Diaminopurine-acridine heterodimers for specific recognition of abasic site containing DNA. Influence on the biological activity of the position of the linker on the purine ring

Article abstract of DOI:10.1016/S0960-894X(01)00310-9

Three acridine-diaminopurine heterodimers tethered by a linker containing an N,N′-substituted guanidine were prepared. The molecules differ by the site of introduction of the linker on the 2,6-diaminopurine. The interactions of the new heterodimers with abasic site containing oligonucleotide were compared, and their cytotoxicity was measured in the presence or absence of the antitumor alkylating agent BCNU.

Full text of DOI:10.1016/S0960-894X(01)00310-9

Bioorganic & Medicinal Chemistry Letters 11 (2001) 1855–1858
Diaminopurine-acridine Heterodimers for Specific Recognition of
Abasic Site Containing DNA. Influence on the Biological Activity
of the Position of the Linker on the Purine Ring
a
a,
a
Karine Alarcon, Martine Demeunynck, * Jean Lhomme,
b
b
Daniele Carrez and Alain Croisy
`
aLEDSS, Chimie Bioorganique, UMR CNRS 5616, Universite´ Joseph Fourier, BP53, 38041 Grenoble cedex 9, France
bINSERM U350, Institut Curie Recherche, Laboratoire Raymond Latarjet, Centre Universitaire, 91405 Orsay cedex, France
Received 17January 2001; revised 21 March 2001; accepted 30 April 2001
0
Abstract—Three acridine-diaminopurine heterodimers tethered by a linker containing an N,N -substituted guanidine were prepared.
The molecules differ by the site of introduction of the linker on the 2,6-diaminopurine. The interactions of the new heterodimers
with abasic site containing oligonucleotide were compared, and their cytotoxicity was measured in the presence or absence of the
antitumor alkylating agent BCNU. # 2001 Elsevier Science Ltd. All rights reserved.
The mode of action of many antitumor drugs
involves the modification (mainly alkylation) of nucleic
1
bases in DNA. Drugs that interact specifically with
such lesions, and inhibit the DNA repair system, may
2
potentiate the action of antitumor drugs. 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 modified or abnormal bases by
3
hydrolysis of the N-glycosidic bond. We have described
in preceding papers the synthesis of a family of mole-
cules that have been optimized for specific recognition
4ꢀ7
of the abasic site in DNA.
They incorporate in their
structure an intercalator with a high affinity for DNA,
tethered to a nucleic base via a polyamine or guanidine
containing linker. We recently reported on the synthesis
and biological properties of 1 that contains two guani-
dine functions in the linker and that was shown to
0
potentiate the action of the anticancer agent N,N -bis(2-
chloroethyl)-N-nitrosourea (BCNU) in vitro and in
5
vivo. High-field NMR measurements of the interaction
of compound 1 with an oligonucleotide containing a
stable analogue of the abasic site indicated that the
purine ring of 1 is inserted inside the abasic pocket and
forms hydrogen bonds with the unpaired base (thymine)
located on the strand opposite to the abasic lesion.5
However, one limitation with compound 1 is a curare-
like acute toxicity in vivo which led us to design new
analogues containing basically the same structural units
in an effort to modulate its biological activity and
*Corresponding author. Tel.:+33-476-514429; fax: +33-467-514382;
e-mail: martine.demeunynck@ujf-grenoble.fr
0
960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00310-9

1856
K. Alarcon et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1855–1858
toxicity. We report herein on the synthesis of hetero-
0
alkylation of the purine with 3-bromopropylphthal-
imide in the presence of sodium hydride. The reaction
takes place regioselectively at position 9 (53% yield), no
product alkylated at the N-7position was observed.
Deprotection of the amine was achieved in 72% yield by
hydrolysis of the phthalimide group in acidic conditions
dimers 2–4 in which the linker contains one N,N -gua-
nidine function. The polymethylene units (butyl and
propyl) that link the guanidine moiety to the hetero-
cyclic units were chosen to adjust the length of the linker
to 11 atoms, that is the same number of atoms as in 1.
The molecules differ by the position of attachment of
the linker on the 2,6-diaminopurine ring. The linker was
tethered at the N-9 position (2), in the same position as in
compound 1, but was also incorporated as substituent of
the two exocyclic amines, at positions 2 and 6 (respec-
tively, 3 and 4). Varying the position of alkylation of the
purine may modify the affinity and/or the specificity for
abasic site containing DNA, as it may alter the formation
of hydrogen bonds between the drug and the unpaired
base located in the abasic site. The interaction with aba-
sic site containing oligonucleotide and some pharmaco-
logical properties will be also presented in this paper.
(HCl/AcOH/H O). Reaction of the amine 6 with the
2
protected thiourea 5 in the presence of coupling agent
EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride) gave the Boc,Troc protected guanidine 7
(79% yield). Deprotections of the guanidino and amino
protecting groups were achieved by one-pot successive
treatments with Zn in acetic acid and HCl 1 N in acetic
acid and gave 8 as a gum. The acridine moiety was
introduced by reaction of 8 with 6-chloro-2-methoxy-9-
7
phenoxyacridine (PhOAcr) as previously described.
Synthesis
The molecules were synthesized according to the retro-
synthetic pathway shown in Figure 1.
Figure 1.
In this strategy, the linker is built step-by-step by coupling
in a key-step, a propylamino substituted 2,6-diamino-
purine (DAP) with the protected amino thiourea 5 to
0
8
form the N,N -substituted guanidine. The Troc pro-
tected thiourea 5 was prepared by the one-pot reaction
9
published by Atkins. Successive addition of 2,2,2-
trichloroethylchloroformate (Troc-Cl) and mono-Boc
protected diaminobutane to a solution of potassium
thiocyanate gave 5 in 86% yield.
The syntheses of the molecules 2–4 are outlined in
Scheme 1.
The 9-substituted-DAP analogue 2 was prepared from
the 2,6-diaminopurine. The 9-aminopropyl-2,6-diamino-
purines 6 was obtained in two steps following the method
reported by Leonard for the alkylation of adenine.
Introduction of the aminoalkyl chain was achieved by
Scheme 1. (i) 3-Bromopropylphthalimide, DMF, NaH, rt, 6 days; (ii)
ꢁ
1
rt, 4 h; (iv) Zn, AcOH/H
2 N HCl/H
2
O/AcOH 1:1:1 mixture, 100 C, 24 h; (iii) 5, EDC, DMF,
ꢁ
2
O, 0 C, 4 h; (v) 1 N HCl in AcOH, rt, 1 h;
ꢁ
(
NEt
vi) PhOAcr, NEt
3
, PhOH, 80 C, 5 h; (vii) para-methoxybenzylamine
ꢁ
1
0
ꢁ
3
, DMF, 80 C, 2 h; (viii) 1,3-diaminopropane, 140 C, 2 h; (ix)
ꢁ
ꢁ
TFA, 70 C, 5 h; (x) BocNH–(CH ) –NH , 140 C, 1.5 days.
2
3
2

K. Alarcon et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1855–1858
1857
2
,6-Dichloropurine was chosen as starting material to
significantly stabilize the TA duplex, as indicated by
ÁTm values that increase regularly when the drug to
DNA ratio r is increased. Compound 2 appeared
slightly more efficient than 3 and 4. The stabilization is
much more pronounced however on the TX duplex,
with similar magnitudes for the three drugs. The shapes
of the curves reflect the mode of binding. With the TX
duplex, the slopes of the curves are steeper at low drug
to DNA ratios (r<1) than at ratios superior to 1. This
effect suggests preferred formation of 1:1 complexes
between the drugs and the abasic site containing oligo-
nucleotides. Indeed a comparable curve was previously
observed in the interaction of molecule 1 with the same
prepare molecule 3, in which the linker is attached on
the 2-amino group of DAP. Regioselective substitution
of the chlorine atom in position 6 with para-methoxy-
benzylamine gave the PMB protected 6-amino-2-chloro-
purine 9 in 76% yield. Higher temperature was required to
introduce the second substituent, 1,3-diaminopropane.
Compound 10 was thus obtained in 71% yield. Con-
densation with thiourea 5 in the presence of EDC gave the
protected guanidine 11 (76% yield). The Troc group was
first removed by treatment with Zn in acetic acid. Reflux-
ing in trifluoroacetic acid achieved complete deprotection
of the molecule, giving 12 as a gummy residue. Reaction of
12 with PhOAcr gave 3. The third molecule, in which the
linker is connected to the 6-amino group of DAP, was
prepared using 2-amino-6-triazolylpurine 13, which was
prepared in one step from DAP. Reaction of 13 with
mono-Boc protected diaminopropane, followed by depro-
tection of the primary amine afforded 14 (40% yield from
5
TX duplex. In this case, the high field NMR study had
revealed formation of a very specific 1:1 complex. From
these results, it appears that the site of tethering of the
linker does not significantly modify the binding to aba-
sic DNA although the geometry of the complex is
probably different for the three drugs. The H-bonding
capacities of the diaminopurine are preserved in all
three analogues.
11
13). Condensation with thiourea 5, one-pot deprotection
of the two protecting groups followed by the introduction
of the acridine moiety gave molecule 4.
12
Pharmacological Studies5
Interaction with Synthetic Oligonucleotides Containing
Abasic Sites
The three molecules were shown to be moderately cyto-
toxic on L1210 cells (IC50 of 36 mM for the most cyto-
toxic compound 3) but displayed relatively high
cytotoxicity on A549 cells (IC50 of 5–6.5 mM for the
three compounds) (Table 1).
In preceding papers,5,6 we used denaturation studies
(Tm measurements) to assay binding of drugs to abasic
DNA. In these experiments, the effect of adding
increased concentrations of a drug on the thermal
denaturation of a synthetic oligonucleotide (11-mer, TX
duplex) containing a stable analogue of the abasic site
[3-hydroxy-2-(hydroxymethyl)tetrahydrofuran, noted
X] was measured (Fig. 2).
Tested in combination with BCNU, compound 2 led to
a small but significant synergy (+16% increase). The
efficiency of compound 2 to potentiate in vitro the
activity of BCNU appeared much lower than the effect
obtained previously with compound 1. The observation
is a new indication that the nature of the linker con-
necting the two heterocycles is determinant for the bio-
logical activities.
The same study was also performed on the correspond-
ing natural duplex (TA duplex) in which the abasic site
is replaced by adenine.
To compare the relative binding affinities of the drug,
we used ÁTm measurements (ÁT =T (duplex in the
As a conclusion, the new heterodimers 2–4 interact spe-
cifically with abasic sites. The different sites of attachment
m
m
presence of the drug)ꢀT (duplex alone)). Results are
m
given in Figure 3.
As reported previously for comparable heterodimers
5
,6
containing a DNA intercalator, the three compounds
m
Figure 3. Influence of the drugs on the ÁT of the TA duplex (open
symbols) and TX duplex (black symbols). Experiments were con-
ducted at pH 7.0 in a buffer containing 10 mM sodium phosphate/
1
mM EDTA/20 mM NaCl. The drugs tested were 2 (*/*), 3 (&/&)
and 4 (~/~). ÁT is defined as the difference between the T of the
duplex in the presence of the drug and the T of the duplex alone. T
were measured for various ratios r of drugs (r=[drug]/[duplex]).
m
m
m
m
Figure 2. Sequences of the synthetic oligonucleotides.

1858
K. Alarcon et al. / Bioorg. Med. Chem. Lett. 11 (2001) 1855–1858
Table 1. Cytotoxicity of compounds 1–4 alone and in association
with BCNU
10. Leonard, N. J.; Lambert, R. F. J. Org. Chem. 1969, 34,
3240.
1
1. Alarcon, K.; Martelli, A.; Demeunynck, M.; Lhomme, J.
Compd
L1210
Cytotoxicity
IC50 (mM)
A549
Cytotoxicity
IC50 (mM)
Synergy with
a
Tetrahedron Lett. 2000, 41, 7211.
1
BCNU (%)
ꢁ
2. 2: Mp 210–220 C. H NMR (200 MHz, CD OD) (free
3
1
base): d ppm 8.20 (1H, d, J=9.2 Hz, Acr–H); 7.77 (1H, s, H-
); 7.72 (2H, m, Acr–H); 7.46 (1H, d, J=1.7Hz, Acr– H); 7.32
(1H, dd, J=9.2, 2.4 Hz, Acr–H); 7.20 (1H, dd, J=9.2, 2.1 Hz,
8
1
2
3
4
33
60
36
>100
10
6.5
5
+ 94%
+ 16%
—
Acr–H); 3.99 (2H, t, DAP-CH ); 3.89 (3H, s, O–CH ); 3.79
2
2
(2H, t, CH ); 3.01 (4H, m, 2 CH
3
5
—
); 1.90 (4H, m, 2 CH
2
); 1.45
2
1
2 2
3
(
2H, m, CH ). C NMR (50 MHz, D O): d ppm 155.8; 155.6;
aSimultaneous exposure of A549 cells to each tested compound (1–4)
10 mM) and BCNU (10 mM) for 24 h. Apparent synergy was measured
1
1
54.0; 151.4; 150.8; 148.7; 141.8; 140.6; 132.9; 127.5; 127.0;
24.5; 119.8; 116.8; 112.6; 110.0; 56.2 (O–CH ); 47.8 (NH–
(
3
when the cytotoxicity of the drug in association with BCNU was sig-
nificantly higher than the addition of the toxic effect of both drugs
alone.
CH
FAB(+), glycerol): M=561, m/z: 562 ((M+1) ). UV (H
lmax (e): 423 (6000), 278 (36,900), 217 (32,900) nm. HR-MS
2 2 2
); 40.5 (NH–CH ); 40.4 (NH–CH ); 38.0; 26.5; 25.6. MS
+
(
2
O):
(
5
3
FAB(+), NBA): found: 562.2552; calcd for C ClH N O+H:
27 32 11
of the linker tested in this study do not influence to a
large extent the binding to abasic site containing oligo-
nucleotides. One molecule, 2, in which the linker is teth-
ered to position 9, displayed a synergy effect with BCNU.
62.2558.
.
ꢁ
1
4HCl: Mp >350 C. H NMR (300 MHz, D
2
O): d ppm 7.85
(
(
1H, d, J=9.6 Hz, Acr–H); 7.50 (1H, s, Acr–H); 7.30–7.08
5H, m, Acr–H and H-8); 3.83 (2H, t, DAP-CH ); 3.75 (3H, s,
); 3.23 (2H, t, CH ); 3.07(2H, t, C H ); 2.87(2H, t,
); 1.87(2H, m, CH –CH ); 1.69 (2H, m, CH –CH
–CH ); 1.54 (2H, m, CH ). C NMR
2
O–CH
CH
CH
3
2
2
2
2
2
–CH
2
2
2
–
1
3
References and Notes
2
2
–CH –CH –CH
2 2 2 2
(
75 MHz, D
126.5; 124.0; 119.5; 116.5; 55.8 (O–CH
(NH–CH ); 37.9 (NH–CH ); 29.8 (CH
2
O): d ppm 155.5; 155.3; 154.1; 150.8; 140.5; 140.2;
); 47.4 (NH–CH ); 39.9
–CH –CH ); 27.1
1
7
2
. Hemminki, K.; Ludlum, D. B. J. Natl. Cancer Inst. 1984,
3, 1021.
. Barret, J. M.; Etievant, C.; Fahy, J.; Lhomme, J.; Hill, B. T.
3
2
2
2
2
2
2
(CH –CH –CH ); 26.2 (CH –CH –CH ); 25.2 (CH –CH –
2
CH
2
2
2
2
2
2
2
+
Anticancer Drugs 1999, 10, 55.
. Lhomme, J.; Constant, J. F.; Demeunynck, M. Biopoly-
mers, Nucleic Acid Science 1999, 52, 65.
. Belmont, P.; Demeunynck, M.; Constant, J.-F.; Lhomme,
J. Bioorg. Med. Chem. Lett. 2000, 10, 293.
. Belmont, P.; Jourdan, M.; Demeunynck, M.; Constant, J.-
2
). MS (FAB(+), glycerol) m/z: 562 ((M+1) ). UV (H
2
O):
3
l
max (e): 423 (6000), 279 (39,800), 217 (36,200) nm.
.
ꢁ
1
4 HCl: Mp 220 C. H NMR (300 MHz, D O): d ppm 7.82
2
4
(1H, d, J=9.2 Hz, Acr–H); 7.66 (1H, s, Acr–H); 7.24–7.06
(5H, m, Acr–H and H-8); 3.80 (2H, t, DAP-CH
3
O–CH ); 3.21 (2H, t, CH ); 3.06 (4H, m, 2 CH ); 1.82 (2H, m,
2
); 3.73 (3H, s,
5
2
2
F.; Garcia, J.; Lhomme, J.; Croisy, A.; Carrez, D. J. Med.
Chem. 1999, 42, 5153.
CH –CH –CH ); 1.65 (2H, m, CH –CH –CH –CH ); 1.58
2
2
2
2
2
2
2
1
3
(2H, m, CH
ppm 156.2; 154.9; 153.3; 152.3; 145.5; 141.2; 141.0; 139.0;
2 2 2 2 2
–CH –CH –CH ). C NMR (75 MHz, D O): d
6
. Berthet, N.; Constant, J.-F.; Demeunynck, M.; Michon, P.;
Lhomme, J. J. Med. Chem. 1997, 40, 3346.
. Fkyerat, A.; Demeunynck, M.; Constant, J.-F.; Michon, P.;
Lhomme, J. J. Am. Chem. Soc. 1993, 115, 9952.
. Poss, M. A.; Iwanowicz, E. J.; Reid, J. A.; Lin, J.; Gu, Z.
Tetrahedron Lett. 1992, 33, 5933.
. Atkins, P. R.; Glue, S. E. J.; Kay, I. T. J. Chem. Soc., Perkin
Trans. 1 1973, 22, 2644.
127.3; 124.8; 120.2; 117.2; 113.4; 109.1; 106.0; 56.5 (O–CH );
3
7
48.1 (NH–CH ); 40.7(NH– CH ); 38.8 (NH–CH ); 38.3 (NH–
2
2
2
CH
(CH
UV (H O): l
2
); 27.8 (CH
2
–CH
2
–CH
2
2 2 2
); 27.1 (CH –CH –CH ); 25.9
+
8
2
–CH
2
–CH
2
). MS (FAB(+), NBA) m/z: 562 ((M+1) ).
(e): 423 (4900), 278 (32,100), 221 (27,900) nm.
max
2
9
HR-MS (FAB(+), NBA): found: 562.2558; calcd for
C
27ClH32N11O+H: 562.2558.