Synthesis and cytotoxic effect of 1,3-dihydroxy-9,10-anthraquinone derivatives

Article abstract of DOI:10.1016/S0223-5234(00)01190-9

1,3-Dihydroxy-9,10-anthraquinone (4) was reacted with epichlorohydrin or 1,ω-dibromo-alkane to yield 1-hydroxy-3-(2,3-epoxypropoxy)-9,10-anthraquinone (5) and 1-hydroxy-3-(3-chloro-2-hydroxypropoxy)-9,10-anthraquinone (6) or 1-hydroxy-3-(ω-bromoalkoxy)-9,10-anthraquinone. Ring-opening of the epoxide (5) or 1-hydroxy-3-(ω-bromoalkoxy)-9,10-anthraquinones with appropriate amines, afforded various 1-hydroxy-3-(3-alkylamino-2-hydroxypropoxy)-9,10-anthraquinones. The synthetic compounds were tested in vitro inhibition of human T-24, Hep 3B, Hep G2, SiHa, HT-3, PLC/PRF/5 and 212 cells. Almost all compounds showed significant inhibitory activity against several different cancer cell lines. Structure - activity analysis indicated epoxidation of the hydroxyanthraquinone increased cytotoxicity against tumour cells, but ring-opening of the epoxide group with amine did not enhance the cytotoxic activity. The phosphatidylserine (PS) externalization and DNA fragmentation in SiHa cells were significantly observed after 48 h incubation with selected compound 19. The results show that 19 cause cell death by apoptosis.

Full text of DOI:10.1016/S0223-5234(00)01190-9

Eur. J. Med. Chem. 35 (2000) 1089–1098
´
© 2000 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved
PII: S0223-5234(00)01190-9/FLA
Original article
Synthesis and cytotoxic effect of 1,3-dihydroxy-9,10-anthraquinone derivatives
Bai-Luh Weia, Szu-Huei Wub, Mei-Ing Chungb, Shen-Jeu Wonc, Chun-Nan Linb*
aNational University Kaohsiung, Kaohsiung, Taiwan 811, ROC
bSchool of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan 807, ROC
cDepartment of Microbiology and Immunology, Medical College, National Cheng Kung University, Taiwan 701, ROC
Received 25 May 2000; revised 5 September 2000; accepted 5 September 2000
Abstract – 1,3-Dihydroxy-9,10-anthraquinone (4) was reacted with epichlorohydrin or 1,v-dibromo-alkane to yield 1-hydroxy-3-
(2,3-epoxypropoxy)-9,10-anthraquinone (5) and 1-hydroxy-3-(3-chloro-2-hydroxypropoxy)-9,10-anthraquinone (6) or 1-hydroxy-3-
(v-bromoalkoxy)-9,10-anthraquinone. Ring-opening of the epoxide (5) or 1-hydroxy-3-(v-bromoalkoxy)-9,10-anthraquinones with
appropriate amines, afforded various 1-hydroxy-3-(3-alkylamino-2-hydroxypropoxy)-9,10-anthraquinones. The synthetic compounds
were tested in vitro inhibition of human T-24, Hep 3B, Hep G2, SiHa, HT-3, PLC/PRF/5 and 212 cells. Almost all compounds
showed significant inhibitory activity against several different cancer cell lines. Structure–activity analysis indicated epoxidation of
the hydroxyanthraquinone increased cytotoxicity against tumour cells, but ring-opening of the epoxide group with amine did not
enhance the cytotoxic activity. The phosphatidylserine (PS) externalization and DNA fragmentation in SiHa cells were significantly
´
observed after 48 h incubation with selected compound 19. The results show that 19 cause cell death by apoptosis. © 2000 Editions
scientifiques et me´dicales Elsevier SAS
anthraquinone / antitumour / apoptosis
1. Introduction
2. Chemistry
Apoptosis is considered to be the major process
responsible for cell death in various physiological
events. It acts as a regulating mechanism of tissue
growth, where it balances cell proliferation [1]. Re-
cently, apoptosis has become a focus of interest in
oncology because a disregulation of the apoptotic
process can prompt malignancy of tumours [2, 3]. As
part of our efforts to continually develop potent anti-
tumour agents which may lead to tumour cell apopto-
sis, we have designed and synthesized a new series of
1,3-dihydroxy-9,10-anthraquinone (DHA) deriva-
tives. In the present paper, we report the synthesis
and cytotoxic effects against several different cancer
cell lines in vitro and cytotoxicity through apoptosis,
of various DHA derivatives and discuss their struc-
ture–activity relationships.
A series of new DHA derivatives were synthesized
by general synthetic routes, as shown in figure 1. In
general, the best method available for the synthesis of
oxygenated anthraquinones is the condensation of
phthalic anhydride (1) with 1,3-dimethoxybenzene (2)
to form a mixture of 1,3-dimethoxybenzoylbenzoic
acid (3a) and 1-hydroxy-3-methoxybenzoylbenzoic
acid (3b). A mixture of 3a and 3b were dehydrated
with a molten mixture of anhydrous aluminum chlo-
ride and sodium chloride to give DHA (4) [4]. 1-Hy-
droxy-3-(2,3-epoxypropoxy)-9,10-anthraquinone (5),
1-hydroxy-3-(3-alkylamino-2-hydroxypropoxy)-9,10-
anthraquinones, and 1-hydroxy-3-(v-alkylamino-
alkoxy)-9,10-anthraquinones were synthesized by a
method described elsewhere [5]. Briefly, DHA (4), was
allowed to react with 1 eq. of sodium hydroxide in
2-propanol and excess of epichlorohydrin to yield the
epoxide 5 as the major product and 1-hydroxy-3-(3-
chloro-2-hydroxypropoxy)-9,10-anthraquinone (6) as
* Correspondence and reprints.
E-mail address: lincna@cc.kmu.edu.tw (C.-N. Lin).

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B.-L. Wei et al. / European Journal of Medicinal Chemistry 35 (2000) 1089–1098
the side product. Ring opening of 5 with appropri-
ate amines in refluxing appropriate solvent afforded
cytotoxic effects against T-24, Hep 3B, Hep G2,
PLC/PRF/5 and 212 cells in vitro.
various
1-hydroxy-3-(3-alkylamino-2-hydroxy-pro-
In order to study the cytotoxic mechanism of the
DHA derivatives on tumour cells, changes of phos-
phatidylserine (PS) externalization and DNA frag-
mentation in SiHa cells after the selected 19
treatment were analysed. Growing evidence indicates
that an early event in apoptosis is the migration of
the PS from the inner to the outer leaflet of plasma
membrane [6–8]. In the study, 19 (4 mg/mL) were
incubated with SiHa cells for 48 h. The percentage
of apoptotic cells was quantified by flow cytometry.
As shown in figure 2, the percentage of apoptotic
cells for 19, obtained from the flow cytometric his-
tograms was 66.6%, indicating that SiHa cytoplasma
did indeed exhibit PS externalization in response to
19. In addition, DNA fragmentation is generally
used to characterize cell death by apoptosis [9–11].
Thus, apoptosis of the SiHa cells after 19 treatment
was also studied by DNA fragmentation. DNA
fragmentation in SiHa cells was significantly ob-
served after 48 h incubation with 19 (4 mg/mL)
(figure 3).
poxy)-9,10-anthraquines. Potassium salt of 4 was al-
lowed to react with 1, v-dibromoalkane in
appropriate solvent and then animated with appro-
priate amines to give various 1-hydroxy-3-(v-alkyl-
aminoalkoxy)-9,10-anthraquines (figure 1).
3. Biological results and discussion
Cytotoxic activities of a series of DHA derivatives
were studied against a number of cancer cell types.
The results are listed in table I and table II. All the
synthetic DHA derivatives, shown in table II, indi-
cated significant inhibitory activities against all can-
cer cell types, except that 15 indicated insignificant
inhibitory activities against Hep3B, PLC/PRF/5 and
212 cells in vitro. As shown in table II, an increase
in the length of the N-substituted alkyl chain did
not significantly enhance the cytotoxicity against all
the cell lines in table II, except that 13 significantly
enhanced the cytotoxicity against SiHa cells due to
an increase of the N-substituted alkyl chain from
three to four carbons. N-substitution with a more
bulky group of three or four carbons significantly
enhanced the cytotoxicity, except for the cytotoxic-
ity against T-24 cells. It clearly indicated that
lipophilicity did not affect the cytotoxicity of com-
pounds with different N-substituted carbon side
chains, while it did affect the cytotoxicity of com-
pounds with same N-substituted carbon side chains.
DHA (4) did not show cytotoxic effects against
T-24, Hep 3B, Hep G2, SiHa, HT-3, PLC/PRF/5 or
212 cells in vitro. As shown in table II, epoxidation
of DHA markedly increased the in vitro cytotoxic
effects against T-24, SiHa, HT-3, PLC/PRF/5 and
212 cells, while ring-opening of the epoxide with
dimethylamine and tert-butylamine enhanced the cy-
totoxic effects against SiHa cells. As shown in table
II1 - hydroxy - 3 - (v - alkylaminoalkoxy) - 9,10 - anthra-
quinones did not show stronger cytotoxic effects
against the cancer cell lines used in table II than
those of 1-hydroxy-3-(3-alkylamino - 2 - hydroxypro-
poxy) - 9,10 - anthraquinones except for the cytotoxic
effect against SiHa cells. It suggested that the alco-
holic group of 1-hydroxy-3-(3-alkylamino-2-hydroxy-
propoxy) - 9,10 - anthraquinones is required for the
4. Conclusions
These results indicate that 19 causes cell death by
apoptosis and the other compound of this series of
DHA derivatives may also induce cell death by
apoptosis. Further experiments are needed to eluci-
date its mechanism of action. Thus, this series of
DHA derivatives with novel cell apoptosis might be
developed as anti-cancer agents.
5. Experimental protocols
Melting points (uncorrected) were determined
with a Yanaco micro-melting point apparatus. IR
spectra were determined with a Hitachi model 260-
1
30 IR spectrophotometer. H- and 13C-NMR spectra
[l (ppm), J (Hz)] were determined with a Varian
Unity-400 spectrometer. Mass spectra were deter-
mined with a Jeol JMS-D-100 mass spectrometer.
Elemental analyses were within 90.4% of the theo-
retical values, unless otherwise noted. Chromatogra-
phy was performed using a flash-column technique
on silica gel 60 supplied by E. Merck.

B.-L. Wei et al. / European Journal of Medicinal Chemistry 35 (2000) 1089–1098
1091
Figure 1. Synthesis of DHA derivatives.

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B.-L. Wei et al. / European Journal of Medicinal Chemistry 35 (2000) 1089–1098
Table I. DHA derivatives
Compd
R
R%
n
mp °C
Rcrystn solvent
% Yield
Formula
Anal.
5
181–183
148–149
109–111
amorph
amorph
200–203
CHCl3
CHCl3
EtOAc
57
24
65
71
70
44
C17H12O5
C17H13ClO5
C19H19NO5·1/3H2O
C21H23NO5
C20H21NO5·HCl·1/2H2O
C20H21NO5·HCl
C, H
C, H
C, H, N
C, H, N
C, H, N
C, H, N
6
7
CH3
C2H5
H
CH3
C2H5
CH(CH3)2
(CH2)2CH3
8
9
10
H
MeOH
11
H
amorph
73
C20H19NO5·HCl
C, H, N
12
13
14
H
H
H
CH(CH3)CH2CH3
C(CH3)3
(CH2)3CH3
amorph
amorph
amorph
83
64
78
C21H23NO5·HCl·1/3H2O
C21H23NO5·HCl·1/2H2O
C21H23NO5·HCl
C, H, N
C, H, N
C, H, N
15
H
amorph
61
C23H25NO5·HCl·1/3H2O
C, H, N
18
19
20
C2H5
CH3
C2H5
C2H5
CH3
C2H5
2
3
3
amorph
amorph
amorph
63
78
91
C20H21NO4·HCl·1/3H2O
C19H19NO4
C21H23NO4·HCl·H2O
C, H, N
C, H, N
C, H, N
(Me2CO), 5.6 g (23.33 mmol, 57.6%), mp 265–269 °C.
The IR, MS and NMR were identified with an authentic
sample of DHA [13].
5.1. 1,3-Dihydroxy-9,10-anthraquinone (DHA) (4)
To a stirred solution of phthalic anhydride (1) (6.0 g,
40.5 mmol) in anhydrous CH2Cl2 (500 mL) was added
anhydrous AlCl3 (15 g, 112.5 mmol) in small portions
and then dropwise 1,3-dimethoxybenzene (2) (6.7 g, 48.5
mmol) at r.t. for 18 h. The reaction mixture was poured
into ice water (500 mL) containing concentrated HCl
(500 mL) and extracted with CHCl3. Removal of the
solvent gave a colourless powder (mixture of 3a and 3b)
(9.66 g).
To a stirred molten mixture of anhydrous AlCl3 (80 g,
600 mmol) and NaCl (50 g, 855.4 mmol) was added
mixture of 3a and 3b (9.66 g) in small portions at
140–150 °C. The reaction temperature was raised to
160–165 °C and maintained for 6 h with continuous
stirring. After cooling, the melt mass was poured into a
mixture of ice water (500 mL) and concentrated HCl
(500 mL) and allowed to stand overnight. The resulting
precipitate was extracted with EtOAc [12]. The EtOAC
extracts was purified by column chromatography (silica
gel and C6H6-EtOAc (2:1) to give 4, orange needles
5.2. 1-Hydroxy-3-(2,3-epoxypropoxy)-9,10-anthra-
quinone (5) and 1-hydroxy-3-(3-chloro-2-hydroxypro-
poxy)-9,10-anthraquinone (6)
To a solution of 0.56 g (10 mmol) sodium hydroxide
in water (1 mL) was added 2-propanol (100 mL) and 4
(2.4 g, 10 mmol). To the above mixture was then added
an excess of epichlorohydrin (18.5 g, 199.9 mmol) and
the mixture was refluxed for 18 h with stirring. The
product was purified by column chromatography (silica
gel–CHCl3) and crystallized from CHCl3 to give two
yellow needles 5 (1.70 g, 5.74 mmol) and 6 (0.79 g, 2.38
1
mmol). Compound 5: IR (KBr) 1680, 1640 cm−1; H-
NMR(CDCl3) l2.80 (dd, J=2.8, 4.8 Hz, 1H, CHH in
the epoxide ring), 2.95 (t, J=4.8 Hz, 1H, CHH), 3.39–
3.43 (m, 1H, CH in the epoxide ring), 4.05 (dd, J=6.0,
11.2 Hz, 1H, OCHH), 4.40 (dd, J=2.8, 11.2 Hz, 1H,
OCHH), 6.73 (d, J=2.8 Hz, 1H, H-2), 7.38 (d, J=2.8

B.-L. Wei et al. / European Journal of Medicinal Chemistry 35 (2000) 1089–1098
Table II. Cytotoxicity of various DHA derivatives (ED50 values in mg/mL).
1093
Compound
Cell linea
T-24
0.51
1.66
1.20
1.44
1.55
1.75
1.40
1.96
2.36
1.96
3.11
1.54
1.31
2.33
Hep 3B
Hep G2
SiHa
HT-3
PLC/PRF/5
212
5
n.d.
n.d.
1.95
2.31
n.d.
1.97
1.76
1.86
1.88
2.59
7.95
2.05
2.31
2.57
n.d.
n.d.
0.72
0.83
n.d.
n.d.
n.d.
n.d.
0.72
n.d.
n.d.
n.d.
0.83
n.d.
0.80
4.18
0.67
0.85
1.18
1.00
1.13
1.17
0.65
1.47
1.99
1.20
0.36
1.57
0.53
1.98
n.d.
n.d.
1.47
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.51
2.08
1.71
2.01
1.67
2.02
1.58
1.85
1.51
2.91
4.77
2.12
1.71
2.60
5.3
0.16
0.63
1.14
1.32
0.38
0.90
0.88
1.04
0.70
1.58
5.76
1.28
1.21
1.31
1.3
6
7
8
9
10
11
12
13
14
15
18
19
20
Cisplatin
Actinomycin D
1.5×10−3
8×10−4
5.6×10−4
1.4×10−3
a For significant activity of the DHA derivatives, an ED50B4.0 mg/mL is required; n.d., not determined.
Hz, 1H, H-4), 7.77–7.80 (m, 2H, H-6 and H-7), 8.25–
8.29 (m, 2H, H-5 and H-8), 12.84 (s, 1H, OH-1);
13CNMR (CDCl3) l44.5 (CH2 in the epoxide ring), 49.6
(CH in the epoxide ring), 69.3 (OCH2), 107.5 (C-2),
107.8 (C-4), 111.2 (C-9a), 126.8 (C-8), 127.4 (C-5), 133.5
(C-8a and C-10a), 134.2 (C-7), 134.3 (C-6), 135.1 (C-4a),
165.0 (C-1), 165.3 (C-3), 182.3 (C-10), 186.8 (C-9); MS
m/z 296 (M+, 75). Compound 6: IR (KBr) 3505, 1676,
quinones or their hydrochloride salts. Treatment of 5
(0.55 g, 1.85 mmol) by this procedure yielding various
1-hydroxy-3-(3-alkylamino-2-hydroxypropoxy)-9,10-
1
1641 cm−1; H-NMR (acetone-d6) l3.76–3.86 (m, 2H,
CH2Cl), 4.24–4.37 (m, 3H, CHOH and OCH2), 4.84 (d,
J=5 Hz, 1H, CHOH), 6.85 (d, J=2.6 Hz, 1H, H-2),
7.31 (d, J=2.6 Hz, 1H, H-4), 7.87–7.97 (m, 2H, H-6
and H-7), 8.21–8.31 (m, 2H, H-5 and H-8), 12.85 (s,
1H, OH-1); 13C NMR (acetone-d6) l47.3 (CH2Cl), 71.0
(CHOH), 71.7 (OCH2), 108.2 (C-2), 109.4 (C-4), 112.2
(C-9a), 128.1 (C-8), 128.6 (C-5), 134.9 (C-8a), 135.0
(C-10a), 136.1 (C-6 and C-7), 136.7 (C-4a), 167.1 (C-1),
167.2 (C-3), 183.2 (C-10), 188.5 (C-9); MS m/z 332 (M+,
10).
5.3. General procedure for obtaining 7–15 [5]
Figure 2. Apoptosis of the SiHa cells after 19 treatment was
analysed by flow cytometric analysis. The quantitative analysis
of apoptotic populations of SiHa cell by MC-540 staining.
Cells (2×105) were seeded into six-well trays. After treatment
with medium (A) or 19 (4 mg/mL) (B), cell pellets were
resuspended in HBS buffer and stained with dye MC-540.
Diluted cell suspension was assayed by FACScan. The per-
centage of apoptotic cells is shown in the panels.
To 5 was added various appropriate amines and
ethanol. The mixture was refluxed for 2 h with stirring.
The product was purified by column chromatography
and crystallized from appropriate solvent or dissolved in
10% HCl–EtOAc solution, yielding various 1-hydroxy-
3 - (3 - alkylamino - 2 - hydroxypropoxy) - 9,10 - anthra-

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B.-L. Wei et al. / European Journal of Medicinal Chemistry 35 (2000) 1089–1098
and C-10a), 134.1 (C-7), 134.3 (C-6), 135.0 (C-4a), 165.4
(C-1 and C-3), 182.4 (C-10), 186.8 (C-9); MS m/z 341
(M+, 5).
5.5. 1-Hydroxy-3-[3-(diethylamino)-2-
hydroxypropoxy]-9,10-anthraquinone (8)
Yellow powder: IR (KBr) 3116, 1681, 1621 cm−1
;
1H-NMR (CDCl3) l1.06 [t, J=7.1 Hz, 6H, 2×CH3),
2.49–2.78 (m, 4H, N(CH2)2], 4.01–4.11 (m, 3H, OCH2
and CHOH), 6.72 (d, J=2.6 Hz, 1H, H-2), 7.38 (d,
J=2.6 Hz, 1H, H-4), 7.71–7.82 (m, 2H, H-6 and H-7),
8.20–8.32 (m, 2H, H-5 and H-8), 12.81 (bs, 1H, OH-1);
13C-NMR (CDCl3) l11.9 (CH3), 47.4 (CH2CH3), 55.7
(CH2N), 67.2 (CHOH), 71.9 (OCH2), 106.7 (C-2), 107.8
(C-4), 110.2 (C-9a), 126.5 (C-8), 127.0 (C-5), 133.0 (C-8a
and C-10a), 134.7 (C-6 and C-7), 134.8 (C-4a), 164.7
(C-1), 165.6 (C-3), 181.7 (C-10), 186.3 (C-9); MS m/z
369 (M+, 1).
5.6. 1-Hydroxy-3-[3-(isopropylamino)-2-
hydroxypropoxy]-9,10-anthraquinone
hydrochloride·1/2 H2O (9)
Orange powder: IR (KBr) 3475, 1681, 1626 cm−1
;
1H-NMR (DMSO-d6) l1.27 [d, J=5.6 Hz, 3H,
CH(CH3)2], 1.28 [d, J=5.6 Hz, 3H, CH(CH3)2], 3.00
š
(dd, J=8.8, 12.4 Hz, H, CHHNH2), 3.15 (dd, J=2.8,
š
Figure 3. Apoptosis of the SiHa cells after 19 treatment was
analysed by DNA fragmentation. Cells (1.5×106) cultured in
culture medium were treated with 19 for 48 h. C, control; lane
1, cells with 19 (2 mg/mL) treatment, lane 2, cells with 19 (4
mg/mL) treatment; M, 100 bp marker.
12.4 Hz, 1H, CHHNH2), 3.34 [m, 1H, NHCH(CH3)2],
4.16–4.23 (m, 2H, OCH2), 4.24–4.28 (m, 1H, CHOH),
5.99 (s, 1H, CHOH), 6.91 (d, J=2.4 Hz, 1H, H-2), 7.23
(d, J=2.4 Hz, 1H, H-4), 7.91–7.95 (m, 2H, H-6 and
H-7), 8.15–8.22 (m, 2H, H-5 and H-8); 13C-NMR
(DMSO-d6) l18.3 and 18.7 (CH3×2), 46.4 (CH2NH),
49.9 [CH(CH3)2], 65.0 (CHOH), 70.7 (OCH2), 106.8
(C-2), 107.8 (C-4), 110.4 (C-9a), 126.5 (C-8), 127.0 (C-5),
132.9 (C-8a and C-10a), 134.7 (C-7), 134.8 (C-6), 134.9
(C-4a), 164.6 (C-1), 165.0 (C-3), 181.6 (C-10), 186.3
(C-9); MS m/z 355 (M+, 2).
anthraquinones: 7 (0.41 g, 1.20 mmol), 8 (0.52 g, 1.41
mmol), 9 (0.49 g, 1.25 mmol), 10 (0.34 g, 0.87 mmol), 11
(0.53 g, 1.50 mmol), 12 (0.71 g, 1.74 mmol), 13 (0.27 g,
0.66 mmol), 14 (0.31 g, 0.76 mmol) and 15 (0.22 g, 0.51
mmol) (table I).
5.4. 1-Hydroxy-3-[3-(dimethylamino)-2-
hydroxypropoxy]-9,10-anthraquinone·1/3 H2O (7)
5.7. 1-Hydroxy-3-[3-(n-propylamino)-2-hydroxypro-
poxy]-9,10-anthraquinone·hydrochloride (10)
Yellow powder: IR (KBr) 3106, 1686, 1626 cm−1
;
1H-NMR (CDCl3) l2.36 [s, 6H, N(CH3)2], 2.39–2.45
(m, 1H, CHHN), 2.52–2.65 (m, 1H, CHHN), 4.07–4.16
(m, 3H, OCH2 and CHOH), 6.73 (d, J=2.5 Hz, 1H,
H-2), 7.39 (d, J=2.5 Hz, 1H, H-4), 7.71–7.82 (m, 2H,
H-6 and H-7), 8.21–8.31 (m, 2H, H-5 and H-8), 12.83
(bs, 1H, OH-1); 13C-NMR (CDCl3) l45.5 (CH3), 61.5
(CH2N), 65.7 (CHOH), 70.9 (OCH2), 107.3 (C-2), 108.0
(C-4), 111.0 (C-9a), 126.8 (C-8), 127.4 (C-5), 133.5 (C-8a
Red prisms: IR (KBr) 3465, 1696, 1621 cm−1; H-
NMR (DMSO-d6) l0.92 (t, J=7.6 Hz, 3H, CH3), 1.63–
1.72 (m, 2H, CH2CH3), 2.87–2.94 (m, 2H,
1
NH2CH2CH2), 2.98–3.05 (m, 1H, CHHNH2), 3.14–
š
3.19 (m, 1H, CHHNH2), 4.15–4.22 (m, 2H, OCH2),
4.23–4.28 (m, 1H, CHOH), 5.98 (s, 1H, CHOH), 6.91
(d, J=2.4 Hz, 1H, H-2), 7.24 (d, J=2.4 Hz, 1H, H-4),
7.90–7.97 (m, 2H, H-6 and H-7), 8.16–8.24 (m, 2H, H-5

B.-L. Wei et al. / European Journal of Medicinal Chemistry 35 (2000) 1089–1098
1095
š
š
and H-8), 8.72 (bs, 1H, NHH), 8.87 (bs, 1N, NHH),
5.10. 1-Hydroxy-3-[3-(tert-butylamino)-2-
hydroxypropoxy]-9,10-anthraquinone
hydrochloride·1/2 H2O (13)
12.75 (s, 1H, OH-1); 13C-NMR (DMSO-d6) l11.5
(CH3), 20.7 (CH2CH3), 51.0 (NH2CH2), 51.1
(CHOHCH2NH2), 66.7 (CHOH), 72.0 (OCH2), 108.3
(C-2), 109.0 (C-4), 128.1 (C-8), 128.5 (C-5), 135.0 (C-8a
and C-10a), 135.9 (C-7), 136.0 (C-6), 136.9 (C-4a), 166.8
(C-1), 181.6 (C-10), 186.3 (C-9); MS m/z 355 (M+, 2).
š
Yellow powder: IR (KBr) 3335, 1681, 1636 cm−1
1H-NMR (DMSO-d6) l1.33 [s, 9H, C(CH3)3], 2.93–3.00
;
š
š
(m, 1H, CHHNH2), 3.14–3.19 (m, 1H, CHHNH2),
4.16–4.20 (m, 1H, CHOH), 4.23–4.26 (m, 1H, OCH2),
6.00 (bs, 1H, CHOH), 6.90 (d, J=2.4 Hz, 1H, H-2),
7.23 (d, J=2.4 Hz, 1H, H-4), 7.89–7.96 (m, 2H, H-6
and H-7), 8.14–8.21 (m, 2H, H-5 and H-8), 8.55 (bs,
5.8. 1-Hydroxy-3-[3-(cyclopropylamino)-2-
hydroxypropoxy]-9,10-anthraquinone·hydrochloride (11)
š
š
1H, CH2NNHH), 8.94 (bs, 1H, CH2NHH), 12.74 (s,
1H, OH-1); 13C-NMR (DMSO-d6) l25.0 (CH3×3),
43.8 (CH2NH2), 56.5 [C(CH3)3], 65.2 (CHOH), 70.6
(OCH2), 106.8 (C-2), 107.7 (C-4), 110.4 (C-9a), 126.5
(C-8), 126.9 (C-5), 132.9 (C-8a and C-10a), 134.6 (C-4a),
134.7 (C-7), 134.8 (C-6), 164.6 (C-1), 165.0 (C-3), 181.6
(C-10), 186.3 (C-9); MS m/z 369 (M+, 1).
Yellow powder: IR (KBr) 3465, 1691, 1631 cm−1
;
1H-NMR (DMSO-d6) l 0.75–0.93 (m, 4H, CH2 in the
cyclopropyl ring), 2.76 (bs, 1H, CH in the cyclopropyl
ring), 3.09–3.14 (m, 1H, CHHNH), 3.24–3.7 (m, 1H,
CHHNH), 4.16–4.25 (m, 3H, OCH2 and CHOH), 5.98
(s, 1H, CHOH), 6.94 (d, J=2.4 Hz, 1H, H-2), 7.27 (d,
J=2.4 Hz, 1H, H-4), 7.91–7.98 (m, 2H, H-6 and H-7),
š
8.18–8.25 (m, 2H, H-5 and H-8), 8.91 (bs, 2H, NH2),
5.11. 1-Hydroxy-3-[3-(n-butylamino)-2-
hydroxypropoxy]-9,10-anthraquinone hydrochloride (14)
12.78 (s, 1H, OH-1); 13C-NMR (DMSO-d6) l3.1 and 3.2
(CH2 in the cyclopropyl ring), 30.0 (CH in the cyclo-
propyl ring), 50.0 (CH2NH2), 64.5 (CHOH), 70.7
(OCH2), 106.8 (C-2), 107.8 (C-4), 110.4 (C-9a), 126.5
(C-8), 127.0 (C-5), 132.9 (C-8a and C-10a), 134.7 (C-4a),
134.8 (C-7), 134.9 (C-6), 164.6 (C-1), 165.0 (C-3), 181.6
(C-10), 186.3 (C-9); MS m/z 353 (M+, 3).
Yellow powder: IR (KBr) 3445, 1691, 1636 cm−1
;
1H-NMR (DMSO-d6) l 0.90 (t, J=7.3 Hz, 3H, CH3),
1.24–1.43 (m, 2H, CH2CH3), 1.56–1.68 (m, 2H,
š
š
NH2CH2CH2), 2.87–3.00 (m, 2H, NH2CH2CH2), 3.08–
š
3.33 (m, 2H, CH2NH2), 4.17–4.20 (m, 2H, OCH2),
4.25–4.31 (m, 1H, CHOH), 5.97 (bs, 1H, CHOH), 6.91
(d, J=2.6 Hz, 1H, H-2), 7.23 (d, J=2.6 Hz, 1H, H-4),
7.91–7.99 (m, 2H, H-6 and H-7), 8.14–8.25 (m, 2H, H-5
5.9. 1-Hydroxy-3-[3-(1-methylpropylamino)-2-
hydroxypropoxy]-9,10-anthraquinone
hydrochloride·1/3 H2O (12)
š
š
and H-8), 8.75 (bs, 1H, NHH), 8.91 (bs, 1H, NHH),
12.75 (s, 1H, OH-1); 13C-NMR (DMSO-d6) l13.5
(CH3), 19.3 (CH2CH3), 27.3 (NHCH2CH2), 47.0
(CH2NH2), 49.1 (NHCH2CH2), 64.6 (CHOH), 70.7
(OCH2), 106.8 (C-2), 107.8 (C-4), 110.4 (C-9a), 126.5
(C-8), 126.9 (C-5), 132.9 (C-8a and C-10a), 134.7 (C-4a
and C-7), 134.8 (C-6), 164.6 (C-1), 165.0 (C-3), 181.6
(C-10), 186.3 (C-9); MS m/z 369 (M+, 3).
Yellow powder: IR (KBr) 3435, 1705, 1626 cm−1
;
1H-NMR (DMSO-d6) l 0.91 (t, J=7.6 Hz, 3H,
CH2CH3), 1.27 (d, J=6 Hz, 3H, CHCH3), 1.47–1.57
(m, 1H, CHHCH3), 1.83–1.89 (m, 1H, CHHCH3),
2.99–3.06 (m, 1H, CHCH3), 3.12–3.21 (m, 2H,
š
CH2NH2), 4.12–4.20 (m, 2H, OCH2), 4.28–4.36 (m, 1H,
CHOH), 6.01 (s, 1H, CHOH), 6.78 (d, J=2.8 Hz, 1H,
H-2), 7.08 (d, J=2.8 Hz, 1H, H-4), 7.84–7.91 (m, 2H,
H-6 and H-7), 8.05–8.11 (m, 2H, H-5 and H-8), 8.74
5.12. 1-Hydroxy-3-[3-(cyclohexylamino)-2-
hydroxypropoxy]-9,10-anthraquinone
hydrochloride·1/3 H2O (15)
š
š
(bs, 1H, NHH) or 8.81 (bs, 1H, NHH), 9.09 (bs, 1H,
š
š
NHH) or 9.21 (bs, 1H, NHH), 12.64 (s, 1H, OH-1);
13C-NMR (DMSO-d6) l9.7 (CH2CH3), 14.9 (CHCH3),
25.0 (CH2CH3), 46.5 (CH2NH), 55.0 (CH), 64.8
(CHOH), 70.6 (OCH2), 106.7 (C-2), 107.7 (C-4),
110.2 (C-9a), 126.3 (C-8), 126.8 (C-5), 132.7 (C-8a and
C-10a), 134.4 (C-4a), 134.6 (C-7), 134.7 (C-6), 164.5
(C-1), 164.9 (C-3), 181.3 (C-10), 186.1 (C-9); MS m/z
369 (M+, 2).
Yellow powder: IR (KBr) 3475, 1686, 1616 cm−1
;
1H-NMR (CD3OD) l1.29–1.47 (m, 6H, CH2×3 in the
cyclohexane ring), 1.87–2.19 [m, 4H, CH(CH2)2 in the
š
cyclohexane ring], 3.13–3.24 (m, 3H, CH2NH2 and CH
in the cyclohexane ring), 4.19–4.21 (m, 2H, OCH2),
4.24–4.30 (m, 1H, CHOH), 6.86 (d, J=2.6 Hz, 1H,
H-2), 7.40 (d, J=2.6 Hz, 1H, H-4), 7.88–7.90 (m, 2H,

1096
B.-L. Wei et al. / European Journal of Medicinal Chemistry 35 (2000) 1089–1098
H-6 and H-7), 8.24–8.34 (m, 2H, H-5 and H-8); 13C-
NMR (CD3OD) l25.5, 25.6 and 26.1 (CH2×3 in the
cyclohexane ring), 30.3 and 30.5 [CH(CH2)2 in the cy-
clohexane ring], 48.0 (CH2NH2), 58.9 (NH2CH), 66.7
(CHOH), 71.7 (OCH2), 108.1 (C-2), 108.8 (C-4), 127.8
(C-8), 128.2 (C-5), 134.8 (C-8a and C-10a), 135.6 (C-7),
135.7 (C-6), 136.6 (C-4a), 166.5 (C-1 and C-3), 183.5
(C-10), 188.2 (C-9); MS m/z 395 (M+, 5).
5.15. General procedure for obtaining 18–20 [14]
To 16 or 17 was added appropriate amines and
ethanol. The mixture was refluxed for 2 h with stirring.
The product was treated as in the procedure for 7–15 to
give 18 (0.26 g, 0.64 mmol), 19 (0.25 g, 0.32 mmol) and
20 (0.12 g, 0.30 mmol).
5.16. 1-Hydroxy-3-[2-(diethylamino)-ethoxy]-9,10-
anthraquinone hydrochloride·1/3 H2O (18)
5.13. 1-Hydroxy-3-[2-(bromo)-ethoxy]-9,10-anthra-
quinone (16)
Yellow powder: IR (KBr) 3355, 1681, 1644 cm−1
;
1H-NMR (CDCl3) l 1.49 (t, J=7.6 Hz, 6H, CH3×2),
3.22–3.36 (m, 4H, CH2CH3×2), 3.53 (dd, J=4.0, 9.2
Hz, 2H, CH2N), 4.72 (t, J=4.4 Hz, 2H, OCH2), 6.73 (d,
J=2.4 Hz, 1H, H-2), 7.29 (d, J=2.4 Hz, 1H, H-4),
To a solution of KOH (0.31 g, 5.5 mmol) in MeOH
(100 mL) was added 4 (1.22 g, 5.1 mmol) and 1,2-dibro-
moethane (47.27 g, 251.6 mmol) and the mixture was
stirred for 36 h under reflux. The reaction mixture was
evaporated and the organic material was purified by
column chromatography (silica gel; C6H6–EtOAc, 5:1).
The purified product was crystallized from CHCl3 to
give 16, yellow powder (0.63 g, 1.81 mmol, 35.3%).
Compound 16: 1H-NMR (CDCl3) l 3.68 (t, J=
6.1Hz, 2H, CH2Br), 4.43 (t, J=6.1 Hz, 2H, OCH2),
7.26 (d, J=2.5 Hz, 1H, H-2), 7.38 (d, J=2.5 Hz, 1H,
H-4), 7.74–7.84 (m, 2H, H-6 and H-7), 8.25–8.32 (m,
2H, H-5 and H-8), 12.84 (s, 1H, OH-1); 13C-NMR
(CDCl3) l28.1 (CH2Br), 68.3 (OCH2), 107.4 (C-2), 107.6
(C-4), 111.3 (C-9a), 126.8 (C-8), 127.4 (C-5), 133.4 (C-8a
and C-10a), 134.2 (C-6), 134.3 (C-7), 135.2 (C-4a), 164.6
(C-1), 165.3 (C-3), 183.3 (C-10), 186.9 (C-9).
7.75–7.81 (m, 2H, H-6 and H-7), 8.22–8.28 (m, 2H, H-5
š
and H-8), 12.73 (bs, 1H, NH), 12.79 (s, 1H, OH-1);
13C-NMR (CDCl3) l8.56 (CH3×2), 46.4 (CH2CH3×
2), 50.4 (CH2NH), 63.5 (OCH2), 107.3 (C-2), 107.4
(C-4), 111.5 (C-9a), 126.9 (C-8), 127.4 (C-5), 133.3 (C-8a
and C-10a), 134.3 (C-7), 134.4 (C-6), 135.2 (C-4a), 163.5
(C-1), 165.3 (C-3), 182.0 (C-10), 186.8 (C-9); MS m/z
339 (M+, 12).
5.17. 1-Hydroxy-3-[3-(dimethylamino)-propoxy]-9,10-
anthraquinone (19)
1
Brown powder: IR (KBr) 1686, 1621 cm−1; H-NMR
(CDCl3) l1.96–2.03 (m, 2H, CH2), 2.26 (s, 6H, CH3×
2), 2.47 (t, J=6.4 Hz, 2H, CH2N), 4.14 (t, J=6.4 Hz,
2H, OCH2), 6.67 (d, J=2.4 Hz, 1H, H-2), 7.33 (d,
J=2.4 Hz, 1H, H-4), 7.73–7.81 (m, 2H, H-6 and H-7),
8.22–8.28 (m, 2H, H-5 and H-8), 12.83 (bs, 1H, OH-1);
13C-NMR (CDCl3) l27.1 (CH2), 45.4 (CH3), 56.0
(CH2NH), 67.1 (OCH2), 107.2 (C-2), 108.1 (C-4), 110.6
(C-9a), 126.7 (C-8), 127.3 (C-5), 133.4 (C-8a), 133.5
(C-10a), 134.0 (C-7), 134.2 (C-6), 134.9 (C-4a), 165.4
(C-1), 165.7 (C-3), 182.4 (C-10), 186.6 (C-9); MS m/z
325 (M+, 16).
5.14. 1-Hydroxy-3-[2-(bromo)-propoxy]-9,10-
anthraquinone (17)
To a solution of KOH (0.18 g, 3.2 mmol) in MeOH
(50 mL) was added 4 (0.48 g, 2.0 mmol) and 1,3-dibro-
mopropane (20.32 g, 101.1 mmol), and the mixture was
treated as in the procedure of 16 to give 17, yellow
powder (0.29 g, 0.80 mmol, 40.0%).
Compound 17: 1H-NMR (CDCl3) l 2.38 (m, 2H,
CH2), 3.62 (t, J=6.4 Hz, 2H, CH2Br), 4.26 (t, J=6.4
Hz, 2H, OCH2), 6.72 (d, J=2.8 Hz, 1H, H-2), 7.38 (d,
J=2.8 Hz, 1H, H-4), 7.76–7.82 (m, 2H, H-6 and H-7),
8.26–8.31 (m, 2H, H-5 and H-8), 12.87 (s, 1H, OH-1);
13C-NMR (CDCl3) l29.3 (CH2Br), 31.9 (CH2), 66.2
(OCH2), 107.2 (C-2), 108.0 (C-4), 111.0 (C-9a), 126.8
(C-8), 127.4 (C-5), 133.5 (C-8a and C-10a), 134.2 (C-6),
134.3 (C-7), 135.1 (C-4a), 165.3 (C-1), 165.4 (C-3), 182.4
(C-10), 186.8 (C-9).
5.18. 1-Hydroxy-3-[3-(diethylamino)-propoxy]-9,10-
anthraquinone hydrochloride·H2O (20)
Yellow powder: IR (KBr) 3365, 1681, 1626 cm−1
;
1H-NMR (CDCl3) l1.46 (t, J=7.2 Hz, 6H, CH3×2),
2.44–2.51 (m, 2H, CH2), 3.13–3.26 [m, 6H,
CH2N(CH2)2], 4.23 (t, J=6.6 Hz, 2H, OCH2), 6.66 (d,
J=2.4 Hz, 1H, H-2), 7.29 (d, J=2.4 Hz, 1H, H-4),

B.-L. Wei et al. / European Journal of Medicinal Chemistry 35 (2000) 1089–1098
1097
7.77–7.79 (m, 2H, H-6 and H-7), 8.23–8.28 (m, 2H,
H-5 and H-8), 12.38 (bs, 1H, NH), 12.82 (s, 1H, OH-
1); 13C-NMR (CDCl3) l8.51 (CH3×2), 23.8 (CH2),
46.8 (CH2CH3), 49.2 (CH2NH), 65.8 (OCH2), 107.0
(C-2), 107.7 (C-4), 111.2 (C-9a), 126.8 (C-8), 127.4 (C-
5), 134.4 (C-8a and C-10a), 134.2 (C-7), 134.3 (C-6),
135.1 (C-4a), 164.7 (C-1), 165.4 (C-3), 182.2 (C-10),
186.8 (C-9); MS m/z 353 (M+, 35).
with 10 mM HEPES buffer pH 7.3 and incubated at
37 °C in a CO2 incubator.
5.20. Flow cytometry
For cell apoptosis analysis, cells (2×106) were
seeded into six-well trays. The cells were treated with
a tested drug for 48 h and harvested. The cell pellets
were resuspended in Hepes-buffer saline (HBS, 160
mM NaCl, 2.7 mM KCl, 6 mM glucose, 10 mM
Hepes, 0.1% BSA, pH 7.3) and stained with dye Me-
rocyanine 540 (MC540; Acros, Belgium) in the dark
for 10 min. MC540, similar to Annexin V, can bind
phosphatidylserine of extroverted inner membrane
component during cell apoptosis and is used as the
indicator of cell apoptosis [21, 22]. The cell suspension
was diluted with 0.5 mL HBS buffer and 5×103 cells
were assayed by the software CELLFIT of the fluores-
cence-activated cell sorter (FACScan; Becton-Dickin-
son immunocytometry system, CA) [23].
5.19. Tumour cell growth inhibition assays
The cytotoxicity was determined by colourimetric
MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-1H-te-
trazolium bromide) assay as described previously [15].
Briefly, cells (5×103 per well) were plated in the 96-
well plates and incubated in medium for 6 h. Fifty
microlitres of serial tested drug dilutions were added.
The cells were incubated for 5 or 6 days at 37 °C and
then pulsed with 10 mL of MTT (5 mg/mL; Sigma, St.
Louis, MO) and incubated for an additional 4 h at
37 °C. Reduced MTT was measured spectrophotomet-
rically by a Dynatech MR5000 microplate reader
(Dynatech Laboratories, VA) at 550 nm after lysis of
cells with 100 mL of 10% SDS in 0.01 M HCl. Con-
trol wells contained medium plus cells (total ab-
sorbance) or medium alone (background absorbance)
cell death was calculated as the percentage of MTT
inhibition:
5.21. DNA fragmentation assay
After tested drug (8 mg/mL) treatment for 48 h,
cells in 150-mm plates were harvested and washed
with PBS. After the addition of 100 mL lysis buffer
[1% of NP-40 (Sigma) in 20 mM EDTA, 50 mM
Tris–HCl, pH 7.5] and mixing, the cell lysates were
centrifuged at 14 000 rpm for 5 min and the superna-
tants were collected. The supernatants were incubated
with 50 mL of RNase A (20 mg/mL) and 20 mL of
SDS (10%) at 56 °C for 2 h. Then, 35 mL of
proteinase K (20 mg/mL) was added and incubated at
37 °C overnight. DNA fragments were precipitated af-
ter the addition of 150 mL of 10 M NH4OAc and 1.2
mL of 100% ethanol at −20°C overnight. After cen-
trifuging and drying, the DNA pellets were resus-
1−mean experimental absorbance
% inhibition=
×100
mean control absorbance
PLC/PRF/5 cells were established from a human
hepatoma and known to produce HBs Ag continu-
ously in culture fluids [16]. Hep 3B cells, human hep-
atitis
B surface antigen (HBs Ag) contains an
integrated hepatitis B virus genome [17]. Human hep-
atoma, PLC/PRF/5, Hep 3B and Hep G2, T-24 cells,
human cervical carcinoma, HT-3 and SiHa cells and
212 cells were maintained in Dulbecco’s modified
Eagle medium (DMEM; Gibco BRL, Grand Island,
NY) [18, 19], containing 10% foetal bovine serum
(FBS; Gibco BRL), 2 mM 1-glutamine, 100 U/mL
penicillin and 100 mg/mL streptomycin. The 212 cells
(an inducible Ha-ras oncogene transformed the NIH/
3T3 cell line) were maintained in minimum essential
alpha medium (MEM; Gibco BRL), containing 10%
calf serum (Gibco BRL) and antibiotics [20]. For the
microassay, the growth medium was supplemented
pended
in
15
mL
Tris-EDTA
buffer
and
electrophoresed on a 1% agarose gel in TBE buffer at
30 V for 8 h, DNA ladder was observed after staining
with ethidium bromide solution and exposing to the
UV light [15].
Acknowledgements
This work was supported by a grant from the Na-
tional Science Council of the Republic of China
(NSC89-2314-B037-024).

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B.-L. Wei et al. / European Journal of Medicinal Chemistry 35 (2000) 1089–1098
[13] Chung M.I., Jou S.J., Cheng T.H., Lin C.N., J. Nat. Prod. 57
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