DNA binding to guide the development of tetrahydroindeno[1,2-b]pyrido[4,3, 2-de]quinoline derivatives as cytotoxic agents

Article abstract of DOI:10.1021/jm0400193

The tetrahydroindeno[1,2-b]pyrido[4,3,2-de]quinoline chromophore was initially designed as a DNA intercalating unit because of its planar structure. Unexpectedly, one molecule (15d) bearing two N-methylpiperazine chains on both sides of this condensed pen

Full text of DOI:10.1021/jm0400193

J . Med. Chem. 2004, 47, 3665-3673
3665
DNA Bin d in g To Gu id e th e Develop m en t of
Tetr a h yd r oin d en o[1,2-b]p yr id o[4,3,2-d e]qu in olin e Der iva tives a s Cytotoxic
Agen ts
Sarah Catoen-Chackal,§,† Michael Facompre´,‡ Raymond Houssin,§ Nicole Pommery,§ J ean-Franc¸ois Goossens,§
Pierre Colson,# Christian Bailly,‡, and J ean-Pierre He´nichart*,§
Institut de Chimie Pharmaceutique Albert Lespagnol, EA 2692, Universite´ de Lille 2, Rue du Professeur Laguesse,
BP 83, 59006 Lille, France, Biospectroscopy and Physical Chemistry Unit, University of Liege, Sart-Tilman,
4000 Liege, Belgium, and INSERM U-524 and Laboratoire de Pharmacologie Antitumorale du Centre Oscar Lambret,
IRCL, Place de Verdun, 59045 Lille, France
Received J anuary 23, 2004
The tetrahydroindeno[1,2-b]pyrido[4,3,2-de]quinoline chromophore was initially designed as
a DNA intercalating unit because of its planar structure. Unexpectedly, one molecule (15d )
bearing two N-methylpiperazine chains on both sides of this condensed pentacyclic skeleton
fits into the minor groove of DNA and preferentially recognizes AT-rich sequences. The
monosubstituted compound 16d was identified as a potent cytotoxic DNA intercalator, whereas
the disubstituted analogue 15d represents a new structural motif for the development of DNA
sequence-reading small molecules.
Ch a r t 1
In tr od u ction
The majority of the drugs used today in the treatment
of cancer bind reversibly or irreversibly to DNA or
induce DNA damage, either directly or via topo-
isomerase inhibition. DNA and associated proteins
remain valid targets for cancer chemotherapy.1,2 Novel
DNA intercalating (acridines,3 indolocarbazoles,4 naph-
thalimides5) and alkylating agents (benzoacronycines,6
pyrrolobenzodiazepines,7 distamycin conjugates8) are
still being developed. DNA, whether with two, three, or
four strands, still represents one of the most challenging
bioreceptors for small molecules and a target of choice
for the control of gene expression.9 The design of
cytotoxic agents interfering with DNA metabolism is
actively pursued.
Although it is well established that DNA binding is
not sufficient to confer cytotoxic activities, interaction
with DNA is often considered as a necessary criterion
to maintain a cytotoxic effect, at least for some series
of planar intercalating chromophores such as acridines
and ellipticines. On the basis of this assumption, we
have recently reported the design of DNA-targeted
benzo[c]pyrido[2,3,4-kl]acridines10 (BPA) combining the
structural architecture (Chart 1) of the topoisomerase
I inhibitor 5,6-dihydro-8-desmethylcoralyne11 (DHDMC)
and the topoisomerase II poison ascididemin12 (ASC).
The combination of the pyrido[2,3,4-kl]acridine skeleton
of ASC with the isoquinoline core of DHDMC led to the
synthesis of the BPA pentacyclic system,13 which pro-
vided the basis for the development of potent cytotoxic
agents. Indeed, among the numerous BPA recently
synthesized, we identified three compounds, 1-3 (Chart
1), highly toxic to PC 3 prostate cancer cells. These
compounds showed little or no effects on the catalytic
activities of topoisomerases, but they bound to DNA. We
thought that drug-DNA interaction could be used as a
guide to develop more active compounds in this series.
The BPA chromophore is not planar because of its
unsaturated six-membered ring, which introduces a
kink in the structure. Considering that a planar struc-
ture may be more favorable for stacking interactions
between the DNA base pairs, we envisaged the replace-
ment of the six-membered ring with a smaller five-
membered ring in order to produce a more flattened
conformation. Here, we report the synthesis of a series
of tetrahydroindeno[1,2-b]pyrido[4,3,2-de]quinoline de-
rivatives (IPQ) bearing various carbon side chains or
* To whom correspondence should be addressed. Phone:
+33-3-2096-4374. Fax:
pharma.univ-lille2.fr.
§ Universite´ de Lille 2.
+33-3-2096-4906. E-mail:
henicha@
† Present address: Department of Chemistry, Georgia State Uni-
versity, Atlanta, GA 30303-3083.
‡ INSERM U-524 and Laboratoire de Pharmacologie Antitumorale
du Centre Oscar Lambret.
#
University of Liege.
Present address: De´partement de Cance´rologie Expe´rimentale,
Centre de Recherche Pierre Fabre, Avenue J ean Moulin, 81106
Castres, France.
10.1021/jm0400193 CCC: $27.50 © 2004 American Chemical Society
Published on Web 06/03/2004

3666 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Catoen-Chackal et al.
Sch em e 1a
a
Reagents and conditions: (a) K2CO3, DMF, 80 °C; (b) EDCI, HOBt, NMM, CH2Cl2-DMF.
Sch em e 2a
a
Reagents and conditions: (a) (i) PPTS, butan-1-ol, reflux, (ii) HCl/MeOH; (b) (i) PPTS, toluene/EtOH (2:1), reflux, (ii) HCl/MeOH; (c)
(i) PPTS, toluene, reflux, (ii) TFA; (d) (i) HCOONH4, Pd/C, MeOH, reflux, (ii) HCl/MeOH; (e) (i) 6 N HCl, reflux, (ii) HCl/MeOH.
substituents (Chart 1) corresponding to those of the BPA
series. The cytotoxicity of the different compounds was
evaluated and their DNA binding strength was mea-
sured by complementary biochemical and spectroscopic
methods.
o-aminoketone 12 or 13. The structure of indanones 9
is such that only one regioisomer could be formed during
the ring closure.
The preparation of 14a ,c,g,h was conveniently carried
out using toluene as solvent and pyridinium p-toluene-
sulfonate (PPTS), with distillation of solvent-water
azeotrope. In the case of 14d ,i, a toluene/ethanol (2:1)
mixture was necessary, whereas the reaction leading
to 14b and 15d was achieved using butan-1-ol. The
choice of these different solvents depends on the azeo-
trope temperature, which must be near 90 °C (to
increase the rate and yield of the reaction),13 and on the
solubility of the starting and target molecules in the
medium. Condensed pentacycles 14e,f and 15e,f were
obtained by reacting aminoketones 12 and 13 with
indanones 9e,f in the presence of PPTS and butan-1-ol;
the purity of the crude products is sufficient to allow
immediately the catalytic cleavage of the Cbz group by
ammonium formate-Pd/C and produced 14j,k and
15j,k , respectively. Finally, acid hydrolysis (6 N HCl)
of amides 14a -d ,g,h ,j,k yielded the secondary amines
16a -d ,g,h ,j,k .
Resu lts a n d Discu ssion
Ch em istr y. Indanones 9a -c,g,h were commercially
available, whereas indanones 9d -f were synthesized
(Scheme 1) in one step by treating 5-hydroxyindanone
5 with the appropriate ω-chloroalkylamines 6-8 (pro-
tected as carbamates for 7,8 in order to prevent imine
formation) according to the Williamson conditions (po-
tassium carbonate). Indanone 9i was obtained from
5-methoxy-1-indanone-3-acetic acid 10 and primary
amine 11 using peptidic coupling conditions (EDCI,
HOBt).
5-Aminoquinolinones 12 and 13 were synthesized, as
previously described,13 in seven and eight steps, respec-
tively. All of the tetrahydroindeno[1,2-b]pyrido[4,3,2-de]-
quinolines 14a -i and 15d -f were prepared (Scheme 2)
by acid-catalyzed Friedla¨nder cyclization14 between the
bicyclic enolizable ketones 9a -i and the substituted
Biologica l Da ta . DNA In ter a ction . Melting tem-
perature (Tm) and fluorescence measurements were

DNA-Binding Indenopyridoquinoline Derivatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3667
Ta ble 1. DNA Binding of the IPQ Derivatives
equivalent BPA compounds. The spectral absorption
titrations for 16b and its analogue 4 in the BPA series
showed important spectral changes (marked hypo-
chromism at 360 nm and hyperchromism at 400 nm)
but quantitative Tm and fluorescence measurements
revealed a higher affinity for the IPQ compound 16b
compared to the BPA analogue 4. Apparent binding
constants were determined by a conventional fluores-
cence method based on quenching ethidium
b
compd
∆Tm (°C)a
Kapp (105 M-1
)
4
1.1
ndd
3.1
ndd
3.8
7.0
6.0
32.5
19.6
22.5
0
4.0
0
10.0
14.8
15.8
0.016
ndd
14a c
14b
6.77
14cc
14d e
14je
14k e
15d e
15je
15k e
16a e
16be
16ce
16d e
16je
16k e
ndd
35.80
9.90
2.93
30.40
5.03
27.80
0.031
6.72
0.017
3.01
51.40
61.30
drug-poly(dAT)2complex
bromide bound to DNA. The ∆Tm (Tm
- Tmpoly(dAT) alone) and Kapp values are collated in Table
2
1. Compound 16b in the IPQ series binds to DNA 400
times more strongly than the related BPA compound 4
(Kapp ) 6.72 × 105 and 0.016 × 105 M-1, respectively).
The difference is less pronounced with compounds
bearing one or two cationic side chains, but replacement
of the six-membered ring for a five-membered ring
almost always reinforces the DNA binding capacity of
the molecules. For example, compound 15d substituted
with two N-methylpiperazine side chains gave a ∆Tm
value of 32.5 °C compared to 9.9 °C for the analogue
BPA derivative (compound 3d in ref 10). Unsurpris-
ingly, the BPA f IPQ conversion reinforces DNA
interaction most probably because of the planarity of
the IPQ chromophore.
a
drug-DNAcomplex
Variation of the ∆Tm (Tm
- TmDNAalone) of the
b
complexes between DNA and the test compounds. Apparent
binding constant measured by fluorescence. c Trifluoroacetate.
nd: not determined. e Hydrochloride.
d
carried out to evaluate the relative DNA binding affini-
ties of the different compounds. Tm analyses were
performed with the polynucleotide poly(dAT)2, which
melts at a low temperature (42 °C) under our experi-
mental conditions. Calf thymus (CT) DNA, used for the
fluorescence assay, melts at a higher temperature (66
°C) and is therefore less convenient in Tm analyses. In
nearly all cases, compounds in the designed IPQ series
showed superior DNA binding affinities compared to the
The DNA binding measurements were compared to
the cytotoxicity data determined by a colorimetric assay
using the prostate PC 3 cell line. Unfortunately, all
compounds were found to be noncytotoxic (IC50 > 1 µM)
F igu r e 1. Binding mode of 15d to DNA studied by electric linear dichroism. (a) ELD spectrum of 15d bound to (O) calf thymus
DNA, (0) poly(dAT)2, and (4) poly(dGC)2. Dependence of the reduced dichroism ∆A/A on electric field strength for the binding of
15d to (b) calf thymus DNA, (c) poly(dAT)2, and (d) poly(dGC)2. Conditions are as follows: (a) 13.6 kV/cm, P/D ) 25 (250 µM
DNA, 10 µM drug), (b, c, d) P/D ) 25, 410 nm for the DNA-drug complexes (open symbols) and 260 nm for DNA alone (filled
symbols) in 1 mM sodium cacodylate buffer, pH 7.0.

3668 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Catoen-Chackal et al.
three active compounds 1-3 identified in the BPA series
(Chart 1), only one remains active in the IPQ series,
16d (IC50 ) 6.5 nM), which has the same substitution
pattern as 2 (IC50 ) 19.9 nM). The slight gain in activity
may be due to its improved DNA binding capacity, but
there is no direct correlation between DNA binding and
cytotoxicity. In the IPQ series, some of the compounds,
such as 15d , exhibit a high affinity for DNA, but this
improved binding is not reflected in cytotoxic potential.
The substitution of the pentacyclic ring does not confer
superior activity in terms of DNA binding or cytotoxic-
ity. Therefore, it seems reasonable to leave this position
unsubstituted.
F igu r e 2. Circular dichroism spectral titration of 15d with
poly(dAT)2. Strong induced signals are observed with increas-
ing ratios of compound to DNA at 290, 375, and 415 nm. The
ratios of compounds to DNA (nucleotide) increase from 0 to
10 (bottom to top lines at 295 nm).
All the BPA and IPQ compounds are supposed to
intercalate into DNA. The binding mode was studied
by electric linear dichroism. This electro-optical method
has proved to be most useful in determining the
orientation of drugs bound to DNA. An additional
advantage is that it senses only the orientation of the
polymer-bound ligand; free ligand is isotropic and does
not contribute to the signal.15 Negative reduced dichro-
with the unique exception of compound 16d substituted
with an N-methylpiperazine side chain, which main-
tained high antiproliferative activity. It is also worth
mentioning that these compounds, as in the BPA series,
showed no effect on DNA topoisomerases I or II. Of the
F igu r e 3. Sequence selective binding of 15d , 14a , 16a (micromolar concentrations). The gels show DNase I footprinting with
two DNA restriction fragments of (A) 117, (B) 198, and (C) 265 base pairs. The 117-mer PvuII-EcoRI fragment was cut from the
plasmid pBS. The 198-mer HindIII-XbaI fragment was obtained from plasmid pMS1. In each case, DNA was 3′-end-labeled at
the EcoRI or HindIII site with [R-32P]dATP in the presence of AMV reverse transcriptase. The products of nuclease digestion
were resolved on an 8% polyacrylamide gel containing 7 M urea. Control tracks (marked Cont) contained no drug. Guanine-
specific sequence markers obtained by treatment of DNA with dimethyl sulfate followed by piperidine were run in the lanes
marked G. Numbers on the side of the gels refer to the standard numbering scheme for the nucleotide sequence of the DNA
fragment.

DNA-Binding Indenopyridoquinoline Derivatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3669
F igu r e 4. Differential cleavage plots comparing the susceptibility of the three DNA fragments of (A) 117, (B) 265, and (C) 198
base pairs to DNase I cutting in the presence of 15d at 20 µM. Negative values correspond to a ligand-protected site, and positive
values represent enhanced cleavage. Vertical scales are in units of ln(fa) - ln(fc), where fa is the fractional cleavage at any bond
in the presence of the drug and fc is the fractional cleavage of the same bond in the control, given closely similar extents of overall
digestion. Each line drawn represents a three-bond running average of individual data points, calculated by averaging the value
of ln(fa) - ln(fc) at any bond with those of its two nearest neighbors. Only the region of the restriction fragments analyzed by
densitometry is shown.
ism (∆A/A) signals were recorded with all the IPQ
compounds bound to calf thymus DNA, and this behav-
ior is typical of intercalating agents. However, here
again we observed an unusual effect with one molecule,
the bis-N-methylpiperazine derivative 15d . This com-
pound (Figure 1) gave reduced ∆A/A signals with calf
thymus DNA and poly(dGC)2 but positive signals with
poly(dAT)2.
Positive ELD data are commonly obtained with minor
groove binders that are positioned at about 45° relative
to the long axis of the DNA molecule (i.e., the electric
field direction).15 The binding mode of 15d was inves-
tigated further by circular dichroism. The CD spectral
titration for 15d (Figure 2) with poly(dAT)2 (ratios 15d /
DNA ranging from 0 to 10) gives a strong induced CD
signal in the compound absorption region above 300 nm
and large changes in the DNA absorption region.
Positive CD and ELD values with the AT DNA are
consistent with a dominant minor groove binding mode
for 15d .
compound 15d protects certain sequences from cleavage
by the nuclease, and the densitometric analyses of the
gels indicate that the protected sequences mainly cor-
respond to AT sites. The differential cleavage plots
indicate (Figure 4) selective binding of 15d to the
following sites: 5′-TAATA, 5′-TAAAA, 5′-TTTT, 5′-
ATTAA, and 5′-AAATTAA.
Footprints were detected only with 15d , the other
compounds showing no inhibition of DNase I cleavage.
The compounds having no side chain (16a , 16b) or only
one side chain (14d , 16d ) intercalate into DNA and have
no sequence preference. The incorporation of a second
cationic side chain confers AT sequence recognition by
virtue of the minor groove binding mode. In other words,
the substitution of the quinoline nitrogen by an N-
methylpiperazine chain changes the intercalative bind-
ing mode of 16d for a minor groove binding process
detected with 15d .
Con clu sion s
All newly synthesized compounds have been tested
for DNA binding by spectroscopic and footprinting
methods. None of the intercalating agents give any
footprint, whereas the minor groove binder 15d was
shown to protect DNA from cleavage by Dnase I at
certain sites. This minor groove mode binder 15d
recognizes preferentially AT-rich DNA sequences (Fig-
ure 3), as revealed by the DNase I footprinting experi-
ments performed with three different DNA radiolabeled
fragments of 117-bp, 178-bp, and 265-bp. It is clear that
We have designed a new DNA binding unit, the
tetrahydroindeno[1,2-b]pyrido[4,3,2-de]quinoline (IPQ)
chromophore, which is derived from the benzo[c]pyrido-
[2,3,4-kl]acridine (BPA) pentacyclic system recently
studied.10,13 The IPQ system is more planar than the
BPA system, and consequently, binding to DNA is
generally reinforced. These two pentacyclic heterocycles
perform more or less efficiently in terms of cytotoxicity.
No direct relationship between DNA binding strength

3670 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Catoen-Chackal et al.
1
IR 1702, 3331 cm-1; H NMR (CDCl3) δ 2.66 (t, J ) 6.1, 2H),
3.07 (t, J ) 6.1, 2H), 3.65 (q, J ) 5.5, 2H), 4.11 (t, J ) 5.2,
2H), 5.12 (s, 2H), 5.33 (bs, 1H), 6.86 (s, 1H), 6.88 (d, J ) 6.6,
1H), 7.35 (s, 5H), 7.67 (d, J ) 9.2, 1H).
and cytotoxicity to PC 3 prostate cancer cells can be
established, but a few highly cytotoxic molecules have
been identified in both the BPA and IPQ series. These
compounds were initially designed as DNA intercalating
agents, but a compound that fits into the minor groove
of AT-rich DNA sequences has been identified in the
IPQ series. The extended molecule 15d bearing two
N-methylpiperazine chains on both sides of the IPQ
chromophore provides a new motif for duplex DNA
recognition but also for more complex nucleic acid
structures. Indeed, recent preliminary studies (unpub-
lished data) reveal that this molecule binds to the
telomer quadruplex sequence AG3(TTAG3)3 and exhibits
a slight inhibitory activity against human telomerase.
This novel activity opens new perspectives for the design
of antitumor agents in this series.
5-[3-(N-Ben zyloxyca r b on yl)a m in op r op oxy]in d a n -1-
on e (9f). Mp 93 °C (EtOH/H2O); Rf ) 0.70 (CH2Cl2/MeOH 9:1);
IR 1685, 1716, 3298 cm-1; 1H NMR (CDCl3) δ 2.05 (p, J ) 5.6,
2H), 2.67 (t, J ) 6.0, 2H), 3.08 (t, J ) 5.6, 2H), 3.43 (p, J )
6.0, 2H), 4.10 (t, J ) 5.6, 2H), 5.01 (bs, 1H), 5.11 (s, 2H), 6.88-
6.90 (m, 2H), 7.35 (s, 5H), 7.68 (d, J ) 9.3, 1H).
2-(6-Meth oxy-2-oxo-2,3-d ih yd r o-1H-1-in d en yl)-N-[3-(4-
m eth ylp ip er a zin o)p r op yl]a ceta m id e (9i). EDCI (530 mg,
3.4 mmol), HOBt (460 mg, 3.4 mmol), and 4-methylmorpholine
(0.38 mL, 3.4 mmol) were added to a solution of 5-methoxy-
1-indanone-3-acetic acid (500 mg, 2.2 mmol) in 10 mL of a
mixture of dichloromethane/dimethylformamide (1:1). After
agitation for 1 h at room temperature, 1-(3-aminopropyl)-4-
methylpiperazine (360 mg, 2.2 mmol) was added and the
mixture was stirred for another 12 h. The residue obtained
after evaporation of the solvents was dissolved in dichlo-
romethane before extraction with a 10% potassium carbonate
solution. The organic layer was washed with a saturated
aqueous sodium chloride solution and dried over magnesium
sulfate. Concentration followed by column chromatography
(silica gel, 70% dichloromethane-ammoniacal methanol) gave
561 mg (71% yield) of 9i as a brown oil. Rf ) 0.33 (CH2Cl2/
MeOH 7:3, NH3); IR 1648, 1704, 3313 cm-1; 1H NMR (CDCl3)
δ 1.63 (p, J ) 6.2, 2H), 2.19 (s, 3H), 2.27-2.43 (m, 10H), 2.52
(dd, J ) 6.5, J ) 7.8, 2H), 2.90 (dd, J ) 7.4, J ) 7.8, 1H), 3.21
(bs, 2H), 3.32 (q, J ) 5.8, 2H), 3.40 (s, 3H), 6.87 (dd, J ) 2.3,
J ) 8.4, 1H), 6.90 (d, J ) 2.3, 1H), 7.44 (t, 1H), 7.62 (d, J )
8.4, 1H). Anal. (C20H29N3O3) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
14a ,c,g,h . A solution of 12 (500 mg, 1.89 mmol), PPTS (470
mg, 1.89 mmol), and appropriate indanone 9a ,c,g, or h (2.84
mmol) in toluene (10 mL) was heated at reflux for 24 h using
a Dean-Stark trap. The precipitate obtained after cooling was
collected by filtration and washed successively with toluene
and ether. The crude product was subjected to column chro-
matography (silica gel, 97% dichloromethane-methanol-TFA
(5 drops)). The solid was then recrystallized.
3-Acetyl-4,5-d im eth oxy-1,2,3,12-tetr a h yd r oin d en o[1,2-
b]p yr id o[4,3,2-d e]qu in olin e Tr iflu or oa ceta te (14a ). Yel-
low solid (340 mg, 50% yield); Rf ) 0.58 (CH2Cl2/MeOH 9:1);
mp 247 °C (EtOH 95%); IR 1609, 1667 cm-1; 1H NMR (CDCl3)
δ 2.28 (s, 3H), 3.36-3.51 (m, 3H), 3.99 (s, 3H), 4.12 (s, 2H),
4.14 (s, 3H), 5.32 (bs, 1H), 7.51 (s, 1H), 7.65-7.80 (m, 3H),
8.25 (d, J ) 8.0, 1H); MS (EI) m/z 360 (M+, 81), 318 (37), 303
(100). Anal. (C22H20N2O3‚TFA‚0.5H2O) C, H, N.
3-Acetyl-4,5,10-tr im eth oxy-1,2,3,12-tetr a h yd r oin d en o-
[1,2-b]p yr id o[4,3,2-d e]qu in olin e Tr iflu or oa ceta te (14c).
Brilliant-yellow solid (317 mg, 43% yield); Rf ) 0.45 (CH2Cl2/
MeOH 9:1); mp >250 °C (EtOH 95%); IR 1607, 1667 cm-1; 1H
NMR (DMSO-d6) δ 2.27 (s, 3H), 3.29-3.45 (m, 3H), 3.97 (s,
3H), 3.99 (s, 3H), 4.04 (s, 2H), 4.11 (s, 3H), 5.33 (bs, 1H), 7.18
(d, J ) 9.1, 1H), 7.25 (s, 1H), 7.44 (s, 1H), 8.17 (d, J ) 8.7,
1H); MS (EI) m/z 390 (M+, 44), 348 (10), 333 (100). Anal.
(C23H22N2O4‚TFA‚0.5H2O) C, H, N.
Exp er im en ta l Section
Ch em istr y. Analytical thin-layer chromatography (TLC)
was performed on precoated Kieselgel 60F254 plates (Merck).
Compounds were visualized by UV and/or with iodine, and Rf
are given for guidance. Melting points were determined with
a capillary melting point apparatus and remain uncorrected.
The structures of all compounds were supported by IR (KBr
1
pellets, FT-Bruker Vector 22 instrument) and H NMR at 300
MHz on a Bruker DPX-300 spectrometer. Chemical shifts δ
are reported in ppm downfield from tetramethylsilane, J
values are in hertz, and the splitting patterns were designated
as follows: s, singlet; d, doublet; t, triplet; q, quartet; m,
multiplet; b, broad. Electron impact (EI) mass spectra were
obtained on a quadripolar Finningan Mat SSQ710 spectrom-
eter. Elemental analyses were performed by the “Service
Central d’Analyses” at the CNRS, Vernaison (France). Dim-
ethylformamide was distilled from CaH2 and stored over 3Å
molecular sieves.
Ma ter ia ls. The preparation of aminoketones 12 and 13 has
been previously described.13 Compounds 9a -c,g,h , 10, and
PPTS were purchased from Sigma Aldrich (Saint Quentin
Fallavier, France).
5-[3-(4-Meth yl)piper azin -1-ylpr opoxy]in dan -1-on e (9d).
Potassium carbonate (3.73 g, 27 mmol) was added to a solution
of 5-hydroxyindanone 5 (1 g, 6.7 mmol) in dry DMF (10 mL),
and the mixture was stirred for 30 min at room temperature.
Then 1-(3-chloropropyl)-4-methylpiperazine dihydrochloride
(1.35 g, 5.4 mmol) was added. The mixture was stirred for 3 h
at 80 °C, then diluted with water and extracted several times
with ethyl acetate. The organic layers were washed with a 10%
potassium carbonate solution and a saturated aqueous sodium
chloride solution and dried over magnesium sulfate. Concen-
tration followed by column chromatography (silica gel, 15%
methanol-ammoniacal dichloromethane) gave 940 mg (60%
yield) of 9d as a brown oil: Rf ) 0.62 (CH2Cl2/MeOH 85:15,
NH3); IR 1703 cm-1; 1H NMR (CDCl3) δ 1.97 (p, J ) 6.4, 2H),
2.27 (s, 3H), 2.49-2.66 (m, 12H), 3.05 (t, J ) 6.4, 2H), 4.06 (t,
J ) 6.4, 2H), 6.83 (dd, J ) 2.0, J ) 7.3, 1H), 6.88 (d, J ) 2.0,
1H), 7.64 (d, J ) 7.3, 1H).
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
9e,f. Potassium carbonate (5.6 g, 40 mmol) was added to a
solution of 5-hydroxyindanone 5 (3 g, 20 mmol) in dry DMF
(20 mL), and the mixture was stirred for 30 min at room
temperature. Then the appropriate ω-chloroalkylcarbamate 716
or 817 (22 mmol) was added. The mixture was stirred for 3 h
at 80 °C, then diluted with water and extracted several times
with ethyl acetate. The organic layers were washed with
saturated aqueous sodium chloride and dried over magnesium
sulfate. Concentration followed by column chromatography
(silica gel, 50% heptane-ethyl acetate) and trituration in
isopropyl ether gave 3.3 g (50% yield) of 9e or 4.6 g (66% yield)
of 9f as white powders.
3-Acetyl-4,5-dim eth oxy-12-m eth yl-1,2,3,12-tetr ah ydr oin -
d en o[1,2-b]p yr id o[4,3,2-d e]q u in olin e Tr iflu or oa cet a t e
(14g). Light-yellow solid (590 mg, 71% yield); Rf ) 0.45 (CH2-
Cl2/MeOH 9:1); mp >250 °C (EtOH 95%); IR 1608, 1667 cm-1
;
1H NMR (CDCl3, TFA-d1) δ 2.27 (s, 3H), 3.32-3.48 (m, 3H),
3.94 (s, 3H), 3.99 (s, 3H), 4.01 (s, 1H), 4.15 (s, 3H), 5.33 (bs,
1H), 7.32 (dd, J ) 2.3, J ) 8.3, 1H), 7.66 (t, J ) 8.8, 2H), 7.70
(s, 1H), 7.87 (dd, J ) 2.3, J ) 8.3, 1H); MS (EI) m/z 374 (M+,
52), 332 (34), 317 (100). Anal. (C23H22N2O3‚TFA‚0.5H2O) C, H,
N.
3-Acetyl-4,5-d im eth oxy-1,2,3,12-tetr a h yd r oin d en o[1,2-
b]p yr id o[4,3,2-d e]qu in olin -12-on e Tr iflu or oa ceta te (14h ).
Yellow solid (533 mg, 62% yield); Rf ) 0.40 (CH2Cl2/MeOH 9:1);
mp 213 °C (EtOH 95%); IR 1612, 1664, 1707 cm-1; H NMR
1
5-[2-(N -Be n zyloxyca r b on yl)a m in oe t h oxy]in d a n -1-
on e (9e). Mp 72 °C (EtOH/H2O); Rf ) 0.62 (CH2Cl2/MeOH 9:1);
(CDCl3) δ 2.09 (s, 3H), 3.24-3.33 (m, 2H), 3.69 (bs, 1H), 3.79
(s, 3H), 4.01 (s, 3H), 5.06 (bs, 1H), 7.31 (s, 1H), 7.42 (t, J )

DNA-Binding Indenopyridoquinoline Derivatives
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14 3671
7.6, 1H), 7.69 (d, J ) 7.6, 1H), 7.78 (t, J ) 7.6, 1H), 7.93 (d, J
) 7.6, 1H); MS (EI) m/z 374 (M+, 84), 332 (52), 317 (100). Anal.
(C22H18N2O4‚0.5TFA‚0.5H2O) C, H, N.
HCl was added to a solution of these solids in methanol to
yield 14j or 14k as yellow solids that were then recrystallized.
3-Acet yl-10-(2-a m in oet h oxy)-4,5-d im et h oxy-1,2,3,12-
tetr a h yd r oin d en o[1,2-b]p yr id o[4,3,2-d e]qu in olin e Dih y-
d r och lor id e (14j). Yellow solid; mp >250 °C (MeOH/Et2O);
3-Acet yl-10-h yd r oxy-4,5-d im et h oxy-1,2,3,12-t et r a h y-
d r oin d en o[1,2-b]p yr id o[4,3,2-d e]qu in olin e (14b). A solu-
tion of 12 (500 mg, 1.89 mmol), PPTS (710 mg, 2.84 mmol),
and indanone 9b (840 mg, 5.68 mmol) in butan-1-ol (10 mL)
was heated under reflux for 3 h using a Dean-Stark trap.
After the mixture cooled, the solvent was evaporated and the
residue was triturated with ether. After the precipitate was
washed with ether, the crude product was subjected to column
chromatography (silica gel, 95% dichloromethane-methanol).
Evaporation of the solvents gave 534 mg (75% yield) of pure
14b as a pale-yellow solid. Mp >250 °C (MeOH/Et2O); Rf )
Rf ) 0.46 (CH2Cl2/MeOH 9:1, NH3); IR 1617, 1645, 3423 cm-1
;
1H NMR (DMSO-d6) δ 2.09 (s, 3H), 3.33-3.39 (m, 4H), 3.60
(bs, 1H), 3.83 (s, 3H), 4.04 (s, 3H), 4.13 (s, 2H), 4.35 (t, J )
4.7, 2H), 4.61 (bs, 1H), 7.25 (d, J ) 8.7, 1H), 7.43 (s, 1H), 7.86
(s, 1H), 8.31 (bs, 3H), 8.67 (d, J ) 8.7, 1H); MS (EI) m/z 419
(M+, 80), 362 (48), 319 (100). Anal. (C24H25N3O4‚2HCl‚0.5H2O)
C, H, N, Cl.
3-Acetyl-10-(3-a m in op r op oxy)-4,5-d im eth oxy-1,2,3,12-
tetr a h yd r oin d en o[1,2-b]p yr id o[4,3,2-d e]qu in olin e Dih y-
d r och lor id e (14k ). Yellow solid; mp >250 °C (MeOH/Et2O);
1
0.45 (CH2Cl2/MeOH 9:1); IR 1649, 1667, 3423 cm-1; H NMR
Rf ) 0.56 (CH2Cl2/MeOH 9:1, NH3); IR 1645, 1673, 3421 cm-1
;
(DMSO-d6) δ 2.28 (s, 3H), 3.29 (bs, 2H), 3.63 (bs, 1H), 3.84 (s,
3H), 4.01 (s, 2H), 4.05 (s, 3H), 4.63 (bs, 1H), 7.00 (d, J ) 8.0,
1H), 7.10 (s, 2H), 7.13 (s, 1H), 8.12 (d, J ) 8.4, 1H), 10.81 (bs,
1H); MS (EI) m/z 376 (M+, 43), 334 (64), 319 (100). Anal.
(C22H20N2O4‚2.25H2O) C, H, N.
1H NMR (DMSO-d6) δ 2.10 (s, 5H), 2.99 (bs, 2H), 3.30-3.36
(m, 3H), 3.82 (s, 3H), 4.04 (s, 3H), 4.10 (s, 2H), 4.20 (t, J )
4.7, 2H), 4.61 (bs, 1H), 7.16 (d, J ) 7.6, 1H), 7.35 (s, 1H), 7.64
(s, 1H), 8.13 (bs, 3H), 8.43 (d, J ) 8.2, 1H); MS (EI) m/z 433
(M+, 100), 376 (100), 319 (60). Anal. (C25H27N3O4‚2HCl‚2.5H2O)
C, H, N, Cl.
3-Acetyl-4,5-d im eth oxy-10-[3-(4-m eth yl)p ip er a zin -1-yl-
p r op oxy]-1,2,3,12-t et r a h yd r oin d en o[1,2-b]p yr id o[4,3,2-
d e]qu in olin e Tr ih yd r och lor id e (14d ). A solution of 12 (2
g, 7.57 mmol), PPTS (950 mg, 3.79 mmol), and indanone 9d
(1.09 g, 3.79 mmol) in 15 mL of a mixture of toluene/ethanol
(2:1) was heated under reflux for 5 days using a Dean-Stark
trap. The precipitate formed after cooling to room temperature
was collected by filtration and washed successively with
toluene and ether. The crude product was subjected first to
column chromatography (silica gel, 90% dichloromethane-
methanol), then to preparative HPLC (C18, 4.6 mm × 150 mm,
5 µm, 100 Å, 34% MeOH-66% H2O-0.1% TFA). Evaporation
of the appropriate fraction (tR ) 15.05 min) gave 710 mg (42%
yield) of a beige solid. Methanol saturated with HCl was added
to a methanolic solution of this solid to yield 14d as a pale-
yellow powder. Mp 250 °C (EtOH/Et2O); Rf ) 0.12 (CH2Cl2/
4,5-Dim eth oxy-3-[2-(4-m eth yl)p ip er a zin -1-yla cetyl]-10-
[3-(4-m eth yl)piper azin -1-ylpr opoxy]-1,2,3,12-tetr ah ydr oin -
d en o[1,2-b]p yr id o[4,3,2-d e]q u in olin e P en t a h yd r och lo-
r id e (15d ). A solution of 13 (500 mg, 1.38 mmol), PPTS (520
mg, 2.07 mmol), and indanone 9d (1.19 g, 4.14 mmol) in butan-
1-ol (10 mL) was heated under reflux for 12 h using a Dean-
Stark trap. The mixture was cooled to room temperature, made
basic with 10% aqueous potassium carbonate, and diluted with
ethyl acetate, and the layers were separated. The dark-red
organic layer was washed with saturated aqueous sodium
chloride and dried over magnesium sulfate. Removal of the
solvent gave an oily product that precipitated in ether. After
filtration and washing with ether, the crude product was
subjected to column chromatography (silica gel, 10% methanol-
dichloromethane to 50% methanol-1% ammoniacal dichlo-
romethane). Evaporation of the appropriate fraction gave 659
mg (63% yield) of pure base. Dichloromethane saturated with
HCl was added to a solution of the base in dichloromethane
to yield 15d as a yellow solid. Mp >250 °C (EtOH/Et2O/H2O);
1
MeOH 9:1, NH3); IR 1647, 1670 cm-1; H NMR (DMSO-d6) δ
2.02 (s, 3H), 2.61-3.67 (m, 18H), 3.83 (s, 3H), 4.03 (s, 3H),
4.08 (s, 2H), 4.21 (bt, 2H), 4.63 (bs, 1H), 7.18 (d, J ) 8.6, 1H),
7.29 (s, 1H), 7.61 (s, 1H), 8.42 (d, J ) 8.6, 1H); MS (EI) m/z
516 (M+, 72), 446 (40), 388 (20), 376 (100), 141 (78), 113 (69),
43 (92). Anal. (C30H36N4O4‚3HCl‚5H2O) C, H, N, Cl.
1
Rf ) 0.63 (CH2Cl2/MeOH 7:3, NH3); IR 1644, 1684 cm-1; H
NMR, insufficiently soluble in usual solvents; MS (EI) m/z 614
(M+, 5), 583 (6), 113 (100). Anal. (C35H46N6O4‚5HCl‚8H2O) C,
H, N, Cl.
2-(3-Acetyl-4,5,10-tr im eth oxy-1,2,3,12-tetr ah ydr oin den o-
[1,2-b]p yr id o[4,3,2-d e]qu in olin -12-yl)-N-[3-(4-m eth ylp ip -
er a zin o)p r op yl]a ceta m id e Tr ih yd r och lor id e (14i). Com-
pound 14i was prepared from 12 and 9i according to the
procedure described for 14d . Methanol saturated with HCl was
added to a methanolic solution of the crude material to yield
14i as a pale-yellow powder (1.55 g, 54% yield). Mp 225 °C
(EtOH/Et2O); Rf ) 0.15 (CH2Cl2/MeOH 9:1, NH3); IR 1645,
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
15j,k . A solution of 13 (500 mg, 1.38 mmol), PPTS (520 mg,
2.07 mmol), and the appropriate indanone 9e or 9f (4.14 mmol)
in butan-1-ol (10 mL) was heated under reflux for 12 h using
a Dean-Stark trap. The mixture was cooled to room temper-
ature, made basic with 10% aqueous potassium carbonate, and
diluted with ethyl acetate, and the layers were separated. The
dark-red organic layer was washed with saturated aqueous
sodium chloride and dried over magnesium sulfate. Removal
of the solvent gave an oily product that precipitated in ether.
After filtration and washing with ether, the crude product
corresponding to 15e or 15f was subjected to chromatography
(silica gel, 95% dichloromethane-methanol). The solid ob-
tained after evaporation of the solvents was dissolved in MeOH
(10 mL), and Pd on C was added. The mixture was heated to
reflux for 30 min before ammonium formate (10 equiv) was
added. After 2 h, the warmed solution was filtered through
Celite and washed with MeOH and the filtrate was evaporated.
The concentrate was subjected to column chromatography
(silica gel, 30% methanol-1% ammoniacal dichloromethane).
Evaporation of the solvents gave 414 mg (58% yield) or 403
mg (55% yield) of pure solids. Methanol saturated with HCl
was added to a solution of these solids in methanol to yield
15j or 15k as yellow powders that were then recrystallized.
1
3422 cm-1; H NMR (DMSO-d6) δ 2.02 (s, 3H), 2.61-3.67 (m,
20H), 3.83 (s, 3H), 3.91 (s, 3H), 4.03 (s, 3H), 4.70 (s, 1H), 4.95
(bs, 1H), 7.18 (d, J ) 8.6, 1H), 7.24 (s, 1H), 7.89 (s, 1H), 8.63
(d, J ) 8.6, 1H), 11.98 (bs, 1H); MS (EI) m/z 588 (M + 1, 5),
586 (48), 531 (100), 419 (14), 391 (91), 332 (33), 276 (58), 207
(42). Anal. (C33H41N5O5‚3HCl‚3.5H2O) C, H, N, Cl.
Gen er a l P r oced u r e for th e Syn th esis of Com p ou n d s
14j,k . A solution of 12 (500 mg, 1.89 mmol), PPTS (710 mg,
2.84 mmol), and the appropriate indanone 9e or 9f (5.68 mmol)
in butan-1-ol (10 mL) was heated under reflux for 5 h using a
Dean-Stark trap. After the mixture cooled, the solvent was
evaporated and the residue was triturated with ether. After
the precipitate was washed with ether, the product was
subjected to column chromatography (silica gel, 97% dichlo-
romethane-methanol). The crude solid 14e or 14f obtained
after evaporation of the solvents was dissolved in MeOH (10
mL), and Pd on C was added. The mixture was heated at reflux
for 30 min before addition of ammonium formate (10 equiv).
After 2 h, the warmed solution was filtered through Celite and
washed with MeOH, and the filtrate was evaporated. The
concentrate was subjected to column chromatography (silica
gel, 20% methanol-1% ammoniacal dichloromethane). Evapo-
ration of the solvents gave respectively 436 mg (55% yield) or
344 mg (42% yield) of pure bases. Methanol saturated with
10-(2-Am in oeth oxy)-4,5-d im eth oxy-3-[2-(4-m eth yl)p ip -
er a zin -1-yl]-1,2,3,12-tetr a h yd r oin d en o[1,2-b]p yr id o[4,3,2-
d e]qu in olin e Tr ih yd r och lor id e (15j). Yellow solid; mp
>250 °C (MeOH/Et2O/H2O); Rf ) 0.77 (CH2Cl2/MeOH 7:3,
NH3); IR 1643, 1682, 3423 cm-1; 1H NMR (DMSO-d6/60 °C) δ

3672 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 14
Catoen-Chackal et al.
2.71 (s, 3H), 3.27-3.39 (m, 12H), 3.82 (s, 3H), 4.02-4.05 (m,
6H), 4.09 (s, 2H), 4.40 (bt, J ) 5.2, 3H), 7.10 (s, 1H), 7.12 (dd,
J ) 2.1, J ) 8.6, 1H), 8.01 (s, 1H), 8.41 (bs, 3H), 8.72 (d, J )
8.6, 1H); MS (EI) m/z 517 (M+, 12), 485 (20), 113 (100), 70 (82).
Anal. (C29H35N5O4‚3.75HCl‚5H2O) C, H, N, Cl.
10-(3-Am in op r op oxy)-4,5-d im et h oxy-3-[2-(4-m et h yl)-
p ip er a zin -1-yl]-1,2,3,12-t et r a h yd r oin d en o[1,2-b]p yr id o-
[4,3,2-d e]qu in olin e Tr ih yd r och lor id e (15k ). Yellow solid;
mp >250 °C (MeOH/Et2O/H2O); Rf ) 0.76 (CH2Cl2/MeOH 7:3,
NH3); IR 1642, 1681, 3423 cm-1; 1H NMR (DMSO-d6/60 °C) δ
2.15 (p, J ) 6.5, 2H), 2.71 (s, 3H), 3.00 (bs, 2H), 3.27-3.39 (m,
10H), 3.81-3.86 (m, 6H), 4.01 (s, 3H), 4.09 (s, 2H), 4.23 (bt, J
) 6.5, 3H), 7.08 (s, 1H), 7.14 (dd, J ) 2.1, J ) 7.6, 1H), 7.99
(s, 1H), 8.13 (bs, 3H), 8.55 (d, J ) 7.6, 1H); MS (EI) m/z 531
(M+, 2), 499 (6), 113 (100), 70 (32). Anal. (C30H37N5O4‚3.75HCl‚
8H2O) C, H, N, Cl.
4,5-Dim eth oxy-1,2,3,12-tetr a h yd r oin d en o[1,2-b]p yr id o-
[4,3,2-d e]qu in olin -12-on e Hyd r och lor id e (16h ). Red solid
(269 mg, 60% yield); mp 250 °C (EtOH 95%); Rf ) 0.89 (CH2-
Cl2/MeOH 9:1); IR 1650, 1709, 3407 cm-1 1H NMR (CDCl3,
;
TFA-d1) δ 3.73 (t, J ) 6.1, 2H), 3.83 (t, J ) 6.1, 2H), 4.00 (s,
3H), 4.12 (s, 3H), 7.47 (s, 1H), 7.73-8.19 (m, 3H), 8.54 (d, J )
7.6, 1H), 10.34 (bs, 2H); MS (EI) m/z 332 (M+, 75), 317 (100).
Anal. (C20H16N2O3‚HCl‚H2O) C, H, N.
10-(2-Am in oet h oxy)-4,5-d im et h oxy-1,2,3,12-t et r a h y-
d r oin d en o[1,2-b]p yr id o[4,3,2-d e]qu in olin e Dih yd r och lo-
r id e (16j). Red solid (233 mg, 50% yield); mp >250 °C (MeOH/
Et2O); Rf ) 0.11 (CH2Cl2/MeOH 9:1, NH3); IR 1642, 3419 cm-1
;
1H NMR (DMSO-d6) δ 3.26-3.28 (m, 4H), 3.46 (t, J ) 6.5, 2H),
3.75 (s, 3H), 3.98 (s, 3H), 4.09 (s, 2H), 4.33 (t, J ) 4.7, 2H),
6.95 (s, 1H), 7.25 (d, J ) 8.8, 1H), 7.42 (s, 1H), 8.40 (d, J )
8.5, 1H); MS (EI) m/z 377 (M+, 96), 362 (64), 319 (100). Anal.
(C22H23N3O3‚2HCl‚3H2O) C, H, N, Cl.
Gen er a l P r oced u r e for Hyd r olysis of Am id es 14a -
d ,g,h ,j,k . A solution of 500 mg of quinolines 14 in 10 mL of
aqueous 6 N HCl was refluxed for 4 h. After cooling to room
temperature, the mixture was made basic using 10% aqueous
potassium carbonate and extracted with ethyl acetate. The red
organic layer was washed with saturated aqueous sodium
chloride and dried over magnesium sulfate. After removal of
the solvent, the crude oily product was subjected to column
chromatography: silica gel; 98% dichloromethane-methanol
(14a,c,g,h ), 97% dichloromethane-methanol (14b), 10% metha-
nol-1% ammoniacal dichloromethane (14d ,j,k ). Evaporation
of the solvents gave pure bases. Methanol saturated with HCl
was added to a solution of these solids in methanol to yield
secondary amines 16 as hydrochlorides, which were then
recrystallized.
10-(3-Am in op r op oxy)-4,5-d im et h oxy-1,2,3,12-t et r a h y-
d r oin d en o[1,2-b]p yr id o[4,3,2-d e]qu in olin e Dih yd r och lo-
r id e (16k ). Red solid (253 mg, 56% yield); mp >250 °C (MeOH/
Et2O); Rf ) 0.08 (CH2Cl2/MeOH 9:1, NH3); IR 1644, 3419 cm-1
;
1H NMR (DMSO-d6) δ 2.08 (p, 2H), 2.98 (t, J ) 6.8, 2H), 3.26
(t, 2H), 3.47 (t, 2H), 3.74 (s, 3H), 3.97 (s, 3H), 4.06 (s, 2H),
4.19 (t, J ) 4.7, 2H), 6.92 (s, 1H), 7.17 (d, J ) 8.8, 1H), 7.34 (s,
1H), 8.33 (d, J ) 8.5, 1H); MS (EI) m/z 391 (M+, 100), 376
(84), 319 (100), 243 (88). Anal. (C23H25N3O3‚2HCl‚2H2O) C, H,
N, Cl.
Biologica l Meth od s. Meltin g Tem p er a tu r e Mea su r e-
m en ts. Melting curves were measured using an Uvikon 943
spectrophotometer coupled to a Neslab RTE111 cryostat. For
each series of measurements, 12 samples were placed in a
thermostatically controlled cell holder, and the quartz cuvettes
(10 mm path length) were heated by circulating water.
Measurements were performed using 20 µM poly(dAT)2 in BPE
buffer, pH 7.1 (6 mM Na2HPO4, 2 mM NaH2PO4, 1 mM EDTA).
The temperature inside the cuvette was measured with a
platinum probe; it was increased over the range 20-100 °C
with a heating rate of 1 °C/min. The “melting” temperature
Tm was taken as the midpoint of the hyperchromic transition.
The Uvikon 943 spectrophotometer was also used to record
absorption spectra.
4,5-Dim eth oxy-1,2,3,12-tetr a h yd r oin d en o[1,2-b]p yr id o-
[4,3,2-d e]qu in olin e Hyd r och lor id e (16a ). Red solid (307
mg, 47% yield); mp >250 °C (EtOH 95%); Rf ) 0.59 (CH2Cl2/
1
MeOH 9:1); IR 1645, 3314 cm-1; H NMR (CDCl3, TFA-d1) δ
3.70-3.75 (m, 4H), 4.05 (s, 3H), 4.13 (s, 3H), 4.17 (s, 2H), 7.66
(s, 1H), 7.65-7.78 (m, 3H), 8.46 (s, 1H), 11.07 (bs, 2H); MS
(EI) m/z 318 (M+, 65), 303 (100). Anal. (C20H18N2O2‚HCl‚
1.5H2O) C, H, N.
10-Hyd r oxy-4,5-d im eth oxy-1,2,3,12-tetr a h yd r oin d en o-
[1,2-b]p yr id o[4,3,2-d e]qu in olin e Hyd r och lor id e (16b). Or-
ange solid (235 mg, 53% yield); mp >250 °C (MeOH/Et2O/H2O);
F lu or escen ce Mea su r em en ts. Binding studies were car-
ried out through a competitive displacement fluorometric assay
with DNA-bound ethidium.18,19 Fluorescence data were re-
corded at room temperature with a SPEX Fluorolog fluorom-
eter. Excitation was set at 515 nm, and fluorescence emission
was monitored over the range 550-700 nm. Experiments were
performed with an [ethidium]/[DNA] molar ratio of 12.6:10 and
a drug concentration range of 0.01-100 µM in a BPE buffer,
pH 7.1. C50 values for ethidium displacement were calculated
using a fitting function incorporated into Prism 3.0, and the
apparent equilibrium binding constants (Kapp) were calculated
as follows: Kapp ) (1.26 µM/C50)Kethidium, with Kethidium ) 107
1
Rf ) 0.49 (CH2Cl2/MeOH 9:1); IR 1649, 3396 cm-1; H NMR
(DMSO-d6) δ 3.23-3.46 (m, 4H), 3.75 (s, 3H), 3.95 (s, 3H), 4.00
(s, 2H), 6.72 (bs, 2H), 7.00 (d, J ) 8.3, 1H), 7.06 (s, 1H), 7.13
(s, 1H), 8.44 (d, J ) 8.3, 1H), 10.71 (s, 1H); MS (EI) m/z 334
(M+, 58), 319 (100). Anal. (C20H18N2O3‚HCl‚2H2O) C, H, N, Cl.
4,5,10-Tr im eth oxy-1,2,3,12-tetr a h yd r oin d en o[1,2-b]p y-
r id o[4,3,2-d e]qu in olin e Hyd r och lor id e (16c). Red solid
(276 mg, 62% yield); mp >250 °C (EtOH 95%); Rf ) 0.65 (CH2-
Cl2/MeOH 9:1); IR 1650, 3321 cm-1; 1H NMR (CDCl3, TFA-d1)
δ 3.67 (bt, 2H), 3.98 (s, 3H), 4.03 (s, 4H), 4.10 (s, 3H), 4.15 (s,
3H), 7.18 (d, J ) 8.5, 1H), 7.21 (s, 1H), 7.72 (s, 1H), 8.36 (d, J
) 8.5, 1H); MS (EI) m/z 348 (M+, 61), 333 (100). Anal.
(C21H20N2O3‚HCl‚0.2H2O) C, H, N.
4,5-Dim eth oxy-10-[3-(4-m eth yl)piper azin -1-ylpr opoxy]-
1,2,3,12-t et r a h yd r oin d en o[1,2-b]p yr id o[4,3,2-d e]q u in o-
lin e Tetr a h yd r och lor id e (16d ). Orange solid (193 mg, 42%
yield); mp >250 °C (EtOH/Et2O); Rf ) 0.06 (CH2Cl2/MeOH 9:1,
NH3); IR 1649, 3143 cm-1; 1H NMR (DMSO-d6) δ 2.02 (p, J )
5.4, 2H), 2.63-3.59 (m, 17H), 3.83 (s, 3H), 4.03 (s, 3H), 4.08
(s, 2H), 4.12 (t, J ) 5.4, 2H), 7.16 (d, J ) 8.7, 1H), 7.30 (s,
1H), 7.59 (s, 1H), 8.41 (d, J ) 8.7, 1H); MS (EI) m/z 474 (M+,
100), 404 (68), 334 (36), 141 (78). Anal. (C28H34N4O3‚4HCl‚
2H2O) C, H, N, Cl.
M-1
.
Cir cu la r Dich r oism . The CD spectra were obtained with
a J -810 J asco spectropolarimeter at 20 °C controlled by a PTC-
424S/L Peltier type cell changer (J asco). A quartz cell of 10
mm path length was used to obtain spectra from 500 to 230
nm with a resolution of 0.1 nm. The desired ratios of compound
to DNA were obtained by adding DNA to the cell containing a
constant amount of the drug (20 µM). The CD measurements
were performed in a 1 mM Na cacodylate, buffer pH 7.0.
Electr ic Lin ea r Dich r oism . ELD measurements were
performed with a computerized optical measurement system
using the previously outlined procedures.20,21 All experiments
were conducted with a 10 mm path length Kerr cell having a
1.5 mm electrode separation. The samples were guided under
an electric field strength varying from 1 to 14 kV/cm. The drug
under test was present at 10 µM together with the DNA at
250 µM unless otherwise stated. The ELD measurements were
performed in a 1 mM Na cacodylate buffer, pH 7.0.
4,5-Dim et h oxy-12-m et h yl-1,2,3,12-t et r a h yd r oin d en o-
[1,2-b]p yr id o[4,3,2-d e]q u in olin e H yd r och lor id e (16g).
Light-red solid (233 mg, 52% yield); mp >250 °C (EtOH 95%);
1
Rf ) 0.57 (CH2Cl2/MeOH 9:1); IR 1650, 3442 cm-1; H NMR
(CDCl3) δ 1.55 (d, J ) 7.1, 3H), 3.38-3.50 (m, 2H), 3.60-3.75
(m, 2H), 3.89 (s, 3H), 4.07 (s, 3H), 4.29 (q, J ) 7.5, 1H), 5.11
(bs, 2H), 7.52 (s, 3H), 7.83 (s, 1H), 9.12 (s, 1H); MS (EI) m/z
332 (M+, 70), 317 (100). Anal. (C21H20N2O2‚HCl‚H2O) C, H, N.
DNa se I F ootp r in tin g. The experimental procedure has
been previously described.22,23

DNA-Binding Indenopyridoquinoline Derivatives
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