New propylamine oligopyrrole carboxamides linked to a heterocyclic or anthraquinone system: Synthesis, DNA binding, topoisomerase I inhibition and cytotoxicity

Article abstract of DOI:10.1016/S0223-5234(02)01441-1

Continuing our studies on combilexines, compounds consisting of a DNA intercalator linked to a minor groove ligand, new results are presented. The synthesis of a series of new propylamine oligopyrrole carboxamides closely related to netropsin and distamycin A, linked to a heterocyclic or anthraquinone system is reported. The cytotoxic activity in vitro, the DNA binding characteristics and the inhibition of the topoisomerase I of the compounds were studied in order to explain the biological mechanism of action of these new potential combilexines. Some of the synthesised compounds showed cytotoxic activity against human tumour cell lines, as well as DNA binding and topoisomerase I inhibiting properties.

Full text of DOI:10.1016/S0223-5234(02)01441-1

European Journal of Medicinal Chemistry 38 (2003) 189Á197
/
www.elsevier.com/locate/ejmech
Original article
New propylamine oligopyrrole carboxamides linked to a heterocyclic
or anthraquinone system: synthesis, DNA binding, topoisomerase I
inhibition and cytotoxicity
Christian Hotzel, Annalisa Marotto, Ulf Pindur *
Department of Pharmacy, Johannes Gutenberg-University, Staudingerweg 5, 55099 Mainz, Germany
Received 10 June 2002; received in revised form 4 November 2002; accepted 20 November 2002
Abstract
Continuing our studies on combilexines, compounds consisting of a DNA intercalator linked to a minor groove ligand, new
results are presented. The synthesis of a series of new propylamine oligopyrrole carboxamides closely related to netropsin and
distamycin A, linked to a heterocyclic or anthraquinone system is reported. The cytotoxic activity in vitro, the DNA binding
characteristics and the inhibition of the topoisomerase I of the compounds were studied in order to explain the biological mechanism
of action of these new potential combilexines. Some of the synthesised compounds showed cytotoxic activity against human tumour
cell lines, as well as DNA binding and topoisomerase I inhibiting properties.
´
# 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: Combilexines; DNA-binding; Topoisomerase I inhibition; Cytotoxic antitumour agents
1. Introduction
with different ligands for the C- and N-terminal ends
and reported DNA binding and cytotoxicity effects of
some new compounds [7]. Encouraged by the results of
this basic study and from the perspective of a more
systematic structure activity profile, we continued our
synthetic efforts. Thus, we now report the preparation
of new potential combilexines of the pyrrole carbox-
amide series with an N,N-dimethylamino-propylamidic
group at the C-terminal end and a heterocyclic, a
quinone or a nucleic base moiety linked via an aliphatic
chain at the N-terminal end (see Fig. 2). This amino-
propylamidic group should represent a structural
equivalent of the amidine group presented in distamycin
A and netropsin. In addition, the DNA binding, the
topoisomerase I inhibition and the antitumour activity
were investigated.
Molecular recognition is a fundamental principle in
biology and pharmacology. In this context creating
DNA-binding compounds which additionally recognise
specific sequences is a central goal in the development of
DNA-targeted drugs [1,2]. Some highly interesting lead
compounds are the naturally occurring antibiotics
netropsin and distamycin A (see Fig. 1) [3,4], which
bind in the minor groove of the DNA with high AT base
selectivity [5]. Meanwhile the oligopyrrole carboxamide
chain of these natural antibiotics has been used as
sequence selective carriers for other cytotoxic DNA
ligands such as intercalators in compounds called
combilexines [6]. Among this group of compounds,
some promising candidates have been described from
several laboratories*for example NetAmsa (see Fig. 1)
/
[6]. Starting from this background, we conducted
systematic studies in the pyrrole carboxamide chemistry
2. Chemistry
Following the protocols recently described [7], a
variety of new N,N-dimethylamino-propylamide-based
* Corresponding author.
E-mail address: pindur@mail.uni-mainz.de (U. Pindur).
´
0223-5234/02/$ - see front matter # 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
doi:10.1016/S0223-5234(02)01441-1

190
C. Hotzel et al. / European Journal of Medicinal Chemistry 38 (2003) 189Á197
/
Fig. 1. The minor groove binding ligands distamycin A and netropsin and, as a typical example, the combilexine NetAmsa [6].
compounds with a variable number of pyrrole units and
different ligands at the N-terminal side were synthesised
by a multi-step procedure closely related to classical
peptide synthesis (see Fig. 2). The synthesis started with
commercially available N-methylpyrrole 1, which was
acylated with trichloroacetylchloride [8] to compound 2
(for detaild see Ref. [7]). After nitration of compound 2
to 3, N,N-dimethyl-1,3-diaminopropane was added to
produce the C-terminal side chain 4. The bispyrrole 5
was synthesised by reduction of compound 4 over Pd/
charcoal and successive additions of building block 3.
The trispyrrole (6) (see Fig. 2) was prepared by the same
procedure starting from bispyrrole 5 [9]. The N-acety-
lated compound 16 was synthesised by reduction of
compound 5 and successive acetylation. The indole and
carbazole acids were synthesised by the same method as
3. Biochemical and biological results
3.1. Cytotoxicity assay
The newly synthesised potential combilexines re-
ported in Fig. 2 were submitted for cytotoxicity testing
to the Developmental Therapeutics Program of the
National Cancer Institute (USA) [7,13,14]. In the
prescreening assay for the determination of the cyto-
toxicity in a three cell line panel consisting of the highly
sensitive cell lines NCI-H460 (lung carcinoma), MCF-7
(breast carcinoma) and SF 268 (glioma) most of the
substances are inactive [15]. The weak effect noticed
with the anthraquinone derivative 14 and its analogue
15 has not been confirmed in the NCI 60-cell lines panel
screening with the sulphorhodamine B-assay in a doseÁ
/
response analysis (concentration range from 10ꢀ4 to
described in earlier publications [7,10Á12]. The final
/
10ꢀ8 M) except for the marked selectivity of compound
combilexines were all synthesised by the classic DCC-
method starting from the reduced form of compounds 4
or 5 (see Fig. 3) [7]. The anthraquinone derivatives were
synthesised starting from sodium-anthraquinone sul-
phonate (13a). This compound was melted with phos-
phorpentachloride to yield compound 13b (yield: 91%)
and successively coupled with the mono- or the bispyr-
role units after their reduction to 4a and 5a to get
compounds 14 and 15, respectively (see Fig. 4). The
yield was 52 and 19%, respectively. The constitution of
15 for the ovarian cell line SK-OV-3 (GI50B
10ꢀ8 M).
/
3.2. DNA-binding studies and topoisomerase I inhibition
To explain the cytotoxicity and the mechanism of
action of the new compounds in the given series, we
studied their interactions with the DNA and the
topoisomerase I as possible molecular targets [16,17].
The DNA-binding was evaluated by the spectrophoto-
metric determination of the melting curve of dsDNA in
the presence of the potential ligands [7,18,19] and
additionally with the ethidium bromide displacement
assay, which comprises a fluorimetric titration [20,21].
the N-methyl-pyrrole derivatives 6Á
/
12 and 14Á16 were
/
unambigously clarified by the combination of several
NMR techniques (1H-NOE, proton decoupling experi-
ments, 13C-spin echo experiments).

C. Hotzel et al. / European Journal of Medicinal Chemistry 38 (2003) 189Á
/197
191
somerase I inhibition assay are reported in Table 1.
Compounds 6Á9, 11, 14 and 16 showed a relevant DNA
binding expressed in the DTm-values in the range of 8Á
/
/
13 8C. Interestingly, seven of the compounds revealed
probably non-specific inhibition of the catalytic activity
of topoisomerase I in different concentrations. How-
ever, the good DNA binding compounds 11 and 16
showed no inhibition of the topoisomerase I according
to the procedures established in our laboratory [7].
4. Discussion of the results and conclusion
Our presumption of the importance of H-bond donor
and acceptor groups in the lexine chain for DNA
interactions, which were recently described [7], has
been confirmed with the compounds of this second
series. Almost all N,N-dimethylamino-propylamide de-
rivatives showed DNA binding according to the melting
experiments. The cytotoxic activity of these compounds
can be explained by their ability to bind to the DNA
and/or to inhibit the catalytic activity of the topoisome-
rase I. A convincing determination of structureÁactivity
/
relationships of the new combilexines is not possible at
the moment. However, the overall results demonstrate
that not only the dimethylamino-propylamide side chain
but also the presence of a minimum of three carbonylic
acceptor groups are necessary for binding to the DNA.
In summarizing all data available, it becomes obvious
that the compounds 14 and 15 with the anthraquinone
moiety should be very promising candidates for further
studies. The lack of cytotoxicity in the NCI antitumour
screening assay for a variety of the compounds which
showed discrete DNA binding and topoisomerase I
inhibiting activity can perhaps be attributed to a
diminished absorption of the substances into the cells.
Our synthetic and analytical studies to develop
potential combilexines including nucleobase derivatives
and to obtain more information about systematic
structureÁactivity relationships will be continued.
/
5. Experimental
5.1. Chemistry
Fig. 2. Synthesised oligopyrrole carboxamides.
Melting points were measured with a Buchi 510
¨
instrument and are uncorrected. IR spectra were re-
The inhibition of the catalytic activity of the topoisome-
rase I was determined by analysis of the different
population distributions of DNA topoisomers after
incubation of a DNA-topoisomerase I mixture in the
absence of and in the presence of the test drugs in three
corded on a PerkinÁElmer 1310 IR-spectrometer using
/
KBr pellets (n in cmꢀ1). H-, 13C-NMR spectra includ-
ing NOE were recorded on a Bruker AC-300 apparatus
(300 MHz). The samples were dissolved in DMSO-d6.
The chemical shift values are reported in parts per
1
different concentrations (5, 20 and 50 mM) [22Á
/
24]. For
details about these three assays, see Refs. [7,25,26].
million (ppm, d units) and spinÁspin coupling J were
listed in Hz. Seventy electron volts EI-mass spectra were
obtained with a Mascom 311-A apparatus and FD mass
/
Results of the melting experiments and of the topoi-

192
C. Hotzel et al. / European Journal of Medicinal Chemistry 38 (2003) 189Á197
/
Fig. 3. Final step in the synthesis of the different combilexines (see Fig. 2).
Table 1
spectra. with a Finnigan MAT 7 instrument. C,H,N-
analysis was performed using a Haereus CHN rapid
apparatus. Analyses indicated by the symbols of the
Results of the determination of the melting temperature of dsDNA
(reported as DTm, the difference between the melting temperature of
the dsDNA in the presence and in the absence of the ligands) and of
topoisomerase I inhibition assay (reported as the concentration by
elements or functions were within 90.4% of the
/
which an inhibition of the enzyme was detected) the for compounds 6Á
/
theoretical values.
9, 11, 12 and 14Á
/16
5.1.1. 2,2,2-Trichloro-1-(1-methyl-1H-2-pyrrolyl)-1-
ethanone (2)
Compound
DTm (8C)
TOPO I-inhibition (mM)
6
9.09
/
0.5
0.5
50
50
To a solution of trichloroacetyl chloride (4.55 g, 28
mmol) in CH2Cl2 (25 mL), was added a solution of N-
methylpyrrole (1, 2.03 g, 25 mmol) in CH2Cl2 (15 mL)
over a period of 3 h. During this time, the reaction
mixture was stirred vigorously and nitrogen was swept
through to remove HCl as it was formed. The solution
was stirred overnight and then evaporated. The dark red
residue was dissolved in CHCl3 and filtered through a
short column of silica gel. Evaporation of the solvent
gave pale yellow needles (2.3 g) (10.16 mmol) (41%),
7
8.49
/
8
10.49
7.690.5
14.09
/
0.5
5
50
9
/
11
12
14
15
16
/
0.5
0.5
0.5
no inhibition
5
0
15.09
/
20
50
0
12.79
/
no inhibition
2850, 1600, 1400, 1350, 1300, 1280, 1200, 1160, 1050,
1
1020, 920, 820, 800, 760, 700, 650; H-NMR (DMSO-
m.p. 62 8C (lit. m.p.: 62Á63 8C [8]); IR: 3350, 3000,
/
Fig. 4. Synthesis of the anthraquinone combilexines.

C. Hotzel et al. / European Journal of Medicinal Chemistry 38 (2003) 189Á
/197
193
d6): d 3.89 (s, 3H, CH3), 6.27 (dd, 1H, pyrrole-H), 7.40
4
was removed in vacuo, and the residue was treated with
i-PrOH. The resultant crystalline solid was collected and
washed with i-PrOH. The filtrate was concentrated, and
the residue was purified by column chromatography on
4
(d, 1H, Jꢁ
/
1.56 Hz, pyrrole-H), 7.42 (d, 1H, Jꢁ1.71
/
Hz). MS: m/z 226 [Mꢂ].
5.1.2. 2,2,2-Trichloro-1-(1-methyl-4-nitro-1H-2-
pyrrolyl)-1-ethanone
silica gel with MeOHÁ
(655 mg) (1.74 mmol) (22.14%), m.p. 190 8C (lit. m.p.:
190Á
191 8C [9]); IR (KBr, cmꢀ1): 3200, 3000, 2850,
2720, 2700, 1610, 1560, 1520, 1410, 1380, 1250, 1200,
1150, 1100, 1060, 1000, 980, 880, 840, 800, 760, 720, 700,
/
NH3 (25%, 97:3); (yellow solid)
To a suspension of 2 (1 g, 4.43 mmol) in Ac2O (6 mL)
at ꢀ40 8C, a mixture of 4 mL Ac2O and HNO3 (70%,
0.8 mL) was added over a period of 30 min and then
/
/
1
3
warmed up to room temperature (r.t.). After stirring at
650; H-NMR (DMSO-d6): d 1.62 (quint, 2H, Jꢁ
/
7.14
3
r.t. for 2 h, the mixture was cooled to ꢀ
/
20 8C. Isopropyl
Hz, CH2), 2.10 (s, 6H, 2-CH3), 2.23 (t, 2H, Jꢁ
CH2), 3.14 (q, 2H, 3Jꢁ
CH3), 3.91 (s, 3H, CH3), 6.79 (d, 1H, 4Jꢁ
pyrrole-H3), 7.19 (d, 1H, 4Jꢁ
1.65, pyrrole-H5), 7.55 (d,
/3.0 Hz,
alcohol (10 mL) was added and the resultant colourless
solid was collected, washed with cold isopropyl alcohol,
and dried under reduced pressure (colourless solid) (640
/
6.95 Hz, CH2), 3.79 (s, 3H,
1.94 Hz,
/
/
4
4
mg) (2.35 mmol) (53.3%), m.p. 134 8C (lit. m.p.: 135Á
/
1H, Jꢁ
/
1.92, pyrrole-H3?), 8.10 (t, 1H, Jꢁ
/1.63 Hz,
140 8C [8]); IR: 3250, 3020, 2970, 1630, 1480, 1440,
1380, 1350, 1260, 1180, 1060, 1030, 950, 810, 800, 760,
NH), 8.16 (d, 1H, 4Jꢁ
/
1.71 Hz, pyrrole-H5?), 10.22
(s, 1H, NH); 13C-NMR (DMSO-d6): d 27.3 (s), 36.3 (p),
37.3 (s), 37.6 (p), 45.5 (p), 45.5 (p), 57.7 (s), 104.1 (t),
107.7 (t), 107.8 (t), 117.5 (t), 121.7 (q), 123.6 (q), 126.6
(q), 134.1 (q), 157.1 (q), 161.4 (q). MS: m/z 376 [Mꢂ];
Anal. C17H24N6O4 (C,H,N).
750, 700, 660, 640; 1H-NMR (DMSO-d6): d 3.98 (s, 3H,
4
CH3), 7.78 (d, 1H, Jꢁ
/
1.84 Hz, pyrrole-H3), 8.55 (d,
1H, Jꢁ
1.7 Hz). MS: m/z 271 [Mꢂ].
/
4
5.1.3. N2-[3-(Dimethylamino)propyl]-1-methyl-4-nitro-
1H-2-pyrrole carboxamide (4)
5.1.5. N2-[5-({[3-
A solution of 3-dimethylaminopropylamine (0.7 g,
6.86 mmol) in CH2Cl2 (15 mL) was added dropwise to a
stirred solution of 3 (1.4 g, 5.16 mmol) in CH2Cl2 (15
mL) of 0 8C. The reaction mixture was warmed up to r.t.
and stirring was continued for 1 h. The solvent was
removed under vacuum and the residual solid was
recrystallised from EtOH. The residue was chromato-
(Dimethylamino)propyl]amino}carbonyl)-1-methyl-1-
H-3-pyrrolyl]-1-methyl-4-{[(1-methyl-4-nitro-1H-
pyrrolyl)carbonyl]amino}-1H-2-pyrrole carboxamide
(6)
This compound was synthesised by the same proce-
dure as for 5; instead of 4 compound 5 (2 g, 5.3 mmol)
was used; (yellow solid) (900 mg) (1.8 mmol) (34.0%),
graphed on neutral Al2O3 (MeOHÁ
/
EtOAc (1:4)). Col-
ourless crystals were obtained (688 mg) (2.7 mmol)
m.p. 127 8C (lit. m.p.: 136Á137 8C [9]); IR: 3380, 3250,
/
3100, 2900, 2820, 2800, 1640, 1620, 1560, 1500, 1450,
1420, 1380, 1290, 1240, 1200, 1150, 1100, 1050, 1020,
1000, 980, 880, 800, 760, 740, 700, 650; 1H-NMR
(66%), m.p. 125 8C (lit. m.p.: 125.5Á127 8C [9]); IR
/
(KBr, cmꢀ1): 3420, 2720, 2340, 1650, 1540, 1530, 1490,
1
1470, 1410, 1305, 1265, 1215, 1035, 820, 755; H-NMR
(DMSO-d6): d 1.60 (quint, 2H, 3Jꢁ
/
6.9 Hz, CH2),
(300 MHz) (DMSO-d6): d 1.6 (m, 2H, 3Jꢁ
/
6.5 Hz,
6.7 Hz,
2.15 (s, 6H, 2-CH3), 2.26 (t, 2H, 3Jꢁ
/6.9 Hz, CH2),
CH2), 2.05 (s, 6H, 2-CH3), 2.2 (t, 2H, 3Jꢁ
CH2N(Me)2), 3.18 (q, 2H, 3Jꢁ
(s, 3H, CH3), 7.38 (d, 1H, 4Jꢁ
/
3.17 (q, 2H, Jꢁ
/
6.0 Hz, CH2), 3.78 (s, 3H, CH3), 3.84
(s, 3H, CH3), 3.9 (s, 3H, CH3), 6.81 (d, 1H, 4Jꢁ
3
/6.7 Hz, CH2N), 3.77
/
1.6 Hz,
1.6 Hz, pyrrole-H), 7.18 (d,
/1.6 Hz, pyrrole-H3), 8.09
pyrrole-H), 7.02 (d, 1H, 4Jꢁ
/
4
3
4
4
(1H, d, Jꢁ
/
1.6 Hz, pyrrole-H5), 8.40 (t, 1H, Jꢁ
/
7.4
1H, Jꢁ
pyrrole-H), 7.58 (d, 1H4, Jꢁ
(t, 1H, 3Jꢁ5.3 Hz, NH), 8.17 (d, 1H, 4Jꢁ
/
1.5 Hz, pyrrole-H), 7.26 (d, 1H, Jꢁ
1.5 Hz, pyrrole-H), 8.08
1.5 Hz,
/
1.6 Hz,
Hz, NH). MS: m/z 254 [Mꢂ].
/
/
/
5.1.4. N2-[5-({[3-
pyrrole-H), 9.94 (s, 1H, NH), 10.30 (s, 1H, NH); 13C-
NMR (DMSO-d6): d 26.6 (s), 36.2 (p), 36.3 (p), 37.7 (s),
38.9 (p), 45.5 (p), 45.5 (p), 57.4 (s), 104.2 (t), 104.2 (t),
104.3 (t), 104.7 (t), 107.8 (t), 107.8 (t), 121.7 (q), 122.3
(q), 123.3 (q), 123.6 (q), 126.6 (q), 134.0 (q), 157.2 (q),
158.6 (q), 161.4 (q). MS: m/z 498 [Mꢂ]; Anal.
C23H30N8O5 (C,H,N).
(Dimethylamino)propyl]amino}carbonyl)-1-methyl-1-
H-3-pyrrolyl]-1-methyl-4-nitro-1H-2-pyrrole
carboxamide (5)
A solution of 4 (2 g, 7.86 mmol) in dioxane (20 mL)
was hydrogenated under reduced pressure over Pd/C
(800 mg) for 12 h. The catalyst was removed by
filtration and then the solvent was removed in vacuo.
The residual solid was dissolved in DMF (20 mL) and
concentrated to half of its original volume under
reduced pressure to remove dioxane completely. A
solution of 3 (2 g, 7.36 mmol) in DMF (15 mL) was
added with stirring at 0 8C. The temperature was
allowed to rise to ambient temperature. The solvent
5.1.6. General procedure for the synthesis of the pyrrole
and the oligopyrrole carboxamides
Carboxylic acid (1 mmol) was dissolved in 20 mL
dioxane. Then 1 mmol of the pyrrole amine (prepared
from the nitropyrrole analogues by hydrogenation in
MeOH over Pd on charcoal) and 0.1 mmol (12 mg) of

194
C. Hotzel et al. / European Journal of Medicinal Chemistry 38 (2003) 189Á197
/
dimethylaminopyridine (DMAP) were added. The mix-
ture was stirred at r.t.; a solution of dicyclohexylcarbo-
diimide (DCC) (288 mg) 1.4 mmol) in dioxane (10 mL)
was added dropwise. After stirring for 24 h the
precipitate, a dicyclohexyl urea, was removed by filtra-
tion. The solution was evaporated to dryness, and the
27.5 (s), 33.4 (s), 36.2 (p), 36.3 (p), 37.3 (s), 45.5 (p), 45.5
(p), 57.4 (s), 104.2 (t), 109.2 (q), 116.6 (t), 111.6 (t), 118.6
(t), 118.6 (t), 119.0 (t), 121.2 (t), 121.2 (t), 122.3 (q),
122.3 (q), 123.0 (q), 123.3 (q), 124.0 (t), 127.5 (q), 136.4
(q), 158.6 (q), 161.4 (q), 168.3 (q). MS: m/z 517 [Mꢂ];
Anal. C28H35N7O3 (C,H,N).
residue was chromatographed on silica gel (MeOHÁ
/
NH3 (25%, 97:3)).
5.1.6.3. N2-[5-({[3-
(Dimethylamino)propyl]amino}carbonyl)-1-methyl-1-
H-3-pyrrolyl]-4-{[2-(5-methyl-2,4-dioxo-1,2,3,4-
tetrahydro-1-pyrimidinyl)acetyl]amino}-1H-2-pyrrole
carboxamide (9). Yellow solid (180 mg) (0.35 mmol)
(35.1%), m.p. 206 8C; IR (KBr, cmꢀ1): 3400, 3250,
3100, 2900, 2800, 1650, 1600, 1560, 1500, 1410, 1380,
5.1.6.1. N2-[5-({[3-
(Dimethylamino)propyl]amino}carbonyl)-1-methyl-1-
H-3-pyrrolyl]-4-{[2-(1H-3-indolyl)acetyl]amino}-1-
methyl-1H-2-pyrrole carboxamide (7). Yellow solid (140
mg) (0.28 mmol) (27.8%), m.p. 96 8C; IR (KBr, cmꢀ1):
3360, 3250, 3080, 2900, 2830, 2800, 1620, 1560, 1500,
1450, 1420, 1380, 1350, 1290, 1250, 1190, 1130, 1090,
1050, 1000, 800, 760, 730, 650; 1H-NMR (DMSO-d6): d
1340, 1210, 1190, 1150, 1000, 950, 880, 790, 750, 650;
1H-NMR (DMSO-d6): d 1.60 (quint, 2H, Jꢁ
3
/
6.99 Hz,
CH2), 1.76 (s, 3H, CH3), 2.10 (s, 6H, 2-CH3), 2.24
3
3
1.6 (quint, 2H, 3Jꢁ
/
7.0 Hz, CH2), 2.1 (s, 6H, 2-CH3), 2.3
5.8 Hz, CH2), 3.7 (s, 3H, CH3), 3.8 (s, 3H,
(t, 2H, Jꢁ
CH2), 3.77 (s, 3H, CH3), 3.81 (s, 3H, CH3), 4.43 (s, 2H,
CH2), 5.7 (s, 1H, thymine-H), 6.79 (d, 1H, 4Jꢁ
4.67 Hz,
pyrrole-H3), 6.89 (d, 1H, 4Jꢁ
1.68 Hz, pyrrole-H5), 7.13
/
6.97 Hz, CH2), 3.17 (q, 2H, Jꢁ7.2 Hz,
/
3
(t, 2H, Jꢁ
/
CH3), 5.7 (s, 2H, CH2), 6.8 (d, 1H, 4Jꢁ
/
1.65 Hz,
1.63 Hz, pyrrole-H5),
/
pyrrole-H3), 6.9 (d, 1H, 4Jꢁ
/
/
3
4
4
6.95 (pt, 1H, Jꢁ
/
7.1 Hz, indole-H 5 or 6), 7.04 (pt,
(d, 1H, Jꢁ
/
1.4 Hz, pyrrole-H3?), 7.16 (d, 1H, Jꢁ
/1.5
3
1H, 3Jꢁ
/
7.0 Hz, indole-H 5 or 6), 7.13 (d, 1H, 4Jꢁ
/1.41
Hz, pyrrole-H5?), 8.05 (t, 1H, Jꢁ
/
5.1 Hz, NH), 9.83
Hz, pyrrole-H3?), 7.15 (d, 1H, 3Jꢁ
H5?), 7.21 (s, 1H, indole-H2), 7.33 (d, 1H, Jꢁ
indole-H7), 7.57 (d, 1H, 7.9 Hz, indole-H4), 8.05 (t, 1H,
3Jꢁ
5.03 Hz, NH), 9.8 (s, 1H, NH), 9.9 (s, 1H, NH),
/
1.51 Hz, pyrrole-
(s, 1H, NH), 10.16 (s, 1H, NH), 10.28 (s, 1H, NH); 13C-
NMR (DMSO-d6): d 12.2 (p), 27.4 (s), 36.2 (p), 36.4 (p),
37.3 (s), 45.4 (p), 45.4 (p), 49.8 (s), 57.3 (s), 104.2 (t),
104.2 (t), 104.3 (t), 104.7 (t), 108.2 (q), 121.5 (q), 122.2
(q), 123.2 (q), 131.9 (q), 142.7 (t), 151.4 (q), 158.5 (q),
161.4 (q), 164.3. MS: m/z 512 [Mꢂ]; Anal. C24H32N8O5
(C,H,N).
3
/
8.0 Hz,
/
10.87 (s, 1H, NH); 13C-NMR (DMSO-d6); d 27.5 (s),
33.4 (s), 36.2 (p), 36.3 (p), 37.3 (s), 45.5 (p), 45.5 (p), 57.4
(s), 104.2 (t), 109.2 (q), 111.6 (t), 111.6 (t), 118.6 (t),
118.6 (t), 119.0 (t), 121.2 (t), 121.2 (t), 122.3 (q), 122.3
(q), 123.0 (q), 123.3 (q), 124.0 (t), 127.5 (q), 136.4 (q),
158.6 (q), 161.4 (q), 168.3 (q). MS: m/z 503 [Mꢂ]; Anal.
C27H33N7O3 (C,H,N).
5.1.6.4. N2-[5-({[3-
(Dimethylamino)propyl]amino}carbonyl)-1-methyl-1-
H-3-pyrrolyl]-4-{[9H-9-carbazolyl)butanoyl]amino}-1-
methyl-1H-2-pyrrole carboxamide (10). Yellow solid
(180 mg) (0.3 mmol) (48.9%), m.p. 99 8C; IR (KBr,
cmꢀ1): 3250, 3100, 3000, 2900, 2820, 2790, 1640, 1600,
1560, 1500, 1450, 1380, 1290, 1240, 1190, 1090, 1050,
5.1.6.2. N2-[5-({[3-
(Dimethylamino)propyl]amino}carbonyl)-1-methyl-1-
H-3-pyrrolyl]-4-{[2-(1H-3-indolyl)propanoyl]amino}-
1-methyl-1H-2-pyrrole carboxamide (8). Yellow solid
(120 mg) (0.23 mmol) (23.1%), m.p. 75 8C; IR (KBr,
cmꢀ1): 3400, 3250, 3100, 2920, 2840, 2800, 2750, 1620,
1560, 1520, 1450, 1420, 1380, 1300, 1240, 1200, 1150,
1130, 1100, 1080, 1050, 1030, 980, 880, 800, 760, 740,
1
880, 800, 740, 700, 650; H-NMR (DMSO-d6): d 1.6
3
(quint, 2H, Jꢁ
/
6.8 Hz, CH2), 2.18 (s, 6H, 2-CH3), 2.0
(quint, 2H, 3Jꢁ
2.27 (t, 2H, 3Jꢁ
CH2), 3.75 (s, 3H, CH3), 3.79 (s, 3H, CH3), 4.4 (t, 2H,
/
6.5 Hz), 2.23 (t, 2H, Jꢁ
/
7.0 Hz, CH2),
6.4 Hz,
3
/
6.6 Hz, CH2), 3.11 (q, 2H, 3Jꢁ
/
1
3
4
650; H-NMR (DMSO-d6): d 1.57 (quint, 2H, Jꢁ
/
6.96
7.11 Hz,
3Jꢁ
H3), 6.84 (d, 1H, Jꢁ
4Jꢁ
/
6.7 Hz, CH2), 6.80 (d, 1H, Jꢁ
/1.55 Hz, pyrrole-
4
Hz, CH2), 2.12 (s, 6H, 2-CH3), 2.22 (t, 2H, 3Jꢁ
/
/
1.6 Hz, pyrrole-H5), 7.13 (d, 1H,
4
3
3
CH2), 2.59 (t, 2H, Jꢁ
7.5 Hz, CH2), 3.15 (t, 2H, Jꢁ
3H, CH3), 3.80 (s, 3H, CH3), 6.79 (d, 1H, Jꢁ
/
7.4 Hz, CH2), 2.98 (t, 2H, Jꢁ
/
/
1.62 Hz, pyrrole-H3?), 7.16 (d, 1H, Jꢁ
/
1.66 Hz,
7.3 Hz, carbazole-H2,8),
3
3
/
6.15 Hz, CH2), 3.77 (s,
pyrrole-H5?), 7.19 (d, 2H, Jꢁ
/
4
3
/
1.66 Hz,
7.4 (pt, 2H, Jꢁ
3Jꢁ
NH), 8.1 (d, 2H, Jꢁ
/
7.5 Hz, carbazole-H2,7), 7.6 (pt, 2H,
4
3
pyrrole-H3), 6.83 (d, 1H, Jꢁ
/
1.4 Hz, pyrrole-H5), 6.97
/
8.1 Hz, carbazole-H3,8), 8.05 (t, 1H, Jꢁ
/5.2 Hz,
3
3
(pt, 1H, Jꢁ
3Jꢁ
7.8 Hz, indole-H5 or 6), 7.13 (d, 1H, Jꢁ
pyrrole-H3?), 7.16 (d, 1H, Jꢁ
/
7.65 Hz, indole-H5 or 6), 7.04 (pt, 1H,
/
7.6 Hz, carbazole-H4,5), 9.8 (s,
4
/
/1.62 Hz,
1H, NH), 9.85 (s, 1H, NH), 10.25 (s, 1H, NH); 13C-
NMR (DMSO-d6): d 24.7 (s), 27.4 (s), 30.6 (s), 31.7 (s),
36.2 (p), 36.3 (p), 37.2 (s), 45.3 (p), 45.3 (p), 57.2 (s),
104.2 (t), 104.3 (t), 109.5 (t), 109.5 (t), 119.0 (t), 119.0 (t),
120.5 (t), 120.5 (t), 120.6 (t), 121.7 (t), 122.3 (q), 122.4
(q), 122.4 (q), 123.0 (q), 124.0 (q), 125.9 (t), 125.9 (t),
4
/
1.66 Hz, pyrrole-H5?),
7.8 Hz, indole-
8.4 Hz, indole-H4), 8.05 (t, 1H,
7.19 (s, 1H, indole-H2), 7.30 (d, 1H, 3Jꢁ
H7), 7.54 (d, 1H, Jꢁ
5.05 Hz, NH), 9.82 (s, 1H, NH), 10.22 (s, 1H, NH),
10.82 (s, 1H, NH); 13C-NMR (DMSO-d6): d 21.3 (s),
/
3
/
3Jꢁ
/

C. Hotzel et al. / European Journal of Medicinal Chemistry 38 (2003) 189Á
/197
195
140.3 (q), 140.4 (q), 153.5 (q), 158.7 (q), 161.5 (q), 169.1
(q), 169.2 (q). MS: m/z 582 [Mꢂ]; Anal. C33H39N7O3
(C,H,N).
5.1.7. 9,10-Dioxo-9,10-dihydro-2-anthracensulphonyl
chloride (13b)
Dry sodium anthraquinonesulphonate (13a) (1 g, 3.95
mmol) was mixed with PCl5 (2 g, 9.6 mmol). The
mixture was heated under reflux for 30 min (120 8C).
After cooling to ambient temperature, C6H3CH3 (20
mL) was added, heated to boiling point and filtered after
cooling to ambient temperature. The C6H3CH3 was
removed under vacuum; (yellow solid) (930 mg) (3.66
mmol) (91.7%), m.p. 197 8C; IR (KBr, cmꢀ1): 3400,
3050, 2950, 1710, 1560, 1360, 1310, 1270, 1170, 1140,
1050, 950, 910, 840, 800, 780, 690, 650; 1H-NMR
(DMSO-d6): d 7.89 (m, 2H, aromatic-H), 8.09 (pd,
1H, aromatic-H), 8.19 (m, 3H, aromatic-H), 8.38 (ps,
1H, aromatic-H). MS: m/z 306 [Mꢂ].
5.1.6.5. N2-[5-({[3-
(Dimethylamino)propyl]amino}carbonyl)-1-methyl-1-
H-3-pyrrolyl]-4-{[2-(1H-1-indolyl)acetyl]amino}-1-
methyl-1H-2-pyrrole carboxamide (11). Yellow solid
(340 mg) (0.73 mmol) (80.6%), m.p. 98 8C; IR (KBr,
cmꢀ1): 3400, 3250, 3080, 2900, 2850, 2800, 1620, 1560,
1510, 1450, 1420, 1380, 1340, 1250, 1190, 1140, 1090,
1
1050, 1000, 950, 800, 750, 730, 650; H-NMR (DMSO-
3
d6): d 1.56 (quint, 2H, Jꢁ
/
7.0 Hz, CH2), 2.1 (s, 6H, 2-
3
3
CH3), 2.22 (t, 2H, Jꢁ
/
7.0 Hz, CH2), 3.16 (q, 2H, Jꢁ
/
6.3 Hz, CH2), 3.77 (s, 3H, CH3), 3.79 (s, 3H, CH3), 4.96
3
5.1.8. N2-[5-({[3-
(s, 2H, CH2), 6.44 (d, 1H, Jꢁ3.1 Hz, indole-H3), 6.78
/
(Dimethylamino)propyl]amino}carbonyl)-1-methyl-1-
H-3-pyrrolyl]-4-{8,9,10-dioxo-9,10-dihydro-2-
anthracenyl)sulphonyl]amino}-1-methyl-1H-2-pyrrole
carboxamide (15)
4
4
(d, 1H, Jꢁ
/
1.7 Hz, pyrrole-H3), 6.89 (d, 1H, Jꢁ
6.9 Hz, indole-H 5 or
6.9 Hz, indole-H 5 or 6), 7.13 (d,
/
2.0
Hz, pyrrole-H5), 7.02 (pt, 1H, 3Jꢁ
6), 7.11 (pt, 1H, Jꢁ
/
3
/
4
4
1H, Jꢁ
pyrrole-H5?), 7.36 (d, 1H, Jꢁ
(d, 1H, 3Jꢁ
indole-H4), 8.06 (t, 1H, Jꢁ
/
1.4 Hz, pyrrole-H3?), 7.16 (d, 1H, Jꢁ
/
1.7 Hz,
Compound 5 (376 mg, 1 mmol) was dissolved in
3
/
3.0 Hz, indole-H2), 7.38
dioxaneÁDMF (1:1) and reduced with Pd/C under
/
/
8.1 Hz, indole-H7), 7.54 (d, 1H, 3Jꢁ
/7.9 Hz,
hydrogen atmosphere (18 h). After removing the cata-
lyst anthraquinonesulphonylchloride 13b (50.1 mg, 0.2
3
/
5.3 Hz, NH), 9.85 (s, 1H,
NH), 10.29 (s, 1H, NH); 13C-NMR (DMSO-d6): d 27.4
(s), 36.2 (p), 36.4 (p), 37.3 (s), 45.4 (p), 45.4 (p), 49.2 (s),
57.3 (s), 101.0 (t), 104.2 (t), 104.3 (t), 110.0 (t), 119.4 (t),
119.4 (t), 120.6 (t), 120.6 (t), 121.3 (t), 121.3 (t), 122.3
(q), 123.3 (q), 128.4 (q), 128.4 (q), 130.2 (q), 136.6 (q),
158.6 (q), 161.5 (q), 164.9 (q). MS: m/z 517 [Mꢂ]; Anal.
C28H35N7O3 (C,H,N).
mmol) was dissolved in dioxaneÁ
dropwise at r.t. The precipitate was chromatographed
/
DMF and added
over silica gel (MeOHÁNH3 (25%); (brown solid) (120
/
mg) (0.2 mmol) (19.4%), m.p. 110 8C; IR (KBr, cmꢀ1):
3890, 2900, 1620, 1560, 1520, 1450, 1420, 1380, 1270,
1240, 1140, 950, 800, 760, 650; 1H-NMR (DMSO-d6): d
3
1.6 (quint, 2H, Jꢁ
/
7.0 Hz, CH2), 2.11 (s, 6H, 2-CH3),
3
3
2.27 (t, 2H, Jꢁ
/
7.19 Hz, CH2), 3.16 (q, 2H, Jꢁ5.75
/
Hz, CH2), 3.72 (s, 3H, CH3), 3.74 (s, 3H, CH3), 6.68 (d,
4
4
1H, Jꢁ
/
1.60 Hz, pyrrole-H3), 6.70 (d, 1H, Jꢁ
1.66 Hz, pyrrole-
1.65 Hz, pyrrole-H5?), 7.93 (m,
/
1.56
5.1.6.6. N2-[3-(Dimethylamino)propyl]-4-{[2-(1H-3-
indolyl)acetyl]amino}-1-methyl-1H-2-pyrrole
Hz, pyrrole-H5), 6.74 (d, 1H, 4Jꢁ
H3?), 7.09 (d, 1H, Jꢁ
2H, anthraquinone-H), 8.05 (t, 1H, Jꢁ
8.10 (pd, 1H, anthraquinone-H), 8.20 (m, 3H, anthra-
/
4
/
carboxamide (12). Yellow solid (170 mg) (0.45 mmol)
(44.6%), m.p. 65 8C; IR (KBr, cmꢀ1): 3350, 3250, 3080,
2900, 2820, 2800, 1640, 1620, 1560, 1500, 1440, 1420,
1380, 1270, 1240, 1210, 1140, 1090, 1000, 980, 950, 800,
3
/
5.1 Hz, NH),
3
quinone-H), 8.32 (ps, 1H, Jꢁ8.1 Hz, anthraquinone-
/
H), 9.78 (s, 1H, NH), 10.08 (s, 1H, NH); 13C-NMR
(DMSO-d6): d 27.05 (s), 36.2 (p), 36.3 (p), 37.3 (s), 45.5
(p), 45.5 (p), 57.1 (s), 104.2 (t), 107.4 (t), 118.7 (q), 118.7
(q), 121.2 (t), 123.0 (q), 124.0 (t), 124.6 (q), 125.1 (t),
127.2 (t), 127.2 (t), 128.3 (t), 132.0 (t), 133.3 (q), 133.9
(q), 135.0 (t), 135.0 (t), 136.6 (q), 145.1 (q), 159.2 (q),
161.0 (q), 181.8 (q), 182.0 (q). MS: m/z 616 [Mꢂ]; Anal.
C31H32N6O6S (C,H,N,S).
1
770, 730, 600; H-NMR (DMSO-d6): d 1.57 (quint, 2H,
3Jꢁ
/
6.8 Hz, CH2), 2.11 (s, 6H, 2-CH3), 2.21 (t, 2H, 3Jꢁ
/
3
7.13 Hz, CH2), 3.14 (q, 2H, Jꢁ
3H, CH3), 5.71 (s, 2H, CH2), 6.63 (d, 1H, Jꢁ
pyrrole-H3), 6.95 (pt, 1H, Jꢁ
/
6.0 Hz, CH2), 3.73 (s,
4
/
1.7 Hz,
7.2 Hz, indole-H5 or 6),
3
/
3
7.05 (pt, 1H, Jꢁ
4Jꢁ
1.4 Hz, pyrrole-H5), 7.20 (s, 1H, indole-H2), 7.32
(d, 1H, 3Jꢁ
indole-H4), 8.03 (t, 1H, Jꢁ
/
6.2 Hz, indole-H5 or 6), 7.07 (d, 1H,
/
/
8.0 Hz, indole-H7), 7.56 (d, 1H, 3Jꢁ
/
7.8 Hz,
3
/
5.3 Hz, NH), 9.89 (s, 1H,
5.1.9. N2-[3-(Dimethylamino)propyl]-4-{[(9,10-dioxo-
9,10-dihydro-2-anthracenyl)sulphonyl]amino}-1-methyl-
1H-2-pyrrole carboxamide (14)
This compound was synthesised by the same proce-
dure as 15; instead of 5 compound 4 (254 mg, 1 mmol)
was used (yellow amorphous solid) (510 mg) (1.03
mmol) (52.4%), m.p. 195 8C; IR (KBr, cmꢀ1): 3360,
NH), 10.87 (s, 1H, NH); 13C-NMR (DMSO-d6): d 27.4
(s), 33.4 (s), 36.2 (p), 37.3 (s), 45.4 (p), 45.4 (p), 57.3 (s),
103.6 (t), 109.2 (q), 111.6 (t), 117.7 (t), 118.6 (t), 119.0
(t), 121.2 (t), 122.3 (q), 124.0 (q), 127.4 (q), 136.4 (q),
161.4 (q), 168.3 (q). MS: m/z 381 [Mꢂ]; Anal.
C21H27N5O2 (C,H,N).

196
C. Hotzel et al. / European Journal of Medicinal Chemistry 38 (2003) 189Á197
/
3050, 2900, 2850, 2800, 2750, 1650, 1600, 1570, 1500,
1450, 1420, 1380, 1360, 1310, 1270, 1170, 1140, 1060,
temperature inside the cuvette was increased over the
range 25Á
90 8C with a heating rate of 1.5 8C minꢀ1
The absorption data were registered and plotted against
the temperature. The melting curve was analysed using
the program ORIGIN for data analysis and technical
graphics. The melting temperature (Tm) was taken as the
midpoint of the hyperchromic transition [19].
/
.
1
1000, 950, 900, 850, 750, 650; H-NMR (DMSO-d6): d
3
1.55 (quint, 2H, Jꢁ
/
6.9 Hz, CH2), 2.17 (s, 6H, 2-CH3),
6.2 Hz,
2.24 (t, 2H, 3Jꢁ
/
7.0 Hz, CH2), 3.07 (q, 2H, 3Jꢁ
/
CH2), 3.67 (s, 3H, CH3), 6.43 (d, 1H, 4Jꢁ
/
1.6 Hz,
1.6 Hz, pyrrole-H5), 7.9
4
pyrrole-H3), 6.62 (d, 1H, Jꢁ
/
(m, 2H, anthraquinone-H), 8.05 (t, 1H, 3Jꢁ
5.4 Hz,
/
NH), 8.10 (pd, 1H, anthraquinone-H), 8.19 (m, 3H,
anthraquinone-H), 8.32 (ps, 1H, anthraquinone-H), 9.3
(s, 1H, NH); 13C-NMR (DMSO-d6): d 27.0 (s), 36.5 (p),
37.1 (p), 45.1 (p), 45.1 (p) 57.0 (s), 107.4 (t), 118.7 (q),
118.7 (q), 121.2 (t), 124.6 (q), 125.1 (t), 127.2 (t), 127.2
(t), 128.3 (t), 132.0 (t), 133.3 (q), 133.8 (q), 135.0 (q),
135.0 (q), 136.6 (q), 145.1 (q), 160.9 (q), 181.8 (q), 181.9
(q). MS: m/z 494 [Mꢂ]; Anal. C25H36N4O5 (C,H,N,S).
5.2.2. Ethidium bromide displacement assay
All fluorescence measurements were conducted on an
Hitachi F-2000 spectrofluorometer. Calf thymus DNA
(Sigma, Type I; 1.0ꢃ
in small aliquots to ethidium bromide (Invitrogen; 5.0ꢃ
10ꢀ6 M) resulting in a 2:1 ratio of basepairÁ
ethidium in
2 mL of a 10 mM TrisÁHCl (pH 7.4), 75 mM NaCl
buffer solution. The fluorescence of the DNAÁethidium
/
10ꢀ5 M in base pairs) was added
/
/
/
/
buffer solution was calibrated at r.t. to 100% fluores-
cence and that of the ethidium buffer solution to 0%
5.1.10. N2-[5-({[3-
(Dimethylamino)propyl]amino}carbonyl)-1-methyl-1-
H-3-pyrrolyl]-4-(acetylamino)-1-methyl-1H-2-pyrrole
carboxamide (16)
Compound 5 (376 mg, 1 mmol) was dissolved in dry
dioxane. Palladium-on-carbon (500 mg) was added and
the reduction ran under a hydrogen atmosphere. After
removing the catalyst by filtration, the yellow solution
fluorescence. The premixed DNAÁethidium solution
/
was titrated with 3 mL aliquots of the stock solution of
the test substances (3 mM drug in DMSO) and stirred at
r.t. for 30 min prior to each fluorescence measurement.
The fluorescence was measured with lꢁ545 nm excita-
/
tion and 595 nm emission with a slit width of 10 nm. The
binding affinity was determined at 50% ethidium
bromide displacement and measured as a drop in
fluorescence to 50%. Results were calculated with the
Data Analysis and Graphics Program GRAFIT and with
the graphical program ORIGIN. Distamycin A as refer-
ence was tested in the same way [25].
was cooled on ice. Hunig’s Base was added (1 mL).
¨
Distilled acetylchloride (157 mg, 2 mmol) was added
dropwise and stirred for 12 h. After removing dioxane
under vacuum, the brown solid was chromatographed
over silica gel (MeOHÁNH3 (25%, 97:3); (yellow, solid)
/
(180 mg) (0.46 mmol) (46.34%), m.p. 77 8C; IR (KBr,
cmꢀ1): 3400, 3250, 3080, 2900, 2830, 2800, 1620, 1560,
1510, 1450, 1420, 1380, 1250, 1200, 1150, 1090, 1050,
5.3. Topoisomerase I inhibition
1
1020, 980, 800, 760, 650; H-NMR (DMSO-d6): d 1.6
Plasmid DNA (pUC 19, Invitrogen; 0.033 mg mLꢀ1
)
3
(quint, 2H, Jꢁ
/
7.1 Hz, CH2), 1.9 (s, 3H, CH3), 2.10 (s,
7.2 Hz, CH2), 3.17 (q, 2H,
5.8 Hz, CH2), 3.77 (s, 3H, CH3), 3.79 (s, 3H, CH3),
6.79 (d, 1H, 4Jꢁ1.85 Hz, pyrrole-H3), 6.82 (d, 1H, 4Jꢁ
1.87 Hz, pyrrole-H5), 7.12 (d, 1H, Jꢁ
was incubated for 15 min at 35 8C with different
concentrations of the tested drugs (5, 20 and 50 mM)
/
6H, 2-CH3), 2.26 (t, 2H, 3Jꢁ
/
3Jꢁ
/
in 1ꢃ
/
topoisomerase I reaction buffer (50 mM TrisÁ
/
/
HCl pH 7.5, 50 mM KCl, 10 mM MgCl, 0.5 mM DTT,
0.1 mM EDTA, 30 mg mLꢀ1 BSA) to ensure equilibra-
tion. The reaction was initiated by adding topoisome-
rase I (Topogen; 0.25 U mLꢀ1) and the samples were
reincubated for 15 min at 35 8C. Reactions were stopped
by addition of SDS to a final concentration of 0.25%
and proteinase K to 250 mg mLꢀ1, followed by
incubation for 30 min at 50 8C. After addition of 2 mL
denaturing loading buffer (Invitrogen), samples were
loaded onto a 1% agarose gel in TBE buffer. Electro-
phoresis was conducted at 120 V for 2 h.
4
/
1.6 Hz, pyrrole-
4
H3?), 7.16 (d, 1H, Jꢁ
/
1.71 Hz, pyrrole-H5?), 8.05 (t,
1H, 3Jꢁ
/
5.0 Hz, NH), 9.8 (s, 1H, NH), 9.9 (s, 1H, NH);
13C-NMR (DMSO-d6): d 23.3 (s), 27.4 (s), 36.2 (p), 36.3
(p), 37.3 (s), 45.4 (p), 45.4 (p), 57.3 (s), 104.0 (t), 104.0
(t), 104.3 (t), 104.3 (t), 122.3 (q), 122.4 (q), 123.0 (q),
123.3 (q), 158.6 (q), 161.5 (q), 166.8 (q). MS: m/z 388
[Mꢂ]; Anal. C19H28N6O3 (C,H,N).
5.2. DNA-binding methods
5.2.1. Melting experiments
Melting curves were measured using an Hitachi U-
3200-spectrophotometer coupled to a Julabo thermo-
stat. The measurements were performed in BPE buffer,
pH 7 (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA)
Acknowledgements
We thank Professor Dr C.B. and his group at the
Centre Oscar Lambret pour la Reserche sur le Cancer,
Lille, France, for their cooperation in developing the
biological assays and the Developmental Therapeutic
with a drugÁ
/
DNA ratio of 1:1, using [Poly(dAdT)×
/
Poly(dAdT)] as DNA (Amersham Pharmacia). The

C. Hotzel et al. / European Journal of Medicinal Chemistry 38 (2003) 189Á
/197
197
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/
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