Sequence selective recognition of DNA by hairpin conjugates of a racemic seco-cyclopropaneindoline-2-benzofurancarboxamide and polyamides.

Article abstract of DOI:10.1016/S0960-894X(02)00341-4

Conjugates of racemic seco-cyclopropaneindoline-2-benzofurancarboxamide (CI-Bf) and four diamides (ImIm 1, ImPy 2, PyIm 3, and PyPy 4, where Py is pyrrole, and Im is imidazole), linked by a gamma-aminobutyrate group were synthesized. In addition to alkyla

Full text of DOI:10.1016/S0960-894X(02)00341-4

Bioorganic & Medicinal Chemistry Letters 12 (2002) 2245–2248
Sequence Selective Recognition of DNA by Hairpin Conjugates of
a Racemic seco-Cyclopropaneindoline-2-benzofurancarboxamide
and Polyamides
James L. Toth,a Carly A. Price,a Erik C. Madsen,a Heather L. Handl,a
Stephen J. Hudson,b Richard B. Hubbard, III,a,c J. Phillip Bowen,c
Konstantinos Kiakos,d John A. Hartleyd and Moses Leea,*
aDepartment of Chemistry, Furman University, Greenville, SC 29613, USA
bDepartment of Biology, Furman University, Greenville, SC 29613, USA
cDepartment of Chemistry, University of Georgia, Athens, GA 30602, USA
dDepartment of Oncology, Royal Free University College Medical School, London W1W 7BS, UK
Received 15 January 2002; accepted 27 March 2002
This paper is dedicated to Professor J. William Lown on the occasion of his retirement
Abstract—Conjugates of racemic seco-cyclopropaneindoline-2-benzofurancarboxamide (CI-Bf) and four diamides (ImIm 1, ImPy
2, PyIm 3, and PyPy 4, where Py is pyrrole, and Im is imidazole), linked by a g-aminobutyrate group were synthesized. In addition
to alkylating at adenine-N3 positions within an A5 sequence, the imidazole-containing compounds 1 and 2 were found to also
alkylate purine-N3 positions within a sequence 30-GGGGGGA(888)CTGCTC(894)-50. A model for the binding of hairpin con-
jugates 1 and 2 with the 30-GACT-50 sequence is proposed. # 2002 Elsevier Science Ltd. All rights reserved.
The human genome project, which has sequenced the
genome of 3 billion base pairs, has led to the identifica-
tion of many new genes and their associated control
sequences.1 Small molecules that are tailored to bind to
predetermined DNA sequences, can permeate cell
membranes, interfere with DNA replication and tran-
scription are potentially powerful tools for molecular
biology2,3 as well as diagnosis and treatment of diseases
at the DNA level.2,4
been reported.8ꢀ10 In addition, polyamides that fold
into a hairpin conformation thereby creating an ‘inter-
nal side-by-side’ binding motif have been shown to be
able to inhibit the expression of the 5S RNA genes by
RNA polymerase III11 and the Ets-1, LEF-1 and TBP
in the HIV-1 enhancer/promoter element in cells.12
Even though the non-covalently reactive polyamide
hairpin compounds were biologically active, the ques-
tion arises whether a covalently reactive hairpin would
be endowed with improved biological properties. In this
regard, a heterodimer of duocarmycin A and distamycin
was found to alkylate within the minor groove of DNA
in a side-by-side manner.13 The dimer bound pre-
ferentially to a sequence different from either one of the
compounds alone. While both distamycin and duo-
carmycin A bind strictly to AT-rich sequences, the
dimer alkylates GN3 position in GC-rich sequences,
such as 50-AGGTG-30. More recently, we demonstrated
that imidazole-containing triamides, such as compound
5, dramatically modulated the sequence-specificity of
duocarmycin A alkylating guanine-N3 atoms at GC
sequences.14 These findings indicate that hairpin con-
jugates of polyamides and duocarmycin A might pro-
vide an attractive direction for the discovery of a new
The DNA minor groove is an attractive target for the
design and development of sequence-specific ligands for
biological activity.5 This is due partly to the observation
that covalent adducts in the minor groove are generally
poorly repaired compared to those in the major
groove.6 The minor groove binding agent distamycin,7 a
polyamide, and its imidazole-containing analogues have
been extensively studied for their ability to bind to spe-
cific DNA sequences in a 2:1 or side-by-side binding motif.
A set of rules for their sequence specific recognition have
*Corresponding author. Tel.: +1-864-294-3368; fax: +1-864-294-
3559; e-mail: moses.lee@furman.edu
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00341-4

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J. L. Toth et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2245–2248
class of gene specific ‘knockout’ agents. To date, several
conjugates of the duocarmycin alkylating pharmaco-
phore and its analogues with polyamides, including
hairpin polyamides, have been reported.8c,15 However,
the sequence specific alkylation exhibited by these con-
jugates were directed by the imidazole/pyrrole pairings
and side-by-side binding motif of the polyamides.8ꢀ10
Conjugates of CC-1065 analogues with oligodeoxy-
nucleotides have also been reported.16 In this paper, we
report the synthesis and sequence selectivity of a series
of conjugates of racemic seco-cyclopropaneindoline-2-
benzofurancarboxamide (CI-Bf) with four different
polyamides (ImIm 1, PyIm 2, ImPy 3, and PyPy 4), in
which the DNA sequence recognition of the side-by-side
CI-Bf/polyamide pairing will be investigated (Fig. 1).
were readily prepared from N-butoxycarbonyl (BOC)
protected diaminobutane with the acid chlorides of
1-methyl-4-nitroimidazole-2-carboxylic acid or 1-methyl-
4-nitropyrrole-2-carboxylic acid.18 Reduction of the
nitro group of compounds 6–9 gave the corresponding
amines, which were reacted with glutaric anhydride in
the presence of N,N-dimethylaminopyridine (DMAP) in
pyridine. The corresponding acids 10–13 were obtained
in 11–98% yields after silica gel column chromato-
graphy. Synthesis of the racemic seco-cyclopropane-
indoline-2-benzofurancarboxamido (CI-Bf) moiety, a
duocarmycin analogue, started from dichloride 14 that
was prepared from the corresponding diol19 in quanti-
tative yield with triphenylphosphine and CCl4. Using a
method that was recently reported from our laboratory,
the dichloride was reduced with stannous chloride in
hydrochloric acid, which after work up with sodium
hydroxide gave an indoline intermediate.20 Coupling of
the indoline with 5-nitrobenzofuran-2-carboxylic acid in
the presence EDCI in DMF gave the desired amide 15
in 71% yield. Selective reduction of the nitro moiety in
compound 15 was readily achieved using Adams cata-
lyst and hydrogenation at a pressure of 55 PSI. Subse-
quently, reaction of the amine with carboxylic acids 10–
13 gave the protected hairpin compounds. The phenolic
moieties in compounds 17–20 were unmasked by
hydrogenation over 10% palladium on carbon, and the
products were obtained in 30–78% yields after silica gel
column chromatography. The BOC protecting group
was removed using 3 M hydrochloric acid in dry ethyl
acetate. The desired off-white precipitates of products
1–4 were collected, washed with dry ethyl acetate, and
dried in vacuo at room temperature. All the compounds
prepared in this study were characterized by 500 MHz
1H NMR, FT-IR, FABmass spectrometry and accurate
mass measurements.
The synthetic strategy for preparation of the target
hairpin molecules 1–4 is depicted in Scheme 1. Using
published methods,17 the diheterocyclic polyamides 6–9
Figure 1. Structures of the target compounds 1–4, duocarmycin A
(DuoA), and AR-1-144.
The covalent sequence specificity of compounds 1–4 was
assessed by a thermally induced DNA strand cleavage
experiment, which is commonly used to probe sequence
specific covalent interactions with purine-N3 in the
minor groove.21 The DNA fragment used in these stud-
ies was obtained from PCR amplification of base pairs
741–940 of the pUC18 plasmid that was linearized with
Hind III. A 50-32P labeled 50-CTGTCGGGTTT-30 pri-
mer was used as the forward primer so that each final
probe copy was singly end-labeled. Results from the
thermally induced DNA strand break experiment, as
depicted in Figure 2, indicate that compounds 1–4
retain a memory for alkylating adenine-N3 within an A5
sequence, a site that is preferred by CC-1065, adozele-
sin, and seco-CI-trimethoxyindole (or seco-CI-TMI)
(data not shown). Interestingly, minor, but unique,
alkylation bands at 30-GGGGGGA(888)CTGC-
TC(894)-50 (alkylation at the purine-N3 of the under-
lined bases) were also be observed for compounds 1 and
2, indicating their ability to recognize a GC-containing
sequence. To do so, the seco-CI-Bf and polyamide sub-
units fold to form a hairpin structure, and the resulting
pairing of an imidazole unit side-by-side with the benzo-
furan moiety then interacts with a GC base pair. These
results are consistent with our earlier findings that imi-
dazole-containing polyamides are capable of altering
Scheme 1. Synthesis of the target compounds 1–4: (a) (i) hydrogen,
5% Pd/C, MeOH; (ii) glutaric anhydride, DMAP, pyridine; (b) (i)
SnCl2, HCl; (ii) NaOH; (iii) 5-nitrobenzofuran-2-carboxylic acid,
EDCI, DMAP, DMF; (c) hydrogen, PtO2, 55PSI; (d) polyamide acid,
EDCI, HOBt, DMF; (e) hydrogen, 10% Pd/C, THF; (f) 3 M HCl,
EtOAc.

J. L. Toth et al. / Bioorg. Med. Chem. Lett. 12 (2002) 2245–2248
2247
the sequence selectivity of duocarmycin A from AT to
GC-rich sequences.
because in that case the pyrrole moiety in compound 2
would have to be unfavorably placed next to a G(891)/
C base pair.22,23 In addition, an extended conformation
of compounds 1 and 2 would require the benzofuran
group to bind unfavorably to a C(889)/G base pair. We
have found that like seco-CI-TMI, seco-CI-Bf also
bound to the A(865) cluster and not to GC-containing
sites (data not shown). Further support of the hairpin
conformation of compounds 1 and 2 came from a
report that an aliphatic dicarboxamide of polyamides,
such as a bis-linked netropsin with pimelic acid, was
capable of forming a hairpin structure.24 Structural
analyses of the binding of compound 1 using NMR and
molecular modeling studies are on-going and the results
will be reported in due course.
In a proposed model of the hairpin complex of com-
pound (S)-1 with the 30-GGGA(888)CTGCTC-50
sequence, given in Figure 3, the imidazole would stack
with the benzofuran to form a complex similar to the
pyrrole/imidazole pairing that is capable of recognizing a
C/G base pair.8,9 It is not likely that compounds 1 and 2
would bind to this sequence in an extended conformation,
The cytotoxicity of the hairpin compounds 1–4 was
determined against L1210 (murine leukemia) and P815
(murine mastocytoma) cell lines using a MTT assay.22
Following a 3-day continuous exposure of compounds
1–4, the experimental IC50 values against the growth of
L1210 cells were determined to be 6.8, 6.7, 45, and
28 mM, respectively. It is worthy to note that com-
pounds 1 and 2 were more cytotoxic to the L1210 cells
than compounds 3 and 4, and that might be related to
their different DNA sequence selectivity profiles. For
P815 cells, the IC50 values are 52, 9.1, 77, and 41 mM,
respectively.
In summary, the studies described in this communi-
cation show that conjugates of duocarmycins and poly-
amides that can fold into a hairpin conformation are
capable of recognizing specific sequences of DNA. They
provide a feasible platform for the design of sequence
specific probes for therapeutic and diagnostic applica-
tions. Studies aimed at improving the sequence specifi-
city and increasing the DNA binding site size are
currently underway in the investigators’ laboratories
and the results will be reported in due course.
Acknowledgements
Figure 2. Thermally induced and sequence selective DNA strand
break study on compounds 1–4.
Financial support from Research Corporation, NSF-
REU (to M.L.), the Arnold and Mabel Arnold Beck-
man Foundation (to M.L. and E.C.M.), and the Cancer
Research Campaign (to J.A.H.) is gratefully acknowl-
edged. The authors also thank Dr. Robert Kelly at
Pharmacia & Upjohn for a generous gift of CC-1065.
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