8,11-Dihydroxy-6-[(aminoalkyl)amino]-7H-benzo[e]perimidin-7-ones with activity in multidrug-resistant cell lines: Synthesis and antitumor evaluation

Article abstract of DOI:10.1021/jm980682p

The synthesis of dihydroxybenzoperimidine derivatives, which are chromophore-modified dihydroxyanthracenediones with an additional pyrimidine ring incorporated into the chromophore, is reported. These derivatives are structurally related to the antitumor

Full text of DOI:10.1021/jm980682p

3494
J . Med. Chem. 1999, 42, 3494-3501
8,11-Dih yd r oxy-6-[(a m in oa lk yl)a m in o]-7H-ben zo[e]p er im id in -7-on es w ith
Activity in Mu ltid r u g-Resista n t Cell Lin es: Syn th esis a n d An titu m or
Eva lu a tion §
Barbara Stefan´ska, Maria Dzieduszycka, Maria M. Bontemps-Gracz, and Edward Borowski*
Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdan˜sk, 11/ 12 G. Narutowicza St,
80-952 Gdan˜sk, Poland
Sante Martelli
Department of Chemistry, University of Camerino, Via S. Agostino, 1, 62032 Camerino, Italy
Rosanna Supino, Graziella Pratesi, MichelAndrea De Cesare, and Franco Zunino
Experimental Oncology B, Istituto Nazionale Tumori, Via Venezian, 1, 20133 Milano, Italy
Halina Kus´nierczyk and Czesław Radzikowski
Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 12 R. Weigla St, 53-114 Wrocław, Poland
Received April 26, 1999
The synthesis of dihydroxybenzoperimidine derivatives, which are chromophore-modified
dihydroxyanthracenediones with an additional pyrimidine ring incorporated into the chro-
mophore, is reported. These derivatives are structurally related to the antitumor agent
mitoxantrone. Their synthesis was carried out by the reaction of 6-amino-8,11-dihydroxy-7H-
benzo[e]perimidin-7-one (5) or 6,8,11-trihydroxy-7H-benzo[e]perimidin-7-one (10) with a number
of respective (alkylamino)alkylamines. The dihydroxybenzoperimidine derivatives exhibited
in vitro cytotoxic activity against murine leukemia L1210 and human leukemia HL60 cell lines
comparable to that of mitoxantrone. These compounds also exhibited a range of in vitro activity
against the human MDR-type resistant leukemia K562R cell line with the MDR phenotype.
The most active compound of this series, namely 6a , exhibited potent in vitro cytotoxic activity
against a panel of human cell lines. Furthermore, in contrast to both mitoxantrone and
doxorubicin, it displayed little cross-resistance in cell lines characterized by a MDR phenotype.
Cell cycle analysis in the sensitive HT-29 and mitoxantrone-resistant HT-29/Mx (not identified
resistance mechanism) cell lines has revealed that both mitoxantrone and 6a induce a G2/M
block. However, while the proportion of apoptotic cells after mitoxantrone treatment is similar
for both sensitive and resistant cell lines, it is much lower for 6a . Compound 6a tested against
P388 murine leukemia in vivo displayed a significant antitumor effect (%T/C 196 at an optimal
dose of 10 mg/kg). The property of overcoming the cross-resistance was maintained also in in
vivo efficacy studies, where no difference was observed in the antitumor activity of compound
6a against the A2780 human tumor xenograft and its MDR A2780/Dx subline. We conclude
that benzoperimidines, if properly substituted, constitute a novel class of compounds that can
overcome multidrug resistance.
In tr od u ction
classes have been identified. Derivatives with unsym-
metrical side chains4 and aza analogues containing
pyridine5 or pyridazine6 rings are noteworthy. It ap-
pears that the most common type of anthracenedione
modification, which targets overcoming multidrug re-
sistance, is the incorporation into the chromophore of a
five- or six-membered heterocyclic ring. This has re-
sulted in the design of anthrapyrazoles7 and their aza
analogues,8 anthrapyridones and anthrapyridazones.9
It is of interest that a similar positive effect on overcom-
ing multidrug resistance has been reported for the
structurally related acridones, such as the imidazo-
acridones,10 triazoloacridones,11 pyrazoloacridones,12 and
pyrimidoacridones.13
Mitoxantrone (1) (Chart 1), a synthetic anthracene-
9,10-dione derivative, is an important antitumor agent
with demonstrated clinical efficacy in the treatment of
leukemia, lymphomas, and breast cancer.1 However,
some undesired side effects have been reported, prin-
cipally cardiotoxicity, which is thought to be one con-
sequence of the ability of anthracenediones to generate
oxygen radicals.2 In recent years the problem of multi-
drug resistance (MDR) toward numerous antitumor
compounds has also become important.3 Many efforts
have been directed toward the design of new antitumor
anthracenedione derivatives with increased effective-
ness against MDR tumor cell lines. Several structural
We have previously reported on the synthesis and
biological activity of a novel group of antineoplastic
agents, the benzoperimidines 2 (Chart 1), which are
§ Polish Patent Application P317493, Dec 12, 1996.
* To whom correspondence should be addressed.
10.1021/jm980682p CCC: $18.00 © 1999 American Chemical Society
Published on Web 08/21/1999

Synthesis of Dihydroxybenzoperimidines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3495
(phenylmethoxy)-9,10-anthracenedione (7)19 (method B)
as starting materials (see Scheme 1). 6-Amino-8,11-bis-
(phenylmethoxy)-7H-benzo[e]perimidin-7-one (4) was
obtained by refluxing 1,4-diamino-5,8-bis(phenylmeth-
oxy)-9,10-anthracenedione (3) with formamide in the
presence of ammonium metavanadate in dimethylac-
etamide (method A). After removal of the benzyl groups
with trifluoroacetic acid, the isolated product 5 was
heated with appropriate aminoalkylamines, in a solvent
mixture of N,N,N′,N′-tetramethylethylenediamine and
generally a small amount of water.
Ch a r t 1
1-Amino-4-hydroxy-5,8-bis(phenylmethoxy)-9,10-an-
thracenedione (8) (method B) was prepared by reduction
of 1-hydroxy-4-nitro-5,8-bis(phenylmethoxy)-9,10-an-
thracenedione (7) using hydrazine and Pd/C. The prod-
uct was cyclized with formamide in phenol to give 9.
After removal of the 8,11-benzyl groups, the derived
6,8,11-trihydroxy-7H-benzo[e]perimidin-7-one (10) was
reacted with appropriate amines under similar condi-
tions as described in method A.
The transamination reaction of 5 with ethylenedi-
amine or N-monosubstituted ethylenediamines may be
accompanied by a subsequent cyclization step to form
side products.14 However, when using 6,8,11-trihydroxy-
benzoperimidine as starting material (method B), we
found that this reaction proceeds under milder condi-
tions, thus diminishing the amount of the cyclized side
product (reaction conditions are indicated in Table 1).
chromophore-modified anthracenediones with a fused
pyrimidine ring.14 This class of compounds did not
stimulate free radical formation, due to their poor
enzyme substrate properties toward NADH dehydroge-
nase.15 Our preliminary results have shown that these
compounds exhibit in vitro cytotoxic activity toward
multidrug resistant cell lines.9b
In this paper we present the synthesis and in vitro
and in vivo evaluation of newer benzoperimidines along
with data on their ability to overcome multidrug resis-
tance. These studies include 6-[(aminoalkyl)amino]-7H-
benzo[e]perimid-7-one derivatives 6a -g with additional
hydroxy groups incorporated into the C-8 and C-11
positions of the ring system. As noted earlier, the
presence of hydroxy groups in related ring systems such
as anthracenediones,1a,b anthrapyrazoles,7a and acridine
derivatives16,17 increases antitumor activity in these
classes of compounds.
Biologica l Resu lts a n d Discu ssion
The 8,11-dihydroxy-6-[(aminoalkyl)amino]-substituted
benzoperimidines 6a -g were tested for the growth
inhibition of L1210 murine and HL60 human leukemia
cells in vitro. The results, including nonhydroxylated
benzoperimidine 2 and reference compounds (mitox-
antrone, ametantrone, and doxorubicin), are presented
in Table 2. All the evaluated compounds 6a -g exhibited
significant in vitro cytotoxicity. Among them 6a was
shown to be the most active. Moreover all benzoperi-
midine derivatives demonstrated activity against the
MDR cell line K562R (Table 2).
Ch em istr y
8,11-Dihydroxy-6-[(aminoalkyl)amino]-substituted ben-
zoperimidines 6a -g were synthesized by two methods
using 1,4-diamino-5,8-bis(phenylmethoxy)-9,10-anthra-
cenedione (3)18 (method A) or 1-hydroxy-4-nitro-5,8-bis-
The compound most effective as a cytotoxic agent (6a )
was tested in the NCI panel of human tumor cell lines.
Sch em e 1

3496 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Stefan´ska et al.
Ta ble 1. Synthesis and Physicochemical Properties of 8,11-Dihydroxybenzoperimidines 6a -ga
reaction conditions
compd
R
method
time (h)
temp of the bath (°C)
yield (%)
mp (°C dec)
molecular formula
6a
CH2CH2N(CH3)2
A
B
A
A
A
B
A
B
A
B
A
B
5
4
3
130
125
160
135
65
65
100
80-85
125-130
110
135
100
36
40
50
42
24
26
24
30
55
65
30
40
>300
C19H18N4O3‚HCl
6b
6c
6d
CH2CH2N(C2H5)2
CH2CH2CH2N(CH3)2
CH2CH2NH2
>300
295-300
280-285
C21H22N4O3‚HCl
3
C20H20N4O3‚HCl
1.5
1.5
12
6.5
14
2.5
4
C17H14N4O3‚HCl‚H2Ob
6e
6f
CH2CH2NHC2H5
>300
>300
>300
C19H18N4O3‚HCl‚0.5H2O
C20H20N4O3‚HCl‚0.5H2O
C19H18N4O4‚HCl‚H2Oc
CH2CH2NHCH(CH3)2
CH2CH2NHCH2CH2OH
6g
2
a
The structures of compounds 6a -g were confirmed by their spectral data (1H NMR, IR, UV-vis, MS-FD) and by elemental analysis.
N: calcd, 14.87; found, 14.38. c H: calcd, 5.03; found, 5.56.
b
Ta ble 2. In Vitro Cytotoxic Activity of 6-[(Aminoalkyl)amino]-7H-benzo[e]perimidine Derivatives
cell line IC50 (nM) ( SEM
compd
L1210a
HL60b
K562Sb
K562Rb (RI)
6a
6b
6c
6d
6e
6f
6g
2
1.29 ( 0.26
19.52 ( 6.27
12.97 ( 6.49
9.02 ( 1.86
3.79 ( 1.52
6.59 ( 2.68
3.55 ( 0.95
203 ( 22
1.67 ( 0.12
16.58 ( 0.63
21.04 ( 0.91
11.68 ( 0.45
14.45 ( 1.16
8.38 ( 0.28
6.62 ( 0.31
587 ( 34
5.56 ( 0.21
49.97 ( 1.71
67.90 ( 5.46
16.83 ( 1.57
48.91 ( 2.15
23.52 ( 0.48
21.35 ( 1.30
1700 ( 340
181 ( 18
10.41 ( 1.07 (1.87)
78.31 ( 6.46 (1.57)
140.0 ( 9.6 (2.06)
45.41 ( 3.45 (2.70)
89.12 ( 9.09 (1.82)
53.41 ( 5.15 (2.27)
42.51 ( 3.38 (1.99)
1756 ( 316 (1.03)
12392 ( 886 (68.5)
133.5 ( 7.1 (18.9)
2474 ( 126 (158.6)
ametantrone
mitoxantrone
doxorubicin
146 ( 47
15.9 ( 0.9
11.4 ( 5.7
0.81 ( 0.06
7.8 ( 0.8
7.06 ( 0.32
15.6 ( 0.8
NTc
a
b
48-h exposure to drug, method I. 72-h exposure to drug, method II. c NT, not tested in these experiments.
It showed significant cytotoxic activity not only against
several leukemic cell lines but also against a number
of other tumor cell lines, including solid tumors. Of 60
cell lines tested against, the response parameter log GI50
was <-8.00 (0.01 µM) for 19 cell lines and -8.00 ÷
-5.23 (0.01-5.9 µM) for the remaining cell lines (Table
3).
A comparison of the pattern of cytotoxic activity of
the compound 6a against the reference agents mitox-
antrone, ametantrone, and doxorubicin in a panel of
sensitive and resistant human cell lines of different
origin (ovarian, colon, and prostate) is given in Table 4.
Compound 6a showed cytotoxic activity similar to that
of mitoxantrone against the sensitive cell lines and
retained activity toward the HT29 cells resistant to
mitoxantrone (Mx). Moreover, 6a showed a similar effect
toward the LoVo/Dx and A2780/Dx cell lines.
The comparative effects of 6a and mitoxantrone on
the cell cycle parameters and apoptosis were studied in
HT29-sensitive and HT29/Mx-resistant cell lines. As
shown in Figure 1, in the sensitive cell line 6a and
mitoxantrone induced similar levels of toxicity, and at
the IC80, both drugs induced 34-37% of apoptosis. In
contrast, in the resistant cell line treated at the IC80
concentrations of drugs, 6a induced a lower apoptotic
response than mitoxantrone (15% and 37%, respec-
tively). Both drugs induced a cell cycle accumulation in
the G2/M phase in both the sensitive and resistant cell
lines (Table 5). Moreover, there is an increase also in
the S phase without a complete depletion of cells in G1
phase, suggesting that HT29 and HT29/Mx cells are
actively cycling after 72 h of treatment.
Ta ble 3. Selected Growth Inhibition Data for 6a from the NCI
in Vitro Screen
panel/cell line
leukemia
log GI50
panel/cell line
melanoma
log GI50
CCRF-CEM <-8.00
LOX IMVI
MALME-3M
M14
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
<-8.00
-7.31
-7.65
-6.00
-5.73
-7.47
-6.28
-7.54
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
<-8.00
<-8.00
<-8.00
-7.60
<-8.00
prostate cancer
DU-145
-7.83
-7.42 ovarian cancer
PC-3
CNS cancer
SF-268
IGROV1
-7.45
-6.21
-6.00
-6.51
-7.46
-7.86
-7.50
<-8.00
-7.61
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
SF-295
SF-539
SNB-19
SNB-75
-7.88
<-8.00
-8.00 breast cancer
U251
non-small-cell
lung cancer
BT-549
HS 578T
MCF7
MCF7/ADR-RES
MDA-MB-231/ATCC
MDA-MB-435
MDA-N
-6.79
-7.38
<-8.00
-6.62
-5.90
-6.78
-6.54
-6.86
A549/ATCC <-8.00
EKVX
HOP-62
HOP-92
-6.24
<-8.00
-7.26
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
<-8.00
-7.59
T-47D
-6.37 renal cancer
<-8.00
-7.51
786-0
A498
<-8.00
-5.74
<-8.00
<-8.00
-6.18
<-8.00
-6.04
<-8.00
ACHN
CAKI-1
RXF-393
SN12C
TK-10
UO-31
<-8.00
-7.05
-7.89
-7.53
-7.61
-6.51
-7.88
HT29
KM12
SW-620
On the basis of the results of cytotoxicity studies,
compound 6a was selected for in vivo evaluation. The

Synthesis of Dihydroxybenzoperimidines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3497
Ta ble 4. In Vitro Cytotoxic Activity of Compound 6a in Comparison with Reference Compounds in a Panel of Human Sensitive and
Resistant Cell Lines
compound IC50 (nM) ( SEM
cell line
6a
ametantrone
mitoxantrone
doxorubicin
DU145a
5.7 ( 0.8
1020 ( 278
1848 ( 143
NTd
5.6 ( 1.6
52.8 ( 2.8
102.8 ( 9.1
56.4 ( 8.1
231.2 ( 15.5 (4.1)
NT
PC3a
11.6 ( 2.6
7.1 ( 0.7
HT/29a
49.1 ( 25.6
56.9 ( 20.7 (1.2)
11.1 ( 2.3
12.7 ( 2.1 (1.1)
8.3 ( 1.2
9.5 ( 1.4 (1.1)
19.6 ( 0.4
2025 ( 890 (103)
17.1 ( 3.8
461.6 ( 87.4 (27)
6.11 ( 0.55
25.1 ( 4.1 (4.1)
HT29/Mxa (RI)
LoVob
NT
641 ( 134
36802 ( 4835 (57.4)
180 ( 30
LoVo/Dxb (RI)
A2780a
NTc
19.9 ( 5.4
466 ( 109 (23.4)
A2780/Dxa (RI)
1108 ( 20 (6.2)
a
b
72-h exposure to drug following 24 h of preincubation, method II. 72-h exposure to drug, method I. c The RI ) 30.4 after 24-h exposure
to drug.25 NT, not tested in these experiments.
d
F igu r e 1. Apoptosis and toxicity of compound 6a and mitoxantrone in HT29 and HT29/Mx cell lines. The percent apoptosis for
the control (not treated cells) was 8 ( 1 and 4 ( 1 for HT29 and HT29/Mx cell lines, respectively. Apoptosis was assessed by
fluorescence microscopy. The percentage of the apoptotic cells refers to the cell number of the whole population (floating + adherent
cells). Toxicity was measured by cell counting. The percentage of toxicity refers to the cell number in control, not treated, cells.
Ta ble 5. Effects of Compound 6a and Mitoxantrone in HT29
and HT29/Mx Cell Lines
dose administration of mitoxantrone; however, it dis-
played activity at much lower doses (Table 6). Prelimi-
cell cycle
%S
nary evaluation of the toxicity of compound 6a (ip)
showed that 50% lethality in healthy CD2F1 mice was
attained at the single dose of ca. 46 mg/kg (in the range
of 43-49 mg/kg).20 This LD50 value was 2 times higher
than that observed for mitoxantrone (20-22 mg/kg).
Compound 6a was also tested in subcutaneously
growing human ovarian carcinoma xenografts (Table 7).
Against the A2780 tumor, good inhibition was achieved
after 3 iv injections of 10 mg/kg. In keeping with the in
vitro results, the antitumor efficacy of compound 6a was
maintained even in the MDR A2780/Dx tumor. Under
the same experimental conditions, the activity of 6a was
comparable to that of mitoxantrone. In contrast, com-
pound 6a showed no activity in the human colon tumor
HT29/Mx, even though higher doses were used (14 and
20 mg/kg). This was not surprising since the parent line
of this tumor is known for being generally resistant to
cytotoxic drugs. Thus, despite the low resistance index
shown by this compound in the in vitro cell system
(Table 4), no activity could be detected in the in vivo
studies.
cell line,
compd (ng/mL)
% survival
%G1
%G2/M
HT29 (72 h)
control
6a
25
100
56 ( 1
18 ( 1
25 ( 1
53 ( 5
20 ( 4
48 ( 3
23 ( 3
19 ( 2
21 ( 2
29 ( 4
57 ( 2
mitoxantrone
25
100
51 ( 9
22 ( 1
46 ( 1
29 ( 2
20 ( 1
24 ( 1
34 ( 1
47 ( 1
HT29/Mx (72 h)
control
6a
10
20
100
mitoxantrone
250
1000
62 ( 1
12 ( 4
26 ( 4
100
61 ( 3
12 ( 3
63
58 ( 2
25 ( 3
7
30
31 ( 3
58 ( 5
11 ( 3
17 ( 3
80 ( 5
45 ( 10
17
62 ( 1
47 ( 7
8 ( 1
17 ( 5
ND
30 ( 2
37 ( 7
ND
3000
NDa
a
ND, not detectable.
antitumor activity of 6a was examined initially in
intraperitoneally (ip) growing murine P388 leukemia.
When given as a single dose on day 1 or in a divided-
dose schedule (5 consecutive days), 6a resulted in
significant life prolongation, which in the total dose
range of 5-20 mg/kg was comparable in both applied
schedules. Similar effects were observed upon single-
We have carried out mechanistic studies showing that
the ability of benzoperimidine derivatives to overcome
multidrug resistance in tumor cells is due to increased
drug uptake relative to active P-gp-dependent and
MRP1-dependent efflux. As a consequence, the cellular

3498 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Stefan´ska et al.
Ta ble 6. In Vivo Antileukemic Activity of Compound 6a toward P388 Murine Leukemia in Comparison with Mitoxantrone
compd
schedule
D1 only
total dose (mg/kg)
%T/Ca
BWCb
toxic deathsc
LTSd
6a
1.0
5.0
118
147
172
205
171
-110
145
167
196
207
215
173
174
158
229
1.05-1.06
1.01-1.04
0.98
0.90
0.88
0/12
1/18
0/12
0/6
0/6
0/6
0/12
1/18
0/18
0/6
0/6
0/12
0/6
10.0
20.0
30.0
35.0
2.5
2/6
5/6
0.73
QD1-5
0.98-1.04
1.02-1.04
0.98-1.00
0.94
0.88
0.98
1.02
1.05
1.02
5.0
10.0
20.0
22.5
0.1
0.2
0.4
1/6
mitoxantrone
D1 only
0/12
0/6
0.8
a
The ratio of average survival times of treated to control mice, expressed as a percentage. Long-term survivors were not included in
b
the calculation. Relative change in mean body weight on day 5, expressed as a fraction of pretreatment body weight (day 1). Control
mice BWC ranged from 1.07 to 1.1 in separate experiments. c Number of toxic deaths/total number of mice. Number of long-term survivors
d
(cures)/total number of mice.
Ta ble 7. Antitumor Effects of Compound 6a Administered iv
to Nude Mice Bearing sc Human Tumor Xenografts
Exp er im en ta l Section
Melting points were determined with a Boeticus PHMK05
apparatus and are uncorrected. Analyses are within (0.4% of
treatmenta
the theoretical values and were carried out on a Carlo Erba
CHNS-O-EA1108 instrument. A Beckman 3600 spectropho-
tometer was used for UV spectral determination. IR spectra
were recorded on a UR 10 Zeiss spectrometer in KBr pellets;
1H NMR spectra were taken on a Varian 300-MHz spectrom-
eter using tetramethylsilane as an internal standard. Molec-
ular weights were determined by mass spectrometry (field
desorption technique, MS-FD) on a Varian MAT 711 instru-
ment. Column chromatography was performed on silica gel
Merck (70-230 mesh) and on Sephadex LH-20 (Pharmacia).
6-Am in o-8,11-bis(p h en ylm eth oxy)-7H-ben zo[e]p er im i-
d in -7-on e (4). A sample of 0.45 g (1 mmol) of 1,4-diamino-
5,8-bis(phenylmethoxy)-9,10-anthracenedione (3),18 0.45 g of
ammonium metavanadate, and 1.5 mL of formamide in 15 mL
of N,N-dimethylacetamide was stirred under reflux for 30 min.
The course of the reaction was followed by TLC in the solvent
system toluene-acetone, 2:1. The reaction mixture was cooled,
the crude product was precipitated with cold water, and the
precipitate was collected and washed well with hot water. The
crude product was purified using silica gel column chroma-
tography (CHCl3/MeOH, 50:1) to give the pure product 4 as
an orange powder: yield 140 mg, 31%; mp 231-233 °C. 1H
NMR (CDCl3): δ 5.22 (s, 2H); 5.3 (s, 2H); 7.25 (d, 1H, J ) 9.19
Hz); 7.3-7.5 (m, 10H); 7.6-7.7 (m, 2H); 8.05 (d, 1H, J ) 9.24
Hz); 9.4 (s, 1H). MS-FD m/z (relative intensity, %): 458 ([M -
1]+, 50); 459 ([M]+, 100); 460 ([M + 1]+, 50). Anal. (C29H21N3O3)
C,H,N.
schedule dose (single)
toxic
tumor
A2780
ovarian carcinoma
A2780/Dx
ovarian carcinoma
HT29/Mx
colon carcinoma
days
(mg/kg)
%TVIb deathsc
4, 11, 18
6.7
10
32
62
46
80
7
0/4
0/4
0/5
0/4
0/4
0/4
7, 14, 21
4, 11, 18
6.7
10
14
20
22
a
Treatments started with just measurable tumors (80-100
b
mm3) in all tested models. Tumor volume inhibition percent in
treated over control tumors, assessed 3-10 days after the last
treatment. c Number of toxic deaths/total number of mice.
accumulation of benzoperimidines is not reduced in
resistant cells compared to sensitive parent lines.21
Compound 6a has also been evaluated as a substrate
toward the enzyme NADH dehydrogenase, a property
which correlates with the peroxidating ability of a
compound. The generation of toxic oxygen radicals is
thought to be responsible for the cardiotoxicity of
anthraquinone antitumor drugs. We found that 6a
exhibits low efficiency in stimulating oxygen radical
formation due to its poor enzyme substrate properties.22
The obtained results allow us to draw the following
conclusions. One can postulate that the structural
feature of the benzoperimidine class that is essential
for the appearance of high cytotoxic activity against
multidrug resistant tumor cell lines (resistance index
close to 1) is the condensed pyrimidine heterocycle.
Anthracenediones not bearing this moiety and having
the same side chains (e.g., mitoxantrone) do not exhibit
this highly desirable property. The kind of substitution
at the benzoperimidine chromophore (side chain, phe-
nolic groups) does not influence the ability of compounds
to overcome multidrug resistance but affects their
cytotoxic potency.
6-Am in o-8,11-dih ydr oxy-7H-ben zo[e]per im idin -7on e (5).
A solution of 460 mg of 4 in 5 mL of trifluoroacetic acid was
left at room temperature for 10 h. The TFA was removed under
reduced pressure by coevaporation with benzene to afford
crude 6-amino-8,11-dihydroxy-7H-benzo[e]perimidin-7-one (265
mg, 95%). An analytical sample was purified by use of column
chromatography (silica gel) using the solvent system CHCl3/
MeOH (50:1). The pure compound was obtained as a dark-
1
cherry powder: mp > 300 °C. H NMR (DMSO-d6): δ 7.2 (d,
1H, J ) 9.0 Hz); 7.35 (d, 1H, J ) 9.05 Hz); 7.65 (d, 1H, J )
8.0 Hz); 8.1 (d, 1H, J ) 8.1 Hz); 9.2 (s, 1H); 13.5 (s, 1H,
exchangeable with D2O); 13.82 (s, 1H, exchangeable with D2O).
MS-FD m/z (relative intensity, %): 279 ([M]+, 100). Anal.
(C15H9N3O3) C,H,N.
1-Am in o-4-h yd r oxy-5,8-b is(p h en ylm et h oxy)-9,10-a n -
th r a cen ed ion e (8). To a stirred solution of 2.4 g (5 mmol) of
1-hydroxy-4-nitro-5,8-bis(phenylmethoxy)-9,10-anthracenedi-
one (7) 19 in 100 mL of CHCl3/MeOH (1:1) was added 30 mg of
Pd/C (10%); then the reaction mixture was cooled to 0 °C and
treated dropwise with 15 mL (0.5 mol) of hydrazine hydrate
98% during 20 min. After additional stirring for 0.5 h at room
temperature the reaction mixture was filtered, diluted with
The overcoming multidrug resistance by benzoperi-
midine derivatives augments our suggestion, put for-
ward in the introductory part of this paper, that the
presence of a heterocyclic ring condensed with the
anthracenedione or the related acridone chromophore
determines cytotoxic activity toward the multidrug
resistant cell lines.

Synthesis of Dihydroxybenzoperimidines
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18 3499
mented with 5% FBS (fetal bovine serum), penicillin
CHCl3, and extracted with diluted HCl and water. The CHCl3
layer was dried over Na2SO4, evaporated under reduced
pressure to a small volume, and chromatographed utilizing
CHCl3/MeOH (100:1) as eluant. Crystallization from CHCl3/
petroleum ether afforded 1.7 g (80%) of 8: mp 127-130 °C.
1H NMR (CDCl3): δ 5.21 (s, 2H); 5.26 (s, 2H); 6.80 (br s,
exchangeable with D2O), 6.97 (d, 1H, J ) 9.4 Hz), 7.14 (d, 1H,
J ) 9.4 Hz), 7.2-7.6 (m, 12H), 13.7 (s, 1H, exchangeable with
D2O). Anal. (C28H21NO5) C,H,N.
G
(100 000 units/L), and streptomycin (100 mg/L). Human HL60
promyelocytic leukemia cells were grown in RPMI 1640
medium supplemented with 10% FBS. Human myelogenous
leukemia-sensitive cell line K562S and doxorubicine-resistant
subline K562R (ICIG, Villejuif, France) were grown in RPMI
1640 medium supplemented with 10% FBS, penicillin G
(100 000 units/L), streptomycin (100 mg/L), and 2 mM L-
glutamine. Human colon adenocarcinoma-sensitive cell line
LoVo and doxorubicin-resistant subline LoVo/Dx (Farmitalia
Carlo Erba, Nerviano, Italy) were grown as monolayer cultures
in Ham’s F-12 medium supplemented with 10% FBS, penicillin
G (100 000 units/L), streptomycin (100 mg/L), 1% of a 200 mM
L-glutamine solution, and 1% of a MEM vitamin solution 100X.
Human colon adenocarcinoma-sensitive cell line HT29 and
mitoxantrone-resistant subline HT29/Mx (Boehringer Man-
nheim, Monza, Italy) were grown as monolayer cultures in
McCoy’s 5A medium supplemented with 10% FBS, and 1% 1
M Hepes. Human ovarian carcinoma-sensitive cell line A2780
and doxorubicin-resistant subline A2780/Dx, human prostate
carcinoma DU145, and human prostate adenocarcinoma PC3
were grown as monolayer cultures in RPMI 1640 medium
supplemented with 10% FBS. Cell lines were grown in a
controlled (air-5% CO2) humidified atmosphere at 37 °C and
were transplanted twice a week. For the experiments the cells
in logarithmic growth were suspended in the growth medium
to give a final required density.
6-Hyd r oxy-8,11-bis(p h en ylm eth oxy)-7H-ben zo[e]p er i-
m id in -7-on e (9). A mixture of 225 mg (0.5 mmol) of 8 and 1
mL (25 mmol) of formamide was heated in 5 g of phenol for
30-40 min at 165 °C. The course of the reaction was monitored
by TLC (CHCl3/MeOH, 10:1). The reaction mixture was diluted
with CHCl3 and extracted several times with 2 N NaOH and
then with water. The CHCl3 layer was dried over Na2SO4,
evaporated to a small volume, and purified by column chro-
matography utilizing the solvent system CHCl3/MeOH (50:1).
For analytical data a sample of 9 was further purified by
additional chromatography on a silica gel column (benzene/
acetone, 10:1). The product 9 was obtained in the yield of 30
1
mg (12%): mp 225-227 °C. H NMR (CDCl3): δ 5.25 (s, 2H);
5.3 (s, 2H); 7.2 (d, 1H, J ) 9.0 Hz); 7.3-7.5 (m, 10H); 7.6-7.7
(m, 2H); 8.0 (d, 1H, J ) 9.1 Hz); 9.35 (s, 1H); 13.6 (s, 1H,
exchangeable with D2O). MS-FD m/z (relative intensity, %):
459 ([M - 1]+, 30), 460 ([M]+). Anal. (C29H20N2O4) C,H,N.
6,8,11-Tr ih ydr oxy-7H-ben zo[e]per im idin -7-on e (10). 460
mg (1 mmol) of 9 in 5 mL of trifluoroacetic acid was left at
room temperature for 20 h. The product was isolated and
purified similarly as described for 5 to give 260 mg (95%) of
the crude product 10. An analytical sample was purified on a
silica gel column utilizing the solvent system CHCl3/MeOH
2. In Vitr o Cytotoxic Eva lu a tion . Cells of required
density were seeded, and different concentrations of the drugs
were added after or without preincubation of the cells in drug-
free medium. The experiments were carried out at times
indicated in a controlled (air-5% CO2) humidified atmosphere
at 37 °C. The cytotoxic activity (IC50 values) of the compounds
were defined as their in vitro concentrations causing 50%
inhibition of cell growth after exposure to the drug, as
measured by the protein content of the cells according to the
Lowry method as described previously23 (method I) or by cell
counting with a ZbI Coulter Counter (Coulter Electronics, Ltd.,
U.K.) (method II). Results are given as the mean of at least
three independent experiments ( standard error of the mean
(SEM). The resistance index was defined as the ratio of IC50
value for the resistant cell line to IC50 value for the sensitive
cell line.
3. An titu m or Activity Eva lu a tion . Murine leukemia
P388 was maintained by ip passages of tumor cells in DBA/2
mice. Studies were conducted according to standard protocols
of the U.S. National Cancer Institute.24 For experimental
purposes, 106 cells were injected ip to CD2F1 (Balb/c × DBA/
2) mice. The medium survival time (MST) of the treated (T)
and control (C) groups was determined, and the percent of T/C
was calculated by using the following formula: %T/C ) [(MST
treated)/(MST control)] × 100. Human tumor xenografts were
maintained by sc passages of tumor fragments in Swiss nude
athymic mice (Charles River Laboratories, Calco, Italy). For
experimental purposes, tumor fragments were grafted sc in
both flanks of athymic mice. Each group included 4-5 mice
(for a total of 8-10 tumors). Tumor growth was followed by
biweekly measurements of tumor diameters. Tumor volume
(TV) was calculated according to the formula: TV (mm3) ) d2
× D/2, where d and D are the shortest and longest diameters,
respectively. Drug treatment started when mean TV was ca.
80-100 mm3.
1
(50:1): mp > 300 °C dec. H NMR (TFA): δ 7.32 (d, 2H, J )
1.4 Hz); 7.61 (d, 1H, J ) 9.4 Hz); 8.03 (d, 1H, J ) 9.4 Hz);
8.85 (s, 1H).
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
6a -g. Meth od A. 8,11-Dih yd r oxy-6-[[2-(d im eth yla m in o)-
et h yl]a m in o]-7H -b en zo[e]p er im id in -7-on e H yd r och lo-
r id e (6a ). A sample of 280 mg (1 mmol) of 6-amino-8,11-
dihydroxy-7H-benzo[e]perimidin-7-one (5) in a mixture of 6 mL
of 2-(dimethylamino)ethylamine, 2 mL of N,N,N′,N′-tetram-
ethylethylenediamine, and 0.8 mL of water was stirred and
refluxed under a nitrogen atmosphere for 5 h. The progress of
the reaction was followed by TLC (silica gel) in the solvent
system CHCl3/MeOH (10:1). The reaction mixture was diluted
with CHCl3 and washed with diluted HCl followed by water
to remove the excess of amines. The organic layer was dried
over Na2SO4 and evaporated under reduced pressure, and the
residue was chromatographed on a silica gel column using
gradient elution with CHCl3/MeOH (10:1, 5:1) and then with
CHCl3/MeOH-25% NH4OH (5:1:0.1). The pure 8,11-dihydroxy-
6-[[2-(dimethylamino)ethyl]amino]-7H-benzo[e]perimidin-7-
one (6a ) was converted into its hydrochloride salt, using an
etheral solution of hydrogen chloride, which was then crystal-
lized from MeOH/H2O (30:1), to give 6a as a dark brown
powder (150 mg, 36%): mp > 300 °C dec. UV-vis (MeOH):
λmax nm (ꢀ) 540 (17343). IR (KBr, major peaks, cm-1): 1460,
1
1565, 1620. H NMR (as free base CDCl3): δ 2.4 (s, 6H); 2.8
(t, 2H, J ) 6.5 Hz); 3.65 (q, 2H, J ) 6.5 Hz); 7.16 (d, 1H, J )
9.0 Hz); 7.3 (d, 1H, J ) 9.0 Hz); 7.5 (d, 1H, J ) 9.1 Hz); 8.0 (d,
1H, J ) 9.5 Hz); 9.1 (s, 1H); 10.8 (m, 1H, D2O exchangeable);
13.4 (s, 1H, exchangeable with D2O); 14.0 (s, 1H, exchangeable
with D2O). MS-FD m/z (relative intensity, %): 349 ([M - 1]+,
30); 350 ([M]+, 100). Anal. (C19H18N4O3‚HCl) C,H,N.
4. Assessm en t of Ap op tosis. Apoptosis was assessed by
fluorescence microscopy. After 72 h of treatment (HT29 and
HT29/Mx cell lines), floating and adherent cells were collected,
washed in PBS, and fixed in 70% ice-cold ethanol. Samples
were then stored at -20 °C until analysis (2-5 days). After
rehydratation in PBS, cells were stained with propidium iodide
solution (30 µg/mL propidium iodide and 66 units/mL RNase
A in PBS) and stored in the dark for 30 min. At least 100 cells
in two different smears were examined for their nuclear
morphology changes (condensed nuclei and/or fragmented
chromatin). The percentage of the apoptotic cells refers to the
cell number of the whole population (floating + adherent cells).
Meth od B. A sample of 10 (280 mg, 1 mmol) in 5 mL of
2-(dimethylamino)ethylamine, 5 mL of N,N,N′,N′-tetrameth-
ylethylenediamine, and 0.4 mL of water was stirred and heated
at 125° C under nitrogen for 4 h. The course of the reaction
was monitored by TLC (silica gel) in the solvent system CHCl3/
MeOH/25% NH4OH (5:1:0.1). The isolation and purification
procedures of the crude products were the same as described
in method A.
Biologica l Tests. 1. Cell Lin es. Murine L1210 lymphocytic
leukemia cells were grown in RPMI 1640 medium supple-

3500 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 18
Stefan´ska et al.
pp 201-243. (c) Klohs, W. D.; Steinkampf, R. W.; Havlick, M.
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Reversal by calcium Blockers and Calmodulin Antagonists.
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5. Cell Cycle An a lysis. Cell cycle perturbations were
studied with a FACScan flow cytometer (Becton Dickinson,
CA), equipped with an argon laser. In brief, after 72 h of
treatment fixed cells (HT29 and HT29/Mx cell lines) were
washed twice and resuspended in PBS containing 30 µg/mL
propidium iodide and 66 units/mL RNase A. At least 104 cells
were collected and evaluated for the DNA content. The cell
cycle distributions were calculated by LYSYS II software
(Becton Dickinson, CA).
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Ack n ow led gm en t. Financial support from the State
Committee for Scientific Research, Warszawa, Poland
(Grant No. PBZ 040-07), and, in part, Italian MURST
(Fondi 40%), Roma, Italy, and the Chemical Faculty
Technical University of Gdan˜sk, Poland, is gratefully
acknowledged. Part of the in vitro work was supported
by a fellowship (M.M.B.-G.) from the International
Union Against Cancer (UICC). The authors thank Dr.
V. L. Narayanan from the National Cancer Institute,
Bethesda, MD, for arranging the preliminary in vitro
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