Structure-Based Design and Synthesis of Regioisomeric Disubstituted Aminoanthraquinone Derivatives as Potential Anticancer Agents

Article abstract of DOI:10.1002/hlca.200490093

Continuing our ongoing studies on cytotoxic substances, we report the synthesis and cytotoxic properties of a series of symmetric 1,5-diamino-9,10-anthraquinones with potentially bioreducible groups. Symmetric amination of 1,5-dichloro-9,10-anthraquinone with the appropriate primary amines in the presence of DMF furnished the structurally related aminoanthraquinone analogs 1-19. Their in vitro cytotoxic activity was evaluated using rat glioma C6 cells, human hepatoma G2 cells, and 2.2.15 cells. Several compounds exhibited very high antitumor activities in these assays. Compound 4 efficiently inhibited C6 cells, human hepatoma G2 cells, and 2.2.15 cells, as determined by means of the XTT colorimetric assay. The antiproliferative activity of 4 was markedly enhanced, reaching a potency comparable to those of the powerful anticancer agents mitoxantrone, adriamycin, and cisplatin. Biological evaluations and structure/activity relationships within this class of novel aminoanthraquinones are discussed.

Full text of DOI:10.1002/hlca.200490093

Helvetica Chimica Acta Vol. 87 (2004)
999
Structure-Based Design and Synthesis of Regioisomeric Disubstituted
Aminoanthraquinone Derivatives as Potential Anticancer Agents
by Hsu-Shan Huang*a), Hui-Fen Chiub), Pen-Fong Yehc), and Chun-Lung Yuana)
a) School of Pharmacy, National Defense Medical Center, Taipei, Taiwan, Republic of China
(phone: ‡886-2-8792 64 54; fax: ‡886-2-8792 31 69; e-mail: huanghs@ndmctsgh.edu.tw)
b) Department of Pharmacology, Kaohsiung Medical University, Kaohsiung, R.O.C.
c) Cheng-Hsin Medical Center, Taipei, Taiwan, R.O.C.
Continuing our ongoing studies on cytotoxic substances, we report the synthesis and cytotoxic properties of
a series of symmetric 1,5-diamino-9,10-anthraquinones with potentially bioreducible groups. Symmetric
amination of 1,5-dichloro-9,10-anthraquinone with the appropriate primary amines in the presence of DMF
furnished the structurally related aminoanthraquinone analogs 1 19. Their in vitro cytotoxic activity was
evaluated using rat glioma C6 cells, human hepatoma G2 cells, and 2.2.15 cells. Several compounds exhibited
very high antitumor activities in these assays. Compound 4 efficiently inhibited C6 cells, human hepatoma G2
cells, and 2.2.15 cells, as determined by means of the XTT colorimetric assay. The antiproliferative activity of 4
was markedly enhanced, reaching a potency comparable to those of the powerful anticancer agents
mitoxantrone, adriamycin, and cisplatin. Biological evaluations and structure/activity relationships within this
class of novel aminoanthraquinones are discussed.
Introduction. In recent years, intermolecular triple helices have found consid-
erable interest as potential antigene agents that are able to inhibit the expression of
particular genes in a sequence-specific fashion [1]. Several strategies have been
proposed to increase the strength of such interactions [2], including 1) covalent
attachment of intercalating groups either at the ends [3 6] or within the third strand
[7][8], 2) covalent attachment of a positively charged moiety [9][10], or 3)
modification of the phosphodiester backbone [11][12].
As part of the work in our laboratory on the use of anthraquinone derivatives as
anticancer agents or human telomerase activity agents, a number of doubly substituted
9,10-anthraquinones have been synthesized and biologically evaluated [13 16].
However, the mode of antitumor action of anthraquinone derivatives (e.g., mitoxan-
trone) has not been clearly established yet. The possible modes include, among others,
intercalation and binding to DNA, bioreduction, and aerobic redox cycling [17]. The
9,10-anthraquinone skeleton is a central constituent of the anthracyclines doxorubicin
and daunorubicin, and of the anthracycline derivatives mitoxantrone and anthrapyr-
azole [18]. 9,10-Anthraquinone-based compounds currently occupy a prominent
position in cancer chemotherapy, with the naturally occurring aminoglycoside-type
anthracycline doxorubicin and the aminoanthraquinone mitoxantrone both in clinical
use [19]. Mitoxantrone is an important synthetic aminoanthraquinone used in clinical
management of leukemia and lymphoma, as well as in combination therapy for
advanced breast and ovarian cancer [20]. Molecular modelling has been carried out for
a number of amine-functionalized anthraquinone derivatives to determine their extent

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of binding to G-tetraplex DNA, and their ability to inhibit the enzymes telomerase and
Taq polymerase [21]. The planarity of the anthraquinone system allows intercalation
into DNA, and its redox properties are linked to the production of radical species in
biological systems [22]. Drug structures have been based upon the DNA-binding
anticancer topoisomerase-II poison mitoxantrone with modified alkylamino side chains
[23].
The anthracycline antibiotic adriamycin is a broad-spectrum antineoplastic agent
with activity against a variety of tumors. Many adriamycin analogs, consisting of the
anthraquinone skeleton with amino, alkylamino, and aminoalkylamino side chains,
have been synthesized [24]. Diaminoanthraquinones were discovered initially as
protein-kinase-C inhibitors, with IC50 values in the range of 50 100 mm with respect to
in vitro tumor-cell growth, without cross-resistance to adriamycin [25]. Adamson
proposed the removal of the aminosugar moiety from daunomycin and adriamycin and
its replacement with an appropriate amino group to enhance antitumor activity [26].
To gain a better understanding of the cytotoxicity of aminoanthraquinone-derived
agents, several structurally related doubly substituted compounds, bearing selected
characteristic functional groups, were designed. The approach was to develop structure/
activity relationships (SAR) for aminoanthraquinone derivatives with redox-active
centers attached to the anthraquinone skeleton through spacer side chains at positions
1 and 5.
Results and Discussion. The 1,5-disubstituted 9,10-aminoanthraquinones 1 19
were readily prepared by nucleophilic substitution of 1,5-dichloro-9,10-anthraquinone
(20)1). Upon heating 20 with a large excess of a primary amine (RNH2) in a glass mini-
reactor in the presence of DMF for 30 min, the symmetrically disubstituted products
1 19 were obtained in 40 80% yield (see the Table). Prolonged heating favored side
reactions, leading to tedious purification, but was required for the preparation of higher
homologs (data not shown). The purities of the products were generally higher after
1
)
Compound 20 is a suitable starting material also for the synthesis of more-complex molecules because of
the juxtaposition of the Cl-atoms and the carbonyl groups [27]. A key to developing such compounds lies
in the stepwise nucleophilic substitution of the Cl-atoms. The characteristic structural feature of the
derivatives is a potential redox-active center in the side chain rather than in the intercalative part of the
molecule. Doubly bioreductive agents such as −bis-nitroimidazoles× [28] or N,N'-dioxides [29] have
previously demonstrated enhanced cytotoxicity and selectivity towards hypoxic tumor cells.

Helvetica Chimica Acta Vol. 87 (2004)
1001
Table. Chemical Yields of the Disubstituted 9,10-Anthraquinones 1 19, and Their Cytotoxicities against
Suspended Murine and Human Tumor Cell Lines
Compound
R
Yield [%]
IC50 [mm]a)
Hep G2b)
C6c)
2.2.15d)
1
2
3
4
5
6
7
8
Et
80
7825.2
63
780.09
70
73
55
74
55
72
23.2 Æ 1.1
Æ 1.1
94.4 Æ 1.7
29.6 Æ 1.0
(CH2)2OH
i-Pr
(CH2)2NMe2
(CH2)2NH(CH2)2OH
Pr
49.37 Æ 1.2
Æ 1.5
19.26 Æ 2.2
18.98 Æ 2.4
0.13 Æ 0.01
7.01 Æ 0.11
Æ 0.02
7.64 Æ 2.3831.06
Æ 0.01
1.20 Æ 0.02
1.74 Æ 0.14
14.27 Æ 1.54
27.6 Æ 1.1
0.12 Æ 0.01
1.17 Æ 0.03
16.03 Æ 0.681.94
12.67 Æ 0.37
94.7 Æ 1.7
i-Bu
32.5 Æ 1.1
28.6 Æ 0.9
7.48 Æ 0.09
(CH2)3OH
(CH2)3NH2
Bu
9
10
11.67 Æ 0.09
12.18 Æ 0.04
> 200
> 200
> 200
11
12
13
14
15
16
17
18
(CH2)4OH0
(CH2)4NH2
(CH2)5NH2
(CH2)5Me
Cyclopentyl
cHexe)
4-(OH)-C6H4
CH2C6H5
(CH2)2C6H5
65
65
40
65
55
46
56
75
133.0 Æ 3.1
11.61 Æ 0.02
17.37 Æ 0.74
192.25 Æ 4.1
12.56 Æ 0.16
8.36 Æ 0.13
15.5 Æ 0.24
10.07 Æ 0.58
18.7 Æ 0.7
> 200
> 200
> 200
36.56 Æ 1.1
56.67 Æ 2.1
4.20 Æ 0.76
185.5
181.5 Æ 7.5
2.00 Æ 0.5
0.90 Æ 0.01
1.48 Æ 0.62
98.76 Æ 2.1
134.6 Æ 4.1
14.21 Æ 0.0832.5
130.9 Æ 3.7
217.1 Æ 4.2
0.07 Æ 0.01
1.00 Æ 0.16
> 1
39.75 Æ 1.1
134.6 Æ 3.5
Æ 2.5
327 Æ 5
19
64
182.5 Æ 5.1
0.40 Æ 0.02
1.60 Æ 0.04
2.0 Æ 0.54
Mitoxantrone
Adriamycin
Cisplatin
a) Drug concentration inhibiting 50% of cell growth after 48h of exposure. The variance for IC50 was less than
Æ20%. Inhibition of cell growth was significantly different relative to that of the controls; n ꢀ 3, P < 0.01.
b) Human hepatoma G2 cells. c) Rat glioma C6 cells. d) Hepatitis B virus transfected hepatoma cell lines.
e) 2,3-Dimethylcyclohexyl.
crystallization. Concerning the choice of substituents, alkylamino groups were chosen
such that the overall reduction potential of the anthraquinone nucleus moved to more-
negative values [30]. The structures of 1 19 were corroborated spectroscopically
(1H-NMR, 13C-NMR, MS).
The anthraquinone moiety is known to undergo redox processes that can give rise to
cytotoxic effects [22][31][32]. We have initiated a structure-based approach towards
discovering non-nucleosidic compounds that selectively inhibit tumor cell lines. Both
the toxicity and cytotoxicity of 9,10-anthraquinone derivatives are greatly affected by
substituents [33]. Mitoxantrone, an aminoanthraquinone first synthesized in 1979,
displays antiviral, antibacterial, immunomodulatory, and antitumor activities. Its

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Helvetica Chimica Acta Vol. 87 (2004)
antitumor activity is attributed to its interaction with DNA topoisomerase II, and its
interaction with human cells may also involve nonintercalating, electrostatic inter-
actions [33]. The current series of aminoanthraquinones were also designed as
telomerase inhibitors (or activators) [16][34]. These include antisense compounds,
reverse transcriptase inhibitors, and compounds capable of interacting with high-order
telomeric-DNA-tetraplex (−G-quadruplex×) structures, so as to prevent enzyme access
to the necessary linear telomere substrate [35]. The cytotoxic in vitro activity of such
drugs, as revealed by colorimetric assays, is mainly due to chromosome damage [36].
Aminoanthraquinones may represent a novel class of polyamine-binding-site ligands
with a unique pharmacophore and may facilitate the rational design of novel NMDA
(N-methyl-d-aspartate)-receptor modulators [37].
As shown in the Table, half of the compounds studied did show significant
cytotoxicities (IC50 < 20 mm) towards tumor cells. Compound 4 showed substantial in
vitro growth delays of Hep G2, 2.2.15, and C6 cells, the mechanism of action not being
clear so far. Preliminary SAR studies on protein-kinase-C activity have suggested that
the terminal Me2N groups of 4 is crucial for PKC inhibition [25].
Compounds 4 6, 9, and 12 were tested in vitro against murine and human tumor
cells, whose growth was inhibited at micromolar level (Table). When compound 4 was
tested in vitro against Hep-G2 and hepatitis-B-virus-transfected hepatoma cell lines
(2.2.15 cells), IC50 values of 0.09 and 0.13 mm were determined, respectively.
Compounds 4 6 were very active, with IC50 values of 0.09, 1.20, and 1.74 mm,
respectively, for the inhibition of cultured Hep-G2 cell growth, as determined by the
XTT colorimetric assay (see Exper. Part), and their antiproliferative activity was
markedly enhanced, comparable to that of mitoxantrone, adriamycin, and cisplatin,
which had IC50 values of 2.00, 0.90 and 1.48 mm, respectively (positive controls).
Indeed, compound 4 was more effective by a factor of ca. 22 than mitoxantrone, ten
times more effective than adriamycin, and sixteen times more effective than cisplatin
towards Hep-G2 cells. Thus, it appears that the anthraquinone chromophore itself
causes the cytotoxicity, although the exact mechanism is still unclear. Further SAR
studies of amino-substituted 9,10-anthraquinones are now underway to investigate the
structural requirements of these interesting pharmacophores.
Experimental Part
General. All reactions were monitored by thin-layer chromatography (TLC; silica gel 60 F254). Flash-
column-chromatographic (FC) purifications were performed on silica gel (70 230 mesh; Merck) with CH2Cl2
as the eluent. Melting points (m.p.) were determined on a B¸chi B-545 apparatus and are uncorrected. FT-IR
Spectra (KBr): Perkin-Elmer 983G spectrometer; in cmÀ1. UV Spectra: Shimadzu UV-160A; lmax in nm (log e).
1H-NMR: Varian Gemini-300 (300 MHz) and Brucker AM-500 (500 MHz); chemical shifts d in ppm relative to
SiMe4 as internal standard, coupling constants J in Hz. Mass spectra (EI-MS; 70 eV, unless otherwise stated):
Finnigan MAT TSQ-46, Finnigan MAT TSQ-700 (Universit‰t Regensburg, Germany), and Finnigan MAT
LCQ (National Research Institute of Chinese Medicine, Taipei, Taiwan); in m/z (rel. %).
General Procedure for the Preparation of the 1,5-Disubstituted 9,10-Anthraquinones 1 19. A mixture of 1,5-
dichloro-9,10-anthraquinone (20; 1.0 g, 3.6 mmol) and N,N-dimethylformamide (DMF; 20 ml), containing the
appropriate amine (8.0 mmol), was heated to 908 in a glass mini-reactor for 30 min under N2 gas. After cooling,
the mixture was treated with crushed ice. The resulting precipitate was collected by filtration, washed with
plenty of H2O, and purified by recrystallization from AcOEt/hexane to afford the final products as red
needles.

Helvetica Chimica Acta Vol. 87 (2004)
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1,5-Bis(ethylamino)-9,10-anthraquinone (1). Yield: 80%. M.p. 193 1958 (AcOEt/hexane). UV (MeOH):
254 (4.50), 514 (1.49). IR (KBr): 3289, 2929, 1649. 1H-NMR (CDCl3): 1.41 1.47 (m, 6 H); 3.39 (q, J ˆ 5.2, 4 H);
6.98( d, J ˆ 7.5, 2 H); 7.53 7.58( m, 2 H); 8.31 (d, J ˆ 3.0, 2 H); 9.65 (br., 2 H). 13C-NMR (CDCl3): 14.4; 37.4;
‡
115.7; 116.2; 126.2; 133.7; 135.0; 151.3; 185.4. EI-MS: 294 (M ), 264.
1,5-Bis[(2-hydroxyethyl)amino]-9,10-anthraquinone (2). Yield: 78%. M.p. 229 2308 (AcOEt/hexane).
UV (MeOH): 521 (0.69), 282 (0.63). IR (KBr): 3370, 1646. 1H-NMR ((D6)DMSO): 3.63 (d, J ˆ 2.4, 4 H); 4.06
(s, 2 H); 5.35 (d, J ˆ 3.6, 4 H); 7.53 (d, J ˆ 8.4, 2 H); 7.79 (d, J ˆ 7.2, 2 H); 7.96 (t, J ˆ 8.1, 2 H); 10.16 (br., 2 H).
‡
13C-NMR ((D6)DMSO): 49.99; 64.59; 117.26; 119.40; 122.32; 140.65; 140.74; 156.39; 185.36. EI-MS: 326 (M ),
295, 264.
1,5-Bis(isopropylamino)-9,10-anthraquinone (3). Yield: 65%. M.p. 170 1728 (AcOEt/hexane). UV
(MeOH): 527 (0.33), 284 (0.26). IR (KBr): 3289, 1648. 1H-NMR (CDCl3): 1.38( d, J ˆ 6.0, 12 H); 3.85 (q,
J ˆ 6.6, 2 H); 7.01 (d, J ˆ 3.3, 2 H); 7.51 (d, J ˆ 7.5, 2 H); 7.55 (t, J ˆ 3.7, 2 H); 9.79 (d, J ˆ 6.0, 2 H). 13C-NMR
‡
(CDCl3): 22.45; 43.30; 112.48; 114.05; 116.25; 134.68; 136.24; 150.29; 185.00. EI-MS: 322 ( M ), 312, 264.
1,5-Bis{[2-(dimethylamino)ethyl]amino}-9,10-anthraquinone (4). Yield: 78%. M.p. 187 1888 (AcOEt/
hexane; lit. [38]: 188 1908). UV (MeOH): 522 (0.42), 282 (0.38). IR (KBr): 3277, 1642. 1H-NMR (CDCl3): 2.39
(s, 12 H); 2.69 (t, J ˆ 6.4, 4 H); 3.46 (q, J ˆ 6.0, 4 H); 6.99 (d, J ˆ 8.4, 2 H); 7.52 7.63 (m, 4 H); 9.80 (br., 2 H).
13C-NMR ((D6)DMSO): 41.02; 45.52; 58.12; 113.21; 114.87; 116.20; 135.05; 136.34; 151.18; 185.35. EI-MS: 380
‡
(M ), 335, 264.
1,5-Bis({2-[(2-hydroxyethyl)amino]ethyl}amino)-9,10-anthraquinone (5). Yield: 70%. M.p. 185 1868
(AcOEt/hexane; lit. [38]: 1858). UV (MeOH): 522 (1.07), 281 (1.15). IR (KBr): 3400, 3288, 1640, 1590.
1H-NMR ((D6)DMSO): 2.66 (d, J ˆ 5.7, 4 H); 3.34 (t, J ˆ 10.8, 8 H); 3.50 (s, 2 H); 4.54 (s, 2 H); 7.18 (d, J ˆ 6.0,
2 H); 7.41 (t, J ˆ 7.0, 2 H); 7.61 (t, J ˆ 7.0, 2 H); 9.79 (s, 2 H). 13C-NMR ((D6)DMSO): 42.45; 48.00; 51.48; 60.54;
‡
112.15; 114.31; 117.23; 135.60; 135.70; 151.18; 184.26. EI-MS: 412 ( M ), 351, 339, 278(100), 265, 252.
1,5-Bis(propylamino)-9,10-anthraquinone (6). Yield: 73%. M.p. 153 1548 (AcOEt/hexane). UV
(MeOH): 524 (0.16), 283 (0.13). IR (KBr): 3330, 1640. 1H-NMR (CDCl3): 1.12 (t, J ˆ 7.5, 6 H); 1.81 (q, J ˆ
7.1, 4 H); 3.38( q, J ˆ 6.4, 4 H); 7.01 (d, J ˆ 7.8, 2 H); 7.54 (t, J ˆ 7.3, 2 H); 7.60 (t, J ˆ 2.4, 2 H); 9.79 (br., 2 H).
‡
13C-NMR (CDCl3): 11.70; 22.27; 45.27; 113.33; 115.43; 117.22; 135.21; 136.20; 150.82; 185.34. EI-MS: 322 (M ),
293 (100), 264, 236.
1,5-Bis(isobutylamino)-9,10-anthraquinone (7). Yield: 55%. M.p. 167 1688 (AcOEt/hexane). UV
1
(MeOH): 527 (0.35), 284 (0.26). IR (KBr): 3359, 1624. H-NMR (CDCl3): 1.11 (d, J ˆ 6.3, 12 H); 2.00 2.10
(m, 2 H); 3.20 (q, J ˆ 6.3, 4 H); 6.98( d, J ˆ 7.8, 2 H); 7.51 (d, J ˆ 7.8, 2 H); 7.56 7.60 (m, 2 H); 9.88 (br., 2 H).
13C-NMR (CDCl3): 20.56; 28.08; 50.74; 112.85; 114.56; 116.35; 135.08; 136.33; 151.66; 185.42. APCI-MS: 351
‡
(M ), 352 (24), 295, 279, 254.
1,5-Bis[(3-hydroxypropyl)amino]-9,10-anthraquinone (8). Yield: 74%. M.p. 178 180 8 (AcOEt/hexane).
UV (MeOH): 522 (0.41), 283 (0.41). IR (KBr): 3359, 1624. 1H-NMR ((D6)DMSO): 1.74 1.83 (m, 4 H); 3.37 (t,
J ˆ 6.6, 4 H); 3.51 (q, J ˆ 5.6, 4 H); 4.63 (t, J ˆ 4.6, 2 H); 7.12 (d, J ˆ 8.7, 2 H); 7.38 (d, J ˆ 7.5, 2 H); 7.57 (t, J ˆ
7.9, 2 H); 9.65 (t, J ˆ 5.2, 2 H). 13C-NMR ((D6)DMSO): 31.96; 38.35; 58.41; 112.08; 114.33; 117.10; 135.55;
‡
135.66; 151.27; 184.37. APCI-MS: 355 (M ), 356 (25).
1,5-Bis[(3-aminopropyl)amino]-9,10-anthraquinone (9). Yield: 55%. M.p. 153 1558 (AcOEt/hexane).
UV (MeOH): 514 (1.95), 281 (1.52). IR (KBr): 3330, 1640. 1H-NMR (CDCl3): 1.92 (t, J ˆ 4.8, 4 H); 1.97 (q, J ˆ
4.5, 4 H); 2.96 (q, J ˆ 4.5, 4 H); 3.44 (q, J ˆ 6.9, 4 H); 7.04 (t, J ˆ 3.7, 2 H); 7.52 7.62 (m, 4 H); 9.79 (s, 2 H).
13C-NMR (CDCl3): 32.93; 39.84; 40.52; 114.74; 115.88; 116.29; 135.12; 136.33; 151.47; 185.44. APCI-MS: 323
‡
‡
(100, [M ‡ 1] ]), 322 (M ).
1,5-Bis(butylamino)-9,10-anthraquinone (10). Yield: 72%. M.p. 152 1538 (AcOEt/hexane). UV (MeOH):
527 (0.25), 283 (0.19). IR (KBr): 3299, 2947, 1623, 1500. 1H-NMR (CDCl3): 1.02 (q, J ˆ 4.9, 6 H); 1.50 1.60 (m,
4 H); 1.74 1.84 (m, 4 H); 3.33 (q, J ˆ 6.4, 4 H); 7.00 (d, J ˆ 7.5, 2 H); 7.52 (t, J ˆ 9.0, 2 H); 7.57 (q, J ˆ 3.0, 2 H);
9.79 (br., 2 H). 13C-NMR (CDCl3): 13.75; 20.32; 31.22; 42.62; 112.92; 114.55; 116.25; 135.05; 136.38; 151.54;
‡
185.41. APCI-MS: 352 (M ), 351 (100).
1,5-Bis[(4-hydroxybutyl)amino]-9,10-anthraquinone (11). Yield: 65%. M.p. 120 1228 (AcOEt/hexane).
UV (MeOH): 523 (1.30), 284 (1.10). IR (KBr): 3384, 1643. 1H-NMR ((D6)DMSO): 1.78( q, J ˆ 4.6, 4 H); 1.86
(q, J ˆ 4.3, 4 H); 3.39 (t, J ˆ 6.6, 4 H); 3.76 (t, J ˆ 6.1, 4 H); 4.18( s, 2 H); 7.00 (d, J ˆ 6.3, 2 H); 7.52 7.61 (m,
4 H); 9.77 (br., 2 H). 13C-NMR ((D6)DMSO): 25.50; 30.10; 60.58; 63.60; 112.08; 114.38; 117.20; 135.55; 135.69;
‡
151.26; 184.42. EI-MS: 382 (M ), 365, 291, 253 (100).
1,5-Bis[(4-aminobutyl)amino]-9,10-anthraquinone (12). Yield: 65%. M.p. 208 210 8 (AcOEt/hexane). UV
1
(MeOH): 514 (1.17), 280 (1.02). IR (KBr): 3282, 1641, 1594. H-NMR (CDCl3): 1.66 1.73 (q, J ˆ 10.6, 4 H);

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Helvetica Chimica Acta Vol. 87 (2004)
1.82 (q, J ˆ 6.3, 4 H); 2.73 2.83 (m, 4 H); 2.84 (t, J ˆ 3.4, 4 H); 3.36 (t, J ˆ 6.3, 4 H); 6.99 (d, J ˆ 7.5, 2 H); 7.52
‡
7.60 (m, 4 H); 9.76 (s, 2 H). APCI-MS: 381 (M ), 364, 309.
1,5-Bis[(5-aminopentyl)amino]-9,10-anthraquinone (13). Yield: 40%. M.p. 113 1158 (AcOEt/hexane).
UV (MeOH): 524 (0.47), 283 (0.45). IR (KBr): 3285, 1646. 1H-NMR (CDCl3): 0.87 (t, J ˆ 7.6, 4 H); 1.56 (d, J ˆ
3.3, 8H); 1.80 ( d, J ˆ 7.5, 8H); 2.75 ( t, J ˆ 6.6, 4 H); 3.34 (q, J ˆ 6.3, 4 H); 6.98( q, J ˆ 3.1, 2 H); 7.52 7.59 (m,
‡
‡
4 H); 9.75 (s, 2 H). APCI-MS: 410 (20, [M ‡ 1] ), 409 (M ).
1,5-Bis(hexylamino)-9,10-anthraquinone (14). Yield: 65%. M.p. 142 1448 (AcOEt/hexane). UV (MeOH):
518 (0.15). IR (KBr): 3388, 1626. 1H-NMR (CDCl3): 0.95 (t, J ˆ 3.5, 6 H); 1.37 (q, J ˆ 3.5, 4 H); 1.47 (t, J ˆ 7.2,
4 H); 1.75 (t, J ˆ 7.3, 4 H); 1.82 (d, J ˆ 6.9, 4 H); 3.32 (t, J ˆ 5.4, 4 H); 6.99 (d, J ˆ 6.0, 2 H); 7.52 (t, J ˆ 7.5, 2 H);
7.58( q, J ˆ 2.2, 2 H); 9.74 (br., 2 H). 13C-NMR (CDCl3): 13.92; 22.50; 26.75; 29.29; 31.52; 42.97; 114.53; 116.23;
‡
‡
127.86; 135.03; 136.38; 151.51; 185.39. APCI-MS: 407 (100, [M ‡ 1] ), 406 (M ).
1,5-Bis(cyclopentylamino)-9,10-anthraquinone (15). Yield: 55%. M.p. 198 200 8 (AcOEt/hexane). UV
(MeOH): 528 (0.39), 285 (0.30). IR (KBr): 3388, 1640. 1H-NMR (CDCl3): 1.72 1.78( m, 8 H); 1.84 (t, J ˆ 5.7,
4 H); 2.11 (t, J ˆ 5.8, 4 H); 4.04 (d, J ˆ 4.8, 2 H); 7.03 (q, J ˆ 3.2, 2 H); 7.51 (d, J ˆ 7.5, 2 H); 7.56 (t, J ˆ 3.7, 2 H);
9.86 (d, J ˆ 5.4, 2 H). 13C-NMR (CDCl3): 24.08; 33.58; 53.88; 112.92; 114.46; 117.10; 134.89; 136.44; 151.0;
‡
185.29. FAB-MS: 375 (M ), 307 (28).
1,5-Bis[(2,3-dimethylcyclohex-1-yl)amino]-9,10-anthraquinone (16). Yield: 46%. M.p. 188 1908 (AcOEt/
1
hexane). UV (MeOH): 527 (0.47), 281 (0.36). IR (KBr): 3273, 1676. H-NMR (CDCl3): 2.00 (s, 12 H); 2.14
3.67 (m, 16 H); 3.90 (s, 2 H); 7.00 9.99 (m, 6 H); 10.20 (br., 2 H). 13C-NMR (CDCl3): 17.05; 20.72; 25.09; 33.61;
‡
35.11; 38.57; 45.44; 57.46; 114.28; 116.50; 116.83; 134.97; 136.67; 151.57; 185.31. APCI-MS: 459 (M ), 460 (25).
1,5-Bis[(4-hydroxyphenyl)amino]-9,10-anthraquinone (17). Yield: 56%. M.p. 211 2128 (AcOEt/hexane).
1
IR (KBr): 3420, 3323, 1642. UV (MeOH): 517 (0.28), 252 (1.49). H-NMR (CDCl3): 4.90 (s, 4 H); 6.92 (d, J ˆ
8.7, 2 H); 7.21 (t, 4 H); 7.48( t, J ˆ 8.1, 2 H); 7.65 7.84 (m, 2 H); 8.30 8.38 (m, 2 H); 11.07 (s, 2 H). APCI-MS:
‡
423 (M ), 352 (35).
1,5-Bis[(phenylmethyl)amino]-9,10-anthraquinone (18). Yield: 75%. M.p. 218 220 8 (AcOEt/hexane). UV
(MeOH): 518(0.59), 281 (0.71). IR (KBr): 3270, 1640. 1H-NMR (CDCl3): 4.61 (d, J ˆ 6.0, 4 H); 6.95 (d, J ˆ 8.4,
2 H); 7.31 7.40 (m, 6 H); 7.41 (t, J ˆ 3.9, 2 H); 7.63 (d, J ˆ 3.0, 2 H); 10.15 (br., 2 H). 13C-NMR (CDCl3): 47.0;
‡
115.27; 116.84; 127.0; 127.31; 128.74; 135.12; 136.24; 138.12; 151.21; 185.38. EI-MS: 418 (M ), 347 (60).
1,5-Bis[(phenylethyl)amino]-9,10-anthraquinone (19). Yield: 64%. M.p. 205 2078 (AcOEt/hexane). UV
(MeOH): 527 (0.13), 285 (0.28). IR (KBr): 3270, 1640. 1H-NMR (CDCl3): 3.07 (t, J ˆ 7.5, 2 H); 3.58 3.65 ( m,
J ˆ 5.1, 2 H); 7.00 (d, J ˆ 8.1, 2 H); 7.31 (t, J ˆ 6.0, 2 H); 7.36 7.41 (m, 4 H); 7.55 (t, J ˆ 5.3, 2 H); 7.59 (d, J ˆ 5.7,
2 H); 9.83 (br., 2 H). 13C-NMR (CDCl3): 35.71; 44.61; 113.13; 114.88; 116.20; 126.52; 128.69; 135.07; 136.32;
‡
‡
138.87; 151.15; 185.36. APCI-MS: 448 (35, [M ‡ 1] ), 447 (M ).
Cell Cultures. Various cancer-cell lines (G2, 2.2.15, and C6 cells) were cultured in minimum essential
medium (MEM), supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 mg/ml)
in a humidified atmosphere with 5% CO2 at 378. Cell-culture media were renewed every 3 d, up to the
confluence of the monolayer. When the cells had formed confluent cultures, they were detached from the
culture flasks or dishes by means of the trypsin/EDTA (−ethylenediamine tetraacetate×) reagent. Test
compounds were stored at À708 before dissolved in neat DMSO. All drug solns. were prepared immediately
before evaluation, and were transfered into complete medium before addition to the cell cultures. The
−inhibitory concentration fifty× (IC50) values (see the Table) were determined after 48h of drug exposure and
represent the mean values of independent measurements performed at least in triplicate, showing the same
pattern of expression.
XTT Color Assay. The reagent XTT (ˆ2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)car-
bonyl]-2H-tetrazolium hydroxide) forms a suitably colored, water-soluble, nontoxic formazan upon metabolic
reduction by viable cells. A total of ca. 2 Â 103 cells, suspended in MEM, were plated onto each well of a 96-well
plate, and incubated in 5% CO2 at 378 for 24 h. Test compounds were then added to the culture medium for each
designated concentration. After 72 h, fresh XTT (50 ml) and the electron-coupling reagent PMS (ˆ−N-methyl
dibenzopyrazin methyl sulfate×; 1 ml) were mixed together and then added to each well. After incubation at 378
for 6 h, the absorbance at 490 nm was measured with an ELISA reader.

Helvetica Chimica Acta Vol. 87 (2004)
1005
This research was partially supported by grants from the National Defense Medical Center. The authors are
indebted to Dr. Klaus K. Mayer (Universit‰t Regensburg, Germany) and Prof. K. M¸ller (Universit‰t M¸nster,
Germany) for mass spectrometric and analytical determinations.
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Received November 17,2003