Benzothiopyranoindole-based antiproliferative agents: Synthesis, cytotoxicity, nucleic acids interaction, and topoisomerases inhibition properties

Article abstract of DOI:10.1021/jm900627v

Novel benzo[3′,2′:5,6]thiopyrano[3,2-b]indol-10(11H)-ones 1a-v were synthesized and evaluated for their antiproliferative activity in an in vitro assay of human tumor cell lines (HL-60 and HeLa). Compounds 1e-v, substituted at the 11-position with a basic

Full text of DOI:10.1021/jm900627v

J. Med. Chem. 2009, 52, 5429–5441 5429
DOI: 10.1021/jm900627v
Benzothiopyranoindole-Based Antiproliferative Agents: Synthesis, Cytotoxicity, Nucleic Acids
Interaction, and Topoisomerases Inhibition Properties
Lisa Dalla Via,*,† Sebastiano Marciani Magno,† Ornella Gia,† Anna Maria Marini,*,‡ Federico Da Settimo,‡ Silvia Salerno,‡
Concettina La Motta,‡ Francesca Simorini,‡ Sabrina Taliani,‡ Antonio Lavecchia,§ Carmen Di Giovanni,§ Giuseppe Brancato,§
Vincenzo Barone, and Ettore Novellino§
†
‡
Dipartimento di Scienze Farmaceutiche, Universita di Padova, via Marzolo 5, I-35131 Padova, Italy, Dipartimento di Scienze Farmaceutiche,
ꢀ
§
ꢀ
ꢀ
Universita di Pisa, via Bonanno 6, I-56126 Pisa, Italy, Dipartimento di Chimica Farmaceutica e Tossicologica, Universita di Napoli Federico II,
Via D. Montesano 40, I-80131 Napoli, Italy, and Scuola Normale Superiore, Piazza dei Cavalieri 7, I-56100, Pisa and Istituto per i Processi
Chimico-Fisici del Consiglio Nazionale delle Ricerche (IPCF-CNR), Area della Ricerca, Via G. Moruzzi 1, I-56124 Pisa, Italy
Received May 12, 2009
Novel benzo[30,20:5,6]thiopyrano[3,2-b]indol-10(11H)-ones 1a-v were synthesizedand evaluated for their
antiproliferative activity in an in vitro assay of human tumor cell lines (HL-60 and HeLa). Compounds
1e-v, substituted at the 11-position with a basic side chain, showed a significant ability to inhibit cell
growth with IC50 values in the low micromolar range. Linear dichroism measurements showed that all
11-dialkylaminoalkyl substituted derivatives 1e-v behave as DNA-intercalating agents. Fluorimetric
titrations demonstrated their specificity in binding to A-T rich regions, and molecular modeling studies
were performed on the most active derivatives (1e, 1i, 1p) to characterize in detail the complexation
mechanism of these benzothiopyranoindoles to DNA. A relaxation assay evidenced a dose-dependent
inhibition of topoisomerase II activity that appeared in accordance with the antiproliferative capacity.
Finally, for the most cytotoxic derivative, 1e, a topoisomerase II poisoning effect was also demonstrated,
along with a weak inhibition of topoisomerase I-mediated relaxation.
Introduction
topology. The enzymes relieve torsional stresses caused during
DNA replication and transcription, cutting and rejoining of
single, topoisomerase I, or double, topoisomerase II, DNA
strands.15-19 Topoisomerase-targeting agents have long been
considered an attractive target for the design of cancer chemo-
therapeutics because they can cause permanent DNA damage
that triggers a series of cellular events, inducing apoptosis and
finally causing cell death.20-26 However, despite their antitu-
mor potency, several drugs that act as intercalators and
topoisomerase targeting agents suffer from poor solubility,
dose-limiting toxicity, reversibility of cleavage-complex for-
mation, and resistance mechanism development;27-29 for these
reasons, novel molecules are being developed to overcome
such limitations.30-33 From a structural point of view, DNA-
intercalating ligands are characterized by a planar (hetero)-
aromatic polycyclic moiety, a common pharmacophoric fea-
ture, and are able to form molecular complexes between DNA
base pairs, which are stabilized via hydrophobic interactions,
hydrogen bonding, and/or van der Waal’s forces. Moreover, in
spite of the undoubted importance of the planar chromophore,
the presence of charged groups or of protonable nitrogen
atom-bearing side chains in an appropriate position on the
planar core improves the binding affinity, allowing further
interactions of the ligands with other important architectural
features of DNA such as its major or minor grooves. Finally,
an enhanced solubility under physiological conditions can be
envisaged.34-36
The incidence of cancer, the main cause of mortality after
cardiovascular diseases, has significantly increased in the past
few years; therefore, an important goal of today’s research is
the development of new drugs characterized by promising
antiproliferative properties, in particular against the most
aggressive tumors.1-4 Anticancer agents that target DNA
are among the most effective and have produced significant
increases in the survival rates of patients, especially when used
in combination protocols with drugs that have different
mechanisms of action.5,6 Among chemotherapeutic drugs,
DNA-intercalating agents, such as anthracyclines, m-amsa-
crine (m-AMSAa), or ellipticines, represent a peculiar group
with excellent antitumor activity.7,8 The intercalators give rise
to an effective nuclear DNA binding complex, affording a
distorted helix and affecting the activity of processing enzy-
mes, thus compromising the structure and physiological
functions of the macromolecule.9,10 More interestingly, seve-
ral DNA-intercalating agents are able to affect topoisome-
rases I and/or II catalytic activity by forming a stable ternary
complex involving the drug, the enzyme, and DNA and
producing permanent DNA strand breaks.11-14
In actively replicating cells, topoisomerases I and II are
involved in many aspects of DNA metabolism and chromatin
*To whom correspondence should be addressed. Phone: þ39 049
8275712. Fax: þ39 049 8275366. E-mail: lisa.dallavia@unipd.it.
a Abbreviations: m-AMSA, m-amsacrine (40-(9-acridinylamino)metha-
nesulfon-m-anisidide); LD, linear flow dichroism; DFT, density functional
theory; PCM, polarizable continuum model; SDS, sodium dodecyl sulfate;
BSA, bovine serum albumin.
As part of our research program devoted to the preparation
and evaluation of new antiproliferative agents, we extensively
studied several polycyclic chromophores that are structurally
r
2009 American Chemical Society
Published on Web 08/19/2009
pubs.acs.org/jmc

5430 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Chart 1. Structures of Compounds I-VI
Dalla Via et al.
correlated to classes of DNA-intercalating agents, among
which we discovered new compounds (Chart 1) incorporating
the purine (I), benzimidazole (II), and indole (III) moieties
that are endowed with a significant cytotoxic activity, which is
comparable to that of ellipticine. These compounds exhibited
the ability to intercalate with DNA and in some cases to
inhibit topoisomerase II, properties which were found to
be affected by the presence, the nature, or the spatial orienta-
tion of the pendant side alkyl chains.37-39 We then extended
our interest to the design and synthesis of new heterocycles,
obtained by using as “synthetic tools” the pyridothiopyrane
and the benzothiopyrane moieties, which demonstrated to
be versatile intermediates. Several compounds (IV and V,
Chart 1) evaluated on human tumor cell lines showed a
detectable cytotoxic activity, thus appearing to be interesting
chromophore systems.40,41
These results prompted us to synthesize a series of the new
tetracyclic benzothiopyranoindoles of the general formula VI
(Chart1), bearing a methoxygroup or a chlorine ineither the 3
or the 7 position of the planar moiety; furthermore, the NH
group (position 11) of the indole moiety allowed us the
possibility to insert suitable pendant protonable dialkyl-
aminoalkyl side chains.
The abilityofthe newbenzothiopyranoindole derivatives to
exert an antiproliferative activity was evaluated by means of
an inhibition growth assay on two human tumor cell lines,
human promyelocitic leukemic (HL-60) and human cervix
adenocarcinoma (HeLa) cells. Linear flow dichroism (LD)
experiments were performed to assess the occurrence of a
molecular complex with DNA. Then the thermodynamic
parameters for the binding process were calculated by fluori-
metric titration. To further understand the binding mode of
these molecules to DNA at the atomic level, a molecular
modeling study was carried out using both flexible docking
and ab initio quantum mechanical calculations as suggested
by the biophysical measurements. Finally, the ability of the
compounds to interfere with the activity of both topoisome-
rases I and II was also evaluated.
performed following the synthetic procedure described in
Scheme 1. The preparation of the key starting 7-methoxy-
and7-chloro-3-hydroxymethylenebenzothiopyranonederiva-
tives 2a-b was accomplished from the suitable 7-methoxy- or
7-chloro-2,3-dihydrobenzo[30,20:5,6]thiopyran-4(4H)-one,
following previously described procedures.41 Compounds
2a-b, containing a methine active group, gave the desired 3-
phenylhydrazono intermediates 3a-d, in particularly good
yields, by coupling with the appropriately p-substituted dia-
zonium salt using the Japp-Klingemann reaction.42 Com-
pounds 3a-d were directly converted to the desired indoles
1a-d using the Fischer cyclization, by refluxing in ethanolic
hydrogen chloride solution, with good to high yields. All the
structures proposed for the final compounds agreed with IR
1
and H NMR spectral data, in which an interesting feature
was the low field signal (δ ≈ 12 ppm), which was exchangeable
with D2O and was assigned to the proton of the indole
NH group.
The target 11-N-dialkylaminoalkyl substituted derivatives
1e-v were obtained by reaction of compounds 1a-d with the
appropriate dialkylaminoalkyliodide hydroiodides in anhy-
drous DMF solution in the presence of sodium hydride.
Dialkylaminoalkyliodide hydroiodides were prepared from
the corresponding commercially available dialkylaminoalkyl-
chloride hydrochorides,43 as the latter gave only poor yields in
1
the alkylation reactions. IR and H NMR spectral data of
compounds 1e-v were consistent with the proposed structure.
Finally, for the biological assays, compounds 1e-v were
transformed into their hydrochlorides by treatment with
hydrogen chloride-saturated ethanol solution.
Results and Discussion
Antiproliferative Activity. The ability of new derivatives to
inhibit cell growth was evaluated by means of an in vitro
assay performed on two human tumor cell lines, HeLa
(cervix adenocarcinoma) and HL-60 (promyelocytic
leukemia). The results, expressed as IC50 values, i.e., the
concentration (μM) of compound able to produce 50% cell
death with respect to the control culture, are shown in
Table 1. Ellipticine was used as a reference compound.39
The results obtained demonstrate that the unsubstituted
tetracyclic chromophore (1a-d), independent of the group X
Chemistry
The preparation of the 7- and/or 3-substituted benzo-
[30,20:5,6]thiopyrano[3,2-b]indol-10(11H)-ones 1a-d and of
the 11-dialkylaminoalkyl substituted derivatives 1e-v was

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5431
Scheme 1. Preparation of 3,7-Substituted-11-dialkylaminoalkyl-benzo[30,20:5,6]thiopyrano[3,2-b]indol-10(11H)-ones 1a-va
a Reagents and conditions: (i) AcONa, MeOH; (ii) EtOH HCl, Δ; (iii) RI HI, NaH, DMF,Δ.
The ability of the derivatives characterized by the inser-
tion of an alkylaminoalkyl side chain (1e-v) to exert
Table 1. Chemical Structure and Cell Growth Inhibition in the Presence
of Examined Compounds
a significant antiproliferative effect on both cell lines
is demonstrated by IC50 values ranging from 0.41 to
14.05 μM, thus suggesting the important role played by
the protonable dialkylamino side chains; moreover, the HL-
60 cells are more sensitive to the effect exerted by all
the active compounds than HeLa cells. Furthermore, com-
paring the antiproliferative effect exerted by analogous
compounds having the same substituents X and X0 on
the tetracyclic nucleus, but carrying different dialkyl-
aminoalkyl side chains, it can be affirmed that within
the subseries characterized by a methoxy group or a chlorine
cell lines IC50 (μM)
compd
1a
1b
X
X0
R
HL-60
HeLa
in the 7 (X) and a hydrogen or a methoxy in the 3 (X0)
position, the insertion of the dimethylaminoethyl chain
confers the higher cytotoxic ability (compounds 1e, 1i,
and 1s). In particular, compound 1e, characterized by a
methoxy group and a hydrogen in positions 7 and 3,
respectively, is the most active on HL-60 cells, showing an
IC50 value lower than that of ellipticine; a notable antipro-
liferative ability, comparable to that of the reference com-
pound, is also shown by the 3,7-dimethoxy substituted
compound 1i. On the other hand, the derivatives carrying
the dimethylaminopropyl or diethylaminoalkyl side chains,
show, within each class of analogues, a lower cytotoxic
capacity, thus indicating that an increase in length and steric
hindrance of the side chain seems to be detrimental on the
antiproliferative activity.
Derivatives 1o-r bearing a chlorine in the 3- position and
a 7-methoxy group, displayed similar cytotoxic potencies,
ranging from 0.71 μM (1p) to 1.17 μM (1o), indicating that in
this group of analogues the dependence of cellular effects on
the type of the basic chain appears less emphasized. Thus,
to exert a significant antiproliferative effect, the benzothio-
pyranoindole moiety requires the presence of a basic side
chain, preferably the dimethylaminoethyl one, at the 11-
position of the heterocyclic scaffold.
OCH3
H
H
H
H
H
>20
>20
OCH3 OCH3
OCH3 Cl
>20
>20
>20
>20
>20
>20
1c
1d
Cl
H
H
H
H
H
1e
OCH3
OCH3
OCH3
OCH3
(CH2)2N(CH3)2 0.41 ( 0.08 1.30 ( 0.10
(CH2)2N(C2H5)2 1.80 ( 0.06 2.70 ( 0.05
(CH2)3N(CH3)2 1.90 ( 0.08 2.90 ( 0.3
(CH2)3N(C2H5)2 2.40 ( 0.08 3.90 ( 0.6
1f
1g
1h
1i
OCH3 OCH3 (CH2)2N(CH3)2 0.66 ( 0.17 2.15 ( 0.50
OCH3 OCH3 (CH2)2N(C2H5)2 1.10 ( 0.06 2.30 ( 0.40
OCH3 OCH3 (CH2)3N(CH3)2 1.35 ( 0.10 2.60 ( 0.30
OCH3 OCH3 (CH2)3N(C2H5)2 1.50 ( 0.12 2.50 ( 0.30
OCH3 Cl
OCH3 Cl
OCH3 Cl
OCH3 Cl
1l
1m
1n
1o
(CH2)2N(CH3)2 1.17 ( 0.12 1.82 ( 0.87
(CH2)2N(C2H5)2 0.71 ( 0.09 1.12 ( 0.20
(CH2)3N(CH3)2 1.07 ( 0.24 1.85 ( 0.21
(CH2)3N(C2H5)2 1.02 ( 0.18 1.68 ( 0.25
(CH2)2N(CH3)2 1.31 ( 0.17 1.53 ( 0.24
(CH2)2N(C2H5)2 3.21 ( 0.39 4.33 ( 0.80
(CH2)3N(CH3)2 7.19 ( 0.69 14.05 ( 1.76
(CH2)3N(C2H5)2 2.64 ( 0.60 2.91 ( 0.11
0.66 ( 0.02 0.29 ( 0.01
1p
1q
1r
1s
Cl
Cl
Cl
Cl
H
H
H
H
1t
1u
1v
ellipticinea
a Taken from ref 39.
or X0 inserted in the 7 and/or 3 positions, is ineffective in
inducing cell death.

5432 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Dalla Via et al.
Figure 1. LD spectra for compounds 1a and 1e-h at [drug]/[DNA] = 0.04. [DNA] = 1.9 ꢀ 10-3 M. Four other experiments gave similar
results.
Interaction with DNA. The study of the mechanism of
action responsible for the antiproliferative activity of the new
derivatives prompted us to investigate on the ability of the
heterocyclic nucleus to form a molecular complex with
DNA. To pursue this question, LD experiments were per-
formed in the presence of salmon testes DNA at different
[drug]/[DNA] ratios. The spectra of DNA solutions in the
presence of the structurally related compounds 1a and 1e-h
are reported in Figure 1. The DNA spectrum presents the
typical negative dichroic signal at 260 nm, while in the
presence of new benzothiopyranoindoles, a further negative
signal at higher wavelength occurs. Nevertheless, the inten-
sity of this signal is significantly reduced for 1a, which is
characterized by the absence of the protonable side chain.
The same behavior was also observed for the other com-
pounds, i.e., a small negative induced signal for the unsub-
with derivative 1i, one of the most active in inducing the
antiproliferative effect, and characterized by a constant
intensity in fluorescence emission throughout the duration
of the experiment. Figure 2A shows that the different
aliquots of 1i that lead to the concentration [B], in mmol of
1i per mol of nucleic acid, of a complex with the double helix,
plotted versus the free quantity [F ], in mmol of 1i per mol
of nucleic acid, tend to saturation. Moreover, the binding
data were plotted as a dependence of [B]/[F ] on [B], thus in
the Scatchard data representation framework, and analyzed
by a general thermodynamic model described elsewhere
(Figure 2B).44a,44b The simulated curves (lines) that satisfac-
torily fitted the experimental data (symbols) are typical for
two binding sites, named S1 and S2, both with monocoordi-
nation.44a,44b The obtained binding results are summarized
in Table 2. The total binding site concentration (Bmax) varies
significantly between the different nucleic acids and, inter-
estingly, points out a certain preference of the benzothio-
pyranoindole ligand for the thymine-adenine base pair.
Moreover, the 1i distribution between S1 and S2 sites is
very different. Indeed, the maximum binding concentration
of 1i on the S1 site, Bmax1, is notably lower with respect to that
of the S2 site, Bmax2, and does not show any significant
variation depending on the base pair composition. In con-
trast, Bmax2 varies according to base composition, in agree-
ment with the behavior evidenced for Bmax. These differences
in binding capacity of 1i between S1 and S2, suggest different
roles for them and the contribution of two binding events to
the overall binding process. Furthermore, taking into con-
sideration that 1i is characterized by two main structural
motifs, consisting in a heteropolycyclic nucleus and a basic
side chain, it could be hypothesized that the interaction
between 1i and the nucleic acid occurs owing to two distinct
processes. The first consists in electrostatic interactions that
stituted benzothiopyranoindoles (1b-d) and
a large
significant signal for the corresponding derivatives carrying
the dialkylaminoalkyl side chains (1i-v) (spectra not
shown). These results suggest that the tetracyclic chromo-
phore may insert between base pairs and that this capacity
becomes significantly more pronounced if a dialkylami-
noalkyl chain is inserted. It is reasonable that the presence
of a basic nitrogen, protonable at physiological pH, facil-
itates the interaction between the nucleic acid and the
charged compound, thus making the intercalative process
more effective.
Binding Studies. To investigate the interaction of the new
benzothiopyranoindoles with nucleic acid base pairs more in
depth, fluorimetric titrations in the presence of DNA
characterized by a different base composition and of syn-
thetic polyDNAs, poly[dA-dT]poly[dA-dT], and poly[dG-
dC]poly[dG-dC] were performed. On the basis of prelimin-
ary data, the fluorimetric determinations were carried out

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5433
take place between the side chain of 1i and the polyanion
skeleton of the nucleic acid, and the second process deals
with an intercalative complexation, mediated by weak inter-
actions. In more detail, the dependence of Bmax2 on base
composition suggests the participation of this site at the
intercalative event, while the independence of Bmax1 could
indicate that S1 is the site involved in the ion pair interac-
tions. The calculated values for the association constants, k1
and k2, corresponding to S1 and S2 sites, respectively,
reinforced this assumption. Indeed, the significantly higher
values of k1 with respect to k2 could reflect the involvement of
strong and weak binding forces for the binding to S1 and S2
sites, respectively.
Molecular Modeling. To rationalize the biological results
obtained with this new series of benzothiopyranoindoles, a
molecular modeling study was carried out. Insight into the
mode of binding of derivatives 1e, 1i, and 1p to DNA and the
stability of selected DNA duplex-intercalator complexes was
gained through molecular docking and ab initio quan-
tum mechanical calculations using the DNA hexamer
d(TATATA)2. This sequence was selected according to the
experimental binding preference demonstrated for this type of
compound in the fluorimetric determinations reported above.
The DNA hexamer was obtained by molecular replacement
using the crystal structure of deglycosylated pepleomycin
bound to the self-complementary hexamer d(CGTACG)2 as
a starting model (PDB code 1a01)45 with the d(CpG) base
pairs (underlined) replaced with d(TpA) ones. Then pepleo-
mycin was removed and the central d(TpA) intercalation site
was utilized for docking simulations. Docking was carried out
using the automated docking program AutoDock 4.0,46
which allows torsional flexibility in the ligand and incorpo-
rates an efficient Lamarckian genetic search algorithm along
with an empirical free energy function.
Out of a total of 200 independent docking runs, AutoDock
provided well-clustered results, which were chosen according
to the following criteria: (i) the most favorable docking
energy (ΔGbind), (ii) the highest frequency of occurrence
(focc), and (iii) the formation of a direct intermolecular
interaction between the alkyl-ammonium side chain of the
ligand and DNA, as suggested by the SAR data. Docking
results are summarized in Table 3, and a graphical repre-
sentation of the binding modes of the most structurally
representative derivatives 1e and 1i is given in Figure 3.
Two possible binding poses were found for the most active
compounds 1e and 1i. As depicted in Figure 3, the planar
benzothiopyranoindole moiety was sandwiched between
adjacent A-T base pairs, while the charged side chain could
sit equally well in major or minor groove. In solution 1
(ΔGbind = -9.9 kcal/mol, focc= 22/200 for 1e; ΔGbind
=
-10.4 kcal/mol, focc= 65/200 for 1i), the protonated
Table 3. Results of 200 Independent Docking Runs for Compounds 1e,
1i, 1p, and Corresponding Binding Energies as Calculated by ab Initio
Methods
molecular docking
ab initio calculations
compd Ntot clusterb focc.
ΔGbind
ΔEbind ΔEbind (PCM)
a
c
d
1e
1i
10
8
1
2
1
2
1
22/200
22/200
-9.9
-9.9
-162.84
-151.11
-49.70
-65.40
-53.93
-67.04
-55.70
65/200 -10.4 -163.87
55/200 -10.2 -184.58
39/200 -10.0 -153.98
1p
16
a Ntot is the total number of clusters. b The assessment of conforma-
tional cluster sizes was determined as a function of the root-mean-square
deviation (rmsd) of 0.3 A between conformers. c The number of results
Figure 2. (A) Binding of 1i to nucleic acids as a function of F
quantity. (B) Binding analysis with the thermodynamic treatment of
the Scatchard method. The experimental data (symbols) were fitted
using the equation shown in Experimental Section, and the theore-
tical curves (lines) indicate the result of the fitting.
contained in the clusters is given by the frequency of occurrence, focc
.
d ΔGbind is the estimated free energy of binding for the clusters. Energies
are in kcal/mol.
Table 2. Binding Parameters for Compound 1i
Bmax
(mmol/mol nucleic acid)
Bmax1
(mmol/mol nucleic acid)
Bmax2
(mmol/mol nucleic acid)
nucleic acid
k1
k2
poly[dA-dT]poly[dA-dT]
Clostridium perfringens DNA
salmon testes DNA
93.93(2)a
41.47(3)
68.89(4)
14.81(3)
18.32(6)
1.49(1)
1.57(4)
1.72(2)
1.74(2)
1.45(2)
92.44(2)
39.89(3)
67.17(3)
13.06(4)
16.87(3)
62.98(3)
31.40(6)
8.22(4)
7.42(2)
NDb
0.0036(4)
0.0076(3)
0.0058(2)
0.013(3)
Mycrococcus lysodeikticus DNA
poly[dG-dC]poly[dG-dC]
0.0157(2)
a Standard deviations in the least significant digits are given in parentheses. b Fitting not satisfactory. Not detected.

5434 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Dalla Via et al.
Figure 3. Intercalation site of the two complexes between compounds 1e (orange, (a) and (b)) and 1i (magenta, (c) and (d)) and hexamer
d(TATATA)2 viewed in a projection plane orthogonal to the helix axis. Left: complexes with the protonated dimethylammonium group in the
major groove ((a) and (c)). Right: complexes with the protonated dimethylammonium group in the minor groove ((b) and (d)). Nonpolar
hydrogens were removed for clarity. H-bonds are represented with yellow dashed lines.
dimethylammonium group of the side chain sat in the DNA
major groove, participating in an ionic interaction with
the negatively charged phosphate backbone of adenine
A10 (Figure 3a,c). On the contrary, in solution 2 (ΔGbind
=
-9.9 kcal/mol, focc= 22/200 for 1e; ΔGbind=-10.2 kcal/mol,
focc= 55/200 for 1i), the charged side chain projected into the
minor groove, establishing H-bonds with both the deoxyr-
ibose O40 and the phosphate backbone of adenine A10
(Figure 3b,d). Notably, in the latter case, a better super-
position of the ligand benzothiopyranoindole moiety with
the DNA base pairs is observed, suggesting stronger π-π
stacking interactions for this binding mode. Such a favorable
arrangement seems to be determined by the length of
the alkylammonium side chain, which is firmly anchored to
the phosphate backbone in both solutions and drives the
extended planar moiety into the DNA double helix.
Figure 4. Binding orientation of compound 1e in the DNA inter-
calation site, before (magenta) and after (cyano) energy-minimiza-
tion. H-bonds are represented with yellow dashed lines.
A well-clustered binding pose was found by AutoDock for
compound 1p (focc = 39/200; ΔGbind = -10.0 kcal/mol),
which strongly resembled the solution 2 found for 1e and
1i (Figure 3b,d). The ligand was found to intercalate between
the d(TpA) base pairs with the side chain protruding into the
DNA minor groove, where the protonated diethylamino
group engaged in H-bonding interactions with both
the deoxyribose O40 and the phosphate backbone of
adenine A10. It is of note that the replacement of dimethyl-
ammonium by the diethylammonium group, as well as the
introduction of a chlorine atom in X0, does not lead to
significant changes in ΔGbind when compared with 1e and 1i.
To estimate the binding energy of the DNA-ligand com-
plexes more accurately, as well as the relative contribution of
specific interactions (π-π stacking, H-bonding, etc.) and the
effects of substituents, we performed ab initio quantum
mechanical calculations focusing on the most active com-
pounds 1e, 1i, and 1p. A full molecular mechanics minimiza-
tion, keeping all degrees of freedom, of the five complex
structures considered (two for 1e and 1i, and one for 1p) was
carried out with the AMBER 9 software package,47 model-
ing the DNA hexamer and the ligands with the AMBER9948
and GAFF49 force fields, respectively. Solvent effects were
also included using the generalized Born/surface area (GB/
SA) model, as implemented in AMBER. In this step, the
DNA-ligand structures underwent minor but still signifi-
cant, rearrangements with respect to the docking structures.
In particular, the aromatic moieties of the DNA base pairs
assumed a more planar conformation with respect to the
ligands, compatible with stronger π-π stacking interactions
and an overall better match of the ligands inside the DNA
strands, as shown in Figure 4. In the second step, we
performed quantum mechanical calculations on a relatively
large fragment of the previously optimized DNA-ligand
complexes, including the whole ligand and the two proximal
nucleic acid base pairs with the connecting sugar rings and
phosphate groups. Hydrogen atoms were added to cap the
terminating oxygen atoms on the deoxyribose. On the basis
of the above considerations, for such calculations we decided
to rely on a well-trusted and effective density functional
theory (DFT) based method, namely B3LYP,50a,50b using a
medium size basis set, 6-31þG(d,p). Because of the known
underestimation of the dispersion forces by DFT, a long-
range correction term was included according to the scheme
proposed by Grimme51 and recently implemented52 in a
modified version of the Gaussian03 software package.53

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5435
Indeed, such correction has shown significant improvements
in the treatment of aromatic ring interactions.52 Finally, the
effects of the aqueous environment were included via an
implicit solvent model, the conductor-like version54a,54b of
the polarizable continuum model (PCM).55 Results on the
binding energy, ΔEbind, where ΔEbind=ΔEcomplex - ΔEDNA
- ΔEligand of the complexes are reported in Table 3.
As expected, we noted that the solvent effect decreases
the absolute binding energy by a significant amount (see
ΔEbind PCM), changing the relative order of the complex
stability. Not surprisingly, the complexes between DNA and
solution 2 of both 1e and 1i were the most stable ones. As
noted above, we observed a strong H-bond between the
dialkyl-ammonium chain and the negatively charged phos-
phate group and, more importantly, more favorable π-π
stacking interactions between the extended planar moiety of
the ligands and the DNA bases, as shown in Figure 3. On the
contrary, 1p, which has a bulkier side chain, possessed a
weaker interaction of the ammonium group with the DNA
phosphate group. Therefore, the present results consistently
support the same binding mode as the preferred one for the
DNA intercalation of the most active compounds considered
in this study, 1e and 1i.
To evaluate the possible contribution of other terms to the
total binding energy with respect to the usual intermolecular
interactions, e.g., π-π stacking, H-bonding, etc., we com-
puted the charge-transfer occurring between the ligand and
the DNA base pairs. Considering the complex between
solution 2 of 1e and DNA, we obtained a net change of the
total electrostatic charge of the ligand of less than 0.07 au
in going from vacuum to the intercalation site, thus showing
a negligible charge transfer, as found in previous computa-
tional studies of other intercalating compounds.56 To
proceed further, the fundamental role of the dialkyl-ammo-
nium side chain was analyzed in terms of the decrease
of the binding energy, ΔEbind, after removing the cationic
headgroup in the DNA/1e complex. By changing
the -(CH2)2NHþ(CH3)2 side chain to a neutral and apolar
terminal group (CH2CH3), we obtained a destabilization
energy of the complex of about 24 kcal/mol, which amounts
to almost 40% of the original binding energy. At the same
time, this result confirms that the predominant contribution
to the DNA/ligand binding is represented by the extended
π-π stacking interactions (about 60%). Note that the
positively charged ammonium group not only significantly
stabilizes the complex but also that it might play an impor-
tant role in anchoring the ligand to the DNA duplex at
the moment of complex formation, making the insertion of
the planar moiety into the DNA intercalation site easier.
Indeed, as observed in Table 1, when the dialkyl-ammonium
side chain is elongated and/or becomes bulkier in compari-
son with 1e and 1i, the cytotoxic activity decreases in every
case, with the exception of 1p. Remarkably, this seems to be
consistent with the proposed DNA-ligand binding mode,
where the side chain specifically binds to the phosphate
group, considering that a longer and more sterically hindered
chain makes the present interaction less favorable.
solution 2 of 1i and DNA (X0=CH3O, ΔEbind=-67.0 kcal/
mol; X0=Cl, ΔEbind=-66.6 kcal/mol).
Effect on Topoisomerase Activity. DNA topoisomerases
have been shown to be the molecular target of many antic-
ancer drugs, including known DNA intercalators.57 Thus,
the ability of new benzothiopyranoindole derivatives to form
an intercalative complex with DNA suggested that their
antiproliferative effect might be related to the interference
with nuclear enzymes involved in DNA processing, such as
topoisomerases.15
Figure 5A shows the effect of 1i and 1e on the relaxation of
plasmid pBR322 DNA, mediated by topoisomerase II. The
supercoiled DNA (DNA) was converted to relaxed forms by
the enzyme (Topo II). The inhibition by 1i and 1e on the
relaxation activity was demonstrated by the appearance of
supercoiled DNA and by a concurrent decrease in relaxed
products in the presence of the enzyme. In detail, for both
derivatives a dose-dependent effect is evident at concentra-
tion ranging from 1 to 5 μM; interestingly, the inhibition
induced at the higher concentration (5 μM) is similar to that
obtained by 8 μM m-AMSA (m-AMSA), which was used as
reference drug. A comparison between 1i and 1e showed a
lower inhibitory effect on enzyme activity for the first
derivative, which was especially evident at 2.5 μM concen-
tration. This behavior appears in accordance with the anti-
proliferative data reported in Table 1. The results of Table 1
further suggested a crucial contribution to the cytotoxicity of
the dialkylaminoalkyl side chain and in particular pointed
out to the unfavorable role of its length and hindrance. This
effect is particularly clear within the 1e-h group of deriva-
tives, for both cell lines tested. On the basis of these con-
siderations, it appeared interesting to evaluate the effects of
1e-h, along with the parent compound 1a, on topoisomerase
II relaxation ability (Figure 5B). The occurrence of super-
coiled DNA, indicative of inhibition of the enzyme activity,
decreases from 1e to 1h, in agreement with the cellular effect.
Regarding the unsubstituted 1a, which showed no cellular
effect up to 20 μM concentration, it stimulates a very low
formation of supercoiled DNA like 1h, but in contrast it
shows the maintenance of the relaxed form as was observed
with the enzyme alone. This result could be a consequence of
the difference in their abilities to interact with DNA, which is
negligible for 1a and notable for 1h, as evidenced by the LD
spectra showed in Figure 1.
Many intercalative agents, such as adriamycin, m-AMSA,
and mitoxantrone, which are able to stabilize a biological
intermediate in topoisomerase II activity, also known as
cleavage complex,20,57,58 into a lethal one, are called topo-
isomerase II poisons. The occurrence of the cleavage com-
plex can be demonstrated experimentally by the enzyme-
dependent formation of linear DNA from supercoiled DNA.
Figure 6A shows a cleavage complex assay performed in the
presence of increasing concentrations of the most active
derivative 1e and 8 μM m-AMSA, which was used as a
reference compound. The supercoiled pBR322 DNA (DNA
lane) was relaxed by the catalytic activity of the enzyme
(Topo II), and as expected, m-AMSA, a well-known topo-
isomerase II poison, induces the formation of a significant
amount of linear DNA. The benzothiopyranoindole deriva-
tive 1e also appears able to stabilize the formation of
cleavage complexes, although at all concentrations tested
(up to 20 μM) it induces less linear DNA formation than
m-AMSA. Experiments performed with 1i showed a very
weak capacity to form linear DNA (results not shown). The
The effect of the substituent group in position X was
analyzed by replacing the methoxy group with a hydrogen
and a chlorine atom. Results showed little change in ΔEbind
in both cases (X = H, ΔEbind = -62.7 kcal/mol; X = Cl,
ΔEbind=-66.0 kcal/mol). Similarly, we observed a negligible
difference in the binding energy by replacing the methoxy
group in position X0 with chlorine in the complex between

5436 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Dalla Via et al.
Figure 5. Effect on relaxation of supercoiled pBR322 DNA of human recombinant topoisomerase II. (A) Supercoiled DNA (DNA) was
incubated with topoisomerase II in the absence (Topo II) and presence of 1i and 1e at indicated concentration (μM). Eight micromolar
m-AMSA was used as reference. (B) Supercoiled DNA (DNA) was incubated with topoisomerase II in the absence (Topo II) and presence of 1a
and 1e-h at 2.5 μM concentration; 8 μM m-AMSA was used as reference. The images are representative of three experiments.
Figure 6. Effect of compound 1e on the stabilization of covalent DNA-topoisomerase II complex. (A) Supercoiled pBR322 DNA (DNA) was
incubated as reported in Experimental Section with topoisomerase II in the absence (Topo II) and presence of 1e at indicated concentration
(μM); 8 μM m-AMSA was used as reference. DNA samples were separated by electrophoresis on agarose gels containing 0.5 μg/mL ethidium
bromide. (B) Experimental conditions as in (A), except where indicated (ATP). The images are representative of four experiments.
poisoning effect shown in Figure 6A appears quite weak with
respect to the inhibition on topoisomerase II relaxation
ability (Figure 5A), nevertheless, the same experiment, per-
formed in the absence of ATP, to avoid any relaxation

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5437
Conclusions
The synthesis of new benzothiopyranoindole derivatives
carrying a dialkylaminoalkyl side group and exhibiting an
antiproliferative effect at low micromolar concentrations on
HeLa and HL-60 human cell lines was described. In parti-
cular, the chromophore scaffold itself (1a-d) does not possess
any significant antiproliferative effect, even in the presence of
substituents, such as a methoxy group or a chlorine atom in
the 3 and/or 7 positions of the system; on the contrary, the
presence of the dialkylaminoalkyl side chains, inserted at the
11-position on the indole nitrogen, seems to be required for
the cytotoxicity (1e-v). In particular, the most notable effect
was shown by the compound carrying a dimethylaminoethyl
side chain, along with a methoxy group in the 7 position
and hydrogen in the 3 position (1e), which is able to exert
a cytotoxic effect higher than that of ellipticine on HL-60
cell line.
Figure 7. Effect of compound 1e on relaxation of supercoiled
pBR322 DNA by human recombinant topoisomerase I. Super-
coiled DNA (DNA) was incubated with topoisomerase I in the
absence (Topo I) and presence of 1e at indicated concentrations
(μM). The image is representative of three experiments.
LD studies revealed that compounds 1e-v are able to form
a complex with DNA, which is consistent with an intercalative
binding mode. Fluorimetric titrations in the presence of DNA
characterized by different base compositions and synthetic
polyDNAs interestingly, demonstrated a high sequence spe-
cificity of the benzothiopyranoindoles for AT rich regions.
These studies allowed us to identify two binding sites, S1 and
S2. The binding parameters permit the hypothesis that S1
participates in a binding event independent of base composi-
tion and mediated by the chargedsidechain, while S2 accounts
for an intercalative complexation to AT rich regions,
mediated by weak interactions. Molecular docking and high
level quantum-mechanical calculations, including implicit
solvent and a proper treatment of dispersion forces, allowed
a further understanding at the molecular level of which
specific interactions provide an optimal intercalative model,
in agreement with experimental data, for the ligand-DNA
complexes of the three most active compounds 1e, 1i, and 1p.
This model showed a strong ion pair interaction between the
protonated dimethylaminoethyl side chain and the phosphate
backbone (about 40% of ΔEbind), with the side chain sitting in
the DNA minor groove, and an extended π-π stacking
interaction between the benzothiopyranoindole moiety and
the two adjacent DNA base pairs (about 60% of ΔEbind).
Hence, the crucial presence of the dialkylaminoalkyl side
chain for achieving significant cytotoxic effects was corro-
borated by the present model, which also suggests thatthe side
chain may play the important role of favoring the complex
formation by anchoring the ligand firmly to the DNA during
the insertion of the benzothiopyranoindole moiety into the
nucleic acid strands.
activity, confirmed the capacity of 1e to induce the formation of
linear DNA (Figure 6B). The topoisomerase II-mediated
cleavage was further demonstrated by cleavage assay per-
formed with end-labeled DNA in the presence of 1e at 5 μM.
By increasing the concentrations of 1e up to 200 μM, a
concurrent inhibition of the cleavage is observed and this
behavior, typical of DNA intercalators,59 confirms again the
capacity of the new derivative to form an effective intercalative
complex with DNA (see Supporting Information Figure S1).
To investigate the role of topoisomerase II on the anti-
proliferative effect exerted by 1e, cell growth inhibition assay
was performed by using HL-60/MX2 cells. These cells dis-
play altered topoisomerase II catalytic activity and reduced
levels of topoisomerase II R and β and are approximately
30-fold less sensitive to m-AMSA and mitoxantrone than the
HL-60 parental cells.60 HL-60/MX2 cells incubated in the
presence of the most active 1e showed IC50 values ranging
from 2.35 to 2.65 μM, thus indicating a degree of resistance,
i.e., the ratio of the IC50 values calculated for resistant and
sensitive cells (HL-60/MX2:HL-60), of about 6-fold.
The rather weak topoisomerase II poisoning effect, along
with the obtained degree of resistance, suggest that, in
addition to the inhibition on the topoisomerase II activity,
other molecular events could contribute to the notable
cytotoxicity induced by the new benzothiopyranoindoles.
In particular, considering the significant intercalative capa-
city of the new compounds, a further interference with other
DNA-related enzymes could occur.
On the basis of the above consideration and in view of the
structural similarities of the investigated compounds with
some dual topoisomerase I/II inhibitors, in particular the
benzopyridoindole derivative Intoplicine,61 the effect on the
nuclear enzyme topoisomerase I was also assayed. Figure 7
shows the relaxation of supercoiled pBR322 DNA catalyzed
by the topoisomerase I, in the presence of increasing
amounts of 1e. The addition of the benzothiopyranoindole
derivative 1e induces a concentration-dependent inhibition
of the relaxation capacity and the effect becomes evident at
10 μM. A comparison between this result and that from
topoisomerase II studies (shown in Figure 5) indicates a
certain preference of action toward topoisomerase II. In-
deed, while 5 μM of 1e completely inhibits the relaxation
catalyzed by this latter enzyme (Figure 5A), 1 order of
magnitude higher of compound has to be used to evoke a
comparable effect on topoisomerase I (Figure 7).
The ability of the new 11-substituted benzothiopyranoin-
doles to intercalate between DNA base pairs prompted
further investigations into possible interference with the ac-
tivity of the nuclear enzymes topoisomerases I and II. The
derivatives showed the capacity to inhibit the relaxation of
supercoiled plasmid DNA mediated by topoisomerase II in a
dose-dependent manner, and interestingly, this ability ap-
peared in accordance with the antiproliferative data. More-
over, for the most cytotoxic derivative 1e,
a weak
topoisomerase II poisoning effect and the capacity to inhibit
the topoisomerase I-mediated DNA relaxation, was also
demonstrated.
In conclusion, the ability to intercalate effectively between AT
rich regions of nucleic acids, together with a noteworthy
cytotoxic effect, render this new 11-substituted heteropolycyclic

5438 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Dalla Via et al.
moiety a very interesting structure inside the field of DNA-
targeted antiproliferative agents. Further modifications of both
the 7 or/and 3 position of the tetracyclic chromophore and of the
geometry of the distal end of the protonable side chains could
improve the biological properties, thus allowing the develop-
ment of new potential antitumor drugs.
penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphoter-
icin B (Sigma Chemical Co.) were added to the media. The cells
were cultured at 37 ꢀC in a moist atmosphere of 5% carbon
dioxide in air.
HL-60 and HL-60/MX2 cells (4 ꢀ 104) were seeded into each
well of a 24-well cell culture plate. After incubation for 24 h,
various concentrations of the test agents were added to
the complete medium and incubated for a further 72 h. HeLa
(4 ꢀ 104) cells were seeded into each well of a 24-well cell culture
plate. After incubation for 24 h, the medium was replaced with
an equal volume of fresh medium, and various concentrations of
the test agents were added. The cells were then incubated in
standard conditions for a further 72 h.
Experimental Section
Chemistry. Melting points were determined using a Reichert
Kofler hot-stage apparatus and are uncorrected. Infrared spec-
tra (IR) were obtained on a NICOLET/AVATAR, 360 FT
spectrophotometer as Nujol mulls. Nuclear magnetic resonance
spectra (1H NMR) were recorded on a Varian Gemini 200
spectrometer in DMSO-d6 solution using TMS as the internal
standard. Coupling constants are given in Hz. Magnesium
sulfate was always used as the drying agent. Evaporations were
made in vacuo (rotating evaporator). Analytical thin-layer
chromatography (TLC) was carried out on Merck 0.2 mm
precoated silica gel aluminum sheets (60 F-254). Reagents,
starting materials, and solvents were purchased from commer-
cial suppliers and used as received. Combustion analyses on
target compounds were performed by our Analytical Labora-
tory in Pisa. All compounds showed g95% purity.
A trypan blue assay was performed to determine cell viability.
Cytotoxicity data were expressed as IC50 values, i.e., the con-
centration of the test agent inducing 50% reduction in cell
number compared with control cultures.
The degree of resistance is expressed as the ratio of the IC50
values calculated for resistant and sensitive cells (HL-60/MX2:
HL-60).
Nucleic Acids. Salmon testes DNA (cat. D-1626, AT% about
60) was purchased from Sigma Chemical Company. Its hypo-
chromicity, determined according to Marmur and Doty,62 was
over 35%. DNA from Micrococcus lysodeikticus (cat. D-8259,
AT% about 30%) and Clostridium perfringens (cat. D-1760,
General Procedure for the Synthesis of 7-Methoxy-2,3-dihy-
dro-3-p-substituted-phenylhydrazonobenzo[30,20:5,6]thiopyran-
4(4H)-ones 3a-c and 7-Chloro derivative 3d. To a solution of
4.5 mmol of compound 2a or 2b in 30 mL of methanol was added
an aqueous saturated solution of 14.0 mmol of sodium acetate.
After cooling at 0 ꢀC, a solution of the suitable diazonium salt,
obtained from the appropriately p-substituted aniline in 18%
hydrochloridric acid and sodium nitrite, was added dropwise in
slight excess (6.0 mmol). An orange precipitate was immediately
formed, and the mixture was stirred for 30 min at room
temperature. The solid was collected and washed with water
to give crude compounds 3a-d, whose purity was assessed by
TLC analysis (Supporting Information Tables 1-2 ).
General Procedure for the Synthesis of 7-Methoxy-3-substi-
tuted Benzo[30,20:5,6]thiopyrano[3,2-b]indol-10(11H)-ones 1a-c
and 7-Chloro-Benzo[30,20:5,6]thiopyrano[3,2-b]indol-10(11H)-
one 1d. A solution of 4 mmol of the suitable p-substituted
phenylhydrazono derivative 3a-d in 20 mL of ethanolic hydro-
gen chloride solution was refluxed for 20 min. After cooling the
yellow/light-brown precipitate was collected and washed with
ethanol to give crude indoles 1a-d, which were purified by
crystallization (Supporting Information Tables 3-4).
General Procedure for the Synthesis of 3,7-Substituted 11-
Dialkylaminoalkyl-benzo[30,20:5,6]thiopyrano[3,2-b]indol-10(11H)-
ones 1e-v. 0.70 mmol of the appropriate compound 1a-d was
added in small portions to a stirred suspension of sodium
hydride in 60% dispersion in mineral oil (0.104 g, 2.60 mmol)
in 10 mL of anhydrous DMF, under nitrogen atmosphere. The
reaction mixture was stirred at room temperature for 2 h and
then supplemented with 1.03 mmol of the appropriate dialky-
laminoalkyl iodide hydroiodide and left at room temperature
for 24 h; then the mixture was heated at 100 ꢀC for 12-24 h
(TLC analysis). After cooling, the reaction mixture was diluted
with water and left to stand at room temperature over-
night. The crude products precipitated were collected and puri-
fied by crystallization from DMF (Supporting Information
Tables 5-12).
AT% about 70), poly[dA-dT] poly[dA-dT] (cat. D-0883), and
3
poly[dG-dC] poly[dG-dC] (cat. D-9389) also came from Sigma.
3
pBR322 DNA was purchased from Fermentas Life Sciences.
Linear Flow Dichroism. LD measurements were performed on
a Jasco J500A circular dichroism spectropolarimeter converted
for LD and equipped with an IBM PC and a Jasco J interface.
Linear dichroism is defined as:
LD ¼ A -A^ðλÞ
ðλÞ
ðλÞ
where A and A^ correspond to the absorbances of the sample
when polarized light is oriented parallel or perpendicular to the
flow direction, respectively. The orientation is produced by a
device designed by Wada and Kozawa63 at a shear gradient of
500-700 rpm and each spectrum was accumulated four times.
A solution of salmon testes DNA (1.9 ꢀ 10-3 M) in ETN
buffer (containing 10 mM TRIS, 10 mM NaCl, and 1 mM
EDTA, pH=7) was used. Spectra were recorded at 25 ꢀC at
different [drug]/[DNA] ratios.
Fluorimetric Determinations. Fluorescence spectra were re-
corded on a Jasco FP-6500 fluorescence spectrophotometer
equipped with a stirred Peltier thermostatted cell holder at
λ
ecc=341 nm. Experiments were performed by addition of small
amounts of free ligand to a solution containing nucleic acid-
ligand complex at [nucleic acid]/[drug]=700. The fluorescence
emission of the complex is significantly higher with respect to
that of free ligand. The concentration of the free ligand was the
same as that of the complex: in this way different [nucleic
acid]/[drug] were obtained at a constant ligand concentration.
All measurements were carried out in ETN buffer at 25 ꢀC. The
amounts of bound and free ligand were determined from emi-
ssion reading at a fixed wavelength, corresponding to the maxi-
mum of emission, in accordance with the equations:
fi ¼ ðFi -F0Þ=ðFmax -F0Þ
B ¼ fi½compoundꢁ
Inhibition Growth Assay. HL-60 (human myeloid leukemic
cells) and HeLa (human cervix adenocarcinoma cells) were
grown in RPMI 1640 (Sigma Chemical Co.) supplemented with
15% heat-inactivated fetal calf serum (Biological Industries)
and in Nutrient Mixture F-12 [HAM] (Sigma Chemical Co.)
supplemented with 10% heat-inactivated fetal calf serum
(Biological Industries), respectively. HL-60/MX2 cells
(ATCC, Manassas, Va) were grown in RPMI 1640 supplemen-
ted with 10% heat-inactivated fetal calf serum. 100 U/mL
Bn ¼ B=½nucleic acidꢁ
F ¼ ð1 -fiÞ½compoundꢁ
Fn ¼ F=½nucleic acidꢁ
where Fi is the ith emission fluorescence, F0 is the emi-
ssion fluorescence of the free compound, Fmax is the emission

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17 5439
fluorescence of the complex, B is the ith concentration of bound,
Bn is the ith concentration of bound normalized to the nucleic
acid concentration, F is the ith concentration of free, and Fn is
the ith concentration of free normalized to the nuclei acid
concentration.
centered on the central d(TpA) intercalation site, with 0.375 A
grid-point spacing applying a standard protocol with an initial
population of 150 randomly placed individuals and a maximum
number of 2.5 ꢀ 107 energy evaluations per run. All other
parameters were maintained at their default settings. A total
of 200 binding conformations were carried out for each ligand
generated by the program and then clusterized by means
of AutoDock Tools.68a,68b Cutoffs for clustering were set at
0.8 A rmsd and were represented by the result with the most
favorable free energy binding (ΔGbind). The fitness score is taken
as the negative of the sum of the energy terms so that larger
fitness scores indicate a better binding. The docked structures
were analyzed using AutoDock Tools for hydrogen bonding
and hydrophobic interactions.
Binding Parameters. The binding data were evaluated by
applying a thermodynamic treatment of ligand-receptor inter-
actions.44 Scatchard analysis was performed using the following
equation:
"
#
s
X
½Bꢁ
½Fꢁ
1
¼
f½Bmax ,iꢁ -½Biꢁg
þεiðFÞ
Ki,1ðtÞ
i ¼1
where
Molecular Mechanics Optimizations and ab Initio Calcula-
tions. Selected complexes of the DNA hexamer with the 1e,
1i, and 1p were further optimized at molecular mechanics
(MM) level by using the AMBER 9 software package,47
including solvent effects via the generalized Born/surface area
(GB/SA) model. The general AMBER force field (GAFF),49
specifically developed for rational drug design, was employed
to model the ligands and, for consistency, the correspond-
ing atomic charges were computed by using the quantum
mechanical RESP charge method at HF/6-31G* level.
The DNA fragment was modeled according to the AMBER-
99 force field and molecular optimizations were per-
formed with the steepest descent method, switching to the
conjugate gradient method every 100 cycles. Once optimized
at MM level, full ab initio calculations were carried out
at B3LYP/6-31þG(d,p) level of the complexes formed be-
tween the intercalating ligands and the two proximal nucleic
acid base pairs, including the chemically relevant deoxyribose
and phosphate groups (see Figure 4). Solvent effects were
eventually included using the conductor-like version54a,54b of
the polarizable continuum model.55 Because of the generally
poor description of van der Waals interactions by DFT based
methods, we included long-range corrections of the London
dispersion forces according to the scheme proposed by
Grimme.51 All quantum mechanical calculations were per-
formed with a modified version52 of the Gaussian03 software
package.53
Topoisomerases-Mediated DNA Relaxation. Supercoiled
pBR322 plasmid DNA (0.25 μg, Fermentas Life Sciences) was
incubated with 1U topoisomerase II (human recombinant to-
poisomerase II R, USB) or 2U topoisomerase I (calf thymus
topoisomerase I, USB) and the test compounds as indicated for
60 min at 37 ꢀC in 20 μL of reaction buffer.
Reactions were stopped by adding 4 μL of stop buffer (5%
sodium dodecyl sulfate (SDS), 0.125% bromophenol blue, and
25% glycerol) and 50 μg/mL proteinase K (Sigma) and incubat-
ing for a further 30 min at 37 ꢀC. The samples were separated
by electrophoresis on a 1% agarose gel at room temperature.
The gels were stained with ethidium bromide 1 μg/mL in
TAE buffer (0.04 M Tris-acetate and 0.001 M EDTA), transil-
luminated by UV light, and fluorescence emission was visual-
ized by a CCD camera coupled to a Bio-Rad Gel Doc XR
apparatus.
ni
k -1
X
½Fꢁ
εiðFÞ ¼
k
k ¼2
P Ki,jðtÞ
j ¼1
The quantity εi(F) represents an appropriate measure of
the extent of multiple coordination on the ith site. [Bmax,i] is
the maximum concentration of the ith site that may be bound by
the ligand, [Bi] is the concentration of ith sites bound by the
ligand, [Bmax] is the maximum receptor-bound ligand concen-
tration, [F] is the free ligand concentration, Ki,j(t) is the affinity
constant of the ligand for the ith site, j is the occupancy number,
and t is time. Fitting was performed using Igor pro 5.0.4.8
program.
Computational Chemistry. Molecular modeling and graphics
manipulations were performedusingthe Maestro GraphicalUser
Interface version 8.0.314 for Linux operating systems,64a,64b
running it on a quad-core QX 6700 processor (8GB RAM).
Model building and energy minimizations of compounds 1e, 1i,
and 1p were realized by employing the MacroModel 9.0 module
of Maestro selecting the OPLS_2005 force field.65a,65b Pymol
version 0.966 were used to create Figures 3 and 4.
Ligand and DNA Setup. The core structure of compounds 1e,
1i, and 1p were retrieved from the Cambridge Structural Data-
base (CSD)67 and modified by using standard bond lengths and
bond angles of the fragment library of Maestro. Geometry
ꢀ
optimizations were realized with the Polak-Ribiere conjugate
gradient (PRCG) method until a convergence to the gradient
threshold of 0.05 kJ/(mol A). The atomic charges were com-
puted using the OPLS_2005 force field. The compounds were
considered in their protonated form.
The d(TATATA)2 hexamer was built by molecular replace-
ment using as a starting model the crystal structure of deglyco-
sylated pepleomycin bound to the self-complementary hexamer
d(CGTACG)2 (PDB code 1a01).45 Pepleomycin was then re-
moved and the central d(TpA) intercalation site was utilized for
docking experiments.
Docking Studies. Docking simulations for 1e, 1i, and 1p were
carried out by means of the AutoDock software (version 4.0).68a,68b
To search the low-energy docked conformation of the ligand on
the DNA, the empirical free energy function and the Lamarckian
genetic algorithm were used. In this approach, the ligand per-
forms a random walk around the static nucleic acid. At each time
step, the ligand is moved by small increments in global trans-
lation and orientation at each of the rotational torsion angles.
Depending on the input parameter values for the number of
generations and the total number of energy evaluations, configura-
tions are generated for grid points on the nucleic acid surface. The
energy of the configurations is calculated based on a previously
defined grid surface. Interaction energies are calculated with a free-
energy-based expression. The docked energies are listed in increas-
ing order of energy.
Topoisomerase II-Mediated DNA Cleavage. Reaction mix-
tures (20 μL) containing 10 mM Tris-HCl (pH=7.9), 50 mM
NaCl, 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 15 μg/mL
bovine serum albumine (BSA), 1 mM ATP, 0.25 μg pBR322
plasmid DNA, 10 U topoisomerase II (human recombinant
topoisomerase II R, USB), and test compounds were incubated
for 60 min at 37 ꢀC. Reactions were stopped by adding 4 μL of
stop buffer (5% SDS, 0.125% bromophenol blue and 25%
glycerol) and 50 μg/mL proteinase K (Sigma) and incubating
for a further 30 min at 37 ꢀC. The samples were separated by
electrophoresis on a 1% agarose gel containing ethidium bro-
mide 0.5 μg/mL at room temperature in TBE buffer (0.09 M
Tris-borate and 0.002 M EDTA).
For the DNA hexamer, polar hydrogens were added and
Kollman charges were assigned, whereas the Gasteiger-Marsili
partial charges were used for the ligands. The ligand binding site
was defined using a grid map of 60 ꢀ 60 ꢀ 60 points (A3),

5440 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 17
Dalla Via et al.
Acknowledgment. This work was supported by MIUR
(cofin 2006).
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Supporting Information Available: Tables including physical
properties, analytical, and spectral data of compounds 1a-v
and 3a-d. Results and experimental procedure for topo-
isomerase II-mediated cleavage of end-labeled DNA induced by
1e. This material is available free of charge via the Internet at
http://pubs.acs.org.
(30) Ryckebusch, A.; Garcin, D.; Lansiaux, A.; Goossens, J. F.;
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Baldeyrou, B.; Houssin, R.; Bailly, C.; Henichart, J. P. Synthesis,
Cytotoxicity, DNA Interaction, and Topoisomerase II Inhibition
Properties of Novel Indeno[2,1-c]quinolin-7-one and Indeno[1,2-
c]isoquinolin-5,11-dione Derivatives. J. Med. Chem. 2008, 51 (12),
3617–3629.
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