Formaldehyde-induced DNA cross-link of indolizino[1,2-b]quinolines derived from the A-D rings of camptothecin

Article abstract of DOI:10.1021/jm020235g

Camptothecin consists of a lactone E ring adjacent to tetracyclic A-D rings of a planar chromophore, which are essential for topoisomerase I inhibition and DNA interaction. The A-D rings can be exploited to develop DNA-sequence-reading molecules. Indolizi

Full text of DOI:10.1021/jm020235g

J . Med. Chem. 2002, 45, 5809-5812
5809
F or m a ld eh yd e-In d u ced DNA Cr oss-Lin k of In d olizin o[1,2-b]qu in olin es Der ived
fr om th e A-D Rin gs of Ca m p toth ecin
Aurore Perzyna,† Carine Marty,‡ Michael Facompre´,‡ J ean-Franc¸ois Goossens,† Nicole Pommery,† Pierre Colson,§
Claude Houssier,§ Raymond Houssin,† J ean-Pierre He´nichart,† and Christian Bailly*,‡
Institut de Chimie Pharmaceutique Albert Lespagnol, EA 2692, Universite´ de Lille 2, BP 83, 59006 Lille, France,
INSERM U-524 et Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret, IRCL, Place de Verdun,
59045 Lille, France, and Unite´ de Biospectroscopie, Universite´ de Lie`ge au Sart-Tilman, 4000 Lie`ge, Belgium
Received May 29, 2002
Camptothecin consists of a lactone E ring adjacent to tetracyclic A-D rings of a planar
chromophore, which are essential for topoisomerase I inhibition and DNA interaction. The
A-D rings can be exploited to develop DNA-sequence-reading molecules. Indolizino[1,2-b]-
quinoline derivatives substituted with a piperidinoethyloxy side chain and an aminomethyl
function on rings A and D, respectively, were synthesized, and their DNA binding and
formaldehyde-mediated bonding properties were investigated.
In tr od u ction
Resu lts
The designed drugs lack the lactone E ring of CPT
Camptothecin (CPT) is a monoterpene indole alkaloid
that inhibits topoisomerase I, an essential enzyme for
the regulation of DNA structures and functions. CPT
binds strongly to the covalent topoisomerase I-DNA
complex but shows little interaction with duplex DNA
or topoisomerase I alone. However, CPT was initially
reported to intercalate into poly(dGC),1 and studies with
the positively charged topotecan (TPT) and irinotecan
(CPT-11) congeners, i.e., two clinically useful anticancer
drugs of the CPT family, have established that DNA
interactions play a role in their biological activity.2,3 TPT
displays a sequence preference of (dG-dC)15 over (dA-
dT)15 and behaves as an intercalating agent in the
absence of topoisomerase I.2 Moreover, photoactivated
CPT interacts specifically and intimately with guanine
residues in double-stranded DNA.4
but preserve the A-D indolizino[1,2-b]quinoline rings,
which can be considered as the DNA binding unit of
CPT. Two types of substitutions were considered to
potentially promote the drug-DNA interaction. On one
hand, the A quinoline ring was substituted at position
2 or 3 (Scheme 1) with a positively charged piperidine
side chain susceptible to interaction with the DNA
phosphates. This side chain also confers hydrosolubility,
and compounds 10a ,b and 12a ,b are fully water-soluble.
An ether linkage was chosen to mimic the hydroxyl
group found in TPT or SN38 (the active metabolite of
CPT-11). On the other hand, the D ring was substituted
with a methyl group at position 7 and either an
aminomethyl or an amide group at position 8. The
amide was selected for its hydrogen binding capacity,
whereas the amine was incorporated to generate form-
aldehyde-mediated drug-DNA cross-links.
Ch em istr y. The 9,11-dihydroindolizino[1,2-b]quino-
lines 10 and 11 were synthesized on the basis of the
Friedla¨nder reaction, requiring the appropriate 2-amino-
benzaldehydes 3a ,b (and the imine surrogate 48,9) and
the enolizable indolizinones 8 and 9 (Scheme 1). The
2-aminobenzaldehydes 3a ,b were obtained in two steps
by O-alkylation of phenols 1a 10 and 1b (K2CO3, DMF)
followed by a Bechamp reduction of the nitro group of
2a ,b. The synthesis of indolizines 6 and 7 was found to
be effective from indolizine 5,11,12 whose cyano group led
to carboxamide 6 by hydrolysis in basic medium (with-
out affecting the lactame moiety). The protected amine
7 resulted from the catalytic hydrogenation of 5 in
acylating medium. These reaction conditions prevent
overhydrolysis of the carboxamide function into car-
boxylic acid and nonregioselective reaction of the pri-
mary amine during the Friedla¨nder cyclization. More-
over, the acetal group in 5 was found to be essential for
protecting the ketone during the hydrogenation process.
Deketalization of 6 and 7 enabled the Friedla¨nder
cyclization13 of aldehydes 3a ,b (and 4) with indolizines
8 and 9 to produce the tetracyclic compounds 10 and
11; acetic acid acts as solvent and catalyst as well as a
In the ternary topoisomerase I-CPT-DNA complex,
the drug also engages direct contacts with the double
helix at the cleavage site. Different models have been
proposed for the configuration of the ternary complex,5-7
and in all cases, the indolizino[1,2-b]quinoline moiety
of CPT, represented by the A-D rings, provides the
necessary framework for the DNA interaction whereas
the lactone E ring interacts essentially, if not exclu-
sively, with the enzyme through the Arg364 and Asp533
residues of human topoisomerase I.5
Thus far, all drug design approaches in the CPT series
have been oriented toward the discovery of potent
topoisomerase I poisons, generally with little or no
consideration of the DNA binding aspect. Here, we
report a different approach that consists of deleting the
lactone E ring, therefore prohibiting the targeting of
topoisomerase I but exploiting the indolizino[1,2-b]-
quinoline structure to develop DNA-sequence-reading
molecules (Chart 1).
* To whom correspondence should be addressed. Phone: +33-3-
2016-9218. Fax: +33-3-2016-9229. E-mail: bailly@lille.inserm.fr.
† Universite´ de Lille 2.
‡ IRCL.
§ Universite´ de Lie`ge au Sart-Tilman.
10.1021/jm020235g CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/22/2002

5810 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26
Brief Articles
Ch a r t 1
Sch em e 1a
a
Reagents and conditions: (a) N-2-chloroethylpiperidine, K2CO3, DMF, 80 °C; (b) Fe, HCl, AcOH, EtOH, H2O, reflux; (c) NaOH, MeOH,
H2O, reflux; (d) Raney Ni, Ac2O, AcOH, H2 at 45 °C and 50 psi; (e) TFA 80%, room temp; (f) AcOH, reflux; (g) 6 N HCl, reflux. b Hydrochloride.
Ta ble 1. DNA Binding Parameters
deprotective agent of the aldehydic function originating
from 4. Compounds 12a -c were classically obtained
from amides 11a -c by acidic hydrolysis.
a
∆Tm (°C)
Absb
∆λ (nm)
c
CT
dAT
H (%)
Kapp × 106 (M-1
)
DNA In ter a ction . Addition of DNA induces marked
changes of the absorption spectra of compounds 12a -c
(Figure S1 in Supporting Information). Good isosbestic
behavior and large bathochromic and hypochromic shifts
were observed with compounds 12a -c (Table 1). With
12a , the absorption maximum is shifted from 381 to 392
nm and the red shift amounts to 9 nm with 12b (from
377 to 386 nm). The amide compounds 10a -c showed
weaker spectral changes compared to the aminomethyl
analogues (Table 1). Thermal denaturation analyses,
carried out with calf thymus DNA and the polynucleo-
10a
10b
10c
12a
12b
12c
4.6
1.2
0
19.4
13.6
1.9
12.8
5.8
0
28.6
19.8
3
8
9
0
11
9
8
35.8
33.0
2.7
38.6
35.3
33.1
0.20 ( 0.002
0.29 ( 0.003
d
8.4 ( 0.12
2.5 ( 0.08
d
Variation in melting temperature (∆Tm ) Tmcomplex - TmDNA).
Tm measurements were performed in BPE buffer, pH 7.1 (6 mM
Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA), using 10 µM drug and
20 µM calf thymus DNA (CT) or poly(dAdT)2 (dAT) at 260 nm with
a
b
a heating rate of 1 °C/min. Absorption spectral changes recorded
upon addition of 200 µM calf thymus DNA to a drug solution at
20 µM in 1 mM Na cacodylate buffer, pH 7.0. H and ∆λ refer to
the hypochromic and bathochromic shifts, respectively. c Binding
constants calculated from the concentration required to reduce by
50% the fluorescence of ethidium bromide bound to calf thymus
DNA. No displacement of DNA-bound ethidium bromide at 50
µM.
complex
tide poly(dAT)2, gave the ∆Tm values (∆Tm ) Tm
- TmDNA) collated in Table 1. There are large differences
between the compounds. In the aminomethyl series,
12a ,b stabilize duplex DNA against heat denaturation
much more strongly than 12c, indicating that the side
chain plays a significant role in the drug-DNA interac-
tion. The position of the side chain on the chromophore
is also important because the Tm shifts are more
pronounced with 12a than with 12b. The side chain
introduced on the chromophore at position 3 is preferred
over position 2. Binding affinities were determined by
fluorescence using an ethidium bromide displacement
assay. 12a was found to bind strongly to calf thymus
DNA with an apparent binding constant about 4 times
d
higher than that determined with 12b and 40 times
superior to those calculated with the amides 10a ,b
(Table 1). The mode of binding of 12a -c to DNA was
probed by electric linear dichroism (ELD) using calf
thymus DNA and the polynucleotides poly(dAT)2 and
poly(dGC)2 (Figure S2 in Supporting Information). In
all cases, the reduced dichroism ∆A/A was negative in
the drug absorption band that reflects the orientation

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26 5811
likely the 2-amino group of guanine exposed in the
minor groove.
Con clu sion
This study shows that the A-D rings of CPT is
particularly well adapted for potent and selective rec-
ognition of GC-rich DNA sequences. The DNA binding
data can be rationalized by a model in which the
indolizino[1,2-b]quinoline chromophore stacks on G-C
pairs with the 8-aminomethyl substituent located a
short distance from an amino group on the DNA base
pair so that a covalent drug-DNA methylene bridge can
be created in the presence of formaldehyde. Incorpora-
tion of a cationic piperidinoethyloxy side chain reinforces
significantly the DNA interaction as is the case with
IRT. Beyond the camptothecins, the present data pro-
vide important information to better understand the
mechanism of action of indolizino[1,2-b]quinolines such
as batracyclins, which are highly potent antitumor
agents.17
F igu r e 1. Mobility shifts of DNA cross-linked to 12a -c in
the presence of formaldehyde. The 117 base pair DNA frag-
ment was reacted with the test drugs (20 or 50 µM) in the
presence of 1% HCHO in 50 mM borate buffer at pH 8.2. After
1 h of incubation at 37 °C, the samples were precipitated with
ethanol and the cross-linked DNA samples were subjected to
electrophoresis on 6% polyacrylamide. The lane marked DNA
contained no drug. Daunomycin (lane Dauno.) and the amino-
rebeccamycin derivative (lane Reb-NH2) (20 µM each) cross-
link the DNA in the presence of HCHO only, and 1%
formaldehyde has no effect on the mobility of the DNA in the
absence of the drug. The free (f) and bound (b) DNA forms are
indicated.
Exp er im en ta l Section
Gen er a l P r oced u r e for th e Syn th esis of 7-Meth yl-1,5-
dioxo-1,2,3,5-tetr ah ydr oin dolizin e-6-car boxam ide (8) an d
N-(7-Met h yl-1,5-d ioxo-1,2,3,5-t et r a h yd r oin d olizin -6-yl-
m eth yl)a ceta m id e (9). A solution of 6 or 7 (4 mmol) in 80%
aqueous TFA (10 mL) was stirred at room temperature for 3
h in a nitrogen atmosphere. Removal of the solvent gave an
oil that crystallized from Et2O. 8: yellow solid (715 mg, 85%
yield). Rf ) 0.38 (CH2Cl2/MeOH 9:1). Mp: 185 °C. IR: 3301
of the chromophore perpendicular to the helix axis, as
expected for an intercalative binding. Unwinding of
supercoiled DNA was observed with 12a , thus confirm-
ing that this compound is a DNA intercalator.
DNA Sequ en ce Recogn ition . Footprinting experi-
ments were performed with four restriction fragments
(Figure S3 in Supporting Information). Clear modifica-
tions of the patterns of cleavage by DNase I were
observed with 12a , whereas 12b and 12c showed little,
if any, effect on the enzyme activity. A few sites of
protection from DNase I cutting (i.e., footprints) adja-
cent to regions of enhanced cleavage can be detected
with 12a as the ligand concentration is raised but not
with 12b ,c. 12a was found to bind selectively to
many sequences with a high GC content, such as 5′-
GGCCAGT, 5′-CGCC, and 5′-GCGTG. Binding to a few
AT-GC mixed sequences, such as 5′-ACGT and 5′-AGTG,
was also detected (Figure S4 in Supporting Informa-
tion). In contrast, DNase I cutting at AT-rich sequences
was found to be enhanced in the presence of 12a , and
this effect can be attributed to intercalation-induced
perturbations of the double helical structure of DNA.
It appears that 12a is sensitive to GC-rich sequences
and those containing GpT (ApC) and TpG (CpA) steps.
(NH), 1735 (CdO ketone), 1647 (CdO amide, pyridone) cm-1
EI MS m/z (relative intensity): 206 (M+, 66%). Anal. (C10H10
.
-
N2O3) C, H, N. 9: pale solid (770 mg, 93% yield). Rf ) 0.46
(CH2Cl2/MeOH 9:1). Mp: 161 °C. IR: 3328 (NH2), 3130 (NH2),
1745 (CdO ketone), 1672 (CdO amide, pyridone) cm-1. EI MS
m/z (relative intensity): 234 (M+, 70%). Anal. (C12H14N2O3) C,
H, N.
Gen er a l P r oced u r e for th e Syn th esis of 9,11-Dih y-
d r oin d olizin o[1,2-b]qu in olin es 10a -c a n d 11a -c. Tetra-
hydroindolizine 8 or 9 and 2-aminobenzaldehyde 3a , 3b, or
48,9 were added to acetic acid (20 mL) and heated while stirring
under reflux for 8 h. The reaction mixture was cooled to room
temperature, and the solvent was removed under reduced
pressure. Methanol saturated HCl was added to the residual
solid dissolved in MeOH. The crude product was purified by
flash chromatography (CH2Cl2/MeOH 8:2 f 7:3).
7-Met h yl-9-oxo-3-(2-p ip er id in -1-ylet h oxy)-9,11-d ih y-
dr oin dolizin o[1,2-b]qu in olin e-8-car boxam ide Hydr och lo-
r id e (10a ). Pale solid (340 mg, 51% yield) from 8 (320 mg, 1.6
mmol) and 3a (385 mg, 1.6 mmol). Rf ) 0.31 (CH2Cl2/MeOH
9:1, ammonia 1%). Mp: >250 °C (EtOH/H2O). IR: 3300 (NH),
1676 (CdO pyridone, amide), 1619 (CdN) cm-1
(C24H26N4O3‚1HCl‚2.5H2O) C, H, N, Cl.
. Anal.
F or m a ld eh yd e-Med ia ted Cr oss-Lin k in g. Form-
aldehyde can be used to cross-link drugs to DNA,
providing that they contain a reactive amino function
that is located adjacent to an amino group of the DNA
bases. For example, the anticancer drugs daunomycin,14
mitoxantrone,15 and a rebeccamycin derivative16 can be
efficiently cross-linked to DNA in the presence of
formaldehyde. This strategy was used here to probe the
reactivity of the 8-aminomethyl function of compounds
12a -c upon binding to DNA. The results of the gel
mobility shift assay presented in Figure 1 indicate
clearly that formaldehyde does promote the formation
of covalent complexes that migrate more slowly into the
gel. We can therefore conclude that in the drug-DNA
complex, the amino group of compound 12 is located at
a short distance from an amino group of the bases, most
7-Met h yl-9-oxo-2-(2-p ip er id in -1-ylet h oxy)-9,11-d ih y-
dr oin dolizin o[1,2-b]qu in olin e-8-car boxam ide Hydr och lo-
r id e (10b). Pale solid (330 mg, 49% yield) from 8 (320 mg, 1.6
mmol) and 3b (385 mg, 1.6 mmol). Rf ) 0.48 (CH2Cl2/MeOH
9:1, ammonia 1%). Mp: >250 °C (EtOH/H2O). IR: 3360 (NH),
1658 (CdO pyridone, amide) cm-1. Anal. (C24H26N4O3‚1HCl‚
2.5H2O) C, H, N, Cl.
7-Meth yl-9-oxo-9,11-dih ydr oin dolizin o[1,2-b]qu in olin e-
8-ca r boxa m id e (10c). Brown solid (290 mg, 42% yield) from
8 (500 mg, 2.4 mmol) and 4 (510 mg, 2.4 mmol). Rf ) 0.49
(CH2Cl2/MeOH 9:1, ammonia 1%). Mp: >250 °C (EtOH/H2O).
IR: 3275 (NH), 1668 (CdO pyridone, amide) cm-1. Anal.
(C17H13N3O2‚0.5HCl‚0.5H2O) C, H, N.
N-[7-Met h yl-9-oxo-3-(2-p ip er id in -1-ylet h oxy)-9,11-d i-
h ydr oin dolizin o[1,2-b]qu in olin -8-ylm eth yl]acetam ide Hy-
d r och lor id e (11a ). Orange solid (1.0 g, 45% yield) from 3a
(1.2 g, 5 mmol) and 9 (1.2 g, 5 mmol). Rf ) 0.39 (CH2Cl2/MeOH
9:1, ammonia 1%). Mp: >250 °C (EtOH/H2O). IR: 3300 (NH),

5812 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 26
Brief Articles
1664 (CdO pyridone, amide), 1618 (CdN) cm-1. EI MS m/z
(relative intensity): 446 (M+, 14%), 403 (M+ - COCH3, 22%),
(2) Yang, D.; Strode, J . T.; Spielman, H. P.; Wang, A. H.-J .; Burke,
T. G. DNA interactions of two clinical camptothecin drugs
stabilize their active lactone forms. J . Am. Chem. Soc. 1998, 120,
2979-2980.
(3) Yao, S.; Murali, D.; Seetharamulu, P.; Haridas, K.; Petluru, P.
N. V.; Reddy, D. G.; Hausheer, F. H. Topotecan lactone selec-
tively binds to double- and single-stranded DNA in the absence
of topoisomerase I. Cancer Res. 1998, 58, 3782-3786.
(4) Leteurtre, F.; Fesen, M.; Kohlagen, G.; Kohn, K. W.; Pommier,
304 (M+ - COCH3 - piperidine, 17%), 275 (M+ - COCH3
piperidine - CH2NH, 14%).
-
N-[7-Met h yl-9-oxo-2-(2-p ip er id in -1-ylet h oxy)-9,11-d i-
h ydr oin dolizin o[1,2-b]qu in olin -8-ylm eth yl]acetam ide Hy-
d r och lor id e (11b). Brown solid (1.0 g, 44% yield) from 3b
(1.2 g, 5 mmol) and 9 (1.2 g, 5 mmol). Rf ) 0.22 (CH2Cl2/MeOH
9:1, ammonia 1%). Mp: >250 °C (EtOH/H2O). IR: 3418 (NH),
1655 (CdO pyridone, amide), 1621 (CdN) cm-1. EI MS m/z
(relative intensity): 446 (M+, 15%), 403 (M+ - COCH3, 20%),
Y. Specific interaction of camptothecin,
a topoisomerase I
inhibitor, with guanine residues of DNA detected by photo-
activation at 365 nm. Biochemistry 1993, 32, 8955-8962.
(5) Redinbo, M. R.; Champoux, J . J .; Hol, W. G. J . Novel insights
into catalytic mechanism from a crystal structure of human
topoisomerase I complex with DNA. Biochemistry 2000, 39,
6832-6840.
304 (M+ - COCH3 - piperidine, 8%), 275 (M+ - COCH3
piperidine - CH2NH, 6%).
-
N-[7-Meth yl-9-oxo-9,11-d ih yd r oin d olizin o[1,2-b]qu in o-
lin -8-ylm eth yl]a ceta m id e (11c). Brown solid (290 mg, 48%
yield) from 4 (450 mg, 1.9 mmol) and 9 (400 mg, 1.9 mmol). Rf
) 0.63 (CH2Cl2/MeOH 9:1, ammonia 1%). Mp: >250 °C (EtOH/
H2O). IR: 3329 (NH), 1666 (CdO pyridone, amide) cm-1. EI
MS m/z (relative intensity): 319 (M+, 10%), 276 (M+ - COCH3,
100%).
Gen er a l P r oced u r e for th e Syn th esis of 9,11-Dih yd r o-
in d olizin o[1,2-b]qu in olin es 12a -c. Quinoline 11a , 11b, or
11c was added to 6 N HCl, and the mixture was heated with
stirring under reflux for 24 h. After the mixture was cooled to
room temperature and after removal of the solvent under
reduced pressure, the crude product was purified by flash
chromatography (CH2Cl2/MeOH 7:3).
8-Am in om e t h yl-7-m e t h yl-9-oxo-3-(2-p ip e r id in -1-yl-
eth oxy)-9,11-d ih yd r oin d olizin o[1,2-b]qu in olin e Dih yd r o-
ch lor id e (12a ). Yellow solid (130 mg, 76% yield) from 11a
(150 mg, 3.4 mmol) and HCl (15 mL). Rf ) 0.39 (CH2Cl2/MeOH
9:1, ammonia 1%). Mp: >250 °C (EtOH/H2O). IR: 3386
(NH3+), 1653 (CdO), 1617 (CdN) cm-1. Anal. (C24H28N4O2‚
2HCl‚3H2O) C, H, N, Cl.
8-Am in om e t h yl-7-m e t h yl-9-oxo-2-(2-p ip e r id in -1-yl-
eth oxy)-9,11-d ih yd r oin d olizin o[1,2-b]qu in olin e Dih yd r o-
ch lor id e (12b). Yellow solid. (150 mg, 70% yield) from 11b
(200 mg, 4.4 mmol) and HCl (20 mL). Rf ) 0.36 (CH2Cl2/MeOH
9:1, ammonia 1%). Mp: >250 °C (EtOH/H2O). IR: 3417
(NH3+), 1655 (CdO), 1621 (CdN) cm-1. Anal. (C24H28N4O2‚
2HCl‚3.5H2O) C, H, N, Cl.
8-Am in om eth yl-7-m eth yl-9-oxo-9,11-dih ydr oin dolizin o-
[1,2-b]qu in olin e Hyd r och lor id e (12c). Pale solid (850 mg,
58% yield) from 11c (150 mg, 4.7 mmol) and HCl (15 mL). Rf
) 0.59 (CH2Cl2/MeOH 9:1, ammonia 1%). Mp: >250 °C (EtOH/
H2O). IR: 3422 (NH3+), 1656 (CdO), 1609 (CdN) cm-1. Anal.
(C17H15N3O1‚1.5HCl‚2.25H2O) C, H, N, Cl.
Bioch em ica l a n d Sp ectr op h otom etr ic Mea su r em en ts.
Absorption and melting temperature studies were performed
as previously described.18 Binding constants were determined
using a competitive displacement fluorometric assay with
DNA-bound ethidium.19 The experimental procedures for the
electric linear dichroism20 and DNase I footprinting21 and
formaldehyde-mediated cross-linking16 experiments have been
previously reported.
(6) Kerrigan, J . E.; Pilch, D. S. A structural model for the ternary
cleavable complex formed between human topoisomerase I,
DNA, and camptothecin. Biochemistry 2001, 40, 9792-9798.
(7) Laco, G. S.; Collins, J . R.; Luke, B. T.; Kroth, H.; Sayer, J . M.;
J erina, D. M.; Pommier, Y. Human topoisomerase I inhibition:
docking camptothecin and derivatives into a structure-based
active site model. Biochemistry 2002, 41, 1428-1435.
(8) Borsche, W.; Doeller, W.; Wagner-Roemmich, M. Uber tri-
cyclische lactone aus chinaldincarbonsa¨ure-(3)-ester und u¨ber
[3-carboxychinolyl-(2)]-brenztraubensa¨uredia¨thylester (About the
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carboxylic acid and about ethyl 2-(2-ethoxycarbonyl-2-oxo-
ethyl)quinoline-3-carboxylate). Chem. Ber. 1943, 76, 1099-1102.
(9) J onhston, D.; Smith, D. M.; Sheperd, T.; Thompson, D. O-
Nitrobenzylidene compounds. Part 3. Formation of 4-arylamino-
3-methoxycinnoline-1-oxydes from N-o-nitrobenzylideneanilines,
cyanide ion, and methanol: the intermediacy of 2-aryl-3-cyano-
2H-indazol-1-oxides. J . Chem. Soc., Perkin Trans. 1987, 1, 495-
500.
(10) Suvorov, N. N.; Fedotova, M. V.; Orlova, L. M.; Ogareva, O. B.
Indole derivatives. XVI. Synthesis of 6- and 4-substituted
tryptamines. J . Gen. Chem. USSR (Engl. Transl.) 1962, 32,
2325-2331.
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antitumor agents. 18. Synthesis and biological activity of camp-
tothecin analogues. J . Med. Chem. 1980, 23, 554-560.
(12) Wall, M. E.; Wani, M. Camptothecin analogs as potent inhibitors
of human colorectal cancer. PCT Int. Appl. WO 91/05556, 1991;
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(13) Sugimori, M.; Ejima, A.; Ohsuki, S.; Uoto, K.; Mitsui, I.;
Matsumoto, K.; Kawato, Y.; Hirota, Y.; Sato, K.; Terasawa, H.
Synthesis and antitumor activity of ring A- and F-modified
hexacyclic camptothecin analogues. J . Med. Chem. 1998, 41,
2308-2318.
(14) Leng, F.; Savkur, R.; Fokt, I.; Przewloka, T.; Priebe, W.; Chaires,
J . B. Base specific and regioselective chemical cross-linking of
daunorubicin to DNA. J . Am. Chem. Soc. 1996, 118, 4731-4738.
(15) Parker, B. S.; Cullinane, C.; Phillips, D. R. Formation of DNA
adducts by formaldehyde-activated mitoxantrone. Nucleic Acids
Res. 1999, 27, 2918-2923.
(16) Bailly, C.; Goossens, J .-F.; Laine, W.; Anizon, F.; Prudhomme,
M.; Ren, J .; Chaires, J . B. Formaldehyde cross-linking of a 2′-
aminoglucose rebeccamycin derivative to both A‚T and G‚C base
pairs in DNA. J . Med. Chem. 2000, 43, 4711-4720.
Ack n ow led gm en t. This work was done with the
support of research grants to C.B. and J .-P.H. from the
Association pour la Recherche sur le Cancer and to C.H.
and P.C. from the Actions de Recherches Concerte´es,
Contract 95/00-93. The authors thank W. Laine and C.
Tardy for expert technical assistance.
(17) Wang, H.-K.; Morris-Natschke, S. L.; Lee, K.-H. Recent advances
in the discovery and development of topoisomerase inhibitors
as antitumor agents. Med. Res. Rev. 1997, 17, 367-425.
(18) Goossens, J .-F.; Bouey-Bencteux, E.; Houssin, R.; He´nichart, J .-
P.; Colson, P.; Houssier, C.; Laine, W.; Baldeyrou, B.; Bailly, C.
DNA interaction of the tyrosine protein kinase inhibitor
PD153035 and its N-methyl analogue. Biochemistry 2001, 40,
4663-4671.
(19) Baguley, B. C.; Denny, W. A.; Atwell, G. J .; Cain, B. F. Potential
antitumor agent. 34. Quantitative relationships between DNA
binding and molecular structure for 9-anilino-acridines substi-
tuted in the anilino ring. J . Med. Chem. 1981, 24, 170-177.
(20) Colson, P.; Bailly, C.; Houssier, C. Electric linear dichroism as
a new tool to study sequence preference in drug binding to DNA.
Biophys. Chem. 1996, 58, 125-140.
Su p p or tin g In for m a tion Ava ila ble: Synthesis of 2a ,b,
3a ,b, 6, and 7; 1H NMR data for 2a ,b, 3a ,b, 6-9, 10a -c, 12a -
c, and four figures showing spectrophotometric DNA titration,
electric linear dichroism data, DNase I footprinting gels, and
differential cleavage plots for 12a -c. This material is available
free of charge via the Internet at http://pubs.acs.org.
(21) Bailly, C.; Waring, M. J . Comparison of different footprinting
methodologies for detecting binding sites for a small ligand on
DNA. J . Biomol. Struct. Dyn. 1995, 12, 869-898.
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