Synthesis and in vitro cytotoxic activities of 2-alkyl-2,3-dihydro-1H-2,6- diazacyclopenta[b]anthracene-5,10-diones

Article abstract of DOI:10.1007/s12272-010-0503-z

A series of 2-alkyl-2,3-dihydro-1H-2,6-diazacyclopenta[b]anthracene-5,10- diones (4a-f) was synthesized and their in vitro cytotoxic activities were evaluated against six human cancer cell lines (HCT15, SK-OV-3, SNB19, A549, MCF7 and MCF7/ADR). They all appeared to be less potent than doxorubicin against all doxorubicin sensitive human cancer cell lines tested. However, these compounds retained considerable cytotoxic activity against the doxorubicin-resistant cell line MCF7/ADR, implying their therapeutic potential to treat doxorubicin-resistant tumors. The most active compound 4c was equipotent with doxorubicin against HCT15 cell line.

Full text of DOI:10.1007/s12272-010-0503-z

Arch Pharm Res Vol 33, No 5, 663-667, 2010
DOI 10.1007/s12272-010-0503-z
Synthesis and In Vitro Cytotoxic Activities of 2-Alkyl-2,3-dihydro-1H-
2,6-diazacyclopenta[b]anthracene-5,10-diones
Jae-Hwan Kwak1, Kwon Namgoong1, Jae-Kyung Jung1, Sang-Bae Han1, Jungsook Cho2, Hwan-Mook Kim3,
Song-Gyu Park3, Kiho Lee3, Jong Soon Kang3, and Heesoon Lee1
2
1College of Pharmacy, Chungbuk National University, Cheongju 361-763, Korea, College of Medicine, Dongguk Univer-
3
sity, Gyeongju 780-714, Korea, and Korea Research Institute of Bioscience and Biotechnology, Chungbuk 363-883, Korea
(Rceived March 9, 2010/Revised March 12, 2010/Accepted March 14, 2010)
A series of 2-alkyl-2,3-dihydro-1H-2,6-diazacyclopenta[b]anthracene-5,10-diones (4a-f) was
synthesized and their in vitro cytotoxic activities were evaluated against six human cancer
cell lines (HCT15, SK-OV-3, SNB19, A549, MCF7 and MCF7/ADR). They all appeared to be
less potent than doxorubicin against all doxorubicin sensitive human cancer cell lines tested.
However, these compounds retained considerable cytotoxic activity against the doxorubicin-
resistant cell line MCF7/ADR, implying their therapeutic potential to treat doxorubicin-resis-
tant tumors. The most active compound 4c was equipotent with doxorubicin against HCT15
cell line.
Key words: Cytotoxic activity, Diazacyclopenta[b]anthracene-5,10-diones
novel antitumor agents that can overcome the short-
comings of anthracyclines, a series of azaanthraquinone
INTRODUCTION
The anthraquinone ring system is a key structural derivatives were recently reported (Lee et al., 2003,
feature of anthracycline antibiotics such as dauno- 2004). The previous study revealed that the incor-
mycin, ametantrone, mitoxantrone and doxorubicin poration of nitrogen into anthraquinone ring system
(Brana et al., 2001). Doxorubicin (1) (Fig. 1) is the improved cytotoxic activities and a basic nitrogen with
most commonly prescribed anthracycline antibiotic for hydrophobic alkyl side chain at 3-position of azaanthr-
the treatment of cancer (Wakelin and Waring, 1990). aquinone is also important for the activity. We also re-
However, its effectiveness is restricted, due to cardio- ported anthraquinones with additional azacyclopentane
toxicity (Priebe, 1995). The mechanism of its cytotoxic ring system in which triazacyclopentaanthracene-
activity is multimodal and involves interaction with 5,10-dione derivatives (
DNA, interaction with nucleic acid condensation, much more potent than monoazacyclopentaanthracene-
inhibition of topoisomerase II activity leading to DNA 5,10-dione derivatives ( ) (Kwak et al., 2009). This
cleavage, and production of DNA damaging free radi- prompted us to prepare diazacyclopentaanthracene-
cals as a consequence of its redox properties (Brana et 5,10-dione derivatives ( ). The same series of alkyl or
2) (Kwak et al., 2008) were
3
4
al., 2001). Topoisomerase II-dependent DNA cleavage cycloalkyl substituents were introduced to the target
is a critical event responsible for the anticancer compounds in order to delineate the structure activity
activity, whereas free radical mediated DNA cleavage relationship.
is more closely related to the cardiotoxic side effects
(Brana et al., 2001).
As part of our continuous interest in the developing
MATERIALS AND METHODS
The melting points were recorded on a Electro-
thermal IA9100 digital melting point apparatus and
were uncorrected. The IR spectra were obtained using
a Jasco FT/IR-300E spectrophotometer and absorbance
peaks are reported as cm-1. The 1H-NMR spectra were
Correspondence to: Heesoon Lee, College of Pharmacy, Chung-
buk National University, Cheongju 361-763, Korea
Tel: 82-43-261-2811, Fax: 82-43-268-2732
E-mail: medchem@chungbuk.ac.kr
663

664
J.-H. Kwak et al.
Fig. 1. Structures of compound 1-4
recorded on a Bruker DPS300 NMR spectrometer dichloromethane (from 1:99 to 3:97) as the eluent.
using TMS as the internal standard and the chemical
shifts are reported as ppm (
the commercially available reagents and solvents anthracene-5,10-dione (4a)
δ). Unless otherwise stated, 2-Butyl-2,3-dihydro-1H-2,6-diazacyclopenta[b]
were used as received without further purification. The product was obtained as a yellow solid (15 mg,
1
The RPMI1640 medium was obtained from Gibco 19%); mp 146-147oC; IR (KBr) 1682, 1313 cm-1; H-
BRL. Dimethyl sulfoxide(DMSO) and the other chemi- NMR (CDCl3, 300 MHz)
cals were purchased from Sigma. Hz, 1 H, hetAr-H), 8.63 (dd,
H, hetAr-H), 8.36 (s, 1H, Ar-H), 8.09 (s, 1H, Ar-H),
7.73 (dd, = 7.9 Hz, = 4.6 Hz, 1H, hetAr-H), 2.74 (
t, = 7.0 Hz, 2H, CH2), 2.55 (s, 4H, CH2), 1.62-1.52
]quinoline-5,10- (m, 2H, CH2), 1.44-1.36 (m, 2H, CH2), 0.94 (t, = 7.2
) (500 mg, 2.11 mmol) in 1,2-dichloroethane Hz, 3H, CH3); MS m/z FAB 307 (M++1).
δ
9.11 (dd,
J
= 4.6 Hz,
J = 1.7
J
= 7.9 Hz,
J
= 1.7 Hz, 1
J
J
7,8-Bis(bromomethyl)benzo[g]quinoline-5,10-
J
dione (8)
To a solution of 7,8-dimethylbenzo[
g
J
dione (
7
(50 mL) was added NBS (1.502 g, 8.44 mmol) and
benzoylperoxide (53 mg, 0.45 mmol). The reaction 2-Cyclopropyl-2,3-dihydro-1H-2,6-diazacyclopenta
mixture was refluxed for 18 h under nitrogen atmos-
[b]anthracene-5,10-dione (4b)
phere with the irradiation of W-lamp (60-70 W). The The product was obtained as a yellow solid (48 mg,
1
solvent was evaporated in vacuo to dryness and the 65%); mp 208-209oC; IR (KBr) 1683, 1308 cm-1; H-
residue was purified by flash column chromatography NMR (CDCl3, 300 MHz)
(CH2Cl2) to give the compound as a yellow solid (580 Hz, 1 H, hetAr-H), 8.65 (dd,
mg, 69.7 %); IR (KBr) 3032, 2978, 1689, 1195 cm-1; 1H H, hetAr-H), 8.15 (s, 1H, Ar-H), 7.74 (dd,
δ
9.11 (dd,
J
= 4.5 Hz,
J
= 1.4
= 1.4 Hz, 1
= 7.8 Hz,
8
J
= 7.8 Hz,
J
J
J
NMR (acetone-d6, 300MHz)
1.7 Hz, 1 H, hetAr-H), 8.90 (s, 1 H, Ar-H), 8.69 (dd,
= 7.9 Hz,
H), 7.93 (dd,
4.90 (s, 4 H, CH2Br).
δ
9.33 (dd,
J
= 4.7 Hz,
J
=
J
= 4.5 Hz, 1H, hetAr-H), 4.22 (s, 4H, CH2), 2.11-2.03
(m, 1H, CH), 0.59-0.57 (m, 4H, CH2); 13C-NMR (CDCl3,
J
= 1.7 Hz, 1 H, hetAr-H), 8.35 (s, 1 H, Ar- 125MHz) δ 182.7, 181.7, 155.0, 149.0, 148.2, 148.0,
J
= 7.9 Hz,
J
= 4.7 Hz, 1 H, hetAr-H), 135.5, 133.0, 132.3, 130.5, 127.9, 122.0, 121.2, 58.7,
53.4, 35.4, 29.7, 6.09; MS m/z FAB 291 (M++1).
2-Cyclobutyl-2,3-dihydro-1
]anthracene-5,10-dione (4c)
The product was obtained as a dark red solid (22 mg,
H-2,6-diazacyclopenta
General procedure for 2-alkyl-2,3-dihydro-1H-
[b
2,6-diazacyclopenta[b]anthracene-5,10-diones
(4a-f)
1
A solution of 7,8-bis(bromomethyl)benzo[
g
]quinoline- 29%); mp 163-164oC; IR (KBr) 1681, 1308 cm-1; H-
5,10-dione ( ) (100 mg, 0.253 mmol) in anhydrous NMR (CDCl3, 300 MHz)
8
δ 9.12-9.10 (m, 1H, hetAr-H),
THF (5 mL) was treated with anhydrous Na2CO3 (300 8.65-8.61 (m, 1 H, hetAr-H), 8.34 (s, 1H, Ar-H), 8.08 (s,
mg) and alkylamine (0.253 mmol). The mixture was 1H, Ar-H), 7.75-7.71 (m, 1H, hetAr-H), 4.30 (s, 4H,
stirred at reflux for 4 h under nitrogen atmosphere. CH2), 3.39-3.36 (m, 1H, CH), 2.40-2.02 (m, 6H, CH2);
The mixture was diluted with water (50 mL) and ex- 13C-NMR (CDCl3, 125MHz)
δ
183.4, 182.3, 168.4, 155.5,
tracted with dichloromethane (3
layer was dried over anhydrous Na2SO4 and concen- 66.3, 54.8, 49.1, 30.1, 20.3, 15.5; MS m/z FAB 305
× 20 mL). The organic 149.8, 145.3, 136.1, 133.0, 132.0, 131.6, 129.5, 128.2,
trated in vacuo. The residue was subjected to flash (M++1).
silica gel column chromatography using methanol/

2-Alkyl-2,3-dihydro-1H-2,6-diazacyclopenta[b]anthracene-5,10-diones
665
2-Cyclopentyl-2,3-dihydro-1
H-2,6-diazacyclopenta
In vitro cytotoxicity assay
The number of cells was measured indirectly using
[b]anthracene-5,10-dione (4d)
The product was obtained as a dark red solid (27 mg, the sulforhodamine B (SRB) method according to the
1
34%); mp 167-168oC; IR (KBr) 1683, 1308 cm-1; H- NCI (USA) protocol (Skehan et al., 1990). Briefly, the
NMR (CDCl3, 300 MHz)
H), 8.66-8.65 (m, 1 H, hetAr-H), 8.26 (s, 1 H, Ar-H),
δ
9.12 - 9.11 (m, 1 H, hetAr- cells were plated into a 96 well plate at a density of 2
×
103 cells per well. On the next day (day 0), the com-
8.15 (s, 1 H, Ar-H), 7.77-7.72 (m, 1 H, hetAr-H), 4.13 pounds of interest dissolved in DMSO/media were
(s, 4 H, CH2), 3.02-2.87 (m, 1 H, CH), 1.42-1.25 (m, 8 added in quadruplicate. The final concentration of
H, CH2); MS m/z FAB 319 (M++1).
each compound ranged from 1 nM - 10 µM and the
final concentration of DMSO was < 0.1%. Seventy-two
hours later, the cells were fixed with 10% trichloroace-
tic acid (TCA) overnight at 4oC. The TCA-treated cells
2-Cyclohexyl-2,3-dihydro-1
H-2,6-diazacyclopenta
[b]anthracene-5,10-dione (4e)
The product was obtained as a yellow solid (26 mg, were washed extensively with distilled water and
1
31%); mp 163-164oC; IR (KBr) 1683, 1310 cm-1; H- dried in air. A SRB solution (0.4% in 1% acetic acid)
NMR (CDCl3, 300 MHz)
δ 9.10-9.05 (m, 1H, hetAr-H), was then added to the well at room temperature for
8.74-8.68 (m, 1H, hetAr-H), 8.28 (s, 1H, Ar-H), 8.24 (s, one hour. The bound dye was dissolved in 10 mM Tris
1H, Ar-H), 7.94-7.90 (m, 1H, hetAr-H), 4.20 (s, 4H, CH2), after washing the wells with 1% acetic acid. The absor-
2.62-2.57 (m, 1H, CH), 1.03-0.90 (m, 10H, CH2); MS bances were measured at 690 nm using a microplate
m/z FAB 333 (M++1).
reader. The absorbance of the day 0 sample was sub-
tracted from the absorbance of the day 3 sample.
2-(2-Diethylaminoethyl)-2,3-dihydro-1H-2,6-diaza-
cyclopenta[b]anthracene-5,10-dione (4f)
RESULTS AND DISCUSSION
The product was obtained as a dark red solid (33 mg,
37 %); mp 116-117oC; IR (KBr) 3418, 1683, 1311 cm-1;
The target compounds were prepared using our
1H-NMR (CDCl3, 300 MHz)
δ
9.13-9.11 (m, 1 H, hetAr- previous synthetic rout developed for triaza analogues
H), 8.66-8.64 (m, 1 H, hetAr-H), 8.27 (s, 1 H, Ar-H), (Kwak et al., 2008) and outlined in Scheme 1. Diels-
8.18 (s, 1 H, Ar-H), 7.75-7.73 (m, 1 H, hetAr-H), 2.97- Alder cycloaddition of quinoline-5,8-dione ( ) (Pratt and
2.88 (m, 4 H, CH2), 2.54 (s, 4H, CH2), 2.02 (q, = 7.1 Drake, 1960) with 2,3-dimethylbutadiene ( ) was carried
= 7.1 Hz, 6 H, CH3); MS m/z out according to the reported procedure (Lee et al.,
2002). The starting compound was treated with 2,3-
dimethylbutadiene ( ) to give the cycloaddition adduct
which was directly aromatized by aerial oxidation in
ethanolic KOH at reflux to give the 6,7-dimethyl-
SNB19, A549, MCF7 and MCF7/ADR were used in benzo[g]quinoline-5,10-dione ( ). The intermediate
this study. All the cells were obtained from the Na- was then treated with -bromosuccinimide (NBS) and
5
6
J
Hz, 4 H, CH2), 1.27 (t,
FAB 350 (M++1).
J
5
6
Cells
Six human cancer cell lines, HCT15, SK-OV-3,
5N
7
7
N
tional Cancer Institute, U.S.A. These cells were main- a catalytic amount of benzoylperoxide in anhydrous
tained in RPMI1640 media supplemented with 10% 1,2-dichloroethane at reflux for 48 h with the irradia-
fetal calf serum at 37oC under a humidified atmos- tion of tungsten lamp to give the bisbromomethyl pro-
phere of 5% CO2.
duct 8 in 42% yield. Treatment of the compound 8
Scheme 1. Synthesis of 2-alkyl-2,3-dihydro-1H-2,6-diazacyclopenta[b]anthracene-5,10-diones (4a-f)

666
J.-H. Kwak et al.
with the appropriate primary alkyl or cycloalkyl amines activity. This result was consistent with our previous
in anhydrous DMF afforded n-butyl- 4a (19%), cyclo- observation with triaza analogues (Kwak et al., 2008).
propyl- 4b (65%), cyclobutyl- 4c (29%), cyclopentyl- 4d Compound 4a bearing
(34%), cyclohexyl- 4e (31%), and diethylaminoethyl- 4f times less potent than the other analogues (4b-e
(37%) substituted diazacyclopenta[ ]anthracene-5,10- containing cycloalkyl side chain. The ring size of the
dione derivatives respectively. All the spectroscopic side chain was not important for the cytotoxic activi-
data were consistent with the expected structures. ties. The most active compound 4c showed comparable
In continuation of our effort to identify novel azaan- cytotoxic activity against HCT15 cancer cell line to
thraquinone derivatives, the target compounds ( that of doxorubicin. With respect to the number of
n-butyl side chain was 2-4
)
b
4
)
were designed to have nitrogen at 2 and 6 position of ring nitrogen, the activity decreased in the order of
cyclopentaanthracene ring system. Various alkyl or 2,6,9-triaza series (Kwak et al., 2008) > 2,6-diaza
cycloalkyl substituents were introduced on 2-nitrogen series (4) > 2-aza series (Kwak et al., 2009). This
of the target compounds (4) to delineate the structure result suggests that the aromatic ring nitrogen plays
activity relationship.
an important role for the cytotoxic activity.
The newly synthesized 2-alkyl-2,3-dihydro-1
diazacyclopenta[ ]anthracene-5,10-dionederivatives
4a-f) were initially evaluated for their in vitro (MCF7/ADR) with > 146-fold resistant to doxorubicin.
H-2,6-
The analogues were also screened in vitro against a
b
human breast cancer cell line (MCF7) and a subline
(
cytotoxicity against four human cancer cell lines Their respective IC50 values and the resistance index
originating from colon (HCT-15), ovarian (SK-OV-3), (RI), the IC50 against the resistant cell line divided by
CNS (SNB19), and lung (A549) cancer according to the IC50 against the sensitive cell line, are shown in
the protocols developed by the National Cancer Insti- Table I. All analogues were less potent than doxorubi-
tute (Skehan et al., 1990). The concentrations of the cin against the sensitive MCF7 cancer cell line with
target compounds 4a-f inhibiting cellular growth by the cyclobutyl compound 4c and the cyclopentyl com-
50%, IC50 values, are shown in Table I. The compara- pound 4d being the most potent. However, it should
tive data for doxorubicin are also shown for compari- be noted that compounds 4a-e retained much of their
son.
activity against the doxorubicin-resistant MCF7/ADR
All analogues tested in this study exhibited signi- cancer cell line. Compound 4a and 4e possessed the
ficant cytotoxic activity except for the compound 4f most favorable resistance index.
(IC50 = 18.539 against A549 and IC50 > 30
HCT15, SK-OV-3, and SNB19). Compounds 4a-e were zacyclopenta[
less potent than doxorubicin against all doxorubicin ) and evaluated their in vitro cytotoxic activities
µM against
In summary, we have synthesized a series of dia-
b
]anthracene-5,10-dione derivatives (4a
-
f
sensitive human cancer cell lines tested. Compounds against six human cancer cell lines (HCT15, SK-OV-3,
4a-e have hydrophobic alkyl or cycloalkyl side chain SNB19, A549, MCF7 and MCF7/ADR). The most active
while compound 4f contains hydrophilic side chain compound 4c bearing cyclobutyl side chain exhibited
containing basic nitrogen, resulting poor cytotoxic comparable cytotoxicity against HCT15 cancer cell
Table I. In vitro cytotoxic activity of 2-alkyl-2,3-dihydro-1
H-2,6-diazacyclopenta[
b]anthracene-5,10-diones (4a-f) against
human cancer cell lines
IC50
(
µ
M)a of cell linesb
Comp.
-R
RIc
HCT15
SK-OV-3
SNB19
A549
MCF7
MCF7/ADR
4a
4b
4c
4d
4e
4f
-n-Butyl
3.886
1.508
0.911
1.441
1.710
>30
6.871
4.760
3.564
5.189
5.068
>30
6.795
3.731
2.492
3.363
4.100
>30
4.035
1.386
1.036
1.156
1.430
4.522
1.981
1.747
1.749
2.984
>30
4.777
4.180
2.534
3.498
3.323
>30
001.1
002.1
001.5
002.0
001.1
-
-Cyclopropyl
-Cyclobutyl
-Cyclopentyl
-Cyclohexyl
-
N,N-diethylaminoethyl
Doxorubicin (
aIC50 = concentration of compound (
18.5390
1)
0.583
0.274
0.315
0.175
0.205
>30
146.4
µ
M) required to inhibit the cellular growth by 50% after 72 h of drug exposure, as
determined by the SRB assay. Each experiment was run at least three times, and the results are presented as an
average value.
bHuman cancer cell lines: HCT-15 (colon cancer), SK-OV-3 (ovarian carcinoma), SNB-19 (CNS), A549 (lung cancer),
MCF7 and MCF7/ADR (breast cancer).
cRI: Resistance Index IC50 (MCF7/ADR) / IC50 (MCF7)

2-Alkyl-2,3-dihydro-1H-2,6-diazacyclopenta[b]anthracene-5,10-diones
667
agents. Arch. Pharm. Res., 25, 416-420 (2002).
line to that of doxorubicin. The Resistance Index IC50
(MCF7/ADR)/IC50 (MCF7) (1.1-2.1) of compound 4a
implies their therapeutic potential to treat doxorubi-
cin-resistant tumors. This may warrant further in
depth biological evaluations.
Lee, H., Lee, S-. I., Cho, J., Choi, S-. U., and Yang, S-. I., Syn-
thesis and in vitro evaluation of 1,8-diazaanthraquinones
bearing 3-dialkylaminomethyl or 3-(N-alkyl- or N-aryl)
carbamoyloxymethyl substituent. Eur. J. Med. Chem., 38,
695-702 (2003).
-e
Lee, H., Cho, S., Namgoong, K., Jung, J-. K., Cho, J., and
Yang, S-. I., Synthesis and in vitro evaluation of 7-di-
alkylaminomethylbenzo[g]quinoxaline-5,10-diones. Bioorg.
Med. Chem. Lett., 14, 1235-1237 (2004).
ACKNOWLEDGEMENTS
This work was supported by Chungbuk National
University Grant in 2008.
Pratt Y. T. and Drake, N. L., Quinolinequinones. V. 6-chloro-
and 7-chloro-5,8-quinolinequinones. J. Am. Chem. Soc.
82, 1155-1161 (1960).
,
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