Design of site specific DNA damaging agents for generation of multiply damaged sites

Article abstract of DOI:10.1016/S0040-4020(02)00345-9

We describe the synthesis and DNA damaging activities of hybrid molecules in which a purine (adenine) is linked to an intercalating chromophore (acridine) by a polyamino linker. A DNA damaging agent, phenanthroline or para-nitrobenzamide, is tethered to the acridine moiety at various positions. Our goal is to induce upon activation other lesions in close proximity to the abasic site and therefore create cytotoxic multiply damaged sites.

Full text of DOI:10.1016/S0040-4020(02)00345-9

TETRAHEDRON
Pergamon
Tetrahedron 58 *2002) 4291±4298
Design of site speci®c DNA damaging agents for generation
of multiply damaged sites
Alain Martelli, Jean-Francois Constant,p Martine Demeunynck,p Jean Lhomme and Pascal Dumy
Â
LEDSS, CNRS/Universite J. Fourier, BP53 38041 Grenoble Cedex 9, France
Received 21 January 2002;revised 11 March 2002;accepted 27 March 2002
AbstractÐWe describe the synthesis and DNA damaging activities of hybrid molecules in which a purine *adenine) is linked to an
intercalating chromophore *acridine) by a polyamino linker. A DNA damaging agent, phenanthroline or para-nitrobenzamide, is tethered
to the acridine moiety at various positions. Our goal is to induce upon activation other lesions in close proximity to the abasic site and
therefore create cytotoxic multiply damaged sites. q 2002 Elsevier Science Ltd. All rights reserved.
A number of cytotoxic agents are known to produce DNA
damage. Several classes of agents, mainly those involving
radical processes *neocarzinostatin, bleomycin, ionizing
radiations),1±3 generate clustered lesions or multiply
damaged sites *MDS) in tracks of a few base pairs. Such
multiple damage have a high biological impact as they
present a more challenging repair problem for the cell.4,5
The strategy we developed to induce MDS is to target a
DNA lesion *the abasic site) with a drug able to create
other damage in close vicinity *Fig. 1). Abasic site6 results
from the loss of a base in DNA, either spontaneously
or enzymatically as intermediate in the repair of modi®ed
bases.
by a non-protonated secondary amine of the linking chain of
the drug acting as a b-elimination catalyst. NMR studies of
the interaction of I with a duplex DNA undecamer contain-
ing a stable analogue of the abasic site have revealed that the
drug ®ts perfectly the abasic site: the purine moiety being
docked in the abasic pocket and the acridine moiety being
intercalated at a two base pairs distance on the 50 side of the
lesion.8
Starting from this optimized structure, we have modi®ed the
acridine nucleus to endow the heterodimer with a photo-
damaging or cleavage activity. Our goal was to induce
upon activation other lesions in close proximity to the abasic
site. In a ®rst approach we have introduced various sub-
stituents *3-nitro, 3-amino, 3-amino-4-iodo) on the acridine
These lesions are chemically unstable, and cleavage of the
DNA strand at these sites may occur via a b-elimination
reaction. We previously described the synthesis and the
study of acridine-purine heterodimers such as I *see Fig. 2)
designed to speci®cally interact and cleave DNA at abasic
sites.7 These molecules behave as `arti®cial nucleases', and
cleavage is triggered in the pre-formed drug±DNA complex
Figure 1. Targeting abasic sites for generation of multiply damage in DNA.
Keywords: DNA damaging agents;acridines;abasic site.
Corresponding authors. Tel.: 133-476-514429;fax: 133-476-514946;
p
e-mail: jean-francois.constant@ujf-grenoble.fr;
martine.demeunynck@ujf-grenoble.fr
Figure 2.
0040±4020/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020*02)00345-9

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A. Martelli et al. / Tetrahedron 58 02002) 4291±4298
Scheme 3. *i) DMF, NEt3, 08C;*ii) PhOH, NEt 3, 708C.
Scheme 1. *i) DMF, NEt3;*ii) PhOH, NEt , 708C.
3
chromophore *Fig. 2), and studied the photodamaging
activity of the corresponding heterodimers II±V.9
Two derivatives, IV and V, showed signi®cant photo-
chemical reactivity in close proximity to the abasic site. In
order to increase both the selectivity and the ef®ciency of
the cleavage, we designed new heterodimers in which the
group responsible for DNA cleavage is tethered to the
acridine chromophore. Denny and co-workers10 have
developed a large series of asymmetrically substituted acri-
dines that binds by positioning a side chain in each groove
of DNA. This mode of DNA binding, known as threading
intercalation, gives to the molecules moderate DNA
af®nities but slow dissociation kinetics. In the design of
`triggered' DNA cleavage agents, this slow dissociation
parameter should be advantageous as it confers to the
molecules longer residence time in DNA that is likely to
promote higher cleavage ratio. Introduction of the two
chains at acridine 9 and 4 positions should place the
two side chains in opposite grooves when the acridine
ring intercalates with maximum overlap with the base pairs.
We therefore prepared new heterodimers *5, 10 and 13)
derived from molecule I, in which the damaging agents
*1,10-phenanthroline that cleaves DNA in the presence of
copper and a reducing agent, and the photochemically active
para-nitrobenzamide group) were introduced as functional
groups on the 4 position. The two acridine±phenanthroline
conjugates differ in the absence *5) or presence *10) of a
linker between the two chromophores. To modify the mode
of binding we also synthesized two conjugates *18a and b)
in which the para-nitrobenzamide group was tethered at
the acridine 2 position. We describe here the synthesis of
the conjugates and the preliminary studies of their DNA
damaging activities.
Scheme 2. *i) N-*bromopropyl) phthalimide;K 2CO3, DMF, rt;
*ii) NH2NH2±H2O, EtOH, 608C;*iii) DMF, NEt ;*iv) PhOH, NEt 3, 708C.
3

A. Martelli et al. / Tetrahedron 58 02002) 4291±4298
4293
Reaction of 5-amino[1,10]phenanthroline *1) with the acri-
dine 211 gave the carboxamido derivative 3 in 62% yield.
Introduction of the amino-substituent at the 9 position was
achieved by reacting 3 with the adenine±polyamine synthon
47 in phenol and in the presence of triethylamine. To prepare
10, the 5-aminophenanthroline 1 was ®rst functionalized
with the amino chain in three steps *tosylation to give 6,12
alkylation and deprotection of the primary amino group of
the linker). Synthesis of conjugate 10 was then achieved as
described above by formation of the carboxamide 9 and
subsequent substitution of chlorine at the 9 position of the
acridine ring by synthon 4.
1.1.2. Nitrobenzamide±acridine conjugates. In compound
13 the nitrobenzamido group was attached at position 4 of
the acridine by formation of a carboxamide. In compounds
18a, b, the reactive group was introduced at the acridine 2
position via an ether function.
1.1.3. 4-Carboxamido acridine. As depicted in Scheme 3,
we used the strategy described above to synthesize 13. The
nitrobenzamide synthon 1113 was reacted with acridine 2 to
give 12 in good yield *80%). Substitution at position 9 was
achieved by adding the adenine±polyamine synthon 4 to a
solution of 12 in phenol. The ®nal conjugate 13 was isolated
in 42% yield.
1.1.4. 2-Substituted acridines. The synthesis of com-
pounds 18a and b is described in Scheme 4. We used the
strategy we reported previously for selective alkylation
of 2-hydroxy-9-phenoxyacridine.14 Reaction of O-silyl
protected 9-phenoxyacridine 16 with methanesulfonate
activated synthons 15a,b *prepared in two steps from
v-aminoalcohols and para-nitrobenzoylchloride) gave the
intermediates 17a, b in 51 and 48% yields, respectively.
Substitution of the phenoxy group with adenine±polyamine
synthon 4 afforded the desired conjugates 18a and b in 52
and 74% yields, respectively.
Scheme 4. *i) CH2Cl2, 0.2N NaOH, 08C;*ii) MsCl, pyridine, 0 8C;*iii) 1 M
TBAF in THF;*iv) PhOH, NEt 3, 808C.
1.2. DNA damaging activity
1. Results
Due to the chemical instability of abasic sites, the damaging
activities of the new conjugates were examined on a
synthetic 23-mer duplex DNA containing the stable tetra-
hydrofurane analogue of the abasic site *X) in strand 1. This
analogue was previously shown to be a good model of
abasic site.15 The oligonucleotide sequence and the structure
of the analogue are shown in Fig. 3. Thymine T35 faces the
abasic site. Each strand was successively 32P 50-end labeled
using g-32P-ATP and T4 polynucleotide kinase.
1.1. Synthesis
1.1.1. Phenanthroline±acridine conjugates. Two phenan-
throline±acridine conjugates, 5 and 10, have been synthe-
sized. In both compounds the aminophenanthroline
substituent was attached at the acridine 4 position. As
described in Schemes 1 and 2, the strategy designed by
Denny11 was used to prepare both compounds, i.e. intro-
duction of the ®rst amino substituent at the 4 position of
9-choro-acridine-4-carbonyl chloride *2), followed by
introduction of the aminoalkyl side chain at the 9 position.
1.2.1. Phenanthroline±acridine conjugates. The 23-mer
duplex containing the abasic site was incubated with
Figure 3.

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A. Martelli et al. / Tetrahedron 58 02002) 4291±4298
Figure 5. Autoradiogram of 20% denaturing polyacrylamide gel showing
the photocleavage activity of the nitrobenzamide±acridine conjugates.
Lanes G1A: Maxam±Gilbert sequencing;lane 1: strand 1 alone;lane 2:
strand 1118a;lane 3: strand 1 118b;lane 4: strand 2 alone;lane 5: strand
2118a;lane 6: strand 2 118b.
Figure 4. Autoradiogram of 20% denaturing polyacrylamide gel showing
the DNA cleavage activity of the phenanthroline±acridine conjugates. Lane
1: strand 1 alone;lanes 2±5: strand 1 in the presence of added drug *lane 2:
Cu;lane 3: phenanthroline±Cu;lane 4: 5;lane 5: 10);lane 6: strand 2 alone;
lanes 7±10: strand 2 in the presence of added drug *lane 7: Cu;lane 8:
phenanthroline±Cu;lane 9: 5;10: 10).
agent is linked at the acridine 4 position, does not induce
any alkali-labile site on either strand *data not shown). As
shown in Fig. 5, compounds 18a and b induce cleavage on
both strands following irradiation and piperidine treatment.
The cleavage is highly selective. The preferential sites are
located at the abasic site X12 and at T35 on the opposite
strand. Minor sites are also observed on G36, G34, G32
and G8.
phenanthroline or with compound 5 or 10, in the presence of
copper and mercaptopropionic acid *MPA) as a reducing
agent. Incubation was carried out at 258C for 40 min. The
autoradiogram of the gel is shown in Fig. 4.
Two controls were done: *1) the duplex in the absence of
any added drug *lanes 1 and 6) and *2) the duplex in the
presence of Cu and MPA *lanes 2 and 7) showed no
cleavage. The duplex in the presence of 1,10-phenanthro-
line, Cu and MPA *lanes 3 and 8) showed cleavage at all
bases on each strand. However, slightly preferential sites
were observed on both strands on the 30-side of the lesion.
This might suggest that the distorted abasic site containing
sequence is a preferential binding structure for *Phen)2Cu1.
For instance, it has been reported that 1,10-phenanthroline
copper complexes show preferential activity on particular
nucleic acid structures.16 Therefore, the preferential sites
observed in this study might suggest the presence of
distorted structures *such as kink) in the vicinity of the
abasic site. In the presence of compound 5, Cu and MPA
*lanes 4 and 9), no signi®cant cleavage was observed on
either strand. Interestingly, in the presence of compound
10, Cu and MPA *lanes 5 and 10) no cleavage was observed
on strand 1, but a highly preferential site, corresponding to
the unpaired base T35 facing the abasic site, appeared on
strand 2. A secondary site of cleavage was also observed
at the ¯anking G36 base.
2. Conclusion
We designed and prepared ®ve new conjugates in which
a reactive group capable of inducing DNA damage or
cleavage was tethered to a vector targeting abasic lesions.
From the three nitrobenzamide±acridine conjugates tested
as photocleavage agents, two molecules, 18a and b, have
been shown to damage DNA upon illumination. As illus-
trated by molecular modeling calculations,17 the two
molecules possibly interact with DNA in a way very similar
to that of the original arti®cial nucleases I and II. Both side
chains of the intercalated acridine appear to be positioned in
the minor groove. The acridine±phenanthroline conjugate
10 is very promising too as it cleaves ef®ciently and selec-
tively the DNA strand at the base opposite the abasic site.
This conjugate probably interacts with DNA by threading
intercalation, i.e. by positioning a side chain in each groove
of DNA.10,18 The presence of the chain joining the phenan-
throline to the acridine 4 position confers to the conjugate
the degree of freedom required for adequate positioning of
the DNA cleavage agent inside one groove. This compound
seems a good candidate for the generation of double-
stranded DNA cleavage *via its intrinsic b-elimination
cleaving activity on the abasic strand and its radicalar
cleavage activity on the opposite strand).
1.2.2. Nitrobenzamide±acridine conjugates. The oligo-
nucleotides were incubated with compounds 13 and 18a,b
and irradiated *l.310 nm) at 48C for 3 h. After irradiation,
the damaged sites were revealed by hot piperidine treatment
*908C, 10 min). Compound 13 in which the photodamaging

A. Martelli et al. / Tetrahedron 58 02002) 4291±4298
4295
3. Experimental
MS *FAB, NBA): Mˆ690; m/z: 691 *M11)1. HRMS:
*M1H)1 calculated for C39H39N12O: 691.3370;Found:
691.3365.
3.1. General
3.1.3. 5-[N-33-Phthalimidopropyl)-N-p-toluenesulfonyl]-
amino[1,10]phenanthroline 37). A mixture of 612 *0.2 g,
0.57 mmol) and potassium carbonate *0.117 g, 0.86 mmol)
was stirred in dry DMF *5 mL) at room temperature for
30 min under nitrogen. N-*3-Bromopropyl)-phthalimide
*0.23 g, 0.86 mmol) was then added and the mixture was
stirred overnight at 608C. After cooling down to room
temperature, the mixture was poured into a large volume
of water. Compound 7 precipitated, and was ®ltered off.
Puri®cation was achieved by crystallization from ethanol±
water. Compound 7 was obtained in 57% yield *0.174 g).
Melting points were determined using a Reicher Thermovar
apparatus and are uncorrected. NMR spectra were recorded
on Bruker AC 200 and AM 300 spectrometers using solvent
as the internal reference;the chemical shifts are reported in
ppm, in d units. The mass spectra were recorded on Varian
Mat 311 and AET MS 30 instruments. High resolution mass
spectra were obtained from `Centre Regional de Mesures
Â
Physiques de l'Ouest', Universite de Rennes. Absorption
spectra were obtained on a Perkin±Elmer Lambda UV/Vis
spectrometer. Microanalyses were performed by the
`Service Central de Microanalyses du CNRS', Lyon.
Puri®cations were generally performed by chromatography
on silica gel or alumina. The purity of the compounds
was assessed by reversed-phase HPLC, performed on a
m-bondapak C18 analytical column *Waters Associates).
The chromatographic system is equipped with two M-510
pumps and a photodiode array detector Waters 996 using
Millenium 32 software. A linear gradient from 0 to 100%
methanol in H2O pH 2.5 *phosphoric acid), 2 mL min21
¯ow rate, was used. Products were characterized by their
retention time and UV/Vis absorption.
1
Mp 190±1938C. H NMR *200 MHz, CDCl3): d *ppm)ˆ
9.18 *2H, m, 2Phen-H);8.60 *1H, dd, Jˆ2 and 8.5 Hz,
Phen-H);8.04 *1H, dd, Jˆ2 and 8.5 Hz, Phen-H);7.77±
7.52 *10H, m, 2Phen-H, Ph-H and Phth-H);7.27 *1H, s,
Phen-H);3.96 *1H, m, CH);3.72±3.56 *3H, m, CH
CH);2.43 *3H, s, Ph-CH );1.82 *2H, m, CH ±CH2±CH2).
and
2
3
2
MS *FAB, NBA): Mˆ536; m/z: 537 *100, *M11)1).
3.1.4. 5-[N-33-Aminopropyl)-N-p-toluenesulfonyl]amino-
[1,10]phenanthroline 38). Hydrazine hydrate *0.15 g,
3 mmol) was added dropwise to a solution of 3 *0.16 g,
0.3 mmol) in ethanol *10 mL). The mixture was stirred at
608C for 2 h. The solvent was then cooled down to room
temperature, and the precipitate was removed by ®ltration.
The solvent was evaporated to dryness under vacuum. The
solid residue was stirred again in ethanol *10 mL). The
insoluble part was removed by ®ltration and the ®ltrate
was evaporated to dryness. This procedure was repeated
three times to eliminate most of the phthalhydrazide. The
solid was ®nally dissolved in 10% aqueous NaHCO3±
CH2Cl2 mixture. Organic layer was separated, washed
with water and dried over Na2SO4. After evaporation of
the solvent, 8 was obtained in 41% yield *0.049 g): mp
3.1.1. 9-Chloro-4-[N-3[1,10]phenantrolin-5-yl)carboxa-
mido]-acridine 33). 5-Amino[1,10]-phenanthroline *1)
*0.146 g, 0.75 mmol) was added to a ice cooled solution
of 211 *0.2 g, 0.72 mmol) and triethylamine *0.2 mL,
1.44 mmol) in dry DMF *5 mL) kept under nitrogen. The
mixture was kept at 48C for 30 min, and then stirred at room
temperature overnight. The solution was poured into cold
10% NaHCO3. The solid was ®ltered off, and washed with
cold water and cold methanol. Compound 3 was thus
obtained in 62% yield *0.194 g) and was used without
1
further puri®cation: mp 185±1908C. H NMR *200 MHz,
CDCl3): d *ppm)ˆ14.51 *1H, s, CO±NH);9.29±7.60
*14H, m, Acr-H and Phen-H). MS *FAB, glycerol): Mˆ
434; m/z: 435 *100, *M11)1).
1
162±1658C. H NMR *200 MHz, CDCl3): d *ppm)ˆ9.09
*2H, m, Phen-H);8.53 *1H, m, Phen-H);7.95 *1H, m,
Phen-H);7.58±7.15 *7H, m, Phen-H and Ph-H);4.00 *1H,
m, CH±NTsPhen);3.65 *1H, m, C H±NTsPhen);2.79 *2H,
m, CH2±NH2);2.41 *3H, s, C H3±Ph);1.60 *2H, m, C H2±
CH2±NH2). MS *FAB, NBA): Mˆ406; m/z: 407 *M11)1.
3.1.2. Acridine±phenanthroline conjugate 35). A solution
of 3 *0.1 g, 0.23 mmol) in freshly distilled phenol *5 mL)
was stirred at 608C under nitrogen for 30 min. Compound 47
*0.1 g, 0.23 mmol) and NEt3 *0.046 mL, 0.33 mmol) were
then added. The mixture was stirred at 708C for 6 h. The
mixture was cooled down to room temperature and poured
into cold 1N NaOH. The precipitate was isolated by centri-
fugation, and washed several times with water. The solid
was dissolved in methanol±methylene chloride mixture
*1:1). 12N HCl was slowly added to allow formation and
precipitation of 5 as the tetrahydrochloride *0.068 g, 35%):
mp 245±2508C. 1H NMR *300 MHz, DMSO-d6: d
*ppm)ˆ12.30 *0.5H, m, NH);10.69 *0.5H, m, N H);10.56
*0.5H, m, NH);9.95 *1H, m, Acr-H or Phen-H);9.65 *3H,
m, Acr-H or Phen-H);9.21 *1H, m, Acr-H or Phen-H);
9.15±8.70 *3H, m Acr-H or Phen-H);8.61 *1H, s,
Ade-H);8.58 *1H, s, Ade-H);8.52 *1H, m, Acr-H or
Phen-H);8.20±8.00 *2H, m, Acr-H or Phen-H);7.70 *1H,
m, Acr-H or Phen-H);7.61 *2H, m, Acr-H or Phen-H);7.39
*0.7H, m, NH);4.67 *2H, m, C H2±Ade);4.27 *2H, m, C H2±
NHAcr);3.47 *2H, m, C H2±CH2±Ade);3.12 *6H, m,
3CH2±NH);2.70±2.30 *2H, m, CH );2.12 *2H, m, CH ).
3.1.5. 9-Chloro-4-[N-33-3N-3[1,10]phenanthrolin-5-yl)-N-
p-toluenesulfonylamino)proyl)-carboxamido]-acridine
39). Compound 8 *0.063 g, 0.15 mmol) was added to a ice
cooled solution of acridine 211 *0.043 g, 0.15 mmol) and
NEt3 *0.021 mL, 0.15 mmol) in anhydrous DMF *3 mL)
under nitrogen. The reaction was stirred for 10 min at 48C
and then the solution was poured into cold 10% aqueous
NaHCO3. The precipitate was ®ltered off, washed with
water and dried to give compound 7 *0.023 g, 23%): mp
1
150±1558C. H NMR *200 MHz, CDCl3): d *ppm)ˆ11.68
*1H, m, CO±NH);9.14 *2H, m);8.89 *1H, dd, Jˆ2 and
8.5 Hz);8.65±8.57 *2H, m);8.42 *1H, dd,
Jˆ2 and
8.5 Hz);7.97 *1H, dd, Jˆ1 and 7.5 Hz);7.93 *1H, dd, Jˆ
2 and 8.5 Hz);7.77±7.47 *8H, m);7.18 *1H, s);7.14 *1H, s);
4.16 *1H, m, CH±NTsPhen);3.79±3.71 *3H, m, C H2±
NHCOAcr and CH±NTsPhen);2.38 *3H, s, Ph-C H3);2.02
*2H, m, CH2±CH2±CH2). MS *FAB, NBA): Mˆ645.5; m/z:
646 *100, *M11)1).
2
2

4296
A. Martelli et al. / Tetrahedron 58 02002) 4291±4298
3.1.6. Acridine±phenanthroline conjugate 310). The
procedure described above for 5 was used for the synthesis
of 10, starting from 9 *0.052 g, 0.085 mmol), 47 *0.045 g,
0.102 mmol) and NEt3 *0.035 mL, 0.25 mmol): mp 220±
144.85;140.09;136.46;134.99;128.17;123.49;119.35;
118.73;46.91;46.31;45.39;45.23;44.94;41.12;40.15;
40.12;26.22;25.82;25.35;22.90. UV *H 2O): l *1): 423.1
*7900);263.7 *47,000). MS *FAB, NBA):
**M11)1). HRMS: *M1H)1 calculated for C38H45N12O4
733.3687;Found: 733.3685.
m/zˆ733
1
2258C. H NMR *200 MHz, DMSO-d6): d *ppm)ˆ10.55
*1H, m, NH);9.62±8.67 *9H, N H and Acr-H and
Phen-H);8.58 *1H, s, Ade-H);8.55 *1H, s, Ade-H);
8.42±7.81 *6H, Acr-H and Phen-H);7.61±7.41 *5H, Ts- H
and NH);4.66 *2H, m, C H2±Ade);4.27 *2H, m,
CH2±NHCOAcr);4.10±3.70 *2H, m, C H2±NTsPhen);
3.42±3.35 *4H, m, 2CH2);3.10 *6H, m, 3C H2);2.40 *5H,
m, CH3±Ph and CH2);2.09 *2H, m, C H2);1.82 *2H,
m, CH2). MS *FAB, NBA): Mˆ901; m/z: 902 *100,
*M11)1). HRMS: *M1H)1 C49H52N13O3: M calculatedˆ
902.4037;M found ˆ902.4036.
3.1.9. 4-34-Nitrobenzamido)butanol 314a). A solution of
p-nitrobenzoyl chloride *0.500 g, 2.7 mmol) in CH2Cl2
*30 mL) was added to a cold solution of 4-amino-butanol
*0.300 g, 3.36 mmol) in 0.2N NaOH solution *30 mL). The
mixture was stirred at 08C for 6 h. The organic layer was
then separated, washed successively with 0.5N NaOH,
water and brine, and ®nally dried over sodium sulfate.
After evaporation of the solvent, compound 14a was
isolated as an oil in 32% yield *204 mg): 1H NMR
*200 MHz, CDCl3): d *ppm)ˆ8.24±8.20 *2H, m, Ph-H);
7.94±7.89 *2H, m, Ph-H);7.09 *1H, m, NH);3.71 *2H, t,
Jˆ6 Hz, CH2±OH);3.47 *2H, q, Jˆ6 Hz, CH2±NH);2.28
*1H, m, OH);1.74±1.51 *4H, m, 2CH 2). 13C NMR
*200 MHz, CDCl3): d *ppm)ˆ165.83;149.59;140.45;
130.87;123.88;62.46;40.36;29.92;26.28.
3.1.7. N-[4-34-Nitrobenzamido)butyl]-9-chloro-acridine-
4-carboxamide 312). The acridine derivative 211 *0.212 g,
0.88 mmol) was added to a solution of amine 1113 *0.210 g,
0.85 mmol) and NEt3 *0.319 mL, 2.29 mmol) in DMF
*10 mL) kept at 08C under nitrogen. The mixture was stirred
at 08C for 20 min, and then allowed to warm up to rt until
complete solubilization of the reactants. The mixture was
then poured into cold NaHCO3. The solid was ®ltered off
and washed with water. Compound 12 was thus obtained in
80% yield *0.325 g, 0.68 mmol). It was crystallized from
3.1.10. 6-34-Nitrobenzamido)hexanol 314b). The pro-
cedure described above for 14a was used for the synthesis
of 14b, starting from 6-amino-hexanol. Compound 14b was
isolated as a white solid in 67% yield *1.09 g, 3.60 mmol):
mp 72±738C; 1H NMR *200 MHz, CDCl3): d *ppm)ˆ7.87±
8.28 *4H, m, Ph-H);6.27 *1H, s, N± H);3.63 *2H, t, Jˆ6 Hz,
CH2±OH);3.43 *2H, q, Jˆ6 Hz, NH±CH2);1.38±1.80 *9H,
m, 4 CH2 and OH).; 13C NMR *50 MHz, CDCl3): d *ppm)ˆ
165.57;149.33;140.22;128.06;123.68;62.48;40.15;
1
ethanol: mp 124±1288C. H NMR *200 MHz, CDCl3): d
ppmˆ11.86 *1H, m, NH±CO±Acr);8.98 *1H, dd, Jˆ2
and 9 Hz, Acr-H);8.64 *1H, dd, Jˆ2 and 9 Hz, Acr-H);
8.46 *1H, d, Jˆ9 Hz, Acr-H);8.24 *2H, m, Ph-H);8.15
*2H, d, Jˆ9 Hz, Acr-H);8.07 *2H, m, Ph-H);7.90±7.65
*3H, m, 3Acr-H);7.55 *1H, m, N H±CO±Ph);3.74 *2H, q,
Jˆ6 Hz, CH2±NH);3.64 *2H, q, Jˆ6 Hz, CH2±NH);1.90
*4H, m, 2CH2). 13C NMR *50 MHz, CDCl3): d *ppm)ˆ
165.75;149.56;147.35;146.37;140.51;135.84;
32.34;29.32;26.45;25.21. MS *CI, NH
31isobutane)
m/zˆ267 *100, *M11)1);Analysis: Calcd for
C13H18N2O4: C 58.63, H 6.81, N 10.52%;Found: C 58.76,
H 6.85, N 10.56%.
132.02;129.30;129.06;128.65;127.85;126.67;
125.07;124.71;123.73;108.74;84.51;40.45;39.30;
28.20;26.13. MS *CI, NH 31isobutane): Mˆ476.1;
m/zˆ477 **M11)1). Analysis: Found: C 62.32, H 4.44, N
10.71%;Calcd for C 25H21ClN4O4 *0.5 EtOH): C 62.46, H
4.84, N 11.21%.
3.1.11. 34-34-Nitrobenzamido)butyl)methane-sulfonate
315a). Methanesulfonyl chloride *0.133 mL, 1.71 mmol)
was added to a cold solution *48C) of 14a *0.204 g,
0.86 mmol) in pyridine *5 mL). After 4 h of stirring, the
solution was diluted with water and extracted with
CH2Cl2. The organic layer was separated, successively
washed with 1N HCl, water and brine. After evaporation
of the solvent, 15a was isolated as an oil in 47% yield
3.1.8. 6-Amino-9-[11-3N-34-34-nitrobenzamido)-butyl)-
acridin-9-yl-4-carboxamido)-3,7,11-triazaundecyl]-9H-
purine trihydrochloride 313). A solution of 12 *0.100 g,
0.21 mmol) in phenol was stirred under nitrogen at 608C for
30 min. Compound 47 *0.092 g, 0.21 mmol) and NEt3
*0.030 mL, 0.21 mmol) were then added, and the mixture
was stirred at 708C for 7 h. The solution was slowly poured
into cold 1N NaOH under vigorous stirring. The yellow
solid was ®ltered off, washed several times with 1N
NaOH and then water. The solid residue was dissolved in
methanol and 12N HCl was added to allow isolation of 14 as
1
*0.126 g): H NMR *200 MHz, CDCl3): d *ppm)ˆ8.21±
8.17 *2H, m, Ph-H);7.92±7.87 *2H, m, Ph-H);6.86 *1H,
m, NH);4.23 *2H, t, Jˆ6 Hz, CH2±OMs);3.45 *2H, q,
Jˆ6 Hz, CH2±NH);2.98 *3H, s, SO ±CH3);1.77 *4H, m,
2
2CH2). MS *FAB, NBA) m/zˆ317 *100, *M11)1).
3.1.12. 36-34-Nitrobenzamido)hexyl)methane-sulfonate
315b). Methanesulfonyl chloride *0.051 mL, 0.66 mmol)
was added to a cold solution *48C) of 14b *0.100 g,
0.33 mmol) in pyridine *5 mL). After 6 h of stirring at
48C, water was added to the solution. The solid was ®ltered
off, washed with cold water and dried. 15b was thus isolated
1
the hydrochloride *78 mg, 42%): mp 192±1958C. H NMR
*200 MHz, D2O): d ppmˆ8.17 *1H, s, Ade-H);8.11±8.07
*2H, m, Ade-H and Acr-H);7.91 *1H, m, Acr-H);7.63 *1H,
m, Acr-H);7.48±7.43 *2H, m, Ph-H);7.28±7.18 *5H, m,
3Acr-H and Ph-H);4.47 *2H, t, Jˆ6 Hz, CH2±NH±Acr);
3.90 *2H, t, Jˆ7 Hz, CH2±CO±Ph);3.43 *2H, t, Jˆ7 Hz,
1
in 80% yield *0.100 g, 0.26 mmol): mp 808C; H NMR
*200 MHz, CDCl3): d *ppm)ˆ7.89±8.29 *4H, m, Ph-H);
6.25 *1H, s, NH);4.23 *2H, t, Jˆ6 Hz, CH2±OMs);3.47
*2H, q, Jˆ6 Hz, NH±CH2);1.40±1.85 *11H, m, 4C H2 and
±SO2±CH3); 13C NMR *50 MHz, CDCl3): d *ppm)ˆ
165.72;149.71;140.45;128.32;124.00;70.01;40.22;
CH2±Ade);3.16 *4H, m, CH ±CH2±Ade and CH2±NH±
2
CO±Acr);3.09±2.90 *6H, m, 3CH );2.09 *2H, m, CH );
2
2
1.91 *2H, m, CH2);1.55 *4H, m, 2CH 2). 13C NMR *75 MHz,
D2O): d *ppm)ˆ150.98;150.56;149.35;148.71;145.52;

A. Martelli et al. / Tetrahedron 58 02002) 4291±4298
4297
37.61;29.45;29.09;26.25;25.13. MS *CI, NH 31isobutane)
m/z: 345 *100, *M11)1);Analysis: Calcd for C 14H20N2O6S:
C 48.83, H 5.85, N 8.13%. Found: C 49.11, H 5.89, N
8.24%.
3.1.16. 6-Amino-9-[11-36-chloro-2-36-34-nitrobenzamido)-
hexyloxy)acridin-9-yl)-3,7,11-triaza-undecyl]-9H-purine
318b). The procedure described above for 18a was used for
the synthesis of 18b, starting from 17b *0.092 g, 0.16 mmol)
and 4 *0.072 g, 0.16 mmol). 18b was isolated as a yellow
1
3.1.13.
6-Chloro-2-[4-34-nitrobenzamido)butyloxy]-9-
solid in 74% yield *0.120 g): mp 170±1738C; H NMR
phenoxy-acridine 317a). A solution of TBAF in THF
*1 M, 0.370 mL, 0.37 mmol) was added to a mixture of
2-silyloxyacridine 1614 *0.157 g, 0.36 mmol) and 15a
*0.126 g, 0.40 mmol) in dry THF *5 mL). The resulting
mixture was stirred for 10 days at room temperature. The
solvent was then evaporated to dryness and the residue was
triturated in diethylether. Compound 17a was ®ltered off
and was thus obtained in 51% yield *0.100 g, 0.18 mmol):
*300 MHz, D2O): d *ppm)ˆ8.39 *1H, s, Ade-H);8.33
*1H, s, Ade-H);8.10 *1H, d, Jˆ9.4 Hz, Acr-H);7.67±7.64
*2H, m, Ph-H);7.48±7.29 *7H, m, Acr-H and Ph-H);4.69
*2H, t, Jˆ6.4 Hz, Ade-CH2);4.12 *4H, m, Acr-O±CH 2 and
Acr-NH±CH2);3.66 *2H, t, Jˆ6 Hz, CH2±NH±CO);3.38
*2H, m, Ade-CH2±CH2);3.26±3.16 *6H, m, 3CH );2.33
2
*2H, m, CH2);2.14 *2H, m, CH );1.88 *2H, m, CH );
2
2
1.66 *2H, m, CH2);1.57 *2H, m, CH 2);1.45 *2H, m,
CH2); 13C NMR *75 MHz, D2O): d *ppm)ˆ168.19;
156.07;155.68;151.04;149.41;148.40;146.14;144.70;
141.23;139.76;139.55;134.18;128.21;127.46;124.83;
123.42;120.49;118.79;117.62;113.80;109.76;104.36;
68.63;46.97;45.93;45.41;45.23;44.98;41.12;39.47;
27.97;27.73;26.50;24.40;24.30;22.95. MS *FAB, NBA)
1
mp 140±1428C; H NMR *200 MHz, CDCl3): d *ppm)ˆ
8.25±8.20 *3H, m, Ph-H and Acr-H);8.09 *1H, d, Jˆ
9 Hz, Acr-H);7.93 *1H, d, Jˆ9 Hz, Acr-H);7.89±7.85
*2H, m, Ph-H);7.45 *1H, dd, Jˆ2 and 9 Hz, Acr-H);7.34
*1H, dd, Jˆ2 and 9 Hz, Acr-H);7.26±7.22 *2H, m, Ph-H);
7.13 *1H, d, Jˆ2 Hz, Acr-H);7.11±7.03 *1H, m,
Ph-H);6.84±6.79 *2H, m, Ph-H);6.28 *1H, m, NH);3.99
*2H, t, Jˆ6 Hz, Acr-O±CH2);3.55 *2H, q, Jˆ6 Hz,
CH2±NH±CO±);1.85 *4H, m, 2CH 2). MS *FAB, glycerol)
m/zˆ542 *100, *M11)1);Analysis: Calcd for
C30H24ClN3O5: C 66.48, H 4.46, N 7.75%;Found: C
66.40, H 4.68, N 7.57%.
m/zˆ768 *24, *M11)1);UV *H O): l *1): 446.0 *6250);
2
427.0 *6340);344.4 *4000);269.0 *49900). HRMS Calcd
for C39H47N11O435Cl 768.3501 *M1H)1;Found: 768.3503.
3.2. DNA cleavage activities
The DNA damaging and cleavage activities of the drugs
were tested on 50-32P end-labeled 23-mer oligonucleotide
containing a tetrahydrofurane analogue of abasic site.
3.1.14. 6-Chloro-2-[6-34-nitrobenzamido)hexyloxy]-9-
phenoxy-acridine 317b). The procedure described above
for 17a was used for to the synthesis of 17b, starting from
16 *0.100 g, 0.23 mmol) and 15b *0.095 g, 0.28 mmol). 17b
was isolated as a yellow solid in 48% *0.063 mg,
0.11 mmol): 1H NMR *200 MHz;CDCl 3): d *ppm)ˆ
8.27±8.22 *2H, m, Ph-H);8.20 *1H, d, Jˆ2 Hz, Acr-H);
8.09 *1H, d, Jˆ9 Hz, Acr-H);7.93 *1H, d, Jˆ9 Hz,
Acr-H);7.90±7.86 *2H, m, Ph-H);7.43 *1H, dd, Jˆ2 and
9 Hz, Acr-H);7.34 *1H, dd, Jˆ2 and 9 Hz, Acr-H);
7.26±7.22 *2H, m, Ph-H);7.12 *1H, d, Jˆ2 Hz, Acr-H);
7.11±7.03 *1H, m, Ph-H);6.84±6.79 *2H, m, Ph-H);6.17
*1H, m, NH);3.92 *2H, t, Jˆ6 Hz, Acr-O±CH2);3.47 *2H,
q, Jˆ6 Hz, CH2±NH±CO±);1.90±1.40 *8H, m, 4CH 2). MS
*CI, NH31isobutane) m/z: 570 *100, *M11)1).
The 23-mer abasic duplex was incubated with compound 5
or 10 *2 mM) in buffered solution *Tris buffer, 50 mM,
pH 8), in the presence of Cu *1025 mol L21) and MPA
*1024 mol L21). Incubation was carried out at 258C
for 40 min. The DNA cleavage was quenched by adding
neocuproine *3 mM solution) to the mixtures.
The abasic 23-mer *0.5 mM) was incubated with the nitro-
benzamide±acridine conjugates 13, 18a,b *2 mM) in
buffered solution *10 mM sodium phosphate buffer, pH 7,
20 mM NaCl, 1 mM EDTA. The solutions were then
irradiated *with an ORIEL Xe/Hg 200 W lamp) for 3 h at
48C. The resulting solution was treated with piperidine
*1 M) at 908C for 10 min, followed by BuOH precipitation.
3.1.15. 6-Amino-9-[11-36-chloro-2-34-34-nitrobenzamido)-
butyloxy)acridin-9-yl)-3,7,11-triaza-undecyl]-9H-purine
318a). A mixture of 17a *0.060 g, 0.11 mmol), 8 *0.053 g,
0.12 mmol) and triethylamine *0.017 mL, 0.12 mmol) in
phenol *5 mL) was stirred at 808C for 4 h. The solution
was then added dropwise to ice-cold 1N NaOH. The solid
thus formed was ®ltered off, washed with water. Compound
18a was puri®ed as the hydrochloride by crystallization in
methanol acidi®ed with 11N HCl. 18a was isolated in 52%
After electrophoresis, the gels were exposed overnight at
2308C on Kodak BioMax MR-2 ®lms. The ®lms were
scanned with an AGFA STUDIOscan IIsi scanner.
References
1
yield *42 mg): mp 190±1958C; H NMR *300 MHz, D2O):
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d, Jˆ9.8 Hz, Acr-H);7.52 *2H, m, Ph-H);7.40±7.34 *3H,
m, Acr-H);7.24±7.14 *4H, m, Ph-H and Acr-H);4.52 *2H,
m, CH2±NH±Acr);4.09 *2H, m, Acr-O±CH 2);3.90 *2H, m,
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