Synthesis and study of a new adenine-acridine tandem, inhibitor of exonuclease III

Article abstract of DOI:10.1016/S0960-894X(99)00681-2

A new heterodimer adenine-chain-acridine containing a mixed amido-guanidinium linker chain was synthesized. To achieve the synthesis a new method of introduction of aminoalkyl chain at position 9 of adenine was designed. The heterodimer interacts specifically with the abasic sites in DNA and inhibits the major base excision repair enzyme in Escherichia coli, Exonuclease III. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(99)00681-2

Bioorganic & Medicinal Chemistry Letters 10 (2000) 293±295
Synthesis and Study of a New Adenine±Acridine Tandem,
Inhibitor of Exonuclease III
Philippe Belmont, Martine Demeunynck,* Jean-Francois Constant and Jean Lhomme
Â
LEDSS, Chimie Bioorganique, UMR CNRS 5616, Universite Joseph Fourier, BP 53, 38041 Grenoble cedex 9, France
Received 15 October 1999; accepted 6 December 1999
AbstractÐA new heterodimer adenine-chain-acridine containing a mixed amido-guanidinium linker chain was synthesized. To
achieve the synthesis a new method of introduction of aminoalkyl chain at position 9 of adenine was designed. The heterodimer
interacts speci®cally with the abasic sites in DNA and inhibits the major base excision repair enzyme in Escherichia coli, Exonu-
clease III. # 2000 Elsevier Science Ltd. All rights reserved.
The biological mode of action of many antitumor drugs
involves the modi®cation (mainly alkylation) of nucleic
bases in DNA.1 Cells possess a speci®c machinery to
recognize and repair these lesions. This process is cri-
tical because the damaged DNA, if unrepaired, can lead
to mutation or cell death, which is the ultimate goal in
chemotherapy treatment.2 Therefore, designing drugs
which could interact speci®cally with these lesions, thus
inhibiting the DNA repair system, would potentiate the
action of antitumor drugs.3 One major intermediate in
the DNA repair cycle is the abasic site or AP-site
(apurinic or apyrimidinic site) which results from the
removal of heterocyclic base after hydrolysis of the N-
glycosidic bond.4±6 AP-site formation occurs sponta-
neously7 and is markedly increased by chemicals8
(alkylating agents, carcinogens,...) or by physical
agents9 (UV, g-radiation,...). One family of enzymes
involved in cell repair are the AP-endonucleases which
recognize and cleave the 30-phosphodiester bond of the
abasic site by a b-elimination mechanism.10±12 We have
described in preceding papers13±18 the synthesis of a
family of molecules which have been optimized for spe-
ci®c recognition of the abasic site in DNA (e.g. 1±3; Fig.
1). They incorporate in their structure three units: (a) an
intercalator for targeting DNA, (b) a nucleic base for
the recognition of the abasic site, and (c) a linker which
is responsible of the type of activity encountered. The
®rst class of compounds containing a polyamino linker
was shown to mimic AP-endonuclease activity. The
most ecient derivatives 1 and 2 cleave the AP-site
present in the DNA molecule at a nanomolar con-
centration.13 We recently reported on the synthesis and
biological properties of 3 that contains two guanidinium
moieties in the linker and which does not cleave the AP-
site but was shown to potentiate the action of the
anticancer agent N,N0-bis(2-chloroethyl)-N-nitrosourea
(BCNU) in vitro and in vivo.18
Compound 3 speci®cally recognizes abasic sites in DNA
as shown by high-®eld NMR measurements.18 The
guanidinium moieties are important for this activity as
they greatly increase the anity towards DNA and do
not cleave the base sensitive abasic site. However, one
limitation with compound 3 is a curare-like acute toxi-
city in vivo which led us to design a new compound with
a structure close to that of 3 in an e€ort to modulate its
biological activity and toxicity. We report herein on the
synthesis of heterodimer 4 in which the linker contains
one guanidinium function, the second guanidine being
replaced by one amido function.19 We also investigated
some biological properties of compound 4.
Synthesis
The synthesis of monoguanidine derivative 4 required
the synthesis of the new hybrid molecule 12 (Schemes 1
and 2). The linker was built step by step from the adenine
functionalized in position 9 by an aminoethyl group.
Leonard's method20 of alkylation of adenine with 2-
bromoethylphthalimide yields compound 6 in only 18%
yield. Similarly, reaction of potassium phthalimide on 2-
bromoethyladenine also gave the substitution product 6
in low yield, the major side-reaction being product 7
resulting from an elimination reaction.
*Corresponding author. Tel.: +33-4-7651-4429; fax: +33-4-7651-
4382; e-mail: martine.demeunynck@ujf-grenoble.fr
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(99)00681-2

294
P. Belmont et al. / Bioorg. Med. Chem. Lett. 10 (2000) 293±295
Figure 1.
Scheme 1.
Scheme 2.
We greatly improved the yield of 6 (up to 73%) by using
a silyl-protected phthalimide21 which is deprotected and
activated for the nucleophilic substitution in the pres-
ence of ¯uoride ions. Deprotection of the amino group
was achieved by hydrazinolysis and gave the amine 8 in
quantitative yield.
yield. Once again the removal of Boc groups was
accomplished by acidic treatment and the mono-
guanidinium derivative 4 was ®nally obtained in 71%
yield.23 We must point out the remarkably selective and
ecient `post-functionalization' strategy used for the
synthesis of 4 from 12.
The synthesis of the linking chain 9 containing two Boc
protected amino groups and one terminal carboxylic
acid group has been previously described.14 It was
introduced onto 8 by formation of an amide linkage
(Scheme 2). The protected compound 10 was isolated in
32% yield. After deprotection of the amino groups, the
primary amine was arylated with the acridine nucleus
giving 12 in 86% yield. The aliphatic amine of the linker
in 12 reacted with bis-tert-butoxycarbonylthiourea22 to
yield the protected monoguanidine derivative 13 in 71%
Interaction with DNA
Binding to DNA is a prerequisite condition for mol-
ecules targeting abasic sites. The anity constant for
native calf-thymus DNA of 4 was determined using
competition experiments with ethidium bromide (EB).
The new compound 4 interacts strongly with native
DNA with a binding anity (K=2Â105 M 1) similar to
that of the AP-endonuclease mimic drug 2.13

P. Belmont et al. / Bioorg. Med. Chem. Lett. 10 (2000) 293±295
295
guanidinium function in the linker, displays a relatively
high cytotoxicity in vitro but no synergistic e€ect with
BCNU. Further modi®cations of the parent molecule
are in progress to modulate the biological activities.
Figure 2. Agarose gel electrophoresis of abasic site containing pBR322
plasmid: T1, pBR322 plasmid; T2, depurinated pBR322; T3, in the
presence of ExoIII without drug; (1)±(8): ExoIII in the presence of
drug at various concentrations: (1) 8.7 Â 10 6 M; (2) 4.4 Â 10 6 M; (3)
Acknowledgements
This work was supported by the Association pour la
Recherche sur le Cancer (ARC) and the Ligue Nation-
ale Francaise Contre le Cancer (LNFCC). Dr. Pierre
Michon is gratefully acknowledged for his assistance in
DNA binding studies and Drs. Alain Croisy and
Daniele Carez for pharmacological studies.
6
6
7
7
2.2 Â 10 M; (4) 1.1 Â 10 M; (5) 5.5 Â 10 M; (6) 4.4 Â 10 M;
7
(7) 3.3 Â 10 M; (8) 1.6 Â 10 7 M.
In vitro Exonuclease III Inhibition Experiments
We tested the activity on pBR322 plasmid containing an
average of two AP-sites per plasmid.13 We ®rst checked
that 4 was not able to induce abasic site cleavage even at
concentrations 10 to 100 times higher than concentra-
tions leading to 100% cleavage by 1. To test the inhibi-
tion of Escherichia coli Exonuclease III (Fig. 2), various
concentrations of compound 4 were incubated with the
plasmid 30 min at 0 ꢀC. To this mixture was added the
enzyme (0.4 u/ml) and incubation was continued for
30 min (in these conditions, the enzyme cleaves 70% of
the plasmid, in the absence of any drug).
References and Notes
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13. Fkyerat, A.; Demeunynck, M.; Constant, J.-F.; Michon,
P.; Lhomme, J. J. Am. Chem. Soc. 1993, 115, 9952±9959.
14. Fkyerat, A.; Demeunynck, M.; Constant, J.-F.; Lhomme,
J. Tetrahedron 1993, 49, 11237±11252.
After densitometry of the gel we noted 50% inhibition
of the enzyme activity for a drug concentration corre-
sponding to one molecule of 4 for 5±6 bases pairs. Tes-
ted in the same conditions (results not shown) ethidium
bromine, a classical intercalator, requires the higher
concentration of one drug for 1±2 bases pairs to achieve
the same 50% inhibition.
Pharmacological Studies
15. Berthet, N.; Boudali, A.; Constant, J.-F.; Decout, J.-L.;
Demeunynck, M.; Fkyerat, A.; Garcia, J.; Laayoun, A.;
Michon, P.; Lhomme, J. J. Molecular Recogn 1994, 7, 99±107.
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Cytostatic/cytotoxic activities of compound 4 have been
determined on murine leukemia L1210 and on the
human pulmonary adenocarcinoma A549 cell lines.
Concentrations inhibiting the growth (L1210) or the
survival (A549) of the cells were measured. Compound
4 is marginally potent on L1210 and more toxic on
A549 (IC50 of more than 100 and 4 mM, respectively).
Unlike bis-guanidine drug 3, tested simultaneously with
the anticancer agent BCNU, compound 4 gives only a
simple additivity of the toxic e€ects.
17. Coppel, Y.; Constant, J.-F.; Coulombeau, C.; Demeunynck,
M.; Garcia, J.; Lhomme, J. Biochemistry 1997, 36, 4831±4843.
18. Belmont, P.; Jourdan, M.; Demeunynck M.; Constant, J-
F; Garcia, J. ; Lhomme, J.; Carez, D.; Croisy. A. J. Med
Chem. 1999, 42, 5153±5159.
19. As described previously,14 a linker with two amido func-
tions does not induce DNA cleavage at the AP-site but anity
1
for DNA falls dramatically (K=1.2Â104
M
compared to
1
K=2Â105 M in polyamino compound 1) due to the lack of
ionisation of the linker at physiological pH.
20. Leonard, N. J.; Lambert, R. F. J. Org. Chem. 1969, 34,
3240±3248.
Conclusion
We have synthesized a new hybrid adenine±acridine
compound 4, which interacts at abasic sites in DNA.
This compound inhibits the repair activity of Exonu-
clease III in vitro. This observation led us to consider a
new approach for anticancer activity where drugs such
as 4 could potentiate the action of antitumor agents,
thus allowing a decrease of the critical amount of
chemotherapeutic drug used. Lead compound 3 was
found to act in line with this strategy showing a syner-
gistic action with BCNU both in vivo and in vitro.
Compound 4 which di€ers by the presence of only one
21. Kita, Y.; Haratu, J. I.; Fujii, T.; Segawa, J. Y. Synthesis
1981, 451±452.
22. Iwanowicz, E. J.; Poss, M. A.; Lin, J. Synthetic Commun.
1993, 23, 1443±1445.
23. Compound 4: mp 185 ꢀC; 1H NMR (300 MHz, D2O) d
ppm 8.30 (1H, s), 8.24 (1H, s), 8.07 (1H, m), 7.60±7.40 (5H,
m), 4.30 (2H, m), 4.10 (2H, m), 4.01 (3H, s, OCH3), 3.60 (4H,
m), 3.47 (2H, m), 2.53 (2H, m), 2.24 (2H, m); UV/vis (H2O)
lmax (E) 226.3 (46,500), 278.8 (47,270), 349.9 (4440), 423
(8690), 444.5 (8341) nm; HRMS (positive FAB) calcd for
C28H33N11O2Cl 590.2507, found 590.2523.