Synthesis and antitumor activity of 1,8-diaminoanthraquinone derivatives

Article abstract of DOI:10.1248/cpb.53.1136

Continuing our ongoing studies on cytotoxic substances, a series of regioisomeric disubstituted aminoanthraquinone (DAAQ) derivatives have been synthesized as cytotoxic activity based on a proposed bioactive amino conformation. To assess the biological activity of amino-substitution in the side-chains of anthraquinone located at positions 1 and 8 of the anthraquinone ring system. The aim of the study was to determine if members of the anthraquinone family could be used as adjuncts to increase the growth inhibiting effect of anticancer agents in rat glioma C6 cells, human hepatoma G2 cells and 2.2.15 cells. In vitro cytotoxicity data is reported for the compounds and some indications of structure-activity relationships have been discerned. A number of compounds were found to have good cytotoxicity against proliferation in these three cell lines. This has led to the discovery some of the DAAQ as a conformationally constrained structure possessing anticancer properties that displays cytotoxicity for these above cell lines and is being investigated further.

Full text of DOI:10.1248/cpb.53.1136

1136
Chem. Pharm. Bull. 53(9) 1136—1139 (2005)
Vol. 53, No. 9
Synthesis and Antitumor Activity of 1,8-Diaminoanthraquinone
Derivatives
a
Hsu-Shan HUANG,*,a Hui-Fen CHIU,b Wei-Chih LU,c and Chun-Lung YUAN
a School of Pharmacy, National Defense Medical Center; No. 161, Minquan E. Rd., Neihu, Taipei 11490, Taiwan, R.O.C.:
b Department of Pharmacology, Kaohsiung Medical University; Kaohsiung: and c Cheng-Hsin Medical Center; Taipei,
Taiwan, R.O.C. Received April 6, 2005; accepted June 17, 2005
Continuing our ongoing studies on cytotoxic substances, a series of regioisomeric disubstituted aminoan-
thraquinone (DAAQ) derivatives have been synthesized as cytotoxic activity based on a proposed bioactive amino
conformation. To assess the biological activity of amino-substitution in the side-chains of anthraquinone located
at positions 1 and 8 of the anthraquinone ring system. The aim of the study was to determine if members of the
anthraquinone family could be used as adjuncts to increase the growth inhibiting effect of anticancer agents in
rat glioma C6 cells, human hepatoma G2 cells and 2.2.15 cells. In vitro cytotoxicity data is reported for the com-
pounds and some indications of structure–activity relationships have been discerned. A number of compounds
were found to have good cytotoxicity against proliferation in these three cell lines. This has led to the discovery
some of the DAAQ as a conformationally constrained structure possessing anticancer properties that displays cy-
totoxicity for these above cell lines and is being investigated further.
Key words aminoanthraquinone; anthraquinone; cytotoxicity; rat glioma C6 cell; human hepatoma G2 cell; 2.2.15 cell
cytotoxicity.19)
The aminoalkyl-functionalized anthraquinone series of
As part of our efforts to discover new chemotherapeutic
agents, we have synthesized a series of 1,8-diaminoan-
thraquinones that have different profiles with cytotoxicity
and other biological evaluation. Cytotoxicity and SARs stud-
ies demonstrated that 1,8-disubstituted aminoanthraquinones
and not their 1,5-isomers can selectively inhibit some of the
tumor cell lines. Previously, we have developed a series of
structurally related symmetrical and asymmetrical substi-
tuted or disubstituted anthraquinones as potential chemother-
apeutic agents.5—11) The positional attachment of the amide
and amino side chains have been shown to profoundly influ-
ence their cytotoxicity. For example, 1,4-diamidoan-
thraquinones have been shown more efficiently inhibited
Hepa G2 cells, whereas their 1,5- and 1,8-disubstituted re-
gioisomer, in which the functionalized side chains may si-
multaneously occupy both the DNA major and minor
grooves, with intercalation of the planar chromophore.2,20,21)
Evidence supporting these distinct binding mechanisms and
activity has been provided by their in vitro cytotoxicity and
structure–activity relationships for the anthraquinone skele-
ton through spacer side chains at these different positions and
results are compared with experimental data with these
agents.
synthetic compounds has been the subject of much study in
the quest for more active and less toxic analogs of the anthra-
cycline antitumor antibiotics, daunomycin and adriamycin.1—4
)
In an attempt to increase therapeutic effectiveness and reduce
toxicity by the anticancer drugs anthracyclines and an-
thraquinone during chemotherapy, we have developed a
series of regioisomeric disubstituted aminoanthraquinone
(DAAQ) derivatives by modifying the chromophore and the
side arms on their anthraquinone skeleton.5—11) The quinoid
anthracycline-related anti-cancer agents represent an impor-
tant group of antitumour drugs with a wide spectrum of ac-
tivity.12) On the basis of previously established structure–ac-
tivity relationships (SARs) for compounds that are able to in-
hibit some of the tumor cell lines proliferation and dual mod-
ulation of the telomerase to varying degrees. A series of di-
aminoanthraquinones were discovered initially as protein ki-
nase C inhibitors, and they also exhibited potent tumor cell
growth inhibitory activity in vitro without cross resistance to
adriamycin.13) Recent reports suggest that triplex stabiliza-
tion could be achieved with a series of DAAQs, which are
able to discriminate between duplex and triplex DNA by
virtue of their structural characteristics.14,15) Mitoxantrone, a
synthetic aminoanthraquinone belongs to chemical class of
agents known as the anthraquinones which antitumor activity
is attributed to its interaction with DNA topoisomerase II,
and its interaction with human cells may also involve nonin-
tercalary, electrostatic interactions.16) The mitoxantrone-de-
rived free radicals or their further oxidative activation of mi-
toxantrone to a DNA-damaging species may contribute to the
mechanism of action of this antitumour agent.17) Aminoan-
thraquinones may represent a class of polyamine binding site
ligands with pharmacophore and may facilitate the rational
design of N-methyl-D-aspartate (NMDA)-receptor modula-
tors.18) Aliphatic amine N-oxides have long been identified as
non-toxic metabolites of a large number of tertiary amines
drugs. Bioreduction of such N-oxides will generate the active
parent amine. This principle has been adopted to develop
AQ4N, a di-N-oxide anticancer prodrug with little intrinsic
Chemistry
As a part of our program aimed at exploring the biological
activity of symmetrical substitution of side chains into the
anthraquinone chromophore, we have synthesized a series of
diaminoanthraquinones that are related to the antitumor
agent mitoxantrone. Based upon the anthraquinone skeleton,
the symmetrical diamino derivatives can be readily prepared
using the method of nucleophilic substitution reaction in-
cluding substitution of 1,8-dichloroanthraquinone. It offers a
number of possibilities as a starting material for the synthesis
of more complex molecules because of the juxtaposition of
the chlorine atoms and the carbonyl groups of the central
ring. A key to developing such synthesis lies in differential
nucleophilic substitution of the chlorine atoms in starting
∗ To whom correspondence should be addressed. e-mail: huanghs@ndmctsgh.edu.tw
© 2005 Pharmaceutical Society of Japan

September 2005
1137
Table 1. Cytotoxic Activity of 1,8-Diaminoanthraquinone Derivatives
IC50 (mM)a)
Compd
R
No.
C6 cellsc)
Hep G2b)
2.2.15d)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
CH2CH3
25.6ꢀ0.5
0.61ꢀ0.01
11.4ꢀ0.4
14.57ꢀ1.1
47.5ꢀ0.8
1.24ꢀ0.01
0.02ꢀ0.01
41.82ꢀ1.0
0.19ꢀ0.01
25.6ꢀ0.6
59.47ꢀ0.9
1.06ꢀ0.03
97.9ꢀ1.5
CH2CH2CH3
CH2CH2CH2CH3
CH2CH(CH3)2
(CH2)5CH3
34.0ꢀ0.9 115.27ꢀ1.8
102.9ꢀ1.0
24.25ꢀ0.7
16.0ꢀ0.1
107.5ꢀ1.3
92.30ꢀ0.9
91.25ꢀ0.8
99.94ꢀ0.9
13.2ꢀ0.7
8.55ꢀ0.09
57.0ꢀ0.6
Chart 1
CH(CH3)2
CH2CH2OH
CH2CH2CH2OH
(C2H5)CH(CH2OH)
CH2CH2N(CH3)2
CH2CH2CH2NH2
CH2CH2CH2CH2NH2
CH2CH2CH2CH2CH2NH2
Cyclopentane
CH2C6H5
1.00ꢀ0.01 102.67ꢀ0.9
0.41ꢀ0.02
0.15ꢀ0.04
5.4ꢀ0.1
1.65ꢀ0.13
0.16ꢀ0.04
11.43ꢀ0.17
80.0ꢀ0.9
0.11ꢀ0.01
88.75ꢀ0.9
1.75ꢀ0.09
70.9ꢀ0.7
0.07ꢀ0.01
1.00ꢀ0.16
ꢁ1.0
12.47ꢀ0.34 81.53ꢀ0.8
0.09ꢀ0.01
126.0ꢀ1.5
46.45ꢀ0.5
1.29ꢀ0.06
131.0ꢀ1.3
41.48ꢀ0.5
CH2CH2C6H5
Mitoxantrone
Adriamycin
133.08ꢀ1.6 115.33ꢀ1.5
Chart 2
2.0ꢀ0.50
0.90ꢀ0.01
1.48ꢀ0.62
0.40ꢀ0.02
1.60ꢀ0.04
2.0ꢀ0.54
Cisplatin
material. The anthraquinones were synthesized by condens-
ing an excess of the appropriate amine with commercially
available 1,8-dichloroanthraquinone and gave rise to the de-
sired diaminoanthraquinones 1—16 in good overall yield.
a) IC50, drug concentration inhibiting 50% of cellular growth following 48 h of drug
exposure. Values are in mM and represent an average of 3 experiments. The variance for
the IC50 values was less than ꢀ20%. Inhibition of cell growth was significantly differ-
ent with respect to that of the control; nꢂ3 or more, pꢃ0.01. Inhibition was compared
Such a reaction is not likely to be general for simpler substi- with that of the control (mitoxantrone–HCl and adriamycin; mM), and standard errors.
b) Hep G2, human hepatoma G2 cells. c) C6 cells, rat glioma C6 cells.
d) 2.2.15 cells, hepatitis B virus transfected hepatoma cell lines, HepG 2.2.15 cells.
tuted anthraquinones except for heating in the miniclave.
More commonly, disubstitution are noted in reactions of
starting material (1,8-dichloroanthraquinone) with nitrogen
nucleophiles, and the problem becomes one of separation active in vitro compounds 2, 10, and 13 displayed cytotoxic-
and purification of products.
ity comparable with that of both mitoxantrone and adri-
amycin in HepG2 cells and 2.2.15 cells. In addition, com-
pounds bearing alkyl substituents and terminal amine moi-
Biological Activity and Discussion
Several classes of planar aromatic compounds have been eties 11 and 12 were found to be inactive, but 13 was found
shown to act as telomerase inhibitors or activators.8,22) In superior activity in these three cell lines. Among these com-
connection with our interest in the development of new chro- pounds, compound 13 displayed the most potent inhibitory
mophoric anthraquinone isomers, we investigated the prepa- activity in Hep G2 cell line. This also correlates with an ob-
ration of well defined anthraquinone structural motif that served 13 had IC50 values of 0.09 mM, 22-fold reduction in
could be represent an attractive target for the rational design cytotoxicity for HepG2 cells compared to the mitoxantrone
of new anticancer agents. The factors that control small mol- and 10-fold to adriamycin. Compound 13 with 5-amino-
ecule intercalation in DNA continue to be the focus of study, pentylamino substituent, which were chosen as typical alkyl-
which the hydrophobicity of the small molecule and net amino group, had IC50 values of 0.11 mM in C6 cells and IC50
charge are usually the most significant.23) Additional factor values of 1.29 mM in 2.2.15 cells, respectively. This result
specific to the side chains include their steric bulk, isohelic- prompted us to introduce this kind of simple long side chain
ity of the side chains with the minor groove, and phasing of on other regioisomers. In conclusion, within a series of dis-
the ligand subunits with the edges of the base pairs.24—26) We ubstituted amidoanthraquinones, Neidle et al. had found that
have recently shown that activation of human telomerase can the 2,7-regioisomer afforded the best stabilization of the TG
be achieved with appropriately disubstituted anthraqui- triplex, though the 1,8-isomers also stabilized the interaction
nones.8,11)
to some extent.27) Expanding or contracting the ring puts the
Comparisons with analogs anthraquinone-based com- binding groups in different positions relative to each other
pounds reveal a general reduction in the level of cellular and may lead to better interactions with the binding site.
cytotoxicity. Growth inhibition was determined in rat glioma 6,6,6 Ring system has a good interaction with both hy-
C6 cells, human hepatoma G2 cells, and 2.2.15 cells using drophobic regions.28) With the aim to provide second-genera-
XTT assay. In vitro cytotoxicity and cell growth inhibitory tion anthraquinone analogs endowed with reduced side ef-
data for the functionalized anthraquinones are collected in fects and a wider spectrum of action than mitoxantrone and
Table 1 and are compared with data for the corresponding doxorubicin, a large number of new molecules bearing nitro-
mitoxantrone, adriamycin and cisplatin where commercial gen atoms in the chromophore were synthesized and
available. All compounds with the exception of compounds screened in vitro. All analogs showed in vitro cytotoxic activ-
2, 7, 9, 10, and 13 are remarkably non-toxic in these selected ity against rat glioma C6 cells, human hepatoma G2 cells and
cell lines used. One of the most active in vitro compounds 7 2.2.15 cell lines. From this screening, compounds 2, 7, 9, 10,
displayed better cytotoxicity comparable with that of both and 13 emerged as the most interesting analogs which were
mitoxantrone and adriamycin in C6 cells. Three of the most tested in vitro on several murine and human tumor cell lines

1138
Vol. 53, No. 9
1,8-Bis(isopropylamino)anthraquinone (6): 74% yield. mp 198—200 °C
(EA/n-hexane). UV lmax (MeOH) nm (log e): 554 (1.13). 1H-NMR (CDCl3)
d: 0.87—0.93 (6H, m), 1.30—1.43 (6H, m), 3.86—3.92 (2H, m), 7.06 (2H,
d, Jꢂ8.4 Hz), 7.47—7.57 (4H, m), 9.64 (2H, br). 13C-NMR (CDCl3) d:
22.86 (CH3), 43.68 (CH), 114.33 (CH), 114.62 (CH), 118.08 (C), 133.97
(CH), 134.55 (C), 150.34 (C), 184.70 (C), 189.00 (C). APCI-MS: 323 (Mꢄ),
324 (24, [Mꢄ1]ꢄ).
and showed cytotoxic potency lower than that of mitoxan-
trone and adriamycin. In particular, those compounds with
methylene links in each side chain separating the amine and
terminal amine moieties have superior cytotoxicity and, in
general, enhanced anticancer characteristics. Based on this
correlation, the most likely models of cytotoxicity complexes
were postulated: (i) mitoxantrone and one derivative of
bis(aminopentylamino)-substituted anthraquinone 13 were
found superior activity in these three cell lines, both with the
same distance of two long side chains, maybe intercalate
from the minor groove of DNA and bind with both chains in
this groove; (ii) propylamino disubstituted 2 and 2-dimethyl-
aminoethylamino disubstituted 10 both with two methylene
links in each side chain also have superior cytotoxicity.
1,8-Bis(ethanolamino)anthraquinone (7): 80% yield. mp 214—215 °C
(EA/n-hexane); lit.29): 260—265 °C, EtOH). UV lmax (MeOH) nm (log e):
1
546 (0.70), 282 (0.83). H-NMR (CDCl3) d: 3.21 (4H, m, CH2), 3.66 (4H,
m, CH2), 4.93 (2H, t, Jꢂ2.7 Hz), 7.20 (2H, d, Jꢂ8.7 Hz), 7.35 (2H, t,
Jꢂ3.9 Hz), 7.52 (2H, t, Jꢂ8.2 Hz), 9.67 (2H, br, NH). 13C-NMR (CDCl3) d:
45.13 (CH2), 59.54 (CH2), 113.66 (CH), 114.33 (CH), 118.63 (C), 133.75
(CH), 134.53 (C), 151.15 (C), 183.67 (C), 187.95 (C). PI-EI-MS m/z: 326
(Mꢄ), 295.
1,8-Bis(propanolamino)anthraquinone (8): 71% yield. mp 211—212 °C
(EA/n-hexane); lit.29): 215—220 °C, EtOH). UV lmax (MeOH) nm (log e):
552 (2.77), 282 (2.31). 1H-NMR (DMSO) d: 1.79 (4H, quint, CH2), 3.21
(4H, m, CH2), 3.55 (4H, m, CH2), 4.62 (4H, t, Jꢂ3.0 Hz, OH), 7.19 (2H, d,
Jꢂ8.7 Hz), 7.34 (2H, d, Jꢂ3.9 Hz), 7.53 (2H, t, Jꢂ8.2 Hz). 9.59 (2H, br).
PI-EI-MS m/z: 354 (Mꢄ), 309, 277.
1,8-Bis(2-amino-1-butanol)anthraquinone (9): 68% yield. mp 180—
181 °C (EA/n-hexane). UV lmax (MeOH) nm (log e): 552 (1.38), 283 (1.51).
1H-NMR (DMSO) d: 0.92—0.97 (6H, m), 1.54—1.59 (4H, m), 1.70—1.73
Experimental
Melting points were determined with a Buchi B-545 melting point appa-
ratus and are uncorrected. All reactions were monitored by TLC (silica gel
60 F254). 1H-NMR: Varian GEMINI-300 (300 MHz) and Brucker AM-500
(500 MHz); d values are in ppm relative to TMS as an internal standard.
Fourier-transform IR spectra (KBr): Perkin-Elmer 983G spectrometer. The (4H, m), 3.32—3.59 (2H, m), 4.90 (2H, s), 7.23 (2H, d, Jꢂ9.3 Hz), 7.32—
UV spectra were recorded on a Shimadzu UV-160A. Mass spectra (PI-EI- 7.35 (2H, m), 7.49 (2H, t, Jꢂ7.9 Hz), 9.67 (2H, d, Jꢂ7.2 Hz). EI-MS m/z:
MS, 70 eV, unless otherwise stated): Finnigan MAT TSQ-46, Finnigan MAT 383 (Mꢄ), 291, 253.
TSQ-700 (Universitat Regensburg, Germany) and Finnigan MAT LCQ-MS
1,8-Bis{[2-(dimethylamino)ethyl]amino}anthraquinone (10)30)
:
68%
(National Research Institute of Chinese Medicine, Taipei, Taiwan). Typical yield. mp 118—120 °C (EA/n-hexane). UV lmax (MeOH) nm (log e): 513
experiments illustrating the general procedures for the preparation of the an-
thraquinones are described below.
(0.61), 256 (3.31). 1H-NMR (CDCl3) d: 0.87—1.02 (6H, m), 1.02—1.34
(6H, m), 2.28—2.40 (4H, m), 3.44—3.52 (4H, m), 7.12—7.15 (2H, m),
General Procedure for the Preparation of the 1,8-Diaminoanthraqui- 7.57—7.62 (2H, m), 7.64—7.86 (2H, m), 9.70 (2H, br). 13C-NMR (CDCl3)
nones A mixture of 1,8-dichloroanthraquinone (1.0 g, 3.6 mmol) and DMF
(20 ml) containing an appropriate amine (8.0 mmol) was heated in a mini-
clave (Büchi®) for 30 min. After cooling and the reaction mixture was
treated with crushed ice. The resulting precipitate was collected by filtration,
washed well with water and further purified by recrystallization from ethyl-
acetate (EA)/n-hexane afforded the final product as red needles.
d: 41.33, 45.60, 58.11, 114.99, 118.10, 126.24, 134.88, 137.61, 151.37,
182.99, 183.92. EI-MS m/z: 381 (Mꢄ), 329.
1,8-Bis[(3-aminopropyl)amino]anthraquinone (11): 80% yield. mp 162—
163 °C (EA/n-hexane); lit.30): 165 °C). UV lmax (MeOH) nm (log e): 544
(0.49), 281 (0.56). 1H-NMR (CDCl3) d: 1.94—1.99 (4H, m), 2.82—2.84
(4H, m), 2.97—2.99 (4H, m), 3.42—3.46 (4H, m), 7.07—7.10 (2H, m),
7.48—7.51 (2H, m), 7.57—7.60 (2H, m), 9.67 (2H, br). APCI-MS: 353
1,8-Bis(ethylamino)anthraquinone (1): 46% yield. mp 152—154 °C
(EA/n-hexane). UV lmax (MeOH) nm (log e): 522 (0.78). 1H-NMR (CDCl3) (Mꢄ), 354 (20, [Mꢄ1]ꢄ).
d: 1.42 (6H, t, Jꢂ7.2 Hz), 3.39—3.48 (4H, m), 7.12 (2H, d, Jꢂ5.1 Hz),
1,8-Bis[(4-aminobutyl)amino]anthraquinone (12)30)
: Yield 43%; mp
7.48—7.62 (4H, m), 9.60 (2H, br). 13C-NMR (CDCl3) d: 14.50 (CH3), 37.60 154—155 °C (EA/n-hexane). UV lmax (MeOH) nm (log e): 551 (0.21), 282
(CH2), 114.93 (CH), 118.16 (CH), 126.32 (C), 134.41 (CH), 137.89 (C), (0.25). 1H-NMR (CDCl3) d: 1.45—1.48 (4H, m), 1.57—1.60 (4H, m),
151.35 (C), 182.96 (C), 188.91 (C). PI-EI-MS m/z: 294 (Mꢄ), 285, 270.
1,8-Bis(propylamino)anthraquinone (2): 83% yield. mp 158—160 °C
(EA/n-hexane). UV lmax (MeOH) nm (log e): 554 (0.80), 282 (0.87). 1H-
NMR (DMSO) d: 1.10 (6H, t, Jꢂ7.4 Hz), 1.78—1.90 (4H, m), 3.29—3.36
1.81—1.90 (4H, m), 2.71—2.81 (4H, m), 3.27—3.42 (4H, m), 7.03 (2H, d,
Jꢂ8.4 Hz), 7.48—7.58 (4H, m), 9.64 (2H, br). APCI-MS: 381.1 (Mꢄ),
382.2 (20, [Mꢄ1]ꢄ).
1,8-Bis[(5-aminopentyl)amino]anthraquinone (13): 35% yield. mp 115—
(4H, m), 7.04 (2H, d, Jꢂ8.1 Hz), 7.47—7.58 (4H, m), 9.67 (2H, br). 13C- 116 °C (EA/n-hexane). UV lmax (MeOH) nm (log e): 545 (1.72), 281 (1.89).
NMR (DMSO-d) d: 11.76 (CH3), 22.43 (CH2), 44.85 (CH2), 114.34 (CH),
1H-NMR (CDCl3) d: 0.86—0.92 (4H, m), 1.48—1.52 (4H, m), 1.57—1.58
114.74 (CH), 117.65 (C), 134.04 (CH), 134.37 (C), 151.24 (C), 184.75 (C), (4H, m), 1.71—1.84 (4H, m), 2.71—2.79 (4H, m), 3.33—3.39 (4H, m), 7.03
189.03 (C). EI-MS m/z: 322 (Mꢄ), 279.
(2H, d, Jꢂ8.7 Hz), 7.47—7.58 (4H, m), 9.65 (2H, br). APCI-MS: 409.1
1,8-Bis(butylamino)anthraquinone (3): 71% yield. mp 133—134 °C
(Mꢄ), 410.2 (20, [Mꢄ1]ꢄ).
(EA/n-hexane). UV lmax (MeOH) nm (log e): 565 (1.19), 282 (1.27). 1H-
1,8-Bis(cyclopentylamino)anthraquinone (14): 52% yield. mp 196—
NMR (CDCl3) d: 1.03 (6H, t, Jꢂ7.2 Hz), 1.50—1.59 (4H, m), 1.63—1.81 198 °C (EA/n-hexane). UV lmax (MeOH) nm (log e): 561 (1.70), 284 (1.80).
(4H, m), 3.32—3.39 (4H, m), 7.04 (2H, d, Jꢂ8.1 Hz), 7.47—7.58 (4H, m),
1H-NMR (CDCl3) d: 1.85—1.87 (8H, m), 2.14—2.20 (8H, m), 4.00—4.02
9.64 (2H, br). 13C-NMR (CDCl3) d: 13.88 (CH3), 20.41 (CH2), 31.25 (CH2), (2H, m), 7.07 (2H, d, Jꢂ7.8 Hz), 7.46—7.57 (4H, m), 9.86 (2H, d,
42.80 (CH2), 114.34 (CH), 114.74 (CH), 117.64 (C), 134.04 (CH), 134.38 Jꢂ6.0 Hz). 13C-NMR (CDCl3) d: 24.2, 33.62, 53.96, 114.41, 114.68,
(C), 151.23 (C), 184.76 (C), 189.03 (C). EI-MS m/z: 350 (Mꢄ), 307, 293, 118.58, 133.85, 134.41, 150.73, 184.76, 188.88. APCI-MS: 375.1 (Mꢄ), 376
251.
1,8-Bis(isobutylamino)anthraquinone (4): 70% yield. mp 166—168 °C
(20, [Mꢄ1]ꢄ).
1,8-Bis[(phenylmethyl)amino]anthraquinone (15): 63% yield. mp 188—
(EA/n-hexane). UV lmax (MeOH) nm (log e): 556 (0.52), 283 (0.68). 1H- 190 °C (EA/n-hexane). UV lmax (MeOH) nm (log e): 525 (0.56), 278 (0.93).
NMR (CDCl3) d: 1.11 (12H, d, Jꢂ6.6 Hz), 2.06—2.15 (2H, m), 3.18 (4H, t, 1H-NMR (CDCl3) d: 4.62 (4H, d, Jꢂ6.0 Hz), 6.99 (2H, d, Jꢂ8.4 Hz), 7.30—
Jꢂ6.3 Hz), 7.04 (2H, d, Jꢂ4.2 Hz,), 7.46—7.57 (4H, m), 9.80 (2H, br). 13C- 7.42 (10H, m), 7.47 (2H, d, Jꢂ7.8 Hz), 7.61 (2H, d, Jꢂ7.8 Hz). 10.11 (2H,
NMR (DMSO-d) d: 20.56 (CH3), 28.05 (CH), 50.87 (CH2), 114.33 (CH),
114.71 (CH), 117.68 (C), 134.00 (CH), 134.38 (C), 151.37 (C), 184.70 (C), 134.20, 134.40, 138.36, 151.03, 184.48, 188.89. EI-MS: 418 (Mꢄ), 328.
189.00 (C). APCI-MS: 351 (Mꢄ), 352, (24, [Mꢄ1]ꢄ).
1,8-Bis[(phenylethyl)amino]anthraquinone (16): 54% yield. mp 162—
1,8-Bis(hexylamino)anthraquinone (5): 52% yield. mp 80—82 °C (EA/n- 164 °C (EA/n-hexane). UV lmax (MeOH) nm (log e): 544 (0.55). H-NMR
hexane). UV lmax (MeOH) nm (log e): 556 (0.53), 283 (0.56). 1H-NMR (CDCl3) d: 3.09—3.14 (4H, m), 3.59—3.65 (4H, m), 7.07 (2H, d,
(CDCl3) d: 0.90—0.99 (6H, m), 1.29—1.39 (8H, m), 1.41—1.59 (4H, m), Jꢂ8.7 Hz), 7.30—7.39 (10H, m), 7.51 (2H, d, Jꢂ7.8 Hz), 7.57 (2H, d,
1.76—1.84 (4H, m), 3.31—3.37 (4H, m), 7.04 (2H, d, Jꢂ8.1 Hz), 7.47— Jꢂ7.8 Hz), 9.72 (2H, br). 13C-NMR (CDCl3) d: 35.70, 44.65, 114.58,
7.58 (4H, m), 9.66 (2H, br). 13C-NMR (CDCl3) d: 14.01 (CH3), 22.57 114.99, 117.46, 126.51, 128.73, 134.08, 134.40, 138.99, 150.90, 184.48,
br). 13C-NMR (CDCl3) d: 47.05, 115.42, 118.06, 126.95, 127.26, 128.72,
1
(CH2), 26.87 (CH2), 29.11 (CH2), 31.55 (CH2), 43.11 (CH2), 114.34 (CH),
114.74 (CH), 117.65 (C), 134.05 (CH), 134.39 (C), 151.23 (C), 184.77 (C),
189.02 (C). EI-MS m/z: 406 (Mꢄ), 335, 321, 251.
188.89. EI-MS: 446 (Mꢄ), 356.
Cell Culture Various cancer cell lines (G2, 2.2.15. cells and C6 cells)
were cultured in minimum essential medium (MEM), supplemented with

September 2005
1139
10% fetal calf serum, 100 units/ml penicillin and 100 mg/ml streptomycin in
999—1006 (2004).
a humidified atmosphere in 5% CO2 at 37 °C. Cell culture media were re- 10) Huang H. S., Chiu H. F., Lee A. R., Guo C. L., Yuan C. L., Bioorg.
newed every three days, up to the confluence of the monolayer. Cell culture
was passaged when they had formed confluent cultures, using trypsin-EDTA
to detach the cells from their culture flasks or dishes. Test compounds were
stored at ꢅ70 °C and solublized in 100% DMSO. All the drug solutions
were prepared immediately before the experiments and were diluted into
complete medium before addition to cell cultures. All data presented in this
report are form at least three independent experiments showing the same
pattern of expression.
Med. Chem., 12, 6163—6170 (2004).
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XTT Method The tetrazolium reagent (XTT) was designed to yield a
suitably colored, aqueous-soluble, non-toxic formazan upon metabolic re-
duction by viable cells. Approximately 2ꢆ103 cells, suspended in MEM
medium, were plated onto each well of a 96-well plate and incubated in 5%
CO2 at 37 °C for 24 h. Test compounds were then added to the culture
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and electron coupling reagent (PMS) 1 ml were mixed together, and 50 ml of 17) Fisher G. R., Patterson L. H., J. Pharm. Pharmacol., 43, 65—68
this mixture were added to each well. After an appropriate incubation at
37 °C for 6 h, the absorbency at 490 nm was measured with the ELISA
reader.
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Acknowledgments This research was partially supported by National
Science Council Grants (NSC94-2113-M-016-003) and TTY Biopharm
Company Limited. The authors are indebted to Mr. Jung-Chin Lin (TTY)
and Dr. Klaus K. Mayer (Universität Regensburg, Germany) for the mass 21) Perry P. J., Gowan S. M., Reszka A. P., Polucci P., Jenkins T. C., Kel-
spectrometry and analytical determinations.
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