Synthesis and cytotoxic evaluation of two novel anthraquinone derivatives

Article abstract of DOI:10.1016/j.farmac.2004.03.006

The antitumor activity of dihydroxyanthracenediones such as mitoxantrone on a panel of cancer cell lines during the last 30 years, led investigators to synthesize thousands of anthracycline analogs and test their cytotoxicity to identify compounds superior to the parent drugs in terms of increased therapeutic effectiveness, reduced toxicity or both. To achieve this, new synthesized congeners either have different side arms or have extra rings on their skeletons. Following these studies, we proposed total synthesis of 2-amino-N-[4-(2-amino-3-hydroxy-propionylamino)-9,10-dioxo-9, 10-dihydroanthracene-1-yl]-3-hydroxy-propionamide (V) and 6-amino-hexanoic acid [4-(5-amino-pentanoylamino)-9,10-dioxo-9,10-dihydro-anthracen-1-yl]-amide (VI). Acetylation of 1,4-diaminobenzene using acetyl chloride and reaction with phthalic anhydride under a Friedel-Crafts reaction and then cyclization gave 1, 4-diamino-anthraquinone. This compound was reacted with two amino acids (L-serine and 6-amino hexanoic acid) in their ester forms, using ethyl chloroformate as a coupling agent. Hydrolyzing esterified compounds gave their amino substituted derivatives. These compounds with diamine side arms are supposed to provide better intercalation with DNA. Synthesized novel ametantrone derivatives were tested against a panel of cancer cells (KB, Hela, MDA-MB-468 and K562), using MTT assay. The results showed that tested compounds inhibited the growth of cancer cells at micromolar concentrations. However, compound (VI) was more cytotoxic than compound (V) probably because of its longer side chains and better intercalation with DNA.

Full text of DOI:10.1016/j.farmac.2004.03.006

IL FARMACO 59 (2004) 645–649
www.elsevier.com/locate/farmac
Synthesis and cytotoxic evaluation of two novel anthraquinone derivatives
a,
b
c
Hojjat Sadeghi-Aliabadi *, Maryam Tabarzadi , Afshin Zarghi
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran
b
School of Pharmacy, Tehran Open University, Tehran, Iran
c
Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences,
Shahid Beheshti University of Medical Sciences, Tehran, Iran
Received 9 February 2004; accepted 4 March 2004
Available online 20 April 2004
Abstract
The antitumor activity of dihydroxyanthracenediones such as mitoxantrone on a panel of cancer cell lines during the last 30 years, led
investigators to synthesize thousands of anthracycline analogs and test their cytotoxicity to identify compounds superior to the parent drugs in
terms of increased therapeutic effectiveness, reduced toxicity or both. To achieve this, new synthesized congeners either have different side
arms or have extra rings on their skeletons. Following these studies, we proposed total synthesis of 2-amino-N-[4-(2-amino-3-hydroxy-
propionylamino)-9,10-dioxo-9,10-dihydroanthracene-1-yl]-3-hydroxy-propionamide (V) and 6-amino-hexanoic acid [4-(5-amino-
pentanoylamino)-9,10-dioxo-9,10-dihydro-anthracen-1-yl]-amide (VI).Acetylation of 1,4-diaminobenzene using acetyl chloride and reaction
with phthalic anhydride under a Friedel–Crafts reaction and then cyclization gave 1, 4-diamino-anthraquinone. This compound was reacted
with two amino acids (L-serine and 6-amino hexanoic acid) in their ester forms, using ethyl chloroformate as a coupling agent. Hydrolyzing
esterified compounds gave their amino substituted derivatives. These compounds with diamine side arms are supposed to provide better
intercalation with DNA. Synthesized novel ametantrone derivatives were tested against a panel of cancer cells (KB, Hela, MDA-MB-468 and
K562), using MTT assay. The results showed that tested compounds inhibited the growth of cancer cells at micromolar concentrations.
However, compound (VI) was more cytotoxic than compound (V) probably because of its longer side chains and better intercalation with
DNA.
©
2004 Elsevier SAS. All rights reserved.
Keywords: Anthraquinone synthesis; Ametantrone derivatives; Cytotoxicity; Cancer cells
1
. Introduction
lates with DNA, although some researchers showed that
non-intercalative mechanisms were involved in its cytotoxic-
ity against L1210 leukemia cells [3]. In the case of breast
cancer, Katsumata et al. [4] in their studies concluded that
combination treatment with mitoxantrone hydrochloride,
vincristine sulfate and prednisolone is an effective treatment
for breast cancer.
Anthraquinones have been used as useful compounds in
treating all kinds of cancers especially breast cancer tumors
for a long time. The discovery of the antitumor activity of
1,4-bis[(aminoalkyl)amino]anthracene-9,10-diones such as
ametantrone (a) and mitoxantrone (b) has led to numerous
synthetic and pharmacological studies on the tumoricidal
mechanisms of these anthraquinone congeners [1]. Mitox-
antrone is used clinically either alone or in combination with
other chemotherapeutic agents to treat a variety of human
cancers, particularly lung carcinoma, leukemia, melanoma
and lymphoma, Hodgkin’s disease and breast cancer [2].
Mitoxantrone inhibits RNA and DNA synthesis and interca-
*
Corresponding author.
E-mail address: sadeghi@pharm.mui.ac.ir (H. Sadeghi-Aliabadi).
©
2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2004.03.006

646
H. Sadeghi-Aliabadi et al. / IL FARMACO 59 (2004) 645–649
Scheme 1. Total synthesis of ametantrone derivatives; a) CH COCl, CH COOH, CH COONa, room temperature, 4 h; b) phthalic anhydride, H BO , H SO ; c)
3
3
3
3
3
2
4
H BO , H SO ;d) NaOH (15%), reflux 3 h; e) C H OCOCH(NH )CH OH to give “V” in which R=CONH(NH )CH OH; f) C H OCO(CH ) NH to give “VI”
3
3
2
4
2
5
2
2
2
2
2
5
2 5
2
in which R=CO(CH ) NH ; for other reaction condition see methods and materials.
2
5
2
Mitoxantrone and its derivatives are loosely related to the
anthracyclines such as doxorubicin and daunorubicin which
bind strongly to DNA and present their antitumor activity by
this mechanism. Unlike anthracyclines, which display a
dose-dependent cardiotoxicity, dihydroxyanthraquinones
previous studies [6], here we decided to synthesize some
novel anthraquinone derivatives related to ametantrone with
a different synthetic strategy. In this study, benzene-1,4-
diamine and phthalic anhydride were used to synthesize the
novel compounds as shown in Scheme 1.
(DHAQ) show a very low incidence of cardiac failure when
used clinically [2]. Low incidence of cardiotoxicity of
DHAQ derivatives led researchers to conduct more studies
on the structure activity relationship (SAR) of these conge-
ners. Introduction of an extra ring (D) into this system was
designed to introduce a non-polar moiety into the molecule
and thus provide a substituted benz(a)anthracene ring sys-
tem. This compound displayed anticancer activity against
both murine and human tumor cell lines [2]. All these new
analogs have been synthesized and tested to identify com-
pounds superior to the parent drugs in terms of increased
therapeutic effectiveness, reduced toxicity or both [5]. Mor-
real et al. in their studies used phthalic anhydride and
2
. Experimental
2
.1. Reagent and instruments
All chemicals were obtained from Aldrich Chemical
Company Ltd. (Dorset) except for doxorubicin which was
obtained from (David Bull Labs.), and all solvents were
purchased from Fisons (Loughborough, UK). Reagents were
0
used as purchased and solvents were dried using type 4-A
molecular sieves. Proof of chemical purity was established
by spectral (infrared IR, elemental analysis and MS) and
analytical (TLC and chemical analysis) techniques. Polyes-
ter coated by silica gel GF254 as TLC plates (Aldrich, Dorset)
were pre-washed with methanol/dichloromethane (50:50)
and activated in an oven at 110 °C for 1 h before use and
visualized by UV light at 254 nm developed by 50% conc.
H SO in methanol or by iodine vapor. Melting points (m.p.)
were determined on an electrothermal apparatus, IA9000
series, (Essex, UK) and are uncorrected. IR spectra were
obtained in a 1% KBr disc using a Perkin Elmer 297 IR
spectrophotometer. Mass spectra were recorded on an AEI
(Kratos) MS902 updated with an MSS Console and data
6-methoxy-1,2,3,4-tetrahydronaphthalene in a reaction to
synthesize 8,11-bis[[2-[(2-hydroxyethyl)amino]
ethyl]amino]-1,2,3,4-tetrahydro-6-methoxy-7,12-bena[a]
anthraquinone in five steps [2]. On the other hand, Krapcho
et al. [1] synthesized and evaluated symmetrical and unsym-
metrical substituted 1,4-bis[(aminoalkyl)amino]-anthra-
cene-9,10-diones and 1,4-bis[(aminoalkyl)amino]-5,8-dihy-
droxyanthracene-9,10-diones starting from 1,4-difluoro-
anthracene-9,10-dione and 1,4-difluoro-5,8-dihydroxy-
anthracene-9,10-dione to substitute fluoride groups by di-
amines at room temperature in pyridine leading to the desired
compounds. Biological evaluation of synthesized com-
pounds by this group showed that some of congeners were
active against cancer cells in a concentration of <24 ng/ml
which is somehow similar to doxorubicin [1]. Following our
2
4
0
system, Acc V 8 kV, 70 EV, 300 µA emission; fast atom
bombardment (FAB) spectra were obtained using a Kratos
MS50 spectrometer. Elemental analysis for C, H and N were
obtained using a Carlo Erba 1106 Elemental Analyzer and
weighed using a Mettler MT 5 microbalance.

H. Sadeghi-Aliabadi et al. / IL FARMACO 59 (2004) 645–649
647
2
2
.2. Chemistry
2.2.5. Synthesis of 2-amino-3-hydroxy-propionic acid ethyl
ester (HCl)
.2.1. Synthesis of N-(4-acetylamino-phenyl)-acetamide (I)
Acetyl chloride (7 ml, 98.1 mmol) was added to a solution
L-Serine (1 g, 9.5 mmol) was dissolved in ethanol (10 ml)
under dry HCl gas and stirred for 30 min. Extra ethanol was
then removed in vacuo and the residual thick syrup triturated
with diethyl ether to give title compound (1.25 g, 98% yield);
of benzene-1,4-diamine (5 g, 46.3 mmol) and anhydrous
sodium acetate (3 g, 36.6 mmol) in acetic acid (50 ml),
dropwise and the resulting mixture was stirred for 4 h at room
temperature. The reaction mixture was poured slowly into a
mixture of ice/water (200 ml) and kept at 4 °C overnight.
Then the precipitated product was filtered and recrystallized
from methanol to give the title compound (6.6 g, 74% yield)
–
1
m.p. 130 °C (130–131 °C) [7]. IR (KBr)/cm : 3400–3200
(
OH, NH ), 1650 (C=O). Elemental analysis: (C H NO ),
2 5 11 5
C, H, N.
2
.2.6. Synthesis of 2-amino-N-[4-(2-amino-3-hydroxy-pro-
as pink-brown flaky crystals; m.p. 303–304 °C; R 0.78
f
pionylamino)-9,10-dioxo-9,10-dihydroanthracene-1-yl]-3-
hydroxy-propionamide (V)
–
1
(
(
chloroform/ethanol, 1:2). IR (KBr)/cm , 3200 (NH), 1700
C=O). Elemental analysis: (C H N O ) C, H, N.
1
0 12 2 2
Compound IV (0.2 g, 0.84 mmol) was dissolved in cold
(0 °C) and dry dimethyl formamide (DMF) (5 ml) and tri-
ethylamine (0.224 ml, 1.6 mmol) was added and stirred to
mix at –10 °C under nitrogen. Ethylchloroformate (0.12 ml,
1.25 mmol) was added dropwise and stirred. Finally a solu-
tion of 2-amino-3-hydroxy-propionic acid ethyl ester (0.14 g,
2
.2.2. Synthesis of 2-(2,5-bis-acetylamino-benzoyl)-benzoic
acid (II)
Compound I (2.0 g, 10.4 mmol) was added to a mixture of
boric acid (H BO ) (2 g, 32.3 mmol) and phthalic anhydride
3
3
(2 g, 13.5 mmol) in H SO (7 ml, 98% purity). The reaction
2 4
mixture was stirred at 130–140 °C in an oil bath for almost
2
2
1
.05 mmol) in dry DMF (5 ml) was added to the reaction
h. The reaction was stopped by adding cold water (0 °C,
0 ml) and precipitated was filtered and recrystallized from
mixture dropwise and stirred at –10 °C for 30 min, followed
by another 30 min stirring at room temperature. The reaction
mixture was diluted with cold water and extracted with
CH Cl (3 × 50 ml), washed with 0.2 M HCl, brine and water,
ethanol to give the title compound (1.44 g, 41% yield) as
–1
yellowish flaky crystals. M.p. 220 °C, IR (KBr)/cm , 3400–
500 carboxylic groups (COOH), 3200 (NH), 1700 (C=O),
m/z, 295 (M–COOH, 5%), 282 (M–NH–CO–CH , 20%),
2
2
2
respectively and dried over MgSO . Removal of the solvent
4
3
and recrystallization from benzene/water (90:10) gave the
+
+
218 (M -C H –COOH, 38%), 191 [M -C H (CO)–COOH,
6 5 6 5
title compound (170 mg, 50% yield) as brown-orange crys-
1
00%] 177 [M-C H (C=O)–COOH–CH , 50%], 135 (M-
–1
6
4
3
tals. M.p. 200 °C, IR (KBr)/cm 3400–3200 (NH , OH),
2
C H –NHCOCH , 40%), 105 [C H (C=O), 15%]. Elemen-
tal analysis: (C H N O ) C, H, N.
+
6
5
3
6 5
1
1
700 (C=O), m/z: 413 (M , 40%), 250 (C H NO , 95%),
1
5
8
3
1
8 16 2 5
64 (C H N O , 100%), 149 (C H N O, 45%). Elemental
8
8
2
2
8 10 2
analysis: (C H N O ), C, H, N.
2
0 20 4 6
2.2.3. Synthesis of N-(4-acetylamino-9,10-dioxo-9,10-dihy-
dro-anthracene-1-yl)-acetamide (III)
2
.2.7. Synthesis of 6-amino-hexanoic acid ethyl ester
Compound II (4 g, 11.7 mmol) and H BO (3.71 g,
3
3
6
0 mmol) were dissolved in H SO (98%, 10 ml) and stirred
2 4
6-Aminohexanoic acid (1 g, 7.6 mmol) was dissolved in
at a temperature of 130–140 °C for 20 min. The reaction was
stopped by slowly pouring into a mixture of ice/water
absolute ethanol (10 ml) under dry HCl gas and stirred for
0 min. Extra ethanol was then removed to provide colorless
3
(200 ml). The precipitate was then filtered and crystallized
–1
oil. Boiling point 83 °C (81–82 °C) [8]. IR (cm ): 3200
from methanol to give pure title compound (2 g, 54% yield)
as white crystals. M.p. 200 °C, IR (KBr)/cm ; 3200 (–NH),
1
(
N.
NH ), 1650 (C=O). Elemental analysis: (C H NO ), C, H,
2 8 17 2
–1
+
+
2
700, 1650 (C=O), m/z, 322 (M , 5%), 238 [MH -(–CO–
CH ) , 20%], 148 (C H O , 30%), 105 (C H O, 100%).
3
2
8
4
3
7 5
2
.2.8. Synthesis of 6-amino-hexanoic acid [4-(5-amino-
Elemental analysis: (C H N O ), C, H, N.
18 14 2 4
pentanoylamino)-9,10-dioxo-9,10-dihydro-anthracene-1-
yl]-amide (VI)
2.2.4. Synthesis of 1,4-diamino-anthraquinone (IV)
Compound III (1 g, 3.1 mmol) was mixed with sodium
Title compound was prepared in the same procedure men-
tioned for compound (V), using a solution of compound IV
hydroxide (15%, 50 ml) and refluxed for 3 h. The product
was extracted with a mixture of isopropyl alcohol/
chloroform (2:1), (3 × 30 ml). Solvents were then evaporated
(
(
0.2 g, 0.84 mmol) and 6-amino-hexanoic acid ethyl ester
0.14 g, 0.88 mmol) in DMF to provide 190 mg (49% yield)
in vacuum and the crude solid was heated in CHCl , filtered
3
–
1
as brown solid. M.p. 320 °C, IR (KBr)/cm 3380 (NH ),
1700, 1650 (C=O), m/z: 464 (M , 5%), 378 [M -(CH ) –
NH
and pentane was added to the filtrate. On cooling, the title
compound (0.32 g, 43% yield) was obtained as brown crys-
2
+
+
2 5
–
1
+
tals. M.p. 268–270 °C. IR (KBr)/cm : 3200–3400 (NH ),
, 28%], 362 (M -C H N , 50%), 208 (9,10-
2 5 14 2
2
+
1
650 (C=O), m/z: 238 (M , 5%), 224 (M–NH , 10%), 179
anthracenedione, 30%), 148 (C H O , 28%), 132 (C H O ,
2
4
4
3
4 4 2
(
C H N O , 36%), 162 (C H N O , 100%), 93 (C H N,
58%),108 (C H N , 100%). Elemental analysis:
6 8 2
8
7
2
3
8
6
2
2
6
7
9
0%). Elemental analysis: (C H N O ), C, H, N.
(C H N O ), C, H, N.
26 32 4 4
8
6
2
2

648
H. Sadeghi-Aliabadi et al. / IL FARMACO 59 (2004) 645–649
2
2
.3. Biological studies
Table 1
In vitro cytotoxic activity of compounds V and VI in comparison to doxoru-
bicin
.3.1. Cell lines
Hela (human cervix carcinoma), KB (human caucasian/
Compounds
IC50 (µM)
KB
Hela
MDA-MB-468 K562
epidermal carcinoma), MDA-MB-468 (human breast adeno-
carcinoma) and K562 (human leukemia) cell lines were pur-
chased from Pasture Institute of Iran in Tehran. They were
grown in RPMI-1640 [each 500 ml of RPMI-1640 was
supplemented with 10% of fetal calf serum, 5 ml of
penicillin/streptomycin (50 IU ml and 500 µg ml , respec-
tively), 5 ml of sodium pyruvate (1 mM), NaHCO (1 g) and
5
Doxorubicin
Compound V
14.1 ± 1.5
22.3 ± 2.1
24.7 ± 1.1
25.2 ± 1.8
20.3 ± 1.9
4.2 ± 1.1
15.6 ± 1.6
10.7 ± 1.1
3.8 ± 0.7
12.5 ± 2.3
7.8 ± 0.9
Compound VI 16.2 ± 1.9
material and methods section. The biological effect of final
synthesized compounds against a panel of cancer cell lines
was evaluated via the MTT cytotoxicity assay and the results
are summarized in Table 1. As it shown in this table, these
compounds are cytotoxic in micromolar concentrations,
though they are almost as effective as doxorubicin, as a
positive control in this study.
–1
–1
3
ml of L-glutamine (2 mM)]. Completed media was steril-
ized by 0.22 µm microbiological filters after preparation and
kept at 4 °C before using.
2
.3.2. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
To perform the first step of these reactions, acetic anhy-
dride and acetic acid were used for acetylation of benzene-
(MTT)-based cytotoxicity assay
The cytotoxic effects of synthesized compounds against
1
,4-diamine. Analysis of the obtained product revealed that
previously mentioned human tumor cell lines were deter-
mined by a rapid colorimetric assay, using MTT bromide and
compared with untreated controls [9]. This assay is based on
the metabolic reduction of soluble MTT by mitochondrial
enzyme activity of viable tumor cells, into an insoluble col-
ored formazan product, which can be measured spectropho-
the product contain three acetyl groups and is not N-(4-
acetylamino-phenyl)-acetamide. Therefore, these reagents
were replaced by acetyl chloride and the reaction was suc-
cessfully completed.
The synthesis of the anthraquinone backbone was carried
out by Friedel–Crafts intramolecular cyclization reaction
with the aid of H BO and conc. H SO . AlCl , as a usual
reagent for Friedel–Crafts reaction, reacts in benzene as
solvent which was not a good solvent for dissolving N-(4-
acetylamino-phenyl)-acetamide. Triflic acid was used by
other researchers for this purpose [11]. Polyphosphoric acid
tometrically after dissolution in dimethyl sulfoxide (DMSO)
4
3
3
2
4
3
[10]. Briefly, 200 µl of cells (5 × 10 cells per ml of media)
were seeded in 96-well microplates and incubated for 24 h
37 °C, 5% CO air humidified). Then 20 µl of final concen-
(
2
tration of each compound was added and incubated for an-
other 72 h in the same condition. Doxorubicin was used as a
positive control. The first column of each microplate was
used as negative control (containing no compound or doxo-
rubicin). To evaluate cell survival, 20 µl of MTT solution
(PPA) is usually used for cyclization, but in this study the
longer time needed for completion of reaction with PPA
causes polymerization and low yield. Therefore, H BO , in
the presence of conc. H SO [2] was used to catalyze this
reaction which gave higher product yield and reduced the
reaction time to 20 min.
Certain amino acids (L-serine and 6-aminohexanoic acid)
were used as specific side arms in the synthesis of an-
thraquinone derivatives. These side arms, which are weakly
related to mitoxantrone and ametantrone side chains, not
only provide the proper length (4–12 atoms) to produce
hydrogenic or covalent bonds with DNA base pairs, but
3
3
(5 mg/ml in phosphate buffer solution) was added to each
2
4
well and incubated for 3 h. Then gently 150 µl of old medium
containing MTT was replaced by DMSO and pipetted to
dissolve any formed formazan crystals. Absorbance was then
determined at 540 nm by ELISA plate reader. Each concen-
tration was assayed in eight wells and repeated 4–6 times.
The drug concentration which inhibited 50% of tumor
growth (IC ) was determined. Percent of cell survival in
5
0
negative control was assumed 100%.
possess nucleophilic groups such as OH or NH to intercalate
2
2
.3.3. Statistical analysis
All results are expressed as the mean ± S.D. of at least
more easily with DNA three dimensional structures. Al-
though some researchers have applied simultaneous deriva-
tization of functional groups by an aqueous-phase
chloroformate-mediated reaction for determination of amino
acids [12,13].Amino acids solubility was increased by esteri-
fication prior to coupling reaction.
three experiments. P values for significance were determined
using the two-tailed Students t-test. Significance was as-
sumed at 5% level.
The final step in the reaction of these compounds is an
amidation process. To increase the yield, the reaction was
3. Results and discussion
performed in dry solvents under N gas and ethyl chlorofor-
2
The total synthesis of anthraquinones with proposed side
mate was used as coupling agent. Carbodiimides such as
[1-ethy1-3 (3-dimethylaminopropyl) carbodiimide hydro-
chloride] (EDC) or alkyl chloroformates (e.g. ethyl chloro-
formate) activate the COOH in the carboxylic acid moiety
arms was performed successfully. In this study m.p., IR and
elemental analysis were used to verify the structures of the
synthesized compounds. All synthetic data are shown in

H. Sadeghi-Aliabadi et al. / IL FARMACO 59 (2004) 645–649
649
and forms the intermediate O-acylisourea, which can chemi-
cally bind to exposed amino groups, such as those in com-
pound IV. In this procedure, the product obtained is essen-
tially pure and in quantitative yield [14]. In the similar way,
Antonini et al. [15] used ethyl chloroformate to synthesize
pyrimido-acridine derivatives as potential anticancer agents.
Synthesized compounds V and VI both have a similar
structure to ametantrone but compound VI, which has longer
side chains, is more cytotoxic than compound V, suggesting
that this structure is more suitable for DNA intercalating.
Although the efficacies of these compounds are almost the
same as doxorubicin, more research should be carried out to
evaluate their cardiotoxicity in comparison to doxorubicin.
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Acknowledgements
We thank Food and Drug Control Laboratory (FDCL) of
Ministry of Health, and School of Pharmacy, Tehran Univer-
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