF-7 as compared to
HeLa cell lines (Ali et al., 2000). In order to further
understand the role of these anthraquinones in their
mechanism leading to cell death of anti-proliferative
activity towards cancer cell lines, a programme to syn-
thesise these compounds along with their analogues was
conducted with the aim of selecting the best candidate for
its further larger scale preparation and biological evalua-
tion. We herein report the synthesis of a number of
substituted simple 9,10-anthraquinone analogues, followed
by the evaluation of their cytotoxic and antioxidant activ-
ities, and finally to assess their structure–activity
relationship.
3
H
OH
Br
H
4
OH
H
H
5
H
OH
H
H
6
Br
H
OH
OH
H
H
7
OH
H
H
8
OH
H
OH
CH3
H
9
OH
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
OH
CH3
OCH3
H
OH
H
H
H
OH
H
OCH3
OCH3
CH3
Br
H
OCH3
OCH3
OH
H
H
H
H
CH3
CH3
CH3
CH3
CH2Br
CH2Br
CHBr2
CH2Br
CH2OEt
CH2OEt
CH2OEt
CHO
CHO
CHO
CHO
OH
OAc
OAc
OCH3
OAc
OCH3
OCH3
OCH3
OH
OCH3
OAc
OH
OH
OCH3
OH
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OH
H
H
H
H
H
H
Results and discussion
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
H
H
The structure of anthraquinone analogues is represented in
Fig. 1. Friedel–Crafts condensation was selected as method
of choice for the preparation of the anthraquinone skeleton
due to the simplicity of the reaction and less demanding
conditions (Wei et al., 2000; Werner et al., 1997; Zhang
et al., 1996). Phthalic anhydride and suitable substituted
benzenes were used as the starting materials for the desired
anthraquinones (1–10). Methylated hydroxyanthraquinones
11–14 as well as 17–18, were prepared from the respective
5 and 8–10 using CH3I in the presence of NaH or
(CH3)2SO4 in the presence of anhydrous K2CO3 (Enger
and Iyenrar, 1998; Roberts et al., 1997), in appropriate
reaction conditions. Acetylation of hydroxyanthraquinones
was accomplished by treating the respective starting
materials with Ac2O with K2CO3 used as base. Compound
10 with free unprotected hydroxyl was converted to the
unexpected 15 in good yield, upon bromination using
N-bromosuccinimide in the presence of benzoyl peroxide.
However, upon bromination using the same reagent,
compounds 16 and 17 gave the expected bromomethyl and
dibromomethyl derivatives (19–21) (Cambie et al., 1992)
H
H
Br
H
H
Fig. 1 Structure of synthesized anthraquinones
in good yields. Compound 20 was further converted to 22
by demethylation using anhydrous aluminium chloride
(Zacharie et al., 1997).
The bromomethyl derivatives, 19 and 20 were success-
fully converted to the respective ethoxymethyl derivatives
23 and 24 by treating them with ethanol in the presence
of aqueous sodium hydroxide. 2-Formylanthraquinones,
26–28 were obtained when the respective ethoxymethyl
derivatives 23–25 were brominated with N-bromosuccini-
mide in the presence of benzoyl peroxide followed by
hydrolysis with aqueous acetic acid (Roberts et al., 1997).
123

Med Chem Res (2013) 22:2093–2104
2095
This may be related to the increase in the calculated lipo-
philicity (ALOGPs) with the predicted value of 4.44. The
addition of the second bromo atom in the bromoalkyl group
could result in the increase in the hydrophobic character,
which is crucial in determining the capability of the com-
pound to cross the cell membranes. Another derivative of
bromomethylanthraquinone 19 exhibited strong cytotoxic-
ity against MCF7 (IC50: 6 lM) and DU145 (IC50: 5 lM)
cell lines. On the other hand, compound 22, which also
bears bromomethyl group at position-2 but with hydroxyl
group at position-1, showed a degree of selectivity against
human uterine sarcoma (MES-SA) cell line with IC50 value
of 4 lM. However, both the compounds 19 and 22 failed to
match the activity of compound 20. It is noteworthy that
the 2-formyl analogues of these compounds also showed
some degree of selectivity towards cancer cell lines tested
Further inspection on the solubility (ALOGpS) of
compounds 20, 21, 19 and 22 demonstrated that they are
less sufficient for fast absorption due to the lower (calcu-
lated) solubility. According to the study on the rate-limit-
ing steps of human oral absorption of 238 drugs, the
absorption is usually very low if the calculated solubility is
less than 0.00001 mg/L (Zhao et al., 2002). Therefore, it is
critical to improve the solubility of these compounds to
permit dissolution and absorption in the future studies.
Compound 22 showed promising results by displaying only
moderate activity against other cell lines and could be a
potential candidate for further drug development. Other
compounds including 14, 21, 28 and 29 also showed
selectivity against MES-SA cell line. Damnacanthal (26),
although exhibited only moderate activity against DU145
and H460, displayed some selectivity against MCF7 with
IC50 value 11 lM.
Table 1 Cytotoxic activity of anthraquinones (IC50 in lM)
Compound MCF7 MES-SA MES-SA/DX5 DU145 H460
1
–
–
–
–
–
2
36
–
14
–
20
–
27
–
20
–
3
4
–
–
–
–
–
5
69
27
55
–
–
–
72
44
29
–
66
43
23
–
6
56
30
–
29
21
–
7
8
9
–
–
–
–
–
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
56
68
79
70
55
28
56
26
–
38
–
35
–
32
70
74
69
48
31
37
27
–
42
68
82
71
54
19
68
–
–
–
–
–
19
20
–
–
30
–
–
–
–
–
–
6
–
–
5
26
5
8
2
2
4
27
34
35
–
10
4
7
28
26
50
–
23
30
–
–
–
–
–
–
–
30
11
19
55
36
–
–
23
26
34
32
42
–
–
–
25
32
65
40
–
–
5
–
18
–
–, not active with IC50 of [100 lM
The overall results indicated that anthraquinones con-
taining bromomethyl group at C-2 with dimethoxy or
methoxy and acetoxy at C-1 and C-3, respectively, (19 and
20) markedly increased cytotoxic activity while the pres-
ence of hydroxyl at C-1 instead of methoxy reduced the
cytotoxicity but increased the selectivity (22). The pres-
ence of a formyl group at C-2 with methoxy at C-1 and
hydroxy at C-3 also caused significant cytotoxicity (26 and
27). Comprehensively, the theoretical study of these com-
pounds showed that they fulfilled Lipinski rule-of-5 and
drug-like properties. Another important physicochemical
criterion is the polar surface area (PSA), which is based on
the summation of tabulated surface contributions of polar
fragments. In this experiment, we calculated the polar
surface area of the compounds using the topological
PSA (TPSA) procedure (see Table 2). All the compounds
were generally within the desired limits of TPSA in the
Compound 28 was converted to 29 by demethylation using
anhydrous aluminium chloride.
The anthraquinone analogues were evaluated for their
cytotoxic activity against five different cell lines: breast
cancer (MCF7), human uterine sarcoma (MES-SA), mul-
tidrug-resistant variant human uterine sarcoma (MES-SA/
DX5), prostate cancer (DU145) and lung cancer (H460).
The highest concentration of the compound used for the
MTT assay was 100 lM with the lowest 0.1 lM using
10-fold serial dilution. Table 1 shows the overall results of
cytotoxic activity of the analogues.
2-Bromomethyl-1,3-dimethoxyanthraquinone 20 was
found to be the most cytotoxic compound against all cell
lines tested. The IC50 values for this compound range
between 2 and 8 lM. However, this compound did not
show any selectivity against any particular cell line.
Additional bromine in dibromo derivative 21 seemed to
decrease the cytotoxicity of 20 in all the cancer cell lines.
2
˚
range of 50–100 A , suggesting a potentially good intes-
tinal absorption, blood–brain barrier penetration and
permeability across cell membranes.
123

2096
Med Chem Res (2013) 22:2093–2104
Table 2 Calculated physicochemical properties for compounds 1–29
Compound
MW
Volume
TPSA
HBA
HBD
ALOGPs
ALOGpS
ALOGpS (g/L)
1
303.111
303.111
303.111
224.215
224.215
287.112
240.214
240.214
238.242
254.241
238.242
254.241
268.268
252.269
333.130
296.278
310.305
282.295
389.201
361.191
440.087
347.164
312.321
326.348
354.358
282.251
361.147
296.278
268.224
208.481
208.481
208.481
190.595
190.595
200.463
198.613
198.613
207.156
215.174
208.123
216.141
233.669
224.684
233.059
251.685
269.213
250.230
287.339
268.356
286.267
250.828
275.290
292.818
311.802
235.124
253.010
252.652
217.596
54.370
54.370
54.370
54.370
54.370
34.142
74.598
74.598
54.370
74.598
43.376
63.604
52.610
43.376
74.598
80.675
69.681
52.610
69.681
52.610
52.610
63.604
72.838
61.844
78.915
80.675
80.675
69.681
91.669
3
3
3
3
3
2
4
4
3
4
3
4
4
3
4
5
5
4
5
4
4
4
5
5
6
5
5
5
5
1
1
1
1
1
0
2
2
1
2
0
1
0
0
2
1
0
0
0
0
0
1
1
0
0
1
1
0
2
3.92 ( 0.41)
3.93 ( 0.43)
3.61 ( 0.39)
3.13 ( 0.38)
2.89 ( 0.32)
3.90 ( 0.35)
3.13 ( 0.37)
2.93 ( 0.69)
3.52 ( 0.46)
3.13 ( 0.46)
3.13 ( 0.37)
3.08 ( 0.43)
3.01 ( 0.44)
3.45 ( 0.42)
3.82 ( 0.60)
3.23 ( 0.39)
3.20 ( 0.54)
3.34 ( 0.54)
3.55 ( 0.34)
3.71 ( 0.48)
4.44 ( 0.64)
3.75 ( 0.46)
2.99 ( 0.57)
3.16 ( 0.55)
3.00 ( 0.41)
2.83 ( 0.54)
3.52 ( 0.69)
2.72 ( 0.53)
2.93 ( 0.89)
-4.01
-4.05
-3.97
-3.12
-3.37
-4.52
-4.55
-2.92
-3.23
-3.23
-4.55
-3.34
-3.85
-4.69
-3.61
-4.05
-4.46
-4.30
-5.15
-5.05
-5.51
-4.57
-3.97
-4.33
-4.40
-3.64
-4.32
-4.28
-3.20
0.029
0.027
0.032
0.170
0.095
0.009
0.007
0.290
0.052
0.150
0.007
0.120
0.038
0.005
0.081
0.026
0.012
0.014
0.003
0.003
0.001
0.009
0.033
0.015
0.014
0.065
0.017
0.002
0.170
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
Antioxidant activity of the anthraquinones was mea-
sured using ferric thiocyanate (FTC) and thiobarbituric
acid (TBA) methods. The FTC method measures the
amount of peroxide in the initial stage of lipid peroxida-
tion. Low absorbance value in the FTC method indicates
high level of antioxidant activity. Only three anthraqui-
nones, damnacanthal (26), nordamnacanthal (29) and
commercial alizarin displayed stronger antioxidant activity
than vitamin E, although these compounds showed lower
activity than butylated hydroxy toluene (BHT). Alizarin
exhibited stronger activity than compound 26 and 29
(Fig. 2). All other anthraquinones were found to be
inactive.
3
2.5
2
1.5
1
0.5
0
0
1
2
3
4
5
6
7
8
Day
During the oxidation process, peroxide is gradually
decomposed to lower molecular compounds and the rela-
tive concentrations are measured using TBA method.
Figure 3 shows the absorbance value of active
Control
BHT
26
Vit.E
Alizarin
29
Fig. 2 Antioxidant activity using FTC method
123

Med Chem Res (2013) 22:2093–2104
2097
2.5
2
under reflux and filtered after cooling. The crude residue
was extracted with ethyl acetate and the compounds were
purified from ethyl acetate extract by using column
chromatography.
1.5
1
3-Bromo-1-hydroxyanthraquinone (1), 4-bromo-1-
hydroxyanthraquinone (2), 3-bromo-2-
hydroxyanthraquinone (3), 1-hydroxyanthraquinone (4)
and 2-hydroxyanthraquinone (5)
0.5
0
Phthalic anhydride (6.7 g, 45 mmol) and 4-bromophenol
(7.05 g, 41 mmol) were reacted according to the general
procedure to produce 1 (2.97 g, 24 %), 2 (1.36 g, 11 %), 3
(0.99 g, 8 %), 4 (0.37 g, 4 %) and 5 (0.28 g, 3 %) with the
total yield of 50 %.
Sample
Fig. 3 Antioxidant activity using TBA method
3-Bromo-1-hydroxyanthraquinone (1) Yellow crystals;
mp 188–189 °C; UV (CHCl3) kmax 403, 334, 284, 257 nm;
IR (KBr disc) m 3437 (OH), 2931, 1674 (C=O, unchelated),
1638 (C=O, chelated), 1591 (C=C, aromatic), 1466, 1355,
1291, 1253, 1033 cm-1; 1H NMR (CDCl3) d 12.63 (1H, s,
1-OH), 8.34–8.30 (2H, m, H-5 & H-8), 7.96 (1H, d,
J = 2.0 Hz, H-4), 7.87–7.85 (2H, m, H-6 & H-7), 7.52
(1H, d, J = 2.0 Hz, H-2); 13C NMR (CDCl3) d 188.4
(C-9), 181.6 (C-10), 163.2 (C-1), 135.2 (C-7), 134.8 (C-6),
134.3 (C-14), 133.3 (C-12), 133.2 (C-11), 131.8 (C-13),
127.9 (C-8), 127.3 (C-5), 127.1 (C-2), 123.1 (C-4), 115.3
(C-3); MS m/z (rel. int.) 304 ([M ? 2]?, 66), 302 (M?, 63),
276 (7), 274 (6), 248 (11), 246 (14), 223 (18), 195 (15), 167
(21), 139 (100), 113 (14), 97 (33), 83 (29), 69 (96).
anthraquinones and the standards. The absorbance value
was measured on the final day (8th day) of FTC assay. The
results were found to be consistent with the results of the
assay using FTC method. Anthraquinones containing
ortho-dihydroxy moiety exhibited antioxidant activity, e.g.
alizarin. The presence of formyl group next to a hydroxyl
in anthraquinone derivative also plays an important role in
their antioxidant activity (26 and 29).
Experimental
Melting points were determined on a hot stage melting point
apparatus XSP-12 model 500X and are uncorrected. UV
spectra were recorded on a CARY 100 Conc UV–Visible
spectrophotometer in CHCl3 or MeOH. IR spectra were
recorded on a Perkin Elmer RXI FT-IR spectrometer as KBr
disc. Mass spectra were measured on Finnigan Mat SSQ 710
spectrometer with ionization induced by electron impact at
70 eV. NMR spectra were recorded in CDCl3 or DMSO-d6
using Varian 500 MHz NMR spectrometer. Column chro-
matography was performed on Silica gel 60 Merck 9385
(230–400 mesh ASTM).
4-Bromo-1-hydroxyanthraquinone (2) Orange needles;
mp 195–196 °C [lit. 197–198 °C, Dictionary of Organic
Compounds (1965)]; UV (CHCl3) kmax 414, 330, 272,
255 nm; IR (KBr disc) m 3438 (OH), 1670 (C=O, unch-
elated), 1636 (C=O, chelated), 1590 (C=C, aromatic),
1447, 1410, 1348, 1244, 1115, 788 cm-1 1H NMR
;
(CDCl3) d 13.29 (1H, s, 1-OH), 8.31–8.28 (2H, m, H-5 &
H-8), 7.90 (1H, d, J = 9.0 Hz, H-3), 7.85–7.81 (2H, m,
H-6 & H-7), 7.17 (1H, d, J = 9.0 Hz, H-2); 13C NMR
(CDCl3) d 188.3 (C-9), 181.4 (C-10), 163.2 (C-1), 144.1
(C-3), 135.4 (C-7), 134.3 (C-12), 134.2 (C-6), 132.3
(C-11), 130.2 (C-14), 128.0 (C-8), 126.8 (C-5), 125.4
(C-2), 118.0 (C-13), 113.4 (C-4); MS m/z (rel. int.) 304
([M ? 2]?, 93), 302 (M?, 100), 276 (14), 274 (15), 248
(20), 246 (21), 223 (5), 195 (5), 167 (6), 139 (69), 113 (12),
97 (6), 69 (28).
General procedure for anthraquinone synthesis
A mixture of anhydrous aluminium chloride (60 g,
450 mmol) and sodium chloride (12 g, 205 mmol) was
heated until it completely melted (external temperature,
125–130 °C). Phthalic anhydride (6.7 g, 45 mmol) and
benzene derivative (41 mmol) were mixed well and slowly
introduced into the melt of aluminium chloride and
sodium chloride. The mixture was heated with stirring at
165–170 °C for 45 min. After cooling, the reaction mixture
was decomposed by adding a mixture of ice and hydro-
chloric acid. The acidic mixture was then briefly heated
3-Bromo-2-hydroxyanthraquinone (3) Yellow amor-
phous compound; mp 262–263 °C [lit. 267–268 °C, Dic-
tionary of Organic Compounds (1965)]; UV (MeOH) kmax
465, 311, 284, 247 nm; IR (KBr disc) m 3413 (OH), 1668
(C=O, unchelated), 1572 (C=C, aromatic), 1336, 1304,
123

2098
Med Chem Res (2013) 22:2093–2104
1278, 718 cm-1; H NMR (DMSO-d6) d 12.00 (1H, br.s,
2-OH), 8.24 (1H, s, H-1), 8.17–8.15 (2H, m, H-5 &H-8),
7.92–7.90 (2H, m, H-6 & H-7), 7.65 (1H, s, H-4); 13C
NMR (DMSO-d6) d 182.8 (C-10), 181.2 (C-9), 160.4
(C-2), 135.4 (C-6), 135.0 (C-7), 134.8 (C-13), 133.7
(C-11), 133.6 (C-12), 132.9 (C-1), 127.4 (C-5 & C-8),
126.6 (C-14), 117.5 (C-3), 113.5 (C-4); MS m/z (rel. int.)
304 ([M ? 2]?, 98), 302 (M?, 100), 276 (19), 274 (18),
248 (17), 246 (18), 223 (15), 195 (18), 167 (21), 139 (89),
123 (14), 83 (36), 69 (94), 50 (25).
of organic compounds (1965)]; UV (MeOH) kmax 366, 277,
243 nm; IR (KBr disc) m 3393 (OH), 1655 (C=O, unch-
elated), 1576 (C=C, aromatic), 1329, 1290, 1245,
715 cm-1; 1H NMR (DMSO-d6) d 11.50 (1H, br.s, 3-OH),
8.16–8.11 (2H, m, H-5 & H-8), 7.93–7.86 (2H, m, H-6 &
H-7), 7.59 (1H, d, J = 2.5 Hz, H-4), 7.46 (1H, d,
J = 2.5 Hz, H-2); 13C NMR (DMSO-d6) d 182.3 (C-10),
180.6 (C-9), 162.6 (C-3), 138.2 (C-14), 135.5 (C-6), 134.8
(C-11), 134.5 (C-7), 132.8 (C-12), 128.1 (C-2), 127.6
(C-5), 126.9 (C-8), 124.2 (C-1), 122.9 (C-13), 114.3 (C-4);
MS m/z (rel. int.) 304 ([M ? 2]?, 22), 302 (M?, 24), 276
(11), 274 (11), 248 (8), 246 (9), 223 (6), 195 (14), 167 (17),
139 (100), 113 (13), 87 (12), 69 (23), 50 (38).
1
1-Hydroxyanthraquinone (4) Yellow-orange needles; mp
190–191 °C [lit. 195–196 °C, Dictionary of Natural Prod-
ucts (2003)]; UV (CHCl3) kmax 405, 330, 270, 253 nm; IR
(KBr disc) m 3436 (OH), 1674 (C=O, unchelated), 1638
(C=O, chelated), 1592 (C=C, aromatic), 1452, 1259, 1227,
1,3-Dihydroxyanthraquinone or xanthopurpurin (7)
When phthalic anhydride (6.7 g, 45 mmol) and resorcinol
(4.51 g, 41 mmol) were used as starting material, two
products 7 (0.98 g, 11 %) and 3,3-di(20,40-dihydroxy-
phenyl)phthalide (2.29 g, 31 %) were found with total
yield of 42 %.
762 cm-1 1H NMR (CDCl3) d 12.64 (1H, s, 1-OH),
;
8.35–8.32 (2H, m, H-5 & H-8), 7.86 (1H, dd, J = 7.5 &
1.0 Hz, H-4), 7.85–7.83 (2H, m, H-6 & H-7), 7.71 (1H, t,
J = 8.0 & 7.5 Hz, H-3), 7.34 (1H, dd, J = 8.0 & 1.0 Hz,
H-2); 13C NMR (CDCl3) d 188.9 (C-9), 182.7 (C-10),
162.8 (C-1), 137.0 (C-3), 134.9 (C-7), 134.4 (C-6), 133.9
(C-12), 133.7 (C-14), 133.5 (C-11), 127.7 (C-8), 127.2
(C-5), 124.6 (C-2), 119.8 (C-4), 116.4 (C-13); MS m/z (rel.
int.) 224 (M?, 100), 196 (19), 168 (36), 139 (51), 113 (7),
98 (10), 84 (13), 70 (44), 50 (19).
1,3-Dihydroxyanthraquinone or xanthopurpurin (7) Yel-
low amorphous compound; mp 268 °C [lit. 268–270 °C,
Dictionary of Natural Products (2003)]; UV (MeOH) kmax
413, 280, 241 nm; IR (KBr disc) m 3394 (OH), 1630
(C=O), 1340, 1320, 1161, 779, 712 cm-1 1H NMR
;
(DMSO-d6) d 12.75 (1H, s, 1-OH), 11.35 (1H, br.s, 3-OH),
8.22–8.15 (2H, m, H-5 & H-8), 7.95–7.89 (2H, m, H-6 &
H-7), 7.16 (1H, d, J = 2.0 Hz, H-4), 6.63 (1H, d,
J = 2.0 Hz, H-2); MS m/z (rel. int.) 240 (M?, 100), 212
(17), 184 (27), 155 (10), 128 (21), 106 (11), 92 (11), 77
(17), 69 (21), 51 (23).
2-Hydroxyanthraquinone (5) Yellow amorphous solid;
mp 298–299 °C [lit. 302–303 °C, dictionary of natural
products (2003)]; UV (MeOH) kmax 373, 329, 270,
242 nm; IR (KBr disc) m 3370 (OH), 1672 (C=O, unch-
elated), 1578 (C=C, aromatic), 1341, 1305, 1281,
720 cm-1; 1H NMR (DMSO-d6) d 11.08 (1H, br.s, 2-OH),
8.18-8.16 (2H, m, H-5 & H-8), 8.10 (1H, d, J = 8.5 Hz,
H-4), 7.91–7.88 (2H, m, H-6 & H-7), 7.51 (1H, d,
J = 2.5 Hz, H-1), 7.25 (1H, dd, J = 8.5 & 2.5 Hz, H-3);
13C NMR (DMSO-d6) d 183.4 (C-9), 181.9 (C-10), 163.8
(C-2), 135.9 (C-13), 135.3 (C-7), 134.7 (C-6), 133.9
(C-12), 133.8 (C-11), 130.6 (C-4), 127.3 (C-8), 127.2
(C-5), 125.9 (C-14), 122.3 (C-3), 112.9 (C-1); MS m/z (rel.
int.) 224 (M?, 100), 196 (35), 168 (35), 139 (78), 113 (9),
98 (7), 84 (12), 63 (21).
1,4-Dihydroxyanthraquinone or quinizarin (8)
Reaction between phthalic anhydride (6.7 g, 45 mmol) and
hydroquinone (4.51 g, 41 mmol) produced 8 (4.33 g) with
44 % yield.
1,4-Dihydroxyanthraquinone or quinizarin (8) Orange-
red crystals; mp 197–198 °C [lit. 194 °C, Dictionary of
Natural Products (2003)]; UV (CHCl3) kmax 476, 328, 280,
250 nm; IR (KBr disc) m 3437 (OH), 2926, 1630 (C=O,
chelated), 1591 (C=C, aromatic), 1453, 1358, 1257, 1226,
1-Bromo-3-hydroxyanthraquinone (6)
790 cm-1 1H NMR (CDCl3) d 12.92 (2H, s, 1-OH &
;
Reaction between phthalic anhydride (6.7 g, 45 mmol) and
3-bromophenol (7.05 g, 41 mmol) gave four products
including 1 (3.96 g, 32 %), 2 (0.12 g, 1 %), 4 (0.46 g,
5 %) and 6 (1.36 g, 11 %). The total yield was 49 %.
4-OH), 8.37–8.35 (2H, m, H-5 & H-8), 7.86–7.85 (2H, m,
H-6 & H-7), 7.33 (2H, s, H-2 & H-3); 13C NMR (CDCl3) d
187.2 (C-9 & C-10), 158.1 (C-1 & C-4), 134.8 (C-6 &
C-7), 133.7 (C-11 & C-12), 129.6 (C-2 & C-3), 127.3 (C-5
& C-8), 113.0 (C-13 & C-14); MS m/z (rel. int.) 240
(M?, 100), 212 (10), 183 (17), 155 (11), 128 (14), 102 (11),
77 (6).
1-Bromo-3-hydroxyanthraquinone (6) Yellow amor-
phous compound; mp 180–181 °C [lit. 187 °C, dictionary
123

Med Chem Res (2013) 22:2093–2104
2099
H-3), 4.01 (3H, s, –OCH3); MS m/z (rel. int.) 238 (M?,
100), 209 (32), 195 (11), 180 (16), 167 (15), 152 (24), 139
(41), 113 (5), 89 (4), 63 (6).
1-Hydroxy-4-methylanthraquinone (9)
Reaction between phthalic anhydride (6.7 g, 45 mmol) and
p-cresol (4.43 g, 41 mmol) gave compound 9 (4.39 g,
45 %).
1-Hydroxy-4-methoxyanthraquinone (12) and 1,4-
dimethoxyanthraquinone (13)
1-Hydroxy-4-methylanthraquinone (9) Yellow crystals;
mp 174–175 °C [lit. 175–176 °C, Dictionary of Organic
compounds (1965)]; UV (CHCl3) kmax 416, 325, 271,
252 nm; IR (KBr disc) m 3435 (OH), 2929, 1638 (C=O),
Dimethyl sulphate (4 mL, 42.3 mmol) was added slowly
into the mixture of 8 (96 mg, 0.4 mmol) and anhydrous
potassium carbonate (1.6 g, 11.8 mmol) in dry acetone
(40 mL) and the reaction mixture was refluxed for 6 h. The
reaction mixture was then poured into crushed ice and
filtered. The product 12 (62 mg, 60 %) and 13 (21 mg,
20 %) were purified by column chromatography.
1365, 1282, 1248, 789, 724 cm-1 1H NMR (CDCl3)
;
d13.20 (1H, s, 1-OH), 8.32–8.28 (2H, m, H-5 & H-8),
7.85–7.79 (2H, m, H-6 & H-7), 7.52 (1H, d, J = 9.0 Hz,
H-2), 7.25 (1H, d, J = 9.0 Hz, H-3), 2.78 (3H, s, -CH3);
MS m/z (rel. int.) 238 (M?, 100), 210 (12), 181 (34), 152
(25), 119 (6), 96 (12), 76 (24), 51 (12).
1-Hydroxy-4-methoxyanthraquinone or quinizarin 4-methyl
ether (12) Orange-red crystals; mp 188–189 °C [lit.
189 °C, Dictionary of Natural Products (2003)]; UV
(CHCl3) kmax 458, 324, 274, 253 nm; IR (KBr disc) m 3436
(OH), 3068, 2928, 1630 (C=O), 1595 (C=C, aromatic),
1,3-Dihydroxy-2-methylanthraquinone or rubiadin (10)
Reaction between phthalic anhydride (6.7 g, 45 mmol) and
2-methylresorcinol (5.08 g, 41 mmol) produced 10
(4.16 g) with 40 % yield.
1
1474, 1352, 1243, 1182, 1015, 785, 724 cm-1; H NMR
(CDCl3) d 13.02 (1H, s, 1-OH), 8.32–8.29 (2H, m, H-5 &
H-8), 7.84–7.76 (2H, m, H-6 & H-7), 7.43 (1H, d,
J = 9.5 Hz, H-2), 7.35 (1H, d, J = 9.5 Hz, H-3), 4.05 (3H,
s, –OCH3); MS m/z (rel. int.) 254 (M?, 74), 225 (100), 211
(17), 197 (28), 183 (27), 152 (30), 139 (16), 127 (21), 105
(7), 77 (8).
1,3-Dihydroxy-2-methylanthraquinone or rubiadin (10) Yel-
low needles; mp 281–282 °C [lit. 280–283 °C, Leistner
(1975)]; UV (MeOH) kmax 411, 279, 245 nm; IR (KBr
disc) m 3402 (OH), 1661 (C=O, unchelated), 1624 (C=O,
chelated), 1591 (C=C, aromatic), 1338, 1310, 1122,
1
712 cm-1; H NMR (DMSO-d6) d 13.05 (1H, s, 1-OH),
11.18 (1H, s, 3-OH), 8.15–8.08 (2H, m, H-5 & H-8),
7.89–7.83 (2H, m, H-6 & H-7), 7.20 (1H, s, H-4), 2.03 (3H,
s, -CH3); MS m/z (rel. int.) 254 (M?, 100), 226 (10), 197
(9), 152 (9), 115 (9), 76 (12).
1,4-Dimethoxyanthraquinone or quinizarin 1,4-dimethyl
ether (13) Yellow amorphous solid; mp 144–145 °C [lit.
143 °C, Dictionary of natural Products (2003)]; UV
(CHCl3) kmax 424, 322, 250 nm; IR (KBr disc) m 2956,
1665 (C=O, unchelated), 1270, 1254, 1055, 976,
2-Methoxyanthraquinone (11)
1
803 cm-1; H NMR (CDCl3) d 8.19–8.17 (2H, m, H-5 &
H-8), 7.73–7.72 (2H, m, H-6 & H-7), 7.37 (2H, s, H-2 &
H-3), 4.02 (6H, s, –OCH3); MS m/z (rel. int.) 268 (M?, 58),
239 (100), 221 (32), 193 (38), 181 (20), 165 (35), 152 (26),
126 (20), 105 (8), 77 (6).
Sodium hydride (39 mg, 1.6 mmol) and 2 molar methyl
iodide solution in tert-butyl methyl ether (4 mL, 8 mmol)
were added successively into the solution of compound 5
(179 mg, 0.8 mmol) in dimethylformamide (10 mL) and
the reaction mixture was stirred at room temperature for
20 h. The reaction mixture was poured into crushed ice and
the product 11 (171 mg, 90 %) was purified by
crystallization.
1-Methoxy-4-methylanthraquinone (14)
Compound 14 (101 mg, 100 %) was formed when 9
(95 mg, 0.4 mmol) was methylated with dimethyl sulphate
(2 mL, 21.2 mmol) and anhydrous potassium carbonate
(0.8 g, 5.9 mmol). The reaction time was 22 h at reflux
condition.
2-Methoxyanthraquinone (11) Yellow crystals; mp
194–196 °C [lit. 195–196 °C, Dictionary of Natural Prod-
ucts (2003)]; UV (CHCl3) kmax 371, 330, 269, 248 nm; IR
(KBr disc) m 3075, 2987, 1675 (C=O, unchelated), 1591
1
(C=C, aromatic), 1330, 1304, 1081, 851, 711 cm-1; H
NMR (CDCl3) d 8.33–8.30 (2H, m, H-5 & H-8), 8.28 (1H,
d, J = 8.5 Hz, H-4), 7.81–7.78 (2H, m, H-6 & H-7), 7.76
(1H, d, J = 2.5 Hz, H-1), 7.30 (1H, dd, J = 8.5 & 2.5 Hz,
1-Methoxy-4-methylanthraquinone (14) Yellow crystals;
mp 127–128 °C [lit. 128 °C, Dictionary of Organic Com-
pounds (1965)]; UV (CHCl3) kmax 389, 321, 254 nm; IR
123

2100
Med Chem Res (2013) 22:2093–2104
1
(KBr disc) m 2970, 2928, 1670 (C=O, unchelated), 1593
1217, 1102, 1014, 781 cm-1; H NMR (CDCl3) d 13.13
1
(C=C, aromatic), 1325, 1255, 1037, 980, 724 cm-1; H
(1H, s, 1-OH), 8.33–8.28 (2H, m, H-5 & H-8), 7.83-7.81
(2H, m, H-6 & H-7), 7.55 (1H, s, H-4), 2.42 (3H, s,
–COCH3), 2.22 (3H, s, -CH3); MS m/z (rel. int.) 296 (M?,
2), 254 (100), 236 (11), 226 (20), 208 (7), 197 (11), 180
(9), 152 (11), 115 (8).
NMR (CDCl3) d 8.21–8.16 (2H, m, H-5 & H-8), 7.74–7.72
(2H, m, H-6 & H-7), 7.52 (1H, d, J = 9.0 Hz, H-2), 7.26
(1H, d, J = 9.0 Hz, H-3), 4.03 (3H, s, –OCH3), 2.76 (3H, s,
–CH3); MS m/z (rel. int.) 252 (M?, 100), 235 (27), 223
(80), 209 (35), 195 (14), 178 (29), 165 (64), 152 (60), 126
(5), 77 (6), 63 (5).
3-Acetoxy-1-methoxy-2-methylanthraquinone (17)
4-Bromo-1,3-dihydroxy-2-methylanthraquinone (15)
Compound 17 (451 mg, 100 %) was formed when 16
(431 mg, 1.46 mmol) was methylated with dimethyl sul-
phate (4.9 ml, 51.1 mmol) and anhydrous potassium car-
bonate (2.01 g, 14.6 mmol) in dry acetone at reflux
condition for 22 h.
A mixture of compound 10 (203 mg, 0.8 mmol), N-bro-
mosuccinimide (370 mg, 2.08 mmol) and benzoyl perox-
ide (20 mg) in carbon tetrachloride (50 mL) was refluxed
for 24 h. The solvent was evaporated and the product was
washed with warm water and then dissolved in a small
amount of acetone. The solution was poured into crushed
ice and the precipitates were collected by filtration. Col-
umn chromatography was used to purify the product 15
(159 mg, 60 %).
3-Acetoxy-1-methoxy-2-methylanthraquinone (17) Pale
yellow crystals; mp 173–174 °C [lit. 174 °C, Roberts et al.,
(1997)]; UV (CHCl3) kmax 406, 336, 280, 260 nm; IR (KBr
disc) m 2940, 1764 (C=O, ester), 1673 (C=O, unchelated),
1582 (C=C, aromatic), 1330, 1279, 1195, 1099, 716 cm-1
;
1H NMR (CDCl3) d 8.30–8.24 (2H, m, H-5 & H-8), 7.86
(1H, s, H-4), 7.83–7.75 (2H, m, H-6 & H-7), 3.97 (3H, s,
–OCH3), 2.42 (3H, s, –COCH3), 2.27 (3H, s, –CH3); MS m/
z (rel. int.) 310 (M?, 8), 268 (100), 250 (84), 239 (56), 222
(48), 194 (39), 165 (44), 152 (38), 139 (22), 115 (12).
4-Bromo-1,3-dihydroxy-2-methylanthraquinone
(15) Yellow-orange amorphous solid; mp 290–292 °C;
UV (CHCl3) kmax 422, 335, 273, 250 nm; IR (KBr disc) m
3409 (OH), 2931, 1672 (C=O, unchelated), 1618 (C=O,
chelated), 1577 (C=C, aromatic), 1420, 1362, 1295, 1197,
1
829, 722 cm-1; H NMR (CDCl3) d 13.87 (1H, s, 1-OH),
1,3-Dimethoxy-2-methylanthraquinone (18)
8.32–8.28 (2H, m, H-5 & H-8), 7.82–7.80 (2H, m, H-6 &
H-7), 7.15 (1H, s, 3-OH), 2.35 (3H, s, –CH3); 13C NMR
(CDCl3) d 187.2 (C-9), 181.8 (C-10), 163.3 (C-1), 157.5
(C-3), 134.8 (C-7), 134.3 (C-6), 134.0 (C-12), 132.7
(C-11), 128.2 (C-14), 127.8 (C-8), 126.7 (C-5), 119.7
(C-2), 112.0 (C-13), 104.2 (C-4), 9.7 (CH3); MS m/z (rel.
int.) 334 ([M ? 2]?, 100), 332 (M?, 98), 306 (16), 304
(16), 292 (17), 290 (17), 253 (67), 225 (86), 197 (28), 169
(16), 139 (26), 115 (15), 77 (6), 55 (5).
Compound 10 (400 mg, 1.57 mmol) was methylated with
dimethyl sulphate (10.4 mL, 110 mmol) in the presence of
anhydrous potassium carbonate (4.26 g, 31.4 mmol) at
reflux condition for 22 h to form 18 (444 mg) as 100 %
product.
1,3-Dimethoxy-2-methylanthraquinone or rubiadin 1,3-
dimethyl ether (18) Yellow crystals; mp 160 °C [lit.
159–160 °C, Roberts et al. (1997)]; UV (CHCl3) kmax 352,
279, 240 nm; IR (KBr disc) m 2941, 1668 (C=O, unch-
elated), 1577 (C=C, aromatic), 1325, 1287, 1135, 979,
3-Acetoxy-1-hydroxy-2-methylanthraquinone (16)
1
Acetic anhydride (1.4 ml, 14.6 mmol) was added dropwise
into the mixture of anhydrous potassium carbonate (0.8 g,
5.84 mmol) and compound 10 (370 mg, 1.46 mmol) in dry
acetone (25 mL) and the reaction mixture was stirred for
20 h at room temperature. The mixture was then poured
into crushed ice and filtered. The product 16 (431 mg,
100 %) was purified by crystallization.
718 cm-1; H NMR (CDCl3) d 8.30–8.23 (2H, m, H-5 &
H-8), 7.80–7.72 (2H, m, H-6 & H-7), 7.65 (1H, s, H-4),
4.04 (3H, s, –OCH3), 3.93 (3H, s, –OCH3), 2.29 (3H, s,
–CH3); MS m/z (rel. int.) 282 (M?, 100), 264 (43), 253
(36), 236 (42), 221 (30), 193 (31), 181 (39), 165 (44), 152
(44), 139 (24), 111 (10), 83 (14), 57 (10).
3-Acetoxy-2-bromomethyl-1-methoxyanthraquinone (19)
3-Acetoxy-1-hydroxy-2-methylanthraquinone (16) Yellow-
ish orange crystals; mp 190–191 °C [lit. 191 °C, Dictio-
nary of Natural Products (2003)]; UV (CHCl3) kmax 409,
331, 285, 262, 248 nm; IR (KBr disc) m 3435 (OH), 2964,
2930, 1764 (C=O, ester), 1667 (C=O, unchelated), 1618
(C=O, chelated), 1594 (C=C, aromatic), 1420, 1330, 1282,
Compound 19 (376 mg, 100 %) was formed when com-
pound 17 (300 mg, 0.97 mmol) was brominated with
N-bromosuccinimide (329 mg, 1.84 mmol) and benzoyl
peroxide (30 mg) in carbon tetrachloride at reflux condi-
tion for 24 h.
123

Med Chem Res (2013) 22:2093–2104
2101
3-Acetoxy-2-bromomethyl-1-methoxyanthraquinone
2-Bromomethyl-1-hydroxy-3-methoxyanthraquinone
(19) Yellow solid; mp 165–166 °C [lit. 169–170 °C,
Hirose (1960)]; UV (CHCl3) kmax 335, 283, 262 nm; IR
(KBr disc) m 3069, 2929, 1775 (C=O, ester), 1677 (C=O,
unchelated), 1578 (C=C, aromatic), 1327, 1285, 1136,
(22) Yellow crystals; mp 193–194 °C; UV (CHCl3) kmax
410, 338, 277, 247 nm; IR (KBr disc) m 3483 (OH), 2930,
1631 (C=O), 1599 (C=C, aromatic), 1376, 1335, 1303,
1134, 718 cm-1; 1H NMR (CDCl3) d 13.15 (1H, s, 1-OH),
8.32–8.29 (2H, m, H-5 & H-8), 7.85–7.80 (2H, m, H-6 &
H-7), 7.45 (1H, s, H-4), 4.89 (2H, s, –CH2Br), 4.00 (3H, s,
–OCH3); MS m/z (rel. int.) 348 ([M ? 2]?, 1), 346 (M?,
1), 284 (22), 269 (60), 267 (35), 255 (100), 253 (11),
238 (13), 223 (20), 208 (16), 181 (20), 152 (20), 139 (34),
77 (8).
1
1076, 987, 916 cm-1; H NMR (CDCl3) d 8.29–8.23 (2H,
m, H-5 & H-8), 7.95 (1H, s, H-4), 7.81–7.77 (2H, m, H-6 &
H-7), 4.62 (2H, s, –CH2Br), 4.11 (3H, s, –OCH3), 2.47 (3H,
s, –COCH3); MS m/z (rel. int.) 390 ([M ? 2]?, 1), 388
(M?, 1), 309 (17), 267 (100), 238 (17), 209 (11), 181 (16),
152 (21), 139 (15), 83 (10).
2-Ethoxymethyl-3-hydroxy-1-methoxyanthraquinone (23)
2-Bromomethyl-1,3-dimethoxyanthraquinone (20) and
2-dibromomethyl-1,3-dimethoxyanthraquinone (21)
Compound 19 (376 mg, 0.97 mmol), ethanol (37 mL) and
10 % aq. NaOH (5 mL) were taken together and stirred the
solution for 24 h at room temperature. Addition of water
(125 mL) and 10 % aq. HCl (6 mL) into the reaction
mixture gave precipitates which were filtered and dried.
The compound 23 (227 mg, 75 %) was finally purified by
column chromatography.
Compound 18 (400 mg, 1.41 mmol) was brominated with
N-bromosuccinimide (479 mg, 2.68 mmol) in the presence
of benzoyl peroxide (40 mg) to form compounds 20
(398 mg, 78 %) and 21 (56 mg, 9 %).
2-Bromomethyl-1,3-dimethoxyanthraquinone (20) Yel-
low crystals; mp 154–155 °C; UV (CHCl3) kmax 366, 340,
280, 244 nm; IR (KBr disc) m 2939, 1670 (C=O, unch-
elated), 1578 (C=C, aromatic), 1330, 1288, 1231, 1161,
2-Ethoxymethyl-3-hydroxy-1-methoxyanthraquinone
(23) Yellow solid; mp 201–202 °C [lit. 202–204 °C,
Roberts et al. (1997)]; UV (CHCl3) kmax 371, 334, 277,
247 nm; IR (KBr disc) m3309 (OH), 2976, 2932, 1672
(C=O, unchelated), 1570 (C=C, aromatic), 1283, 1094,
1
1112, 987, 720 cm-1; H NMR (CDCl3) d 8.31–8.25 (2H,
m, H-5 & H-8), 7.83–7.75 (2H, m, H-6 & H-7), 7.70 (1H, s,
H-4), 4.72 (2H, s, –CH2–), 4.13 (3H, s, –OCH3), 4.11 (3H,
s, –OCH3); MS m/z (rel. int.) 362 ([M ? 2]?, 2), 360 (M?,
3), 281 (100), 267 (6), 236 (7), 223 (4), 181 (5), 165 (7),
152 (9), 139 (6), 76 (2).
977, 716 cm-1 1H NMR (acetone-d6) d 9.90 (1H, s,
;
3-OH), 8.27–8.19 (2H, m, H-5 & H-8), 7.92–7.86 (2H, m,
H-6 & H-7), 7.60 (1H, s, H-4), 4.79 (2H, s, –CH2O–), 3.97
(3H, s, –OCH3), 3.68 (2H, q, J = 7.0 Hz, –OCH2CH3),
1.24 (3H, t, J = 7.0 Hz, –OCH2CH3); MS m/z (rel. int.)
312 (M?, 2), 297 (42), 283 (56), 265 (100), 251 (50), 238
(66), 210 (45), 181 (52), 152 (37), 139 (38).
2-Dibromomethyl-1,3-dimethoxyanthraquinone (21) Yel-
low crystals; mp 145–146 °C; UV (CHCl3) kmax 369, 339,
280, 245 nm; IR (KBr disc) m 3042, 2933, 1674 (C=O,
unchelated), 1577 (C = C, aromatic), 1331, 1287, 1130,
984, 712 cm-1; 1H NMR (CDCl3) d 8.31–8.26 (2H, m, H-5
& H-8), 7.85–7.76 (3H, m, H-4, H-6 & H-7), 7.42 (1H, s,
–CHBr2), 4.26 (3H, s, –OCH3), 4.05 (3H, s, –OCH3); MS
m/z (rel. int.) 442 ([M ? 4]?,\1), 440 ([M ? 2]?, 1), 438
(M?, \1), 361 (100), 359 (99), 280 (65), 251 (50), 222
(12), 194 (13), 165 (16), 151 (15), 138 (9), 76 (4).
1,3-Dimethoxy-2-ethoxymethylanthraquinone (24)
Compound 24 (240 mg, 76 %) was formed when 20
(349 mg, 0.97 mmol) was reacted with ethanol (37 mL)
and 10 % aq. NaOH (5 mL) at room temperature.
1,3-Dimethoxy-2-ethoxymethylanthraquinone (24)
Yellow crystals; mp 139–140 °C [lit. 141–142 °C, Roberts
et al. (1997)]; UV (CHCl3) kmax 364, 336, 277, 245 nm; IR
(KBr disc) m 2929, 2858, 1674 (C=O, unchelated), 1579
(C=C, aromatic), 1459, 1316, 1283, 1133, 1093, 979,
2-Bromomethyl-1-hydroxy-3-methoxyanthraquinone (22)
Anhydrous aluminium chloride (167 mg, 1.12 mmol) was
introduced into the solution of 20 (50 mg, 0.14 mmol) in
dichloromethane (30 mL). Pyridine (0.04 mL, 0.56 mmol)
was then added dropwise and the reaction mixture was
refluxed for 24 h, acidified with dilute hydrochloric acid
and extracted with ethyl acetate. The product 22 (33 mg,
68 %) was purified by column chromatography.
1
714 cm-1; H NMR (CDCl3) d 8.30–8.23 (2H, m, H-5 &
H-8), 7.80–7.74 (2H, m, H-6 & H-7), 7.69 (1H, s, H-4),
4.66 (2H, s, –CH2O–), 4.06 (3H, s, –OCH3), 4.02 (3H, s,
–OCH3), 3.66 (2H, q, J = 7.0 Hz, –CH2CH3), 1.26 (3H, t,
J = 7.0 Hz, –OCH2CH3); MS m/z (rel. int.) 326 (M?, 3),
311 (88), 297 (44), 283 (100), 281 (100), 267 (68), 237
123

2102
Med Chem Res (2013) 22:2093–2104
(43), 209 (30), 181 (42), 165 (34), 152 (45), 139 (30), 83
(14).
hydrolysis with 80 % aq. acetic acid (60 mL) gave com-
pound 27 (228 mg) with 80 % yield.
3-Acetoxy-2-ethoxymethyl-1-methoxyanthraquinone (25)
4-Bromo-2-formyl-3-hydroxy-1-methoxyanthraquinone
(27) Yellowish orange compound; mp 219–220 °C; UV
(CHCl3) kmax 402, 290, 261 nm; IR (KBr disc) m 3433
(OH), 2938, 1655 (C=O, unchelated), 1625 (C=O, che-
lated), 1535 (C=C, aromatic), 1334, 1254, 1193, 994,
726 cm-1; 1H NMR (CDCl3) d 13.28 (1H, s, 3-OH), 10.46
(1H, s, –CHO), 8.24–8.20 (2H, m, H-5 & H-8), 7.82–7.80
(2H, m, H-6 & H-7), 4.17 (3H, s, –OCH3); MS m/z (rel.
int.) 362 ([M ? 2]?, 5), 360 (M?, 10), 333 (94), 331 (100),
304 (30), 302 (36), 252 (79), 224 (86), 196 (28), 178 (24),
150 (36), 138 (80), 82 (47).
Acetylation of compound 23 (227 mg, 0.73 mmol) with
acetic anhydride (0.7 mL, 7.3 mmol) and anhydrous
potassium carbonate (402 mg, 2.92 mmol) yielded com-
pound 25 (257 mg, 100 %).
3-Acetoxy-2-ethoxymethyl-1-methoxyanthraquinone
(25) Yellow amorphous compound; mp 165–166 °C; UV
(CHCl3) kmax 338, 273, 259, 237 nm; IR (KBr disc) m 2976,
2931, 2893, 1764 (C=O, ester), 1666 (C=O, unchelated),
1583 (C=C, aromatic), 1327, 1214, 1079, 972, 717 cm-1
;
1H NMR (CDCl3) d 8.32–8.26 (2H, m, H-5 & H-8), 7.92
(1H, s, H-4), 7.84–7.77 (2H, m, H-6 & H-7), 4.65 (2H, s,
–CH2O–), 4.03 (3H, s, –OCH3), 3.56 (2H, q, J = 7.0 Hz,
–OCH2CH3), 2.41 (3H, s, –COCH3), 1.23 (3H, t,
J = 7.0 Hz, –OCH2CH3); MS m/z (rel. int.) 354 (M?, 1),
339 (4), 312 (30), 297 (18), 283 (34), 265 (100), 238 (64),
210 (36), 181 (30), 252 (20), 139 (22).
1,3-Dimethoxy-2-formylanthraquinone (28)
Bromination of compound 24 (240 mg, 0.74 mmol) with
N-bromosuccinimide (252 mg, 1.41 mmol) followed by
hydrolysis with 80 % aq. acetic acid (60 mL) formed
compound 28 (174 mg, 80 %).
2-Formyl-3-hydroxy-1-methoxyanthraquinone
or damnacanthal (26)
1,3-Dimethoxy-2-formylanthraquinone or damnacanthal
3-methyl ether (28) Yellow amorphous solid; mp
183–184 °C [lit. 184–185 °C, Roberts et al. (1997)]; UV
(MeOH) kmax 366, 329, 274, 243 nm; IR (KBr disc) m
2940, 1673 (C=O, unchelated), 1578 (C=C, aromatic),
Compound 25 (164 mg, 0.46 mmol) was brominated with
N-bromosuccinimide (157 mg, 0.88 mmol) in the presence
of benzoyl peroxide (16 mg) and the brominated product
was washed with warm water and dissolved in 80 %
aqueous acetic acid (40 mL). The reaction mixture was
refluxed for 24 h and poured into crushed ice. The pre-
cipitates were collected by filtration and compound 26
(107 mg, 82 %) was found after purification by column
chromatography.
1
1320, 1283, 1135, 978, 714 cm-1; H NMR (CDCl3) d
10.54 (1H, s, –CHO), 8.32–8.26 (2H, m, H-5 &H-8),
7.86–7.77 (2H, m, H-6 & H-7), 7.75 (1H, s, H-4), 4.12 (3H,
s, –OCH3), 4.08 (3H, s, –OCH3); MS m/z (rel. int.) 296
(M?, 39), 281 (100), 267 (56), 239 (79), 209 (31), 181 (39),
152 (34), 139 (39), 126 (18), 75 (7).
1,3-Dihydroxy-2-formylanthraquinone
or nordamnacanthal (29)
2-Formyl-3-hydroxy-1-methoxyanthraquinone or damn-
acanthal (26) Pale yellow crystals; mp 211 °C [lit.
211–212 °C, Hirose (1960)]; UV (CHCl3) kmax 389, 289,
254 nm; IR (KBr disc) m 3432 (OH), 2957, 2927, 1670
(C=O, unchelated), 1648 (C=O, chelated), 1566 (C=C,
Demethylation of compound 28 (82 mg, 0.28 mmol) with
anhydrous aluminium chloride (334 mg, 2.24 mmol) gave
29 (52 mg) with 70 % yield.
aromatic), 1344, 1260, 1132, 980, 716 cm-1 1H NMR
;
(CDCl3) d 12.31 (1H, s, 3-OH), 10.49 (1H, s, –CHO),
8.32–8.26 (2H, m, H-5 & H-8), 7.87–7.78 (2H, m, H-6 &
H-7), 7.69 (1H, s, H-4), 4.15 (3H, s, –OCH3); MS m/z (rel.
int.) 282 (M?, 5), 267 (12), 254 (100), 237 (16), 225 (56),
208 (24), 197 (24), 180 (18), 168 (20), 152 (32), 139 (36).
1,3-Dihydroxy-2-formylanthraquinone or nordamnacan-
thal (29) Orange crystals; mp 217–218 °C [lit.
220–221 °C, Hirose (1960)]; UV (CHCl3) kmax 422, 292,
263, 249, 234 nm; IR (KBr disc) m 3436 (OH), 2929, 1630
(C=O), 1331, 1192, 1108, 786, 715 cm-1 1H NMR
;
(CDCl3) d 14.09 (1H, s, 1-OH), 12.72 (1H, s, 3-OH), 10.53
(1H, s, –CHO), 8.36–8.31 (2H, m, H-5 & H-8), 7.88–7.85
(2H, m, H-6 & H-7), 7.37 (1H, s, H-4); MS m/z (rel. int.)
268 (M?, 10), 240 (100), 212 (34), 184 (34), 155 (8), 138
(12), 128 (21), 83 (7), 77 (6).
4-Bromo-2-formyl-3-hydroxy-1-methoxyanthraquinone
(27)
Bromination of compound 23 (227 mg, 0.73 mmol) with
N-bromosuccinimide (247 mg, 1.38 mmol) followed by
123

Med Chem Res (2013) 22:2093–2104
Cytotoxic assay
2103
Antioxidant assays
Cell culture
Ferric thiocyanate (FTC) method
The cancer cell lines were cultured in incubator with a 95 %
humidified atmosphere containing 5 % CO2 at 37 °C. Once
cells reached 80 % confluency, the medium was removed
and 1 mL of trypsin–EDTA (concentrated) was added to
detach the cells from the flask. The cells were collected in a
fresh medium as subculture. This subculturing procedure
was done approximately every 3–4 days at a density of
0.64 9 106 cells/ml in a 25 cm2 flask. Cell cultures were
routinely checked for mycoplasmal contamination.
A screw-cap vial (/ 38 9 75 mm) containing a mixture of
2 mg of sample in 4 mL of 99.5 % ethanol, 4.1 mL of
2.51 % linoleic acid in 99.5 % ethanol, 8.0 mL of 0.02 M
phosphate buffer (pH 7.0) and 3.9 mL of water was placed
in an oven at 40 °C in the dark (Kikuzaki and Nakatani,
1993). 0.1 mL of this mixture in a test tube (/
1.5 9 14.5 cm), 9.7 mL of 75 % (v/v) ethanol, 0.1 mL
30 % ammonium thiocyanate and finally, 0.1 mL of
2 9 10-2 M ferrous chloride in 3.5 % hydrochloric acid
were added to the reaction mixture. Three minutes after the
addition of ferrous chloride, the absorbance was measured
at 500 nm. This step was repeated every 24 h until 1 day
after the control reached its maximum absorbance value.
Assay procedure
Rapidly growing cells were harvested by mild trypsiniza-
tion to detach them from the substratum of the culture flask
and fresh medium was added to prepare suspensions of
single cell. The cells were counted using the improved
Newbauer haemacytometer. 4,000–5,000 cells in 180 lL
media were transferred into each well of 96-well plates.
The microtiter plates were incubated overnight for cell
attachment. Subsequently, each well was added with 20 lL
of sample solution diluted in RPMI 1640 medium. Final
concentrations 0.1–100 lM for compounds were used with
tenfold serial dilution. The DMSO concentration was
maintained at 0.1 %. 20 ll of fresh medium alone was
added to control wells to make the final volume of 200 ll
and medium alone (without cells and sample) was used as
blank. Each concentration of the sample was assayed
in quadruplicate and the plate was incubated for 96 h.
The fraction of surviving cells was determined relative
to the untreated cell population by the colorimetric 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) method (Mosmann, 1983). 50 ll of 2 mg/mL MTT
was added to each well after discarding the supernatant and
the plates were incubated for 4 h at 37 °C in 95 % air and
5 % CO2 for the formation of insoluble purple formazan.
Then, the medium with MTT was aspirated and this was
followed by the addition of 100 lL of DMSO to dissolve
the insoluble formazan. The plates were then shaken for
10 min. The absorbance of the formazan solution was read
at 550 nm and the percentage of cell viability is calculated
using the formula:
Thiobarbituric acid (TBA) method
The samples prepared for FTC method were used for this
assay. To 1 mL of 20 % aqueous trichloroacetic acid and
2 mL of 0.67 % aqueous thiobarbituric acid (Mackeen
et al., 2000), 2 mL of the sample solution in a 10 mL
centrifuge tube was added. The mixture was placed in a
boiling water bath for 10 min. After cooling, it was cen-
trifuged at 3,000 rpm for 30 min. Absorbance of the
supernatant was measured at 532 nm. Antioxidant activity
was based on the absorbance of the final day of FTC assay.
All the assays were carried out in triplicate and the
readings were averaged.
Physicochemical calculations
Evaluation on the lipophilicity, solubility, polar surface
area and other ‘rule-of-five’ parameters on the anthraqui-
nones was performed using the online tools Molsinspira-
tion and ALOGPS2.1 (Tetko et al., 2005; Veber et al.,
2002).
Acknowledgments The authors wish to thank the University Putra
Malaysia and the Ministry of Science, Technology and the Environ-
ment for the fund provided under the Intensified Research in Priority
Areas (IRPA) programme. The corresponding author (NHL) also
thanks the Scientific Chairs Unit, Taibah University for its supports.
% of Viability
References
OD of Sample ꢁ OD of Blank ðmedia onlyÞ
¼
ꢂ 100
Ali AM, Ismail NH, Mackeen MM, Yazan LS, Mohamed SM, Ho
ASH, Lajis NH (2000) Antiviral, cyototoxic and antimicrobial
activities of anthraquinones isolated from the roots of Morinda
elliptica. Pharm Biol 38(4):298–301
Cambie RC, Rutledge PS, Woodgate PD (1992) Synthetic anthracy-
clines from anthraquinones. Aust J Chem 45:483–512
OD of Control ꢁ OD of Blank ðmedia onlyÞ
For analysis of the results, 50 % inhibitory concentration
(IC50) was determined from the dose–response cytotoxic
curves.
123

2104
Med Chem Res (2013) 22:2093–2104
Cardia MC, Begala M, DeLogu A, Maccioni E (2001) Synthesis and
biological evaluation of some differently substituted 9,10-
anthracenediones. Il Farmaco 56(8):549–554
Mosmann T (1983) Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxic assays.
J Immunol Methods 65:55–63
Chang P, Lee KH, Shingu T, Hirayama T, Hall IH (1982) Antitumor
agents. 50. Morindaparvin-A, a new antileukemic anthraquinone,
and alizarin-1-methyl ether from Morinda parvifolia, and the
antileukemic activity of the related derivatives. J Nat Prod
45(2):206–210
Enger TA, Iyenrar R (1998) Lewis acid-directed reactions of quinones
with styrenyl systems: the case of 2-methoxy-3-methyl-1,4-
benzoquinone. J Org Chem 63:1929–1934
Roberts JL, Rutledge PS, Trebilcock MJ (1997) Experiments directed
towards the synthesis of anthracyclinones. I Synthesis of
2-formylmethoxyanthraquinones. Aust J Chem 30:1553–1560
Tetko IV, Gasteiger J, Todeschini R, Mauri A, Livingstone D, Ertl P,
Palyulin VA, Radchenko EV, Zefirov NS, Makarenko AS,
Tanchuk VY, Prokopenko VV (2005) Virtual computational
chemistry laboratory: design and description. J Comput Aided
Mol Des 19(6):453–463
Huang SS, Yeh SF, Hong CY (1995) Effect of anthraquinone
derivatives on lipid peroxidation in rat heart mitochondria:
structure-activity relationship. J Nat Prod 58(9):1365–1371
Ismail NH, Ali AM, Aimi N, Kitajima M, Takayama H, Lajis NH
(1997) Anthraquinones from Morinda elliptica. Phytochemistry
45(8):1723–1725
Veber DF, Johnson SR, Cheng HY, Smith BR, Ward KW, Kopple KD
(2002) Molecular properties that influence the oral bioavailabil-
ity of drug candidates. J Med Chem 45(12):2615–2623
Wei BL, Wu SH, Chung MI, Won SJ, Lin CN (2000) Synthesis and
cytotoxic effect of 1,3-dihydroxy-9,10-anthraquinone deriva-
tives. Eur J Med Chem 35(12):1089–1098
Jin G-Z, You Y-J, Ahn B-Z (2001) Esters of 2-(1-hydroxyalkyl)-1,4-
dihydroxy-9,10-anthraquinoneswithmelphalanas multifunctional
anticancer agents. Bioorg Med Chem Lett 11(11):1473–1476
Johnson MG, Kiyokawa H, Tani S, Koyama J, Morris-Natschke SL,
Mauger A, Bowers-Daines MM, Lange BC, Lee KH (1997)
Antitumor agents-CLXVII. Synthesis and structure-activity
correlations of the cytotoxic anthraquinone 1,4-bis-(2,3-epoxy-
propylamino)-9,10-anthracenedione, and of related compounds.
Bioorg Med Chem 5(8):1469–1479
Werner W, Grafe U, Ihn W, Trrsselt D, Winter S, Paulus E (1997)
Synthesis of tolypocladin and isotolypocladin. Tetrahedron
53(1):109–118
Yen G-C, Duh P-D, Chuang D-Y (2000) Antioxidant activity of
anthraquinones and anthrone. Food Chem 70(4):437–441
Zacharie B, Attardo G, Barriault N, Penney C (1997) Regioselective
synthesis of 6-substituted 2-hydroxybenzaldehyde: efficient
synthesis of the immunomodulator tucaresol and related ana-
logues. J Chem Soc Perkin Trans 1:2925–2929
Kikuzaki H, Nakatani N (1993) Antioxidant effect of some ginger
constituents. J Food Sci 58:1407–1410
Zagotto G, Supino R, Favini E, Moro S, Palumbo M (2000) New 1,4-
anthracene-9,10-dione derivatives as potential anticancer agents.
Farmaco 55(1):1–5
Zhang X, Fox BW, Hadfield JA (1996) Preparation of naturally
occurring anthraquinones. Synth Comm 26(1):49–62
Zhao YH, Abraham MH, Le J, Hersey A, Luscombe CN, Beck G,
Sherborne B, Cooper I (2002) Rate-limited steps of human oral
absorption and QSAR studies. Pharm Res 19(10):1446–1457
Mackeen MM, Ali AM, Lajis NH, Kawazu K, Hassan Z, Amran M,
Habsah M, Mooi LY, Mohamed SM (2000) Antimicrobial,
antioxidant, antitumour-promoting and cytotoxic activities of
different plant part extracts of Garcinia atroviridis griff. ex T.
anders. J Ethnopharmacol 72(3):395–402
Mishra G, Gupta N (1982) Chemical investigation of roots of
Morinda tinctoria Roxb. J Inst Chem 54(1):22–25
123