Synthesis of symmetrical 1,5-bisacyloxyanthraquinone derivatives and their dual activity of cytotoxicity and lipid peroxidation

Article abstract of DOI:10.1002/ardp.200290002

Symmetrical bis-substituted anthraquinones were successfully prepared and demonstrated potent cytotoxicity against the growth of suspended murine and human tumors, i.e. rat glioma C6 cells and human hepatoma G2 cells. We report here a convenient synthetic pathway that leads to symmetrically substituted 1,5-bisacyloxyanthraquinone derivatives. Acylation of the hydroxyl group of 1,5-dihydroxyanthraquinone with the appropriate acyl chlorides in the presence of pyridine or sodium hydride, respectively, furnished this structural class of anthraquinones. The bis(butyryloxy) analog 2 b, bis(2-chlorobenzoyl) analog 2 f, and bisphenylpropionyloxy analog 2 n exhibit potent cytotoxicity in inhibition of human hep G2 cell growth in culture, as determined by using XTT colorimetric assay, while their antiproliferative activity is markedly enhanced and is comparable to that of the anticancer agent mitoxantrone. In addition, redox properties of the compounds for the inhibition of lipid peroxidation in model membranes were determined. Compounds 2n also exhibited stronger antioxidant activity than ascorbic acid, (+)-α-tocopherol, and anthrarufin. Biological evaluation and SAR studies of these symmetrical anthraquinones have been performed and the results are discussed.

Full text of DOI:10.1002/ardp.200290002

Arch. Pharm. Pharm. Med. Chem. 2002, 10, 481–486
Symmetrical 1,5-Bisacyloxyanthraquinones 481
Hsu-Shan Huanga,
Synthesis of Symmetrical
1,5-Bisacyloxyanthraquinone Derivatives and Their
Dual Activity of Cytotoxicity and Lipid Peroxidation
Hui-Fen Chiub,
Jeng-Fong Chiouc,
Pen-Fong Yehd,
Chi-Wei Taod,
Wei-Ren Jenga
Symmetrical bis-substituted anthraquinones were successfully prepared and dem-
onstrated potent cytotoxicity against the growth of suspended murine and human
tumors, i.e.rat glioma C6 cells and human hepatoma G2 cells.We report here a con-
venient synthetic pathway that leads to symmetrically substituted 1,5-bisacyloxyan-
thraquinone derivatives. Acylation of the hydroxyl group of 1,5-dihydroxyanthraqui-
none with the appropriate acyl chlorides in the presence of pyridine or sodium hy-
dride, respectively, furnished this structural class of anthraquinones. The bis(buty-
ryloxy) analog 2 b, bis(2-chlorobenzoyl) analog 2 f, and bisphenylpropionyloxy ana-
log 2 n exhibit potent cytotoxicity in inhibition of human hep G2 cell growth in culture,
as determined by using XTT colorimetric assay, while their antiproliferative activity is
markedly enhanced and is comparable to that of the anticancer agent mitoxantrone.
In addition, redox properties of the compounds for the inhibition of lipid peroxidation
in model membranes were determined.Compounds 2n also exhibited stronger anti-
oxidant activity than ascorbic acid, (+)-α-tocopherol, and anthrarufin. Biological
evaluation and SAR studies of these symmetrical anthraquinones have been per-
formed and the results are discussed.
a
School of Pharmacy,
National Defense Medical
Center, Taipei, Taiwan,
R.O.C.
Department of
Pharmacology, Kaohsiung
Medical University,
Kaohsiung, Taiwan, R.O.C.
Department of Radiotherapy
and Oncology, Taipei
Medical University, Taipei,
Taiwan, R.O.C.
b
c
d
Cheng-Hsin Medical Center,
Taipei, Taiwan, R.O.C.
Keywords: Anthraquinone;Cytotoxicity;Rat glioma C6 cells;Human hepatoma G2
cells; XTT colormetric assay; Lipid peroxidation
Received: March 7, 2002 [FP679]
management of antitumor therapy is necessarily com-
plementary and antioxidation is one of the most interest-
Introduction
A number of analogs of anthraquinone have been syn-
thesized and evaluated both for preclinical antitumor effi-
cacy as well as for biochemical pharmacology [1–5]. In-
tercalating agents continue to occupy a prominent posi-
tion in the treatment of malignant diseases and thus the
antitumor and biochemical effects of these compounds
remain subjects of intensive research. The anthraqui-
none (anthracene-9,10-dione) mitoxantrone has been
shown to have outstanding antitumor activities but a
much narrower spectrum of activity in comparison with
those of the anthracyclines [3]. Anthracyclines and an-
thraquinones form ternary complexes with DNA and the
enzyme and stimulate DNA cleavage in a sequence-
specific manner [6]. Consequently, considerable effort
has been invested in the development of structural ana-
logs or other classes of DNA binders which may circum-
vent or at least reduce this disadvantage. Based on
these facts, there are still demands for synthesis of new
analogs of anthraquinone to provide good antitumor ac-
tivities and less toxic side effects. On the other hand,
ing aspects investigated in this context. Previously, we
have developed series of structurally related substituted
1,5-and 1,8-dichloroanthracenes as potential antitumor
analogs.The positional attachment of the side chain has
been shown to profoundly influence their activity.There-
fore, the need still exists for anthraquinone or anthracene
congeners endowed with improved therapeutic efficacy
and less toxic side effects, as well as effectiveness
against multiple drug-resistant (MDR) cell lines [7]. As in
the case of the anthracyclines, the mechanisms of cell
kill by the anthraquinones are poorly understood. The
goal of the present study was an evaluation of the impor-
tance of the side-arm substitution patterns at the 1 and 5
positions of the anthraquinone skeleton: not only would
the nature of the side arms influence the binding or kinet-
ic dissociation rate of the anthraquinone-DNA interca-
lant but they might also be intimately involved in lipid per-
oxidation. This has enabled us to address the issue of
how, and to what extent, the position of side chain sub-
stituents affects biological activity.
Correspondence: Hsu-Shan Huang, School of Pharmacy,
National Defense Medical Center, No.161, Mingchuan East
Road, Section 6, P.O.Box 90048-508, Neihu 114,
Taipei, Taiwan, R.O.C. Phone: +886287926454, Fax:
+886287923169, e-mail: huanghs@ndmctsgh.edu.tw.
Results
The synthesis of 1,5-bisacyloxyanthraquinone deriva-
tives shown in Figure 1 was accomplished using proce-
© 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/10/0481 $ 17.50+.50/0

482 Huang et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 481–486
of controls. The bis(butyryloxy) analogue 2 b demon-
strates that the potent cytotoxicity could be regained
without sacrificing potent antioxidant properties. The
acyl substitution of 2 b leads to the highest activity
against Hep G2 cell line when compared with mitox-
antrone (about 100 times stronger); the bis-aromatic
substitution of 2 f also shows good activity (about
50 times stronger); it is interesting that the differences
between 2 b and 2 n are the end of substituted side
chains, which are bis-butyroyloxy group and bis-(2-chlo-
robenzoyl) group, respectively. Moreover, bis(2-chlo-
robenzoyl) substituted as in 2 f even has slightly less
cytotoxicity;however, the potency against lipid peroxida-
tion was reduced. Introduction of phenylpropionyloxy
groups into the 1,5-position linking the anthraquinone
nucleus resulted in a decrease of cytotoxicity but the po-
tent lipid peroxidation was strongly increased. Com-
pounds 2 n exhibited stronger antioxidant activity than
ascorbic acid, (+)-α-tocopherol and mitoxantrone. In
contrast to (+)-α-tocopherol and ascorbic acid, 2 a, 2 c,
2 d, 2 e, and 2 k do not enhance lipid peroxidation in
model membranes but exhibit prooxidation.The lipid per-
oxidation results show that not all members this series of
compounds, except for 2 n, are good antioxidants; they
may be promoters against lipid peroxidation. However, it
is necessary to further investigate which 2 n was addi-
tionally evaluated at several concentrations against
ascorbic acid and α-tocopherol with lipid peroxidation,
giving the results shown in Table 2. At the concentration
0.01 mM, 2 n retained 50 % activity;the activity of ascor-
bic acid decreased to 10 %; α-tocopherol no longer had
any more effect at 0.1 mM. It seems that 2 n is a better
antioxidant against ascorbic acid and α-tocopherol.
Based on this character, 2 n could be a potent antioxi-
dant to protect normal cells from oxidative damage. By
cross-linkage of the two biological evaluations, we can
screen the compounds which are active in these evalua-
tions. Among these compounds, 2 n is the most active
one, both in its cytotoxicity and in its lipid-peroxidation.
Figure 1. Reagents: (a) Method A, pyridine, CH2Cl2, N2,
R = (CH2)nCH3, n = 1,2,5; C6H5; 2-CH3C6H4; 4-CH3C6H4;
CH2C6H5; Method B, NaH, THF, R = C(CH3)3; 2-ClC6H4;
3-ClC6H4; 4-ClC6H4; 2,4-Cl2C6H3; 3-CH3C6H4.
dures somewhat modified from those described else-
where [8–12].The method of preparation of the symmet-
rical bis-substituted anthraquinones was based on that
of simple acylation involving 1,5-dihydroxyanthraqui-
none (anthrarufin) with an excess of the appropriate acyl
chlorides in the presence of pyridine and dichlorometh-
ane at room temperature for 1 to 2 hours; or in the pres-
ence of NaH and THF at room temperature or under re-
flux for 1 to 2 hours. Accordingly, acylation of the appro-
priate anthraquinones with the appropriate acyl chloride
gave the bis-substituted anthraquinones in essentially
quantitative yield. Structural assignments for the sym-
1
metrical products as 2 a–n are based on H and 13C-
NMR spectra. Compound 2 a shows absorption for the
H-4,8 protons at δ 8.16 (d, 2 H, J = 7.7 Hz), H-3,7 protons
at δ 7.75 (t, 2 H, J = 8.0 Hz), and H-2,6 protons at δ 7.37
(d, 2 H, J = 8.1 Hz), respectively.Various reaction condi-
tions and solvents were used and details are given in the
experimental section. Furthermore, the 13C-NMR spec-
tra of these compounds showed the carbonyl resonance
of anthraquinone chemical shift in the δ 181.1 region for
C-9,10.The structures and biological activity of the sym-
metrical bis-substituted anthraquinones are listed in
Table 1. Their in vitro cytotoxicity and lipid peroxidation
properties are reported and compared with those of mi-
toxantrone, ascorbic acid, (+)-α-tocopherol, and starting
material, respectively. The most active compounds dis-
playing cytotoxicity was 2 b, 2 f, and 2 n with IC50 values
0.02, 0.04, and 0.4 µM against hep G2 cells, compared
to mitoxantrone with IC50 value 2.0 µM, which indicated
about 5 to 100 times the potency of the positive control,
while lipid peroxidation was somewhat weaker than that
Discussion
Based on the results of cytotoxic evaluation, we find that
2 b, with non-polar butyryl side chain at 1,5-positions,
has the highest activity being 100 times stronger than
mitoxantrone. The compound with the aromatic 2-chlo-
robenzoyl group, 2 f, also has good activity, being about
50 times stronger than mitoxantrone.Compared with 2 b,
2 f, and 2 n, the cytotoxicity of 2 n is lower although it is
still 5 times stronger than that of mitoxantrone. Perhaps
both lipophilic and van der Waals forces were needed to
interact with the active site of Hep G2 cell line or helped
to penetrate through the cell membrane; in addition, the

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 481–486
Symmetrical 1,5-Bisacyloxyanthraquinones 483
Table 1. Cytotoxicity against the growth of suspended murine and human tumors and inhibitory effect of anthraquinone
derivatives on iron-induced lipid peroxidation in rat brain homogenates.
Compd
R
IC50 (µM)a
Inhibition of LP %
(10 mM)b
Hep G2c
C6 cellsd
2 a
2 b
2 c
2 d
2 e
2 f
2 g
2 h
2 i
2 j
2 k
2 l
2 m
2 n
CH2CH3
4.1 ± 0.5
0.02 ± 0.01
36.2 ± 2.5
13.7 ± 1.8
47.7 ± 5.5
0.04 ± 0.01
15.1 ± 1.9
48.1 ± 4.5
>50
21.6 ± 2.2
18.1 ± 1.5
9.3 ± 0.9
9.0 ± 1.5
0.4 ± 0.1
2.0 ± 0.5
21.1 ± 1.6
38.5 ± 2.8
39.1 ± 4.1
12.0 ± 1.5
38.7 ± 3.6
40.7 ± 4.7
25.1 ± 2.8
38.6 ± 3.5
38.4 ± 4.4
25.1 ± 2.8
30.1 ± 3.3
37.6 ± 4.1
39.1 ± 6.2
40.1 ± 5.5
0.07 ± 0.01
–100
54 ± 2.2
–55 ± 1.5
–23 ± 1.1
–50 ± 1.9
5 ± 0.5
1 ± 0.1
2 ± 0.2
–1 ± 0.1
23 ± 1.1
–32 ± 1.5
33 ± 1.2
–1 ± 0.2
>100
CH2CH2CH3
CH2CH2CH2CH2CH3
C(CH3)3
C6H5
2-ClC6H4
3-ClC6H4
4-ClC6H4
2,4-Cl2C6H3
2-CH3C6H4
3-CH3C6H4
4-CH3C6H4
CH2C6H5
CH2CH2C6H5
mitoxantrone
ascorbic acid
(+)-α-tocopherol
anthrarufin
>100
>100
>100
–36 ± 1.1
a
IC50, drug concentration inhibiting 50 % of cellular growth following 48 h of drug exposure.Values are in µM and repre-
sent an average of three experiments.The variance for the IC50 was less than ±20 %.Inhibition of cell growth was sig-
nificantly different with respect to that of the control; N = 3 or more, P < 0.01.
Relative percentage of inhibition. Inhibition was compared to that of the control [ascorbic acid, >α-tocopherol and
mitoxantrone-HCl], P < 0.01, Mean ± S.E., n = 4.Values are mean percent inhibition at the indicated concentration
(mM), and standard errors.
b
c
Hep G2: human hepatoma G2 cells.
C6 cells: rat glioma C6 cells.
d
Table 2. Inhibitory effects of 2 n on iron-induced lipid peroxidation in rat brain homogenates.
Compound
Inhibition (%)a
10 mM
1 mM
>100
75 ± 1.5
55 ± 1.7
54 ± 2.1
0.1 mM
0.01 mM
2 n
>100
100
100
100
95 ± 2.0
32 ± 1.2
0
50 ± 0.8
10 ± 0.6
0
ascorbic acid
(+)-α-tocopherol
mitoxantrone-HCl
22 ± 3.5
5 ± 0.3
a
Relative percentage of inhibition. Inhibition was compared to that of the control [ascorbic acid, (+)-α-tocopherol and
mitoxantrone-HCl], P < 0.01, Mean ± S.E., n = 4.Values are mean percent inhibition at the indicated concentration
(mM), and standard errors.

484 Huang et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 481–486
were washed with water and dried (MgSO4), and concentrated.
The resulting precipitate was collected by filtration, washed
with water, and further purified by crystallization and chroma-
tography.
distance between two forces may also be important.
Substituted side chains of the three active compounds
are obviously less polar than that of mitoxantrone. This
means that on Hep G2 cell lines, the three non-polar side
chains showed better affinities than polar side chains of
mitoxantrone. Finally, 2 b, 2 f, and 2 n are potent anti-
tumor agent against human hepatoma cell line. In lipid
peroxidation, 2 n was the best antioxidant, even at a con-
centration 0.01 mM. It is a potent antioxidant protecting
normal cells from oxidative damage, although the exact
mechanism still needs to be investigated.The results of
cytotoxic tests and lipid peroxidation show 2 n to be the
most active among these compounds.The interrelation-
ships between results from the two tests of 2 n are not
very clear and it will be worthwhile undertaking further
investigations.
1,5-Bis(propionyloxy)anthraquinone (2 a)
The title compound was obtained from 1 and acetyl chloride ac-
cording to Method A. Recrystallization from ethanol gave yel-
1
low needles; 55 % yield; mp 230–231 °C; H-NMR (CDCl3) δ
1.34 (t, J = 7.5 Hz, 6 H), 2.80 (q, J = 7.5 Hz, 4 H), 7.37 (d, J =
8.1 Hz, 2 H), 7.75 (t, J = 8.0 Hz, 2 H), 8.16 (d, J = 7.7 Hz, 2 H);
FTIR (KBr): 1759, 1674 cm–1; UV νmax (CHCl3) nm (log ε): 318
(2.48); MS m/z = 352 (4, M+), 296 (23), 240 (100); Anal.
C20H16O6 (C, H).
1,5-Bis(butyryloxy)anthraquinone (2 b)
The title compound was obtained from 1 and butyryl chloride
according to Method A.Recrystallization from ethanol gave yel-
low needles;59 % yield;mp 211–213 °C;1H-NMR (CDCl3) δ 1.1
(t, J = 7.4 Hz, 6 H), 1.83–1.90 (m, 4 H), 2.75 (t, J = 7.5 Hz, 4 H),
7.36 (d, J = 8.0 Hz, 2 H), 7.74 (t, J = 7.9 Hz, 2 H), 8.16 (d, J =
7.8 Hz, 2 H); 13C-NMR (CDCl3) δ 181.14, 172.03, 150.08,
135.93, 134.88, 129.65, 125.63, 124.45, 36.15, 18.04, 13.75;
UV νmax (CHCl3) nm (log ε): 319 (2.51); FTIR (KBr) 1757,
1676 cm–1; MS m/z 380 (3, M+), 310 (22), 240 (100); Anal.
C22H20O6 (C, H).
Acknowledgements
This research was partially supported by grants from
NDMC of the R.O.C. The authors are indebted to Dr.
K. K. Mayer (Universität Regensburg, Germany) for the
mass spectrometric analytical determinations.
1,5-Bis(hexanoyloxy)anthraquinone (2 c)
The title compound was obtained from 1 and hexanoyl chloride
according to Method A.Recrystallization from ethanol gave yel-
1
low needles; 74 % yield; mp 183–184 °C; H-NMR (CDCl3) δ
0.95 (t, J = 7.1 Hz, 6 H), 1.38–1.49 (m, 8 H), 1.84 (q, J = 7.4 Hz,
4 H), 2.76 (t, J = 7.7 Hz, 4 H), 7.40 (dd, J = 7.8, 1.0 Hz, 2 H),7.74
(t, J = 8.1, 7.8 Hz, 2 H), 8.16 (t-like, J = 7.7, 2.3 Hz, 2 H); 13C-
NMR (CDCl3) δ 181.14, 172.22, 150.10, 135.93, 134.87,
129.64, 125.63, 124.45, 34.26, 31.35, 24.18, 22.39, 13.97; UV
Experimental
Materials and apparatus
Melting points were determined with a Büchi B-545 melting
point apparatus and are uncorrected. All reactions were moni-
tored by TLC (silica gel 60 F254), flash column chromatography:
silica gel (E. Merck, 70–230 mesh) with CH2Cl2 as eluent.
1H-NMR: Varian GEMINI-300 (300 MHz) and Brucker AM-500
(500 MHz); δ values are in ppm relative to TMS as an internal
standard. Fourier-transform-IR spectra (KBr): Shimadzu FTIR-
87000 spectrometer. UV spectra: Shimadzu UV-160A spec-
trometer. Mass spectra (EI, 70 eV, unless otherwise stated):
Varian MAT 311A EIMS and Finnigan MAT 95 EIMS (Univer-
sität Regensburg).
ν
max (CHCl3) nm (log ε) 318 (2.44);FTIR (KBr) 1755, 1676 cm–1
;
MS m/z 436 (4, M+), 338 (24), 240 (100); Anal. C26H28O6 (C, H).
1,5-Bis(pivaloyloxy)anthraquinone (2 d)
The title compound was obtained from 1 and pivaloyl chloride
according to Method B.Recrystallization from ethanol gave yel-
1
low needles; 25 % yield; mp 166–167 °C; H-NMR (CDCl3) δ
1.47 (s, 18 H), 7.31 (d, J = 8.1 Hz, 2 H), 7.72 (d, J = 8.0 Hz, 2 H),
8.16 (d, J = 7.5 Hz, 2 H); 13C-NMR (CDCl3) δ 181.00, 176.66,
150.40, 135.98, 134.62, 129.38, 125.59, 124.78, 39.21, 27.23;
UV νmax (EtOH) nm (log ε) 363 (1.40); FTIR (KBr) 1751,
1670 cm–1; MS m/z 408 (3, M+), 324 (23), 240 (100); Anal.
C24H24O6 (C, H).
Synthesis
General procedure for the preparation of 1,5-bisacyloxy an-
thraquinones
1,5-Bis(benzoyloxy)anthraquinone (2 e)
The title compound was obtained from 1 and benzoyl chloride
according to Method A.Recrystallization from ethanol gave yel-
low needles; 76 % yield; mp 336–338 °C (lit.[13] mp 342 °C);
1H-NMR (CDCl3) δ 7.50 (d, J = 7.9 Hz, 2 H), 7.56 (t, J = 7.7 Hz,
4 H), 7.68 (t, J = 7.3 Hz, 2 H), 7.77 (t, J = 7.9 Hz, 2 H), 8.17 (d, J =
7.7 Hz, 2 H), 8.29 (d, J = 7.7 Hz, 4 H);UV νmax (CHCl3) nm (log ε)
340 (0.69); FTIR (KBr) 1734, 1672 cm–1; MS m/z 448 (4, M+),
105 (100); Anal. C28H16O6 (C, H).
Method A:To a solution of anthrarufin (4.25 mmol) and pyridine
(20 mL) in dry CH2Cl2 (150 mL) was added dropwise a solution
of an appropriate acyl chloride (10 mmol) in dry CH2Cl2 (10 mL)
at 0 °C under N2.The reaction mixture was stirred or refluxed for
1–2 hours.Water (250 mL) was added and the mixture then ex-
tracted with dichloromethane. The combined organic extracts
were washed with water and dried (MgSO4), and concentrated.
The resulting precipitate was collected by filtration, washed
with water, and further purified by crystallization and chroma-
tography.
1,5-Bis(2-chlorobenzoyl)anthraquinone (2 f)
The title compound was obtained from 1 and o-chlorobenzoyl
chloride according to Method B. Recrystallization from THF
gave yellow needles; 39 % yield; mp 254–255 °C; 1H-NMR
(CDCl3) δ 7.47–7.55 (m, 8 H), 7.80 (t, J = 7.9 Hz, 2 H), 8.22 (d,
J = 7.8 Hz, 2 H), 8.39 (d, J = 7.7 Hz, 2 H); UV νmax (CHCl3) nm
(log ε) 334 (2.20); FTIR (KBr) 1747, 1672 cm–1; MS m/z 516 (2,
M+), 139 (100); Anal. C28H14Cl2O6 (C, H).
Method B: To a solution of anthrarufin (4.25 mmol) in dry THF
(20 mL) and NaH (12.75 mmol) was added dropwise a solution
of an appropriate acyl chlorides (3 mmol) in dry THF (10 mL) at
0°C under N2. The reaction mixture was stirred or refluxed for
1–2 hours.Water (250 mL) was added and the mixture then ex-
tracted with dichloromethane. The combined organic extracts

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 481–486
Symmetrical 1,5-Bisacyloxyanthraquinones 485
181.06, 170.12, 149.99, 135.87, 134.95, 133.33, 129.76,
129.57, 128.63, 127.31, 125.83, 124.28, 41.13; UV νmax
(CHCl3) nm (log ε) 318 (2.20); FTIR (KBr) 1763, 1670 cm–1; MS
m/z 358 (5, M+), 240 (94), 118 (100); Anal. C30H20O6 (C, H).
1,5-Bis(3-chlorobenzoyl)anthraquinone (2 g)
The title compound was obtained from 1 and m-chlorobenzoyl
chloride according to Method B. Recrystallization from THF
gave yellow needles; 49 % yield; mp 301–302 °C; 1H-NMR
(CDCl3) δ 7.50–7.52 (m, 4 H), 7.65 (d, J = 7.4 Hz, 2 H), 7.79 (t,
J = 7.9 Hz, 2 H), 8.16–8.19 (m, 4 H), 8.26 (s, 2 H); UV νmax
(CHCl3) nm (log ε) 351 (0.33); FTIR (KBr) 1744, 1674 cm–1; MS
m/z 516 (5, M+), 141 (35), 139 (100); Anal. C28H14Cl2O6 (C, H).
1,5-Bis(phenylpropionyloxy)anthraquinone (2 n)
The title compound was obtained from 1 and phenylpropionyl
chloride according to Method A. Recrystallization from THF
gave yellow needles; 62 % yield; mp 219–220 °C; 1H-NMR
(CDCl3) δ 3.10 (s, 4 H), 3.17 (t, J = 7.9, 1.4 Hz, 4 H), 7.22–7.35
(m, 12 H), 7.74 (t, J = 7.9 Hz, 2 H), 8.16 (dd, J = 7.7, 0.8 Hz, 2 H);
13C-NMR (CDCl3) δ 181.08, 171.35, 149.97, 140.39, 135.88,
134.96, 129.62, 128.57, 128.47, 126.36, 125.75, 124.33,
35.86, 30.58;UV νmax (CHCl3) nm (log ε) 318 (1.10);FTIR (KBr)
1761, 1676 cm–1;MS m/z 504 (5, M+), 372 (10), 240 (100);Anal.
C32H24O6 (C, H).
1,5-Bis(4-chlorobenzoyl)anthraquinone (2 h)
The title compound was obtained from 1 and p-chlorobenzoyl
chloride according to Method B. Recrystallization from THF
gave yellow needles; 69 % yield; mp 327–328 °C; 1H-NMR
(CDCl3) δ 7.50 (d, J = 7.9 Hz, 2 H), 7.54 (d, J = 8.4 Hz, 4 H), 7.78
(t, J = 7.9 Hz, 2 H), 8.16 (d, J = 7.9 Hz, 2 H), 8.22 (d, J = 8.4 Hz,
4 H); UV νmax (CHCl3) nm (log ε) 351 (1.77); FTIR (KBr) 1736,
1676 cm–1; MS m/z 516 (2, M+), 139 (100); Anal. C28H14Cl2O6
(C, H).
Pharmacological studies
Cytotoxic evaluations (XTT colormetric assay)
1,5-Bis(2,4-dichlorobenzoyl)anthraquinone (2 i)
Tumor cell lines used were rat glioma C6 cells and human
hepatoma G2 cells. The cells (2.5 × 104 cells/mL) were placed
into 96-well plates and preincubated for 24 to 72 hours in com-
plete medium. The drug concentration inhibiting 50 % of cellu-
lar growth (IC50, mg/mL) was determined by XTT assay follow-
ing 72 h of drug exposure.The results are the means of at least
three independent experiments unless otherwise indicated.
The title compound was obtained from 1 and o,p-dichloroben-
zoyl chloride according to Method B. Recrystallization from
THF gave yellow needles; 38 % yield; mp 310–312 °C; IR (KBr)
1740, 1668 cm–1; MS m/z 586 (4, M+), 421 (25), 240 (56), 173
(100); HRMS m/z: Calcd. for C28H12Cl4O6: 514.1464. Found:
514.1478.
1,5-Bis(2-toluoyloxy)anthraquinone (2 j)
Lipid peroxidation
The title compound was obtained from 1 and o-toluoyl chloride
according to Method A. Recrystallization from THF gave yellow
needles; 68 % yield; mp 262–263 °C; 1H-NMR (CDCl3) δ 2.68
(s, 6 H), 7.35 (d, J = 7.6 Hz, 2 H), 7.39 (t, J = 7.6 Hz, 2 H), 7.49–
7.53 (m, 4 H), 7.78 (t, J = 7.9 Hz, 2 H), 8.19 (dd, J = 8.1, 0.1 Hz,
2 H), 8.35 (t, J = 7.7, 0.7 Hz, 2 H); 13C-NMR (CDCl3) δ 181.15,
165.55, 150.21, 141.57, 136.01, 134.93, 132.86, 131.91,
131.69, 129.89, 128.39, 126.03, 125.84, 124.71, 21.76; UV
Fresh S.D. rat brains were obtained and the residual vessels
were cleaned up.The fresh brains were then homogenized with
Kreb’s buffer. After centrifugation, the upper solution (about
9 mL) was obtained. The 9 mL of solution was then separated
between about 18 vials (500 mL/vial), which are divided into
control and experimental sets.Then Kreb’s buffer, 60 µL, and a
DMSO solution of the tested compounds (30 µL), respectively,
were added to the vials. After 10 minutes, ferrous sulfate solu-
tion was added to the control and experimental sets and main-
tained at 37 °C in water bath. After 30 minutes, the vials were
taken from the water bath and trichloroacetic acid 10 mL (4 %
(w/v) in 0.3 N HCl) added to denature the residual protein.
2-Thiobarbituric acid solution 200 mL (0.5 % (w/v) 2-thiobarbi-
turic acid in 50 % (v/v) acetic acid) was then added to the solu-
tion which was kept at 100 °C in a water bath for 15 minutes.
The effects of the tested compounds on lipid peroxidation were
determined by measuring the percentages of red-colored prod-
uct formed by 2-thiobarbituric acid and malondialdehyde,
which is one of the products formed by lipid peroxidation.
ν
max (CHCl3) nm (log ε) 314 (1.56);FTIR (KBr) 1736, 1674 cm–1
;
MS m/z 476 (2, M+), 119 (100); Anal. C30H20O6 (C, H).
1,5-Bis(3-toluoyloxy)anthraquinone (2 k)
The title compound was obtained from 1 and m-toluoyl chloride
according to Method B. Recrystallization from THF gave yellow
needles; 28 % yield; mp 269–270 °C; 1H-NMR (CDCl3) δ 2.47
(s, 6 H), 7.44 (t, J = 7.6 Hz, 2 H), 7.48–7.50 (m, 4 H), 7.76 (t, J =
8.0 Hz, 2 H), 8.09 (d, J = 6.7 Hz, 4 H), 8.17 (d, J = 7.7 Hz, 2 H);
13C-NMR (CDCl3) δ 181.05, 165.28, 150.25, 138.52, 135.94,
134.91, 134.56, 130.96, 129.78, 129.34, 128.60, 127.67,
125.94, 124.60, 21.36; UV νmax (CHCl3) nm (log ε) 310 (1.84);
FTIR (KBr) 1732, 1674 cm–1; MS m/z 476 (4, M+), 119 (100);
Anal. C30H20O6: (C, H).
Statistics
The mean and standard deviation are designated as “X ± SD”.
The probable level of significance ( p Յ 0.05) between test and
control sample was determined by the Student’s “t”test with the
raw data.
1,5-Bis(4-toluoyloxy)anthraquinone (2 l)
The title compound was obtained from 1 and p-toluoyl chloride
according to Method B. Recrystallization from THF gave yellow
needles; 39 % yield; mp 331–332 °C; 1H-NMR (CDCl3) δ 2.47
(s, 6 H), 7.36 (d, J = 8.0 Hz, 4 H), 7.49 (dd, J = 7.8, 1.0 Hz, 2 H),
7.75 (t, J = 7.9 Hz, 2 H), 8.17 (d, J = 7.8 Hz, 4 H); UV νmax
(CHCl3) nm (log ε) 318 (0.90); FTIR (KBr) 1736, 1672 cm–1; MS
m/z 476 (5, M+), 119 (100); Anal. C30H20O6 (C, H).
References
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