Total synthesis, cytotoxic effects of damnacanthal, nordamnacanthal and related anthraquinone analogues

Article abstract of DOI:10.3390/molecules180810042

Naturally occurring anthraquinones, damnacanthal (1) and nordamnacanthal (2) were synthesized with modified reaction steps and investigated for their cytotoxicity against the MCF-7 and K-562 cancer cell lines, respectively. Intermediate analogues 2-bromomethyl-1,3-dimethoxyanthraquinone (5, IC ₅₀ = 5.70 ± 0.21 and 8.50 ± 1.18 μg/mL), 2-hydroxymethyl-1,3-dimethoxyanthraquinone (6, IC₅₀ = 12.10 ± 0.14 and 14.00 ± 2.13), 2-formyl-1,3-dimethoxyantharquinone (7, IC ₅₀ = 13.10 ± 1.02 and 14.80 ± 0.74), 1,3-dimethoxy-2-methylanthraquinone (4, IC₅₀ = 9.40 ± 3.51 and 28.40 ± 2.33), and 1,3-dihydroxy-2-methylanthraquinone (3, IC ₅₀ = 25.60 ± 0.42 and 28.40 ± 0.79) also exhibited moderate cytotoxicity against MCF-7 and K-562 cancer cell lines, respectively. Other structurally related compounds like 1,3-dihydroxyanthraquinone (13a, IC₅₀ = 19.70 ± 0.35 and 14.50 ± 1.28), 1,3-dimethoxyanthraquinone (13b, IC₅₀ = 6.50 ± 0.66 and 5.90 ± 0.95) were also showed good cytotoxicity. The target compound damnacanthal (1) was found to be the most cytotoxic against the MCF-7 and K-562 cancer cell lines, with IC₅₀ values of 3.80 ± 0.57 and 5.50 ± 1.26, respectively. The structures of all compounds were elucidated with the help of detailed spectroscopic techniques.

Full text of DOI:10.3390/molecules180810042

Molecules 2013, 18, 10042-10055; doi:10.3390/molecules180810042
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Communication
Total Synthesis, Cytotoxic Effects of Damnacanthal,
Nordamnacanthal and Related Anthraquinone Analogues
Muhammad Nadeem Akhtar 1,2,*, Seema Zareen 1, Swee Keong Yeap 2, Wan Yong Ho 3,
Kong Mun Lo 4, Aurangzeb Hasan 4 and Noorjahan Banu Alitheen 5
1
Faculty of Industrial Sciences & Technology, Universiti Malaysia Pahang,
Lebuhraya Tun Razak 26300, Kuantan Pahang, Malaysia; E-Mail: seema_zareen@yahoo.com
2
Institute of Bioscience, Universiti Putra Malaysia, UPM Serdang 43400, Selangor Darul Ehsan,
Malaysia; E-Mail: skyeap2005@yahoo.com
3
School of Biomedical Sciences, The University of Nottingham Malaysia Campus, Jalan Broga,
Semenyih 43500, Selangor, Malaysia; E-Mail: wanyongho@gmail.com (W.Y.H.)
4
Department of Chemistry, University of Malaya, Kuala Lumpur 50603, Malaysia;
E-Mails: kimlo@um.edu.my (K.M.L.); flavonoids@hotmail.com (A.H.)
5
Faculty of Biotechnology and Bimolecular Sciences, Universiti Putra Malaysia,
UPM Serdang 43400, Selangor Darul Ehsan, Malaysia; E-Mail: noorjahan@biotech.upm.edu.my
* Author to whom correspondence should be addressed; E-Mail: nadeem@ump.edu.my;
Tel.: +60-9-5492393; Fax: +60-9-5492766.
Received: 22 May 2013; in revised form: 29 June 2013 / Accepted: 16 July 2013 /
Published: 20 August 2013
Abstract: Naturally occurring anthraquinones, damnacanthal (1) and nordamnacanthal (2)
were synthesized with modified reaction steps and investigated for their cytotoxicity
against the MCF-7 and K-562 cancer cell lines, respectively. Intermediate analogues
2-bromomethyl-1,3-dimethoxyanthraquinone (5, IC50 = 5.70 ± 0.21 and 8.50 ± 1.18 μg/mL),
2-hydroxymethyl-1,3-dimethoxyanthraquinone (6, IC50 = 12.10 ± 0.14 and 14.00 ± 2.13),
2-formyl-1,3-dimethoxyantharquinone (7, IC50 = 13.10 ± 1.02 and 14.80 ± 0.74),
1,3-dimethoxy-2-methylanthraquinone (4, IC50 = 9.40 ± 3.51 and 28.40 ± 2.33), and
1,3-dihydroxy-2-methylanthraquinone (3, IC50 = 25.60 ± 0.42 and 28.40 ± 0.79)
also exhibited moderate cytotoxicity against MCF-7 and K-562 cancer cell lines,
respectively. Other structurally related compounds like 1,3-dihydroxyanthraquinone (13a,
IC50 = 19.70 ± 0.35 and 14.50 ± 1.28), 1,3-dimethoxyanthraquinone (13b, IC50 = 6.50 ± 0.66
and 5.90 ± 0.95) were also showed good cytotoxicity. The target compound damnacanthal (1)
was found to be the most cytotoxic against the MCF-7 and K-562 cancer cell lines, with

Molecules 2013, 18
10043
IC50 values of 3.80 ± 0.57 and 5.50 ± 1.26, respectively. The structures of all compounds
were elucidated with the help of detailed spectroscopic techniques.
Keywords:
Anthraquinone;
damnacanthal;
nordamnacanthal;
MCF-7;
K-562;
cytotoxic effects
1. Introduction
Anthraquinone compounds, especially anthracyclines, have long been used as effective anticancer
drugs. Depending on their chemical structure, anthraquinone drugs can kill tumor cells by diverse
mechanisms, involving different initial intracellular targets that normally contribute to drug-induced
toxicity [1–3]. Anthraquinones are known as “multipotent antioxidants”, as they are molecules that besides
antioxidant activity possess additional pharmacological activities such as inhibition of platelet-aggregation
or display antineoplastic and anticancer activities [4,5]. Many anthraquinones also display various
biological activities such as antimicrobial, antifungal, hypotensive, analgesic, antimalarial [6–11],
antileukemic, mutagenicity and anti-inflammatory properties [12–14]. Natural anthraquinones from
Damnacanthus subspinosus and Morinda parvifolia have long been used in traditional medicine for the
treatment of cancer [15]. The discovery of new compounds with antitumor activity has become one of
the most important challenges in medicinal chemistry. The detail study on anthraquinones has revealed
that a range of DNA-recognizing molecules that act as antitumor agents, including groove binders,
alkylating and intercalator compounds. DNA intercalators have attracted particular attention because
of their antitumor activity. For example, a number of acridine and anthracycline compounds are
excellent DNA intercalators that are now on the market as chemotherapeutic agents [15,16].
Substituted anthraquinones such as rubiadin, subspinosin and morindaparvin are widely distributed in
nature and are known to display various pharmacological activities [17–21]. Previously, we have
reported the antitumor and anti-oxidant activities of anthraquinones isolated from Morinda elliptica [22].
Recently, we have also reported the cytotoxic and immunomodulatory effects of damnacanthal and
nordamnacanthal against different cell lines [23,24]. Damnacanthal and nordamnacanthal were
originally isolated from Damnacanthus major [25]. Previous studies on the synthesis of damnacanthal
and nordmnacanthal were reported by Hirose, Roberts and Saha [26–28]. The current study describes
the total synthesis of damnacanthal (1) and nordamnacanthal (2) with modified reaction steps, their
cytotoxic activities of against MCF-7 and K-562 cancer cell lines and their structure activity
relationships (SARs).
2. Results and Discussion
Anthraquinone skeletons are generally synthesized by Friedel-Crafts acylation condensation
between phthalic anhydride and benzene derivatives [29]. 1,3-Dihydroxy-2-methylanthraquinone (3) was
used as the common precursor for the synthesis of damnacanthal (1) and nordamnacanthal (2). This
was synthesized by mixing of phthalic anhydride and 1,3-dihydroxy-2-methylbenzene in a molten
mixture of AlCl3/NaCl [29,30].

Molecules 2013, 18
10044
The synthesis of nordamnacanthal (2) was accomplished by first acetylating the precursor compound 3
with acetic anhydride and potassium carbonate to afford the monoacetylated intermediate 8.
Upon methylation of compound 8 with K2CO3/(CH3)2SO4 in dry acetone to afforded 1,3-dimethoxy-2-
methylathraquinone (4), which was then brominated with Wohl-Ziegler’s reagent (N-bromosuccinimide) in
dry CCl4 to yield 2-bromomethyl-1,3-dimethoxyathraquinone (5) [25,31,32] and structure was
confirmed by single X-ray diffraction (Figure 1). It is noteworthy that the use of a catalytic amount of
benzoyl peroxide in this reaction gave 2-dibromomethyl-1,3-dimethoxyanthraquinone [26]. Compound
5 was hydrolyzed by refluxing it in acetic acid-water (8:2) to give the desired 2-hydroxymethyl-1,3-
dimethoxyanthraqunone (6) in quantitative yield [26,31]. Compound 6, which contains a
hydroxymethyl moiety, was converted into the corresponding aldehyde 7 in 92.3% yield using a mild
oxidizing agent [pyridinium chlorochromate (PCC) in dry CH2Cl2 at 20–25 °C]. The use of an excess
amount (1.5 equiv.) of PCC also gave the undesired 1-hydoxy-3-methoxyanthraquinone-2-carboxylic
acid. Upon treating compound 7 with AlCl3/CH2Cl2, nordamnacanthal (2) was obtained in 28% yield.
The detailed reaction conditions are shown in Scheme 1.
The synthesis of damnacanthal (1) was accomplished by first acetylated the precursor compound 3
with (CH3)2SO4 and potassium carbonate to afforded monoacetylated derivative 8. Upon methylation of
compound 8 with K2CO3/CH3I compound 9 was obtained, which then brominated with NBS to yield
1-methoxy-3-aectoxy-2-bromomethyl-1-methoxyathraquinone (10) [25,32]. The bromo derivative 10
was converted into ethoxymethyl derivative 11 by dissolving it in a mixture of aq. NaOH/ethanol and
followed by reflux in acidic media. Compound 11 was acetylated using acetic anhydride and K2CO3 to
afforded 12, which on oxidation with PCC in CH2Cl2 to give damnacanthal (1) in good yield. Thus the
synthesis of damnacanthal (1) was achieved through modified steps as shown in Scheme 2.
Figure 1. The ORTEP diagram of 2-bromomethyl-1,3-dimethoxy-9,10-athraquinone (5).

Molecules 2013, 18
Scheme 1. Reactions pathway for synthesis of nordamnacanthal (2).
10045
Reagents and conditions: (a) anhydrous AlCl3, NaCl, 165–175 °C, 1 h; (b) Anhydrous K2CO3 (CH3)2SO4, dry
acetone, refluxed, 22 h; (c) NBS/CCl4 refluxed for 30 h; (d) 80% acetic acid refluxed 24 h; (e) PCC, CH2Cl2
stir. 2–4 h. rt; (f) AlCl3 /CH2Cl2, HCl.
Scheme 2. Reactions pathway for synthesis of damnacanthal (1).
Reagents and conditions: (a) anhydrous K2CO3 /Ac2O, acetone; (b) anhydrous K2CO3/(CH3)2SO4 acetone,
refluxed 22 h; (c) NBS/CCl4 refluxed 30 h; (d) 10% aq. NaOH/EtOH, aq. HCl; (e) Ac2O/ K2CO3 stirred for
24 h; (f) PCC, CH2Cl2 stir 2–4 h. rt.
For the SARs additional anthraquinone analogous 13a–h were synthesized by Friedel-Crafts
acylation of phthalic anhydride with resorcinol or catechol to yielded respective anthraquinones [29,30] as

Molecules 2013, 18
10046
shown in Figure 2. All compounds showed significant in vitro cytotoxicity against two cancer cell
lines, indicating that anthraquinone is an interesting class of compounds for cancer therapy. The
compound damnacanthal (1) contains methoxy, formyl and hydroxyl groups at the 1, 2 and 3 positions
that might be important for the cytotoxicity since the standard drug doxorubicin [33] also possesses an
anthraquinone moiety. However, nordamnacanthal (2), 3, 8, 13a, 13c, 13f and 13h exhibited less
cytotoxic effects on both the MCF-7 and K-562 cell lines, suggesting that a protected OH group at
position 1 and 3 increase the solubility and might make it easy for the compounds to diffuse across the
cellular membrane as investigated by a theoretical study of 188 drug-like compounds by MI-QSAR
analysis [34,35], while for poorly soluble drugs dissolution could be the rate limiting step in the
absorption process. The methylated compound 13b (IC50 = 24.25 ± 2.46 μM and 22.01 ± 3.54 μM)
showed the stronger cytotoxicity than 13a (IC50 = 82.08 ± 1.46 and 60.42 ± 5.33 μM) and 13c
(IC50 = 87.80 ± 2.01 and 64.96 ± 1.57μM). Other derivatives 13e (IC50 = 43.64 ± 2.12 and
46.61 ± 12.37) and 2-hydroxymethylanthraquinone (13d) (IC50 = 49.58 ± 12.61and 50.84±14.12)
showed poor cytotoxicity as shown in Table 1. However, previously synthesized compounds 13f–h
with mono or dihydroxyl groups at the 1 and 2 positions exhibited moderate cytotoxic activity [36].
Further derivatives as exemplified by compounds 13c–h indicated that this series of compounds only
exhibited moderate activities not exceeding that of damnacanthal (1), suggesting that functional groups
such as formyl at C-2, methoxy at C-1, hydroxyl groups at C-3 (damnacanthal) and bromomethyl at
C-2 are important pharmacophores for the anticancer activity. However, the active compound 5
((IC50 = 15.83 ± 0.58 and 23.61 ± 3.28 μM) might involve another mechanism like perfusion. The overall
cytotoxic compounds (1, 5, 13b) showed more selectivity towards cancer cell lines because of their side
chains and might fulfill the Lipinski rule-of-5 and drug-like properties.
Figure 2. Structure of compounds 13a to 13h.

Molecules 2013, 18
Table 1. IC50 values of compounds 1 to 13h against MCF-7 and K-562 cell lines.
10047
IC50 value (μM)
Compound No.
1
Chemical Structure
MCF-7
K562
13.48 ± 2.02
19.50 ± 4.47
2
3
4
60.07 ± 6.46
90.78 ± 1.49
37.01 ± 13.82
32.46 ± 8.73
100.71 ± 2.80
87.81 ± 9.17
O
O
OMe
Br
OMe
5
15.83 ± 0.58
23.61 ± 3.28
6
7
43.54 ± 0.48
43.96 ± 3.42
47.62 ± 7.24
49.66 ± 2.48
8
9
28.38 ± 7.94
59.35 ± 8.23
39.69 ± 6.24
69.59 ± 1.62
46.77 ± 3.81
54.90 ± 7.11
10

Molecules 2013, 18
10048
Table 1. Cont.
IC50 value (μM)
Compound No.
11
Chemical Structure
MCF-7
K562
O
OMe
OEt
59.13 ± 11.59
42.06 ± 6.39
OH
O
12
43.62 ± 5.35
89.36 ± 3.83
13a
13b
82.08 ± 1.46
24.25 ± 2.46
60.42 ± 5.33
22.01 ± 3.54
64.96 ± 1.57
13c
13d
13e
87.80 ± 2.01
49.58 ± 12.61
43.64 ± 2.12
50.84 ± 14.12
46.61 ± 12.37
13f
13g
13h
45.98 ± 2.95
63.84 ± 12.46
52.50 ± 2.63
107.14 ± 0.54
70.54 ± 1.56
94.17 ± 1.88
Doxorubicin
0.94 ± 0.20
0.24 ± 0.07
3. Experimental
3.1. General
Melting points were determined on a XSP-12 500X hot-stage melting point apparatus, and are
uncorrected. UV spectra were recorded on a CARY 100 Conc. UV-visible spectrophotometer in
MeOH. IR spectra were recorded on a Perkin–Elmer RXI Fourier Transform Infrared spectrometer
(FTIR) as KBr discs. Mass spectra were measured on a Finnigan Mat SSQ 710 Spectrometer with
ionization induced by electron impact at 70 eV. NMR spectra were recorded in CDCl3 and acetone-d6
using a Varian 500 MHz NMR spectrometer. Column chromatography was performed on silica gel (60
Merck 9385, 230–400 mesh, ASTM). The molecular structure of the intermediate 1,3-dimethoxy-2-

Molecules 2013, 18
10049
bromomethylanthraquinone (5) was determined by X-ray crystallography. The data were collected at
100K using a Bruker APEXII with a CCD area-detector X-ray diffractometer. The structure was
solved by direct method with SHELXS97 program and refined on F2 by full-matrix least-squares
methods with anisotropic non-hydrogen atoms. The compound crystallizes in the monoclinic P2(1)/c
space group with the crystallographic detail given in Table 2. The molecule exists as a planar molecule
with r.m.s deviation of 0.018Å (Figure 1).
Table 2. Crystal data and structure refinement for 2-bromomethyl-1,3-dimethoxyanthraquinone (5).
Empirical formula
Formula weight
Temperature
C17H13O4Br
361.18
100(2) K
Wavelength
0.71073 Å
Crystal system
Space group
Monoclinic
P2(1)/c
Unit cell dimensions
a = 4.7401(8)Å, α = 90°
b = 17.364(3)Å, β = 94.543(2)°
c = 16.825(3)Å, γ = 90°
1380.5(4) Å3
Volume
Z
4
Calculated density
Absorption coefficient
F(000)
1.738 Mgm−3
2.994 mm−1
728
Crystal size
0.20 × 0.10 × 0.10 mm
1.69–28.26°
θ range for data collection
Limiting indices
−6 ≤ h ≤6, −14 ≤ k ≤23, −22 ≤ l ≤ 16
3404
Reflections collected
Independent reflections
Completeness to θ = 26.00º
Absorption correction
Max. and min. transmission
Refinement method
Data / restraints / parameters
Goodness-of-fit on F2
Final R indices [I>2σ(I)]
R indices (all data)
Largest difference peak and hole
2522 [R (int) = 0.0494]
99.40%
Multi-scan
0.5973 and 0.7457
Full-matrix least-squares on F2
3404/0/201
1.038
R1 = 0.0417, wR2 = 0.0984
R1 = 0.0633, wR2 = 0.1124
0.521 and −0.782 e.Å−3
3.2. General Procedure for Anthraquinone Synthesis
A mixture of anhydrous aluminium chloride (30 g, 225.0 mmol) and sodium chloride (12 g,
205.0 mmole) were melted at 125–130 °C. Phthalic anhydride (6.7 g, 45.0 mmole) and 2-methyl
resorcinol (40.5 mmole) were mixed well and added slowly into the molten mixture of aluminum
chloride and sodium chloride. The reaction temperature was raised till to 165–175 °C and maintained
for 45–60 min. Another portion of anhydrous aluminum chloride (30 g, 225.0 mmole) was added into
the hot mixture of the reactants [28]. After being cooled, the deep red solid product was decomposed

Molecules 2013, 18
10050
by adding a mixture of ice water (250 mL) and conc. hydrochloric acid (250 mL). The crude mixture
was dissolved in distilled water and organic layer was extracted with ethyl acetate, washed with brine
and dried over anhydrous sodium sulfate. The crude products were purified by flash silica gel column
chromatography with elution of the ethyl acetate /hexane as yellow needles.
1,3-Dihydroxy-2-methylanthraquinone (3). Yellow needles: 65% yield: m.p. 280–282 °C; {lit. [27]
280–283 °C}. UV (nm) in MeOH: 408, 279, 251. IR (cm−1): 3402 (OH), 2930, (C-H), 1661 (C=O
non-chelated), 1628, 1591 (C=C< aromatic), 1338, 1310, 1122, 712. 1H-NMR (acetone-d6): δ 13.22 (s,
1H, 1-OH), 8.13 (dd, 1H, J = 1.5, 7.5 Hz, H-8), 8.23 (dd, 1H, J = 1.5, 7.5 Hz, H-5), 7.95–7.90 (m, 2H,
H-6 & H-7), 7.37 (1H, s, H-4), 2.19 (3H, s, CH3). MS m/z (rel.int.): 254 (M+, 100), 226, 152 (10),
128 (21), 105 (11).
2-Bromomethyl-1,3-dimethoxyanthraqinone (5). To a solution of compound 4 (400 mg. 1.4 mmole) in
dry CCl4 (40 mL), N-bromosuccinimide (500 mg, 3.0 mmole) was added portionwise. The whole
mixture was stirred for 30 h. Compound 5 was extracted with ethyl acetate, filtered and purified by
passing through a short column containing anhydrous sodium sulfate. The compound was recrystallized
in a mixture of EtOAc and drops of MeOH as yellow crystals. Yield 90%; m.p.: 154–155 °C; {lit [27]
159–160 °C}. IR (cm−1): 2938 (CH), 1670 (C=O non-chelated), 1589 (C=C< aromatic), 1330, 1288,
1
1231, 1161, 730. H-NMR (CDCl3): δ 8.30 (d, 1H, J = 8.0 HZ, H-8), 8.25 (d, 1H, J = 8.0 Hz, H-5),
7.80–7.75 (m, 2H, H-6 & H-7), 7.26 (s, 1H, H-4), 4.76 (s, 2H, -CH2Br), 4.05 (s, 3H, OCH3), 3.97 (s,
3H, OCH3). MS m/z (rel.int.): 361 (M++H, 38) 362 (M++2, 37), 281 (100), 267 (35), 253 (36),
236 (42), 221 (7), 105 (11), 223 (41), 77 (21).
1,3-Dihydroxy-2-formylanthraquinone (nordamnacanthal) (2). Compound 7 (150 mg, 0.5 mmole) was
suspended in dry CH2Cl2 (10 mL), stirred for 10 min. at rt and anhydrous AlCl3 (500 mg, 3.7 mmole)
was added and stirred for 1h and then reflux for 4–6 h. The reaction mixture was cooled and added
mixture of ice HCl (250 mL) and water (250 mL). The products was extracted by EtOAc and purified
by CC and followed by recrystallized from a mixture of MeOH/EtOAc as orange crystals. Yield 28%;
m.p.: 217–218 °C; {lit. [25] 220–221 °C}. UV (nm) in CHCl3: 422, 292, 263, 250. IR (cm−1): 3436
1
(OH), 2928, 2927, (CH), 1630 (C=O), 1560 (>C=C< aromatic), 1331, 1108, 786, 715. H-NMR
(MeOD): δ 10.34 (s, 1H, CHO), 8.29 (d, 1H, J = 7.0 Hz, H-8), 8.25 (d, 1H, J = 7.0 Hz, H-5), 7.85–7.82
(m, 2H, H-6 & H-7), 7.29 (s, 1H, H-4). MS m/z (rel.int.): 268 (M+, 65) (10), 281 (100), 249 (100),
212 (34), 184 (32), 1197(24), 155 (7), 138 (12), 128 (21), 77 (12).
2-Formyl-3-hydroxy-1-methoxyanthraquinone (1). Compound 12 (200 mg 0.56 mmole) was dissolved
in dry dichloromethane (30 mL) and PCC (125 × 2 mg, 1.39 mmole) was introduced slowly in a stirred
solution. The reaction mixture was stirred at room temperature for 4–6 h. A black brown solid was
obtained, dissolved in aqueous solution and extracted with EtOAc, solvent removed in vacuo on a
rotary evaporator and dried over anhydrous sodium sulfate. The product was purified by column
chromatography using EtOAc/hexane as solvent system. The product was obtained as orange crystals.
Yield 64.5%; m.p.: 209–211 °C; (lit. (25) 211–212 °C. IR (cm−1): 3434 (OH), 2957, 2927, (CH), 1670
(non-chelated C=O), 1648 (C=O non-chelated), 1566 (C=C< aromatic), 1344, 1260, 1231, 1132, 7310.
1
UV (nm) in CHCl3: 412, 284, 264, 254. H-NMR (acetone-d6): δ 10.45 (s , 1H, CHO), 8.32 (dd, 1H,

Molecules 2013, 18
10051
J = 1.5, 7.5 Hz, H-8), 8.26 (dd, 1H, J = 1.5, 7.5 Hz, H-5), 7.87–7.78 (m, 2H, H-6 & H-7) 7.69 (s, 1H,
H-4), 4.11 (s, 3H, OCH3). MS m/z (rel.int.): 282 (M+, 100) 267 (35), 254 (100), 237 (16), 225 (7),
197 (24), 180 (31), 168 (6) 152 (9), 139 (41).
3.3. X-ray Crystallography of Compound 5
A single crystal of compound 5 was obtained by slow evaporation at room temperature from a
mixture of ethyl acetate-MeOH (drops). The crystal structure was solved by direct method with
SHELXS97 programe and refined on F2 by full-matrix least-squares methods with anisotropic
non-hydrogen bond atoms. The compound crystallized as a monoclinic P2(1)/c space group with the
crystallographic data is given in Table 2. The molecule exists as a planar molecule with r.m.s deviation
of 0.018 Å (Figure 1). There is no evidence of any hydrogen bonding interaction or π–π stacking
present in the crystal packing of the compound. Full crystallographic data for structures 5 has been
deposited at the Cambridge Crystallographic Data Center (CCDC) as supplementary publication
number CCDC870566. Copies of the data can be obtained, free of charge, on application to CCDC
12 Union Road, Cambridge CB2 1EZ, UK [Fax: þ44 1223 336033. email: deposit@ccdc.cam.ac.uk or
at www.ccdc.cam.ac.uk].
4. Cytotoxicity Assay
4.1. Preparation of Compounds
The compounds were dissolved in dimethylsulfoxide (DMSO, Sigma, St. Louis., MO, USA) to get
a stock solution of 1 mg/mL and stored at 4 °C.
4.2. Preparation of Cell Lines
Adherent human estrogen dependent breast carcinoma MCF-7 and suspension human chronic
myelogenic leukemia K-562 cell lines were obtained from ATCC, USA. Both MCF-7 and K562 were
cultured in RPMI-1640 supplemented with 10% FBS and incubated at 37 °C, 5% CO2 and 90%
humidity throughout the study. Cell viability was assessed by trypan blue exclusion method where
both MCF-7 and K562 cell lines were collected, washed with PBS and mixed with 1:1 ratio of trypan
blue dye and the cell number and viability were evaluated using haemacytometer under inverted light
microscope. Only cell with viability higher than 95% will be subjected to MTT cell viability assay.
4.3. Cell Viability Assay
The effect of all compounds on cell viability of MCF-7 and K-562 cell lines were determined using
the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [37].
MCF-7 cell were seeded on a 96 well microtiter plate with each well containing 200 µL of culture
media with 10% of FBS per well at a density of 1 × 105 cells/mL and incubated overnight at 37 °C,
5% CO2 at humidified atmosphere of 95% air. When cells were reached 70% confluence then
subjected to the cell viability assay. Medium containing the seeded MCF-7 cell was discarded.
Treatment of both cultures were carried out by first adding 100 µL of RPMI-1640 medium

Molecules 2013, 18
10052
supplemented with 10% of FBS into all wells except row A of the 96 well plates (TPP, Zollstrasse,
Trasadingen, Switzerland). Then, 100 µL of each diluted compound (60 µg/mL) was added into row A
and row B. A series of two-fold dilutions of compounds were carried out from row B to row G to give
concentrations of 30, 15, 7.5, 3.75, 1.375 and 0.687 µg/mL, while row H was left as untreated control
cell. For K-562 culture plate, 100 µL of cells (2 × 105 cells/mL) was added into all wells. For MCF-7
culture plate, 100 µL of RPMI-1640 medium was added into all wells. The plates were incubated in
37 °C, 5% CO2 and 90% humidity incubator for 72 h. After incubation period, 20 µL of MTT (Sigma,
5 mg/mL) was added into all wells and the plates were further incubated for 4 h. Then, 170 µL of
supernatant was aspirated from every well plate. The plates containing K-562 culture were centrifuged
at 200 × g for 5 min prior to medium aspiration. The resulting formazan crystals in each well were
solubilized by 100 µL of DMSO (Fisher Scientific, Waltham, MA, USA) followed by incubation for
20 min. Finally, the plates were read at 570 nm and 630 nm as reference wavelength by using µQuant
ELISA Reader (Bio-tech Instruments, Winooski, VT, USA). The results of the compound-treated cells
were compared with the standard doxorubicin. Each compound and control was assayed in triplicates
in three independent experiments. The percentage of inhibitions were calculated by using Graph pad
and expressed in µg/mL or µM and listed in Table 1.
5. Conclusions
The synthesis of damnacanthal (1) and nordamnacanthal (2) was achieved. The key difference from
the previously synthesis was the conversion of hydroxymethylanthraquinones 7 or 12 into the
corresponding aldehyde, which was achieved by the use of optimized mild conditions with PCC as
oxidant. The synthesis in good yield of 2-ethoxymethyl-3-hydroxy-1-methoxyanthraquinone (11) was
achieved through a short sequence. The structure activity relationship suggested that methoxy, formyl,
bromomethyl and hydroxyl groups are important for the cytotoxicity and selectivity in these
substituted anthraquinones. These side chain modifications give an idea for further efforts to increase
the therapeutic potential of this class of compounds.
Acknowledgments
This project was supported by the Universiti Putra Malaysia, (RUGS research grant no. 05-03-10-0967
RU) and University Malaysia Pahang (internal grant No. RDU 120373). For EI-MS analysis HEJ
Research Institute of Chemistry, Karachi, Pakistan is gratefully acknowledged.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Murphy, G.P.; Lawrence, W.J.; Lenhard, R.E. American Society Textbook of Clinical Oncology,
2nd ed.; American Cancer Society: Atlanta, GA, USA, 1995.

Molecules 2013, 18
10053
2. Doroshow, J.H. Anthracyclines and anthracenediones. In Cancer Chemotherapy and Biotherapy:
Principles and Practice, 2nd ed.; Chabner, B.A., Longo, D.L., Eds.; Lippincott-Raven Publishers:
Philadelphia, PA, USA, 1996; pp. 409–434.
3. Faulds, D.; Balfour, J.A.; Chrisp, P.; Langtry, H.D. Mitoxantrone, a review of its pharmacodynamic
and pharmacokinetic properties, and therapeutic potential in the chemotherapy of cancer. Drugs 1991,
3, 400–449.
4. Kosalec, I.; Kremer, D.; Locatelli, M.; Epifano, F.; Genovese, S.; Carlucci, G.; Randic, M.;
Koncic, Z.M. Anthraquinone profile, antioxidant and antimicrobial activity of bark extracts of
Rhamnus alaternus, R. fallax, R. intermedia and R. pumila. Food Chem. 2013, 136, 335–341.
5. Shi, Y.Q.; Fukai, T.; Sakagami, H.; Kuroda, J.; Miyaoka, R.; Tamura, M.; Yoshida, N.; Nomura,
T. Cytotoxic and DNA damage-inducing activities of low molecular weight phenols from
rhubarb. Anticancer Res. 2001, 21, 2847–2853.
6. Sittie, A.A.; Lemmich, E.; Olsen, C.E.; Hviid, L.; Kharazmi, A.; Nkrumah, F.K.; Christensen, S.B.
Structure-activity studies: in vitro antileishmanial and antimalarial activities of anthraquinones from
Morinda lucida. Planta Med. 1999, 65, 259–261.
7. Rath, G.; Ndonzao, M.; Hostettmann, K. Antifungal anthraquinones from Morinda lucida. Int. J.
Pharmacogol. 1995, 33, 107–114.
8. Younos, C.; Rolland, A.; Fleurentin, J.; Lanhers, M.; Misslin, R.; Mortier, F. Analgesic and
behavioral effects of Morinda citrifolia. Planta Med. 1990, 56, 430–434.
9. Adwankar, M.K.; Chitnis, M.P. In vivo Anti-Cancer Activity of RC-18. Chemotherapy 1982, 28,
291–293.
10. Koumaglo, K.; Gbeassor, M.; Nikabu, O.; de Souza, C.; Werner, W. Effects of three compounds
extracted from Morinda lucida on Plasmodium falciparum. Planta Med. 1992, 58, 533–434.
11. Tripathi, Y.B.; Sharma, M.; Manickam, M. Rubiadin, a new antioxidant from Rubia cordifolia
Indian J. Biochem. Biophys. 1997, 34, 302–306.
12. Chang, P.; Chen, C. Isolation and Characterization of Antitumor Anthraquinones from Morinda
umbellate. Chin. Pharm. J. (Taipei) 1995, 47, 347–353.
13. Ismail, N.H.; Ali, A.M.; Aimi, N.; Kitajima, M.; Takayama, H.; Lajis, N.H. Anthraquinones from
Morinda elliptica. Phytochemistry 1997, 45, 1723–1725.
14. Di Marco, A.; Gaetani, M.; Orezzi, P.; Scarpinato, B. M.; Silvestrini, R.; Soldati, M.; Dasdia, T.;
Valentini, L. Daunomycin, A New Antibiotic of the Rhodomycin Group. Nature 1964, 201, 706–707.
15. Kim, S.J.; Kim, M.C.; Lee, B.J.; Park, D.H.; Hong. S.H.; Um, J.Y. Anti-Inflammatory Activity of
Chrysophanol through the Suppression of NF-κB/Caspase-1 Activation in Vitro and in Vivo.
Molecules 2010, 15, 6436–6451.
16. Preobrazhenskaya, M.N.; Shchekotikhin, A.E.; Shtil, A.A.; Huang, H.S. Antitumor Anthraquinone
Analogues for Multidrug Resistant Tumor Cells. J. Med. Sci. 2006, 26, 1–4.
17. Teng, C.H.; Won, S.J.; Lin, C.N. Design, synthesis and cytotoxic effect of hydroxy- and
3-alkylaminopropoxy-9,10-anthraquinone derivatives. Bioorg. Med. Chem. 2005, 13, 3439–3435.
18. Jin, G.Z.; Song, G.Y.; Zheng, X.G.; Kim, Y.; Sok, D.E.; Ahn, B.Z. 2-(1-Oxyalkyl)-1,4-dioxy-
9,10-anthraquinone: synthesis and Evaluation of Antitumor Activity. Arch. Pharm. Res. 1998, 21,
198–206.

Molecules 2013, 18
10054
19. Cairns, D.; Michalitsi, E.; Jenkins, T.C.; Mackay, S.P. Molecular Modelling and Cytotoxicity of
Substituted Anthraquinones as Inhibitors of Human Telomerase. Bioorg. Med. Chem. 2002, 10,
803–817.
20. Murdock, K.C.; Child, R.C.; Fabio, P.F.; Angier, R.B.; Wallace, R.E.; Durr, F.E.; Chella, R.V.
Antitumor Agents.1.1,4-Bis[(Aminoalkyl)amino]-9,19-Anthracenediones. J. Med. Chem. 1979, 22,
1024–1030.
21. Chang, P.; Lee, K.H.; Shingu, T.; Hirayama, T.; Hall, I.H.; Huang, H.C. 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. 1982,
45, 206–210.
22. Ali, A.M.; Ismail, N.H.; Mackeen, M.M.; Yazan, L.S.; Mohammad, S.M.; Ho, A.S.H.; Lajis, N.H.
Antiviral, Cytotoxic and Antimicrobial Activities of Anthraquinones isolated from the roots of
Morinda elliptca. Pharm. Biol. 2000, 38, 298–301.
23. Alitheen, N.B.; Mashitoh, A.R.; Yeap, S.K.; Shuhaimi, M.; Manaf A.A.; Lajis, N.H. Cytotoxic
effect of damnacanthal, nordamnacanthal, zerumbone and betulinic acid isolated from Malaysian
plant sources. Int. Food Res. J. 2010, 17, 711–719.
24. Noorjahan, B.M.A.; Ali, A.M.; Yeap, S.K.; Suhaimi, M.; Lajis, N.H.; Mashitoh, A.R.; Ho, W.Y.;
Ismail, N.H. Cytotoxicity and Immunomodulatory Effects of Damnacanthal and Nordamnacanthal
Isolated from Roots of Morinda elliptica. J. Agrobiotec. 2010, 1, 29–42.
25. Hirose, Y. Syntheses of Damnacanthal, Damnacanthol, Norjuzunal and Norjuzunol, the Coloring
Matters of Damnacanthus Spp. Chem. Pharm. Bull. 1960, 8, 417–426.
26. Roberts, J.L.; Rutledge, P.S.; Trebilcock, M.J. Experiments Directed Towards the Synthesis
of Anthracyclinones.1 Synthesis of 2-formylmethoxyanthraquinones. Aust. J. Chem. 1977, 30,
1553–1560.
27. Roberts, J.L.; Rutledge, P.S. Experiments Directed Towards the Synthesis of Anthracyclinones.
111 P-Formylation of Hydroxyanthraquinonesby the Claisen Rearrangement. Aust. J. Chem.
1977, 30, 1743–1760.
28. Saha,K.; Lam, K.W.; Abas, F.; Hamzah, A.S.; Stanslas, J.; Hui, L.S.; Lajis, N.H. Synthesis of
damnacanthal, a naturally occurring 9,10-anthraquinone and its analogues, and its biological
evaluation against five cancer cell lines. Med. Chem. Res. 2013, 22, 2093–2104.
29. Wei, B.L.; Wu, S.H.; Chung, M.I.; Won, S.J.; Lin, C.N. Synthesis and cytotoxic effect of
1,3-dihydroxy-9,10-anthrquinone derivatives. Eur. J. Med. Chem. 2000, 35, 1089–1098.
30. Horii, Z.; Hakusui, H.; Momose, T. Synthetic Studies on Anthracyclinones. VII. Synthesis of
Bisanhydroaklavinone. Chem. Pharm. Bull. 1968, 16, 1262–1265.
31. Castonguay, A.; Brassard, P. Total synthesis of (+)-averufin and (+)-bipolarin. Can. J. Chem.
1977, 55, 1324–1332.
32. Ma, H.M.; Liu, Z.Z.; Chen, S.Z. Regioselective Bromination of 3, 4-Dimethoxytoluene with
N-Bromosuccinimide. Chin. Chem. lett. 2003, 14, 371–374.
33. Abraham, S.A.; Waterhouse, D.N.; Mayer, L.D.; Cullis, P.R.; Madden, T.D.; Bally, M.B. The
liposomal formulation of doxorubicin. Meth. Enzymol. 2005, 391, 71–97.

Molecules 2013, 18
10055
34. Iyer, M., Tseng, Y.J., Senese, C.L., Liu, J.; Hopfinger, A.J. Prediction and Mechanistic
Interpretation of Human Oral Drug Absorption Using MI-QSAR Analysis. Mol. Pharm. 2007, 4,
218–231.
35. Zhao, Y.H.; Le, J.; Abraham, M.H.; Hersey, A.; Eddershaw, P.J.; Luscombe, C.N.; Butina, D.;
Beck, G.; Sherborne, B.; Cooper, I.; Platts, J.A. Evaluation of human intestinal absorption data for
use in QSAR studies and quantitative relationship obtained with the Abraham descriptors.
J. Pharm. Sci. 2001, 90, 749–784.
36. Saha, K.; Lajis, N.H.; Abas, F.; Naji, N.A.; Hamzah, A.S.; Shaari, K. Rearrangement in Lewis
Acid-Catalyzed Friedel-Crafts Conditions: Evidence of Competitive Initial Protonation and
Acylation. Aust. J. Chem. 2008, 61, 821–825
37. Mossman, T. Rapid calorimetric assay for cellular growth and survival: Application to
proliferation and cytotoxicity assays. J. Immunol. Meth. 1983, 65, 55–63.
Sample Availability: Samples of the compounds 1–13h are available from the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).