Synthesis, DNA binding, topoisomerases inhibition and cytotoxic properties of 4-arylcarboxamidopyrrolo-2-carboxyanilides

Article abstract of DOI:10.1016/S0960-894X(00)00028-7

Three 4-arylcarboxamidopyrrolo-2-carboxyanilides bearing different substituents on the pyrrole nitrogen were synthesized and evaluated for their capacities to bind to specific sequences within the minor groove of DNA and to inhibit human topoisomerases I and II in vitro. The cytotoxicity of the drugs correlates with their DNA binding affinities. The two drugs bearing a N-methyl or N-benzyl pyrrole stabilize topoisomerase I-DNA complexes. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00028-7

Bioorganic & Medicinal Chemistry Letters 10 (2000) 553±557
Synthesis, DNA Binding, Topoisomerases Inhibition
and Cytotoxic Properties of
4-Arylcarboxamidopyrrolo-2-carboxyanilides
Fabienne Dudouit, a Jean-Francois Goossens, a Raymond Houssin, a
Jean-Pierre Henichart, a Pierre Colson, b Claude Houssier, b Nathalie Gelus c
and Christian Bailly c,*
a
Â
Institut de Chimie Pharmaceutique Albert Lespagnol, Universite de Lille 2, BP 83, 59006, Lille, France
Laboratoire de Chimie Macromoleculaire et Chimie Physique, Universite de Liege, Liege 4000, Belgium
b
Â
Â
Á
Á
cINSERM U-524, IRCL, Place de Verdun 59045 Lille, France
Received 8 October 1999; accepted 14 January 2000
AbstractÐThree 4-arylcarboxamidopyrrolo-2-carboxyanilides bearing di€erent substituents on the pyrrole nitrogen were synthe-
sized and evaluated for their capacities to bind to speci®c sequences within the minor groove of DNA and to inhibit human
topoisomerases I and II in vitro. The cytotoxicity of the drugs correlates with their DNA binding anities. The two drugs bearing a
N-methyl or N-benzyl pyrrole stabilize topoisomerase I-DNA complexes. # 2000 Elsevier Science Ltd. All rights reserved.
The pyrrole-amidine antibiotics netropsin and dista-
mycin (1) represent prototypic DNA minor groove bin-
ders endowed with a pronounced selectivity for AT-rich
sequences but are weakly cytotoxic. A vast number of
lexitropsins analogues and conjugates targeting speci®c
sequences in DNA have been designed.1 Several potent
antitumor drugs have been obtained, such as the dis-
tamycin±nitrogen mustard conjugate tallimustine which
is undergoing clinical trials as an anticancer agent.1
AT-speci®c recognition also occurs with furamidine (2)
and related diphenylfuran derivatives which present
interesting therapeutic pro®les, in particular as anti-
Pneumocystis agents.2,3
activities of nuclear enzymes involved in the regulation
of DNA topology in cells. Topoisomerases I and II are
the primary targets of many anticancer drugs including
the anthracyclines and camptothecins.4,5 Certain DNA
minor groove binding agents related to Hoechst 33342
or bifunctional netropsin analogues act as poisons for
topoisomerase I.6±8 This ubiquitous enzyme is con-
sidered a valid target for the development of antitumor
agents.
These considerations prompted us to design a new series
of potential DNA minor groove binder inhibitors of
topoisomerases. We developed the 4-arylcarboxamido-
pyrrolo-2-carboxyanilide skeleton which combines the
structural features of distamycin (1) and furamidine (2).
The amidine side chains were replaced with more stable
In addition to interacting with speci®c sequences in DNA,
minor groove binders can interfere with the catalytic
*Corresponding author. Tel.: +33-320-16-92-18; fax: +33-320-16-92-
29; e-mail: bailly@lille.inserm.fr
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00028-7

554
F. Dudouit et al. / Bioorg. Med. Chem. Lett. 10 (2000) 553±557
primary amine-containing chains. The central pyrrole
ring was substituted with either a N-methyl group as
found in distamycin or netropsin or with a more bulky
N-benzyl group.
Compound 9 was prepared from 4-nitrobenzonitrile 6
(Scheme 2).12 The nitrile group was reduced (7) with
borane at re¯ux in THF prior to protection of the
resulting amino group with di-tert-butyldicarbonate in
dioxane and water at pH 10 (8).13 Then, the nitro group
of 8 was reduced in the presence of Pd/C.
Here we report the synthesis of the drugs A, B and C
and their DNA biding anity and binding mode char-
acterized by a combination of spectroscopic approaches
including absorption, ¯uorescence, circular dichroism
and electric linear dichroism. A relaxation assay
employing supercoiled plasmid DNA was used to eval-
uate the e€ects of the drugs on human topoisomerases.
Esters 3±5 were saponi®ed overnight at room tempera-
ture with sodium hydroxide in water and THF to a€ord
10,14 1114 and 12, and then coupled with the aniline
derivative 9 using PyBOP as a coupling agent in DMF.
Catalytic hydrogenation of the resulting amides (with
Pd/C at room temperature for 13 and at atmospheric
pressure and Raney nickel for 14 and 15 at 60 ꢀC and
30 bars) gave amines 16, 17 and 18 which are unstable
and therefore were immediately coupled with 19 with
PyBOP in N-methylpyrrolidinone. 4-tert-Butoxycarbonyl-
amino)benzoic acid 1915 was obtained from 4-(amino-
methyl)benzoic acid (commercially available) by
reaction with di-tert-butyldicarbonate in a water/diox-
ane mixture containing NaOH. Finally, protected
amino groups of compounds 20±22 were released in the
presence of TFA to a€ord 4-(4-aminomethylphenyl-
carboxamido)-2-(4-aminomethyl)-1H-pyrrolecarboxamide
(A), its N-methyl (B) and N-benzyl (C) analogues,
respectively (Scheme 3).
DNA Binding Anity
Synthesis
Complex formation between the drugs A, B, C and
DNA results in a signi®cant increase in the Tm values of
helix-to-coil transition (Fig. 1). A 31 ꢀC increase in the
Compounds A, B and C9 were prepared from three dif-
ferently substituted pyrroles 3, 4 and 5. The pyrrole
heterocycle was obtained by nitration of mucobromic
acid 1 (commercially available) with sodium nitrite in
ethyl alcohol at 54 ꢀC, followed by cyclization of the
nitromalonaldehyde carbanion 2 with glycine methyl
ester hydrochloride in sodium hydroxide and methyl
alcohol at 50 ꢀC.10 N-Substituted pyrroles 4 and 5 were
obtained from pyrrole 3 which was alkylated by iodo-
methane or benzyl bromide in DMF at room tempera-
ture (Scheme 1). The N-methylpyrrole 4 can also be
obtained from the nitration of pyrrole-2-carboxylic
acid, as previously described.11
.
Tm of poly(dA-dT) (dA-dT) is observed for compounds
A and B whereas the ÁTm value with C reaches 23 ꢀC.
With calf thymus DNA, under similar conditions, the
measured ÁTm values (Tdmrug-DNA complex-TmDNA) were
16.3, 11.6 and 6.8 ꢀC with A, B and C respectively. The
stabilisation of the duplex structure is weaker with C
than with A and B. DNA binding anity constants
were determined from ¯uorescence titration experi-
ments. As indicated in Table 1, the Ka value for com-
pound C is about 3 times lower than that calculated for
A. The N-benzyl group reduces slightly the strength of
the drug-DNA interaction.
Scheme 1. Reagents and conditions: (a) NaNO2, EtOH, 54 ꢀC; (b) HCl,NH2CH2COOCH3, 50 ꢀC, NaOH, MeOH; (c) NaH, CH3I, DMF; (d) NaH,
BrCH2È, DMF.
Scheme 2. Reagents and conditions: (a) BH3 Me2S, THF; (b) Boc2O, NaOH, dioxane/water; (c) H2, Pd/C, MeOH.

F. Dudouit et al. / Bioorg. Med. Chem. Lett. 10 (2000) 553±557
555
Scheme 3. Reagents and conditions: (a) NaOH, H2O/THF then HCl; (b) 9, PyBOP, DIPEA, DMF; (c) H2, Pd/C or Raney nickel, MeOH; (d) 19,
PyBOP, DIPEA, N-methylpyrrolidinone; (e) TFA, CH2Cl2 then NaHCO3, H2O.
shows the dependence of the reduced dichroism ÁA/A
on the wavelength for compound B. The ELD signal is
always positive in sign in the 300±390 nm absorption
band, consistent with an inclination of the chromophore
at about 45ꢀ with respect to the orientation axis of the
DNA molecules, as expected for a minor groove bin-
der.17 The CD and ELD measurements provide strong
evidences that the 4-arylcarboxamidopyrrolo-2-carboxy-
anilides bind into the minor groove of DNA double
helix.
Sequence Speci®city
.
DNase I footprinting experiments were carried out with
the 160 bp tyrT fragment which has been previously
used to map distamycin and furamidine binding sites.18±20
The gel in Figure 3 shows clearly that the three drugs
recognize preferentially AT-rich sequences. The foot-
prints are less pronounced with C than with A or B
(compare the 2 mM lanes) but however, the introduction
of the N-benzyl group on the central pyrrole does not
abolish the capacity of the drug to bind selectively to
AT sites. Four well-resolved footprints encompassing
the sequences 50-AAAT, 50-TAAA, 50-AAAAAT and
50-TAATTTT can be detected using 5 mM drugs.
Regions protected from DNase I cleavage by the drugs
are con®ned to the same AT-rich sequences as those
detected with distamycin and furamidine.18
Figure 1. Thermal denaturation curves for poly(dA-dT) (dA-dT) in
the absence (&) and presence of A (&), B (*) and C (~) (2 mM each).
Tm measurements were performed in BPE bu€er pH 7.1 (6 mM
Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA) using 20 mM DNA in 3 mL
quartz cuvettes at 260 nm with a heating rate of 1 ꢀC/min. Each drug
concentration was tested in triplicate. The Tm values were obtained
from ®rst-derivative plots.
Table 1. Cytotoxicity and DNA binding anity
1
Compounds
IC50 (mM)
Ka (105 M
)
A
B
C
1.7‹0.3
5.4‹0.3
8.2‹0.6
9.5
6.8
3.4
Topoisomerases Inhibition
DNA Binding Mode
Closed circular DNA was treated with human topo-
isomerase I or II in the presence of the test drugs at 10
or 50 mM. DNA samples resolved in 1% agarose gels
containing ethidium bromide. In both cases, supercoiled
plasmid DNA was fully relaxed by the enzyme (I or II)
in the absence of the drug. As shown in Figure 4 (right),
a band of linear DNA (form III) could be seen with
Figure 2 (left) displays the circular dichroism (CD)
spectrum of B bound to DNA. The positive CD band
around 310±340 nm re¯ects the orientation of the drug
in the minor groove of the double helix.16 The geometry
of the drug±DNA complexes was investigated further
using electric linear dichroism (ELD).17 Figure 2 (right)

556
F. Dudouit et al. / Bioorg. Med. Chem. Lett. 10 (2000) 553±557
Figure 2. (CD) Circular dichroism and (ELD) electric linear dichroism spectra of compound B bound to calf thymus DNA at a P/D ratio of 20, in
1 mM sodium cacodylate bu€er pH 7.0.
The same relaxation assay was used to investigate the
e€ects of the drugs on topoisomerase I (Fig. 4, left). In
the presence of compound C at 10 or 50 mM, the inten-
sity of the band corresponding to the nicked form II of
DNA has increased signi®cantly. This e€ect, observed
with camptothecin (positive control), re¯ects the stabi-
lization of topoisomerase I±DNA cleavable complexes.
Compound A has no e€ect on topoisomerase I whereas
C and to a lower extent B behave as speci®c topo-
isomerase I inhibitors trapping the covalent DNA±
topoisomerase I complexes.
Cytotoxicity
The compounds were evaluated for their anti-
proliferative activities using human HL60 leukemia
cells.21 The results expressed as IC50 values (concentra-
tion reducing by 50% the cell proliferation) are reported
in the Table 1. There is apparently no correlation
between the IC50 values and the e€ects of the drug on
topoisomerase I but interestingly, a link can be estab-
lished with the DNA binding data. The fact that the
N-benzyl compound C is about 4 times less cytotoxic
than compound A likely account for its reduced capa-
city to interact with DNA.
Conclusion
Figure 3. DNase I footprinting of A, B and C bound to the tyrT DNA
fragment. The DNA was 30-end labeled with [a-32P]dATP in the pre-
4-Arylcarboxamidopyrrolo-2-carboxyanilides represent
sence of AMV reverse transcriptase. The drug concentration (mM) is
a new series of sequence-selective minor groove binders.
shown at the top of the appropriate gel lanes. The track labelled ``Ct''
The substitution of the pyrrole nitrogen with a benzyl
contained no drug. The track labeled ``G'' represents a dimethylsul-
group slightly reduces the DNA interaction but however
reinforces the capacity of the drug to inhibit topo-
isomerase I. The benzyl group might serve as a hook to
anchor the enzyme close to the cleavage sites on DNA.
Such a behavior has been reported with the DNA minor
groove binding antitumor drug ecteinascidin22,23 which
is also an inhibitor of topoisomerase I.24 The benzyl
group of compound C likely behaves as the external C
domain of ecteinascidin which protrudes outside the
DNA double helix. The design of DNA minor groove
binders equipped with such a protein-trapping domain
may provide a new avenue for the design of antitumor
drugs.
phate-piperidine marker speci®c for guanines. Numbers on the side of
the gels refer to the numbering scheme of the fragment. DNase I
cleavage products were resuspended in 5 mL 80% formamide contain-
ing 10 mM EDTA and 0.1% tracking dyes. Samples were heated to
90 ꢀC for 4 min and then chilled in an ice-bath just before being loaded
on a sequencing gel (8% polyacrylamide, 7 M urea). The AT-rich
sequences protected from cleavage are indicated.
etoposide which is a well known topoisomerase II poi-
son. No such band of linear DNA was detected with the
4-arylcarboxamidopyrrolo-2-carboxyanilides. They have
no e€ects on topoisomerase II.

F. Dudouit et al. / Bioorg. Med. Chem. Lett. 10 (2000) 553±557
557
Figure 4. E€ect of A, B and C on the relaxation of supercoiled plasmid DNA by topoisomerase I or II. The DNA (0.5 mg) was incubated with 4
units topoisomerase in the absence (lane TopI/II) or presence of drug at 10 or 50 mM. Etoposide (Etop.) and camptothecin (CPT) were used at 50
mM. Reactions were stopped with SDS and proteinase K. DNA samples were electrophoresed on agarose gel containing ethidium bromide. Nck,
nicked; Lin, linear; Rel, relaxed; Sc, supercoiled.
00
H5), 7.38 (d, J=8.37 Hz, 2, H3 ), 7.54 (s, 1, H3), 7.60 (d,
Acknowledgements
0
J=7.87 Hz, 2, H3 ), 7.74 (d, J=8.37 Hz, 2, H2 ), 8.00 (d,
00
0
J=7.87 Hz, 2, H2 ), 8.20 (sl, 2, NH2), 8.38 (sl, 2, NH2), 10.09
Supported by research grants (to C.B. and J.P.H.) from
the Ligue Nationale Francaise Contre le Cancer; (to
C.H. and P.C.) from the Actions de Recherches Con-
certees contract 95/00-193 and the FNRS, Televie 7/
4526/96. Support by the ``convention INSERM-CFB''
is acknowledged.
(s, 1, NH), 10.51 (s, 1, NH); MS (EI) for C27H27N5O2 (M+)
calcd 453, found 453. Anal. (C70.00H5.85N15.30O8.85) CHN.
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9. A: mp >260 ꢀC; IR (KBr) 3500±3250, 1645, 1640, 1600; H
1
NMR (300 MHz; TFA) d 4.45 (sl, 2, CH2), 4.54 (sl, 2, CH2),
7.80 (m, 11, aromatics); MS (CI) for C20H21N5O2, 2H2O
(MH ) calcd 362, found 362. Anal. (C59.73H6.10N17.01O17.16
)
CHN. B: mp 185 ꢀC; IR (KBr) 3310, 1650, 1610; 1H NMR
(300 MHz; TFA) d 4.03 (s, 3, CH3), 4.46 (sl, 2, CH2), 4.54 (sl,
2, CH2), 7.56±06 (m, 10, aromatics); MS (EI) for C21H23N5O2,
2H2O (M+) calcd 377, found 377. Anal. (C61.25H6.41N16.75
O15.59) CHN. C: mp 99±101 ꢀC; IR (KBr) 3420, 3030, 1675,
1605; 1H NMR (300 MHz; TFA) d 3.92 (s, 2, CH2), 4.13 (s, 2,
CH2), 5.63 (sl, 2, CH2), 7.15±34 (m, 5, aromatics), 7.28 (s, 1,
24. Takebayashi, Y.; Pourquier, P.; Yoshida, A.; Kohlagen,
G.; Pommier, Y. Proc. Natl. Acad. Sci. USA 1999, 96, 7196.