Design, synthesis, DNA-binding and cytotoxicity evaluation of new potential combilexines

Article abstract of DOI:10.1016/S0223-5234(02)01349-1

Combilexines, compounds in which a DNA intercalator is linked to a minor groove binding component, interact with the DNA in a sequence specific manner to yield in most cases compounds with anticancer activity. A series of new compounds closely related to netropsin in which the two components were linked by an amide group was synthesised as potential combilexines. As some of these compounds showed cytotoxic activity in vitro, an attempt was made to rationalise their mechanism of action. The DNA binding characteristics of the carboxamides were evaluated by thermal denaturation experiments and by ethidium bromide displacement assay. Their ability to inhibit the topoisomerase I was also determined. It was concluded that the new compounds were only weak DNA ligands although able in some cases to inhibit topoisomerase I.

Full text of DOI:10.1016/S0223-5234(02)01349-1

Eur. J. Med. Chem. 37 (2002) 367–378
www.elsevier.com/locate/ejmech
Original article
Design, synthesis, DNA-binding and cytotoxicity evaluation of
new potential combilexines
Christian Hotzel, Annalisa Marotto, Ulf Pindur *
Department of Pharmacy, Johannes Gutenberg Uni6ersity, Staudingerweg 5, D-55099 Mainz, Germany
Received 5 December 2001; received in revised form 18 February 2002; accepted 20 February 2002
Abstract
Combilexines, compounds in which a DNA intercalator is linked to a minor groove binding component, interact with the DNA
in a sequence specific manner to yield in most cases compounds with anticancer activity. A series of new compounds closely
related to netropsin in which the two components were linked by an amide group was synthesised as potential combilexines. As
some of these compounds showed cytotoxic activity in vitro, an attempt was made to rationalise their mechanism of action. The
DNA binding characteristics of the carboxamides were evaluated by thermal denaturation experiments and by ethidium bromide
displacement assay. Their ability to inhibit the topoisomerase I was also determined. It was concluded that the new compounds
´
were only weak DNA ligands although able in some cases to inhibit topoisomerase I. © 2002 Editions scientifiques et me´dicales
Elsevier SAS. All rights reserved.
Keywords: combilexines; DNA-binding; topoisomerase I inhibition; cytotoxic antitumor agents
this context they can also be used as sequence selective
carriers for other cytotoxic active DNA ligands, such as
intercalators [8]. Incorporation of intercalating hetero-
cycles into the distamycin A–netropsin skeleton leads
to compounds called combilexines which represent an-
other drug series for oncogene inhibition [8]. A success-
ful example of this approach is NetAmsa [8,10] (see
Fig. 1). Starting from these promising candidates we
report here the synthesis, biological evaluation and
DNA binding of new combilexine compounds formally
derived from netropsin and distamycin A.
1. Introduction
Cancer arises from normal cells which are trans-
formed into tumour cells because of genetic alterations
causing inhibition of tumour suppressor or activation
of tumour promotor genes [1–3]. In recent years several
compounds have been synthesized with the aim of
controlling such oncogenes [4,5]. In particular, great
interest has been shown in low molecular-weight agents
interacting with double stranded DNA and which also
show sequence selective binding. Distamycin A and
netropsin (see Fig. 1), naturally occurring polyamide
antibiotics, are among the first drugs discovered that
bind sequence-selectively in the minor groove of the
DNA [3,6,7]. The oligopyrrole carbamoyl framework of
distamycin A and netropsin binds reversibly to the
minor groove of the DNA with a strong preference for
AT-rich sequences containing at least four AT-base
pairs [6]. Such oligopyrrole derivatives, and the
analogous oligohydroxypyrrole and oligoimidazole
derivatives, are also called ‘lexitropines’ because of their
ability to read special sequences on the DNA [8,9]. In
2. Chemistry
Following the combilexine approach to DNA li-
gands, we synthesised a series of new compounds with
differing indole or carbazole heterocycles or a nitro
group as the left side building block (X in Figs. 2 and
3). These functions were linked via an amide group to
pyrrole or oligopyrrole chains terminating with diffe-
rent polar or lipophilic functions (Y in Fig. 2).
The synthesis of the oligopyrrole-amides was carried
out according to the method of Lown and Krowicki.
[11]. The commercially available N-methylpyrrole
* Correspondence and reprints.
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.
PII: S0223-5234(02)01349-1

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C. Hotzel et al. / European Journal of Medicinal Chemistry 37 (2002) 367–378
carboxylic acid (1) was nitrated to compound 2, which
was then chlorinated with thionyl chloride and treated
with propionitrile or N,N-dimethylamino propane-1,3-
diamine to give rise to compounds 7 and 8, respectively
(yield: 79, 66%) [12]. The direct esterification of com-
pound 2 to 6 with methanol–H2SO4 gave an unsatis-
fying yield. However, a better yield was achieved with
another strategy: the pyrrole ester 5 was synthesised by
condensation of nitromalone aldehyde 4 with glycine
methylester 4a in sodium hydroxide aquous solution
and methylated to 6 (39%) [13–15]. Compound 6 was
reduced to 6a by the standard method and then acy-
lated with 3 to give rise to the bis-pyrrole 9. The
tris-pyrrole amide building block 14 was synthesised by
the same procedure.
The heterocyclic carbonic acids 10–12 were prepared
by routine nitrogen alkylation with the appropriate
halogen carbonic ester and subsequent hydrolysis [16–
18] (see Fig. 4). The also used 2-(1H-1-indolyl)acetic
acid and the 3-(1H-1-indolyl)propionic acid as coupling
partners are commercially available.
For the final synthetic step to the respective com-
bilexines the compounds 6–9 were used as lexine com-
ponents. After reducing the pyrrolic nitro group of 6–9
the readily formed primary amine group was acylated
with the respective heterocyclic acids (RCO2H) using
the DCC method [19], (see Fig. 5).
The constitution of the N-methyl-pyrrole derivates
were unambigously clarified by the combination of
1
several NMR techniques (for example, H-NOE, pro-
ton decoupling experiments, 13C-spin echo experi-
ments).
3. Biochemical and biological results and discussion
3.1. Cytotoxicity assay
The new potential combilexines were submitted for
cytotoxicity testing to the Developmental Therapeutics
Program of the National Cancer Institute (USA)
[19,20]. On the basis of structural novelty 11 of them—
compounds 9, 14, 15, 17, 18, 20-23, 25, 26—were
chosen initially for prescreening 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). Results of the SRB-Assay for the
determination of the cytotoxicity are reported in Table
1 [21].
Fig. 1. The minor groove binding ligands distamycin A and netropsin, the intercalator amsacrin and as a typical example, the combilexine
NetAmsa.

C. Hotzel et al. / European Journal of Medicinal Chemistry 37 (2002) 367–378
369
3.2. DNA-binding studies
We applied different methods to study the mechanis-
tic principle of the cytotoxic activity of the synthesised
compounds, starting by analysing the interactions with
DNA as a possible molecular target. The general aim
was to determine the occurrence of DNA-binding, the
affinity of the substance for DNA and the mode of
binding [22,23]. An established method for evaluation
of the binding of a compound to DNA is the determi-
nation of the melting curve of dsDNA in the presence
of the potential ligand [24,25]. Compounds which bind
to dsDNA stabilise the double strand and raise the
melting temperature (Tm) of the DNA. The DTm-value,
the difference between the melting temperature of the
DNA in the presence and in the absence of the drug is
related to the DNA binding affinity of the drug. How-
ever, none of the tested compounds showed a signifi-
cant DTm (\5 °C) thus indicating a weak DNA
affinity. This weak DNA binding was also confirmed by
the ethidium bromide displacement assay, which com-
prises fluorimetric titration [26,27]. Adding a DNA
binding drug to the fluorescent complex of DNA and
ethidium bromide results in a decrease of the fluores-
cence of this complex. The concentration of a drug
leading to 50% reduction in the fluorescence intensity of
the ethidium–DNA complex is defined in the literature
as the C50 value and is inversely proportional to the
apparent binding constant value of the drugs [27]. The
displacement assay was validated and evaluated in our
group using test compounds whose affinity is known
[27–29]. It leads to reliable results for qualitative com-
parisons of the DNA binding abilities of a series of
related compounds. Anyway, the tested pyrrole carbox-
amides showed only a weak fluorescence reduction
which may be attributed to quenching effects (Fig. 6)
[30,31].
Fig. 2. Structure of synthesised combilexines and lexins.
3.3. Topoisomerase I inhibition
Compounds which in a 0.1 mM concentration limi-
ted cell growth to 32% or less of the untreated controls
(compounds 14, 15, 17, 20, 26) were submitted to the
NCI 60-cell lines panel screening with the SRB-Assay
in a concentration range from 10−4 to 10−8 M. The
potency of the tested compounds in inhibiting cell
growth was determined in terms of GI50, the concentra-
tion at which growth is inhibited by 50% [21]. The
mean values of GI50 for compounds 14, 15, 17, 20 and
26 and for amsacrine, a commercially available anti-
cancer drug, as a reference, is reported in Table 2. For
all these compounds the GI50 value is above the limit
for further biological screening at the NCI. Anyway
compound 14 shows an interesting specific cytotoxicity
on the ovarian cancer cell line IGROV1 (GI50 1.47×
10−8 M).
Another interesting target of DNA binding ligands
like distamycin A and netropsin is topoisomerase I
[32,33]. Topoisomerase I introduces transient nicks in
DNA at specific sites, leading to relaxation of the DNA
helix. Incubation of the DNA–topoisomerase I mixture
in the absence of and in the presence of the test drugs
results in different population distributions of topoiso-
mers which can be revealed by agarose gel elec-
trophoresis [34]. Four of the five cytotoxic active
compounds (see Table 1) showed inhibition of the
catalytic activity of the topoisomerase I in different
concentrations. Comparison of the obtained plasmid
DNA forms (supercoiled or relaxed) in Fig. 7 indicated
the ability of some of the oligopyrrole derivatives to
inhibit the enzyme. Inhibition of topoisomerase I is
revealed in lines 3 and 4 (camptothecin as a reference

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C. Hotzel et al. / European Journal of Medicinal Chemistry 37 (2002) 367–378
Fig. 3. Synthesis of the building block bis-pyrrole 9.
compound), in line 7 (compound 26 at the concentra-
tion of 20 mM), in line 9 (compound 17 at the concen-
tration of 5 mM), in line 13 (compound 20 at the
concentration of 20 mM) and in line 18 (compound 14
at the concentration of 5 mM).
Moreover, the substitution of the aldehyde group of
distamycin A with a nitro group as well as the substitu-
tion of the guanidine terminal with an ester prevent
compound 14 from binding to the DNA, showing the
importance of H-bond-donor and acceptor groups for
DNA interactions.
Our research on these compounds will continue by
modifying and developing the most promising cytotoxic
active compounds. Moreover, a variety of new candi-
dates are in synthetic development in our laboratory
including also nucleobase-linked oligopyrroles for more
systematic structure activity studies.
The cytotoxic active compound 15 showed no inhibi-
tion of topoisomerase I (results not reported).
4. Conclusion
None of the new compounds appeared to be real
combilexins on the basis of the present DNA-binding
studies. However, a variety of the new pyrrole carbo-
xamides were probably able to inhibit the catalytic
activity of the topoisomerase I. This characteristic
seems to be in correlation with the cytotoxic activity
determined at the NCI. Four of the five active com-
pounds in the three cell lines preliminary screening
showed topoisomerase I inhibition in concentrations
from 5 to 50 mM. Due to the great structural differences
in the active compounds, a rigorous determination of
structure–activity relationships is not possible at the
moment. However, it seems that cyanoalkyl side chains
are deleterious for any activity (see compounds 18, 19).
Fig. 4. Prepared acids as chromophore building blocks.

C. Hotzel et al. / European Journal of Medicinal Chemistry 37 (2002) 367–378
371
Fig. 5. Preparation of the potential combilexins 16–27 starting from compound 9.
5. Experimental
5.1.2. 1-Methyl-4-nitropyrrole-2-carboxylic acid methyl
ester (6) [12]
Compound 2 (5 g, 29.4 mmol) was dissolved in
MeOH (50 mL) and concentrated H2SO4; the mixture
was heated under reflux for 12 h. Water was added and
the mixture was extracted with CHCl3. The residue was
chromatographed on silica gel (petroleum ether–ethyl
acetate (3:1)). Yellow needles were obtained (2.1 g)
(11.5 mmol) (39%), m.p: 121 °C (lit, m.p.: 120 °C); IR
(KBr) (cm−1): 3200, 3110, 3100, 3040, 1710, 1510,
1485, 1470, 1405, 1395, 1320, 1260, 1240, 1200, 1190,
1050, 1010, 905, 880, 780; 1H NMR (200 MHz)
(CDCl3) l: 3.82 (s, 3H, CH3), 4.04 (s, 3H, CH3), 7.31
5.1. Chemistry
Melting points were measured with a Bu¨chi 510 and
are uncorrected. IR spectra were recorded on a Perkin–
Elmer 1310 infrared-spectrometer using potassium bro-
mide pellets (w in cm−1). 1H NMR, 13C NMR and
NOE- spectra were recorded on a Bruker AC-300 ap-
paratus (300 MHz). The samples were dissolved in
DMSO-d6. The chemical shift values are reported in
parts per million (ppm, l units) and spin–spin coupling
J were listed in Hz. EI-mass spectra were performed
with a Mascom 311-A apparatus. C, H, N-analysis
were performed. Analyses indicated by the symbols of
the elements or functions were within 90.4% of the
theoretical values.
4
4
(d, 1H, J=1.6 Hz, pyrrole-H 3), 8.05 (d, 1H, J=1.6
Hz, pyrrole-H5). MS m/z: 184 [M+].
5.1.3. Nitromalonic aldehyde (4) [13]
Mucochloric acid (33.8 g, 200 mmol) dissolved in
ethanol (100 mL) was added to a solution of sodium
nitrite (55.2 g) in water (100 mL) during a period of 25
5.1.1. 1-Methyl-4-nitropyrrole-2-carboxylic acid (2)
[11]
Acetic anhydride (8 mL) was treated with 1.6 mL of
70% nitric acid and the mixture cooled to −25 °C and
slowly added to a suspension of 2 g (16 mmol) 1-
methylpyrrole-2-carboxylic acid (1) in 12 mL of acetic
anhydride. The mixture was stirred for 0.5 h, then the
temperature was allowed to rise to the ambient and
stirring was continued for 20 min. The mixture was
cooled again to −25 °C and the resulting precipitate
collected in a funnel cooled in dry ice. The solid was
washed with a small quantity of cold acetic anhydride
(−25 °C) and then with acetic anhydride–CCl4 1:1
(−25 °C) and then CCl4 and hexane. The obtained
residue was purified on silica gel (petroleum ether–ethyl
acetate (1:1)). Yellow amorphous compound was ob-
tained (6.5 mmol, 1.1 g) (41%), melting point (m.p.)
204 °C (lit. m.p. 201–201.5 °C); IR (KBr) (cm−1):
3140, 3090, 3010, 2870, 1700, 1580, 1520, 1450, 1420,
1400, 1360, 1320, 1270, 1200, 1130, 1090, 850, 825, 750,
Table 1
Cytotoxic activity of compounds 9, 14, 15, 17, 18, 20-23, 25, 26 in the
primary three cell lines NCI-H460 (lung carcinoma), MCF-7 (breast
carcinoma) and SF 268 (glioma) assay
Compound 0.1 mM
Growth percentages
NCI-H460
MCF7
SF-268
9
104
14
31
14
86
−2
109
102
113
93
74
62
24
31
53
12
90
91
86
84
13
97
59
41
7
76
23
93
62
83
96
36
14 a
15 a
17 a
18
20 a
21
22
23
25
26 a
10
1
610, 480; H NMR (200 MHz) (CDCl3) l: 3.9 (s, 3H,
4
CH3), 7.23 (d, 1H, J=2.0 Hz, pyrrole-H3), 8.22 (d,
The values are reported as cell growth percentages.
a Further screening.
4
1H, J=2.0 Hz, pyrrole-H5). MS m/z: 170 [M+].

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C. Hotzel et al. / European Journal of Medicinal Chemistry 37 (2002) 367–378
Table 2
Mean GI50 of compounds 14, 15, 17, 20, 26 and of amsacrine for comparison, expressed as log 10 of the molar concentration
Compound
Mean GI50
14
15
17
20
26
Amsacrine
−4.27
−4.19
−4.36
−4.47
−4.52
−6.28
Fig. 6. Ethidium bromide displacement effect of the cytotoxic active compounds 14, 15, 17, 20, 26 and distamycin A in relation to DMSO.
Fig. 7. Topoisomerase I inhibition assay for compounds 14, 17, 20, 23, 26. Line 1: Form I DNA, Line 2: Form IV DNA, Lines 3, 4: Camptothecin
20 mM; Lines 5, 6, 7: 26; Lines 8, 9, 10: 17; Lines 11, 12, 13: 20; Lines 14, 15, 16: 23; Lines 17, 18, 19: 14. Each compound was tested at 0.5, 5
and 20 mM concentration.
min. Some external cooling was necessary to keep the
temperature from rising above 60 °C. After stirring for
a further 35 min and cooling overnight, the formed
solid was filtered and extracted with boiling ethanol
(70 mL). After rotating in vacuum the total yield was
3.2 g. Orange amorphous powder (2.8 g, 24 mmol,
12%), m.p: 124 °C (lit. m.p.: 124 °C); IR (KBr)
(cm−1): 3510, 3440, 3370, 3290, 1650, 1600, 1570, 1530,
1480, 1380, 1350, 1270, 930, 820, 760, 530, 490; 1H
NMR (300 MHz) (DMSO-d6) l: 8.4 (s, 1H, enole-H),
9.7 (s, 1H, aldehyde-H), 12.0 (1H, s, OH); MS m/z: 116
[M+].
5.1.4. 4-Nitro-pyrrole-2-carboxylic methyl ester (5)
[14]
Equimolar quantities of sodium nitromalonic alde-
hyde (4) (1.3 g, 11.15 mmol) and of glycine methyl ester
(4a, 1.4 g, 11.15 mmol) were dissolved in 6–7 mL of
warm of 65% ethanol. 40 drops of a 20% sodium
hydroxide solution were added and the red reaction
mixture set aside for half an hour at a constant tempe-
rature of 50 °C. Then the mixture was added to water
and the resulting yellow precipitate was extracted se-
veral times with ether. The diethylether-phase was dried
and concentrated in vacuum. The residue was chro-

C. Hotzel et al. / European Journal of Medicinal Chemistry 37 (2002) 367–378
373
matographed on silica gel (petroleum ether–ethyl ace-
5.1.7. 1-Methyl-4-nitropyrrole-2-carboxamido
(-3-(N,N-dimethyl))propanamine (8) [11]
tate (3:1)). Yellow amorphous powder was obtained
(1.35 g) (7.94 mmol) (71%); m.p. 197 °C (lit. mp.
197 °C); IR (KBr) (cm−1): 3280, 3260, 3140, 1700,
1560, 1500, 1450, 1440, 1410, 1385, 1340, 1325, 1260,
1215, 1180, 1145, 1090, 980, 960, 930, 850, 840, 820,
A solution of 0.7 g (4.1 mmol) 2 and a slight excess
of SOCl2 in dry THF was heated under reflux for 5
min. The excess of SOCl2 and the solvent were removed
under reduced pressure and the evaporation was re-
peated with some dry THF. The residue was dissolved
in THF and cooled to −20 °C. A solution of 0.6 mL
(490 mg, 4.8 mmol) N,N-dimethyl-1,3-propandiamine
and 0.8 mL Hu¨nig’s base in 10 mL THF was added
dropwise. The temperature was allowed to rise to the
ambient and then the solvent was removed in vacuo.
The residue was chromatographed on neutral alu-
minium oxide (methanol–ethyl acetate (1:4)). Colour-
less crystals were obtained (688 mg, 2.7 mmol) (66%),
m.p. 125 °C (lit. m.p: 125.5–127 °C). IR (KBr)
(cm−1): 3420, 2720, 2340, 1650, 1540, 1530, 1490, 1470,
1
800, 780, 750; H NMR (200 MHz) (CDCl3) l: 3.85 (s,
4
3H, CH3), 7.28 (d, 1H, J=1.6 Hz, pyrrole-H3), 8.96
4
(d, 1H, J=1.6 Hz, pyrrole-H5); MS m/z: 170 [M+].
5.1.5. 1-Methyl-4-nitropyrrole-2-carboxylic methyl
ester (6) [15]
A solution of 5 (300 mg, 1.75 mmol) in dry THF (15
mL) was added dropwise to a suspension of hot THF
(20 mL) and potassium (200 mg, 5.1 mmol). After
stirring the mixture on reflux for 1 h, iodomethane (250
mg, 141.91 mmol) (0.1 mL) in dry THF (2 mL) was
added dropwise. The reaction mixture was stirred under
reflux for another 1 h. Methanol was added to destroy
the potassium; the THF was removed in vacuum and
then soluted in diethylether. The diethylether was dried
over magnesium sulfate and removed in vacuum. The
residue was chromatographed on silica gel (petroleum
ether–ethyl acetate (3:1)). Yellow amorphous powder
was obtained (231 mg, 1.25 mmol, 72%); m.p: 121 °C
(lit. m.p.: 120 °C); IR (KBr) (cm−1): 3150, 1710, 1570,
1510, 1500, 1445, 1420, 1400, 1370, 1340, 1320, 1260,
1
1410, 1305, 1265, 1215, 1035, 820, 755; H NMR (300
3
MHz) (DMSO-d6) l: 1.6 (m, 2H, J=6.5 Hz, CH2),
2.05 (s, 6H,
CH2N(Me)2), 3.18 (q, 2H, J=6.7 Hz, CH2N), 3.77 (s,
2
CH3), 2.2 (t, 2H, 3J=6.7 Hz,
3
4
3H, CH3), 7.38 (d, 1H, J=1.6 Hz, pyrrole-H3), 8.09
4
3
(1H, d, J=1.6 Hz, pyrrole-H5), 8.40 (t, 1H, J=7.4
Hz, NH); MS m/z: 254 [M+].
5.1.8. Methyl-1-methyl-4-(1-methyl-4-nitropyrrole-2-
carboxamido)pyrrole-2-carboxylate (9) [12]
A solution of 6 (200 mg) (1.08 mmol) was hydro-
genated over 60 mg of 10% Pd on charcoal in 10 mL of
dry methanol at atmospheric pressure and room tem-
perature (r.t.). After 3 h of reduction the catalyst was
removed by filtration, the solvent evaporated under
reduced pressure, and then 0.5 mL of Hu¨nig’s base in 3
mL of dry THF was added. The mixture was cooled to
−20 °C and treated with a solution of 1-methyl-4-ni-
tropyrrole-2-carboxylic acid chloride (3). Compound 3
was prepared as above by heating 200 mg (1.18 mmol)
of compound 2 under reflux with SOCl2 and dissolved
in THF. After 30 min of stirring at r.t. the mixture was
evaporated to dryness. The residue was chro-
matographed on silica gel (methanol–ethyl acetate
(1:4)). A yellow amorphous solid was obtained. (228
mg, 0.75 mmol) (69%), m.p: 225 °C (lit. m.p.: 225 °C);
IR (KBr) (cm−1): 3370, 3140, 2980, 2500, 1690, 1660,
1565, 1500, 1480, 1440, 1390, 1320, 1250, 1210, 1120,
1100, 845, 815, 780, 750; 1H NMR (300 MHz) (DMSO-
d6) l: 3.72 (s, 3H, CH3), 3.83 (s, 3H, CH3), 3.96 (s, 3H,
1
1200, 1120, 1090, 985, 945, 860, 830, 800, 765, 750; H
NMR (200 MHz) (CDCl3) l: 3.82 (s, 3H, CH3), 4.04 (s,
4
3H, CH3), 7.31 (d, 1H, J=1.6 Hz, pyrrole-H3), 8.05
4
(d, 1H, J=1.6 Hz, pyrrole-H5); MS m/z: 184 [M+].
5.1.6. 1-Methyl-4-nitropyrrole-2-carboxamide
propionitrile (7) [11]
A solution of 7 g (41.14 mmol) of 2 and a slight
excess of SOCl2 in 20 mL of dry THF was heated under
reflux for 5 min. The excess of SOCl2 and the solvent
were removed under reduced pressure and the evapora-
tion was repeated with some anhydrous THF. The
residue was dissolved in THF and cooled to −20 °C.
A solution of 3.12 mL (2.97 g, 42.37 mmol) of
aminopropionitrile and 7.8 mL of Hu¨nig’s base in 10
mL of THF was added. The temperature was allowed
to rise to the ambient, then the solvent was removed in
vacuo, water was added, and the resulting crystalline
solid collected. A yellow amorphous powder was ob-
tained (7.22 g, 32.5 mmol, 79%), m.p.: 132 °C (lit. m.p.:
132–133 °C); IR (KBr) (cm−1): 3310, 3115, 2875,
2240, 1640, 1550, 1525, 1410, 1320, 1140, 1110, 1070,
4
CH3), 6.86 (1H, d, J=1.8 Hz, pyrrole-H3), 7.45 (d,
4
4
1H, J=1.7 Hz, pyrrole-H5), 7.53 (d, 1H, J=1.8Hz,
pyrrole-H3%), 8.18 (d, 1H, 4J=1.7 Hz, pyrrole-H5%),
10.28 (s, 1H, NH); MS m/z: 306 [M+]; Anal. C13H14N4
(C, H, N).
1
855, 750; H NMR (200 MHz) (CDCl3) l: 2.65 (t, 2H,
3
3J=6.5 Hz, CH2ꢀCN), 3.5 (2H, q, J=6,5 Hz), 3.9 (s,
4
3H, CH3), 7.25 (d, 1H, J=1.6 Hz, pyrrole-H3), 7.8 (d,
5.1.9. 2-(1H-1-Indolyl)acetic acid (10) [16]
Molten indole (22 g, 188 mmol) was stirred with
finely powdered potassium hydroxide (17 g, 425.1
4
1H, J=1.6 Hz, pyrrole-H5), 8.05 (s, 1H, NH); MS
m/z: 222 [M+].

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C. Hotzel et al. / European Journal of Medicinal Chemistry 37 (2002) 367–378
mmol) in a fractionating flask (200 mL capacity) in-
serted deeply in a salt bath kept at 200–250 °C. The
stirring was continued until no more water has been
formed (6–8 h). The flask was removed from the bath
and allowed to cool a little. Dry chloroacetate (36 g)
(285 mmol) was added and the flask was heated at
140–150 °C for 4 h with occasional shaking, and
cooled. A solution of potassium hydroxide (25 g) in
water (75 mL) was added, the mixture heated on a
waterbath for 3 h and then poured into water (250 mL).
The formed crystalline product was separated and
cooled, washed, dried, dissolved in ether, and the solu-
tion filtered through a short column of aluminium
oxide and then evaporated to dryness. The crude
product acid was dissolved in hot ethanol; on cooling
the acid light brown needles were obtained (3.95 g,
22.56 mmol, 12%) m.p: 175 °C (lit. mp.: 174–179 °C);.
IR (KBr) (cm−1): 3420, 3200, 3110, 3100, 3040, 1710,
1610, 1510, 1485, 1470, 1405, 1395, 1340, 1320, 1260,
1240, 1200, 1200, 1190, 1125, 1050, 1010, 905, 880, 780,
740; 1H NMR (200 MHz) (CDCl3) l: 4.63 (s, 2H,
mmol, 17%); m.p.: 150 °C (lit. m.p.: 150 °C); IR
(KBR) (cm−1): 3100, 2930, 2860, 2330, 1680, 1565,
1460, 1430, 1380, 1350, 1320, 1300, 1230, 1210, 1190,
1
1150, 1120, 110, 900, 725, 700; H NMR (300 MHz)
3
(DMSO-d6) l: 1.95 (m, 2H, J=6.5 Hz, CH2), 2.25 (t,
3
3
2H, J=6.4 Hz, CH2), 4.4 (t, 2H, J=6.6 Hz, CH2),
3
7.2 (d, 2H, J=6.3 Hz, carbazole-H1,8), 7.4 (pt, 2H,
3J=6.9 Hz, carbazole-H2,7), 7.6 (pt, 2H, J=6.9 Hz,
3
carbazole-H3,6), 8.1 (d, 2H, 3J=7.0 Hz, carbazole-
H4,5), 11.5 (s, 1H, COOH); MS m/z: 253 [M+].
5.1.12. 1-Methyl-4-{[(1-methyl-4-nitro-1H-2-propyl)-
carbonyl] aminoc-1-H-2-pyrrole carboxylic acid (13)
[11]
To 1 g of 9 (3.27 mmol) dissolved in methanol (30
mL) a solution of 50% potassium hydroxide (10 mL)
was added, and the mixture was heated under reflux for
12 h; HCl (conc.) was added dropwise, until pH was 1.
The precipitate was filtered, washed with water, soluted
in CH2Cl2 and evaporated a few times. A yellow amor-
phous solid was obtained, (0.82 g, 2.8 mmol)(86%);
m.p.: 231 °C (lit. m.p.: 233–234 °C); IR (KBr) (cm−1):
3040, 3010, 1640, 1550, 1480, 1430, 1400, 1300, 1280,
1230, 1190, 1100, 800; 740; 1H NMR (300 MHz)
(DMSO-d6) l: 3.9 (s, 3H, CH3), 3.95 (s, 3H, CH3), 6.9
3
CH2), 6.48 (d, 1H, J=7.8 Hz, indole-H3), 7.0 (pt, 1H,
3
3J=7.6 Hz, 1H, indole-H 5 or 6), 7.1 (pt, 1H, J=7.7
3
Hz, indole-H 5 or 6), 7.32 (d, 1H, J=7.6 Hz, indole-
3
H2), 7.34 (d, 1H, J=7.7 Hz, indole-H7), 7.58 (d, 1H,
3J=7.9 Hz, indole-H2); MS m/z: 175 [M+].
(d, 1H, J=1.8 Hz, pyrrole-H3), 7.4 (d, 1H, J=1.7
4
4
4
Hz, pyrrole-H5), 7.5 (d, 1H, J=1.8 Hz, pyrrole-H3%),
5.1.10. 2-(9H-9-Carbazolyl)acetic acid (11) [17]
8.1 (d, 1H, 4J=1.7 Hz, pyrrole-H5%), 10.25 (s, 1H,
COOH); MS m/z: 292 [M+]; Anal. C12H12N4O5 (C, H,
N).
Molten carbazole (20 g, 119.76 mmol) was stirred
with fine powdered potassium hydroxide (15 g, 441.3
mmol) until no more steam was generated. After cool-
ing and powdering the solid, dry ethyl-2-chloroacetate
was added. The mixture was heated at 180 °C for 4 h.
Then water was added (100 mL); HCl (1 N) was added
dropwise until precipitation occurred; the obtained
solid was dissolved in CH2Cl2 and evaporated to dry-
ness a few times. Brown amorphous solid (3.77 g, 16.77
mmol, 14%); m.p.: 192 °C (lit. mp.: 193–194 °C); IR
(KBr) (cm−1): 3480, 3400, 2800, 2350, 1600, 1480,
5.1.13. Methyl-1-methyl-4-{[(1-methyl-4-{[(1-methyl-
4-nitro-1H-2-pyrrol)carbonyl]amino}-1H-2-pyrrolyl)-
carbonyl]amino}-1H-2-pyrrole carboxylate (14) [12]
A solution of 9 (612 mg) (2 mmol) in dry methanol
(10 mL) was hydrogenated over 200 mg of Pd on
charcoal. After 9 was reduced, the catalyst was re-
moved by filtration, the solvent evaporated under re-
duced pressure, and then 1 mL of Hu¨nig’s base in 3 mL
dry THF was added. The mixture was cooled to −
20 °C and treated with a solution of the acid chloride
from 2 (340 mg) (2 mmol) in 5 mL of dry THF. After
30 min of stirring at r.t. the mixture was evaporated to
dryness. The residue was chromatographed on silica gel
(petroleum ether–ethyl acetate (1:1). A yellow amor-
phous solid was obtained (360 mg, 0.84 mmol, 42%),
m.p: 228 °C; IR (KBr) (cm−1): 3030, 2970, 1700, 1640,
1
1450, 1425, 1400, 1305, 1215, 1070, 1000, 750, 725; H
NMR (300 MHz) (DMSO-d6) l: 4.6 (s, 2H, CH2), 7.1
(pt, 2H, 3J=7.2 Hz, carbazole-H1,8), 7.3 (m, 4H,
3
3J=7.3 Hz, carbazole-H2,3,6,7), 8.1 (pt, 2H, J=7.3
Hz, carbazole-H4,5); MS m/z: 225 [M+].
5.1.11. 4-(9H-9-Carbazolyl)butanoic acid (12) [18]
A mixture of carbazole (20 g, 119.76 mmol) and
potassium hydroxide (10 g, 294.2 mmol) was finely
powdered and made molten (260 °C). The solid carba-
zole-potassium was again powdered after cooling. Then
g-butyrolactone (300 g, 3.488 mol) was added and
heated (180–200 °C) for 10 h. The mixture was cooled
in ice, precipitated carbazole was filtered off and the
product was chromatographed on neutral aluminium
1
1550, 1430, 1390, 1300, 1250, 1200, 1100, 1050, 790; H
NMR (300 MHz) (DMSO-d6) l: 3.7 (s, 3H, CH3), 3.8
(s, 3H, CH3), 3.9 (s, 3H, CH3), 4.1 (s, 3H, CH3), 6.9 (d,
4
4
1H, J=1.7 Hz, pyrrole-H), 7.05 (d, 1H, J=1.6 Hz,
4
pyrrole-H), 7.3 (d, 1H, J=1.7 Hz, pyrrole-H), 7.4 (d,
4
4
1H, J=1.6 Hz, pyrrole-H), 7.6 (d, 1H, J=1.7 Hz,
4
pyrrole-H), 8.1 (d, 1H, J=1.6 Hz, pyrrole-H), 9.9 (s,
oxide
(petroleum
ether–ethylacetate–methanol
1H, NH), 10.25 (s, 1H, NH), 10.4 (s, 1H, NH); MS
(10:10:1)); brown needles were obtained, (5.15 g, 20.36
m/z: 428 [M+]; Anal. C19H20N6O6 (C, H, N).

C. Hotzel et al. / European Journal of Medicinal Chemistry 37 (2002) 367–378
375
5.1.14. General procedure for the synthesis of the
pyrrole- and the oligopyrrole carboxamides
phous solid (1.13 g, 3.62 mmol, 78%), m.p.: 195 °C; IR
(KBr) (cm−1): 3380, 3300, 3050, 3010, 2910, 2860,
2800, 1710, 1660, 1630, 1610, 1605, 1595, 1580, 1490,
1460, 1450, 1405, 1380, 1350, 1330, 1265, 1240, 1210,
Four mmol of the carboxylic acid were dissolved in
40 mL CH2Cl2. Then 4.4 mmol of the pyrrole amine
(prepared from the nitropyrrole analogues by hydro-
genation in methanol over Pd on charcoal) and 0.1
mmol (12 mg) of dimethylaminopyridin (DMAP) were
added. The mixture was cooled to −15 °C. A second
solution of 4.8 mmol (1.2 g) dicyclohexylcarbodiimide
(DCC) in 10 mL CH2Cl2 was added dropwise. After
stirring for 24 h the precipitate a dicyclohexyl urea was
removed by filtration. The solution was evaporated to
dryness, and the residue was chromatographed on silica
gel (petroleum ether–ethyl acetate (1:1)).
1
1200, 1150, 1130, 1120, 1100, 1060, 750; H NMR (75
MHz) DMSO-d6): 3.70 (s, 3H, CH3), 3.77 (s, 3H, CH3),
4
5.17 (s, 2H, CH2), 6.7 (d, 1H, J=2.0 Hz, pyrrole-H3),
3
7.20 (pt, 2H, J=7.8 Hz, carbazole-H3,6), 7.29 (d, 1H,
4J=2.8 Hz, pyrrole-H5), 7.42 (pt, 2H, 3J=7.5 Hz,
carbazole-H2,7), 7.5 (d, 2H, 3J=8.2 Hz, carbazole-
3
H1,8), 8.1 (d, 2H, J=7.7 Hz, carbazole-H4,5), 10.4 (s,
1H, NH); 13C NMR (75 MHz) (DMSO-d6) l: 36.5 (p),
44.5 (s), 52.8 (p), 108.0 (t), 109.8 (2t), 119.4 (t), 119.7
(2t), 120.5 (2t), 122.5 (4q), 126.1 (2t), 141.2 (q), 141.3
(q), 164.9 (q), 169.6 (q); MS m/z: 361 [M+]; Anal.
C21H19N3O3 (C, H, N).
5.1.14.1. Methyl-4-{[2-(1H-3-indolyl)acetyl]amino}-1-
methyl-1H-2-pyrrole carboxylate (15). Brown needles
(896 mg, 2.88 mmol, 72%), m.p.: 155 °C; IR (KBr)
(cm−1): 3400, 3330, 3270, 3110, 3040, 2900, 2870, 1700,
1650, 1575, 1480, 1450, 1400, 1350, 1315, 1260, 1215,
5.1.14.4. N2-(2-Cyanoethyl)-4-{[2-(1H-3-indolyl)acet-
yl]amino}-1-methyl-1H-2-pyrrole carboxamide (18). Or-
ange amorphous solid (586 mg, 1.68 mmol, 42%), m.p.:
126 °C; IR (KBr) (cm−1): 3400, 3330, 3270, 3100,
3040, 2920, 2850, 2790, 2400, 2220, 1720, 1650, 1620,
1570, 1520, 1430, 1400, 1310, 1290, 1270, 1240, 1215,
1
1195, 1140, 1100, 1060, 1015, 750; H NMR (300 MHz)
(DMSO-d6) l: 3.67 (s, 2H, CH2), 3.75 (s, 3H, CH3),
4
3.85 (s, 3H, CH3), 6.72 (d, 1H, J=1.88 Hz, pyrrole-
3
1
H3), 6.9 (pt, 1H, J=7 Hz, indole-H5 or 6), 7.0 (pt,
1150, 1085, 1060, 1005, 890, 800, 780, 740; H NMR
3
4
1H, J=6.9 Hz, indole-H5 or 6), 7.2 (d, 1H, J=1.8
(300 MHz) (DMSO-d6) l: 2.7 (t, 2H, 3J=6.4 Hz,
CH2), 3.39 (q, 2H, 3J=6.4 Hz, CH2), 3.67 (s, 2H,
CH2), 3.75 (s, 3H, CH3), 6.7 (d, 1H, 4J=1.8 Hz,
3
Hz, pyrrole-H5), 7.3 (d, 1H, J=7.9 Hz, indole-H7),
7.34 (s, 1H, indole-H2), 7.5 (d, 1H, J=7.9 Hz, indole-
3
H4), 10.0 (s, 1H, NH), 10.8 (s, 1H, indole-NH); 13C
NMR (75 MHz) (DMSO-d6) l: 33.0 (s), 36.5 (p), 51.3
(p), 108.0 (t), 109.5 (q), 111.8 (t), 118.9 (t), 119.0 (t),
120.6 (t), 121.5 (t), 123.6 (q), 124.3 (t), 127.4 (q), 136.8
(q), 160.9 (q), 168.8 (q); MS m/z: 311 [M+]; Anal.
C17H17N3O3 (C, H, N).
pyrrole-H3), 6.96 (pt, 1H, J=7.1 Hz, indole-H5 or 6),
3
3
7.04 (pt, 1H, J=6.9 Hz, indole-H5 or 6), 7.1 (d, 1H,
4J=1.9 Hz, pyrrole-H5), 7.2 (s, 1H, indole-H2), 7.32
(d, 1H, 3J=7.8 Hz, indole-H7), 7.5 (d, 1H, 3J=7.7 Hz,
indole-H4), 9.9 (s, 1H, NH), 10.8 (s, 1H, NH); 13C
NMR (75 MHz) (DMSO-d6) l: 32.0 (s), 32.4 (s), 35.2
(s), 37.4 (p), 107.9 (t), 113.2 (q), 116.1 (t), 123.1 (t),
123.8 (t), 124.0 (q), 126.3 (2t), 127.0 (q), 128.9 (t), 132.5
(q), 140.6 (q), 165.1 (q), 168.1 (q); MS m/z: 349 [M+];
Anal. C19H19N5O2 (C, H, N).
5.1.14.2. Methyl-4-{[2-(1H-1-indolyl)acetyl]amino}-1-
methyl-1H-2-pyrrole carboxylate (16). Orange amor-
phous solid (310 mg, 1 mmol, 25%), m.p. 160 °C; IR
(KBr) (cm−1): 3560, 3480, 3400, 3340, 3270, 3195,
3120, 3050, 2980, 2950, 1700, 1650, 1590, 1505, 1260,
1445, 1400, 1365, 1330, 1310, 1261, 1240, 1200, 1140,
5.1.14.5. N2-(2-Cyanoethyl)-4-{[2-(1H-1-indolyl)acet-
yl]amino}-1-methyl-1H-2-pyrrole carboxamide (19).
Brown amorphous solid (628 mg, 1.8 mmol, 45%),
m.p.: 109 °C; IR (KBr) (cm−1): 3600, 2550, 3450, 3380,
3320, 3010, 2920, 2850, 2770, 2680, 2300, 2220, 1960,
1900, 1720, 1650, 1620, 1570, 1510, 1440, 1400, 1360,
1310, 1270, 1240, 1200, 1150, 1085, 1010, 960, 890, 850,
1
1100, 1060, 1010, 960, 825, 790; H NMR (300 MHz)
(DMSO-d6) l: 3.70 (s, 3H, CH3), 3.78 (s, 3H, CH3), 4.9
4
(s, 2H, CH2), 6.4 (d, 1H, J=2.7 Hz, pyrrole-H3), 6.7
3
3
(d, 1H, J=7.7 Hz, indole-H3), 7.0 (pt, 1H, J=7.3
Hz, indole-H5 or 6), 7.1 (pt, 1H, 3J=7.8 Hz, indole-H5
or 6), 7.35 (d, 1H, J=3.0 Hz, pyrrole-H5), 7.38 (d,
1H, J=7.2, indole-H2), 7.5 (d, 1H, J=7.4 Hz, in-
4
1
805, 780, 760, 750, 710; H NMR (300 MHz) (DMSO-
3
3
d6) l: 2.68 (t, 2H, 3J=6.4 Hz, CH2), 3.37 (q, 2H,
3J=6.4 Hz, CH2), 3.75 (s, 3H, CH3), 4.9 (s, 2H, CH2),
6.42 (d, 1H, 4J=3.1 Hz, pyrrole-H3), 6.76 (d, 1H,
3
dole-H7), 7.54 (d, 1H, J=7.7 Hz, indole-H7), 10.3 (s,
1H, NH); 13C NMR (75 MHz) (DMSO-d6) l: 36.0 (p),
49.3 (s), 51.1 (p), 105.3 (t), 113.0 (t), 115 (t) 121.6 (q),
122.1 (t), 124.2 (t) 125.1 (t), 125.3 (t), 127.0 (q), 133.1
(q), 135.8 (t) 141.1 (q), 166.3 (q), 171.0 (q); MS m/z:
311 [M+]; Anal. C17H17N3O3 (C, H, N).
3
4J=1.6 Hz, indole-H3), 7.0 (pt, 1H, J=7.5 Hz, in-
3
dole-H5 or 6), 7.1 (pt, 1H, J=6.9 Hz,indole-H5 or 6),
7.34 (d, 1H, 4J=1.9 Hz, pyrrole-H5), 7.36 (d, 1H,
3
3J=8.1 Hz, indole-H2), 7.39 (d, 1H, J=7.6 Hz, in-
3
dole-H7) 7.54 (d, 1H, J=7.9 Hz, indole-H4), 8.34 (s,
5.1.14.3. Methyl-4-{[2-(9H-9-carbazolyl)acetyl]amino}-
1-methyl-1H-2-pyrrole carboxylate (17). Brown amor-
1H, NH), 10.25 (s, 1H, NH); 13C NMR (75 MHz)
(cm−1),: 32.0 (s), 32.8 (s), 35.1 (s), 44.1 (s), 107.9 (t),

376
C. Hotzel et al. / European Journal of Medicinal Chemistry 37 (2002) 367–378
113.2 (q), 116.1 (t), 123.1 (t), 123.8 (t), 124.0 (q), 126.3
(2t), 127.0 (q), 128.9 (t), 132.5 (q), 140.6 (q), 165.1 (q),
168.1 (q); MS m/z: 349 [M+]; Anal. C19H19N5O2 (C, H,
N).
1020, 850, 780, 720, 650; 1H NMR (300 MHz) (DMSO-
3
3
d6) l: 2.7 (t, 2H, J=7.4 Hz), 2.9 (t, 2H, J=7.5 Hz,
CH2), 3.8 (s, 3H, CH3), 3.9 (s, 3H, CH3), 6.8 (d, 1H,
4J=1.9 Hz, pyrrole-H3), 6.9 (pt, 1H, 3J=7.8 Hz,
3
indole-H5 or 6), 7.05 (pt, 1H, J=7.9 Hz, indole-H5 or
4
5.1.14.6. Methyl-4-{[4-{[2-(1H-3-indolyl)acetyl]amino}-
1- methyl-1H-2-pyrrolyl)carbonyl}-1-methyl-1H-2-pyr-
role carboxylate (20). Yellow needles (675 mg, 1.56
mmol, 39%), m.p.: 173 °C; IR (KBr) (cm−1): 3600,
3450, 3380, 3320, 3240, 3160, 3080, 3000, 2930, 2850,
2780, 2300, 2100, 2000, 1700, 1650, 1620, 1570, 1530,
1435, 1400, 1350, 1310, 1250, 1200, 1150, 1105, 1080,
6), 7.15 (s, 1H, indole-H2), 7.25 (d, 1H, J=1.8 Hz,
3
pyrrole-H5), 7.3 (d, 1H, J=7.9 Hz, indole-H7), 7.5 (d,
3
1H, J=7.8 Hz, indole-H4), 10.3 (s, 1H, NH), 10.8 (s,
1H, NH); 13C NMR (75 MHz) (DMSO-d6) l: 25.8 (s),
33.2 (s), 36.5 (p), 51.3 (p), 108.0 (t), 109.4 (q), 111.6 (t),
115.1 (q), 118.9 (t), 119.1 (t), 120.5 (t), 121.5 (t), 123.8
(q), 124.5 (t), 127.5 (q), 136.9 (q), 160.6 (q), 171.4 (q);
MS m/z: 325 [M+]; Anal.: C18H19N3O3 (C, H, N).
1
1060, 1005, 960, 890, 820, 795, 755, 745, 660; H NMR
(300 MHz) (cm−1) l: 3.63 (s, 2H, CH2), 3.71 (s, 3H,
CH3), 3.78 (s, 3H, CH3), 3.81 (s, 3H, CH3), 5.5 (d, 1H,
3J=8.0 Hz, indole-H2), 6.8 (d, 1H, 4J=1.8 Hz,
5.1.14.9. Methyl 4-[2-(4-{[3-(1H-indolyl)propynolyl]-
amino}-1-methyl-1H-2-pyrrolyl)-2-oxoethyl]-1-methyl-
1H-2-pyrrole carboxylate (23). Orange amorphous solid
(805 mg, 1.8 mmol, 45%), m.p.: 163 °C; IR (KBr)
(cm−1): 3350, 3260, 1700, 1640, 1600, 1550, 1440, 1400,
1350, 1250, 1200, 1100, 750; 1H NMR (300 MHz)
(DMSO-d6) l: 2.7 (t, 2H, 3J=7.4 Hz, CH2), 2.9 (t, 2H,
3J=7.5 Hz, CH2), 3.7 (s, 3H, CH3), 3.8 (s, 3H, CH3),
3
pyrrole-H3), 6.9 (pt, 1H, J=7.9 Hz, indole-H5 or 6),
3
7.0 (pt, 1H, J=7.9 Hz, indole-H5 or 6), 7.1 (d, 1H,
4J=1.4 Hz, pyrrole-H5), 7.2 (d, 1H, 4J=1.6 Hz,
3
pyrrole-H3%), 7.32 (d, 1H, J=8.0 Hz, indole-H4), 7.4
(d, 1H, 4J=1.6 Hz, pyrrole-H5%), 7.5 (d, 1H, 3J=
7.9Hz, indole-H7), 9.9 (s, 1H, NH), 10.8 (s, 1H, NH);
13C NMR (75 MHz) (DMSO-d6) l: 33.6 (s), 36.4 (p),
47.8 (p), 51.2 (p), 104.4 (t), 108.6 (t) 109.2 (q), 111.6
((t), 115.8 (t), 118.5 ((t), 118.7 (t), 121.2 (t), 121.3 (t),
122.4 (q), 122.8 (q), 124.0 (t), 127.5 (q), 136.4 (q), 158.7
(q), 161.1 (q), 168.3 (q); MS m/z: 433 [M+]; Anal.
C23H23N5O4 (C, H, N).
4
3.9 (s, 3H, CH3), 6.8 (d, 1H, J=1.9 Hz, pyrrole-H3),
3
6.9 (pt, 1H, J=7.8 Hz, indole-H5 or 6), 7.05 (pt, 1H,
3J=7.9 Hz, indole-H5 or 6), 7.15 (s, 1H, indole-H2),
3
4
7.3 (d, 1H, J=7.9 Hz, indole-H7), 7.45 (d, 1H, J=
3
pyrrole-H5), 7.5 (d, 1H, J=7.8 Hz, indole-H4), 7.55
4
4
(d, 1H, J=1.7 Hz, pyrrole-H3%), 8.1 (d, 1H, J=1.7
Hz, pyrrole-H5%), 9.8 (s, 1H, NH), 10.3 (s, 1H, NH),
10.9 (s, 1H, NH); 13C NMR (75 MHz) (DMSO-d6) l:
25.4 (s), 33.0 (s), 36.1 (p), 47.1 (p), 51.0 (p), 104.6 (t),
108.1 (t), 108.3 (q), 111.2 (q), 111.6 (t), 113.9 (q), 115.8
(t), 118.0 (q), 118.7 (t), 119.1 (t), 120.4 (t), 121.6 (t),
124.6 (t), 125.6 (q), 127.4 (q), 135.3 (q), 158.5 (q), 165.4
(q), 170.1 (q); MS m/z: 447 [M+]; Anal.: C24H25N5O4
(C, H, N).
5.1.14.7. Methyl-4-{[4-{[2-(1H-1-indolyl)acetyl]amino}-
1-methyl-1H-2-pyrrolyl)carbonyl}-1-methyl-1H-2-pyr-
role carboxylate (21). Brown amorphous solid (658 mg,
1.52 mmol, 38%), m.p.: 152 °C; IR (KBr) (cm−1):
3550, 3480, 3400, 3330, 2970, 1740, 1625, 1590, 1425,
1
1310, 1215, 1190, 1120, 1090, 755, 725, 600; H NMR
(300 MHz) (DMSO-d6) l: 3.7 (s, 3H, CH3), 3.81 (s, 3H,
CH3), 3.83 (s, 3H, CH3), 4.9 (s, 2H s, CH2), 6.4 (d, 1H,
3J=7.8 Hz, indole-H3), 6.8 (d, 1H, 4J=1.7Hz,
5.1.14.10.
Methyl4-{[4-(9H-9-carbazolyl)butanolyl]
3
pyrrole-H3), 7.0 (pt, 1H, J=7.9 Hz, indole-H5 or 6),
amino}-1-methyl-1H-2-pyrrole carboxylate (24). Yellow
amorphous solid (700 mg, 1.8 mmol, 45%), m.p.:
240 °C; IR (KBr) (cm−1): 3320, 2930, 2850, 1700,
1650, 1620, 1570, 1480, 1450, 1400, 1350, 1260, 1190,
3
7.1 (pt, 1H, J=7.9 Hz, indole-H5 or 6), 7.15 (d, 1H,
4J=1.6 Hz, pyrrole-H5), 7.30 (d, 1H, 3J=7.8 Hz,
3
indole-H2), 7.32 (d, 1H, J=7.9, indole-H7), 7.38 (d,
4
4
1
1H, J=1.9 Hz, pyrrole-H3%), 7.4 (d, 1H, J=1.8 Hz,
1140, 1100, 780, 750; H NMR (300 MHz) (cm−1) l:
3
3
3
pyrrole-H5%), 7.45 (d, 1H, J=7.8 Hz, indole-H4), 9.9
2.0 (t, 2H, J=6.5 Hz), 2.3 (q, 2H, J=6.9 Hz, CH2),
(s, 1H, NH), 10.35 (s, 1H, NH); 13C NMR (75 MHz)
(DMSO-d6) l: 36.8 (p), 46.9 (p), 51.8 (p), 58.9 (s),104.4
(t), 108.9 (t) 109.3 (q), 111.6 (t), 115.8 (t), 118.5 (t),
118.7 (t), 120.6 (t), 121.1 (t), 122.9(t), 123.3 (q), 124.0
(t), 127.9(q), 135.9 (q), 158.6 (q), 161.2 (q), 168.7 (q);
MS m/z: 433 [M+]; Anal. C23H23N5O4 (C, H, N).
3.7 (s, 3H, CH3), 3.8 (s, 3H, CH3), 4.4 (t, 2H, J=6.7
Hz, CH2), 6.6 (d, 1H, J=1.6 Hz, pyrrole-H3), 7.2 (pt,
2H, J=7.3 Hz, carbazole-H2,7), 7.3 (d, 1H, J=1.6
Hz, pyrrole-H5), 7.4 (pt, 2H, J=7.5 Hz, carbazole-
3
4
3
4
3
3
H3,6), 7.6 (d, 2H, J=8.1 Hz, carbazole-H1,8), 8.1 (d,
2H, J=7.6 Hz, carbazole-H4,5), 9.8 (s, 1H, NH); 13C
3
NMR (75 MHz) (DMSO-d6) l: 24.6 (s), 32.6 (s), 36.4
(p), 42.0 (s), 51.2 (p), 108.0 (t), 109.4, (t), 109.5 (t),
118.9 (q), 118.9 (t), 119.0 (t), 120.5 (t), 120.7 (t), 122.3
(q), 122.4 (q) 123.0 (q), 125.8 (t), 125.9 (t), 126.0 (t),
140.2 (q), 140.3 (q), 161.0 (q), 169.1 (q); MS m/z: 389
[M+]; Anal. C23H23N3O3 (C, H, N).
5.1.14.8. Methyl-4-{[3-(1H-3indolyl)propanoyl]amino}-
1-methyl-1H-2-pyrrole carboxylate (22). Brown needles
(546 mg, 1.68 mmol, 42%), m.p.: 116 °C; IR (KBr)
(cm−1): 3350, 3260, 3190, 3100, 3040, 2980, 2800, 1640,
1570, 1550, 1450, 1400, 1350, 1260, 1190, 1130, 1100,

C. Hotzel et al. / European Journal of Medicinal Chemistry 37 (2002) 367–378
377
5.1.14.11. Methyl-4-{[-(9H-carbazolyl)butanoyl]amino}-
1-methyl-1H-2-pyrrolyl)-2-oxoethyl]-1-methyl-1H-2-
pyrrole carboxylate (25). Yellow amorphous solid (531
mg, 1.04 mmol, 26%), m.p.: 104 °C; IR (KBr) (cm−1):
the range 25–90 °C with a heating rate of 1.5 °C
min−1. 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 transi-
tion [25].
1
3410, 3350, 1640, 1560, 1300, 1215, 820; H NMR (300
3
MHz) (DMSO-d6) l: 1.9 (t, 2H, J=6.4 Hz, CH2), 2.3
3
(q, 2H, J=6.9 Hz, CH2), 3.7 (s, 3H, CH3), 3.8 (s, 3H,
3
CH3), 3.9 (s, 3H, CH3), 4.4 (t, 2H, J=6.7 Hz, CH2),
4
3
6.8 (d, 1H, J=1.6 Hz, pyrrole-H3), 7.2 (pt, 2H, J=
5.2.2. Ethidium bromide displacement assay
7.3 Hz, carbazole-H2,7), 7.35 (d, 1H, 4J=1.6 Hz,
All fluorescence measurements were conducted on an
Hitachi F-2000 spectrofluorometer. Calf thymus DNA
(Sigma, Type I; 1.0×10−5 M in base pairs) was added
in small aliquots to ethidium bromide (Invitrogen;
5.0×10−6 M) resulting in a 2:1 ratio of basepair–
ethidium in 2 mL of a 10 mM Tris–HCl (pH 7.4), 75
mM NaCl buffer solution. The fluorescence of the
DNA–ethidium buffer solution was calibrated at r.t. to
100% fluorescence and that of the ethidium buffer
solution to 0% fluorescence, respectively. The premixed
3
pyrrole-H5), 7.4 (pt, 2H, J=7.5 Hz, carbazole-H3,6),
7.45 (d, 1H, 4J=1.7 Hz, pyrrole-H3%), 7.6 (d, 2H,
3J=8.1 Hz, carbazole-H1,8), 8.0 (d, 1H, J=1.6 Hz,
4
3
pyrrole-H5%), 8.1 (d, 2H, J=7.6 Hz, carbazole-H4,5),
9.8 (s, 1H, NH), 10.4 (s, 1H, NH), 10.8 (s, 1H, NH);
13C NMR (75 MHz) (DMSO-d6) l: 24.3 (s), 33.0 (s),
36.5 (p), 39.6 (p), 42.0 (s), 51.5 (p), 104.7 (t), 108.0 (t),
109.1 (t), 109.6 (t), 111.2 (q), 113.9 (q), 115.6 (t), 118.8
(q), 118.9 (t), 119.0 (t), 120.3 (t), 120.6 (t), 122.6 (q),
122.7 (q), 123.0 (q), 126.0 (t), 126.1 (t), 126.2 (t), 140.7
(q), 140.8 (q), 158.9 (q), 161.0 (q), 169.0 (q); MS m/z:
511 [M+]; Anal. C29H29N5O4 (C, H, N).
DNA–ethidium 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 u=545 nm excitation and 595 nm
emission with a slit width of 10 nm. The binding
affinity was determined at 50% ethidium bromide dis-
placement, measured as a drop in fluorescence to 50%.
Results were calculated with the Data Analysis and
Graphics Program GRAFIT and with the graphical pro-
gram ORIGIN. Distamycin A as reference was tested in
the same way [28].
5.1.14.12. N2-[3-(Dimethylamino)propyl]-4-{[4-(9H-9-
carbazolyl)butanolyl]amino}-1-methyl-1H-2-pyrrole car-
boxamide (26). Brown amorphous solid (312 mg, 0.68
mmol, 17%), m.p.: 118 °C; IR (KBr) (cm−1): 3320,
2920, 2845, 1700, 1650, 1550, 1450, 1350, 1260, 780,
1
750; H NMR (300 MHz) (DMSO-d6) l:1.6 (m, 2H,
3
3J=6.5 Hz), 1.9 (m, 2H, J=6.5 Hz, CH2), 2.0 (s, 6H,
3
3
2CH3), 2.2 (t, 2H, J=6.8 Hz, CH2), 2.3 (t, 2H, J=
3
7.6 Hz, CH2), 3.1 (q, 2H, J=6.7 Hz, CH2), 3.8 (s, 3H,
CH3), 4.4 (t, 2H, 3J=6.8 Hz, CH2), 7.2 (pt, 2H,
5.3. Topoisomerase I Inhibition
3J=7.3 Hz, carbazole-H2,7), 7.3 (d, 1H, J=1.6 Hz,
4
3
pyrrole-H3), 7.4 (pt, 2H, J=7.5 Hz, carbazole-H3,6),
Plasmid DNA (pUC 19, Invitrogen; 0.033 mg mL−1
)
3
7.6 (d, 2H, J=8.1 Hz, carbazole-H1,8), 8.05 (d, 1H,
was incubated for 15 min at 35 °C with different
concentrations of the tested drugs (5, 20 and 50 mM) in
1× topoisomerase I reaction buffer (50 mM Tris–HCl
pH 7.5, 50 mM KCl, 10 mM MgCl, 0.5 mM DTT, 0.1
mM EDTA, 30 mg mL−1 BSA) to ensure equilibration.
The reaction was initiated by adding topoisomerase I
(Topogen; 0.25 U mL) and the samples were reincu-
bated for 15 min at 35 °C. Reactions were stopped by
addition of SDS to a final concentration of 0.25% and
proteinase K to 250 mg mL−1, followed by incubation
for 30 min at 50 °C. After addition of 2 mL denaturing
loading buffer (Invitrogen) samples were loaded onto a
1% agarose gel in TBE Buffer. Electrophoresis was
conducted at 120 V for 2 h.
4J=1.6 Hz, pyrrole-H5), 8.1 (d, 2H, J=7.6 Hz, car-
3
3
bazole-H4,5), 8.4 (t, 1H, J=7.4 Hz, NH), 9.9 (s, 1H,
NH); 13C NMR (75 MHz) (DMSO-d6) l: 24.5 (s), 28.0
(s), 32.6 (s), 36.4 (p), 38.1 (s), 42.0 (s), 45.0 (p), 45.1 (p),
59.2 (s), 108.0 (t), 109.3 (t), 109.4 (t), 118.0 (q), 119.0
(t), 119.1 (t), 120.1 (t), 120.6 (t), 122.3 (q), 122.6 (q),
123.0 (q), 125.8 (t), 126.0 (t), 126.1 (t), 140.0 (q), 140.3
(q), 160.3 (q), 171.0 (q); MS m/z 459 [M+]; Anal.
C27H33N5O2 (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 ther-
mostat. The measurements were performed in BPE
buffer, pH 7 (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM
EDTA) with a drug–DNA ratio of 1:1, using [Poly(-
dAdT). Poly(dAdT)] as DNA (Amersham Pharmacia).
The temperature inside the cuvette was increased over
Acknowledgements
We thank Professor Dr. C. Bailly and his group at
the Centre Oscar Lambret pour la Reserche sur le
Cancer, Lille, France, for their cooperation in develop-

378
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