Synthesis of new cytotoxic aminoanthraquinone derivatives via nucleophilic substitution reactions

Article abstract of DOI:10.3390/molecules18078046

Aminoanthraquinones were successfully synthesized via two reaction steps. 1,4-Dihydroxyanthraquinone (1) was first subjected to methylation, reduction and acylation to give an excellent yield of anthracene-1,4-dione (3), 1,4-dimethoxyanthracene- 9,10-dione (5) and 9,10-dioxo-9,10-dihydroanthracene-1, 4-diyl diacetate (7). Treatment of 1, 3, 5 and 7 with BuNH2 in the presence of PhI(OAc)2 as catalyst produced seven aminoanthraquinone derivatives 1a, b, 3a, and 5a-d. Amination of 3 and 5 afforded three new aminoanthraquinones, namely 2-(butylamino)anthracene-1,4-dione (3a), 2-(butylamino)anthracene-9,10-dione (5a) and 2,3-(dibutylamino)anthracene-9,10-dione (5b). All newly synthesised aminoanthraquinones were examined for their cytotoxic activity against MCF-7 (estrogen receptor positive human breast) and Hep-G2 (human hepatocellular liver carcinoma) cancer cells using MTT assay. Aminoanthraquinones 3a, 5a and 5b exhibited strong cytotoxicity towards both cancer cell lines (IC50 1.1-13.0 μg/mL).

Full text of DOI:10.3390/molecules18078046

Molecules 2013, 18, 8046-8062; doi:10.3390/molecules18078046
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of New Cytotoxic Aminoanthraquinone Derivatives
via Nucleophilic Substitution Reactions
Siti Mariam Mohd Nor 1,*, Mohd Aspollah Hj Md Sukari 1, Saripah Salbiah Syed Abdul Azziz 2,
Wong Chee Fah 3, Hasimah Alimon 3 and Siti Fadilah Juhan 1
1
Department of Chemistry, Faculty of Science, Universiti Putra Malaysia, 43400 UPM Serdang,
Selangor, Malaysia
2
Department of Chemistry, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris,
35900 Tanjong Malim, Perak, Malaysia
3
Department of Biology, Faculty of Science and Mathematics, Universiti Pendidikan Sultan Idris,
35900 Tanjong Malim, Perak, Malaysia
* Author to whom correspondence should be addressed; E-Mail: smariam@upm.edu.my;
Tel.: +603-8946-6780; Fax: +603-8943-5380.
Received: 14 May 2013; in revised form: 21 June 2013 / Accepted: 25 June 2013 /
Published: 8 July 2013
Abstract: Aminoanthraquinones were successfully synthesized via two reaction steps.
1,4-Dihydroxyanthraquinone (1) was first subjected to methylation, reduction and
acylation to give an excellent yield of anthracene-1,4-dione (3), 1,4-dimethoxyanthracene-
9,10-dione (5) and 9,10-dioxo-9,10-dihydroanthracene-1,4-diyl diacetate (7). Treatment
of 1, 3, 5 and 7 with BuNH2 in the presence of PhI(OAc)2 as catalyst produced seven
aminoanthraquinone derivatives 1a, b, 3a, and 5a–d. Amination of 3 and 5 afforded
three new aminoanthraquinones, namely 2-(butylamino)anthracene-1,4-dione (3a),
2-(butylamino)anthracene-9,10-dione (5a) and 2,3-(dibutylamino)anthracene-9,10-dione
(5b). All newly synthesised aminoanthraquinones were examined for their cytotoxic
activity against MCF-7 (estrogen receptor positive human breast) and Hep-G2 (human
hepatocellular liver carcinoma) cancer cells using MTT assay. Aminoanthraquinones 3a, 5a
and 5b exhibited strong cytotoxicity towards both cancer cell lines (IC50 1.1–13.0 µg/mL).
Keywords: methylation; reduction; acylation; amination; substitution; aminoanthraquinone;
mechanism; cytotoxic; MCF-7; Hep-G2

Molecules 2013, 18
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1. Introduction
Natural and synthetic anthraquinones have attracted the interest of researchers due to their
significant biological activities such as antitumour [1–4], anti-inflammatory [5], antimalarial [5–7],
antimicrobial [5,8], antifungal [9], antileukemic [10,11], antiviral and anti-HIV properties [12–15].
Anthraquinone and its derivatives are also used as antioxidants [16], dyes [17–20] or in photoimaging [20].
It has also been reported that amino-substituted anthraquinones show significantly increased
antiproliferative activities against human/mammalian cancer cell lines [21,22] and are known to have
potential antitumor activity, but are less toxic to normal cells and display low cardiotoxicity [23–26]. A
study on 4-(N-cyclohexylamino)-emodin implied that it can discriminate well between hepatoma
cells and primary hepatocytes and it retained the capacity to reverse the multi-drug-resistance
phenotype [27]. Other aminoanthraquinone derivatives such as Reactive Blue 2 (RB-2), Acid Blue 25
(AB-25) and Acid Blue 129 (AB-129) also known as a good nucleotide-binding proteins [28] where
RB-2 is one of the most widely used P2-receptor antagonists [29]. The potential of RB-2 was driven by
its hydrophobic interactions of aromatic ꢀ-electron systems, and the hydrogen bonds with nitrogen as
donor and acceptor atoms as these properties could block the P2-recptor.
Based on these promising bioactivities, the aim of this research was therefore to synthesise some
new aminoantharaquinone derivatives with high potential as anticancer and antimicrobial agents. The
effects of substrate amount, catalyst amount, reaction time and reaction temperature were studied.
According to a study, shorter amines would result in lower cytotoxic effects [24] whereas the use of
diamines or longer amine chains allow the possibility of forming side products due to their reactive
properties [30]. Therefore, a simple straight chain amine containing four carbons (BuNH2) was chosen
for this study, while diacetoiodobenzene [PhI(OAc)2] was used as catalyst because of it selectivity
towards the substitution reaction [24]. Synthesised aminoanthraquinones were then further tested for
cytotoxicity and antimicrobial activity.
2. Results and Discussion
The synthesis of aminoanthraquinone derivatives was achieved through two simple reaction steps.
1,4-Dihydroxyanthraquinone (1) was first subjected to methylation, reduction, and acylation to
produce anthracene-1,4-dione (3) 1,4-dimethoxyanthracene-9,10-dione (5) and 9,10-dioxo-9,10-dihydro-
anthracene-1,4-diyl diacetate (7). Compounds 1, 3, 5 and 7 were then treated with butylamine (BuNH2)
using iodobenzenediacetate [PhI(OAc)2] as catalyst to produce the desired aminoanthraquinones. All
the compounds were elucidated using mp, IR, MS, 1D NMR, 2D NMR and comparison with data in
the literature. The proposed mechanisms for the amination are also presented.
2.1. Reduction, Methylation and Acylation
The reduction of compound 1 using NaBH4 (1:1 equiv.) was performed successfully in 30 min to
give a mixture of 4-hydroxyanthracene-1,10-dione (2) and anthracene-1,4-dione (3) in 69% and 21%
yields, respectively. Increasing the amount of NaBH4 to 3 equiv. led to the formation of only
compound 3 in excellent yield (Scheme 1 and Table 1). Further NaBH4 increases resulted in a lower
yield of 3 as the single product obtained (Table 1, Entry I, II and III).

Molecules 2013, 18
8048
The methylation of compound 1 with (CH3)2SO4 in the presence of K2CO3 was achieved in acetone
under reflux at 60 °C [31,32]. The used of (CH3)2SO4 as methylating agent is proven to give higher
yields [33]. A mixture of 1-hydroxy-4-methoxyanthracene-9,10-dione (4) and 1,4-dimethoxy-
anthracene-9,10-dione (5) were obtained as yellow and orange solids, respectively (Table 1, Entry IV
1
and V). The H-NMR spectra of compounds 4 and 5 showed singlets at δ 3.76 and δ 3.94 ppm,
respectively, which were attributed to the methoxy protons.
Scheme 1. Reduction, methylation and acylation of 1.
O
OH
O
O
O
NaBH4, CH3OH
RT
+
O
OH
OH
O
OH
3
1 (1 eq)
2
O
O
OMe
OH
O
OMe
OMe
K2CO3 (11 eq), (CH3)2SO4 (14 eq)
(CH3)2CO, reflux
+
O
OH
OH
O
O
5
4
1 (1 eq)
O
O
OAc
O
OAc
Ac2O (33 eq), NaOAc (1 eq)
+
100°C
O
OH
O
OH
O
OAc
1 (1 eq)
6
7
Table 1. Some effects on the reduction, methylation and acylation of 1.
Entry
I
NaBH4 (equiv.)
Reaction Time
Product (% Yield)
1
3
15
-
30 min
30 min
30 min
3 h
2 (69), 3 (21)
3 (90)
II
III
3 (60)
IV
V
4 (5), 5 (85)
5 (96)
-
4 h
VI
VII
VII
-
2 h
6 (26), 7 (60)
6 (15), 7 (75)
7 (90)
-
3 h
-
9 h
The acylation of compound 1 by using excess acetic acid anhydride in the presence of sodium acetate
was completed in 2 h to obtain a mixture of 4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl acetate (6)
and 9,10-dioxo-9,10-dihydroanthracene-1,4-diyl diacetate (7) (Scheme 1). Increasing the reaction time
for both methylation and acylation seemed to increase the dimethylated and diacetylated products, 5
and 7 (Table 1, Entry V, VI, VII and VIII). It was believed that the product of 3, 5 and 7 were
produced through the intermediate compounds 2, 4 and 6, respectively.

Molecules 2013, 18
8049
2.2. Amination
Amination reactions were attempted using procedure of Teich et al. [24]. It was stated that the formation
of aminoanthraquinones can be achieved in higher yield using 1.1 equiv. of the catalyst PhI(OAc)2.
Compounds 1, 3, 5, and 7 (1 equiv.) were then used in a substitution reaction to produce
aminoanthraquinones derivatives. The reaction was performed with excess BuNH2 in the presence of
PhI(OAc)2 for 15 min to 6 d of reaction, depending on the hindrance of the starting material. BuNH2
also acted as a solvent for the reaction (Scheme 2). The catalytic effect was studied by performing the
reaction either with or without the catalyst to observe what is the possible reaction or product that
might occur or form. Therefore, direct amination of 1 with higher equivalents of butylamine in the
presence of 1.1 equiv. of catalyst at RT gave a mixture of anthraquinone butylamines 1a and 1b in 1:1
ratio (78% yield). Reducing the equivalents of amine to half either with or without the catalyst
produced a single product 1a in higher yield (70%–90%, Table 2, Entry I, II and III). However,
increasing the reaction temperature gave a mixture of products again, where 1a was isolated as the
major one (70%). The butylamino group was observed to substitute at the ortho position or replace the
OH group. The results proved that the amination occurred via a nucleophilic substitution reaction and
1
in agreement with reported works [21,25]. The H- and 13C-NMR spectra of 1a and 1b are also
consistent with their structures and the reported data.
Scheme 2. Proposed mechanism of formation of 1a in the presence of catalyst.
AcOꢀ
Ph
I
Ph
I
H
OAc
OAc
H
O
O
O
O
O
OAc
O
O
O
OAc
Ph
I
BuNH2
OH
O
OH
OH
+ AcOH
1
Ph(OAc)Iꢀ
H
N
O
O
O
OH
OH
O
O
H
NHBu
H
NHBu
Bu
O
OH
O
O
OH
+
PhI(OAc)H
1a

Molecules 2013, 18
8050
Table 2. Effect of amine equivalence, catalyst and temperature on the amination of
1, 3, 5 and 7.
Entry
I
BuNH2 (equiv.)
PhI(OAc)2 (equiv.)
Temperature
RT
Product (% Yield)
1a (40), 1b (38)
1a (90)
450
225
225
225
225
225
112
225
225
225
225
225
1.1
1.1
-
II
RT
III
RT
1a (70)
IV
V
-
80 °C
RT
1a (70), 1b (15)
3a (60)
1.1
-
VI
VII
VIII
IX
X
RT
3a (51)
1.1
1.1
1.1
1.1
-
RT
3a (46)
RT
5a (7), 5b (10), 5 (78)
5c (10), 5d (73)
1a (83)
80 °C
RT
XI
XII
RT
1a (55)
-
80 °C
1a (46)
In the presence of PhI(OAc)2, the reaction proceeded with the interaction of the OH group of
compound 1 with the catalyst to give the intermediate, O-IPh(OAc). Further attack by BuNH2 at the
next carbon was produced C=O and removed the PhIOAc. Proton elimination then restored the double
bond and OH of 1a (Scheme 2). In the absence of catalyst, the electron-donating nature of the OH
group together with the electron delocalization made compound 1 easily attacked by the BuNH2 at the
ortho-position to form 1a (Scheme 3).
Scheme 3. Proposed mechanism of formation of 1a without using a catalyst.
O
OH
OH
O
O
OH
OH
O
O
OH
NHBu
H
NHBu
BuNH2
O
O
H
1
BuNH2
H
N
H
H
O
O
OH
OH
O
O
OH
O
H
NHBu
NHBu
1a
+
CH3CH2CH2CH3 + NH3
The compounds 1a, 1b and the optimised amount of BuNH2 (225 equiv.) and PhI(OAc)2
(1.1 equiv.) were used as a comparison of nucleophilic substitution for amination of compounds 3, 5
and 7. Therefore, the effect of different 1,4-disubstituted of anthraquinones and the mechanisms were
further investigated.

Molecules 2013, 18
2.2.1. Amination of 3
8051
Amination of compound 3 produced a higher yield of 2-(butylamino)anthracene-1,4-dione (3a)
when treated with 255 equiv. of BuNH2 (Scheme 4). It was observed that the yield decreased when the
reaction was performed without a catalyst or if equivalents of amine were reduced (Table 2, Entry V,
VI and VII).
Scheme 4. Amination of 1, 3, 5, and 7.
O
OH
O
OH
O
NHBu
OH
NHBu
BuNH2, PhI(OAc)2
RT, 15 min
+
O
OH
O
O
OH
O
O
1a
1b
1 (1 eq)
NHBu
BuNH2, PhI(OAc)2
RT, 15 min
O
O
3a
3 (1 eq)
O
O
NHBu
+
NHBu
NHBu
BuNH2, PhI(OAc)2
RT, 3 d
O
OMe
OMe
O
OMe
O
5b
5a
O
O
NHBu
O
NHBu
NHBu
BuNH2, PhI(OAc)2
80°C, 3 d
5 (1 eq)
+
O
OMe
OH
O
5d
5c
O
OAc
OAc
O
NHBu
BuNH2, PhI(OAc)2
RT, 6 h
O
O
OH
7 (1 eq)
1a
1
The H-NMR spectrum of 3a showed aromatic proton singlets at ꢁ 8.50 and ꢁ 8.48 ppm that are
attributed to the aromatic carbons at positions C9 and C10, respectively. A proton singlet observed at ꢁ
5.80 ppm indicated the presence of an unsaturated proton, whereas a broad singlet observed at ꢁ 5.99
13
ppm referred to the chelated proton of the amine. The C-NMR spectrum showed the presence of 18
signals correspond to the 18C in the structure. Higher chemical shifts were observed at ꢁ 182.7 and ꢁ
181.4 ppm that represent the two C=O groups, whereas lower signals for C2 and C3 were observed at
ꢁ 130.2 and ꢁ 134.0 ppm. In comparison to the starting material, only one signal was observed for both
C=O (ꢁ 184.7), and for C2 and C3 (ꢁ 140.1) due to the symmetrical structure of 3. The position of the
3
butylamino group of 3a was confirmed by an HMBC experiment which showed a J correlation as
indicated in Figure 1.

Molecules 2013, 18
8052
Figure 1. Selected HMBC correlation of 3a and 5c.
H
O
O
HN
O
O
H
N
H
H
H
H
O
CH3
3a
5c
The proposed mechanism showed that C3 is positively charged and readily attacked by BuNH2 due
to the resonance that drives by the interaction between the oxygen at C1 with the iodine of the catalyst.
Further elimination of a proton at position C3, PhI and AcOH then restored the double bonds between
C-C and C-O to give the product 3a (Scheme 5). A similar resonance effect is assumed to drive the
BuNH2 to attack the C3 to produce a negatively charged oxygen for the reaction without catalyst. The
reaction proceeded through a keto-enol tautomerism followed by elimination of H to give 3a (Scheme 6).
Scheme 5. Proposed mechanism of formation of 3a by using a catalyst.
Ph
I
Ph
I
O
O
OAc
OAc
O
O
O
OAc
O
O
OAc
NHBu
Ph
I
H
Bu
N
H
NHBu
H
O
BuNH2
3
3a
+
AcOH
AcOꢀ
AcOH
+
+
PhI
Scheme 6. Proposed mechanism for the formation of 3a (without catalyst).
H
O
O
O
O
O
H
BuNH2
N
NHBu
NHBu
BuNH2
Bu
H
O
O
O
3
BuNH2
O
O
O
O
NHBu
NHBu
CH3CH2CH2CH3
+
NH3
+
H
BuNH2
H
3a
H
N
H
H
2.2.2. Amination of 5
Treatment of dimethoxyanthraquinone 5 with BuNH2 in the presence of PhI(OAc)2 produced four
aminoathraquinones. At RT, a mixture of 2-(butylamino)-4-methoxyanthracene-9,10-dione (5a) and

Molecules 2013, 18
8053
2,3-(dibutylamino)anthracene-9,10-dione (5b) was obtained in low yields (17%) together with the
unreacted starting material (78%). A mixture of 1-(butylamino)-4-methoxyanthacene-9,10-dione (5c)
and 1,4-(dibutylamino)anthracene-9,10-dione (5d) was produced at 80 °C, in higher combined yield
(83%, Scheme 2). The diaminosubstituted products 5b and 5d were isolated as a major product (Table 2,
Entry VIII and IX). It appears that the higher the temperature, the more the nucleophilic substitution
selectivity. All four amino derivatives were easily separated by column chromatography
(dichloromethane-petroleum ether/3:1). It seems that compound 5a and 5c which had same Rf values
and appearance (Rf 0.33, dichloromethane-petroleum ether, 4:1; dark pink solid) were produced first as
intermediates and then reacted further to form diaminoanthraquinones 5b and 5d. Surprisingly,
compounds 5b and 5d also shared same Rf values 0.67 (dichloromethane-petroleum ether, 4:1) and
both were obtained as dark blue solids.
1
The H-NMR spectra of 5a and 5c displayed signals attributed to methoxy protons at δ 3.98 and δ
3.98 ppm, respectively. A broad singlet signal at δ 7.27 ppm in the spectrum of 5a was assigned to the
non-chelated amine proton, whereas for 5c, the spectrum show a higher chemical shift at δ 9.84 ppm
which is due to the hydrogen bond between the amine hydrogen and carbonyl oxygen. Both
compounds shared the same molecular formulae, C19H19NO3, as the mass spectra exhibited a
molecular ion peak at m/z 309. Selected HMBC correlations were shown in Figure 1.
The 1H-NMR of 5d showed the disappearance of the signal for the methoxy protons, which clearly
indicated that nucleophilic aromatic substitution had occurred. The broad singlet at δ 10.73 ppm was
13
assigned to the chelated amine proton. The C-NMR showed the presence of 11 signals attributed to
22 carbons which are symmetrical. Four quaternary carbons were observed at δ 182.0 (C=O), 146.2
(C-C=O), 134.6 (O=C-C) and 109.5 ppm (C-NH). Three peaks at δ 123.5, 126.0 and 131.8 ppm were
assigned to methine aromatic carbons.
The reaction occurred as the methoxy group interacts with the catalyst. The positive charge on
oxygen is then stabilized by the C1-O bond cleavage driven by the formation of PhI(OAc)(OCH3) and
leaves the aromatic carbocation. With increasing heat, the carbocation is readily attacked by BuNH2 to
produce 5c and 5d. In contrast a slow reaction at RT favored the carbocation rearrangement to produce
5a and 5b (Scheme 7). This aromatic substitution of methoxy groups by amines is also supported by
literature precedents [21,25,28].
2.2.3. Amination of 7
2-(Butylamino)-1,4-dihydroxyanthracene-9,10-dione (1a) was successfully obtained in 83% yield
under a similar amination approach applied to the acylated anthraquinone 7 (Scheme 4). The
mechanism suggested that the acetate groups were reduced by the conditions and reagent used to give
1,4-dihydroxyanthraquinone (1) as an intermediate before it further reacted with BuNH2/PhI(OAc)2 to
give 1a (Scheme 8). It was observed that 1a can still be obtained when the reaction was performed
without catalyst or at 80 °C but gave lower yield. Heat applied to the reaction induced the reaction to
be faster as the reaction time reduced from 6 h to 4 h (Table 2, Entry X, XI and XII).

Molecules 2013, 18
Scheme 7. Proposed mechanism of formation of 5a, 5b, 5c and 5d.
8054
Ph
I
BuNH2
BuNH2
OMe
OAc
OAc
Me
O
Me
Ph
O
OAc
O
O
O
O
O
O
I
RT, slow
heat, fast
O
OMe
OMe
+
O
OMe
ꢀ
5
+
AcO
PhI(OAc)(OMe)
ꢀ
ꢀ
AcO
AcO
H
H
H
Bu
N
Formation of 5b and 5d were followed
the same step of mechanisms.
O
O
H
N
Bu
O
O
O
OMe
O
O
OMe
O
NHBu
NHBu
NHBu
NHBu
NHBu
O
O
NHBu
5b
5d
O
OMe
5b
O
OMe
5a
5c
5d
Scheme 8. Reduction of acyl group of 7 by butylamine.
BuNH2
O
O
O
BuHN
BuH2N
O
O
Me
O
O
Me
O
O
Me
O
O
O
OH
HꢀNH2Bu
O
OAc
O
OAc
O
OAc
O
OAc
O
OAc
7
6
+
BuNH3
+
MeCONHBu
1
The H-NMR for 1a showed the disappearance of the COOCH3 signal which proved that the acyl
groups were reduced. This is also proven by the existence of strong chemical shifts at δ 14.23 and δ
13.90 ppm which represented the two chelated OH. A broad singlet proton at δ 5.60 ppm was
attributed to the amine proton.
All three new aminoanthraquinones 3a, 5a and 5b were subjected to cytotoxic activity assays
against MCF-7 (estrogen receptor positive human breast) and Hep-G2 (human hepatocellular liver
carcinoma) cancer cell lines with compounds 3a and 5a being the most cytotoxic (IC50 1.1–3.0 µg/mL).
Increasing the number of amino substituents (compound 5b) on the structure of anthraquinone
basically reduced the activity against both MCF-7 and Hep-G2 cancer cell lines with IC50 of 3.0 and
13.0 µg/mL, respectively (Table 3). Saha et al. [34] reported that the starting material, quinizarin 1
(free hydroxy groups) was not active towards MCF-7 but the methylated hydroxyl derivative of
quinizarin showed some potential with IC50 values 70–80 µM. The results of this study also supported
their finding where the presence of methoxy or amino groups enhanced the cytotoxic activity of
the compounds.

Molecules 2013, 18
8055
Table 3. Cytotoxic activity of aminoanthraquinones.
Cytotoxic Activity IC50 (µg/mL)
Sample
MCF-7
1.1
Hep-G2
1.2
3a
5a
5b
1.1
3.0
3.0
13.0
Compounds 3a, 5c and 5d have been screened for antimicrobial activity using four different types
of microbe, methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Candida
albicans and Escherichia coli. The other two new compounds, 5a and 5b were replaced by compounds
5c and 5d due to insufficient materials and based on the similarity of their structures. All four
compounds have the same methoxy and amino substituents and only differ at their amino positions.
Therefore, it was assumed that all four compounds might give similar activity results. However, none
of compounds had any effect on the growth of all four bacteria or the fungus.
3. Experimental
3.1. General
All chemicals are commercially available and were of analytical grade and used without
purification unless otherwise stated. All reactions were monitored by TLC, using Silica gel 60 F245
(Merck KGaA) precoated aluminium backed plates and visualised by UV and H2SO4 solution. All
organic extracts were dried over Na2SO4 and evaporated using rotary evaporator. Column
chromatography was performed on silica gel 60. Melting points were recorded by digital melting point
equipment (Electrothermal IA9000 Series). The IR spectra were obtained by using Perkin-Elmer
FT-IR Model Spectrum 100 series spectrophotometer using UATR techniques and the adsorption
bands were measured in the range of 280–4000 cm−1. MS spectra were recorded using equipped
Shimadzu model QP5050A series. The 1D NMR and 2D NMR spectra were run on a JEOL machine at
400 MHz or 500 MHz. All chemical shifts, δ were recorded in ppm relative to TMS signal. The
coupling constants J are given in Hz.
3.2. Reduction
1,4-Dihydroxyanthraquinone (1, 1 equiv.) was dissolved in methanol (3 mL) and stirred
homogenously under a nitrogen atmosphere. Sodium borohydride (1, 3 or 15 equiv.) was added slowly
to the reaction mixture and stirred for 30 min. Water (0.5 mL) was added to the reaction mixture and
the pH was adjusted to 5–6 with 1 M HCl. Methanol was evaporated and the residue of aqueous layer
was extracted with CH2Cl2 (3 × 10 mL). The combined organic layer were dried, filtered and evaporated
under vacuum. The crude product was purified by column chromatography (CH2Cl2-petroleum ether, 3:2)
to obtain compounds 2 (NaBH4: 1 equiv.) and 3 (NaBH4: 1, 3 and 15 equiv.).
4-Hydroxyanthracene-1,10-dione (2). Dark orange powder (65.0 mg, 69%); mp 205.8–206.1 °C; vmax
(UATR) 3317, 3056, 1662, 1568, 1271 cm−1; δH (500 MHz, CDCl3) 13.74 (1H, s, OH), 8.44 (1H, d,
J = 8.0, CH=C), 8.03 (1H, s, CH=C), 7.91 (1H, d, J = 8.0, CH=C), 7.64–7.69 (2H, m, H-aromatic),

Molecules 2013, 18
8056
7.00 (1H, d, J = 10.3, H-aromatic), 6.96 (1H, d, J 10.3, H-aromatic); δC (125 MHz, CDCl3) 189.2,
184.1, 162.6, 140.8, 140.0, 136.0, 131.4, 130.6, 129.3, 127.7, 127.4, 124.9, 121.9, 108.9; m/z (EI) 224
(M+, C14H8O3 requires 224).
Anthracene-1,4-dione (3). Orange powder (78.4 mg, 90%); mp 216.4–217.2 °C; vmax (UATR) 3319,
3053, 1662, 1596 cm−1; δH (400 MHz, CDCl3) 8.55 (2H, s, CH=C), 8.01 (2H, dd, J = 2.8, 6.4,
H-aromatic), 7.66 (2H, dd, J = 2.8, 6.4, H-aromatic), 7.03 (2H, s, H-aromatic); δC (100 MHz, CDCl3)
184.7, 140.1, 134.9, 130.3, 129.7, 128.9, 128.4; m/z (EI) 208 (M+, C14H8O3 requires 208).
3.3. Methylation
A mixture of 1 (1 equiv.), K2CO3 (11 equiv.) and (CH3)2SO4 (14 equiv.) in acetone (5 mL) was refluxed
for 3 h. The reaction mixture was added to 25 mL of 2 M HCl and extracted with EtOAc (3 × 25 mL).
The combined organic layers were dried, filtered and evaporated under vacuum. The crude product was
purified by column chromatography (CH2Cl2-petroleum ether, 4:1) to produce compounds 4 and 5.
1-Hydroxy-4-methoxyanthracene-9,10-dione (4). Orange powder (5.3 mg, 5%); mp 162–163 °C; vmax
(UATR) 3073, 2924, 1667, 1590, 1440, 1234, 1178 cm−1; δH (400 MHz, CDCl3) 12.71 (1H, s, OH),
8.01 (2H, t, J = 6.4, H-aromatic) 7.52 (2H, dt, J = 19.3, 6.4, H-aromatic), 7.15 (1H, d, J = 9.2,
H-aromatic), 7.06 (1H, d, J = 10.1, H-aromatic), 3.76 (3H, s, OCH3); δC 188.8, 181.5, 157.2, 154.1,
134.9, 134.6, 133.1, 132.1, 127.2, 126.2, 126.2, 123.5, 119.0, 115.8, 56.9 (100 MHz, CDCl3); m/z (EI)
254 (M+, C15H10O4 requires 254).
1,4-Dimethoxyanthracene-9,10-dione (5). Yellow powder (108.1 mg, 96%); mp 198–200 °C; vmax
(UATR) 3089, 2998, 1668, 1566, 1404, 1243 cm−1; δH (400 MHz, CDCl3) 8.10 (2H, dd, J = 5.5, 3.6
H-aromatic), 7.65 (2H, dd, J = 5.5, 3.6 H-aromatic), 7.28 (2H, s, H-aromatic), 3.94 (6H, s, OCH3); δC
(100 MHz, CDCl3) 183.5, 154.2, 134.3, 133.4, 126.5, 123.1, 120.3, 57.1; m/z (EI) 268 (M+, C16H12O4
requires 268).
3.4. Acylation
A mixture of compound 1 (1 equiv.), Ac2O (33 equiv.), and NaOAc (1 equiv) was stirred at 100 °C
for 2 h. The reaction mixture were added to ice cold water and extracted with CH2Cl2 (3 × 25 mL). The
combined organic layers was dried and evaporated under vacuum. The crude product was purified by
column chromatography (CH2Cl2-petroleum ether, 3:1) to produce compounds 6 and 7.
4-Hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl acetate (6). Light orange powder (31.8 mg, 26%);
mp 189.1–189.5 °C; vmax (UATR) 3535, 2935, 1772, 1667, 1587, 1360, 1168, 1011 cm−1; δH (400
MHz, CDCl3) 13.04 (1H, s, OH), 8.28 (1H, t, J = 4.6, H-aromatic), 8.21 (1H, t, J = 4.6, H-aromatic),
7.79 (2H, t, J = 3.7, H-aromatic), 7.33 (2H, s, H-aromatic), 2.46 (3H, s, OCH3 ); δC (100 MHz, CDCl3)
188.5, 181.2, 169.9, 161.3, 143.2, 135.0, 134.1, 133.5, 132.5, 131.22, 127.4, 126.8, 126.0, 123.5,
115.9, 21.2; m/z (EI) 282 (M+, C16H10O5 requires 282).

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9,10-Dioxo-9,10-dihydroanthracene-1,4-diyl diacetate (7). Light yellow powder (122.4 mg, 90%); mp
236.2–237.0 °C; vmax (UATR) 3081, 1762, 1667, 1582, 1437, 1177 cm−1; δH (400 MHz, CDCl3) 8.14 (2H, dd,
J = 5.5, 3.7 H-aromatic), 7.73 (2H, dd, J = 5.5, 3.7, H-aromatic), 7.41 (1H, s, H-aromatic), 2.47 (6H, s,
OCCH3 ); δC (100 MHz, CDCl3) 181.6, 169.6, 148.3, 134.2, 133.4, 131.1, 127.0, 126.2, 21.3; m/z (EI)
324 (M+, C16H10O5 requires 324).
3.5. Amination—General Procedure
Butylamine (112, 225 or 450 equiv.) was added dropwise to a mixture of 1, 3, 5, or 7 (1 equiv.) and
PhI(OAc)2 (0.41 mmol, or without catalyst) at RT (or heated at 80 °C) and stirred for 15 min to 3 d.
The reaction mixture, 25 mL 10 M HCl and NaHCO3 (84 mL) were added to ice cold water (84 mL)
successively. The resulting solution was extracted with EtOAc (3 × 25 mL). The organic layer was
washed with H2O (3 × 25 mL), dried and evaporated. The crude product was then chromatographed on
silica gel (DCM-petroleum ether, 4:1) to give a mixture of amine derivatives of anthraquinone 1a, 1b,
3a, 5a–d.
2-(Butylamino)-1,4-dihydroxyanthracene-9,10-dione (1a). Dark pink powder (176.0 mg, 90%); mp
158.0–158.4 °C; vmax (UATR) 3378, 3308, 2949, 1635, 1565, 1516, 1459, 1417, 1260, 1156 cm−1;
δH (500 MHz, CDCl3) 14.23 (1H, s, OH) 13.90 (1H, s, OH), 8.28 (1H, d, J = 6.9, H-aromatic), 8.25
(1H, d, J = 6.9, H-aromatic), 7.75 (1H, t, J = 5.8, H-aromatic), 7.68 (1H, t, J = 5.8, H-aromatic), 6.05
(1H, s, H-aromatic), 5.57 (1H, br. s, NH), 3.21 (2H, q, J = 6.9, NHCH2CH2), 1.65–1.72 (2H, m,
CH2CH2CH2), 1.42–1.50 (2H, m, CH2CH2CH3), 0.98 (3H, t, J 6.9, CH2CH3); δC (125 MHz, CDCl3)
183.5, 178.0, 166.5, 153.6, 147.7, 134.7, 134.0, 132.4, 132.3, 126.5, 126.3, 110.4, 102.9, 100.3, 42.8,
30.7, 20.3, 13.7; m/z (EI) 311 (M+, C18H17NO4 requires 311).
1-(Butylamino)-4-hydroxyanthracene-9,10-dione (1b). Dark purple powder (31.5 mg, 38%); mp
127.2–127.9 °C; vmax (UATR) 3170, 2954, 2926, 2853, 1616, 1580, 1463, 1233, 1162 cm−1; δH
(500 MHz, CDCl3) 13.71 (1H, s, OH) 10.29 (1H, br. s, NH), 8.30 (2H, dd, J = 8.0, 16.0, H-aromatic),
7.77 (1H, t, J = 5.7, H-aromatic), 7.70 (1H, t, J = 5.8, H-aromatic), 7.21 (1H, s, H-aromatic), 7.20 (1H, s,
H-aromatic), 3.36 (2H, t, J = 6.9, NHCH2CH2), 1.70–1.77 (2H, m, CH2CH2CH2), 1.47–1.55 (2H, m,
CH2CH2CH3), 0.99 (3H, t, J = 6.9, CH2CH3); δC (125 MHz, CDCl3) 187.5, 182.0, 156.8, 147.7, 135.5,
134.2, 132.7, 132.5, 129.0, 126.7, 126.4, 124.1, 113.8, 108.4, 42.7, 31.6, 20.4, 13.9; m/z (EI) 311
(M+, C18H17NO3 requires 295).
2-(Butylamino)anthracene-1,4-dione (3a). Yellow powder, (70.1 mg, 60%); mp 173.4–173.9 °C; vmax
(UATR) 3325, 2926, 1677, 1573, 1504, 1456, 1406, 1319, 1250 cm−1; δH (500 MHz, CDCl3) 8.50 (1H, s,
H-aromatic), 8.48 (1H, s, H-aromatic), 7.93 (2H, t, J = 8.0, H-aromatic), 7.59 (1H, t, J = 5.8,
H-aromatic), 7.55 (1H, d, J = 8.1, H-aromatic), 5.99 (1H, br. s, NH), 5.80 (1H, s, CH=C), 3.16 (2H, q,
J = 6.9, NHCH2CH2), 1.62–1.69 (2H, m, CH2CH2CH2), 1.37–1.46 (2H, m, CH2CH2CH3), 0.94 (3H, t,
J = 6.9, CH2CH3); δC (125 MHz, CDCl3) 182.7, 181.4, 149.1, 135.7, 134.0, 130.2, 130.0, 129.8, 129.6,
129.0, 128.7, 127.7, 127.6, 102.6, 42.4, 30.4, 20.3, 13.8; m/z (EI) 279 (M+, C18H17NO2 requires 279).

Molecules 2013, 18
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2-(Butylamino)-4-methoxyanthracene-9,10-dione (5a). Dark purple powder (12.5 mg, 10%);
mp 87.8–88.2 °C; vmax (UATR) 3535, 2936, 1630, 1590, 1506, 1461, 1354, 1250, 1180 cm−1; δH (400
MHz, CDCl3) ) 8.23 (1H, t, J = 2.8, H-aromatic), 8.21 (1H, t, J = 2.8, H-aromatic), 7.70 (2H, t, J = 3.6,
H-aromatic), 7.37 (1H, d, J = 9.2, H-aromatic), 7.27 (1H, d, J = 10.1, H-aromatic), 5.29 (1H, br. s,
NH), 3.98 (3H, s, OCH3), 3.33 (2H, t, J = 7.3, NHCH2CH2), 1.73–1.82 (2H, m, CH2CH2CH2),
1.46–1.57 (2H, m, CH2CH2CH3), 0.99 (3H, t, J = 7.3, CH2CH3); δC (100 MHz, CDCl3) 185.3, 183.3,
152.7, 145.6, 134.5, 134.0, 133.3, 133.2, 126.8, 126.2, 124.1, 121.7, 121.4, 114.0, 57.6, 44.1, 31.1,
20.4, 13.9; m/z (EI) 309 (M+, C19H19NO3 requires 309).
2,3-(Dibutylamino)anthracene-9,10-dione (5b). Dark blue powder (10.6 mg, 7%); mp 113.0–113.6 °C;
v
max (UATR) 3539, 2929, 1669, 1583, 1522, 1364, 1249, 1172 cm−1; δH (500 MHz, CDCl3) 8.29 (2H,
dd, J = 5.7, 3.4, H-aromatic), 7.76 (2H, d, J = 5.7, 3.4, H-aromatic), 7.50 (2H, s, H-aromatic), 5.29
(2H, s, NH), 3.36 (4H, t, J = 6.9, NHCH2CH2), 1.79–1.85 (4H, m, CH2CH2CH2), 1.47–1.54 (4H, m,
CH2CH2CH3), 0.99 (6H, t, J = 6.9, CH2CH3); δC (500 MHz, CDCl3) 184.5, 141.6, 133.8, 133.7, 126.8,
125.4, 114.7 , 45.9, 30.6, 20.3, 13.8; m/z (EI) 350 (M+, C22H26N2O2 requires 350).
1-(Butylamino)-4-methoxyanthacene-9,10-dione (5c). Dark purple powder (11.4 mg, 10%);
mp 96.3–96.9 °C; vmax (UATR) 3536, 2929, 1647, 1591, 1506, 1461, 1356, 1248, 1180 cm−1; δH
(500 MHz, CDCl3) 9.84 (1H, s, NH), 8.17 (2H, dd, J = 16.9, 2.3, H-aromatic), 7.60–7.67 (2H, m,
H-aromatic), 7.27 (1H, d, J = 9.2, H-aromatic), 7.02 (1H, d, J = 9.2, H-aromatic), 3.92. (3H, s, OCH3),
3.23 (2H, q, J = 6.9, NHCH2CH2), 1.66–1.71 (2H, m, CH2CH2CH2), 1.43–1.51 (2H, m, CH2CH2CH3),
0.96 (3H, t, J = 6.9, CH2CH3); δC (125 MHz, CDCl3) 184.8, 183.4, 151.6, 147.5, 134.4, 134.2, 133.0,
132.8, 126.6, 126.1, 124.3, 121.1, 120.2, 112.3, 57.5, 42.8, 31.4, 20.5, 13.9; m/z (EI) 309
(M+, C19H19NO3 requires 309).
1,4-(Dibutylamino)anthracene-9,10-dione (5d). Dark blue powder (94.5 mg, 73%); mp 113.5–114.2 °C;
vmax (UATR) 3747, 2927, 1641, 1570, 1518, 1461, 1364, 1258 cm−1; δH (500 MHz, CDCl3) 10.73 (2H,
br. s, NH), 8.28 (2H, dd, J 5.7, 3.4, H-aromatic), 7.62 (2H, dd, J = 5.7, 3.4, H-aromatic), 7.04 (2H, s,
H-aromatic), 3.25–3.29 (4H, m, NHCH2CH2), 1.65–1.71 (4H, m, CH2CH2CH2), 1.43–1.51 (4H, m,
CH2CH2CH3), 0.96 (6H, t, J = 6.9, CH2CH3); δC (125 MHz, CDCl3) 182.0, 146.2, 134.6, 131.8, 126.0,
123.5, 109.5 , 42.6, 31.8, 20.5, 14.0; m/z (EI) 350 (M+, C22H26N2O2 requires 350).
3.6. Cytotoxic Assays
The cytotoxic assay was carried out according to the method described by Sukari et al. [35]. The
MCF-7 (estrogen receptor positive human breast) and Hep-G2 (human hepatocellular liver carcinoma)
cancer cells were purchased from ATCC. The cells were grown and maintained in RPMI 1640 media,
supplemented with 10% fetal calf serum (FCS) and 1% antibiotic penicillin-streptomycin in an
atmosphere of 5% CO2 at 37 °C. The medium was used to dilute the cells to a concentration of
5 × 105 cells/mL. From this cells suspension, 100 μL of various concentrations of the synthesised
compounds were pipetted into a 96-well micro titer plate and incubated in 37 °C, 5% CO2 incubator
for 72 h. The various concentration used were 100, 50, 25, 12.5, 6.25, 3.125, 1.56 μg/mL. The assay of
each concentration of synthesised compounds was performed in triplicate and the control wells of

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untreated population were also included. After three d, the fraction of surviving cells was determined
relative to the untreated cells population by the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) method where the viability of cells was measured by 20 μL of blue
formazan crystals of MTT solution (5 mg/mL in phosphate-buffered saline, PBS) added to each well
followed by incubation in 37 °C, 5% CO2 incubator for 3–4 h. 100 μL of cells suspension or cells
monolayer in each micro titer was removed from each well. The plate was left at room temperature for
30 min before reading the absorbance. The absorbance was read with the multiwell scanning
spectrophotometer (ELISA reader) test wavelength of 570 nm and reference wavelength of 630 nm.
The cytotoxic index used was IC50 which is the concentration that yield 50% inhibition of the cells
compared with untreated control.
3.7. Antimicrobial Assay
The modified disc diffusion method was followed a procedure by Garba and Okeniyi [36]. Nutrient
agar (20 g) was suspended in 1 L of distilled water and stirred. It was boiled to dissolve homogenously
and autoclaved at 121 °C for 20 min. The agar was allowed to cool to 50 °C and poured into sterile
disposable petri dishes. Four microbes, methicillin-resistant Staphylococcus aureaus (MRSA),
Pseudomonas aeruginosa, Candida albicans and Escherichia coli were inoculated into prepared
nutrient broth and incubated at 37 °C for overnight. The suspension of the microbes in the broth was
inoculated on the nutrient agar using sterile cotton bud. The sterile 6 mm paper discs were impregnated
with the synthesised compounds in concentrations of 20, 10, 5, 2, 1, 0.5 and 0.1 mg/mL and allowed to
soak for 1 min. The paper discs were removed, dried and placed on the surface of agar plates
inoculated with the microbial cultures. Each synthesised compound was tested in triplicate. Paper discs
impregnated with acetone were used as a negative control. The petri dishes were incubated in inverted
position at 37 °C for 24 h. The zones of inhibitions (clear area without bacterial growth) were
measured in cm.
4. Conclusions
Aminoantraquinone derivatives were synthesised through methylation, reduction or acylation
then followed by amination. In total, seven aminoanthraquinones (compounds 1a, 1b, 3a, and 5–d)
were produced, including three new compounds: 2-(butylamino)anthracene-1,4-dione (3a),
2-(butyl- amino)anthracene-9,10-dione (5a) and 2,3-(dibutylamino)anthracene-9,10-dione (5b). All
amino-anthraquinones were produced via nucleophilic substitution mechanisms. Aminoanthraquinones
3a, 5a and 5b were found to demonstrate strong cytotoxic activity against both MCF-7 (IC50 1.1, 1.1
and 3.0 µg/mL respectively) and Hep-G2 cancer cell lines (IC50 1.2, 3.0 and 13.0 µg/mL, respectively).
Acknowledgments
We are grateful to FRGS (Vot. No. 5523918) and RUGS (Vot. No. 9342700) for financial support,
Department of Chemistry, Faculty of Science, UPM for the research facilities, Zainal for MS spectra
and Rusnani for the IR spectra.

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Conflict of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 3a and 5d are available from the authors.
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