Effect of modification of 6-[(aminoalkyl)amino]-7H-benzo[e]-perimidin-7-ones on their cytotoxic activity toward sensitive and multidrug resistant tumor cell lines. Synthesis and biological evaluation

Article abstract of DOI:10.1016/S0968-0896(01)00358-3

Benzoperimidines, a novel group of antitumor anthracenedione analogues, are of interest due to their ability to overcome multidrug resistance of tumor cells (Stefanska, B., Dzieduszyc a, M., Bontemps-Gracz, M. M., Borows i, E., Martelli, S., Supino, R., Pratesi, G., De Cesare, MA., Zunino, F., Kusnierczy , H., Radziowski, Cz. J. Med. Chem. 1999, 42, 3494). Although the structural factor essential for exhibiting this desirable property is the presence in the molecule of a fused heterocyclic ring, the cytotoxicity against resistant cells is highly influenced by the nature and location of the substituents. A series of novel synthetic derivatives, comprising monohydroxylated benzoperimidines and 2-aminobenzoperimidines, allowed the establishment of an invitro structure-activity relationship for a panel of leuemia sensitive, as well as P-gp dependent multidrug resistance (MDR) and multidrug resistance associated protein dependent resistance (MRP) resistant cell lines. The membrane affinity for the compounds has also been determined. Copyright

Full text of DOI:10.1016/S0968-0896(01)00358-3

Bioorganic & Medicinal Chemistry 10 (2002) 1025–1035
Effect of Modification of 6-[(Aminoalkyl)amino]-7H-benzo[e]-
perimidin-7-ones on Their Cytotoxic Activity Toward Sensitive
and Multidrug Resistant Tumor Cell Lines. Synthesis and
Biological Evaluation
Maria Dzieduszycka,a Sante Martelli,b Ma•gorzata Arciemiuk,a
Maria M. Bontemps-Gracz,a Agnieszka Kupieca and Edward Borowskia,*
a
´ ´
Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdansk, 80-952 Gdansk, Poland
bDepartment of Chemistry, University of Camerino, 62033 Camerino, Italy
Received 9 March 2001; accepted 8 October 2001
Abstract—Benzoperimidines, a novel group of antitumor anthracenedione analogues, are of interest due to their ability to over-
come multidrug resistance of tumor cells (Stefanska, B., Dzieduszycka, M., Bontemps-Gracz, M. M., Borowski, E., Martelli, S.,
Supino, R., Pratesi, G., De Cesare, MA., Zunino, F., Kusnierczyk, H., Radzikowski, Cz. J. Med. Chem. 1999, 42, 3494). Although
the structural factor essential for exhibiting this desirable property is the presence in the molecule of a fused heterocyclic ring, the
cytotoxicity against resistant cells is highly influenced by the nature and location of the substituents. A series of novel synthetic
derivatives, comprising monohydroxylated benzoperimidines and 2-aminobenzoperimidines, allowed the establishment of an in
vitro structure–activity relationship for a panel of leukemia sensitive, as well as P-gp dependent multidrug resistance (MDR) and
multidrug resistance associated protein dependent resistance (MRP) resistant cell lines. The membrane affinity for the compounds
has also been determined. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
cell lines is a five- or six-membered heterocyclic ring
fused with the anthracenedione moiety.1 Anthrapyr-
azoles5 and their aza-analogues,6 anthrapyridones,7,8
anthrapyridazones,7 and benzoperimidines,1 all exhibit-
ing activity against resistant cells, fulfill this require-
ment. Although the presence of a fused heterocyclic ring
is essential for overcoming the multidrug resistance by
an anthracenedione analogue, appropriate substituents
are necessary to achieve optimal results. In this paper,
the effect of substituents on benzoperimidines has been
studied. The synthesis and biological activity of several
benzoperimidines, chromophore-modified anthracene-
diones with a fused pyrimidine ring, have been pre-
viously described by us1,9 (2a, 2b, Fig. 1). These
compounds exhibited in vitro cytotoxic activity against
a panel of sensitive and multidrug resistant tumor cell
lines with a good resistance index (RI of 1–2), which
included solid tumors.
The prolonged treatment of cancer patients by chemo-
therapeutic antitumor agents leads to the development
of multidrug resistance toward numerous antitumor
drugs.2ꢀ4 This effect depends on the overexpression of
plasma membrane drug efflux pumps such as P-gp and
multidrug resistance associated protein dependent resis-
tance (MRP), and presents one of the major limitations
in clinical oncology. Its circumvention is a major chal-
lenge in cancer chemotherapy. This serious problem is
also exhibited by mitoxantrone (MIT) (1b, Fig. 1), a
synthetic anthracenedione anticancer agent. Several
structural modifications to avoid or decrease the multi-
drug resistance phenomenon have been done, resulting
in some progress in overcoming multidrug resistance.
We have previously postulated that the structural factor
essentially favouring the activity toward resistant tumor
We have performed the synthesis and biological evaluation
of two new groups of 2a and 2b derivatives: 2-amino-6-
[(aminoalkyl)amino]-7H-benzo[e]-perimidin-7-ones (com-
pounds 3–6, referred to as 2-aminobenzoperimidines) and
*Corresponding author. Tel.: +48-58-347-2523; fax: +48-58-347-
1893; e-mail: borowski@altis.chem.pg.gda.pl
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00358-3

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M. Dzieduszycka et al. / Bioorg. Med. Chem. 10 (2002) 1025–1035
8- or 11-monohydroxy-6-[(aminoalkyl)amino]-7H-ben-
zo[e]-perimidin-7-ones (7, 8). The structures of the syn-
thesized compounds are shown in Figure 1 and Tables 1
and 2. The 2-aminobenzoperimidine derivatives include
compounds with one alkylaminoalkylamino side chain
attached at position 2 (Table 1, 5a, 6b) and position 6
(Table 1, 3b–d, 4c–e) as well as with two basic chains at
position 2 and 6 (Table 1, 5b, 6c). The derivatives with
an unsubstituted 2-amino group have also been
obtained (Table 1, 3a, 4b). Additionally, some of the
synthesized compounds hold hydroxyl groups at the C-8
and C-11 positions of the ring system, while the others
are dehydroxy analogues. Such selection of the deriva-
tives enabled us to examine if, like in the case of related
antitumor agents as anthracenediones,10,11 anthrapyr-
azoles12 and acridine derivatives,13ꢀ15 the position of the
attached side chains, the presence of the second arm, as
well as hydroxyl groups, in the structure of chromo-
phore, are essential for the activity of benzoperimidine
analogues and ability to overcome multidrug resis-
tance.
The
monohydroxybenzoperimidine
derivatives
(hydroxyl groups at position 8 or 11) have been synthe-
sized (Fig. 1, 7 and 8, respectively), following proce-
dures used for anthrapyrazoles,16 where the presence of
one hydroxyl group instead of two, increased the cyto-
toxic activity of the derivatives.
In vitro cytotoxic activity of the obtained compounds,
together with reference compounds was examined
against sensitive murine L1210 and human K562 and
HL-60 leukemia cell lines and resistant P-gp dependent
multidrug resistance (MDR) (K562/DX and HL-60/
VINC) and MRP (HL-60/DX)0sublines. The membrane
determined and related to their structural characteristics.
17,18
affinity of compounds (log k IAM
)
has been also
Chemistry
In the synthesis of derivatives 3–6 (Fig. 1), the starting
compounds were 1,4-dichloro-9,10-anthracenedione
(9)19 or its 5,8-bis(phenylmethoxy) analogue (10)20 (see
Fig. 2).
The cyclization of these compounds to 2-amino benzo-
perimidines (3a or 4a, respectively) with the use of gua-
nidine, in the presence of copper, in DMF solution at an
elevated temperature was performed by following Hol-
lin’s method.21 Surprisingly, under these reaction con-
ditions, not only cyclization to 2-aminopyrimidine ring
occurred, but also the substitution of the chlorine atom
at position 6 of the substrates by the –N(CH3)2 residue
(which comes from DMF) took place. The course of the
reaction was followed by 1H NMR and 13C NMR
Figure 1. The structures of AMET, MIT, benzoperimidines and syn-
thesized compounds.
Table 1. Structure and characteristic of compounds 3–6
Compound
X
R1
R2
R3
Mp (ꢁC)
% Yield
Formula
3a
3b
H
H
H
H
H
CH3
H
H
H
CH3
H
CH3
CH3
H
H
H
CH3
(CH2)2N(CH3)2
(CH2)3NH2
(CH2)2N(CH2)5
CH3
(CH2)2N(CH3)2
CH3
CH3
(CH2)2N(CH3)2
(CH2)2N(CH2)5
(CH2)2N(CH2)4O
CH3
CH3
(CH2)2N(CH3)2
H
H
H
208–210
218–220
276–277
178–179
110–112
213–115 (dec)
189–190
>330
40
86
90
70
25
65
26
95
65
70
94
22
95
80
C17H14N4O
C19H19N5O
3c ꢂ HCl
C18H18N5OCl ꢂ 1.1 H2O
3d
5a
H
(CH2)2N(CH3)2
(CH2)2N(CH3)2
H
C22H23N5O
C21H23N5O
5b ꢂ 2HCl
H
C23H30N6OCl2 ꢂ 2.1 H2O
4a
4b
4c
4d
4e
6a
6b
6c
OCH2C6H5
OH
OH
OH
OH
OCH2C6H5
OH
OH
C31H26N4O3
C17H14N4O3
H
H
H
H
282–285
C19H19N5O3
C22H23N5O3
C21H21N5O4
C35H35N5O3
C21H23N5O3
C23H28N6O3
270–273 (subl)
260–263
170–172
200–202 (dec)
242–244
CH3
CH3
H
(CH2)2N(CH3)2
(CH2)2N(CH3)2
(CH2)2N(CH3)2

M. Dzieduszycka et al. / Bioorg. Med. Chem. 10 (2002) 1025–1035
1027
spectroscopy and by determination of the molecular
weight of resulting compounds. In the reaction of 9 with
guanidine the main product 3a, as well as small
amounts of the 6 substituted derivative 11 were isolated
and identified. The subsequent reaction of 3a or 4b with
the appropriate aminoalkylamines occurred very readily
and led to the desired 3b–d or 4c–e (4b was obtained by
debenzylation of 4a with trifluoroacetic acid). In con-
trast, substitution of chlorine in 11 by amines required
more drastic reaction conditions. The acetylation of
3a to 12 gave us a derivative suitable for spectral
analysis.
Table 2. Structure and characteristic of compounds 7–8
Compound
X
R
Mp (ꢁC)
% Yield
Formula
7a ꢂ HCl
8-OH
8-OH
8-OH
11-OH
11-OH
11-OH
(CH2)2N(CH3)2
(CH2)2N(CH2)4NH
(CH2)2N(CH2)5
(CH2)2N(CH3)2
(CH2)2N(CH2)4NH
(CH2)2N(CH2)5
175–177 (dec)
155–157
135–136
275–280 (dec)
205–207
137–139
30
50
35
43
50
55
C19H19N4O2Cl ꢂ 0.5 H2O
C21H21N5O2
C22H22N4O2
7b
7c
8a
8b
8c
C19H18N4O2
C21H21N5O2
C22H22N4O2
Figure 2. Synthetic route for compounds 3–6. Reagents: (a) for 3a: guanidine dicarbonate or hydrochloride, Cu, NaOH, DMF, 125–130 ꢁC/4 h; for
4a: the same reagents, 145 ꢁC/80 h; (e) NaNH2, toluene, 125 ꢁC/2 h; (f) for 5a: aminoethyl chloride, toluene, 112–115 ꢁC/2 h; for 6a: the same
reagents, 118–120 ꢁC/40–120 min; for 6b: 6a, TFA, room temp/18 h; (g) amine, 120 ꢁC/60 min.

1028
M. Dzieduszycka et al. / Bioorg. Med. Chem. 10 (2002) 1025–1035
According to the literature, the alkylation of the 2-
amino group of pyrimidine derivatives frequently failed
and, if it was successful, only poor yield of the product
were achieved.22 The 2-N-alkylamino derivatives 5a and
6a were obtained by reaction of 3a or 4a with sodium
amide in toluene followed by the treatment of the
obtained sodium salts of 3a or 4a with alkylaminoalkyl
chloride. Debenzylation of 6a with trifluoroacetic acid
gave 6b. The subsequent treatment of 5a or 6b with
aminoalkylamines afforded 5b or 6c. Structures, melting
points, yields and formulas of compounds 3–6 are
reported in Table 1.
reaction with formamide yielded benzoperimidines 15
and 16. Compound 14 was synthesized by modification
of Gabriel’s method in the reaction of 13 with potas-
sium phthalimide, followed by hydrazinolysis of phtha-
lylaminoanthracenedione.25 Then 14 was treated with
formamide in phenol to afford mixture of 15 and 16
(1.4:1, respectively). The ratio of the regioisomers was
1
calculated on the basis of H NMR spectra of 15 and
16, as well as of their debenzyl derivatives (17 and 18,
Fig. 3). The signals at 5.39, 9.4 and 14.2 ppm were
assigned to the –CH2– benzyl group, proton of perimi-
dine ring and hydroxyl group of 15 or 17, while the sig-
nals at 5.4, 9.3 and 13.8 ppm to the appropriate protons
of 16 or 18, respectively. Because of the similar mobility of
15 and 16 on flash silica gel chromatography in eluents
used, their separation required repeated tedious opera-
tions. The removal of benzyl groups by trifluoroacetic acid
afforded 17 and 18, which were reacted with appropriate
amines to provide the desired derivatives 7a–b and 8a–c. It
was noticed that the transamination of an amino group of
11-hydroxy isomer by aminoalkylamines occurred more
easily then a similar reaction of the 8-hydroxy isomer.
Structures, melting points, yields and molecular formulas
of 7 and 8 are presented in Table 2.
The 8- or 11-monohydroxy-6-aminoalkylamino sub-
stituted benzoperimidines (7a–c and 8a–c, Fig. 1) were
obtained starting from 1,4-dichloro-5-(phenylmethoxy)-
9,10-anthracenedione (13)23 (Fig. 3).
All our attempts according to Showalter’s method24 to
obtain
1-amino-4-hydroxy-5-(phenylmethoxy)-9,10-
anthracenedione, the starting material for the cycliza-
tion step, leading to the benzoperimidine ring were
unsuccessful. Instead, we utilized 1,4-diamino-5-(phe-
nylmethoxy)-9,10-anthracenedione (14), which in the
Figure 3. Synthetic route for compounds 7–8. Reagents: (a) (C6H4)2NK, Cu2l2, DMA, reflux/20 h; (b) N2H4, K2CO3, pyridine, 100 ꢁC/10 min; (c)
HCONH2/PhOH, 160 ꢁC/95 min; (d) flash chromatography; (e) TFA, room temp/18 h; (f) amine, Á.

M. Dzieduszycka et al. / Bioorg. Med. Chem. 10 (2002) 1025–1035
1029
For the biological and physicochemical evaluations, the
free bases of 3–8 were converted into their hydrochloride
or dihydrochloride salts by routine methods.
benzoperimidine
derivatives,
including
mono-
hydroxybenzoperimidines (7–8) and the novel subgroup
of 2-aminobenzoperimidines (3–6) have been synthe-
sized. Membrane affinity of the compounds, essential
for the uptake characteristics, and their in vitro effec-
tiveness against a panel of leukemia cell lines have been
studied. The latter determinations comprised sensitive
leukemia cell lines: murine L1210 and human K562 and
HL-60, as well as human multidrug resistant leukemia
cell sublines: K562/doxorubicin (DX) and HL-60/vin-
cristine (VINC) (resistance of P-gp dependent, MDR)
and HL-60/DX (multidrug resistance associated protein
dependent, MRP). The results are presented in Table 3.
Results and Discussion
Benzoperimidines, the novel group of anthracenediones
analogues, are of interest because of their ability to
overcome multidrug resistance of tumor cells.1,9 Of
importance is the determination of the effect of benzo-
perimidines modification on susceptibility to these
compounds of tumor cell lines representing the two
major types of multidrug resistance. They are based on
the existence of two types of xenobiotics exporting
pumps. One of these is P-glycoprotein directly effluxing
the drugs as its substrates (MDR type resistance).26
Another type of exporting pump is multidrug resistance
associated protein (MRP type resistance), exporting
anionic conjugates of neutral or basic drugs with glu-
tathione or with glucuronic acid formed upon the action
of appropriate transferases, or cotransporting the drugs
with mentioned metabolites.27 The present paper is
devoted to fulfillment of these goals.
The membrane affinity characteristic of the examined
compounds has been based on a new method for the
characterization of abilities to diffuse the anthracene-
dione compounds. The affinity of a drug to cytoplas-
matic membrane is an essential factor governing its
diffusion to the cells. The determinations have been
done on HPLC chromatographic column IAM (an
immobilized artificial membrane) constituting chroma-
tographic surfaces prepared by covalently immobilized
cell membrane phospholipids to solid surfaces at
monolayer densities. IAM surfaces mimic the fluid cell
membrane. The retention time for a given compound,
expressed as log k0IAM values, comprises not only lypo-
philicity characteristic but also other interactions with
membrane like hydrogen bond formation, electrostatic
It should be noted that the influence of substituents is
often quite different for different types of analogues. To
establish the structure–activity relationship in this novel
group of anthracenedione analogues, the series of
Table 3. In vitro cytotoxic activity of examined compounds in a panel of sensitive and resistant leukemia cell lines and membrane affinity of tested
compounds
Cell linea/IC50 (nM) ꢃ SEM
log k0IAM
c
Compound
L1210
K562
K562/DX
RIb
HL-60
HL-60/VINC RIb
HL-60/DX
RIb
3a
3b
3c
3d
4b
4c
4d
4e
5a
5b
6b
6c
7a
7b
7c
8a
8b
8c
19,075ꢃ2907 32,662ꢃ2436 33,729ꢃ2279
1.03 26,951ꢃ765 24,975ꢃ887
0.93 25,348ꢃ222
0.94
2.95
1.38
1.66
7.50
ND
359ꢃ53
333ꢃ36
2112ꢃ467
1611ꢃ138
3615ꢃ516
4395ꢃ43
8.0ꢃ1.1
4033ꢃ595
3477ꢃ563
2251ꢃ218
4062ꢃ832
13.8ꢃ2.3
140ꢃ23
1.91
2.16
0.62
0.92
1.73
1.07
1.42
0.37
1.53
2.85
2.16
1.57
1.16
1.18
1.57
1.90
1.15
761ꢃ46
942ꢃ234
1306ꢃ82
216ꢃ49
4.7ꢃ0.4
40ꢃ7
1067ꢃ54
1494ꢃ267
1143ꢃ65
907ꢃ36
1.40
1.59
0.88
4.20
1.47
8.03
6.85
0.95
3.90
4.24
1.68
3.38
2244ꢃ368
1301ꢃ28
2167ꢃ197
1620ꢃ135
1.894
1.166
2.157
ND
1020ꢃ111
382ꢃ25
2.2ꢃ0.1
6.9ꢃ0.1
321ꢃ65
777.0ꢃ107.3 165
2.231
2.817
2.469
1.442
1.765
1.883
2.343
2.080
2.500
2.537
2.170
2.460
2.700
12.7ꢃ1.0
71.7ꢃ4.0
1664ꢃ111
21.7ꢃ2.6
34.3ꢃ3.5
0.87ꢃ0.09
23.1ꢃ3.6
911ꢃ24
131ꢃ22
2102ꢃ580
4122ꢃ1189
1679ꢃ196
8048ꢃ401
1830ꢃ222
752.9ꢃ77.6
741ꢃ33
52.6
39.6
1.04
158
43.6
342
17.6
4.72
10.4
18.3
4.56
6.08
330ꢃ20
469ꢃ116
2828ꢃ218
144ꢃ6
104ꢃ26
1619ꢃ187
51ꢃ14
712ꢃ127
1544ꢃ192
199ꢃ50
7710ꢃ641
94ꢃ17
256ꢃ34
730ꢃ203
15.1ꢃ0.5
237ꢃ55
42ꢃ20
178ꢃ11
7.0ꢃ0.3
2.2ꢃ0.1
42ꢃ7
3.7ꢃ0.2
142ꢃ29
151ꢃ27
7617ꢃ224
488ꢃ30
8808ꢃ758
574ꢃ138
72.5ꢃ15.1
7540ꢃ1080
392ꢃ21
2556ꢃ114
211ꢃ40
23.8ꢃ5.9
2249ꢃ125
176ꢃ11
4257ꢃ242
710ꢃ179
50.8ꢃ9.9
3182ꢃ304
565ꢃ16
1.67 12,068ꢃ1560
568ꢃ90
3.36
2.13
2201ꢃ325
6.5ꢃ0.9
46.3ꢃ3.5
3974ꢃ749
340ꢃ119
434.8ꢃ30.4
841ꢃ95
1.41 10,258ꢃ2407
3.21
67.7ꢃ4.7
1070ꢃ92
2a
2b
203ꢃ22d
1700ꢃ340d
1756ꢃ316d
1.03
1.86
634ꢃ162
5.2ꢃ1.0
619ꢃ39
0.98
2.73
1371ꢃ239
736ꢃ163
2.16
142
1.808
2.464
1.3ꢃ0.3d
5.6ꢃ0.2d
10.4ꢃ1.1d
14.2ꢃ3.0
AMET
MIT
DX
146ꢃ47d
1.4ꢃ0.1
181ꢃ18
21.0ꢃ4.0
42.1ꢃ3.3
12,392ꢃ886
554.0ꢃ49.5
7031.5ꢃ542.0
68.5
26.4
167
36.5ꢃ2.7
2.2ꢃ0.3
20.0ꢃ3.0
5256ꢃ2704
56.5ꢃ4.2
647ꢃ60
144
25.7
32.4
6096ꢃ109
1179ꢃ203
167
536
1.255
1.841
1.434
19.4ꢃ1.3
3928ꢃ262.9 196
ND, not determined.
aL1210, murine lymphocytic leukemia; K562, human myelogenous leukemia and doxorubicin resistant (MDR type) subline K562/DX; HL-60,
human promyelocytic leukemia, and vincristine resistant (MDR type) subline HL-60/VINC, and doxorubicin resistant (MRP type) subline HL-60/
DX.
bRI, resistance index; the ratio of IC50 value for resistant cell line to IC50 value for sensitive cell line.
cLogarithm of HPLC capacity factor determined in an immobilized artificial membrane column with acetonitrile-buffer pH 7.0 eluent.
dAccording to ref 1.

1030
M. Dzieduszycka et al. / Bioorg. Med. Chem. 10 (2002) 1025–1035
interactions and so on. Therefore we prefer to interpret
the log k0IAM values as characterizing the membrane
affinity rather then as commonly used lypophilicity
characteristics. The effect of both factors: lypophilicity
and other interactions, on the membrane affinity,
expressed by the log k0IAM values, is well illustrated
taking into account various examined compounds (for
structures, see Tables 1 and 2). For instance, 3d, 4d, 7c,
8c bearing lypophilic piperidyl moiety exhibit high log
k0IAM. On the other hand, the presence in the molecule
of hydrophilic groups, able to interact with the mem-
b0rane by other mechanisms, also show elevated log
k IAM. The latter case could be exemplified by phenolic
derivatives and compounds bearing hydrophilic side
chains (e.g., 5b, 6c, and 7b). The great impact of hydro-
philic phenolic groups on log k0IAM values can be also
illustrated by comparing date for MIT and ametantrone
(AMET).
retainment in the cells of therapeutic concentration of a
drug.28
Tumor cell lines with multidrug resistance of MRP type
behave quite differently. The data indicate that mem-
brane affinity (log k0IAM) and consequently the ability to
diffuse of the compounds is not a decisive factor in
overcoming MDR-type multidrug resistance. Two
structural factors of benzoperimidines which positively
influence the membrane affinity, dramatically decrease
the activity toward MRP cells. One of these factors is
the presence of phenolic groups. One phenolic group
exerts a negative effect and two such groups increase it.
The same effect of phenolic groups on RI of the com-
pounds is also observed for MIT and AMET. Another
factor also negatively affecting the activity is the pre-
sence of alkylaminoalkylamino side chains at positions
6 and 2. The presence of both chains in both these
positions essentially decreases the cytotoxic activity
toward MRP cell line. The highest RI index exhibits
compound with two phenolic groups and two side
chains (6c). It could be postulated that a possible
explanation of this behaviour is, that the subject com-
pounds are poor substrates for transferases participat-
ing in the formation of anionic drug conjugates (the
substrates of MRP pumps) or are not suitable for their
cotransport with glutathione or glucuronate.
The results obtained in the examination of structure–
activity relationships of benzoperimidines allowed us to
draw the following conclusions. The nature and loca-
tion of substituents essentially influence cytotoxic activ-
ity of the derivatives, ability to overcome multidrug
resistance and to differentiate between tumor cell lines
with the two above mentioned types of drug exporting
pumps (MDR and MRP). In regard to sensitive cell
lines, the presence of two phenolic groups at positions 8
and 11 essentially increases the cytotoxicity. The lack of
these groups causes the dramatic decrease of activity.
The presence of one phenolic group, the best at position
11, gives an intermediate effect. In regard to this, ben-
zoperimidines behave differently than compounds of the
anthrapyrazole group, where the presence of one phe-
nolic group is more beneficial than two.16 The alkyla-
minoalkylamino side chain at position 6 is indispensable
for the high cytotoxicity and the optimal substituent is
dimethylaminoethylamino residue. The introduction of
amino group at position 2 does not change essentially
the activity of the compound. The substitution at this
group of dimethylaminoethyl chain is very beneficial for
the cytotoxicity of the compound providing the simul-
taneous presence of the second dimethylaminoethyla-
mino side chain at position 6 (5b). Moreover, the
simultaneous presence of two hydroxyl groups at posi-
tions 8 and 11 provides compound with very high cyto-
toxic activity (6c). We have noticed the differential effect
of substituents on cytotoxicity against multidrug resis-
tance cell lines exhibiting two types of resistance (MDR,
MRP). All benzoperimidine derivatives exhibit good or
very good resistance indexes (RI) against tumor cell
lines with P-gp dependent efflux pump (MDR), regard-
less of their cytotoxic potency. No marked influence of
substituents on this property coul0d be observed. We
assume that this is due to the log k IAM values of exam-
ined compounds (above 1) optimal f0or their ability to
diffuse to the cells. Much lower log k IAM values would
slow down the drug diffusion into cells and high values
(log k0IAM 3–4) would cause the retainment of the com-
pounds in cytoplasmatic membrane (unpublished data).
We postulated that fast diffusion of drugs into the cells
with simultaneous functioning of drug exporting pump
according to the saturation kinetics, allows for the
Experimental
Chemistry
Melting points, determined with a Boeticus PHMK05
apparatus, are uncorrected. Thin layer chromatography
(TLC) was carried out on Kieselgel 60 plates (Merck),
column chromatography on Kieselgel Merck (minus
200 mesh). NMR spectra were taken on a Varian 300-
MHz spectrometer using tetramethylsilane as an inter-
nal standard. MS spectra were recorded on a Quad-
rupolic Mass Spectrophotometer Trio-3 (FAB
technique). The elemental analyses were performed on a
Carlo Erba CHNS-O-EA1108 instrument for C, H, N.
2-Amino-6-(dimethylamino)-7H-benzo[e]perimidin-7-one
(3a). A sample of 550 mg (2 mmol) of 9,19 400 mg of
guanidine hydrochloride, 180 mg of copper and 240 mg
of NaOH in DMF (14 mL) was stirred at 125–130 ꢁC for
4 h. The course of the reaction was followed by TLC in
toluene, and in CHCl3/MeOH (20:1). After cooling, the
reaction mixture was filtered. The filtrate was diluted
with CHCl3 and Et2O, washed several times with water
and dried over Na2SO4. The solvents were evaporated
and the residue was purified on flash column (eluent
CHCl3/MeOH 100:1) to afford 3a (40%) as a purple
powder and 11 (5%) as a yellow-brown powder. 1H
NMR of 3a (DMSO-d6) 3.4 (s, 6H, 2ꢂCH3), 6.6 (s, 2H,
NH2, ex), 7.68 (d, 1H, ar, J=9.5 Hz), 7.8 (m, 2H, ar),
7.83 (d, 1H, ar, J=9.7 Hz), 8.3 (dd, 1H, ar, J=10,
1.22 Hz), 8.7 (dd, 1H, ar, J=10, 0.73 Hz). 13C NMR
(DMSO-d6) d 177.13; 159.58; 152.58; 150.83; 147.44;
135.5; 133.19; 132.42; 131.14; 127.47; 126.55; 124.04;
115.03; 107.26; 40.0. MS m/z (relative intensity, %): 291

M. Dzieduszycka et al. / Bioorg. Med. Chem. 10 (2002) 1025–1035
1031
([M+1]+, 100). Found: C, 70.1; H, 4.75; N, 19.1; calcd
for C17H14N4O: C, 70.40; H, 4.87; N, 19.32.
followed by washing with Et2O. The subsequent reac-
tion with 2-(dimethylamino)ethyl chloride was per-
formed in toluene at 125 ꢁC for 30–60 min. The reaction
mixture was worked up to give a residue, which was
flash-chromatographed (eluent CHCl3/MeOH 5:1 then
2:1) to afford 5a (yield 25%) as a fluorescing at 366 nm
purple powder. Mp 110–112 ꢁC. 1H NMR (CDCl3) d 2.4
(s, 6H, 2ꢂCH3), 2.7 (t, 2H, J=6.1 Hz), 3.2 (s, 6H,
2ꢂCH3), 3.75 (q, 2H, J=5.8 Hz), 5. 7 (t, 1H, NH,
J=5.3 Hz, ex), 7.4–7.8 (m, 4H, ar), 8.4–8.5 (m, 2H, ar),
8.8–8.9 (m, 2H, ar). MS m/z (relative intensity, %): 362
([M]+, 100). Found: C, 69.41; H, 6.32; N, 18.8; calcd for
C21H23N5O: C, 69.59; H, 6.40; N, 19.31.
11: mp 284–286 ꢁC, MS m/z (relative intensity, %): 282
([M]+, 100).
2-Acetamido-6-(dimethylamino)-7H-benzo[e]perimidin-7-
one (12). A sample of 3a was left with Ac2O at room
temperature for 18 h. The reaction mixture was worked
up to give a residue, which was flash-chromatographed
(eluent CHCl3/MeOH 20:1). 1H NMR (CDCl3) d 2.7 (s,
3H, CH3), 3.4 (s, 6H, 2ꢂCH3), 7.7–7.8 (m, 3H, ar), 7.9
(d, 1H, ar, J=9.8 Hz), 8.25 (br s, 1H, NH), 8.4–8.5 (m,
1H, ar), 8.7–8.8 (m, 1H, ar).
2,6-Di[2-(dimethylamino)ethyl]amino]-7H-benzo[e]perimi-
din-7-one (5b). A sample of 5a in 2-(dimethylami-
2-Amino-6-[(2-dimethylamino)ethyl]amino]-7H-benzo[e]-
perimidin-7-one (3b). A sample of 3a with 2-(dimethyla-
no)ethylamine
and
N,N,N0,N0-tetramethyl-
mino)ethylamine
and
N,N,N0,N0-tetramethyl-
ethylenediamine was stirred under nitrogen atmosphere
at 120 ꢁC for 2 h. The reaction mixture was worked up
to give a residue, which was flash-chromatographed
(eluent CHCl3/MeOH 5:1 then CHCl3/MeOH/NH4OH
5:1:0.1) to afford 5b (yield 65%) as a fluorescing at
366 nm purple powder. 1H NMR (CDCl3) d 2.36 (s, 6H,
2ꢂCH3), 2.39 (s, 6H, 2ꢂCH3), 2.6–2.8 (two overlapping
t, 4H), 3.6–3.8 (two overlapping q, 4H), 5.7 (t, 1H, NH,
J=5.0 Hz, ex), 7.45 (d, 1H, J=9.67 Hz, ar), 7.75–7.95
(m, 3H, ar), 8.6 (m, 1H, ar), 9.0 (m, 1H, ar), 11.2 (t, 1H,
NH, J=4.2 Hz, ex). MS m/z (relative intensity, %): 404
([M]+, 100). Found: C, 53.48; H, 6.64; N, 15.41; calcd
for C23H30N6OCl2ꢂ2.1 H2O: C, 53.60; H, 6.65; N,
16.32.
ethylenediamine was stirred under reflux in a nitrogen
atmosphere for 30 min. The reaction mixture was dilu-
ted with CHCl3 and washed with dilute HCl to remove
excess of amine. The organic layer was flash-chromato-
graphed (eluent CHCl3/MeOH 5:1) to give 3b as a dark
pink powder (yield 86%), with a fluorescence at 366 nm.
Mp 218–220 ꢁC. 1H NMR (CDCl3) d 2.4 (s, 6H,
2ꢂCH3), 2.8 (t, 2H, J=6.5 Hz), 3.65 (q, 2H, J=6.5 Hz),
4.8 (br s, 2H, NH2, ex), 7.5 (d, 1H, ar, J=9.6 Hz), 7.9–
8.0 (m, 3H, ar), 8.4 (dd, 1H, ar, J=6.3, 1.95 Hz), 8.7 (d,
1H, ar, J=7.8 Hz), 11.1 (br, s, 1H, NH, ex). MS m/z
(relative intensity, %): 333 ([M]+, 100). Found: C,
68.20; H, 5.70; N, 19.87; calcd for C19H19N5O: C, 68.40;
H, 5.74; N, 21.01.
2-Amino-6-(dimethylamino)-8,11-bis(phenylmethoxy)-
7H-benzo[e]perimidin-7-one (4a). Compound 4a was
obtained from 1020 by a procedure similar to that
described for 3a. The reaction was carried out for
80 min at 145 ꢁC. 1H NMR (CDCl3) d 3.1 (s, 6H,
2ꢂCH3), 4.9 (br s, 2H, NH2, ex), 5.25 (s, 2H), 5.72 (s,
2H), 7.2–7.7 (m, 14H, ar). MS m/z (relative intensity,
%): 503 ([M+1]+, 100). Found: C, 73.82; H, 5.18; N,
11.01; calcd for C31H26N4O3: C, 74.11; H, 5.21; N,
11.15.
2-Amino-6-[(3-aminopropyl)amino]-7H-benzo[e]perimidin-
7-one (3c). Compound 3c was obtained in the reaction
of 3a with 1,3-diaminopropane by a procedure similar
to that described for 3b, as a dark pink solid yield 90%.
1H NMR (CDCl3) d 2.04 (quintet, 2H, J=6.8 Hz), 2.95
(t, 2H, J=7.2 Hz), 3.7 (q, 2H, J=6.4 Hz), 4.9 (br s, 2H,
NH2, ex), 7.5 (d, 1H, ar, J=9.5 Hz), 7.9–8.0 (m, 3H, ar),
8.2 (br s, 2H, NH2, ex), 8.45 (dd, 1H, ar, J=6.3,
1.95 Hz), 8.68 (d, 1H, ar, J=7.8 Hz), 11.1 (br s, 1H,
NH, ex). MS m/z (relative intensity, %): 319 ([M]+,
100).
2-Amino-6-(dimethylamino)-8,11-dihydroxy-7H-benzo[e]-
perimidin-7-one (4b). A sample of 4a was left in tri-
fluoroacetic acid at room temperature for 18 h. The
TFA was removed under reduced pressure by coeva-
poration with benzene. An analytical sample was dis-
solved in CHCl3 and the solution was carefully washed
with NaHCO3 solution and then with water. The
organic layer was dried over Na2SO4, evaporated under
reduced pressure and the residue was solidified under
Et2O to yield 4b (yield 95%) as blue powder. Mp
2-Amino-6-[2-(1-piperydyl)ethyl]amino]-7H-benzo[e]peri-
midin-7-one (3d). The reaction of 3a with 1-(2-ami-
noethyl)piperidine was carried out as described for 3b
for 2.5 h. The reaction mixture was worked up and
purified by flash-chromatography (eluent CHCl3/
MeOH 20:1) to give 3d (yield 70%), as pink powder
with a fluorescence at 366 nm. Mp 178–179 ꢁC. 1H
NMR (CDCl3) d 1.6–1.7 (m, 6H), 2.5 (t, 4H, J=4.9 Hz),
2.75 (t, 2H, J=7.0 Hz), 3.6 (q, 2H, J=6.6 Hz), 5.2 (s,
2H, NH2, ex), 7.5 (d, 1H, ar, J=9.68 Hz), 7.7–8.0 (m,
3H, ar), 8.56 (m, 1H, ar), 8.7 (m 1H, ar), 11.2 (br s, 1H,
NH, ex). MS m/z (relative intensity, %): 373 ([M]+,
100).
>300 ꢁC. H NMR (DMSO-d6) d 3.15 (s, 6H, 2ꢂCH3),
1
5.2 (br s, 2H, NH2, ex), 7.3–7.7 (m, 4H, ar), 13.2 (s, 1H,
ex), 13.8 (s, 1H, ex). MS m/z (relative intensity, %): 324
([M]+, 100).
2-Amino-6-[(2-dimethylamino)ethyl]amino]-8,11-dihydroxy-
7H-benzo[e]perimidin-7-one (4c). A sample of 4b was
2 - [(2 - Dimethylamino)ethyl]amino] - 6 - (dimethylamino) -
7H-benzo[e]perimidin-7-one (5a). A sample of 3a in dry
toluene was stirred under reflux with sodium amide for
2 h. After cooling the product was isolated by filtration
treated
with
2-(dimethylamino)ethylamine
and
N,N,N0,N0-tetramethylethylenediamine under nitrogen
atmosphere at 100 ꢁC for 30 min. The reaction mixture

1032
M. Dzieduszycka et al. / Bioorg. Med. Chem. 10 (2002) 1025–1035
was worked up and purified by flash chromatography
(eluent CHCl3/MeOH 5:1) to afford 4c (yield 65%) as a
purple powder. Mp 282–285 ꢁC. 1H NMR (CDCl3) d 2.4
(s, 6H, 2ꢂCH3), 2.8 (t, 2H, J=6.2 Hz), 3.62 (q, 2H,
J=6.4 Hz), 5.2 (br s, 2H, NH2, ex), 7.3–7.7 (m, 4H, ar),
11.1 (t, 1H, NH, J=4.5 Hz, ex), 13.2 (s, 1H, ex), 13.8 (s,
1H, ex). MS m/z (relative intensity, %): 365 ([M]+, 100).
Found: C, 62.95; H, 5.18; N, 19.01; calcd for
C19H19N5O3: C, 62.47; H, 5.24; N, 19.19.
2,6-Di[(2-dimethylamino)ethyl]amino]-8,11-dihydroxy-7H-
benzo[e]perimidin-7-one (6c). The reaction of 6b with 2-
(dimethylamino)ethylamine was performed similar to
that described for 5b to afford 6c (yield 80%), as a dark
purple solid. Mp 242–244 ꢁC. H NMR (CDCl3) d 2.33
1
(s, 6H, 2ꢂCH3), 2.38 (s, 6H, 2ꢂCH3), 2.6–2.8 (two
overlapping t, 4H), 3.6 (q, 4H, J=3.2 Hz), 6.0 (m, 1H,
ex), 7.12 (d, 1H, ar, J=9.0 Hz), 7.2–7.28 (m, 1H, ar),
7.36 (d, 1H, ar, J=9.6 Hz), 7.68 (d, 1H, ar, J=9.5 Hz),
10.6 (m, NH, ex), 13.6 (br s, 1H, ex), 14.0 (br s, 1H, ex).
MS m/z (relative intensity, %): 422 ([M]+, 100). Found:
C, 64.87; H, 6.57; N, 16.01; calcd for C23H28N6O3: C,
65.38; H, 6.68; N, 16.58.
2-Amino-6-[2(1-piperidinyl)ethyl]amino]-8,11-dihydroxy-
7H-benzo[e]perimidin-7-one (4d). The reaction of 4b
with 1-(2-aminoethyl)piperidine was carried out as
described for 4c for 30 min. The reaction mixture was
worked up and then purified by chromatographed (elu-
ent CHCl3/MeOH 10:1) to afford 4d (yield 70%) as a
1,4-Diamino-5-(phenylmethoxy)-9,10-anthracenedione (14).
Compound 1324 in the reaction with potassium phthali-
mide, in the presence of Cu2I2, according to literature26
was transformed into 1,4-diphthalyloamino-5-(phe-
nylmethoxy)-9,10-anthracenedione; an orange solid, mp
251–253 ꢁC, MS m/z (relative intensity, %): 712 ([M]+,
100). The following reaction with hydrazine hydrate in
pyridine solution, performed at 110 ꢁC for 10 min, gave
purple solid. Mp 270–273 ꢁC. H NMR (CDCl3) d 1.6
1
(m, 6H), 2.5 (t, 4H, J=4.9 Hz), 2.75 (t, 2H, J=6.5 Hz),
3.6 (q, 2H, J=6.5 Hz), 5.1 (s, 2H, NH2, ex), 7.16 (d, 1H,
ar, J=9.0 Hz), 7.24–7.3 (m, 3H, ar), 7.44 (d, 1H, ar,
J=9.69 Hz), 7.7 (d, 1H, ar, J=9.52 Hz), 10.8 (t, 1H,
NH, J=4.2 Hz, ex), 13.2 (s, 2H, ex). MS m/z (relative
intensity, %): 405 ([M]+, 100).
ꢁ
1
14 as a dark blue solid (yield 45%). Mp 143–144 C. H
NMR (CDCl3) d 5.4 (s, 2H), 6.85 (d, 1H, ar, J=9.1 Hz),
6.9 (d, 1H, ar, J=9.1 Hz), 6.92–6.98 (br s, 4H, NH2, ex),
7.3–7.35 (m, 1H, ar), 7.4 (t, 1H, ar, J=3.2 Hz), 7.54–
7.62 (m, 5H, ar), 8.02 (d, 1H, ar, J=7.6 Hz). MS m/z
(relative intensity, %): 343 ([M]+, 100).
2-Amino-6-[2(4-morpholinyl)ethyl]amino]-8,11-dihydroxy-
7H-benzo[e]perimidin-7-one (4e). Compound 4e was
obtained in the reaction of 4b with 4-(2-amino-
ethyl)morpholine by a procedure similar to that descri-
bed for 4c, as a pink solid (yield 94%). Mp 260–263 ꢁC.
1H NMR (CDCl3) d 2.6 (t, 4H, J=4.68 Hz), 2.75 (t, 2H,
J=6.26 Hz), 3.65 (q, 2H, J=4.19 Hz), 3.8 (t, 4H,
J=4.6 Hz), 5.05 (br s, 2H, NH2, ex), 7.2 (d, 1H, ar,
J=9.65 Hz), 7.25–7.3 (m, 3H, ar), 7.45 (d, 1H, ar,
J=9.65 Hz), 7.75 (d, 1H, ar, J=9.55 Hz), 10.8 (t, 1H,
NH, J=4.1 Hz, ex), 13.6 (s, 2H, ex), 13.6 (s, 2H, ex).
MS (relative intensity, %): 407 ([M]+, 100).
6-Amino-8-(phenylmethoxy)-7H-benzo[e]perimidin-7-one
(isomer 15) and 6-Amino-11-(phenylmethoxy)-7H-ben-
zo[e]perimidin-7-one (isomer 16). A mixture of 205 mg
(0.6 mmol) of 14, 1.8 mL (45 mmol) of formamide and
4.8 g of phenol was heated at 160 ꢁC for about 95 min.
The course of the reaction was monitored by TLC
(CHCl3/MeOH 20:1). The reaction mixture was diluted
with CHCl3 and extracted several times with 2 N NaOH
and then with water. The organic phase was dried over
Na2SO4, the solvent was removed and the residue was
purified by flash chromatography (eluent CHCl3/MeOH
70:1) to afford 115 mg (yield 54 %) of 15 and 16 isomers
mixture, showing by 1H NMR a ca. 1.4:1 ratio of 15:16.
Repeated flash chromatography with using of the same
eluent gave 15 (the slower eluting component) as a yel-
lowꢁsolid with strong fluorescence at 366 nm. Mp 232–
234 C (subl), 1H NMR of 15 (DMSO-d6) d 5.39 (s, 2H),
7.3–77 (m, 8H, ar), 8.06 (d, 1H, ar, J=9.4 Hz), 8.2 (d,
1H, ar, J=6.7 Hz), 9.4 (s, 1H). MS m/z (relative inten-
sity, %): 353 ([M]+, 100). Compound 16 (the faster
eluting component), a dark yellow solid with fluores-
cence at 366 nm. Mp 246–248 ꢁC (subl), 1H NMR of 16
(CDCl3-d6) d 5.41 (s, 2H), 7.3–7.48 (m, 5H, ar), 7.6–7.75
(m, 3H), 8.0 (d, 1H, ar, J=9.32 Hz), 8.74 (dd, 1H, ar,
J=9.7, J=1.1 Hz), 9.3 (s, 1H). MS m/z (relative inten-
sity, %): 353 ([M]+, 100).
2-[(2-Dimethylamino)ethyl]amino]-6-(dimethylamino)-8,11-
bis(phenylmethoxy)-7H-benzo[e]perimidin-7-one
(6a).
The reaction of 4a with sodium amide followed by
treatment of obtained sodium salt of 4a with 2-(dime-
thylamino)ethyl chloride was performed similar to that
described for 5a to afford 6a (yield 22%) as a dark pur-
ple powder. Mp 170–172 ꢁC. H NMR (CDC3) d 2.3 (s,
1
6H, 2ꢂCH3), 2.5 (t, 2H, J=6.1 Hz), 3.1 (s, 6H, 2ꢂCH3),
3.5 (q, 2H, J=5.6 Hz), 5.24 (s, 2H), 5.26 (s, 2H), 5.55 (t,
1H, NH, J=5.4 Hz, ex), 7.4 (d, 1H, ar, J=6.2 Hz),
7.34–7.66 (m, 12H, ar), 7.72 (d, 1H, ar, J=9.5 Hz), MS
m/z (relative intensity, %): 574 ([M+1]+, 100). Found:
C, 72.89; H, 6.05; N, 12.01; calcd for C35H35N5O3: C,
73.28; H, 6.15; N, 12.21.
2-[(2-Dimethylamino)ethyl]amino]-6-(dimethylamino)-8,11-
dihydroxy-7H-benzo[e]peri-midin-7-one (6b). A sample
of 6a was treated with trifluoroacetic acid at room tem-
perature for 18 h. The TFA was removed under reduced
pressure. An analytical sample was worked up as
described forꢁ4b, to afford 6b (yield 95%) as blue solid.
6-Amino-8-hydroxy-7H-benzo[e]perimidin-7-one (17). A
sample of 15 was treated with trifluoroacetic acid at
room temperature for 18 h. The TFA was removed
under reduced pressure by coevaporation with benzene.
An analytical sample was worked up as described for 4b.
The flash chromatography (eluent CHCl3/MeOH 70:1)
gave 17 as an orange solid (yield 95%). Mp 264–265 ꢁC.
1
Mp 200–202 C (dec). H NMR (CDCl3) d 2.3 (s, 6H,
2ꢂCH3), 2.7 (t, 2H, J=6.0 Hz), 3.2 (s, 6H, 2ꢂCH3), 3.72
(q, 2H, J=5.7 Hz), 5.6 (t, 1H, NH, J=5.1 Hz, ex), 7.4–
7.6 (m, 2H, ar), 8.3 (m, 2H, ar), 13.3 (s, 1H, ex), 13.6 (s,
1H, ex). MS m/z (relative intensity, %): 373 ([M]+, 100).

M. Dzieduszycka et al. / Bioorg. Med. Chem. 10 (2002) 1025–1035
1033
1H NMR (DMSO-d6) d 7.34 (dd, 1H, ar, J=8.0,
1.2 Hz), 7.58 (d, 1H, ar, J=9.6 Hz), 7.65 (t, 1H, ar,
J=8.0 Hz), 8.06 (dd, 1H, ar, J=4.6, 1.2 Hz), 8.1 (dd,
1H, ar, J=3.0, 1.2 Hz), 9.2 (s, 1H), 14.2 (s, 1H, ex).
thylamino)ethylamine
and
N,N,N0,N0-tetramethyl-
ethylenediamine was carried out for 12 h under condi-
tions as described for 7a. Then the reaction mixture was
diluted with CHCl3 and carefully washed with diluted
HCl was worked up and purified by flash chromato-
graphy (eluent CHCl3/MeOH 20:1 then 5:1) to afford 8a
as a brown solid (yield 43%). Mp 275–280 ꢁC. 1H NMR
(CDCl3) d 2.4 (s, 6H), 2.8 (t, 2H, J=6.5 Hz), 3.6 (q, 2H,
J=6.5 Hz), 7.18 (dd, 1H, ar, J=9.0, 1.0 Hz), 7.45 (d,
1H, ar, J=9.6 Hz), 7.65 (t, 1H, ar, J=8.0 Hz), 7.98 (d,
1H, ar, J=9.8 Hz), 8.42 (dd, 1H, ar, J=9.0, 1.0 Hz), 9.2
(s, 1H), 11.0 (br s, 1H, ex), 13.6 (s, 1H, ex). Found: C,
67.87; H, 5.20; N, 16.51; calcd for C19H18N4O2: C,
68.19; H, 5.43; N, 16.76.
6-Amino-11-hydroxy-7H-benzo[e]perimidin-7-one (18). A
sample of 16 was treated with trifluoroacetic acid and
worked up in the same manner as described for 17.
Obtained 18 (brown solid), melted at 307–309 ꢁC. H
1
NMR (DMSO-d6) d 7.15 (dd, 1H, ar, J=9.0, 1.0 Hz),
7.4 (d, 1H, ar, J=9.6 Hz), 7.7 (t, 1H, ar, J=8.0 Hz), 8.0
(d, 1H, ar, J=9.8 Hz), 8.43 (dd, 1H, ar, J=9.0, 1.0 Hz),
9.25 (s, 1H), 13.8 (s, 1H, ex).
6-[2-(Dimethylamino)ethyl]amino]-8-hydroxy-7H-benzo[e]-
perimidin-7-one (7a). A crude sample of 17 with 2-
6-[2-(piperazin-1-yl)ethyl]amino]-11-hydroxy-7H-benzo[e]-
perimidin-7-one (8b). The reaction of 18 with 1-(2-ami-
noethyl)-piperazine was carried out for 5 h. The workup
was similar to that described for 7b. Yield 50%, mp
205–207 ꢁC. 1H NMR (CDCl3) d 2.0 (m, 4H), 2.8 (t, 2H,
J=6.5 Hz), 2.9 (m, 4H), 3.55 (q, 2H, J=6.5 Hz), 7.18
(dd, 1H, ar, J=9.0, 1.0 Hz), 7.45 (d, 1H, ar, J=9.6 Hz),
7.65 (t, 1H, ar, J=8.0 Hz), 7.98 (d, 1H, ar, J=9.8 Hz),
8.42 (dd, 1H, ar, J=9.0, 1.0 Hz), 9.2 (s, 1H), 11.0 (br s,
1H, ex), 13.6 (s, 1H, ex). MS m/z (relative intensity, %):
375 ([M]+, 100).
(dimethylamino)ethylamine
and
N,N,N0,N0-tetra-
methylethylenediamine was heated at 115 ꢁC under
nitrogen atmosphere for 16 h. The progress of the reac-
tion was followed by TLC (CHCl3/MeOH 10:1). The
reaction mixture was diluted with CHCl3 and carefully
washed with diluted HCl followed by washed with
water. The organic layer was dried over Na2SO4, evap-
orated under reduced pressure and the residue was flash
chromatographed (eluent CHCl3/MeOH 20:1 then 5:1)
1
to afford 7a as a brown powder (yield 30%). H NMR
(CDCl3) d 2.4 (s, 6H), 2.8 (t, 2H, J=6.5 Hz), 3.6 (q, 2H,
J=6.5 Hz), 7.35 (dd, 1H, ar, J=8.0, 1.2 Hz), 7.58 (d,
1H, ar, J=9.6 Hz), 7.7 (t, 1H, ar, J=8.0 Hz), 8.06 (dd,
1H, ar, J=9.0, 1.0 Hz), 8.1 (dd, 1H, ar, J=9.0, 1.0 Hz),
9.2 (s, 1H), 11.45 (br s, 1H, ex), 14.2 (s, 1H, ex). Found:
C, 59.87; H, 5.28; N, 13.92; calcd for C19H19N4O2Cl ꢂ
0.5 H2O: C, 60.04; H, 5.31; N, 14.76.
6-[2-(1-piperydyl)ethyl]amino]-11-hydroxy-7H-benzo[e]-
perimidin-7-one (8c). The reaction of 18 with 1-(2-ami-
noethyl)-piperidine was carried out for 6 h and then
worked up as described for 7c. Yield 55%, mp. 137–
139 ꢁC. H NMR (CDCl3) d 1.6 (m, 6H), 2.5 (t, 4H,
1
J=4.9 Hz), 2.75 (t, 2H, J=6.5 Hz), 3.6 (q, 2H,
J=6.5 Hz), 7.18 (dd, 1H, ar, J=9.0, 1.0 Hz), 7.45 (d,
1H, ar, J=9.6 Hz), 7.65 (t, 1H, ar, J=8.0 Hz), 7.98 (d,
1H, ar, J=9.8 Hz), 8.42 (dd, 1H, ar, J=9.0, 1.0 Hz), 9.2
(s, 1H), 11.0 (br s, 1H, ex), 13.6 (s, 1H, ex). MS m/z
(relative intensity, %): 374 ([M]+, 100).
6-[2-(piperazin-1-yl)ethyl]amino]-8-hydroxy-7H-benzo[e]-
perimidin-7-one (7b). The reaction of 17 with 1-(2-amino-
ethyl)-piperazine was carried out under conditions
described for 7a (reaction time 9 h). Then the reaction
mixture was worked up and purified by flash chroma-
tography with using of deactivated silica gel (eluent
CHCl3/MeOH 5:1 then CHCl3/MeOH/NH4OH 5:1:0.1)
to afford 7b (yield 50%). Mp 155–157 ꢁC. 1H NMR
(CDCl3) d 2.0 (m, 4H), 2.8 (t, 2H, J=6.5 Hz), 2.9 (m,
4H), 3.55 (q, 2H, J=6.5 Hz), 7.35 (dd, 1H, ar, J=8.0,
1.2 Hz), 7.58 (d, 1H, ar, J=9.6 Hz), 7.7 (t, 1H, ar,
J=8.0 Hz), 8.06 (dd, 1H, ar, J=9.0, 1.0 Hz), 8.1 (dd, 1H,
ar, J=9.0, 1.0 Hz), 9.2 (s, 1H), 11.45 (br s, 1H, ex), 14.2 (s,
1H, ex). MS m/z (relative intensity, %): 375 ([M]+, 100).
Membrane Affinity Measurements
HPLC column containing as stationary phase 1-myr-
istoyl-2-[13-carbonylimidazolide-tridecanoyl]sn-3-gly-
cerophospholine(lecithin-imidazolide) bonded to silica-
propylamine with the unreacted propylamine moieties
end-capped with C10 and C3 alkyl chains
(IAM.PC.DD2) was purchased from Regis Technolo-
gies Inc. (Morton Grove, IL, USA). The IAM.PC.DD2
column was 3 cm ꢂ 4.6 mm; particle diameter 12 mm;
pore diameter 300 A. For all studies the injection
volume was ca. 10 mL of a solute aqueous solution.
Acetonitrile/0.1 M Sorensen buffer (K2HPO4/KH2PO4)
pH 7.2 eluent was used in proportion 50:50, 40:60,
35:65, 30:70 and 25:75 (v/v). The flow rate was 1 mL/
min and solute detection was at 495 nm. The chroma-
tographic system consisted of a Model L-6200 A pump,
Model L-4250 UV–vis detector and a Model D-2500
chromatointegrator (all from Merck-Hitachi, Vienna,
Austria).
6-[2-(1-piperidinyl)ethyl]amino]-8-hydroxy-7H-benzo[e]-
perimidin-7-one (7c). The reaction of 17 with 1-(2-ami-
noethyl)-piperidine was carried out for 9 h. The workup
was similar to that described for 7a (yield 35%). Mp
135–136 ꢁC. 1H NMR (CDCl3) d 1.6 (m, 6H), 2.5 (t, 4H,
J=4.9 Hz), 2.75 (t, 2H, J=6.5 Hz), 3.6 (q, 2H,
J=6.5 Hz), 7.35 (dd, 1H, ar, J=8.0, 1.2 Hz), 7.58 (d,
1H, ar, J=9.6 Hz), 7.7 (t, 1H, ar, J=8.0 Hz), 8.06 (dd,
1H, ar, J=9.0, 1.0 Hz), 8.1 (dd, 1H, ar, J=9.0, 1.0 Hz),
9.2 (s, 1H), 11.45 (br s, 1H, ex), 14.2 (s, 1H, ex). MS m/z
(relative intensity, %): 374 ([M]+, 100).
6-[2-(Dimethylamino)ethyl]amino]-11-hydroxy-7H-benzo[e]-
perimidin-7-one (8a). The reaction of 18 with 2-(dime-
Capacity factors, k0IAM, were calculated assuming that
the dead volume of the column was the signal given by

1034
M. Dzieduszycka et al. / Bioorg. Med. Chem. 10 (2002) 1025–1035
50 mg/mL citric acid solution. A standard, commercially
available statistical package for regression analysis was
employed on a personal computer.
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Murine L1210 lymphocytic leukemia cells were grown in
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The authors acknowledge the financial support of these
studies by the State Committee for Scientific Research
(KBN), Warsaw, Poland (Grant No. 4 PO5F 035 19),
and in part by Italian Ministry of Foreign Affairs and
the Chemical Faculty Technical University of Gdansk,
Poland. We are also indebted to Dr. J. Mazerski for the
valuable discussions.
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