Novel anthraquinone derivatives with redox-active functional groups capable of producing free radicals by metabolism: Are free radicals essential for cytotoxicity?

Article abstract of DOI:10.1016/S0223-5234(00)80029-X

The mode of action of antitumour anthraquinone derivatives (i.e. mitoxantrone) is not clearly established yet. It includes, among others, intercalation and binding to DNA, bioreduction and aerobic redox cycling. A series of anthraquinone derivatives, with potentially bioreducible groups sited in the side chain, have been synthesized and biologically evaluated. Their redox and cytotoxic activities were screened. Derivatives which bear a 2-(dimethylamino)ethylamino substituent, known to confer high DNA affinity, demonstrated cytotoxicity but not redox activity (beside the anthraquinone reduction). Conversely, derivatives which showed redox activity were not cytotoxic toward the P388 cell line. The results suggest that bioreduction is not the main mode of action in the cytotoxicity of anthraquinones.

Full text of DOI:10.1016/S0223-5234(00)80029-X

Eur. J. Med. Chem. 34 (1999) 597−615
1999 Éditions scientifiques et médicales Elsevier SAS. All rights reserved
597
©
Original article
Novel anthraquinone derivatives with redox-active functional groups capable
of producing free radicals by metabolism: are free radicals essential
for cytotoxicity?
a
a,b
b
c
Dinorah Barasch , Omer Zipori , Israel Ringel , Isaac Ginsburg ,
d
a
Amram Samuni , Jehoshua Katzhendler *
a
Department of Pharmaceutical Chemistry, The Hebrew University of Jerusalem, Jerusalem, 91120, Israel
b
Department of Pharmacology, School of Pharmacy, The Hebrew University of Jerusalem, Jerusalem, 91120, Israel
c
Department of Oral Biology, School of Dental Medicine, The Hebrew University of Jerusalem, Jerusalem, 91120, Israel
Department of Molecular Biology, School of Medicine, The Hebrew University of Jerusalem, Jerusalem, 91120, Israel
d
(Received 6 October 1998; accepted 20 January 1999)
Abstract – The mode of action of antitumour anthraquinone derivatives (i.e. mitoxantrone) is not clearly established yet. It includes, among
others, intercalation and binding to DNA, bioreduction and aerobic redox cycling. A series of anthraquinone derivatives, with potentially
bioreducible groups sited in the side chain, have been synthesized and biologically evaluated. Their redox and cytotoxic activities were
screened. Derivatives which bear a 2-(dimethylamino)ethylamino substituent, known to confer high DNA affinity, demonstrated cytotoxicity
but not redox activity (beside the anthraquinone reduction). Conversely, derivatives which showed redox activity were not cytotoxic toward
the P388 cell line. The results suggest that bioreduction is not the main mode of action in the cytotoxicity of anthraquinones. © 1999 Éditions
scientifiques et médicales Elsevier SAS
anthraquinones / bioreduction / cytotoxicity
1
. Introduction
not fully established yet, it includes, inter alia: a) the
involvement of the quinone component in a redox cycle
process [4] leading, through a semiquinone (AQ ) inter-
The broad spectrum of antitumour efficacy of anthra-
•–
cyclines is reflected in their widespread use in cancer
chemotherapy [1–3]. Although the mode of their action is
mediate (by flavin-mediated one-electron reduction), to
alkylating intermediates and the generation of reactive
•–
•
oxygen species (ROS), in particular O2 , H O and OH.
2
2
*
Correspondence and reprints.
These species are capable of producing DNA lesions,
peroxidative injury to membrane lipids and alteration of
subcellular organelles and functional integrity, and b)
their intercalative capacity, which either affects DNA
structure and transcription to mRNA or alternatively
stabilizes the cleavable complex between topoisomerase
II and DNA [5].
Abbreviations: anh., anhydrous; AQ, anthraquinone; aq., aqueous;
CDCl3, deuterochloroform; CV, cyclic voltammetry; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; DCC, dicyclohexylcarbodiimide;
DCU, dicyclohexylurea; δ, chemical shift (ppm); DMF, N,N-
dimethylformamide;
d7-DMF,
heptadeutero-N,N-dimethyl-
formamide; d6-DMSO, hexadeuterodimethylsulfoxide; DMSO, di-
a
c
methylsulfoxide; E , anodic redox potential; E , cathodic redox
potential; EDC, water soluble carbodiimide, 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; Et3N,
triethylamine; EtOAc, ethyl acetate; FAB-MS, Fast Atom Bom-
The anthraquinone (9,10-anthracenedione) skeleton is
a central constituent of the anthracyclines (doxorubicin
and daunorubicin) and other antineoplastic agents such as
mitoxantrone [6, 7], (figure 1) and anthrapyrazole deriva-
tives [8]. To gain a wider understanding of the involve-
ment of radicals in the action of anthraquinone-derived
t
bardment Mass Spectrometry; Florisil (U.S. Silica Co.), magne-
sium silicate particle size 75-150mm; leucoquinizarin, 2,3-dihydro-
9
,10-dihydroxy-1,4-anthraquinone; MeOH, methanol; mp, melting
point; NHS, N-hydroxysuccinimide; Py, pyridine; ROS, reactive
oxygen species; RT, room temperature; TLC, thin layer chromato-
graphy.

5
98
Figure 1. Selected antineoplastic agents having an anthraquinone moeity.
agents, several related compounds bearing selected char-
In order to discriminate between the effects of redox-
acteristic functional groups were designed. The approach
was to develop structure-activity relationships (SAR) of
anthraquinone analogues with redox-active centres at-
tached to the anthraquinone skeleton through spacer side
chains a) at positions 1 and 1,4; and (b) at position 1,
together with substituents with DNA-binding affinity at
position 4.
If radicals mediate the cytotoxic effect, redox-active
groups linked to an anthraquinone skeleton might amplify
the anticancer activity. However, if the cytotoxic activity
relates only to the intercalative capacity of the an-
thraquinone structure, it might be affected by groups that
enhance binding-affinity to DNA. In the present work, the
cytotoxic activity of the derivatives was assessed using
the P388 cell line.
active groups on compounds with high and low DNA-
binding affinity, two classes of derivatives were designed:
derivatives with cationic substituents (with binding affin-
ity to DNA) and derivatives without cationic groups. The
cationic [2-(dimethylamino)ethyl]amino side chain (de-
rivatives 3, 9, 29–32), present in the known cytotoxic
compound 37 (figure 1) was selected to confer DNA-
binding affinity [6]. Unfortunately, part of the non-
cationic derivatives (1, 4–6, 10–15) lack the necessary
solubility in aqueous solutions to be appropriately
screened, therefore soluble derivatives were designed, via
the introduction of the 3-hydroxypropylamino residue
(derivatives 2, 7 and 8).
The derivatives may be classified according to the
redox-active group introduced:
1
) Nitroaromatic derivatives (6–11). Nitroaromatics
2
2
2
. Results and discussion
and nitroheterocycles undergo bioactivation [14, 15], un-
der anaerobic conditions, through a series of single
electron reductions by a range of flavoenzymes leading to
the reduction sequence RNO ⇒ RNO2
RNHOH ⇒ RNH2.
.1. Chemistry
•–
.1.1. Design of synthetic derivatives
A variety of compounds (derivatives 1–36), having an
⇒
RNO ⇒
2
additional redox-active centre with potential to be bioac-
tivated, were designed. The characteristic structural fea-
ture of the designed substances is a potential redox-active
centre in the side chain rather than in the intercalative part
of the molecule (table I). These compounds were ex-
pected to intercalate and undergo activation during their
metabolic pathway and thus serve as electron transfer
mediators. Bis-bioreductive agents such as bis-
nitroimidazoles [9] or N,N≠-dioxides [10] have previ-
ously demonstrated enhanced cytotoxicity and selectivity
towards hypoxic tumour cells. Nitracrine and its
N-oxide [11, 12] have been shown to be DNA-directed
bioreductive drugs, while the N,N≠-dioxide analogue of
mitoxantrone [13] has been proposed to be a prodrug,
which under hypoxia is bioreductively activated to a
DNA-binding agent.
2
) Pyridine, pyridine N-oxide and pyridinium deriva-
tives (16–21, 24–35). Pyridinium derivatives, like
N-methyl-4-phenylpyridinium (MPP ) [16], are known
+
electron acceptors which undergo one-electron reduction
by the following pathways: a) direct one electron transfer
and b) a flavin-mediated hydride transfer followed by one
electron reduction. The pyridine group might lead to the
formation of a pyridyl radical. Pyridine N-oxide deriva-
tives, such as nicotinamide N-oxide, undergo reduction to
the corresponding pyridine derivative [17]. During the
reduction pathway, the pyridine N-oxide group might be
converted to a cytotoxic nitroxide radical intermedi-
ate [18].
3) Polyhaloalkyl derivatives (14–15). Polyhalogenated
alkanes can either undergo oxygenation [19] or reductive

5
99
Table I. Anthraquinone derivatives synthesized.
Derivative R1
R4
R5
R8
1
2
3
4
5
X: n = 3, Y = OC(C H )
H
H
H
H
H
H
H
H
H
H
H
6
5 3
X: n = 3, Y = OC(C H )
X: n = 3, Y = OH
X: n = 2, Y = N(CH3)2
X: n = 3, Y = OC(C H )
6 5 3
6
5 3
X: n = 3, Y = OC(C H )
6
5 3
X: n = 3, Y = OC(C H )
6
5 3
X: n = 3, Y = NHCON(C H )
H
6
5 2
6
X: n = 3, Y =
X: n = 3, Y =
H
H
H
7
X: n = 3, Y = OH
X: n = 3, Y = OH
H
H
8
9
X: n = 3, Y =
X: n = 3, Y =
H
H
H
H
X: n = 2, Y = N(CH3)2
1
0
X: n = 3, Y =
X: n = 3, Y =
H
H
H
11
H
H
H
1
1
1
1
2
3
4
5
X: n = 3, Y = NHCOC H5
X: n = 2, Y = CHO
H
H
H
H
H
H
H
H
H
H
H
H
6
X: n = 3, Y = NHCOOCH CCl3
2
X: n = 3, Y = COOCH CCl3
2
16
17
18
19
X: n = 1, Y =
X: n = 1, Y =
X: n = 1, Y =
X: n = 1, Y =
H
Cl
H
H
H
H
H
X: n = 1, Y =
Cl
H
H
X: n = 1, Y =
2
0
1
H
H
H
H
H
2
OH
2
2
2
3
NHCOC H
H
OH
H
H
H
H
6
5
NHCOC H
6
5

6
00
Table I. Continued.
Derivative R1
R4
H
R5
H
R8
H
24
25
26
27
28
29
30
31
32
X: n = 2, Y =
X: n = 2, Y =
X: n = 2, Y =
X: n = 2, Y =
X: n = 2, Y =
X: n = 1, Y =
X: n = 1, Y =
H
H
Cl
H
X: n = 2, Y =
H
X: n = 2, Y =
H
H
H
H
X: n = 2, Y =
X: n = 2, Y = N(CH3)2
H
H
H
H
H
H
X: n = 2, Y = N(CH3)2
X: n = 2, Y = N(CH3)2
X: n = 2, Y = N(CH3)2
H
H
3
3
4
H
H
H
H
H
3
3
3
5
6
H
H
H
H
+
+
X: n = 3, Y = NH3
X: n = 3, Y = NH3
dehalogenation by microsomal cytochrome P450
20–22]. The proposed reductive pathway involves one-
electron transfer and halide elimination to generate a
carbon-centred radical. The radical formed may either
abstract a hydrogen from the close environment (mem-
brane lipid), or react with oxygen to form a reactive
peroxy radical, or undergo a second dehalogenation.
2.1.2. Synthesis
[
Method A: this method involves nucleophilic displace-
ment of 1-chloro-, 1,5-dichloro- and 1,8-dichloro-
anthraquinone by alkanamine derivatives to form mono-
and bis-substituted anthraquinones [7, 24]. The alkan-
amine derivatives in this class (figure 2) were 1,3-
propanediamine, 3-aminopropanol, 4-aminobutyric acid
and various aminoalkylpyridine derivatives. This method
led to a direct (one step) synthesis of the 1-ethylamino
and 1-methylamino side chains bearing 2≠-pyridyl or
3≠-pyridyl residues (derivatives 16, 18, 24 and 25). The
symmetrically substituted 1,5- and 1,8-bis(pyridyl-
4) Triarylmethyl derivatives (1–4). Triphenylmethyl
radicals are stable carbon-centred radicals, which may be
formed during bioreduction (cytochrome P450) of triaryl-
methane derivatives [23] and may react with oxygen to
form a reactive peroxy radical.

6
01
Figure 2. (a) 1,3-diaminopropane, toluene, reflux, 18 h; (b) diphenylcarbamyl chloride, CH Cl , R.T., 3 h; (c) 4-nitrobenzoic acid,
2
2
DMF, Et N, NHS, water soluble carbodiimide (WSCI), 3 d; (d) benzoyl chloride, Py, R.T., 18 h; (e) 2,2,2-trichloroethyl chloroformate,
3
Et N, CH Cl , R.T., 90 min; (f) 3-aminopropanol, n-butanol, reflux, 4 h; (g) chlorotriphenylmethane, Py, 80 °C, 8 h; (h) methanesul-
3
2
2
fonyl chloride, Py, CH Cl , R.T., 48 h; (i) 4-nitroimidazole, DMF:CH Cl (1:2), DBU, reflux, 12 h; (j) pyridinium chlorochromate /
2
2
2
2
silica gel, CH Cl , 90 min, R.T. in ultrasound bath; (k) 4-aminobutyric acid, DMSO, Et N, 150 °C, 90 min; (l) 2,2,2-trichloroethanol,
2
2
3
DCC, R.T., 3 h; (m) 2-amino-5-nitrothiazole, DMF, Et N, NHS, WSCI, R.T., 3 d; (n) 2-(2-aminoethyl)pyridine, DMSO, 150 °C,
3
1
0 min; (o) 3-(aminomethyl)pyridine (neat), 40–50 °C, 36 h; (p) 2-(aminomethyl)pyridine (neat), 40–50 °C, 36 h; (q) N,N-dimethyl-
ethylenediamine (neat), 55 °C, 72 h.
alkylamino)anthraquinones (derivatives 17, 19, 26 and
nizarin [6, 24, 26, 27] with a two molar excess of the
appropriate amine under nitrogen atmosphere followed
by air oxidation. The derivatives 27 and 36 were thus
prepared in good yield. Derivatives 2 and 4 were prepared
in two steps: synthesis of the intermediate 42 from
leucoquinizarin and 3-aminopropanol, followed by trity-
lation of the hydroxy groups in the side chains.
The non-symmetrically 1,4-substituted analogues were
obtained in a “one pot” sequential treatment of leucoqui-
nizarin [26, 28] with two amines (intermediates 43–45
and derivative 29). The two side chains of the non-
symmetrically substituted compounds thus prepared
serve two goals. One side arm is designed toward its
engagement in bioreductive processes by itself (deriva-
tive 29), or after substitution with bioreductive groups
(derivatives 3, 7–9) and the second enhances solubility
(3-aminopropanol) or DNA-binding affinity (N,N-di-
methylethylenediamine).
2
8) were obtained under the same reaction conditions.
The non-symmetrically substituted derivative 30 was
prepared in a two-step reaction, where the intermediate
derivative 18 was further treated with N,N-dimethyl-
ethylenediamine. Other monosubstituted derivatives (fig-
ure 2) derived from conversion of 3-amino-, 3-hydroxy-
and 3-carboxypropylamino side chains (intermediates
3
8–40) to their final defined moieties. Intermediate 38
was carbamoylated (5), benzoylated (6 and 12) and
formylated (14) via conventional methods. Intermediate
3
9 was tritylated to yield 1. In addition, it was oxidized
by pyridinium chlorochromate (on silica gel) to form
3 [25]. Derivative 11 was obtained in two steps by
1
mesylation of 39 to get 41, which underwent nucleophilic
substitution by 4-nitroimidazole in the presence of DBU
as a strong base. The carboxy group in 40 was amidated
(10) and esterified (15).
Method B: this method is related to the synthesis of
Method C: this method involves an amidation [29, 30]
of 1-amino-, 1,4-diamino-, 1-amino-4-hydroxy- and
1-amino-4-[2-(dimethylamino)ethylamino] anthraquinone
(figure 4). In general, this was accomplished by treatment
of the 1-aminoanthraquinone derivatives with benzoyl
symmetrically and non-symmetrically substituted 1,4-
bis(alkylamino)anthraquinones (figure 3). In the case of
the symmetrically substituted analogues, the usual meth-
odology for synthesis is the treatment of leucoqui-

6
02
Figure 3. (a) N,N-dimethylethylenediamine, MeOH, N , 50 °C, 1 h; (b) 3-(aminomethyl)pyridine, N , 50 °C, 2 h; air 18 h; (c)
2
2
2
3
-(2-aminoethyl)pyridine, MeOH, N , 50 °C, 1 h; air, 18 h; (d) 1,3-diaminopropane, MeOH, N , 50 °C, 2 h; air, 18 h; (e)
2 2
-aminopropanol, MeOH, N , 50 °C, 2 h; air, 18 h; (f) chlorotriphenylmethane, Py, 80 °C, 6 h; (g) 4-nitrobenzoic acid, DMF, Et N,
2
3
NHS, WSCI, RT, 3 d; (h) chlorotriphenylmethane, Py, 80 °C, 3 h; (i) 3-aminopropanol, MeOH, N , 50 °C, 1 h; (j) 4-nitrobenzoyl
2
chloride, DMF, Et N, R.T., 12 h; (k) 2-nitrophenylacetyl chloride, DMF, Et N, R.T., 12 h.
3
3
reduction waves (table II). The electrochemical transi-
tions consisted of two consecutive one-electron reduction
steps [31] of anthraquinone (AQ) to generate first the
chloride (derivatives 22, 23), nicotinoyl chloride (deriva-
tives 20, 21, 31, 34) and nicotinoyl chloride N-oxide
(
derivative 32) in toluene under reflux. The monopyri-
dinium and the bispyridinium salts (derivatives 33 and
5) were obtained from derivatives 20 and 34 respec-
•–
semiquinone radical anion AQ and then the dianion
–2
AQ . Derivatives with an amido group attached to the
anthraquinone skeleton (20–22, 31–35) displayed less
negative values (from –0.48 V to –0.73 V) for the first
reduction wave than the amino derivatives (1–18, 24–30,
3
tively, by a direct methylation of the pyridine residue with
methyl iodide (neat). Intermediate 46, which was em-
ployed as a starting material for the synthesis of 31 and
4
2–44, 46), which displayed values from –0.85 V to
32, was prepared according to the guidelines presented in
–1.20 V. This recurring difference in the redox potential
the synthesis of non-symmetrically 1,4-substituted an-
thraquinones (figure 3): leucoquinizarin was treated first
with N,N-dimethylethylenediamine, then with gaseous
ammonia in one reaction vessel and finally oxidized.
between the two series of compounds can be attributed
either to the difference between the electron withdrawal
effect of the substituents of the two systems, or to
possible perturbation due to the acyl substituent, to
hydrogen bonding between the amino group in position 1
and the quinonic oxygen atom. Concerning the substitu-
ent polar effect, it is known that electron-donating sub-
stituents, such as alkylamino groups, shift more than
electron-withdrawing substituents such as the amido
2.1.3. Redox activity assayed by cyclic voltammetry (CV)
Reduction of the anthraquinone moiety: the reduction
sweep (cathodic) for most of the derivatives in DMF
consisted of two major reversible (or quasi-reversible)

6
03
Figure 4. (a) benzoyl chloride, toluene, reflux, 24 h; (b) nicotinoyl chloride, toluene, reflux, 24 h; (c) iodomethane, reflux, 24 h; (d)
N,N-dimethylethylenediamine, MeOH, N , 50 °C, 1 h; (e) NH (gas) bubbled 5 min, R.T., then sealed for 2 d, R.T.; (f) nicotinoyl
2
3
chloride N-oxide, toluene, reflux, 24 h.
groups, the reduction potential of the anthraquinone to
negative values [32]. Referring to the second explanation
to the above phenomenon, it is assumed that in aprotic
solvents an internal hydrogen bond is formed between the
quinonic oxygen and the amino group at positions 1, 4, 5
and 8. Accordingly, this would result in a more negative
reduction wave potential. Acylation of the amino group at
these positions modulates the function of the hydrogen
bond. In the amide form, the carbonyl group of the side
chain tends to compete with the quinonic oxygen for
hydrogen bonding. Thus, the quinone will be less resis-
tant to reduction.
The results presented in table II for 37 are in accord
with those previously described [31], although the two
sets of experiments were not carried out in the same
solvent (DMF instead of DMSO). However, a third wave
at –1.54 V was observed, in analogy to other alkylamino-
anthraquinone derivatives assayed (table II). It is plau-
sible to assign the peaks at –1.13 V and –1.58 V to the
first and the second electron reduction wave of the
quinone structure. The reversible peak at –0.79 V seems
also to be related to the first reduction process, although
its specific implication is obscure. It can be speculated
that this reduction peak is associated with internal hydro-
gen bonding (NH-----O=C) equilibrium which is faster
than the scan rate of 50 mV/s and thus, the quinone is
reduced in its two forms. An alternative possibility is that
the two less negative peaks (–0.79 V and –1.13 V)
correspond to the first and the second electron reduction
steps, while the third reduction step is attributed to a rapid
complex formation between species involved in the
course of the CV reduction [33]. In the case of hyperi-
cin [34], the two cathodic waves (in DMF) at –1.14 V and
–1.53 V, ascribed to the redox steps of the quinone part,
are in good agreement with the data presented in table II
for the cathodic peaks of alkylaminoanthraquinones.
Electrochemical reduction of anthrapyrazole (in
DMF) [35], leading to the formation of a semiquinone-
type radical, also occurs at a highly negative redox
potential of –1.30 V which is in the range of the tested
alkylaminoanthraquinones.
Redox-active substituent effect: most of the nitro-
derived compounds (6–10) showed an additional quasi-
reversible reduction wave (shoulder) at –1.6 V. This
reduction potential is attributed to the nitro group since a
similar reduction wave pattern was observed for methyl
4-nitrobenzoate (47) (table II). An additional reversible
reduction wave (shoulder) was observed for the nitro
derivatives 7, 9 and 10 at –0.8 V to –1.0 V, which is not
related explicitly to the anthraquinone or the nitro groups.
This wave (shoulder) is presumably related to intra-
molecular redox activity between two reductive linked
species (nitro and quinone groups). Since the reduction
potential of the nitro group resides in the range of the two

6
04
Table II. Redox potential of anthraquinone derivatives measured by cyclic voltammetry.
c
a
a
b
c
a
a
b
c
a
a
b
c
a
a
b
Derivative
E
E
E
E
E
E
E
E
p4
p1
p1
p2
p2
p3
p3
p4
1
1
1
1
1
2
3
4
5
6
7
8
9
1
,4-Ametantrone
,5-Ametantrone
,5-Dichloroanthraquinone
-Aminoanthraquinone
– 0.79
– 1.03
– 0.77
– 0.97
– 0.96
– 1.08
– 1.13
– 1.13
– 1.00
– 0.98
– 1.00
– 1.15
– 1.03sh
– 0.83sh
– 0.95
– 0.95
– 0.73sh
– 0.98
– 0.93
– 0.88
– 0.71
– 0.67sh
– 0.67
– 0.52
– 0.68
– 0.93
– 0.65
– 0.70
– 1.07
– 0.99
– 0.75sh
– 1.09
– 1.53
– 1.40
– 1.52
– 1.58
– 1.55
– 1.59
– 1.62
– 1.53sh
– 1.53sh
– 1.15
– 1.58
– 1.14
– 0.95
– 1.48
– 1.58
– 1.05
– 1.50
– 1.03
– 1.51
– 1.21
– 1.51
– 1.43
– 0.96
– 0.69
– 0.92
– 0.93
– 1.03
– 1.08
– 1.06
– 0.85
– 0.90
– 0.95
– 1.08
– 0.98
– 0.70
– 0.90
– 0.90
– 1.24
– 1.48
– 1.53
– 1.55
– 1.38
d
– 1.63
– 1.65
– 1.58sh
– 1.67
– 1.60sh
– 1.55
– 1.58
– 1.53
– 1.40
– 1.08
– 1.47
– 1.09
– 0.89
– 1.40
– 1.45
– 0.95
– 1.39
– 1.70
– 1.72
– 1.53
– 1.55
– 1.50
0
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
4
5
6
7
8
0
1
2
4
5
6
7
8
9
– 1.75
– 1.45
– 0.93
– 0.88
– 0.80
– 1.47
– 0.93
– 0.87
– 1.27
– 1.07
– 1.27
– 1.38
– 0.90
– 1.05
– 1.53
– 1.48
– 1.05
– 1.38
– 0.89
– 0.80
– 1.22
– 0.98
– 1.20
– 1.31
– 0.80
– 0.97
– 1.43
– 1.35
– 1.03
– 1.60
– 1.46
– 1.53
– 1.38
– 0.61
– 0.44
– 0.62
– 0.84
– 1.35
– 1.40
– 1.32
– 1.50
– 0.98
– 0.90
– 1.60
–
0.85sh
30
31
32
33
35
36
37
42
43
44
45
46
47
– 0.72sh
– 0.77
– 0.72
– 0.61
– 0.40
– 0.70
– 0.79
– 0.80
– 1.08
– 0.93
– 0.55
– 0.76
– 0.95
– 1.02
– 1.01
– 1.24
– 1.01sh
– 0.71
– 0.95
– 1.13
– 1.14
– 1.65
– 1.20
– 1.03
– 1.14
– 1.55sh
– 0.96
– 1.18
– 1.64
– 1.32
– 1.58
– 1.24
– 0.70
– 0.68
– 0.53
– 1.21
– 1.29
– 1.13
– 1.58
– 1.62
– 1.10
– 1.16
– 0.67
– 0.85
– 1.08
– 1.08
– 1.55
– 1.13
– 0.83
– 1.11
– 1.30
c
– 0.75
– 1.05
– 1.50
– 1.53
– 1.62sh
– 1.70
– 1.65
– 1.58
– 1.70
– 1.47
– 1.52
– 0.80
a
c
b
a
c
d
E : cathodic peak potential (V); E : anodic peak potential (V); see scheme 1; sh: shoulder.
p
p
reduction potentials of the anthraquinone molecule, it
seems most plausible that the nitro group plays a role in
some intramolecular electron transfer processes.
Derivative 33 displayed an additional shoulder at –1.0
V, which could be due to the reduction of the pyridinium
ring, in compliance with the reduction potential of
NADP in DMSO [36].
For derivative 5, which contains a diphenylureido
component, an additional quasi-reversible reduction
wave (shoulder) was noticed at –1.5 V. This shoulder
+

6
05
Table III. Concentration of synthetic derivatives effective in inhibiting the growth rate of the P388 murine leukaemia cells and
macrophage-like J774.2 cells by 50 % (ED ).
50
Derivatives
ED50 (µM)
Derivatives
ED50 (µM)
P388
P388
J 774.2
1
2
3
4
5
6
7
8
9
> 10
6.0
2.8
> 10
10
10
> 10
10
17
18
19
20
21
22
23
29
30
31
32
33
35
36
37
46
>> 7
>> 20
>> 4
> 6
> 1.3
>> 3
> 0.8
1.2
8.6
1.6
0.5
0.097
5
1.6
1
0
3.7
> 10
10
0.086
0.043
>> 20
12
1
1
1
1
1
1
1
2
3
4
5
6
3
> 10
2.4
>> 20
0.37
0.044
0.87
0.097
0.5
might be associated with the effect of the ureido group on
the quinone structure since N,N-diphenyl-N≠-propylurea
did not exhibit any detectable peak during the CV sweep.
Other attached functional groups as triphenylmethoxy
The presence of at least one cationic substituent, like
2-(dimethylamino)ethylamino, which confers DNA-
binding affinity, was essential for cytotoxicity. However,
its presence does not account for the differences in
cytotoxicity: a cationic derivative (9) was not cytotoxic,
suggesting that other unknown factors contribute to
cytotoxicity. Derivatives 29 and 31 both possess a cat-
ionic substituent, though 31 is ten times more potent than
29, suggesting that amidoanthraquinones (with less nega-
tive reduction potentials) are more cytotoxic than ami-
noanthraquinones. All the non-cationic derivatives, even
those soluble in aqueous solutions, were not cytotoxic.
These results reaffirm that the presence of at least one
cationic group is required for cytotoxicity and suggest
that the bioreduction is not the predominant mode of
action in the cytotoxicity of anthraquinones.
(
(
1–4), formyl (13), trichloromethyl (14, 15) and pyridine
16–21, 24–31, 34) did not exhibit any additional reduc-
tion wave, which can be assigned exclusively to these
groups. One plausible explanation is that their reduction
wave is hidden by the anthraquinone wave.
2.2. Biological activity
2.2.1. Cytotoxic activity of the anthraquinone derivatives
in cell culture
All the derivatives synthesized were evaluated for
cytotoxic activity against P388 murine leukaemia
cells [37, 38]. Those compounds which have shown
activity were then evaluated against murine macrophage-
like J774.2 cells [39] (table III).
Derivatives 31 and 32, which are non-symmetrically
substituted at the 1,4-positions of the anthraquinone,
showed high cytotoxic activity comparable to 37, as
described in the literature [6, 40]. The N-oxide of the
nicotinoyl group neither increased nor lowered the cyto-
toxicity. The intermediate 46 and derivative 36 had
significant cytotoxic activity. All the other derivatives did
not show any significant activity. Some of them precipi-
tated in the medium due to their low solubility in aqueous
solutions, thus preventing an accurate assessment of their
cytotoxic potential.
In conclusion, new anthraquinone derivatives with
potential bioreducible groups were synthesized. Two
derivatives (31 and 32) showed high cytotoxicity toward
the P388 cell line. The presence of at least one cationic
substituent, like 2-(dimethylamino)ethylamino in these
derivatives, which confers DNA-binding affinity, was
essential for cytotoxicity. However, one cationic deriva-
tive (9) was not cytotoxic, suggesting that other unknown
factors contribute to cytotoxicity. All the non-cationic
derivatives, even those soluble in aqueous solutions, were
not cytotoxic. These results reaffirm that the presence of
at least one cationic group is required for cytotoxicity and
suggest that the bioreduction is not the predominant mode
of action in the cytotoxicity of anthraquinones.

606
3
. Experimental protocols
137.9, 137.4, 136.4, 130.4, 130.2, 122.7, 118.9, 115.9,
•+
4
1
4.0, 43.0, 36.5. MS m/z (rel. intensity, %): 280 ([M] ,
Materials: solvents were purchased from Biolab
•+
•+
5), 238 ([M–C H N] , 100), 237 ([M–C H N] , 96).
2
4
2 5
(
Jerusalem, Israel) and Frutarom (Haifa, Israel). Chemi-
Anal C H N O (C, H, N).
17 16 2 2
cal reagents were bought from Aldrich (Milwaukee, WI).
Unless otherwise stated, the experiments were conducted
at room temperature.
3
.1.2.
N-[3-(9,10-Dihydro-9,10-dioxo-1-anthracenyl-
amino)propyl]-N≠,N≠-diphenylurea (5)
1
13
H NMR (300 MHz) and C NMR spectra (75.429
To a suspension of 0.6 g (2.1 mmol) of 38 in CH Cl
2
2
MHz) were obtained on a Varian VXR-300S (Palo Alto,
CA) spectrometer. Chemical shifts are reported as ppm
using tetramethylsilane (TMS) as internal standard. Mass
spectra were obtained with an LKB 2091 mass spectrom-
eter using electron impact ionization (70 eV). The ion
source was heated to 220 °C and the sample was inserted
by direct inlet without external heating. FAB-MS were
carried out by the Mass Spectra Laboratory, Faculty of
Chemistry, Technion, Haifa. IR spectra were recorded on
a Perkin Elmer FT-2000 model. Solid samples were
pressed into KBr pellets or dispersed in Nujol onto NaCl
plates. Melting points were observed on an Electrother-
mal apparatus (Electrothermal, England) and are uncor-
rected. Elemental analyses were performed by the Mi-
croanalytical Laboratory at the Hebrew University, Givat
Ram, Jerusalem. Analyses indicated by the symbols of
the elements were within ± 0.4% of theoretical values.
(
40 mL), 0.8 g (3.5 mmol) of diphenylcarbamyl chloride
were added and the mixture was stirred at room tempera-
ture for 3 h. The solution was diluted with CH Cl
2
2
(
100 mL), washed with water (2 × 50 mL), dried over
anh. MgSO and evaporated to dryness. The residue was
4
purified by flash chromatography on silica gel (eluent:
EtOAc:CH Cl 10:90) giving a red solid (yield: 0.5 g,
5
2
2
0%), which was recrystallized from EtOAc. M.p.
–1
2
05–208 °C. IR (cm , KBr) 3 315 (NH), 1 657 (C=O).
1
H NMR (CDCl ) δ 9.72 (bt, 1H), 8.22–8.25 (m, 2H),
3
7
.68–7.79 (m, 2H), 7.51–7.61 (m, 2H), 7.30–7.36 (m,
H), 7.24–7.28 (m, 4H), 7.16–7.22 (m, 2H), 7.02–7.05
4
(m, 1H), 4.72 (t, 1H), 3.45 (q, 2H), 3.38 (q, 2H), 1.99 (m,
13
2
1
1
4
H). C NMR (CDCl ) δ 185.8, 184.6, 157.0, 152.3,
3
43.4 (2C), 136.0, 135.6, 135.3, 134.6, 133.6, 130.1 (4C),
28.0 (4C), 127.4, 127.3, 126.9 (2C), 118.4, 116.4, 113.9,
•+
1.3, 39.5, 30.4. MS m/z (rel. intensity, %): 475 ([M] ,
•+
1
1), 307 ([M–C H N] , 97), 300 (100). Anal
12 10
3
3
.1. Chemistry
C H N O (C, H, N).
30
25
3
3
.1.1. 1-[(3-Aminopropyl)amino]-9,10-anthracenedione
(
38)
To a solution of 10 g (41 mmol) of 1-chloro-
anthraquinone in toluene (350 mL), 14 mL (0.17 mol) of
,3-diaminopropane were added and the mixture was
3.1.3.
anthracenedione (6)
1-[3-(4≠-Nitrobenzamido)propylamino]-9,10-
To a solution of 0.67 g (4 mmol) of 4-nitrobenzoic acid
in DMF (100 mL), triethylamine (1 mL), 0.58 g (5 mmol)
of N-hydroxysuccinimide (NHS) and 0.96 g (5 mmol) of
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride (EDC) were added. The solution was stirred at
room temperature for 1 h and then 0.56 g (2 mmol) of 38
were added and the mixture was stirred overnight. Then
0.38 g (2 mmol) of EDC were added and the mixture was
stirred for a further 48 h. The mixture was filtered and the
solid was washed with CH Cl and discarded. The filtrate
1
refluxed for 18 h to a deep red suspension. The reaction
mixture was concentrated under vacuum and the residue
partitioned between CH Cl2 (400 mL) and water
2
(
(
150 mL). The organic layer was washed with water
100 mL) and then the product was extracted with aq.
1
M HCl (2 × 200 mL) to the aqueous layer. The organic
layer was discarded. The acidic aqueous solution was
made alkaline with 10% NaOH and the resulting suspen-
sion was extracted several times with CH Cl (1 L), the
2
2
was successively washed with 0.5 M HCl (2 × 100 mL)
2
2
organic layer was washed once with water, dried over
and water (5 × 50 mL), dried over anh. MgSO and
4
anh. MgSO and evaporated to dryness (yield: 7.2 g,
evaporated to dryness. The residue was purified by flash
chromatography on silica gel (eluent: EtOAc:
CH Cl 20:80) giving a red solid (yield: 0.65 g, 76%),
4
63%). An analytical sample was obtained by column
chromatography either on Florisilt (eluent: MeOH:E-
tOAc 1:1) or neutral alumina grade I (eluent: MeOH:
CH Cl 5:95), followed by recrystallization from EtOAc.
2
2
which was recrystallized from acetone. M.p. 220–222 °C.
–1
IR (cm , KBr) 3 280 (NH), 1 663 (quinone C=O), 1 625
2
2
–
1
1
M.p. 155–158 °C. IR (cm , KBr) 3 327 (NH), 1 668
quinone C=O). H NMR (d -DMSO) δ 9.80 (t, 1H), 8.25
(C=O). H NMR (d -DMSO) δ 9.84 (t, 1H), 9.04 (t, 1H),
6
1
(
8.38 (d, 2H), 8.22 (m, 2H), 8.18 (d, 2H), 7.90–8.00 (m,
6
(
7
dd, 2H), 7.90–8.02 (m, 2H), 7.74 (t, 1H), 7.52 (d, 1H),
2H), 7.74 (t, 1H), 7.52 (d, 1H), 7.37 (d, 1H), 3.56 (q, 4H),
1
3
13
.37 (d, 1H), 3.45 (q, 2H), 2.78 (t, 2H), 1.84 (m, 2H).
C
2.04 (m, 2H). C NMR (d -DMSO) δ 187.8, 186.7,
6
NMR (d -DMSO) δ 187.9, 186.9, 155.5, 139.6, 138.5,
168.6, 155.2, 152.8, 144.1, 139.5, 138.3, 138.2, 137.9,
6

6
07
1
37.3, 136.2, 132.6 (2C), 130.3, 130.1, 127.3 (2C), 122.5,
3-aminopropanol were added and the solution was heated
to reflux for 4 h. The solution was evaporated to dryness.
The residue was purified by flash chromatography on
silica gel (eluent: EtOAc:CH Cl 10:90) giving a red
118.9, 116.0, 44.0, 41.4, 32.3. MS m/z (rel. intensity, %):
4
•+
•+
29([M] , 70), 264 ([M–C H N O ] , 59), 259 (100).
7
5 2 3
Anal C H N O (C, H, N).
2
4
19
3
5
2
2
solid (yield: 2.5 g, 43%), which was recrystallized from
3
.1.4.
1-[(3-Benzamidopropyl)amino]-9,10-anthra-
–1
EtOAc. M.p. 185–188 °C. IR (cm , KBr) 3 300 (OH),
cenedione (12)
1
2
920 (CH ), 2 868 (CH ), 1 664 (quinone C=O). H
2
2
To a solution of 0.56 g (2 mmol) of 38 in Py (20 mL),
NMR(CDCl ) δ 9.78 (bt, 1H), 8.22–8.26 (m, 2H),
3
0
.9 mL (8 mmol) of benzoyl chloride were added drop-
7
.69–7.76 (m, 2H), 7.51–7.58 (m, 2H), 7.10 (d, 1H), 3.88
wise and the mixture was stirred for 18 h at room
temperature. The mixture was then partitioned between
13
(t, 2H), 3.49 (q, 2H), 2.02 (m, 2H). C NMR (CDCl ) δ
3
1
84.6, 183.8, 151.8, 135.3, 135.0, 134.7, 133.9, 133.0,
CH Cl (100 mL) and 1 M HCl (50 mL). The organic
2
2
132.9, 126.7 (2C), 117.8, 115.7, 113.1, 60.4, 39.8, 31.9.
MS m/z (rel. intensity, %): 281([M] , 36), 236
([M–C H O] , 100). Anal C H NO (C, H, N).
layer was successively washed with 1 M HCl (4 ×
0 mL), 5% NaHCO (50 mL) and water, dried over anh.
•+
5
•+
3
2
5
17 15
3
MgSO and evaporated to dryness. The residue was
4
purified by chromatography on Florisilt (eluent:
3
.1.7. 1-[3-(Triphenylmethoxy)propylamino]-9,10-anthra-
EtOAc:CH Cl 1:1) giving a red solid (yield: 0.4 g,
2
2
cenedione (1)
5
2
2
2%), which was recrystallized from EtOAc. M.p.
A solution of 0.56 g (2 mmol) of 39 and 0.67 g
–1
26–229 °C. IR (cm , KBr) 3 306 (NH), 2 920 (CH ),
2
(
2.4 mmol) of chlorotriphenylmethane in Py (30 mL) was
1
853 (CH ), 1 668 (quinone C=O), 1 627 (C=O). H
2
heated to 80 °C for 8 h. The mixture was then partitioned
between CH Cl (100 mL) and 1 M HCl (50 mL). The
NMR (CDCl ) δ 9.84 (bt, 1H), 8.22–8.26 (m, 2H), 7.75
3
2
2
(
d, 2H), 7.70–7.79 (m, 2H), 7.53–7.62 (m, 2H), 7.36–7.46
organic layer was successively washed with 1 M HCl (4
30 mL), 5% NaHCO (30 mL) and water, dried over
(m, 3H), 7.08 (d, 1H), 6.36 (bt, 1H), 3.68 (q, 2H), 3.49 (q,
×
•+
3
2
7
1
H), 2.12 (m, 2H). MS m/z (rel. intensity, %): 384([M] ,
anh. MgSO and evaporated to dryness. The residue was
purified by flash chromatography on silica gel (eluent:
petroleum ether 40–60 °C:CH Cl 80:20) giving a red
•+
•+
4
8), 264 ([M–C H NO] , 71), 236 ([M–C H NO] ,
7
6
9 10
00). Anal C H N O (C, H, N).
24 20 2 3
2
2
solid (yield: 0.68 g, 65%), which was recrystallized from
3
.1.5. 2≠,2≠,2≠-Trichloroethyl 3-[(9,10-dihydro-9,10-
–1
acetone. M.p. 180–183 °C. IR (cm , KBr) 3 059 (arom.
CH), 2 918 (CH ), 2 872 (CH ), 1 667 (quinone C=O).
dioxo-1-anthracenyl)amino]propyl carbamate (14)
To a solution of 0.42 g (1.5 mmol) of 38 and triethy-
lamine (0.3 mL) in CH Cl (50 mL), 0.28 mL (2 mmol)
of 2,2,2-trichloroethyl chloroformate were added, and the
solution was stirred at room temperature for 90 min. The
mixture was washed with water (2 × 50 mL), dried over
anh. MgSO and evaporated to dryness. The residue was
purified by flash chromatography on silica gel (eluent:
CH Cl ) giving a red solid (yield: 0.43 g, 63%), which
2
2
1
H NMR (CDCl ) δ 9.72 (bt, 1H), 8.22–8.25 (m, 2H),
3
2
2
7
1
6
3
1
1
1
.72–7.78 (dt, 1H), 7.66–7.71 (dt, 1H), 7.56–7.59 (dd,
H), 7.47–7.53 (t, 1H), 7.42–7.46 (m, 6H), 7.24–7.29 (m,
H), 7.17–7.22 (m, 3H), 7.06–7.09 (dd, 1H), 3.48 (q, 2H),
13
.26 (t, 2H), 2.03 (m, 2H). C NMR (CDCl ) δ 184.6,
3
4
83.8, 151.8, 144.2 (3C), 135.2, 135.1, 134.7, 133.9,
33.1, 132.8, 128.7 (6C), 127.8 (6C), 126.9 (3C), 126.7,
26.6, 117.9, 115.6, 113.0, 86.4, 60.8, 40.1, 29.9. MS m/z
2
2
was recrystallized from EtOAc. M.p. 140–142 °C. IR
•+
•+
–
1
(rel. intensity, %): 523([M] , 5), 280 ([M–C19H15] ,
100), 274 ([C20
N).
(cm , KBr) 3 348 (NH), 2 949 (CH ), 2 863 (CH ),
2 2
•+
1
H18O] , 100). Anal C36H29NO (C, H,
3
1
9
7
738 (C=O), 1 666 (quinone C=O). H NMR (CDCl ) δ
3
.75 (bt, 1H), 8.22–8.25 (m, 2H), 7.68–7.79 (m, 2H),
.51–7.61 (m, 2H), 7.02–7.05 (d, 1H), 5.22 (bt, 1H), 4.75
13
3.1.8.
anthracenedione (41)
To a solution of 0.8 g (2.8 mmol) of 39 in CH Cl
2
1-[3-[(Methylsulfonyl)oxy]propylamino]-9,10-
(s, 2H), 3.40–3.50 (m, 4H), 2.03 (m, 2H). C NMR
(CDCl ) δ 185.2, 183.8, 154.7, 151.5, 135.4, 134.9,
3
1
7
34.8, 134.0, 133.0, 126.7 (2C), 117.6, 115.9, 113.3, 95.6,
4.6, 40.2, 39.1, 29.4. MS m/z (rel. intensity, %): 454
2
(
50 mL) and Py (1.2 mL), 0.5 mL (6.5 mmol) of meth-
•+
•+
anesulfonyl chloride were added and the solution was
stirred for 48 h. The mixture was washed with 1 M HCl
([M] , 9), 307 ([M–C H Cl O] , 38), 301 (100). Anal
2 2 3
C H Cl N O (C, H, Cl, N).
2
0
17
3 2 4
(2 × 50 mL) and water (50 mL), dried over anh. MgSO4
3
.1.6.
1-[(3-Hydroxypropyl)amino]-9,10-anthracene-
and evaporated to a red solid (yield: 1 g, 100%). M.p.
–1
dione (39)
125–128 °C. IR (cm , KBr) 2 921 (CH ), 2 851 (CH ),
2
2
1
To a solution of 5 g (21 mmol) of 1-chloro-
anthraquinone in n-butanol (100 mL), 6.3 g (85 mmol) of
1 665 (quinone C=O), 1 197 (S=O). H NMR (CDCl ) δ
3
8.22–8.28 (m, 2H), 7.69–7.80 (m, 2H), 7.55–7.65 (m,

608
2
2
1
H), 7.08 (d, 1H), 4.43 (t, 2H), 3.55 (t, 2H), 3.08 (s, 3H),
HCl (250 mL). The organic layer was washed with water
(4 × 100 mL) to extract DMSO and then the product was
extracted with aq. 0.1 M NaOH (2 × 200 mL) to the
aqueous layer. The organic layer was discarded. The basic
aqueous solution was acidified with 1 M HCl and the
resulting suspension was filtered through sintered glass.
The red solid was dried in an oven (yield: 1.4 g, 23%). An
analytical sample was obtained by column chromatogra-
phy on silica gel (eluent: EtOAc:CH Cl 30:70), fol-
•+
.18–2.26 (m, 2H). MS m/z (rel. intensity, %): 359([M] ,
•+
6), 236 ([M–C H O S] , 100).
3
7 3
3
9
.1.9. 1-[3-(4≠-Nitro-1H-imidazol-1≠-yl)propylamino]-
,10-anthracenedione (11)
To a solution of 0.8 g (2.2 mmol) of 41 in CH Cl
2
2
(
40 mL) and DMF (20 mL), 0.75 mL (5 mmol) of DBU
and 0.5 g (4.4 mmol) of 4-nitroimidazole were added and
the mixture was refluxed for 12 h. The mixture was then
partitioned between CH Cl (100 mL) and water
2
2
lowed by recrystallization from EtOAc. M.p.
1
2
2
214–215 °C. H NMR (d -DMSO) δ 9.82 (bt, 1H), 8.27
6
(
3
50 mL). The organic layer was washed with water (5 ×
0 mL), dried over anh. MgSO4 and evaporated to
(
dd, 2H), 7.94–8.01 (m, 2H), 7.76 (t, 1H), 7.54 (d, 1H),
13
7.40 (d, 1H), 3.52 (q, 2H), 2.48 (t, 2H), 1.99 (m, 2H).
C
dryness. The residue was purified by flash chromatogra-
phy on silica gel (eluent: EtOAc) giving a red solid
NMR (d -DMSO) δ 188.0, 186.8, 178.0, 155.3, 139.6,
6
1
38.4, 138.3, 137.9, 137.4, 136.3, 130.4, 130.2, 122.5,
(
yield: 0.6 g, 72%), which was recrystallized from
1
3
19.0, 116.0, 45.4, 35.0, 28.1. MS m/z (rel. intensity, %):
09 ([M] , 82), 291 ([M–H O] , 100), 264
–1
EtOAc. M.p. 201–203 °C. IR (cm , KBr) 1 656 (quinone
•+
•+
2
1
C=O). H NMR (d -DMSO) δ 9.72 (t, 1H), 8.61 (s, 1H),
•+
2 3 5 2
•+
6
([M–CHO ] , 30), 236 ([M–C H O ] , 87).
8
1
2
1
1
.26 (dd, 2H), 8.01 (s, 1H), 7.92–8.04 (m, 2H), 7.75 (t,
H), 7.54 (d, 1H), 7.35 (d, 1H), 4.33 (t, 2H), 3.50 (q, 2H),
.32 (m, 2H). C NMR (d -DMSO) δ 188.0, 186.8,
55.0, 141.3, 139.6, 138.4, 138.3, 137.9, 137.4, 136.3,
30.3, 130.2, 125.5, 122.5, 119.1, 116.2, 49.5, 43.4, 33.2.
3
.1.12.
4-[(9,10-Dihydro-9,10-dioxo-1-anthracenyl)
13
6
amino]-N-(5≠-nitrothiazol-2≠-yl) butanamide (10)
To a solution of 0.46 g (1.5 mmol) of 40 in DMF
(
15 mL), triethylamine (0.5 mL), 0.34 g (3 mmol) of
•+
MS m/z (rel. intensity, %): 376([M] , 20), 236
NHS and 0.38 g (2 mmol) of EDC were added. The
solution was stirred at room temperature for 3 h and then
•+
([M–C H N O ] , 61), 135 (100). Anal C H N O (C,
5 6 3 2 20 16 4 4
H, N).
0
.43 g (3 mmol) of 2-amino-5-nitrothiazole were added
and the mixture was stirred for 18 h. Then 0.38 g
2 mmol) of EDC were added and the mixture was stirred
3
(
.1.10. 1-[(3-Oxopropyl)amino]-9,10-anthracenedione
13)
A finely-ground mixture of 0.86 g (4 mmol) of pyri-
(
for further 48 h. The mixture was partitioned between
EtOAc (150 mL) and water (100 mL). The organic layer
was successively washed with 0.5 M HCl (2 × 50 mL)
dinium chlorochromate and 0.86 g of silica gel was
suspended in CH Cl (30 mL). 0.56 g (2 mmol) of 39
were added and the suspension was kept in an ultrasound
bath for 90 min. The mixture was filtered through a short
column of Celite and silica gel and eluted with CH Cl2
giving a red solid (yield: 0.23 g, 41%), which was
recrystallized from EtOAc. M.p. 165–168 °C. IR (cm ,
KBr) 1 718 (C=O), 1 663 (quinone C=O). H NMR
2
2
and water (4 × 50 mL), dried over anh. MgSO and
4
evaporated to a red solid (yield: 0.6 g, 90%), which was
recrystallized from dioxane-methanol. M.p. 245–247 °C.
2
–1
IR (cm , KBr) 3 225 (NH), 1 661 (quinone C=O) 1 622
1
–1
(C=O). H NMR (d
-DMF) δ 9.80 (t, 1H), 8.42 (s, 1H),
7
1
8.10–8.20 (m, 2H), 7.80–7.94 (m, 2H), 7.67 (t, 1H), 7.48
d, 1H), 7.36 (d, 1H), 3.56 (q, 2H), 2.84 (t, 2H), 2.18 (m,
(
2
(
(
CDCl ) δ 9.90 (t, 1H, J = 1Hz), 9.78 (bt, 1H), 8.22–8.25
m, 2H), 7.68–7.79 (m, 2H), 7.56–7.64 (m, 2H), 7.08 (d,
3
13
H). C NMR (d -DMF) δ 185.0, 183.7, 173.6, 162.9,
7
1
52.4, 142.8, 136.2, 135.3, 135.1, 134.9, 134.0, 133.4,
1
1
H), 3.71 (q, 2H, J = 6Hz), 2.95 (dt, 2H, J = 6Hz, J =
•+
127.1, 126.8, 119.2, 115.8, 113.4, 42.5, 33.6, 24.6. MS
m/z (rel. intensity, %): 436([M] , 16), 292
Hz). MS m/z (rel. intensity, %): 279([M] , 7), 236
•+
•
+
•+
([M–C H O] , 22), 223 ([M–C H O] , 100). Anal
2 3 3 4
•+
3 2 3 2 21 16 4 5
([M–C H N O S] , 25), 232 (100). Anal C H N O S
C H NO (C, H, N).
1
7
13
3
(C, H, N, S).
3
.1.11. 1-[(3-Carboxypropyl)amino]-9,10-anthracene-
dione (40)
suspension
-chloroanthraquinone in DMSO (100 mL) and triethy-
lamine (8 mL) was heated to solution and 4.1 g
40 mmol) of 4-aminobutyric acid were added. The
3.1.13. 1-[3-[(2≠,2≠,2≠-Trichloroethoxy)carbonyl]pro-
pylamino]-9,10-anthracenedione (15)
A
of
4.8 g
(20 mmol)
of
1
To a suspension of 0.31 g (1 mmol) of 40 in CH Cl
2
2
(10 mL) containing Py (0.15 mL) and 0.2 mL (2 mmol)
of 2,2,2-trichloroethanol, were added 0.2 g (1mmol) of
DCC and the mixture was stirred for 3 h at room
temperature. The suspension was filtered to remove DCU
(
mixture was heated to 150 °C for 90 min. After cooling it
was partitioned between CH Cl (300 mL) and aq. 0.1 M
2
2

6
09
and washed with CH Cl (50 mL). The combined filtrates
3.1.15.1. 1-Chloro-5-[(3≠-pyridinyl)methylamino]-9,10-
2
2
were washed successively with 1 M HCl (30 mL), 5%
NaHCO (20 mL) and water, dried over anh. MgSO and
anthracenedione (16)
1
Yield: 52%. M.p. 192 °C. H NMR (d -DMSO) δ 9.95
3
4
6
evaporated to dryness. The residue was purified by flash
chromatography on silica gel (eluent: petroleum ether
(t, 1H, NH-AQ), 8.65 (s, 1H, H-2≠), 8.5 (d, 1H, H-6≠),
8.25 (d, 1H, H-8), 7.9 (m, 2H, H-6,7), 7.8 (d, 1H, H-4),
7.65 (t, 1H, H-3), 7.4 (dd, 2H, H-4≠,5≠), 7.2 (d, 1H, H-2),
4
5
1
1
9
7
0–60 °C:CH Cl 50:50) giving a red solid (yield: 0.23 g,
2 2
2%), which was recrystallized from EtOAc. M.p.
4.7 (d, 2H, NH–CH ). MS m/z (rel. intensity, %) 348
2
–
1
•+
06–109 °C. IR (cm , KBr) 2 954 (CH ), 2 864 (CH ),
([M] , 81), 108 (100). Anal C H ClN O (C, H, Cl, N).
2
2
20 13
2 2
1
754 (C=O), 1 667 (quinone C=O). H NMR (CDCl ) δ
3
3
.1.15.2.
1,5-Bis[(3≠-pyridinyl)methylamino]-9,10-
.80 (bt, 1H), 8.24–8.28 (m, 2H), 7.68–7.79 (m, 2H),
.54–7.62 (m, 2H), 7.06–7.12 (d, 1H), 4.78 (s, 2H), 3.48
anthracenedione (17)
1
13
Yield: 25%. M.p. 225 °C. H NMR (d -DMSO) δ 10.0
(q, 2H), 2.68 (t, 2H), 2.16 (m, 2H). C NMR (CDCl ) δ
6
3
(
7
t, 2H, NH-AQ), 8.65 (s, 2H, H-2≠), 8.5 (d, 2H, H-6≠),
1
1
3
85.2, 183.7, 171.3, 151.6, 135.4, 135.0, 134.8, 134.0,
33.0, 126.8, 126.7, 117.7, 115.9, 113.3, 94.9, 74.1, 41.9,
.8 (d, 2H, H-4,8), 7.6 (t, 2H, H-3,7), 7.5 (d, 2H, H-4≠),
.4 (dd, 2H, H-5≠), 7.15 (d, 2H, H-2,6), 4.7 (d, 4H,
•+
7
1.3, 24.2. MS m/z (rel. intensity, %): 439 ([M] , 7), 292
•+
•+
•+
NH–CH ). MS m/z (rel. intensity, %): 420 ([M] , 58),
([M–C H Cl O] , 10), 236 ([M–C H Cl O ] , 100).
2
2
2
3
5
6
3 2
3
27 (100). Anal C H N O (C, H, N).
Anal C H Cl NO (C, H, Cl, N).
26 20 4 2
2
0
16
3
4
3
.1.15.3. 1-Chloro-5-[(2≠-pyridinyl)methylamino]-9,10-
anthracenedione (18)
3
.1.14. 1-[2-(2≠-Pyridinyl)ethylamino]-9,10-anthracene-
1
Yield: 53%. M.p. 172 °C. H NMR (d -DMSO) δ 10.2
6
dione (24)
(
t, 1H, NH-AQ), 8.6 (d, 1H, H-6≠), 8.25 (d, 1H, H-8),
A suspension of 2.4 g (10 mmol) of 1-chloro-
anthraquinone in DMSO (50 mL) was heated to dissolu-
tion and then 6 mL (50 mmol) of 2-(2-amino-
ethyl)pyridine were added. The mixture was heated to
7.86 (m, 2H, H-6,7), 7.8 (t, 1H, H-5≠), 7.63 (t, 1H, H-3),
7.42 (d, 1H, H-4), 7.37 (d, 1H, H-3≠), 7.33 (dd, 1H,
H-4≠), 7.2 (d, 1H, H-2), 4.75 (d, 2H, NH–CH ). MS m/z
2
•+
(rel. intensity, %): 348 ([M] , 100), 107 (89). Anal
C H ClN O (C, H, Cl, N).
1
(
50 °C for 10 min and, after the addition of cold water
150 mL), the product was collected by filtration. The red
solid was purified by flash chromatography on silica gel
20
13
2 2
3
.1.15.4.
1,5-Bis[(2≠-pyridinyl)methylamino]-9,10-
anthracenedione (19)
(
2
1
eluent: EtOAc:CH Cl 20:80) giving a red solid (yield:
2 2
1
Yield: 30%. M.p. 223–224 °C. H NMR (d -DMSO) δ
g, 60%), which was recrystallized from methanol. M.p.
6
1
1
0.3 (t, 2H, NH-AQ), 8.62 (d, 2H, H-6≠), 7.8 (t, 2H,
22–124 °C. H NMR (d -DMSO) δ 9.75 (bt, 1H), 8.56
6
H-5≠), 7.63 (t, 2H, H-3,7), 7.5 (d, 2H, H-4.8), 7.43 (d,
(
1
d, 1H), 8.15 (d, 2H), 7.86 (m, 2H), 7.74 (t, 1H), 7.67 (t,
H), 7.45 (d, 1H), 7.39 (d, 1H), 7.34 (d, 1H), 7.26 (dd,
2
4
1
H, H-3≠), 7.3 (dd, 2H, H-4≠), 7.15 (d, 2H, H-2,6), 4.7 (d,
•+
H, NH–CH ). MS m/z (rel. intensity,%): 420 ([M] ,
1
H), 3.80 (q, 2H), 3.15 (t, 2H). MS m/z (rel. intensity, %)
2
•+
•+
00), 328 (49).
328 ([M] , 22), 236 ([M–C H N] , 00). Anal
6 6
C H N O (C, H, N).
2
1 16 2 2
3
.1.16. 1-Chloro-5-[2-(2≠-pyridinyl)ethylamino]-9,10-
anthracenedione (25) and 1,5-Bis[2-(2≠-pyridinyl)
ethylamino]-9,10-anthracenedione (26)
3.1.15.
(Pyridinylmethylamino)-9,10-anthracenedione
A
suspension of 2.7 g (10 mmol) of 1,5-
derivatives (16–19)
dichloroanthraquinone in DMSO (50 mL) was heated to
dissolution and then 12 mL (100 mmol) of 2-(2-
aminoethyl)pyridine were added. The mixture was heated
to 150 °C for 10 min and, after the addition of cold water
(150 mL), the product was collected by filtration. The
residue was purified by flash chromatography on silica
gel (first eluent: EtOAc:CH Cl 3:7, second eluent:
The above compounds were prepared according to the
following general method:
10.8 mmol) of 1,5-dichloroanthraquinone in 20 mL of an
appropriate (aminomethyl)pyridine was heated to
0–50 °C for 36 h. Then the mixture was poured into
a suspension of 3 g
(
4
water (100 mL) and the precipitate was collected by
2
2
filtration, dissolved in CH Cl2 and dried over anh.
EtOAc) giving two red products: 25 (yield: 0.8 g, 22%)
2
MgSO . Upon removal of the solvent the products were
and 26 (yield: 1.4 g, 35%), which were recrystallized
4
1
isolated by column chromatography on silica gel (gradi-
ent elution from CH Cl to CH Cl :MeOH 95:5) and
from methanol. 25: M.p. 167–171 °C. H NMR (d -
6
DMSO) δ 9.60 (bt, 1H), 8.55 (d, 1H), 8.20 (d, 1H), 7.87
2
2
2
2
recrystallized from chloroform-methanol (1:1).
(d, 1H), 7.82 (t, 1H), 7.74 (t, 1H), 7.67 (t, 1H), 7.39 (d,

610
–
1
1
2
4
H), 7.37 (d, 1H), 7.32 (d, 1H), 7.26 (dd, 1H), 3.78 (q,
from EtOAc. M.p. 161–162 °C. IR (cm , KBr) 3 300
(OH), 2 934 (CH ), 2 862 (CH ), 1 645 (quinone C=O).
•+
H), 3.15 (t, 2H). MS m/z (rel. intensity, %) 362 ([M] ,
2
2
•+
1
0), 270 ([M–C H N] , 100). Anal C H ClN O (C,
H NMR (CDCl ) δ 10.83 (bt, 2H), 8.30–8.34 (m, 2H),
6
6
21 15
2
2
3
H, Cl, N). 26: M.p. 148–152 °C. Anal C H N O (C, H,
N).
7.66–7.70 (m, 2H), 7.24 (s, 2H), 3.86–3.91 (q, 4H),
3.54–3.60 (q, 4H), 1.98–2.06 (m, 4H).
2
8 24 4 2
3
.1.17. 1-[2-(Dimethylamino)ethylamino]-5-[2≠-(pyri-
dinyl)methylamino]-9,10-anthracenedione (30)
3
.1.20. 1,4-Bis[3-(triphenylmethoxy)propylamino]-9,10-
anthracenedione (4) and 1-[(3-Hydroxypropyl)amino]-
-[3-(triphenylmethoxy)propylamino]-9,10-anthracene-
dione (2)
A solution of 18 (600 mg, 1.72 mmol) in N,
N-dimethylethylenediamine (15 mL) was heated at 55 °C
for 72 h. Then, cold water was added and the resulting
solid was filtered and dissolved in CH Cl . The solution
4
2
2
A solution of 0.41 g (1.1 mmol) of 42 and 0.56 g
2 mmol) of chlorotriphenylmethane in Py (15 mL) was
heated to 80 °C for 6 h. The mixture was then partitioned
between CH Cl (100 mL) and 1 M HCl (50 mL). The
was washed with water (to extract the free amine) and
(
dried over anh. MgSO . After removal of the solvent
4
under reduced pressure the crude material was subjected
to column chromatography on silica gel (gradient elution
from CH Cl to (95:5) CH Cl :MeOH) and recrystallized
2
2
organic layer was successively washed with 1 M HCl (4
50 mL), 5% NaHCO (50 mL) and water, dried over
2
2
2
2
×
from CHCl :MeOH (1:1) (yield: 495 mg, 72%). M.p.
3
3
H
1
anh. MgSO and evaporated to dryness. The residue was
162 °C.
NMR (d -DMSO)
δ
10.3 (s, 1H,
4
6
purified by chromatography on Florisilt (eluent: CH Cl )
NHCH CH ), 9.78 (s, 1H, NH–CH ), 8.63 (d, 1H, H-6≠),
2
2
2
2
2
giving two blue solids 4 (yield: 0.3 g, 32%) and 2 (yield:
.4 g, 61%), which were recrystallized from MeOH. 4:
7
.82 (t, 1H, H-5≠), 7.64 (m, 2H, H-3,7), 7.46 (m, 3H,
H-4,8, H-3≠), 7.34 (dd, 1H, H-4≠), 7.18 (d, 1H, H-7),
.13 (d, 1H, H-2), 4.75 (d, 2H, NHCH ), 3.3 (q, 2H,
0
–
1
M.p. 114–118 °C. IR (cm , KBr) 3 060 (arom. CH),
2
7
2
1
922 (CH ), 2 850 (CH ), 1 644 (quinone C=O). H
NHCH CH ), 2.6 (t, 2H, NHCH CH ), 2.22 (s, 6H,
2 2
2
2
2
2
•+
NMR (CDCl ) δ 10.78 (bt, 2H), 8.30–8.34 (m, 2H),
N–CH ). MS m/z (rel. intensity, %): 400 ([M] , 3), 354
3
3
7
2
.68−7.72 (m, 2H), 7.40–7.44 (d, 12H), 7.17–7.30 (m,
(
100). Anal C H N O (C, H, N).
24 24 4 2
0H), 3.56 (q, 4H), 3.27 (t, 4H), 2.03 (m, 4H). Anal
–
1
3
.1.18. 1,8-Bis[2-(2≠-pyridinyl)ethylamino]-9,10-anthra-
C H N O (C, H, N). 2: M.p. 152–156 °C. IR (cm ,
58 50 2 4
cenedione (28)
KBr) 3 500 (OH), 3 059 (arom. CH), 2 924 (CH ), 2 854
2
1
A
suspension of 2.7 g (10 mmol) of 1,8-
(CH ), 1 643 (quinone C=O). H NMR (CDCl ) δ 10.84
2
3
dichloroanthraquinone in DMSO (50 mL) was heated to
dissolution and then 12 mL (100 mmol) of 2-(2-
aminoethyl)pyridine were added. The mixture was heated
to 150 °C for 10 min and, after the addition of cold water
(bt, 1H), 10.75 (bt, 1H), 8.29–8.35 (m, 2H), 7.66–7.70
(m, 2H), 7.40–7.44 (m, 6H), 7.25–7.30 (m, 6H),
7.17–7.24 (m, 5H), 3.87 (q, 2H), 3.56 (q, 4H), 3.26 (t,
2H), 1.99–2.04 (m, 4H). Anal C H N O (C, H, N).
39 36 2 4
(150 mL), the product was collected by filtration. The
residue was purified by flash chromatography on silica
gel (eluent: EtOAc:CH Cl 20:80) giving a violet solid
2
2
3.1.21. 1,4-Bis[2-(2≠-pyridinyl)ethylamino]-9,10-anthra-
cenedione (27)
(
yield: 1.3 g, 30%), which was recrystalized from metha-
nol. M.p. 182–185 °C. H NMR (d -DMSO) δ 9.62 (bt,
1
6
A suspension of 1 g (4 mmol) of leucoquinizarin in
MeOH (20 mL) was de-gassed by stirring at 5 °C under
N2 and then 4.8 mL (40 mmol) of 2-(2-amino-
ethyl)pyridine were added dropwise. The mixture was
heated to 50 °C for 1 h, cooled and then aerated over-
night. The mixture was evaporated and the residue was
purified by flash chromatography on silica gel (eluent:
MeOH:EtOAc 10:90) giving a blue solid (yield: 0.55 g,
2
7
H), 8.56 (d, 2H), 7.74 (t, 2H), 7.58 (t, 2H), 7.38 (m, 4H),
.27 (m, 4H), 3.72 (q, 4H), 3.15 (t, 4H). Anal
C H N O (C, H, N).
2
8 24 4 2
3
.1.19. 1,4-Bis[(3-hydroxypropyl)amino]-9,10-anthra-
cenedione (42)
A suspension of 2 g (8 mmol) of leucoquinizarin in
MeOH (20 mL) was de-gassed by stirring at 5 °C under
N and then 6.3 mL (82 mmol) of 3-aminopropanol were
30%), which was recrystallized from methanol. M.p.
2
1
added dropwise. The mixture was heated to 50 °C for 1 h,
cooled and then aerated overnight. The mixture was
evaporated and the residue was purified by chromatogra-
phy on Florisilt (eluent: MeOH:EtOAc 5:95) giving a
blue solid (yield: 1 g, 35%), which was recrystallized
115–117 °C. H NMR (d -DMSO) δ 9.82 (bt, 2H), 8.55
6
(d, 2H), 8.19 (d, 2H), 7.77 (m, 2H), 7.72 (dd, 2H), 7.54 (s,
2H), 7.38 (d, 2H), 7.25 (dd, 2H), 3.88 (q, 4H), 3.15 (t,
•+
4H). MS m/z (rel. intensity, %) 448([M] ), 263. Anal
C H N O (C, H, N).
28 24 4 2

6
11
3
.1.22.
1,4-Bis[(3-aminopropyl)amino]-9,10-anthra-
(100 mL), dried over anh. MgSO and evaporated to a
4
cenedione.2HCl (36)
blue solid (yield: 2.5 g, 51%), which was recrystallized
–1
A suspension of 10.5 g (42 mmol) of leucoquinizarin
in MeOH (100 mL) was de-gassed by stirring at 5 °C
under N2 and then 32 mL (0.42 mol) of 1,3-
diaminopropane were added dropwise. The mixture was
heated to 50 °C for 1 h, cooled and then aerated over-
night. After evaporation the residue was dissolved in
CH Cl (100 mL) and 17.9 g (82 mmol) of di-tert-butyl
from EtOAc. M.p. 108–112 °C. IR (cm , KBr) 3 380
(NH), 2 940 (CH ), 2 860 (CH ), 2 819 (CH ), 2 770
2
2
3
1
(CH ), 1 645 (quinone C=O). H NMR (d -DMSO) δ
3
6
10.84 (m, 2H), 8.22–8.26 (m, 2H), 7.77–7.80 (m, 2H),
7.51 (s, 2H), 3.51 (q, 4H), 2.71 (t, 2H), 2.54 (t, 2H), 2.24
(s, 6H), 1.73–1.79 (m, 2H).
2
2
dicarbonate in CH Cl (20 mL) were added dropwise.
The mixture was stirred for 1 h, then evaporated and the
residue was purified by chromatography on Florisilt
3.1.25. 1-[2-(Dimethylamino)ethylamino]-4-[3-(4≠-nitro-
benzamido)propylamino]-9,10-anthracenedione (9)
To a solution of 0.84 g (5 mmol) of 4-nitrobenzoic acid
in DMF (30 mL), triethylamine (2 mL), 0.69 g (6 mmol)
of NHS and 1.9 g (10 mmol) of EDC were added. The
solution was stirred at room temperature for 18 h, then
0.73 g (2 mmol) of 43 in DMF (10 mL) were added and
the mixture was stirred for 24 h. Then 0.38 g (2 mmol) of
EDC were added and the mixture was stirred for a further
48 h. The mixture was partitioned between CH Cl
2
2
(
eluent: EtOAc:CH Cl 10:90) giving a blue solid. The
2 2
solid was dissolved in MeOH (90 mL) containing conc.
HCl (10 mL), refluxed for 1 h and evaporated giving a
blue solid (yield: 4 g, 22%), which was recrystallized
–1
from methanol. M.p. 88–92 °C. IR (cm , KBr) 2 926
+
3 3
+
1
(
(
(
NH ), 1 642 (quinone C=O) 1 594 (NH ). H NMR
CD OD) δ 8.48–8.54 (m, 2H), 7.96–8.00 (m, 2H), 7.72
3
2
2
s, 2H), 3.82 (t, 4H), 3.32 (t, 4H), 2.22 (m, 4H).
(150 mL) and water (100 mL). The organic layer was
successively washed with water (5 × 50 mL) and 5%
NaHCO (50 mL), dried over anh. MgSO and evapo-
3.1.23.
1,4-Bis[2-(dimethylamino)ethylamino]-9,10-
3
4
anthracenedione (37)
rated to dryness. The residue was purified by flash
chromatography on silica gel (eluent: MeOH:EtOAc
10:90) giving a blue solid (yield: 0.65 g, 65%), which
was recrystallized from EtOAc. M.p. 218–221 °C. IR
Compound 37 was prepared according to the procedure
described in the literature [6] (yield: 70%). M.p.
1
1
72–173 °C. H NMR (CDCl ) δ 10.77 (s, 2H,
3
–1
NHCH CH ), 8.38 (d, 2H, H-5,8), 7.7 (m, 2H, H-6,7),
(cm , KBr) 3 280 (NH), 2 920 (CH ), 2 855 (CH ),
2
2
2 2
1
7
4
.26 (s, 2H, H-2,3), 3.50 (q, 4H, NHCH CH ), 2.70 (t,
2 817 (CH ), 2 767 (CH ), 1 640 (quinone C=O). H
3 3
2
2
H, NHCH CH ), 2.40 (s, 12H, N–CH ).
NMR (CDCl ) δ 10.72 (bt, 1H), 10.68 (bt, 1H),
2
2
3
3
8
.22–8.26 (m, 1H), 8.10–8.14 (m, 1H), 8.04 (d, 2H), 7.78
3
.1.24.
1-[(3-Aminopropyl)amino]-4-[[2-(dimethyl-
(d, 2H), 7.58–7.64 (m, 2H), 7.16 (s, 2H), 6.64 (bt, 1H),
3.66 (q, 2H), 3.52 (q, 2H), 3.44 (q, 2H), 2.62 (t, 2H), 2.28
amino)ethyl]amino]-9,10-anthracenedione (43)
(
s, 6H), 2.24–2.32 (m, 2H). Anal C H N O (C, H, N).
A suspension of 3 g (12 mmol) of leucoquinizarin in
MeOH (250 mL) was de-gassed by stirring at 5 °C under
N2 and then 4.9 mL (44 mmol) of N,N-dimethyl-
ethylenediamine in MeOH (10 mL) were added drop-
wise. The de-gassed mixture was heated to 50 °C for 1 h,
cooled and 2 mL (25 mmol) of 1,3-diaminopropane were
added dropwise. The mixture was heated again to 50 °C
for 2 h, cooled and then aerated overnight. After evapo-
ration the residue was dissolved in MeOH (100 mL) and
28 29 5 5
3.1.26. 1-[2-(Dimethylamino)ethylamino]-4-[3-(hydroxy-
propyl)amino]-9,10-anthracenedione (44)
A suspension of 3 g (12 mmol) of leucoquinizarin in
MeOH (200 mL) was de-gassed by stirring at 5 °C under
N2 and then 4.9 mL (44 mmol) of N,N-dimethyl-
ethylenediamine in MeOH (10 mL) were added drop-
wise. The de-gassed mixture was heated to 50 °C for
45 min, cooled and 2 mL (25 mmol) of 3-aminopropanol
were added dropwise. The mixture was heated again to
50 °C for 2 h, cooled and then aerated overnight. After
evaporation the residue was purified by chromatography
on Florisilt (eluent: MeOH:EtOAc 5:95) giving a blue
solid (yield: 2 g, 45%), which was recrystallized from
4
g (18 mmol) of di-tert-butyl dicarbonate in MeOH
(10 mL) were added dropwise. The mixture was stirred
for 8 h, then evaporated and the residue was purified by
chromatography on Florisilt (eluent: MeOH:EtOAc
1
0:90) giving a blue solid. The solid was dissolved in
MeOH (90 mL) containing conc. HCl (10 mL) and the
mixture was refluxed for 1 h, evaporated and dissolved in
water (150 mL). The aqueous solution was washed with
CH Cl (50 mL) to eliminate apolar impurities and then
–1
EtOAc. M.p. 149–150 °C. IR (cm , KBr) 3 400 (OH),
2 943 (CH ), 2 862 (CH ), 2 821 (CH ), 2 770 (CH ),
1 643 (quinone C=O). H NMR (d -DMSO) δ 10.88 (bt,
2
2
3
3
1
2
2
6
basified with 5% NaOH and extracted with EtOAc (4 ×
50 mL). The organic layer was washed with water
1H), 10.84 (bt, 1H), 8.23–8.27 (m, 2H), 7.78–7.81 (m,
2H), 7.50 (s, 2H), 4.69 (t, 1H), 3.58 (q, 2H), 3.54 (q, 4H),
1

612
2
.57 (t, 2H), 2.25 (s, 6H), 1.80–1.84 (m, 2H). MS m/z
(1.6 mmol) of 4-nitrobenzoyl chloride and the mixture
was stirred overnight. The mixture was then partitioned
between CH Cl (100 mL) and water (50 mL). The or-
•+
•+
(
rel. intensity, %): 367 ([M] , 73), 309 ([M–C H O] ,
00), 238 ([M–C H NO] , 25).
3 6
•+
1
7 15
2
2
ganic layer was washed with water (5 × 30 mL), dried
3
.1.27. 1-[2-(Dimethylamino)ethylamino]-4-[3-(triphenyl-
over anh. MgSO and evaporated to dryness. The residue
4
methoxy)propylamino]-9,10-anthracenedione (3)
was purified by flash chromatography on silica gel
A solution of 0.41 g (1.1 mmol) of 44 and 0.72 g
(
eluent: MeOH:EtOAc 5:95) giving a blue solid (yield:
(
2.6 mmol) of chlorotriphenylmethane in Py (20 mL) was
0
1
2
.22 g, 49%), which was recrystallized from EtOAc. M.p.
heated to 80 °C for 3 h. The mixture was then partitioned
between EtOAc (600 mL) and water (200 mL). The
organic layer was successively washed with 1 M HCl
–1
84–186 °C. IR (cm , KBr) 3 420 (OH), 3 323 (NH),
917 (CH ), 2 850 (CH ), 1 642 (quinone C=O), 1 625
2
2
1
(
C=O). H NMR (d -DMSO) δ 10.92 (bt, 1H), 10.89 (bt,
6
(
100 mL) and water (2 × 50 mL), dried over anh. MgSO4
1H), 8.91 (bt, 1H), 8.27 (d, 2H), 8.18–8.24 (m, 2H), 8.06
d, 2H), 7.76–7.79 (m, 2H), 7.51 (s, 2H), 4.66 (t, 2H),
.44–3.59 (m, 6H), 1.95 (m, 2H), 1.81 (m, 2H). Anal
C H N O (C, H, N).
and evaporated to dryness. The residue was purified by
chromatography on Florisilt (eluent: EtOAc) giving a
blue solid (yield: 0.35 g, 52%), which was recrystallized
from CH Cl /petroleum ether. M.p. 101–103 °C. IR
(
3
27 26 4 6
2
2
–
1
(
cm , KBr) 3 060 (arom. CH), 2 944 (CH ), 2 862
2
3.1.30. 1-[(3-Hydroxypropyl)amino]-4-[3-(2≠-nitrophenyl-
acetamido)propylamino]-9,10-anthracenedione (8)
To a solution of 0.46 g (0.9 mmol) of 45 in DMF
(CH ), 2 820 (CH ), 2 769 (CH ), 1 644 (quinone C=O).
2
3
3
1
H NMR (CDCl ) δ 10.76 (bt, 1H), 10.70 (bt, 1H),
3
8
6
3
.29–8.37 (m, 2H), 7.67–7.70 (m, 2H), 7.41–7.45 (m,
H), 7.24–7.29 (m, 8H), 7.17–7.22 (m, 3H), 3.56 (q, 4H),
.26 (t, 2H), 2.74 (t, 2H), 2.40 (s, 6H), 2.02 (m, 2H). Anal
(
2
50 mL) and triethylamine (0.5 mL) 0.44 g (2.2 mmol) of
-nitrophenylacetyl chloride were added and the mixture
was stirred overnight. The mixture was then partitioned
between CH Cl (100 mL) and water (50 mL). The or-
C H N O (C, H, N).
4
0 39 3 3
2
2
ganic layer was washed with water (5 × 30 mL), dried
3
.1.28.
1-[(3-Aminopropyl)amino]-4-[(3-hydroxy-
over anh. MgSO and evaporated to dryness. The residue
4
propyl)amino]-9,10-anthracenedione hydrochloride (45)
A suspension of 6 g (25 mmol) of leucoquinizarin in
MeOH (300 mL) was de-gassed by stirring at 5 °C under
N and then 6.8 mL (89 mmol) of 3-aminopropanol in
MeOH (20 mL) were added dropwise. The de-gassed
mixture was heated to 50 °C for 1 h, cooled and 4.1 mL
was purified by flash chromatography on silica gel
(
eluent: MeOH:EtOAc 5:95) giving a blue solid (yield:
0
1
8
.20 g, 43%), which was recrystallized from EtOAc. M.p.
2
1
82–185 °C. H NMR (d -DMSO) δ 10.89 (bt, 2H),
6
.23–8.27 (m, 2H), 8.21 (bt, 1H), 8.00 (d, 1H), 7.77–7.80
(
4
(
m, 2H), 7.67 (t, 1H), 7.48–7.55 (m, 2H), 7.50 (s, 2H),
.66 (t, 2H), 3.89 (s, 2H), 3.48–3.59 (m, 6H), 1.77–1.82
m, 4H). Anal C H N O (C, H, N).
(50 mmol) of 1,3-diaminopropane were added dropwise.
The mixture was heated again to 50 °C for 2 h, cooled
and then aerated overnight. After evaporation the residue
was dissolved in MeOH (100 mL) and 8 g (37 mmol) of
di-tert-butyl dicarbonate in MeOH (20 mL) were added
dropwise. The mixture was stirred overnight, then evapo-
rated and the residue was purified by chromatography on
Florisilt (eluent: EtOAc) giving a blue solid. The solid
was dissolved in MeOH (90 mL) containing conc. HCl
28 28 4 6
3
.1.31. 1-[2-(Dimethylamino)ethylamino]-4-[(3≠-pyri-
dinyl)methylamino]-9,10-anthracenedione (29)
Compound 29 was prepared from leucoquinizarin
2.5 g, 10.3 mmol) in MeOH (125 mL) according to the
(
procedure outlined for 43 and 44. The first step involved
the addition of N,N-dimethylethylenediamine (4 mL,
(
10 mL) and the mixture was refluxed for 1 h and
3
7.1 mmol) and in the second stage 3-(amino-
evaporated giving a blue solid (yield: 2.5 g, 25%), which
was recrystallized from MeOH. M.p. 193–195 °C. IR
methyl)pyridine (2.1 mL, 20.6 mmol) was added. After
air bubbling (overnight) and removal of the solvent, the
obtained product was purified by column chromatogra-
phy on silica gel (gradient elution from EtOAc to (90:10)
–
1
+
3
(cm , KBr) 3 385 (OH), 2 926 (NH ), 1 630 (quinone
+
3
1
C=O), 1 593 (NH ). H NMR (CD OD) δ 8.15–8.23 (m,
3
2
H), 7.90–7.95 (m, 2H), 7.70 (d, 1H), 7.50 (d, 1H), 4.00
EtOAc:MeOH, followed by EtOAc:MeOH:Et N (88:
(t, 2H), 3.65 (q, 4H), 3.35 (t, 2H), 2.30 (m, 2H), 2.25 (m,
3
1
0:2). The product was recrystallized from (90:10)
2H). Anal C H ClN O (C, H, Cl, N).
20 24 3 3
1
EtOAc:hexane (yield: 70%). M.p. 160–161 °C. H NMR
3
.1.29.
1-[(3-Hydroxypropyl)amino]-4-[3-(4≠-nitro-
(d -DMSO) δ 11.05 (t, 1H, NHCH ), 10.73 (t, 1H,
6
2
benzamido)propylamino]-9,10-anthracenedione (7)
To a solution of 0.46 g (0.9 mmol) of 45 in DMF
NHCH CH ), 8.63 (s, 1H, H-2≠), 8.5 (d, 1H, H-6≠), 8.24
2
2
(m, 2H, H-5,8), 7.76 (m, 3H, H-7,6, H-4≠), 7.45 (s, 2H,
(250 mL) and triethylamine (0.5 mL) was added 0.3 g
H-2,3), 7.39 (dd, 1H, H-5≠), 4.78 (d, 2H, NHCH ), 3.47
2

6
13
(
6
q, 2H, NHCH CH ), 2.54 (t, 2H, NHCH CH ), 2.23 (s,
(18 mmol) nicotinoyl chloride (yield: 23%). M.p. 254 °C.
2
2
2
2
1
H, N–CH ). Anal C H N O (C, H, N).
H NMR (d -DMSO) δ 13.01 (s, 2H, OH; NHCO), 9.24
3
24 24
4
2
6
(
s, 1H, H-2≠), 9.07 (d, 1H, H-2), 8.87 (d, 1H, H-6≠), 8.41
3
.1.32. 1-(Nicotinamido)-9,10-anthracenedione (20)
Nicotinoyl chloride (5.7 g, 40 mmol) was added to a
(
7
d, 1H, H-4≠), 8.31 (m, 2H, H-5,8), 8.01 (m, 2H, H-6,7),
.71 (dd, 1H, H-5≠), 7.6 (d, 1H, H-3). MS m/z (rel.
•+
solution of 1-aminoanthraquinone (3 g, 13.5 mmol) in
toluene (150 mL) and refluxed for 24 h. The solvent was
then removed and the residue was dissolved in CH Cl .
intensity, %): 344 ([M] , 81), 103 (100). Anal
C H N O (C, H, N).
20 12 2 4
2
2
The organic layer was washed with 5% aqueous Na CO3
to remove the excess of acyl chloride, dried over anh.
3.1.36. 1-Benzamido-4-hydroxy-9,10-anthracenedione
(23)
2
MgSO , concentrated and the residue chromatographed
To a solution of 2.5 g (10.45 mmol) of 1-amino-4-
hydroxyanthraquinone in 100 mL toluene, 1.46 mL
(12.54 mmol) of benzoyl chloride were added and re-
fluxed for 24 h. The product was worked up as described
4
on Florisilt column eluting with CH Cl . The product
2
2
was recrystallized from CHCl (yield: 88%). M.p.
3
–1
2
1
1
44 °C. IR (cm , KBr) 3 172, 3 111 (NH), 1 698 (C=O),
637 (quinone C=O). H NMR (d -DMSO) δ 13.2 (s,
1
1
for compound 20 (yield: 92%). M.p. 248–249 °C. H
6
H, NHCO), 9.37 (s, 1H, H-2≠), 9.24 (d, 1H, H-2), 9.00
NMR (CDCl ) δ 13.28 (m, 2H, NHCO, OH), 9.38 (d, 1H,
3
(
d, 1H, H-6≠), 8.53 (d, 1H, H-4≠), 8.36 (d, 2H, H-5,8),
H-2), 8.38 (m, 2H, H-5,8), 8.17 (d, 2H, H-2≠), 7.83 (m,
8
.06–8.18 (m, 4H, H-3,4,6,7), 7.84 (dd, 1H, H-5≠). MS
2H, H-6,7), 7.6 (m, 3H, H-3≠,4≠), 7.42 (d, 1H, H-3). MS
•+
•+
m/z (rel. intensity, %): 328 ([M] , 39), 106 (100). Anal
C H N O (C, H, N).
m/z (rel. intensity, %): 342 ([M] , 37), 105 (100). Anal
C H NO (C, H, N).
2
0
12
2
3
21 13
4
3
.1.33. 1-(Nicotinamido)-9,10-anthracenedione methio-
dide (33)
A suspension of 100 mg (0.3 mmol) of 20 in 10 mL of
3.1.37. 1,4-Bis(nicotinamido)-9,10-anthracenedione (34)
Compound 34 was prepared according to the general
procedure described above for compound 20 employing
nicotinoyl chloride (10.7 g, 76 mmol) and 1,4-
iodomethane was refluxed for 24 h. Acetone (60 mL) was
added and the suspension was stirred for 1 h at room
temperature. The precipitate was filtered off and triturated
several times with diethyl ether to afford the salt (yield
diaminoanthraquinone (3 g, 13 mmol) in 100 mL toluene
1
(yield: 39%). M.p. 140–145 °C. H NMR (d -DMSO) δ
6
13.31 (s, 2H, NHCO), 9.27 (s, 2H, H-2≠), 9.24 (s, 2H,
H-2,3), 8.87 (d, 2H, H-6≠), 8.41 (d, 2H, H-4≠), 8.32 (dd,
2H, H-5,8), 7.97 (dd, 2H, H-6,7), 7.69 (dd, 2H, H-5≠).
–1
6
0%). M.p. 243–245 °C. IR (cm , KBr) 3 185 (NH),
3
031 (CH ), 1 695 (C=O), 1 666 (quinone C=O), 1 634.
3
1
H NMR (d -DMSO) δ 12.98 (s, 1H, NHCO), 9.62 (s,
6
3.1.38.
1,4-Bis(nicotinamido)-9,10-anthracenedione
1
H, H-2≠), 9.25 (d, 1H, H-6≠), 9.11 (d, 1H, H-4≠), 8.95
dimethiodide (35)
(
d, 1H, H-2), 8.44 (m, 1H, H-5≠), 8.23 (m, 2H, H-5,8),
Compound 35 was obtained from 34 by the procedure
8
.08 (m, 2H, H-3,4), 8.00 (m, 2H, H-6,7), 4.5 (s, 3H,
1
+
described for 33 (yield: 85%). M.p. 311 °C. H NMR
NCH ). Anal C H IN O (C, H, N).
3
21 15
2 3
(
d -DMSO) δ 12.92 (s, 2H, NHCO), 9.63 (s, 2H, H-2≠),
6
3.1.34. 1-(Benzamido)-9,10-anthracenedione (22)
9.25 (d, 2H, H-6≠), 9.13 (d, 2H, H-4≠), 8.97 (s, 2H,
To a solution of 1.5 g (6.7 mmol) of 1-amino-
H-2,3), 8.46 (dd, 2H, H-5≠), 8.26 (dd, 2H, H-5,8), 8.15
+
anthraquinone in 100 mL toluene, 2.3 mL (20.1 mmol) of
benzoyl chloride were added and refluxed for 24 h. The
product was worked up as described for compound 20
(dd, 2H, H-6,7), 4.5 (s, 6H, N–CH ). MS (+FAB) m/z
3
•
+
•+
(rel. intensity, %): 605 ([M–I] , 16), 478 ([M–2I] , 53),
477 ([M–I–HI] , 100). Anal C H I N O (C, H, I, N).
28 22 2 4 4
•+
1
(
yield: 54%). M.p. 244 °C. H NMR (CDCl ) δ 13.01 (s,
3
3
9
.1.39.
1-Amino-4-[[2-(dimethylamino)ethyl]amino]-
1
H, NHCO), 9.39 (d, 1H, H-2), 8.35 (d, 2H H-5,8), 8.18
d, 2H, H-2≠,6≠), 8.13 (d, 1H, H-4), 7.85 (m, 3H,
H-3,6,7), 7.62 (m, 3H, H-3≠,4≠,5≠). MS m/z (rel. inten-
,10-anthracenedione (46)
The general procedure described above for compounds
3 and 44 was followed starting with leucoquinizarin
(
•+
4
sity, %): 327 ([M] , 23), 106 (100). Anal C H NO (C,
21
13
3
(
(
5 g, 20.6 mmol) and N,N-dimethylethylenediamine
8.2 mL, 74.3 mmol) in MeOH (150 mL, 50 °C, 45 min).
H, N).
3
.1.35.
1-Hydroxy-4-(nicotinamido)-9,10-anthracene-
After cooling to room temperature, NH (g) was bubbled
3
dione (21)
through the solution for 5 min. The reaction vessel was
sealed and kept at room temperature for 48 h. The desired
product was purified by column chromatography on silica
gel (eluent: EtOAc), followed by recrystallization from
Compound 21 was prepared as described for com-
pound 20, employing 2.8 g (12 mmol) of 1-amino-4-
hydroxyanthraquinone in 100 mL toluene and 2.5 g

6
14
1
EtOAc (yield: 40%). M.p. 143 °C. H NMR (d -DMSO)
measurements to remove interfering oxygen. CV mea-
surements were performed mostly at a scan rate of
50 mV/s.
6
δ 10.8 (s, 1H, NHCH CH ), 8.4 (bs, 2H, NH ), 8.23 (m,
H, H-5,8), 7.8 (m, 2H, H-6,7), 7.3–7.4 (2d 2H, H-2,3),
.5 (q, 2H, NHCH CH ), 2.58 (t, 2H, NHCH CH ), 2.2
2
2
2
2
3
(
2
2
2
2
•+
s, 6H, N–CH ). MS m/z (rel. intensity, %): 309 ([M] ,
3.2. Biological evaluation
3
1
00), 250 (84). Anal C H N O (C, H, N).
18 19 3 2
3
3
.2.1. Cytotoxic activity in cell culture
3
.1.40.
1-[[2-(Dimethylamino)ethyl]amino]-4-(nicoti-
.2.1.1. Cell culture
A: the P388 murine leukaemia cell line was a generous
namido)-9,10-anthracenedione (31)
Nicotinoyl chloride (1.6 g, 11 mmol) was added to a
solution of 46 (3 g, 9.7 mmol) in 100 mL toluene and
refluxed for 24 h. The product was worked up as de-
scribed for compound 20 and purified by column chro-
matography on silica gel (gradient elution from EtOAc to
gift from Dr A. Ramu, Department of Radiation and
Clinical Oncology, Hadassah University Hospital, Jerusa-
lem, Israel. Cells were grown in RPMI 1640 medium
(GIBCO), supplemented with 10% heat-inactivated foetal
calf serum (GIBCO), 10 µM 2-mercaptoethanol, 50
units/mL of penicillin base and 50 µg/mL of streptomycin
base in a controlled air mixture containing 5% CO2
humidified atmosphere at 37 °C. An inoculum of cells
was transferred to fresh medium once every 4 d in order
to maintain growth in the exponential phase. Cell growth
was assessed by measurement of cell density in a Coulter
(
95:5)
EtOAc:MeOH,
followed
by
(94:5:1)
EtOAc:MeOH:Et N) and recrystallization from (90:10)
3
CH Cl -petroleum ether (yield: 45%). M.p. 218–220 °C.
2
2
1
H NMR (d -DMSO) δ 13.26 (s, 1H, AQNHCO), 10.36
6
(
t, 1H, NHCH CH ), 9.23 (s, 1H, H-2≠), 8.96 (d, 1H,
2
2
H-2), 8.85 (d, 1H, H-6≠), 8.38 (d, 1H, H-4≠), 8.24 (dd,
2
7
H, H-5,8), 7.88 (m, 2H, H-6,7), 7.69 (dd, 1H, H-5≠),
5
counter. Initial cell density was 1 × 10 cells/mL and after
4
.55 (d, 1H, H-3), 3.52 (q, 2H, NHCH CH ), 2.65 (t, 2H,
2
2
6
d it became 1–2 × 10 cells/mL.
NHCH CH ), 2.3 (s, 6H, N–CH ). MS m/z (rel. inten-
sity, %): 414 ([M] , 32), 105 (100). Anal C H N O
2
2
3
•+
B: J774.2 macrophage-like cell line. The cells were
2
4 22 4 3
grown in complete DMEM, supplemented with 10%
horse serum according to published procedures [39].
(C, H, N).
3
.1.41. 1-[[2-(Dimethylamino)ethyl]amino]-4-(nicotina-
3
.2.1.2. Sensitivity to anthraquinone derivatives
The anthraquinone derivatives were dissolved in DMF.
mido)-9,10-anthracenedione N-oxide (32)
The product was prepared from nicotinoyl chloride
N-oxide (1.9 g, 12 mmol) and 46 (3 g, 9.7 mmol) as
described for 31 (yield: 59%). M.p. 195–196 °C: H
NMR (d -DMSO) δ 13.02 (s, 1H, AQNHCO), 10.38 (t,
Cells were cultured in the presence of various drug
concentrations for 72 h. The final DMF concentration
1
(
0.2% v/v) was proved not to be cytotoxic against the cell
6
culture. The initial and final cell densities were measured.
The growth rate at each drug concentration was expressed
as the percentage of the control growth rate. The concen-
tration of drug effective in inhibiting the growth rate by
50% (ED ) was determined according to previously
1
8
H, NHCH CH ), 8.84 (d, 1H, H-2), 8.7 (s, 1H, H-2≠),
2
2
.49 (d, 1H, H-4≠), 8.25 (dd, 2H, H-5,8), 7.9 (m, 3H,
H-6,7, H-6≠), 7.7 (t, 1H, H-5≠), 7.54 (d, 1H, H-3), 3.4 (q,
2H, NHCH CH ), 2.58 (t, 2H, NHCH CH ), 2.25 (s, 6H,
2
2
2
2
50
N–CH ). MS (+FAB) m/z (rel. intensity, %): 431 ([M +
H] , 100). MS (+DCI): 431 ([M + H] , 18), 415 ([M +
H–16] , 100). MS (–DCI): 430 (M , 47), 414 (100).
Anal C H N O (C, H, N).
3
described protocols [24].
•+
•+
•+
•–
2
4
22
4
4
Acknowledgements
3
.1.42. Cyclic voltammetry (CV)
The CV measurements were performed using a BAS
This research was partly supported by grant 95-00287
(
A.S.) from the USA-Israel Binational Science Founda-
tion (BSF), by grant 3810 (J.K.) from the Israeli Ministry
of Health and by the Alex Grass Centre for Drug Design
and Synthesis of Novel Therapeutics (D.B.).
model CV-58 cyclic voltammeter (West Lafayette, Indi-
ana) equipped with a glassy carbon working electrode, a
Pt wire auxiliary electrode and an Ag/AgCl reference
electrode. The measurements were carried out between
–
2 V and 2 V. The samples (1 mM) were dissolved in
References
high-purity DMF, containing 0.1 M tetrabutylammonium
perchlorate as supporting electrolyte. The solutions were
bubbled with high-purity nitrogen for 20 min before the
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