Aromatic analogues of DNA minor groove binders - Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2003.11.005

Nine carbocyclic analogues of mono- and bis-lexitropsins and two analogues of pentamidine with unsubstituted N-terminal amine group were synthesized. We have investigated the cytotoxic activity of new aromatic analogues of DNA binding ligands in MCF-7 breast cancer cells and assessed their ability to act as inhibitors of topoisomerase I and II. These studies indicate that aromatic analogues of bis-netropsin contain two identical units tethered by alkyloxyl chains are a potent catalytic inhibitor of both topoisomerases and exhibit moderate cytotoxicity in MCF-7 breast cancer cells.

Full text of DOI:10.1016/j.ejmech.2003.11.005

European Journal of Medicinal Chemistry 39 (2004) 99–105
www.elsevier.com/locate/ejmech
Short communication
Aromatic analogues of DNA minor groove binders—
synthesis and biological evaluation
Anna Puc´kowska a,*, Krzysztof Bielawski b, Anna Bielawska b, Krystyna Midura-Nowaczek a
a Department of Organic Chemistry, Medical University of Białystok, A. Mickiewicza 2a, 15-222 Bialystok, Poland
b Department of Medicinal Chemistry and Drug Technology, Medical University of Białystok, A. Mickiewicza 2a, 15-222 Bialystok, Poland
Received 15 July 2003; received in revised form 13 November 2003 accepted 13 November 2003
Abstract
Nine carbocyclic analogues of mono- and bis-lexitropsins and two analogues of pentamidine with unsubstituted N-terminal amine group
were synthesized. We have investigated the cytotoxic activity of new aromatic analogues of DNA binding ligands in MCF-7 breast cancer cells
and assessed their ability to act as inhibitors of topoisomerase I and II. These studies indicate that aromatic analogues of bis-netropsin contain
two identical units tethered by alkyloxyl chains are a potent catalytic inhibitor of both topoisomerases and exhibit moderate cytotoxicity in
MCF-7 breast cancer cells.
© 2003 Elsevier SAS. All rights reserved.
Keywords: Netropsin; Bis-netropsin; DNA topoisomerase; Cytotoxicity; Minor groove binding
1. Introduction
Netropsin, Hoechst 33258 and pentamidine (Fig. 1) have
long served as model compounds for biochemical and bio-
physical studies of drugs that bind to the DNA minor groove.
Several high resolution crystal and NMR structures have
been determined for these ligands bound to short oligonucle-
otides and provide valuable insights into the details of their
interaction [1,2]. The typical binding sites for these minor
groove binding drugs are four consecutive AT base pairs,
where a particularly narrow groove with a floor lacking
amino groups permits an optimization of van der Waals’
contacts and hydrogen bonding [1,2]. Groove binding typi-
cally exerts only subtle changes in DNA conformation, and
DNA remains essentially in the native form. The discovery of
the details of minor groove binding of these compounds let to
the synthesis of sequence-reading molecule based on them
[1,2].
Recently published data suggest that DNA-binding drugs
might exert their biological effects by interfering with DNA–
protein interactions [1–3]. A number of minor groove bind-
Fig. 1. Structure of Hoechst 33258, netropsin and pentamidine.
ing drugs are dual inhibitors of topoisomerase I and II. These
data suggest that these topological enzymes read DNA struc-
ture at least in part through the minor groove [4]. These
enzymes are crucial for cellular genetic processes, such as
* Corresponding author.
E-mail address: pucanka@amb.edu.pl (A. Puc´kowska).
© 2003 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2003.11.005

100
A. Puc´kowska et al. / European Journal of Medicinal Chemistry 39 (2004) 99–105
experimental conditions [9]. Compounds 2 and 3 were syn-
thesized with good yields by standard chemical transforma-
tions described for compound 1 [5]. The starting material
N-[5-amino-2-methoxy]benzoyl-N′,N′-dimethylpropyl-1,3-
diamine dihydrochloride [5] was condensed with 3-nitro- or
4-nitrobenzoyl chloride respectively, and the obtained prod-
ucts were hydrogenated to aromatic amines 2 and 3. Com-
pound 4 was prepared from 4-nitrobenzoyl chloride by fol-
lowing steps: condensation with N,N-dimethylpropyl-1,3-
diamine, hydrogenation of nitro group, condensation of
obtained amine with (2-methoxy-5-phenyldiazenyl)benzoyl
chloride and hydrogenation of diazenyl group. Compounds
7–12 were prepared like compounds 1–3 from 1,4-bis{4-
amino-2-[(1,5-diaza-5-methylhexyl)carbonyl]phenyloxy}
butane (5) [6] or 1,6-bis{4-amino-2-[(1,5-diaza-5-methyl-
hexyl)carbonyl]phenyloxy}hexane (6). Compound 6 was
obtained according to the procedure described for 5 from
methyl [2-hydroxy-5-phenyldiazenyl]benzoate [6]. All com-
pounds were prepared as hydrochloride salts.
3. Pharmacology
3.1. Cytotoxic activity
Fig. 2. Structure of compounds 1–12.
We studied the effect of compounds 1–12 and Hoechst
33258 on DNA synthesis in human MCF-7 breast cancer
cells (Table 1). Hoechst 33258 was shown to possess activity
against L1210 murine leukemia and several positive studies
in various solid tumors led to the compound being entered
into phase I clinical trials in humans, but toxicity precluded
further advanced trials [10]. This compound was shown to
inhibit binding of TATA-box binding protein to DNA, and
also to be an effective inhibitor of mammalian DNA topoi-
somerase I and II [1,2]. Measurement of [3H]thymidine in-
corporation during DNA synthesis by proliferating MCF-7
breast cancer cells showed that compounds 1–12 inhibited
DNA synthesis in a dose-dependent manner. Among these
derivatives, 5, 6, 8, 10, 11 and 12 proved to be slightly less
potent than Hoechst 33258, with IC50 values of 185 2 µM,
193 2 µM, 57 2 µM, 159 2 µM, 132 2 µM and
182 2 µM, respectively, compared to 53 µM for Hoechst
33258. In contrast, benzene derivatives of netropsin 1–4, and
compounds 7 and 9 are clearly much less active and showed
a very low level of cytotoxic potency (IC50 > 250 µM).
DNA replication, transcription, recombination, and chromo-
some segregation at mitosis [4].
In the course of our investigations of minor groove bind-
ing drugs [5–8], we reported a synthesis and cytotoxicity of
12 aromatic analogues of DNA binding ligands 1–12 (Fig. 2)
in cultured breast cancer MCF-7 cells. Compounds 1–4 are
benzene derivatives of netropsin. Compounds 7–12, in which
tetra- or hexamethylene chains linked two identical units of
1, 2 or 3, are analogues of bis-netropsin. Compounds 5 and 6
are similar to aromatic bis-amidines. We also investigated the
mode of action of compounds 1–12 in a cell-free system by
employing the topoisomerase I/II inhibition assay.
2. Chemistry
The benzene derivatives of lexitropsins are readily avail-
able; they can be easily modified, and are stable under most
Table 1
Cytotoxic effects of 1–12 and Hoechst 33258 on the cultured breast cancer cells as measured by inhibition of [3H]thymidine incorporation into DNA
Concentration
(µM)
[3H]Thymidine incorporation (% of control) a
Hoechst 33258
100
1
2
3
4
5
6
7
8
9
10
100
74
63
53
51
47
11
12
100
95
87
70
60
45
0
100
98
92
87
75
68
100
97
91
89
85
82
100
92
82
78
68
54
100
93
84
79
74
67
100
84
82
80
70
49
100
97
90
84
69
48
100
95
81
79
78
65
100
85
51
43
35
30
100
88
74
71
70
64
100
96
75
66
42
38
25
78
52
25
11
2
2
2
2
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
50
100
150
200
1
a Mean values
S.D. of three independent experiments done in duplicates are presented.

A. Puc´kowska et al. / European Journal of Medicinal Chemistry 39 (2004) 99–105
101
Table 2
Viability of MCF-7 cells treated for 24 h with different concentrations of 1-12 and Hoechst 33258
Concentration
(µM)
Viability of cells (% of control) a
Hoechst 33258
100
1
2
3
4
5
6
7
8
9
10
100
94
86
72
63
58
11
12
100
91
83
65
59
53
0
100
98
97
94
89
82
100
98
88
72
70
69
100
98
89
82
78
72
100
99
92
84
77
75
100
83
78
76
73
69
100
86
83
80
79
68
100
92
88
84
82
78
100
92
78
59
12
11
100
98
94
91
83
75
100
86
83
62
37
10
25
75
67
49
35
24
2
2
2
2
1
2
2
1
1
2
1
2
1
2
1
1
2
3
2
1
2
3
2
2
1
2
1
1
1
1
2
2
1
3
2
1
2
1
2
1
1
2
1
2
1
2
1
1
2
2
1
2
3
2
1
1
2
2
1
2
1
2
3
2
1
50
100
150
200
a Mean values
S.D. of 3 independent experiments done in duplicates are presented.
Cell viability of breast cancer MCF-7 cells was measured
by the method of Carmichael et al. [11] using tetrazolium salt
(Table 2). In terms of reduction in cell viability, the com-
pounds rank in the order Hoechst 33258 >8 >11. The concen-
trations of 11, 8, and Hoechst 33258 needed to 50% reduc-
tion in cell viability in breast cancer MCF-7 (IC50) was found
to be 124 2 µM, 108 2 µM, and 96 2 µM, respectively.
4. Results and discussion
The topoisomerases induce transient cuts in DNA, which
enable the strands to pass through the nicks, and then rejoin
the nicked strands to DNA. During this process, covalent
links are formed between topoisomerase and DNA, which
are called ‘cleavable complexes’ [4]. The topo-targeting
drugs can be classified as either topo poisons, which act by
stabilizing enzyme-DNA cleavable complexes leading to
DNA breaks, or topo catalytic inhibitors, which act at stages
in the catalytic cycle of the enzyme where DNA strands
remain intact and no DNA strand breaks occur [13,14].
Treatment with the classical topo I or II poison (camptoth-
ecin and etoposide, respectively) results in the production of
linear DNA which demonstrates that these compounds stabi-
lize DNA–topo covalent complexes and hence stimulate a
single or double-strand cleavage by the enzymes. Con-
versely, with 5–12 no band corresponding to linear DNA was
detected in the presence of enzyme (data not shown), imply-
ing that these compounds do not act as topo poisons. It is
likely that the ability of compounds 5–12 to inhibit the
activity of topo I and II that we have observed (Table 3) is
simply due to blockade of the binding of these enzymes to
DNA. The increasing the total size occupied by the com-
pounds 5–12 compared to 1–4 could afford greater opportu-
nity for inhibiting topoisomerase activity. It is possible that
compounds 5–12 acts through inducing a conformational
change in the DNA and hindering the formation of the cleav-
able complex. The flexible nature of tetra- or hexamethylene
chains permits the compounds 5–12 to take an optimum
conformation, thus binding effectively to double-stranded
DNA.
3.2. Relaxation assay of topoisomerase I and II
The mechanism of antitumor action of compounds 7–12
may be due to their ability to inhibit the binding of some
transcription factors to their consensus sequences in DNA,
thereby preventing transcription. Several major forms of hu-
man cancer express high levels of topoisomerase enzymes
[12]. To test whether cytotoxic properties were related to
topoisomerase I/II inhibition, the aromatic analogues of
DNA binding ligands 1–12 were evaluated in a cell-free
system.
Purified topoisomerases I and II were incubated with
increasing concentrations (range 10–100 µM) of compounds
1–12 and Hoechst 33258 in the presence of supercoiled DNA
plasmid PBR322. The products of this reaction were sub-
jected to electrophoresis in the presence of ethidium bromide
in order to separate closed and open circular DNA. Compari-
son of 1–12 with Hoechst 33258 as inhibitors of topoi-
somerase I and II demonstrated that several of these com-
pounds had similar potency (Table 3). None of the
compounds 1–4 inhibited the topoisomerases mediated re-
laxation of supercoiled DNA at a concentration of 100 µM.
As shown in Table 3 compounds 5–12 showed potent inhibi-
tory activity on topo I and topo II with concentrations ranging
from 30 to 50 µM. They exhibited similar potency to Hoechst
33258 as topoisomerase I and II inhibitors (Table 3).
Evaluation of the topoisomerases inhibition and cytotox-
icity of 1–12 provided additional insight into the structure–
activity relationships associated with these compounds. Sev-
eral of compounds that were among the more potent
Table 3
Topoisomerase I/II inhibitory effect of compounds 5–12
Enzyme
Concentration (µM) a
Hoechst 33258
5
50
50
6
30
30
7
30
40
8
40
40
9
40
40
10
30
40
11
30
30
12
40
50
Topo I
30
40
Topo II
a The concentrations of the inhibitor that prevented of the supercoiled DNA from being converted into relaxed DNA were determined by averaging the data
from at least three experiments.

102
A. Puc´kowska et al. / European Journal of Medicinal Chemistry 39 (2004) 99–105
topoisomerase I and II inhibitors did exhibit greater cytotox-
icity toward human MCF-7 breast cancer cells. Compounds
1–4 were not a topoisomerase I and II inhibitors and did not
possess cytotoxic activity. The fact that compounds 8 and 11
are more potent than 9 and 12 indicates that cytotoxicity in
MCF-7 cultured breast cancer cells is also sensitive to the
position of the terminal cationic amine groups of the com-
pounds. The cytoxicity observed for 7 and 10 in comparison
to 8 and 11 suggest that steric factors associated with meth-
oxy groups may moderate influence the activity of similar
compounds. Among the compounds evaluated, 7 and 9 were
active as topoisomerase inhibitors, but did not exhibit signifi-
cant cytotoxicity. It should be noted that other factors such as
low penetration into cell, cellular distribution and metabolic
deactivation may also influence the cytoxicity results, but
they are not assessed in the present study.
ture of hexane and acetone (1:2) with 10–40% addition of
methanol.
Catalytic hydrogenation of nitro or diazenyl groups was
carried out in methanol (with addition of HClaq, pH 4) in the
presence of Pd/C (10%). The obtained aromatic amines were
purified by column chromatography using the solvent system
as follow: (1) C6H6/C2H5OH 1:1; (2) CH3OH. Acylations of
amine groups, hydrogenations of nitro or diazenyl groups,
and the purity of final compounds were controlled by TLC
(1% NH3 in CH3OH). The identification of all the compounds
was made by UV and by spraying of chromatograms with
solution of DMAB (4-dimethylaminobenzaldehyde—1 g,
36% HCl—30 ml, EtOH—10 ml, n-ButOH—180 ml). Thin-
layer chromatograms were prepared on precoated plates
(Merck, silica gel 60 F254).
The structures of all the compounds were confirmed by 1H
and 13C NMR spectra recorded on Brucker AC 200F appara-
tus (1H—200 MHz and 13C—50 MHz) in D6-DMSO. Melt-
ing points were determined on Büchi 535 melting-point ap-
paratus and were uncorrected. Elemental analysis of C, H,
and N was performed on a Perkin Elmer 240 analyser, while
Cl was determined by Shuniger method, and satisfactory
results within 0.4% of calculated values were obtained.
5. Conclusions
In this study, we have investigated the cytotoxic activity of
new aromatic analogues of DNA binding ligands 1–12 in
MCF-7 breast cancer cells and assessed their ability to act as
inhibitors of topoisomerases I and II. Among these deriva-
tives, compound 8 proved to be only slightly less potent than
Hoechst 33258, with IC50 value for DNA synthesis of 57 µM,
compared to 53 µM for Hoechst 33258.
6.1.1.
N-[5-(5-Amino-2-methoxy)benzamido-2-methoxy]
benzoyl-N′,N′–dimethylpropyl-1,3-diamine dihydrochloride
(1)
Recent in vitro studies indicate that the inhibitory activity
of minor groove binders as distamycin, netropsin, Hoechst
33258, etc., are rather limited to proteins, which reside
within the minor groove [1–3]. We have shown in the present
report that compounds 5–12 are a potent catalytic inhibitor of
both topoisomerase I and II. These compounds inhibit the
catalytic activity of the topoisomerase at a step prior to the
formation of the topo–DNA complex. This suggests that
DNA-binding may be implicated in the cytotoxicity of com-
pounds 5–12 possibly by inhibiting interactions between
topoisomerases and their DNA targets. Moreover, there
might be other possible targets, such as other enzymes, in-
volved in DNA metabolism and/or transcription factors be-
cause their activities were inhibited by some DNA minor
groove binders [1–3]. Further biological evaluation is under-
way and these results, DNA binding studies and sequence
selective protection of restriction enzyme recognition sites
by described compounds will be described in due course.
This compound was obtained as described earlier [5].
6.1.2.N-[5-(3-Amino)benzamido-2-methoxy]benzoyl-N′,N′–
dimethylpropyl-1,3-diamine dihydrochloride (2)
1
Yield 51%. M.p.: 132–133 °C. Rf = 0.34. H NMR:
1.89–1.96 (m, 2H, CH2), 2.72–2.75 (d, J = 4.3, 6H,
(CH3)2NH+), 3.06–3.08 (m, 2H, CH2NR), 3.32–3.41 (m, 2H,
CONHCH2), 3.89 (s, 3H, OCH3), 7.12–7.17 (d, J = 9.0, 1H,
C-3), 7.44–7.60 (m, 2H, H-4′, H-5′), 7.79–7.93 (m, 3H, H-4,
H-2′, H-6′), 8.14–8.15 (d, J = 2.63, 1H, H-6), 8.39–8.45 (t,
J = 5.7, 1H, ArCONHR), 10.45 (s, 2H, ArCONHAr, H+),
13C NMR: 24.19 (CH2), 36.32 (CONHCH2), 42.00
((CH3)2NH+), 54.40 (CH2NR), 56.18 (OCH3), 112.21 (C-3),
121.07 (C-6), 122.78 (C-2′), 122.97(C-1), 124.50 (C-4),
124.67 (C-4′), 124.98 (C-3′), 129.64 (C-6′), 132.01 (C-5′),
135.37 (C-5), 136.07 (C-1′), 153.35 (C-2), 164.42 (Ar-
CONHR), 165.09 (ArCONHAr). Anal. C20H26N4O3·2HCl
(C, H, N, Cl).
6.1.3. N-[5-(4-Amino)benzamido-2-methoxy]benzoyl-N′,N′-
dimethylpropyl-1,3-diamine dihydrochloride (3)
6. Experimental protocols
1
Yield 57%. M.p.: 140–142 °C, Rf = 0.32. H NMR:
6.1. Chemistry
1.89–1.97 (m, 2H, J = 9, CH2), 2.72–2.74 (d, J = 4.6, 6H,
(CH3)2NH+), 3.01–3.06 (m, 2H, CH2N), 3.32–3.41 (m, 2H,
CONHCH2), 3.89 (s, 3H, OCH3), 7.10–7.14 (d, J = 9, 1H,
H-3), 7.23–7.27 (d, J = 8.4, 2H, H-3′, H-5′), 7.88–7.94, (dd,
J = 2.6, J = 8.9, 1H, H-4), 7.97–8.01 (d, J = 8.4, 2H, H-2′,
H-6′), 8.13–8.15 (d, J = 2.6, 1H, H-6), 8.37–8.42 (t, J = 5.6,
1H, ArCONHR), 10.26 (s, 1H, ArCONHAr), 10.66 (bs, 1H,
Acylations of starting aromatic amines by acid chlorides
were carried out at pyridine/CH2Cl2 solution, in catalytic
presence of 4-(dimethylamino)pyridine (DMAP). The prod-
ucts of these reactions were purified by column chromatog-
raphy (silica gel 60, Merck, 230–400 mesh), using the mix-

A. Puc´kowska et al. / European Journal of Medicinal Chemistry 39 (2004) 99–105
103
H+). 13C NMR: 24.12 (CH2), 36.29 (CONHCH2), 41.95
(N(CH3)2), 54.34 (CH2N), 112.12 (C-3), 119.82 (C-6),
122.73 (C-1), 122.84 (C-3′, C-5′), 124.35 (C-4), 129.17 (C-
2′, C-6′), 130.10 (C-5), 132.28 (C-4′), 140.36 (C-1′), 153.09
(C-2), 164.51 (ArCONHR), 165.10 (ArCONHAr). Anal.
C20H26N4O3·2HCl (C, H, N, Cl).
121.89 (C-3), 123.82 (C-2), 124.41 (C-5′), 125.42 (C-1′),
126.84 (C-6′), 127.18 (C-5), 131.83 (C-4), 145.72 (C-4′),
152.32 (C-1), 155.87 (C-2′), 163.06 (ArCONHR),
165.40ArCONHAr). Anal. C44H58N8O8·4HCl (C, H, N, Cl).
6.1.8.
1,4-Bis{4-[(3-amino)benzamido]-2-[(1,5-diaza-5-
methylhexyl)carbonyl]-phenyloxy}butane bis(dihydrochlo-
ride) (8)
6.1.4. N-[4-(5-Amino-2-methoxybenzamido]benzoyl-N′,N′-
dimethylpropyl-1,3-diamine dihydrochloride (4)
Yield 38%. M.p.: 180–182 °C. Rf = 0.2. 1H NMR: 1.87–
2.03 (m, 4H, CH2), 2.70 (s, 6H, (CH3)2N), 2.88–3.05 (m, 2H,
CH2N), 4.16–4.25 (m, 2H, OCH2), 6.73–6.77 (d, J = 8.48,
1H, H-6), 7.11–7.18 (m, 4H, Ar′), 7.82–7.88 (dd, J = 2.51,
J = 8.96, 1H, H-5), 8.08–8.09 (d, J = 2.57, 1H, H-3), 8.27–
8.30 (t, J = 5.70, 1H, ArCONHR), 10.12 (s, 1H, ArCON-
HAr), 10.84 (bs, 1H, H+). 13C NMR: 24.19 (CH2), 25.28
(CH2), 36.33 (CONHCH2), 41.87 ((CH3)2N), 54.31 (CH2N),
68.45 (OCH2), 113.03 (C-6), 113.29 (C-2′), 114.74 (C-6′),
116.79 (C-3), 122.44 (C-4′), 123.49 (C-5), 124.01 (C-2),
128.72 (C-5′), 132.46 (C-4), 135.65 (C-1′), 148.68 (C-3′),
152.06 (C-1), 165.41 (ArCONHR), 166.01 (ArCONHAr).
Anal. C42H54N8O6·4HCl (C, H, N, Cl).
Yield 51%. M.p.: 178–180 °C. Rf = 0.3. 1H NMR: 1.90–
1.97 (m, 2H, CH2), 2.73 (s, 6H, (CH3)2N), 3.05–3.12 (m, 2H,
CH2N), 3.28–3.38 (m, 2H, CONHCH2), 3.92 (s, 3H, OCH3),
7.27–7.32 (d, J-8.9, 1H, H-3′), 7.52–7.58 (dd, J = 2.7, J = 8.8,
1H, H-4′), 7.66–7.68 (d, J = 2.7, 1H, H-6′), 7.78–7.83 (d,
J = 8.7, 2H, H-3, H-5), 7.89–7.93 (d, J = 8.7, 2H, H-2, H-6),
8.68–8.74 (t, J = 5.8, 1H, ArCONHR), 10.43 (s, 1H, ArCON-
HAr), 10.62 (bs, 1H, H+). 13C NMR: 24.19 (CH2), 36.29
(CONHCH2), 41.91 ((CH3)2N), 54.43 (CH2N), 56.43
(OCH3) 113.29 (C-3′), 118.93 (C-3, C-5), 124.26 (C-6′),
124.78 (C-5′), 125.25 (C-1′), 126.81 (C-2, C-6), 128.08 (C-
4′), 129.21 (C-1), 141.26 (C-4), 155.68 (C-2′), 163.55 (Ar-
CONHR), 165.76 (ArCONHAr). Anal. C20H26N4O3·2HCl
(C, H, N, Cl).
6.1.9.
1,4-Bis{4-[(4-amino)benzamido]-2-[(1,5-diaza-5-
methylhexyl)carbonyl]-phenyloxy}butane bis(dihydrochlo-
ride) (9)
6.1.5. 1,4-Bis{4-amino-2-[(1,5-diaza-5-methylhexyl)carbo-
nyl]phenyloxy}butane bis(dihydrochloride) (5)
Yield 41%. M.p.: 167–169 °C, Rf = 0.2. 1H NMR: 1.91–
2.15 (m, 4H, 2 × CH2), 2.70–2.72 (d, J = 4.55, 6H,
(CH3)2NH+), 3.06–3.16 (m, 2H, CH2N), 3.35–3.38 (m, 2H,
CONHCH2), 4.07–4.32 (m, 2H, OCH2), 7.16–7.20 (d,
J = 8.32, 3H, H-6, H-3′, H-5′), 7.87–7.94 (m, 3H, H-5, H-2′,
H-6′), 8.09–8.11 (d, J = 2.6, 1H, H-3), 8.27–8.32 (t, J = 5.9,
1H, ArCONHR), 10.22 (s, 1H, ArCONHAr), 10.72 (bs, 1H,
H+).13C NMR: 24.19 (CH2), 25.27 (CH2), 36.29 (CON-
HCH2), 41.88 ((CH3)2N), 54.33 (CH2N) 68.45 (OCH2),
113.29 (C-6), 119.08 (C-3), 122.52 (C-3′, C-5′), 123.45 (C-
2), 124.10 (C-5), 129.04 (C-4) 129,16 (C-2′, C-6′), 132.37
(C-4′), 141.50 (C-1′), 152.09 (C-1), 163.53 (ArCONHR),
165.38 (ArCONHAr). Anal. C42H54N8O6·4HCl (C, H, N,
Cl).
This compound was obtained as described earlier [6].
6.1.6. 1,6-Bis{4-amino-2-[(1,5-diaza-5-methylhexyl)carbo-
nyl]phenyloxy}hexane bis(dihydrochloride) (6)
1
Yield 45%. M.p.: 214–215 °C, Rf = 0.22. H NMR:
1.47–1.59 (m, 2H, CH2), 1.82–1.91 (m, 4H, 2XCH2), 2.72 (s,
6H, (CH3)2N), 3.02–3.05 (m, 2H, CH2N), 3.33–3.50 (m, 2H,
CONHCH2), 4.04–4.14 (m, 2H, OCH2), 7.18–7.19 (d,
J = 8.9, 1H, H-6), 7.45–7.51 (dd, J = 2.5, J = 8.8, 1H, H-5),
7.71–7.73 (d, J = 2.6, 1H, H-3), 8.31–8.37 (m, 1H, Ar-
CONHR), 10.75 (bs, 1H, H+). 13C NMR: 24.12 (CH2), 25.04
(CH2), 28.19 (CH2), 36.43 (CONHCH2), 41.92 ((CH3)2N),
54.29 (CH2N) 68.91 (OCH2), 115.54 (C-6), 124.25 (C-4),
124.61 (C-2), 126.70 (C-3), 129.52 (C-5), 155.35 (C-1),
164.43 (ArCONHR). Anal. C30H48N6O4·4HCl (C, H, N, Cl).
6.1.10.
1,6-Bis{4-[(5-amino-2-methoxy)benzamido]-2-
[(1,5-diaza-5-methylhexyl)-carbonyl]phenyloxy}hexane bis
6.1.7. 1,4-Bis{4-[(5-amino-2-methoxy)benzamido]-2-[(1,5-
diaza-5-methylhexyl)-carbonyl]phenyloxy}butane bis(dihy-
drochloride) (7)
(dihydrochloride) (10)
1
Yield 44%. M.p.: 165–167 °C, Rf = 0.19. H NMR:
1.42–1.52 (m, 2H, CH2), 1.83–1.91 (m, 4H, 2 × CH2), 2.73
(s, 6H, (CH3)2N), 3.06–3.18 (m, 2H, CH2N), 3.91 (s, 3H,
OCH3), 4.11–4.18 (m, 2H, OCH2), 6.95–6.99 (d, J = 8.81,
1H, H-6), 7.13–7.17 (d, J = 8.99, 1H, H-3′), 7.48–7.54, (m,
1H, H-5), 7.63–7.65 (d, J = 2.6, 1H, H-3), 7.78–7.83 (m, 1H,
H-4′), 8.04–8.06 (d, J = 2.53, 1H, H-6′), 8.31–8.40 (m, 1H,
ArCONHR), 10.19 (s, 1H, ArCONHAr), 10.58 (bs, 1H, H+).
13C NMR: 24.26 (CH2), 25.17 (CH2), 28.43 (CH2), 36.31
(CONHCH2), 41.95 ((CH3)2N), 54.37 (CH2N), 56.43
(OCH3), 68.76 (OCH2), 113.26 (C-6), 114.13 (C-3′), 122.38
(C-3), 123.89 (C-2), 124.26 (C-5′), 124.58 (C-1′), 125.01
(C-6′), 127.42 (C-5), 131.81 (C-4), 139.72 (C-4′), 152.41
1
Yield 51%. M.p.: 161–163 °C, Rf = 0.15. H NMR:
1.88–1.99 (m, 4H, 2 × CH2), 2.71 (s, 6H, (CH3)2N), 3.06–
3.18 (m, 2H, CH2N), 3.34–3.37 (m, 2H, CONHCH2) 3.91 (s,
3H, OCH3), 4.15–4.24 (m, 2H, OCH2), 7.15–7.20 (d, J = 9.0,
1H, H-6), 7.26–7.31 (d, J = 8.75, 1H, H-3′), 7.52–7.57, (dd,
J = 2.33, J = 8.75, 1H, H-5), 7.67–7.68 (d, J = 2.26, 1H, H-3),
7.78–7.83 (d, J = 8.7, 1H, H-4′), 8.02–8.03 (d, J = 2.28, 1H,
H-6′), 8.31–8.34 (m, 1H, ArCONHR), 10.20 (s, 1H, ArCON-
HAr), 10.81 (bs, 1H, H+). 13C NMR: 24.19 (CH2), 25.27
(CH2), 36.37 (CONHCH2), 41.93 ((CH3)2N), 54.36 (CH2N),
56.46 (OCH3), 68.53 (OCH2), 113.25 (C-6), 113.52 (C-3′),

104
A. Puc´kowska et al. / European Journal of Medicinal Chemistry 39 (2004) 99–105
(C-1), 155.68 (C-2′), 163.02 (ArCONHR), 165.29 ArCON-
6.2.2. MCF-7 cell culture
HAr). Anal. C46H62N8O8·4HCl (C, H, N, Cl).
Stock cultures of breast cancer MCF-7 cells were main-
tained in continuously exponential growth by weekly pas-
sage in DMEM supplemented with 10% FBS, 50 µg/ml
penicillin, 50 µg/ml streptomycin at 37 °C in humid atmo-
sphere containing 5% CO2 in an incubator. Cells were cul-
tured in Costar flasks and subconfluent cells were detached
with 0.05% trypsin, 0.02% EDTA in calcium-free phosphate-
buffered saline. The study was carried out using cells from
passages three to seven, growing as monolayer in 6-well
plates (Nunc) (5 × 106 cells per well).
6.1.11.
1,6-Bis{4-(3-amino)benzamido-2-[(1,5-diaza-5-
methylhexyl)carbonyl]-phenyloxy}hexane
bis(dihydrochloride) (11)
1
Yield 35%. M.p.: 182–184 °C. Rf = 0.25. H NMR:
1.45–1.60 (m, 2H, CH2), 1.88–1.92 (m, 4H, CH2), 2.72–2.74
(d, J = 3.43, 6H, (CH3)2NH+), 3.05–3.18 (m, 2H, CON-
HCH2), 3.35–3.38 (m, 2H, CH2N), 4.05–4.17 (m, 2H,
OCH2), 7.13–7.18 (d, J = 9.03, 1H, H-6), 7.33–7.37 (m, 1H,
H-4′), 7.45–7.53 (m, 1H, H-5′), 7.70 (s, 1H, H-2′), 7.77–7.81
(m, 1H, H-6′), 7.88–7.92 (dd, J = 2.57, J = 8.91, 1H, H-5),
8.18–8.28 (d, J = 2.61, 1H, H-3), 8.29–8.34 (t, J = 5.58, 1H,
ArCONHR), 10.41 (s, 1H, ArCONHAr), 10.84 (bs, 1H, H+).
13C NMR: 24.19 (CH2), 25.28 (CH2), 28.41 (CH2), 36.32
(CONHCH2), 41.94 ((CH3)2N), 54.35 (CH2N), 68.72
(OCH2), 113.28 (C-6), 119.89 (C-2′), 123.17 (C-6′), 123.42
(C-3), 123.47 (C-4′), 124.33 (C-5), 124.44 (C-2), 129.45
(C-5′), 132.06 (C-4), 135.96 (C-1′), 137.48 (C-3′), 152.45
(C-1), 164.64 (ArCONHR), 165.27 (ArCONHAr). Anal.
C44H58N8O6·4HCl (C, H, N, Cl).
6.2.3. DNA synthesis assay
To examine the effect of studied compounds on cells
proliferation MCF-7 cells were seeded in 6-well plates and
grown as described above. Cells culture were incubated with
varying concentrations of compounds 1–12, Hoechst
33258 and 0.5 µC of [3H]thymidine for 24 h at 37 °C. The
cells were then harvested by trypsinization and washed (with
cold phosphate-buffered saline) with centrifugation for
10 min at 1500 g several times (4–5) until the dpm in the
washes were similar to the reagent control. Radioactivity was
determined by liquid scintillation counting. [3H]Thymidine
uptake was expressed as dpm/well.
6.1.12.
1,6-Bis{4-(4-amino)benzamido-2-[(1,5-diaza-5-
methylhexyl)carbonyl]-phenyloxy}hexane
bis(dihydrochloride) (12)
Yield 40%. M.p.: 178–180 °C, Rf = 0.3. 1H NMR: 1.41–
1.59 (m, 2H, CH2), 1.83–1.92 (m, 4H, 2 × CH2), 2.72 (s, 6H,
(CH3)2N), 3.06–3.19 (m, 2H, CH2N), 3.35–3.40 (m, 2H,
CONHCH2), 4.05–4.20 (m, 2H, OCH2), 7.11–7.15 (d,
J = 8.6, 1H, H-6), 7.24–7.28 (d, J = 8.0, 2H, H-3′, H-5′),
7.87–7.91 (d, J = 8.7, 1H, H-5) 7.97–8.01 (d, J = 8.0, 2H,
H-2′, H-6′), 8.11 (s, 1H, H-3), 8.22–8.26 (m, 1H, Ar-
CONHR), 10.28 (s, 1H, ArCONHAr), 10.69 (bs, 1H,
ArCONHAr).13C NMR: 24.21 (CH2), 25.12 (CH2), 28.38
(CH2), 36.29 (CONHCH2), 41.91 ((CH3)2N), 54.34 (CH2N),
68.71 (OCH2), 113.21 (C-6), 120.01 (C-3), 122.66 (C-3′,
C-5′), 123.20 (C-2), 124.25 (C-5), 129.16 (C-2′, C-6′),
130.18 (C-4), 132.23 (C-4′), 139.99 (C-1′), 152.27 (C-1),
164.46 (ArCONHR), 165.29 (ArCONHAr). Anal.
C44H58N8O6·4HCl (C, H, N, Cl).
6.2.4. Cell viability assay
The assay was performed according to the method of
Carmichael using 3-(4,5-dimethylthiazole-2-yl)-2,5-di-
phenyltetrazolium bromide (MTT) [11]. Confluent cells, cul-
tured for 24 h with various concentrations of studied com-
pounds in 6-well plates were washed three times with PBS
and then incubated for 4 h in 1 ml of MTT solution
(0.5 mg/ml of PBS) at 37 °C in 5% CO2 in an incubator. The
medium was removed and 1 ml of 0.1 mol/l HCI in absolute
isopropanol was added to attached cells. Absorbance of con-
verted dye in living cells was measured at a wavelength of
570 nm. Cell viability of breast cancer MCF-7 cells cultured
in the presence of ligands was calculated as a percent of
control cells.
6.2.5. Relaxation assay of topoisomerase I
6.2. Pharmacology
PBR322 plasmid DNA (0.083 µg) was incubated with
1 unit of topoisomerase I in a standard reaction buffer
(50 mM Tris–HCI (pH 7.9),1 mM EDTA, 0.5 M NaCI, 1 mM
dithiothreitol) in the presence of the test compound at con-
centrations ranging from 10 to 100 µM. The mixture was
incubated at 37 °C for 1 h and the reaction was terminated by
addition of 2 µl of 10% SDS and 2 µl of proteinase K
(1 mg/ml). The reaction mixture was subjected to electro-
phoresis through a 0.8% agarose gel containing 0.5 mg/ml
ethidium bromide in TBE buffer (90 mM Tris–borate and
2 mM EDTA). The gels were stained with ethidium bromide
and photographed under UV light using UVI-KS4000i gel
documentation and analysis system (SyngenBiotech, San
Carlos, CA, USA).
6.2.1. Materials
Hoechst 33258, topoisomerase I, ethidium bromide,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) were purchased from Sigma Chemical Co.
(USA). Topoisomerase II was purchased from Amersham
Pharmacia Biotech. Stock cultures of breast cancer MCF-7
were purchased from the American Type Culture Collection,
Rockville, MD. Dulbecco’s minimal essential medium
(DMEM) and fetal bovine serum (FBS) used in cell culture
were products of Gibco (USA). Glutamine, penicillin and
streptomycin were obtained from Quality Biologicals Inc.
(USA). [3H]Thymidine (6.7 Ci/mmol) was the product of
NEN (USA).

A. Puc´kowska et al. / European Journal of Medicinal Chemistry 39 (2004) 99–105
105
[2] S. Neidle, Nat. Prod. Rep. 18 (2001) 291–309.
6.2.6. Relaxation assay of topoisomerase II
PBR322 plasmid DNA (0.083 µg) was incubated with
1 unit of topoisomerase II in a standard reaction buffer
(10 mM Tris–HCI (pH 7.9), 1 mM ATP, 50 mM KCl, 5 mM
MgCI2, 50 mM NaCI, 0.1 mM EDTA, and 15 µg/ml bovine
serum albumin) in the presence of the test compound at
concentrations ranging from 10 to 100 µM. The mixture was
incubated at 37 °C for 1 h and the reaction was terminated by
addition of 2 µl of 10% SDS and 2 µl of proteinase K
(1 mg/ml). The reaction mixture was subjected to electro-
phoresis through a 0.8% agarose gel containing 0.5 mg/ml
ethidium bromide in TBE buffer (90 mM Tris–borate and
2 mM EDTA). The gels were stained with ethidium bromide
and photographed under UV light using UVI-KS4000i gel
documentation and analysis system (SyngenBiotech, San
Carlos, CA, USA).
[3] M. Gniazdowski, M. Czyz˙, Acta Biochim. Pol. 46 (1999) 255–262.
[4] J.J. Champoux, Annu. Rev. Biochem. 70 (2001) 369–413.
[5] D. Bartulewicz, K. Bielawski, A. Markowska, K. Zwierz, A. Puc´k-
owska, A. Róz˙an´ski, Acta Biochim. Pol. 45 (1998) 41–57.
[6] A. Puc´kowska, D. Bartulewicz, A. Markowska, A. Róz˙an´ski, Acta
Polon. Pharm. 56 (Suppl) (1999) 104–111.
[7] A. Puc´kowska, K. Bielawski, A. Bielawska, A. Róz˙an´ski, Acta Bio-
chim. Pol. 49 (2002) 177–183.
[8] D. Bartulewicz, K. Bielawski, A. Bielawska, Arch. Pharm. Pharm.
Med. Chem. 9 (2002) 422–426.
[9] K.E. Rao, V. Sasisekharan, Indian J. Chem. 29B (1990) 508–513.
[10] E.T. Kraut, T. Fleming, M. Segal, N. Neidhart, B.C. Behrens, J. Mac-
Donald, Invest. New Drugs 9 (1999) 95–96.
[11] J. Carmichael, W. Degraff, A. Gazdar, J. Minna, J. Mitchell, Cancer
Res. 47 (1987) 936–942.
[12] H.J. Burstein, C.A. Bunnell, E.P. Winer, Semin. Oncol. 28 (2001)
344–358.
References
[13] J.L. Nitiss, Curr. Opin. Invest. Drugs 3 (2002) 1512–1518.
[1] B.S. Reddy, S.K. Sharma, J.W. Lown, Curr. Med. Chem. 8 (2001)
475–508.
[14] D.A. Burden, N. Osheroff, Biochim. Biophys. Acta 1400 (1998)
139–154.