Design and synthesis of a novel series of pyranonaphthoquinones as topoisomerase II catalytic inhibitors

Article abstract of DOI:10.1021/jm800499x

On the basis of previous pharmacophore modeling studies of naphthoquinones derivatives, we have designed and synthesized a new set of pyranonaphthoquinones. These compounds were obtained through a direct and highly efficient approach based on an intramole

Full text of DOI:10.1021/jm800499x

J. Med. Chem. 2008, 51, 6761–6772
6761
Design and Synthesis of a Novel Series of Pyranonaphthoquinones as Topoisomerase II
Catalytic Inhibitors
Sandra Jime´nez-Alonso,†,‡ Haydee Cha´vez Orellana,†,| Ana Este´vez-Braun,*,†,‡ Angel G. Ravelo,†,‡ Elisa Pe´rez-Sacau,†,‡ and
Felix Mach´ın*,§
Instituto UniVersitario de Bio-Orga´nica “Antonio Gonza´lez”, UniVersidad de La Laguna, AVda. Astrof´ısico Fco, Sa´nchez 2, 38206 La Laguna,
Tenerife, Spain, Instituto Canario de InVestigaciones del Ca´ncer (ICIC) (http://www.icic.es), Spain, Facultad de Farmacia Bioqu´ımica,
UniVersidad San Luis Gonzaga de Ica, Peru, and Unidad de InVestigacio´n, Hospital UniVersitario Nuestra Sen˜ora de Candelaria, Carretera
del Rosario, 145, 38010, Santa Cruz de Tenerife, Spain
ReceiVed April 30, 2008
On the basis of previous pharmacophore modeling studies of naphthoquinones derivatives, we have designed
and synthesized a new set of pyranonaphthoquinones. These compounds were obtained through a direct and
highly efficient approach based on an intramolecular domino Knoevenagel hetero Diels-Alder reaction
from lawsone (2-hydroxynaphthoquinone) and a variety of aldehydes containing an alkene. The synthesized
pyranonaphthoquinones were evaluated against the R isoform of human topoisomerase II (hTopoIIR). Among
the 11 derivatives studied, we found that six of them act as catalytic inhibitors of the enzyme in vitro. These
six derivatives strongly preclude the enzyme from decatenating or relaxing suitable substrates. Finally, we
correlate their active/inactive status with docking studies of these novel compounds into the ATPase domain
of hTopoIIR.
Introduction
the relaxation of positive supercoiling by making single strand
breaks in one of the DNA strands while allowing the other one
to rotate around it. Type II topoisomerases make transient double
strand breaks and allow other DNA molecule to pass through
it. Type II topoisomerasas, like TopoI, can also relax supercoiled
DNA, but they are the only enzymes capable of unknotting the
replicated sister chromatids in order to allow a faithful segrega-
tion of the genetic material to the daughter cells during cell
division.14-17
Mass screening programs of natural products by the National
Cancer Institute have identified the quinone moiety as an
important pharmacophoric element for cytotoxic activity.1 A
representative group of quinonoid compounds are 1,2- and 1,4-
naphthoquinones, which are widely distributed in nature and
play important physiological roles in animals and plants.2 Some
illustrative examples of antitumoral naphthoquinones are plum-
bagin,3 juglone,4 ꢀ-lapachone,5 and rhinacanthone.6 These 1,2-
and 1,4-naphthoquinones are considered privileged structures
in medicinal chemistry on the basis of their numerous biological
activities and structural properties.7 In most cases, the biological
activity is related to the ability of quinones to accept one or
two electrons to form highly reactive radical anion intermediates,
which are responsible for the oxidative stress observed in the
cells.8 But there are several genotoxic properties9 attributed to
quinonoid compounds such as DNA intercalation or alkylation
of DNA that might also contribute to their cytoxicity. Especially
relevant is the fact that quinones may have topoisomerase II as
a target.10,11
Topoisomerases are important targets in antitumoral chemo-
therapy. In fact, after more than 30 years of clinical use, about
50% of current treatment protocols still employ at least one drug
directed against topoisomerases.12
From a physiological point of view, these enzymes catalyze
changes in the topology of the DNA molecule and facilitate
events such as transcription, replication, and physical separation
of sister chromatids in mitosis.13
The key to understanding why topoisomerases are good
antitumoral targets comes from different angles. First of all they
are highly active in cells that are proliferating and therefore
need to replicate and segregate the DNA.13,17-19 Moreover, in
humans, one of the type II topoisomerases, TopoIIR, is
up-regulated in proliferating cells.18 On the other hand, as they
can generate strand breaks, they are potential cytotoxic molec-
ular scissors. In fact, this seems to be the cytotoxic mechanism
for a number of antitumoral drugs that target topoisomerases.20-26
These drugs, known as topoisomerase “poisons”, stabilized the
topoisomerase-induced breaks and therefore generate DNA
damage, the ultimate cause of induced cell death for most cancer
cells.22,27,28 Examples of such poisons can be found for both
topoisomerases. Campthotecin and its derivatives are specific
TopoI poisons,29 whereas epipodophyllotoxins (etoposide),
aminoacridines (amsacrine), and antracyclines (doxorubicin,
daunorubicin, etc.) are potent TopoII poisons.30-34 Aside from
the poisons, other groups of drugs with potent antitumoral
activity have been shown to act as pure catalytic inhibitors of
the enzymes. This is the case of the anti-TopoII agents
merbarone and the family of bisdioxopiperazines.35-38 The
actual mechanism by which these later group exerts their
cytotoxic properties remains controvertial, but it is likely that
cell death occurs through mitotic collapse as cells fail to
segregate chromosomes because of reduced TopoII activity.39-43
Two types of topoisomerases can be differentiated on the basis
of their molecular reactions. Type I topoisomerases catalyze
* To whom correspondence should be addressed. For A.E.-B.: phone,
+ 34 922 318576; fax, + 34 922 318571; e-mail, aestebra@ull.es. For F.M.:
phone, +34 922 602951; fax, +34 922 600562; e-mail, fmacconw@
canarias.org.
Here, we present the synthesis of novel pyranonaphthoquino-
nes based on previous pharmacophore modeling studies.44 We
have also evaluated their potential as drugs that target human
topoisomerase IIR and find that six of them do act as TopoII
†
Universidad de La Laguna.
Instituto Canario de Investigaciones del Ca´ncer.
Universidad San Luis Gonzaga de Ica.
Hospital Universitario Nuestra Sen˜ora de Candelaria.
‡
|
§
10.1021/jm800499x CCC: $40.75
2008 American Chemical Society
Published on Web 09/25/2008

6762 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Jime´nez-Alonso et al.
Scheme 1
catalytic inhibitors. Finally, we correlate their active/inactive
status with docking studies of these novel compounds into the
ATPase domain of hTopoIIR.
and the adducts 1a, 1b, 2a,51b and 2b51b were produced with
excellent selectivity and high yield. Attending to the structural
diversity, the synthetic sequence is very attractive because 1,2-
and 1,4-naphthoquinone adducts are generated in a one-pot
reaction, allowing after biological evaluation the direct com-
parison between 1,2- and 1,4-naphthoquinones in the SAR
(structure-activity relationship) study. The trans stereochemistry
of the adducts 1a, 1b, 2a, and 2b was established on the basis
of coupling constants and ROESY experiments. This stereo-
chemistry agrees with the results by Tietze and co-workers47
for similar pyrano fused systems described. In the formation of
trans-annulated products only the exo-(E)-anti transition state
can be operative, since the exo-(E)-anti form is not possible
for geometrical reasons.47a
Chemistry
Recently we have published the synthesis and pharmacophore
modeling of naphthoquinone derivatives with cytotoxic activity
in human HL-60 cell line.44 In the CoMSIA model the steric
map of prenylpyranonaphthoquinones contains a favorable area
located in the pyran ring, which indicates that bulky substituents
or an extension of the structure in this part of the molecule
produces high cytotoxicity. Continuing with our interest in
synthesizing bioactive naphthoquinone derivatives,44,45 we aim
at achieving complex prenylpyranonaphthoquinones by exten-
sion of the structure through the pyran ring.
One of the most efficient synthetic methodology creating
complexity and diversity are the domino reactions.46 In this
sense, the domino Knoevenagel hetero Diels-Alder reaction
in its intramolecular version has emerged as a powerful process
that not only allows the efficient synthesis of complex com-
pounds such as natural products47 starting from simple substrates
but also permits the preparation of highly diverse molecules.
Representative examples of this methodology using R,ꢀ-
unsaturated carbonyl compounds are the preparation of tetra-
substituted dihydropyrans,48 tetracyclic pyrazoles,49 substituted
2H,5H-pyrano[4,3-b]pyran-5-ones,50 and a variety of R-lapa-
chone derivatives.51
The aromatic aldehydes 3-6 were prepared from the O-
alkylation of the corresponding phenol derivatives, using
1-bromo-3-methylbutene and K2CO3 in dry acetone under reflux.
Only cis adducts were obtained from these aromatic aldehydes,
with overall yields slightly lower than those of 1 and 2. An
endo-(E)-syn orientation can be assumed as transition structure
because an exo-(Z)-syn transition seems less likely owing to
steric interferences.47a,d The cis nature of the bridging hydrogens
of the aducts was strongly supported by their coupling constant
1
(∼4.4 Hz) in the H NMR spectrum and by the NOE effects
detected in the ROESY spectrum. With the aldehyde 5 (entry
5) only one compound 5a was detected. The no formation of
the corresponding orthoquinone is probably due to steric
hindrance of the bromine group in the transition state. The
energetic difference HOMOdienophile - LUMOheterodiene of the two
possible 1-oxa-1,3-butadiene must be similar for all but entry
5 in Table 1, a conclusion following from the ratio of resulting
adducts. Aiming at improving the degree of regioselectivity,
we repeated the reaction under microwave irradiation52 follow-
ing different conditions; however, microwaving only improves
the reaction time (from 20 to 3 min; see Table 1) with no
significant change of the yields corresponding to reflux conditions.
As reported by Ferreira et al.,51b the reaction of 2-hydroxy-
1,4-naphthoquinone with aliphatic aldehydes bearing a double
bond at an appropriate distance generates two new fused rings
next to the naphthoquinone core. Thus, the Knoevenagel
condensation of 2-hydroxy-1,4-naphthoquinone with an unsatur-
ated aldehyde produces the intermediate shown in Scheme 1.
This species presents two heterodiene systems that will provide
the formation of o- or p-naphthoquinones.
We selected 2,6-dimethyl-5-heptenal 1 to search for the best
reactions conditions, and then we use different ratios of reagents,
different solvents (EtOH, MeOH, C7H8, DCE, dioxane, aceto-
nitrile), and different bases (ethylene diammonium diacetate,
triethylamine, N-acetylpiperidine). The best yield was obtained
using 3 equiv of aldehyde, 6% mol of EDDA, and EtOH as
solvent under reflux. In these conditions, the corresponding
adducts were obtained in quantitative yield in 30 min (Table 1,
entry 1). Then we applied these conditions to several aliphatic
and aromatic aldehydes. With the aliphatic aldehydes 2,6-
dimethyl-5-heptenal (entry 1) and citronellal (entry 2), we
obtained high diastereoselectivity since trans-adducts were
obtained. The products were obtained as a mixture of o- and
p-naphthoquinones, easy to separate by chromatography. The
stereogenic center in R-position or ꢀ-position to the carbonyl
in the aldehydes 1 and 2 renders a high asymmetric induction,
Biological Results and Discussion
To evaluate the potential biological activities of the new
compounds, we decided to check their abilities as anti-
topoisomerase II agents. This first and direct approach was
chosen because (i) quinones have already been shown to act
on TopoII without the need to take into account possible, and
more complex, intracelular conversions to other more reactive
species,13 (ii) topoisomerase II is possibly the most important
molecular target of many antitumoral agents in use,12,13 and
(iii) regardless of the cytotoxic potency and mechanism of
quinones in vivo, the knowledge of their antitopoisomerase II
activities has important implications to take into consideration
if they want to be developed for clinical use.13

Pyranonaphthoquinones as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6763
Table 1. Reaction of 2-Hydroxy-1,4-naphthoquinone with Several Unsaturated Aldehydes
a Heating under reflux using EtOH as solvent. b Heating using a CEM-Discover microwave and EtOH as solvent.
To test whether compounds were active against human
topoisomerase IIR, we employed the purified enzyme and a set
of small molecules of circular DNA as substrates in a series of
different in vitro experiments. The use of these small DNA
molecules greatly facilitates the visualization of the topoi-
somerase reactions by simple electrophoretic means in the three
assays described below: “decatenation”, “relaxation”, and
“stabilization of the cleavage complex”.
ia fasciculata, is a network consisting of thousands of inter-
locked closed circular DNA molecules called “minicircles”.
Because of its overall size, kDNA cannot enter a typical agarose
gel during electrophoresis unless minicircles are previously
released. A transient double strand break is the only way to
take a minicircle out of the network and preserve its circular
nature. This can be accomplished through the decatenation
activity of topoisomerase II. In fact, this assay is considered to
be the most specific one for topoisomerase II activity.53
Minicircles, once released, enter the agarose gel and appear as
For the decatenation assay, the kinetoplast DNA (kDNA) was
employed as substrate. Kinetoplast DNA, obtained from Crithid-

6764 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Jime´nez-Alonso et al.
Figure 1. Inhibition of hTopoIIR-mediated kDNA decatenation by pyranonaphthoquinones 3a, 3b, 4a, 5a, 6a, 6b. Catenanes minicircles from
kinetoplast DNA were treated with hTopoIIR in the presence of either just 1% DMSO or 100 µM ellipticine, etoposide, or a novel set of
pyronaphthoquinones in 1%DMSO: (A) agarose gel electrophoresis to separate substrates (kDNA) and products of the hTopoIIR reaction (Nck +
Rel + SC); (B) quantitation of product formation of three independent experiments relative to 1% DMSO. Note how the presence of
pyranonaphthoquinones 3a, 3b, 4b, 5a, 6a, and 6b completely inhibit hTopoIIR (compared to controls without enzyme or with ellipticine): kDNA
(intact kinetoplast DNA), Caten (catenanes subproducts), Nck (nicked minicircles), Lin (linearized minicircles), SC (supercoiled minicircles), Rel
(relaxed minicircles).
a mixture of covalently closed topoisomers and nicked products
(“Nck” band in Figure 1A). To simplify the overall number of
visible products, the electrophoresis is carried out in the presence
of an excess of an intercalative agent (ethidium bromide in these
assays) to covert all covalently closed topoisomers to a unique
band of maximum supercoiled unique topoisomer (“SC” band
in Figure 1A).
Thus, we incubated the purified enzyme with the synthesized
pyranonaphthoquinones and immediately added the kDNA
(Figure 1). Several controls were included to confirm the correct
performance of the assays. First samples with just the final
concentration of the solvent (1% DMSO) were used with or
without the enzyme to set up our 100% or 0% of activity,
respectively. These samples were also used to titrate the amount
of enzyme, kDNA, and incubation time to just complete the
reaction under the presence of the enzyme (“end-point” reaction,
not shown). Two well-known anti-TopoII agents were also
included. Etoposide is a topoisomerase II highly specific
nonintercalative poison that stabilizes the cleavage complex. As
the cleavage step is indeed reversible,26 etoposide does not
inhibit greatly the overall reaction (Figure 1). On the other hand,
ellipticine, while it modestly promotes the formation of more
cleavage intermediates, can strongly inhibit topoisomerase II
decatenation at the same time that it intercalates into the DNA
molecule54 (Figure 1). So far, complete decatenation was seen
in the control without any drugs and the sample with etoposide,
whereas ellipticine inhibited the minicircles release to a great
extent as seen by comparison with the sample that lacked the
enzyme (Figure 1). The same complete absence of decatenation
activity was observed when compounds 3a, 3b, 4b, 5a, 6a, and
6b were present, whereas no inhibition was apparent for the
pyranonaphthoquinones 1a, 1b, 2a, 2b, and 4a.
To further check that decatenation failed by an inhibition of
topoisomerase IIR in the presence of the pyranonaphthoquino-
nes, we performed another assay for TopoII activity. In this
assay we measured the TopoII-dependent relaxation of a plasmid
(pRYG) that contained a hot spot sequence for TopoII.55
Plasmids, as they are in bacteria, are negatively supercoiled in
its natural form (“SC” band in Figure 2A), and this basic form
is preserved during DNA extraction. However, two more species
are marginally obtained as well: a “relaxed” covalently closed
form (“Rel” band in Figure 2A) and a “nicked” single-strand
break form (termed “Nck” in Figure 2A). All these topological
species can be resolved in an agarose gel electrophoresis under

Pyranonaphthoquinones as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6765
Figure 2. Inhibition of hTopoIIR-mediated supercoiled DNA relaxation by pyranonaphthoquinones 3a, 3b, 4a, 5a, 6a, 6b. Covalently closed
negative supercoiled pRYG plasmid (SC form) was treated with hTopoIIR in the presence of either just 1% DMSO or 100 µM ellipticine, etoposide,
or a novel set of pyranonaphthoquinones in 1%DMSO: (A) agarose gel electrophoresis (without ethidium bromide) to separate substrates (SC form)
and products (relaxed topoisomers) of the htopo IIR reaction; (B) quantitation of product formation of three independent experiments relative to 1%
DMSO. Note how the presence of pyronaphthoquinones 3a, 3b, 4b, 5a, 6a, and 6b greatly inhibit hTopoIIR (compared to controls without enzyme
or with ellipticine): Nck (nicked pRYG), Rel (relaxed pRYG), Lin (linearized pRYG), SC (supercoiled pRYG).
specific conditions, where the order of migration is, from fastest
to slowest, SC, Nck, Rel. Either topoisomerase I or II can relax
supercoiling in vitro to an equilibrium of topoisomers. As it is
shown in Figure 2A and quantified in Figure 2B, the SC form
of pRYG is reduced and appeared as a set of variably relaxed
topoisomers that migrate more slowely than the SC form when
the enzyme is present without any drugs. This pattern of
topoisomers is also obtained for etoposide and some of the
compounds studied (1a, 1b, 2a, 2b, and 4a) (Figure 2). By
contrast, ellipticine and the other novel pyranonaphthoquinones
tested (3a, 3b, 4b, 5a, 6a, and 6b) showed a pattern that
resembled that of the control without the enzyme. Interestingly,
this pattern of inhibition of relaxation fully coincides with the
pattern observed in the decatenation assay (Figure 1). Thus, this
double-check of inhibition strongly points out compounds 3a,
3b, 4b, 5a, 6a, and 6b as active drugs against human
topoisomerase IIR.
Although some of the compounds analyzed did not show any
inhibition of TopoIIR in these assays, this does not preclude
that those derivatives do not act against TopoIIR. In fact, as
shown here in the previous assays, etoposide has little effect
on the overall TopoII activity in spite of being a strong
topoisomerase poison (Figures 1 and 2). Therefore, we tested
our compounds to see whether they could poison the TopoII
reaction by increasing the steady state of the cleavage complex,
as etoposide does. To do so, we repeated the assay with pRYG
as substrate. In this case, the tested drug was added once the
relaxation reaction had already started and the electrophoresis
was run under the presence of an excess of ethidium bromide
to fully positively supercoil all the topoisomers (make them run
as a sole band during the electrophoresis) and thus clearly
unmask the linear form (“Lin” band in Figure 3) of the DNA.
This form is the result of the cut by the enzyme, and although
it is normally rather transient and difficult to detect because
the enzyme quickly reseals it, it can be seen with potent
inhibitors of the ligation step of the catalytic cycle of TopoII,
as is the case of etoposide.26 In fact, in our hands etoposide did
show this linear form of the pRYG plasmid (Figure 3). However,
this form was not visible for the other controls, including
ellipticine54 and the novel pyranonaphthoquinones tested.
Therefore, we can conclude that none of the compounds behaved
as the classical poisons of TopoII, at least at the concentration
tested.
Finally, two types of catalytic inhibitors of TopoII can be
differentiated with regard to their action on the DNA molecule
itself: those that can intercalate between the bases of the DNA
and those that cannot. Normally, although not necessarily,
intercalating agents can inhibit topoisomerase II in an indirect
fashion as they overwound DNA, a situation that might change
the efficiency of the topoisomerase II reaction. On the other

6766 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Jime´nez-Alonso et al.
Figure 3. Pyranonaphthoquinones do not enhance the cleavage complex. Covalently closed negative supercoiled pRYG plasmid was treated with
hTopoIIR and then inmediately incubated with just 1% DMSO, 100 µM ellipticine, 100 µM etoposide, or 100 µM of a novel set of
pyranonaphthoquinones, all in 1% (v/v) DMSO. The agarose gel electrophoresis (with ethidium bromide) separates substrates and products of the
hTopoIIR reaction from the linear intermediate (Lin band). Note how none of the pyranonaphthoquinones lead to the presence of the Lin form in
comparison to etoposide.
Figure 4. Pyranonaphthoquinones do not intercalate within the DNA. Covalently closed negative supercoiled pBSKS plasmid was incubated with
just 1% DMSO, 1 µg/mL ethidium bromide, 100 µM ellipticine, 100 µM etoposide, or 100 µM of a novel set of pyranonaphthoquinones, all in 1%
(v/v) DMSO. An agarose gel electrophoresis (without ethidium bromide) allows us to see a retardation for the DNA treated with intercalators such
as ethidium bromide and ellipticine, whereas no such phenomenon is observed for the synthesized pyranonaphthoquinones.
hand, those drugs that do not intercalate in the DNA may indeed
be more specific for the enzyme. To discern between these two
possibilities, we employed a simple but reliable test of intercala-
tion.56 We incubated a negatively supercoiled small circular
DNA (in this case the plasmid pBSKS) with the compounds
used in this paper, plus the intercalative agent ethidium bromide
as an additional control, followed by an electrophoresis without
EthBr in low-field conditions. If any compound intercalates into
the DNA, it would (i) change the degree of supercoling toward
a positive (overwound) molecule and/or (ii) change the apparent
mass of the DNA molecule. For a small circular DNA molecule
negatively supercoiled the result of any of these changes would
be a noticeble retardation in the migration during electrophoresis.
In fact, this can be clearly seen under the presence of the
intercalatives ethidium bromide and ellipticine, whereas etopo-
side does not change the mobility (Figure 4). None of the
pyranonaphthoquinones studied led to DNA retardation (Figure
4). Thus, we conclude that they are not inhibiting the TopoII
reaction by previously intercalating within the DNA bases.
Eukaryotic topoisomerase II are multisubunit proteins that
modulate topology by passing an intact helix through a transient
double-stranded break they create in the DNA backbone.13,57
Six steps in the catalytic cycle of the enzyme can be envisioned.
First, TopoII binds the DNA. Second, it cleaves one of the DNA
molecules under the presence of a divalent cation. Third, ATP
binds the enzyme and allows a conformational change that
directs the strand passage of the second DNA molecule. Fourth,
the enzyme relegates the first DNA. Fifth, ATP hydrolysis opens
the enzyme to liberate the DNA molecule that has just passed
through, and finally, the other DNA is released from the enzyme.
ATP is an important cofactor for the overall catalytic activity,
in spite of its role in the catalytic cycle being restricted to the
latter stages. A number of antitumoral agents have been shown
to act on how the ATP binds to or is hydrolyzed by the enzyme.
Most of them have been shown to be competitive inhibitors of
ATP. Remarkably, they do not enhance dramatically the cleavage
complex even though ATP binding is needed to continue the
catalytic cycle toward the religation step. In fact, they seem to stop
the reaction at the step of ATP hydrolysis, trapping the enzyme in
a closed clamp with the DNA molecules.36 Therefore, it seems
likely that these agents simulate the ATP binding step, in relation
to the conformational changes that allow strand passage and DNA
strand breaks resealing, but they are not easily “excluded” out
of the pocket when needed to finish up the reaction cycle.36
All these would explain why they strongly inhibit decatenation
and relaxation activities while keeping the DNA molecules free
of double strand breaks. In fact, our molecular docking studies
support this hypothesis.
Docking
We first tried docking our pyranonaphthoquinones into
various possible pockets of TopoII. We obtain the best results
into the ATP pocket, so we used an accurate model for this
target site. This fact is consistent with the biological results that
point out that active pyranonaphthoquinones act as catalytic
inhibitors of TopoII. The program AutoDock 3.0.5.58 was used
to dock separately o- and p-pyranonaphthoquinones. The model
shows the compounds acting as hTopoII inhibitors by binding
to the ATP pocket in the ATPase domain. We have found that
all active compounds (3a, 3b,4b, 5a, 6a, and 6b) were
superimposed in the binding site and they showed an effective
π interaction with the magnesium (gray ball) (Figure 5). This
type of interaction seems essential, since the inactive compounds
1a, 1b, 2a, 2b do not have aromatic rings. These docking results
also predicted favorable interactions between the compounds
and key residues at the ATPase domain such as K140, N122,

Pyranonaphthoquinones as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6767
Supporting Information). This interaction is less effective than
the one with the additional aromatic ring observed in the active
compounds. In addition these docking results do not predict
favorable interactions between the compounds and key residues
at the ATPase domain such us K140, N122, and S121, which
were predicted for the active compounds.
Conclusion
In summary, on the basis of previous pharmacophore model-
ing studies of naphthoquinones derivatives, we have designed
and synthesized a novel set of pyranonaphthoquinones. A simple
and rapid protocol of the intramolecular domino Knoevenagel
hetero Diels-Alder reaction was developed for the preparation
of these pyranonaphthoquinones in a single preparative step.
The reactions are very efficient, since two new fused rings are
formed through three new σ-bonds (two C-C σ bonds, and
one O-C σ-bond). The biological activities of the synthesized
naphthoquinones, focused on the action against human topoi-
somerase IIR, revealed that compounds 3a, 3b,4b, 5a, 6a, and
6b act as topoisomerase II catalytic inhibitors. In support of
the proposed action of the active pyranonaphthoquinones, we
show that (i) no linear DNA molecules (result for cleavage
complex enhacement) are seen (Figure 3) and (ii) potent
decatenation and relaxation inhibition are present with the
compounds that better fit within the ATP pocket (Figures 1,
2,5, and 6). We have further checked that the inhibition was
not due to any action of the compound on the DNA itself, which
would inhibit the very first two steps of the catalytic cycle, by
showing that none were DNA intercalators (Figure 4).
Figure 5. Docking model of compounds 3a and 3b binding into the
ATP pocket of htopo II. Both compounds showed a π interaction with
the magnesium (gray ball). The Lys140 is predicted to bind the oxygen
of the pyrane ring. Meanwhile, the residues Asn122 and Ser121 bind
the carbonyl at position 2 for the ortho derivatives or the carbonyl at
position 1 for the p-pyranonaphthoquinones.
Experimental Section
Figure 6. Docking model of compound 4a binding into the ATP pocket
of hTopoII. The relative disposition between the magnesium (gray ball)
and the aromatic ring of the naphthoquinone produces a poor π
interaction. In this case the oxygen atoms of both pyrane rings and the
carbonyl in position 2 were situated at an adequate distance from key
residues such us Asn122, Ser121, Tyr379, and Arg70.
Chemistry. General Methods. All solvents and reagents were
purified by standard techniques reported in Perrin, D. D.; Amarego,
W. L. F. Purification of Laboratory Chemicals, 3rd ed.; Pergamon
Press: Oxford, U.K.,1988 or used as supplied from commercial
sources as appropriate. Reactions were monitored by TLC (on silica
gel POLYGRAM SIL G/UV254 foils). Purification by column flash
chromatography used Merk Kiesel 60-H (0.063-0.2 mm) as
adsorbent and different mixtures of hexanes-ethyl acetate as eluent.
Precoated TLC plates SIL G-100 UV254 (Machery-Nagel) were used
and S121. The model also explains that 1,2- and 1,4-pyranon-
aphthoquinones show similar inhibitory activity. In Figure 5
we can observe how the carbonyl at C-4 of 3a superimposes
with the carbonyl at C-2 of 3b, and both carbonyls bind the
residues Asn122 and Ser 121, respectively. However, com-
pounds 4a, with an additional but nonactivated aromatic ring,
docked into the binding site, adopting different poses compared
with the activated compounds. In this case the naphthoquinone
motif was situated near the magnessium cation but their
interaction is less effective compared with those in the active
compounds (see Figure 6). The additional interactions with the
key residues were fitted only partially. All these results are
consistent with the obtained experimental data, and the generated
model explains the TopoII inhibitory activity of the pyranon-
aphthoquinones synthesized. Interestingly, salvicine, an ATP-
compiting hTopoIIR inhibitor with a naphthoquinone ring gives
a same pattern of in vitro inhibition of the enzyme.59 Remark-
ably, other o- and p-pyranonapthtoquinones such as ꢀ-lapachone
have been shown to be catalytic inhibitors of the hTopoIIR, in
spite of not having the second aromatic ring.60 However, they
inhibited the topoisomerase activity more weakly and required
higher concentrations of the drug. Since no docking models were
available for these naphthoquinones, we included them in our
studies and compared them to our active and inactive compounds
(see Supporting Information Figures S-1, S-2, S-3, and S-4).
Again, in this case the best docking pose into the ATP binding
pocket reveal a π stacking interaction between the aromatic ring
A of the naphthoquinone moiety and the magnesium cation (see
1
for preparative TLC purification. H NMR spectra were recorded
in CDCl3 or C6D6 at 300 and 400 MHz, using Bruker AMX300
and Bruker AMX400 instruments. For 1H spectra, chemical shifts
are given in parts per million (ppm) and are referenced to the
residual solvent peak. The following abbreviations are used: s,
singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad.
Proton assignments and stereochemistry are supported by H-1H
1
COSY and ROESY where necessary. Data are reported in the
following manner: chemical shift (integration, multiplicity, coupling
constant if appropriate). Coupling constants (J) are given in hertz
(Hz) to the nearest 0.5 Hz. 13C NMR spectra were recorded at 75
and 100 MHz using Bruker AMX300 and Bruker AMX400
instruments. Carbon spectra assignments are supported by DEPT-
135 spectra, 13C-1H (HMQC), and 13C-1H (HMBC) correlations
where necessary. Chemical shifts are quoted in ppm and are
referenced to the appropriate residual solvent peak. MS and HRMS
were recorded at VG Micromass ZAB-2F. IR spectra were taken
on a Bruker IFS28/55 spectrophotometer.
Microwave Irradiation Experiments. All microwave irradiation
experiments were carried out in a Discover-CEM monomode
microwave apparatus operating at a frequency of 2.45 GHz with
continuous irradiation power from 0 to 300 W with utilization of
the standard absorbance level of 300 W maximum power. The
reactions were carried out in 10 mL glass tubes sealed with
aluminum/Teflon crimp tops, which can be exposed up to 250 °C
and 20 bar of internal pressure. Temperature was measured with
an IR sensor on the outer surface of the process vial. After the

6768 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Jime´nez-Alonso et al.
irradiation period, the reaction vessel was cooled rapidly to ambient
temperature by air jet cooling.
yield 80.7 mg of adduct 2a (45.7%) and 85.9 mg (48.6%) of adduct
2b. Following the procedure B described above, the reaction mixture
was irradiated for 2.5 min at a preselected temperature of 130 °C,
with an irradiation power of 136 W. The crude was purified by
preparative TLC using hexanes/EtOAc (7:3) to provide 18.1 mg
of 2a (40.8%) and 19.5 mg (44.0%) of 2b.
General Procedure for the Reaction of 2-Hydroxynaphtho-
quinone with the Unsaturated Aldehydes by Heating (Procedure
A). An amount of 100 mg (0.547 mmol) of 2-hydroxy-1,4-
naphthoquinone in 10 mL of EtOH was treated with 3 equiv of
aldehyde and catalitic amounts of EDDA (ethylenediamino diac-
etate, 6% mmol). The reaction mixture was heated under reflux
and checked by TLC until disappearance of the starting naphtho-
quinone. Then the reaction mixture was cooled and the EtOH was
removed under reduced pressure. The crude was purified by silica
gel column chromatography with hexanes/EtOAc as solvent.
General Procedure by Microwave (MW) Irradiation (Proce-
dure B). Twenty-five mg of 2-hydroxy-1,4-naphthoquinone (0.143
mmol), 3 equiv of aldehyde and catalytic amount of EDDA (5.5
mg, 0.03 mmol) were suspended in 2 mL of EtOH in a 10 mL
reaction glass containing a stirring magnet. The vial was sealed
tightly with an aluminum-Teflon crimp top. After the irradiation
period, the reaction vessel was cooled rapidly to ambient temper-
ature by air jet cooling. The solvent was removed under reduced
pressure and the residue was purified by TLC-preparative
chromatography.
Reaction of 2-Hydroxy-1,4-naphthoquinone with 2,6-Dimethyl-
5-heptenal. Following the general procedure A described above,
an amount of 174.96 mg (1.0 mmol) of 2-hydroxy-1,4-naphtho-
quinone in 10 mL of EtOH was treated with 0.47 mL (3.0 mmol)
of 2,6-dimethyl-5-heptenal and 10 mg of EDDA (0.06 mmol). The
reaction mixture was heated under reflux for 30 min. The solvent
was removed under vacuum, and the crude product was purified
by flash chromatography with 10% hexanes/EtOAc to yield 136.9
mg (46.3%) of adduct 1a and 159.3 mg (53.8%) of adduct 1b.
Following the procedure B described above, the reaction mixture
was irradiated for 2 min at a preselected temperature of 130 °C,
with an irradiation power of 150 W. The crude was purified by
preparative TLC using hexanes/EtOAc (7:3) to provide 21.4 mg
of 1a (50.6%) and 20.1 mg (47.4%) of 1b.
Data for 2a.51b It was isolated as an amorphous yellow solid Rf
1
(Hex/AcOEt, 4:1) ) 0.58. [R]2D5 +183 (c 0.57, CHCl3). H NMR
3
(300 MHz, CDCl3) δ 0.68 (dd, JH,H ) 11.7, 12.0 Hz, 1H), 0.92
3
(d, 3H, JH,H ) 6.5 Hz), 1.10 (m, 1H, H-13a), 1.12 (s, 3H, CH3),
1.32 (m, 1H), 1.50 (s, 3H, CH3), 1.65 (m,1H), 1.83 (m, 3H), 2.41
(td, 3JH,H )11.0, 2.7 Hz, 1H), 2.83 (d, 3JH,H ) 12.5 Hz, 1H), 7.64
(m, 2H), 8.00 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3) δ 19.1
(CH3), 22.0 (CH3), 26.7 (CH3), 27.3 (CH2), 32.5 (CH), 34.3 (CH),
35.0 (CH2), 38.6 (CH2), 47.9 (CH), 82.2 (C), 123.6 (C), 125.6 (CH),
125.8 (CH), 130.7 (C), 132.4 (CH), 132.6 (C), 133.6 (CH), 154.0
(C), 179.9 (C), 184.1 (C) ppm. EI-MS m/z (%) 310 (M+, 100),
295 (31), 267 (28), 240 (9). EI-HRMS 310.1566 [(M+) calcd for
C20H22O3 310.1569]. IR (CHCl3) νmax 2923, 2867, 1679, 1600, 1574,
1456, 1372, 1334, 1303, 1270, 1208, 1136, 1089, 1041, 1014, 980,
963, 933, 794, 755,725, 667, 528 cm-1
.
Data for 2b.51b It was isolated as an amorphous orange solid.
[R]2D5 +18 (c 0.39, CHCl3). Rf (Hex/AcOEt, 4:1) ) 0.45. 1H NMR
3
(300 MHz, CDCl3) δ 0.59 (dd, JH,H ) 11.5, 12.0 Hz, 1H), 0.92
(d, 3JH,H ) 6.5 Hz, 3H), 1.05 (m, 1H), 1.18 (s, 3H, CH3), 1.34 (m,
1H), 1.51 (s, 3H, CH3), 1.60 (m, 1H), 1.81 (m, 3H), 2.36 (td, 3JH,H
3
3
) 11.0, 2.7 Hz, 1H), 2.87 (d, JH,H ) 12.6 Hz, 1H), 7.45 (t, JH,H
3
3
) 7.5 Hz, 1H), 7.61 (t, JH,H ) 7.0 Hz, 1H), 7.78 (d, JH,H ) 7.7
3
Hz, 1H), 8.00 (d, JH,H ) 7.6 Hz, 1H) ppm. 13C NMR (75 MHz,
CDCl3) δ 19.18 (CH3), 22.0 (CH3), 26.9 (CH3), 27.1 (CH2), 32.1
(CH), 33.6 (CH), 35.0 (CH2), 37.7 (CH2), 47.98 (CH), 82.3 (C),
116.5 (C), 124.1 (CH), 127.9 (CH), 130.1 (C), 130.3 (CH), 132.6
(C), 134.5 (CH), 161.3 (C), 178.7 (C), 180.1 (C) ppm. EI-MS m/z
(%) 310 (M+, 100), 282 (M+ - CO, 14). EI-HRMS 310.1565
[(M+) calcd for C20H22O3 310.1569]. IR (CHCl3) νmax 2925, 2867,
2360, 1679, 1641, 1594, 1563, 1453, 1378, 1286, 1209, 1136, 1090,
972, 963, 930, 861, 754, 692, 662 cm-1
.
Data for 1a. It was isolated as an amorphous yellow solid. Rf
(Hex/AcOEt, 4:1) ) 0.58. 1H NMR (300 MHz, CDCl3) δ 1.26 (s,
3H), 1.30 (m, 1H), 1.50 (s, 3H), 1.50 (d, 3JH,H ) 5.7 Hz, 3H), 1.55
(m, 1H), 1.66 (m, 1H), 1.83 (m, 1H), 2.07 (m, 1H), 2.20 (m, 2H),
7.63 (m, 2H), 8.00 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3) δ
20.3 (CH3), 23.5 (CH3), 24.6 (CH2), 28.0 (CH3), 34.2 (CH2), 35.3
(CH), 43.5 (CH), 52.6 (CH), 82.5 (C), 124.1 (C), 125.7 (CH × 2),
130.5 (C), 132.2 (C), 132.3 (CH), 133.5 (CH), 154.7 (C), 179.9
Synthesis of 4-Methoxy-2-(3-methylbut-2-enyloxy)benzaldehyde.
An amount of 1 mol (152.15 mg) of 2-hydroxy-4-methoxybenzal-
dehyde was added to a solution of 1-bromo-3-methylbut-2-ene (2
mol, 0.115 mL) and K2CO3 (1.2 mol, 168.85 mg) in dry acetone
(30 mL). The mixture was heated under reflux for 18 h under
nitrogen atmosphere. Then the mixture was filtered and the solvent
removed under vacuum. The residue was treated with an aqueous
solution of 5% NaOH and extracted three times with Et2O. The
organic layers were dried over MgSO4. After removal of solvent
the crude was purified by flash chromatography (silica gel, 9.5:
0.5, hexanes/EtOAc) to provide 187.6 mg (74%) of 4-methoxy-2-
(3-methylbut-2-enyloxy)benzaldehyde. 1H NMR (CDCl3, 300 MHz)
δ 1.69 (s, 3H, CH3), 1.73 (s, 3H, CH3), 3.78 (s, 3H, CH3), 4.53 (d,
(C), 183.8 (C) ppm. EI-MS m/z (%) 296 (M+, 100), 281 (M+
-
CH3, 97), 253 (M+ - CO, 32), 225 (M+ - 2 × CO, 13). EI-
HRMS m/z 296.1402 [(M+) calcd for C19H20O3 296.1412]. IR
(CHCl3) νmax 2948, 2870, 1678, 1649, 1594, 1561, 1460, 1362,
1334, 1303, 1272, 1244, 1207, 1132, 1039, 980, 838, 723 cm-1
.
Data for 1b. It was isolated as an amorphous orange solid. Rf
(Hex/AcOEt, 4:1) ) 0.45. 1H NMR (300 MHz, CDCl3) δ 1.31 (s,
3H), 1.31 (m, 1H), 1.48 (d, 3JH,H ) 5.3 Hz, 3H), 1.55 (s, 3H), 1.68
(m, 2H), 1.85 (m, 1H), 2.07 (m, 3H), 7.46 (t, 3JH,H ) 7.5 Hz, 1H),
3JH,H ) 6.6 Hz, 2H), 5.42 (t, 3JH,H ) 6.6 Hz, 1H), 6.37 (d, 4JH,H
)
2.2 Hz, 1H), 6.45 (dd, 3JH,H ) 8.7, 4JH,H ) 2.0, 1H), 7.71 (d, 3JH,H
)8.7 Hz, 1H), 10.24 (s, 1H). 13C NMR (75 MHz, CDCl3) δ 17.7
(CH3), 25.2 (CH3), 55.0 (CH3), 64.8 (CH2), 98.3 (CH), 105.4 (CH),
118.3 (CH), 118.6 (C), 129.6 (CH), 138.0 (C), 162.5 (C), 165.5
(C), 187.7 (CH). EIMS m/z (%) 220 (M+, 5), 152 (M+ - C5H8,
100), 151 (M+ - C5H9, 78), 69 (C4H5O, 24). EIHRMS 220.1092
[(M+) calcd para C13H16O3 220.1099]. IR (CHCl3) νmax 2856, 1678,
1601, 1501, 1443, 1384, 1260, 1202, 1167, 1113, 1035, 998, 815,
3
7.61 (t, 1H, JH,H ) 7.5 Hz, H-17), 7.82 (d,1H, d, J ) 7.8 Hz,
H-19), 8.01(d,1H, 3JH,H ) 7.5 Hz, H-16) ppm. 13C NMR (75 MHz,
CDCl3) δ 20.5 (CH3), 23.2 (CH3), 24.8 (CH2), 28.2 (CH3), 32.6
(CH2), 34.0 (CH), 39.4 (CH), 52.6 (CH), 83.7 (C), 117.2 (C), 124.6
(CH), 127.9 (CH), 130.0 (C), 130.2 (CH), 132.9 (C), 134.4 (CH),
161.9 (C), 178.3 (C), 180.2 (C) ppm. EI-MS m/z (%) 296 (M+,
100), 281 (M+ - CH3, 91), 253 (M+ - CO, 48), 225 (M+ - 2CO,
33). EI-HRMS 296.1412 [(M+), calcd for C19H20O3 296.1412]. IR
(CHCl3) νmax 2947, 2869, 2359, 1797, 1644, 1590, 1451, 1375,
643 cm-1
.
Reaction of 2-Hydroxy-1,4-naphthoquinone with 4-Methoxy-
2-(3-methylbut-2-enyloxy)benzaldehyde. Following the general
procedure described above, an amount of 100 mg (0.57 mmol) of
2-hydroxy-1,4-naphthoquinone in 10 mL of EtOH was treated with
378.8 mg (1.71 mmol) of 4-methoxy-2-(3-methylbut-2-enyloxy)-
benzaldehyde and 5.5 mg of EDDA. The reaction mixture was
refluxed for 30 min, cooled to room temperature, and freed from
the solvent in vacuum. The residue was purified by preparative TLC
using hexanes/EtOAc (9:1) to yield 74.1 mg (34.6%) of adduct 3a
and 77.0 mg (35.9%) of 3b. Following the procedure B described
above, the reaction mixture was irradiated for 3 min at a preselected
1291, 1231, 1129, 1085, 927, 862, 776, 718, 691, 661, 469 cm-1
.
Reaction of 2-Hydroxy-1,4-naphthoquinone with (s)-(-)-Cit-
ronellal. Following the general procedure described above, an
amount of 100 mg (0.57 mmol) of 2-hydroxy-1,4-naphthoquinone
in 10 mL of EtOH was treated with 0.31 mL (1.71 mmol) of (s)-
(-)-citronellal and 5.5 mg of EDDA (0.03 mol). The reaction
mixture was heated under reflux for 30 min, cooled to room
temperature, and freed from the solvent in vacuo. The residue was
purified by flash chromatography using hexanes/AcOEt (9:1) to

Pyranonaphthoquinones as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6769
temperature of 130 °C, with an irradiation power of 150 W. The
crude was purified by preparative TLC using hexanes/EtOAc (7:3)
to provide 23.6 mg of 3a (43.9%) and 22.0 mg (40.9%) of 3b.
Data for 3a. It was isolated as an amorphous yellow solid. Rf
(Hex/EtOAc, 7:3) ) 0. 37. 1H NMR (300 MHz, CDCl3) δ 1.30 (s,
Hz, 1H), 7.34 (t, 3JH,H ) 7.4 Hz, 1H), 7.50 (m, 2H), 7.65 (m, 2H),
7.74 (d, 3JH,H ) 8.0 Hz, 1H), 7.91 (d, 3JH,H ) 7.8 Hz, 1H), 7.99 (d,
3JH,H ) 7.4 Hz, 1H), 8.13 (d, 3JH,H ) 8.5 Hz, 1H) ppm. 13C NMR
(75 MHz, CDCl3) δ 25.8 (C), 26.1 (CH), 26.8 (C), 38.2 (CH), 63.1
(CH2), 78.7 (C), 113.2 (Carom), 115.2 (Carom), 117.8 (CHarom), 122.8
(CHarom), 123.4 (CHarom), 124.6 (CHarom), 125.5 (CHarom), 127.9
(CHarom), 128.2 (CHarom), 128.8 (Carom), 129.0 (CHarom), 130.0
(Carom), 132.2 (Carom), 134.2 (Carom), 134.6 (CHarom), 151.0 (Carom),
160.5 (Carom), 177.6 (C, CdO), 178.8 (C, CdO) ppm. EI-MS m/z
(%) 396 (M+, 58), 379 (100). EI-HRMS 396.1360 [(M+) calcd
for C26 H20O4 396.1362). IR (CHCl3) νmax ) 2926, 2357, 1698,
1657, 1573, 1513, 1452, 1359, 1290, 1258, 1231, 1135, 1087, 923,
3
3H, CH3), 1.60 (s, 3H, CH3), 2.15 (dd, JH,H ) 9.4, 4.4 Hz, 1H),
3.71 (s, 3H, OCH3), 4.30 (dd, 3JH,H ) 11.9, 4.4 Hz, 1H), 4.46 (dd,
3JH,H ) 12.0, 6.6 Hz, 2H), 6.27 (d, 3JH,H ) 2.5 Hz, 1H), 6.42 (dd,
3
3JH,H ) 8.6, 2.5 Hz, 1H), 7.18 (d, JH,H ) 8.6 Hz, 1H), 7.67 (m,
2H), 8.08 (m, 2H) ppm. 13C NMR (75 MHz, CDCl3) δ 23.8 (CH3),
27.2 (CH3), 28.7 (CH), 37.9 (CH), 54.9 (OCH3), 64.3 (CH2), 80.4
(C), 100.7 (CHarom), 107.1 (CHarom), 112.9 (Carom), 121.3 (Carom),
126.0 (CHarom), 126.1 (CHarom), 130.5 (Carom), 130.8 (CHarom), 132.1
(Carom), 132 8 (CHarom), 133.9 (CHarom), 153.6 (Carom), 154.5 (Carom),
159.2 (Carom), 179.6 (C, CdO), 184.9 (C, CdO) ppm. EI-MS m/z
(%) 376 (M+, 100), 361 (M+ - CH3, 19). EI-HRMS 376.1324
[(M+) calcd for C23H20O5 376.1311]. IR (CHCl3) νmax ) 2982,
1680, 1646, 1578, 1503, 1463, 1364, 1336, 1269, 1240, 1202, 1164,
1129, 1079, 1036, 987, 954, 833, 797, 756, 725, 665, 562, 478
812, 750, 705, 660, 533 cm-1
.
Synthesis of 3,5-Dibromo-2-(3-methylbut-2-enyloxy)benzalde-
hyde (5). An amount of 419.89 mg (1.5 mmol) of 3,5-dibromo-2-
hydroxybenzaldehyde was added to a solution of 1-bromo-3-
methylbut-2-ene (3 mmol, 0.345 mL) and Cs2CO3 (1.8 mmol, 586.7
mg) in dry THF (30 mL). The mixture was heated under reflux for
18 h under nitrogen atmosphere. Then the mixture was filtered and
the solvent removed under vacuum. The residue was treated with
an aqueous solution of 5% NaOH and extracted three times with
Et2O. The organic layers were dried over MgSO4. After removal
of solvent the crude was purified by flash chromatography (silica
gel, 9.5:0.5, hexanes/EtOAc) to provide 441.0 mg (85%) of 3,5-
cm-1
.
Data for 3b. It was isolated as an amorphous orange solid. Rf
(Hex/EtOAc, 7:3) ) 0.45. 1H NMR (300 MHz, CDCl3) δ 1.34 (s,
3
3H, CH3), 1.67 (s, 3H, CH3), 2.21 (dd, JH,H ) 6.7, 4.1 Hz, 1H),
4
3.72 (s, 3H, OCH3), 4.40 (m, 3H), 6.27 (d, JH,H ) 2.6 Hz, 1H),
6.40 (dd, 3JH,H ) 8.6 Hz, 4JH,H ) 2.5 Hz, 1H), 7.16 (d, 3JH,H ) 8.6
dibromo-2-(3-methylbut-2-enyloxy)benzaldehyde. 1H NMR
Hz, 1H), 7.54 (t, 3JH,H ) 7.5 Hz, 1H), 7.65 (t,3JH,H ) 7.5 Hz, 1H),
3
(CDCl3, 300 MHz) δ 1.46 (s, 3H), 1.64 (s, 3H), 4.51 (d, JH,H
)
3
3
3
7.85 (d, JH,H ) 7.8 Hz, 1H), 8.10 (d, JH,H ) 7.6 Hz, 1H) ppm.
13C NMR (75 MHz, CDCl3) δ 24.0 (CH3), 27.7 (CH3), 28.1 (CH),
38.1 (CH), 54.9 (OCH3), 64.4 (CH2), 81.6 (C), 100.8 (CH), 107.1
(CHarom), 113.5 (Carom), 115.1 (Carom), 124.6 (CHarom), 128.3
(CHarom), 130.2 (Carom), 130.5 (CHarom), 130.9 (CHarom), 132.5
(Carom), 134.7 (CHarom), 154.2 (Carom), 159.1 (Carom), 161.5 (Carom),
179.5 (C, CdO), 179.8 (C, CdO) ppm. EI-MS m/z (%) 376 (M+,
100), 361 (M+ - CH3,50). EI-HRMS 376.1304 [(M+) calcd for
C23H20O5 376.1311). IR (CHCl3) νmax ) 2982, 1680, 1646, 1578,
1503, 1463, 1364, 1336, 1269, 1240, 1202, 1164, 1129, 1079, 1036,
6.7 Hz, 2H), 6.82 (t, JH,H ) 6.4 Hz, 1H), 7.21 (s, 1H), 7.80 (d,
3JH,H ) 2.5 Hz, 1H), 10.15 (s, 1H) ppm. 13C NMR (CDCl3, 75
MHz) δ 17.7 (CH3), 25.2 (CH3), 71.8 (CH2), 117.6 (CHarom), 119.5
(Carom), 129.7 (CHarom), 132.2 (Carom), 140.7 (CHarom), 141.9 (C),
157.3 (C), 187.4 (C) ppm.
Reaction with 3,5-Dibromo-2-(3-methylbut-2-enyloxy)benzal-
dehyde. Following the general procedure described above, an
amount of 100 mg (0.57 mmol) of 2-hydroxy-1,4-naphthoquinone
in 10 mL of EtOH was treated with 0.310 mL (1.72 mmol,) of
3,5-dibromo-2-(3-methylbut-2-enyloxy)benzaldehyde and 5 mg of
EDDA (5.5 mg, 0.03 mmol). The reaction mixture was refluxed
for 25 min and cooled to room temperature and the solvent removed
under vacuum. The residue was purified by flash chromatography
using hexanes/EtOAc, 9:1, to yield 180.2 mg of the adduct 5a
(62%). Following the procedure B described above, the reaction
mixture was irradiated for 3 min at a preselected temperature of
130 °C, with an irradiation power of 100 W. The crude was purified
by preparative TLC using hexanes/EtOAc (7:3) to provide 42.3
mg (58.6%) of 5a.
987, 954, 833, 797, 756, 725, 665, 562, 478 cm-1
.
Reaction of 2-Hydroxynaphthoquinone with 4-Methoxy-2-[(3-
methyl-2-butenyl)oxy]-1-naphthaldehyde. Following the general
procedure described above, an amount of 100 mg (0.57 mmol) of
2-hydroxy-1,4-naphthoquinone in 10 mL of EtOH was treated with
413.48 mg (1.722 mmol) of 2-[(3-methyl-2-butenil)oxy]-1-naph-
thaldehyde and 5.5 mg of EDDA. The reaction mixture was refluxed
for 25 min, cooled to room temperature, and freed from solvent in
vacuum. The residue was purified by preparative TLC using
hexanes/EtOAc (9:1) to afford 60.1 mg (26.6%) of 4a and 100 mg
(44.3%) of 4b. Following the procedure B described above, the
reaction mixture was irradiated for 4 min at a preselected temper-
ature of 130 °C, with an irradiation power of 108 W. The crude
was purified by preparative TLC using hexanes/EtOAc (7:3) to
provide 18.7 mg of 4a (33%) and 18.7 mg (33%) of 4b.
Data for 5a. It was isolated as an amorphous yellow solid. Rf
1
(Hex/EtOAc 7:3) ) 0.5. H NMR (300 MHz, CDCl3) δ 1.20 (s,
3H, CH3), 1.62 (s, 3H, CH3), 2.21 (dd, 3JH,H ) 4.2 Hz,3JH,H ) 9.5
2
3
Hz, 1H), 4.48 (m, 3H), 4.56 (dd, JH,H ) 12 Hz, JH,H ) 3.4 Hz,
1H), 7.32 (s, 1H), 7.46 (s, 1H), 7.68 (m, 2H), 8.08 (m, 2H) ppm.
13C NMR (75 MHz, CDCl3) δ 23.6 (CH3), 27.3 (CH3), 29.3 (CH),
37.5 (CH2), 65.2 (CH2), 80.4 (C), 110.8 (Carom), 112.4 (Carom), 119.5
(Carom), 124.2 (Carom), 126.1 (CHarom), 126.3 (CHarom), 130.5 (Carom),
131.9 (CHarom + Carom), 133.0 (CHarom), 133.8 (CHarom), 134.1
(CHarom), 149.4 (Carom), 154.0 (Carom), 179.2 (C, CdO), 184.5 (C,
Data for 4a. It was isolated as an amorphous yellow solid. Rf
1
(Hex/AcOEt 7:3) ) 0.53. H NMR (300 MHz, CDCl3) δ 1.61 (s,
6H), 2.30 (m, 1H), 4.0 (t, 3JH,H )11.0 Hz, 1H), 4.41 (m, 1H), 4.87
(d, 3JH,H ) 4.6 Hz, 1H), 7.01 (d, 3JH,H ) 8.8 Hz, 1H), 7.42 (t, 3JH,H
) 7.1 Hz, 1H), 7.59 (m, 3H), 7.73 (d, 3JH,H ) 6.2 Hz, 2H), 7.80 (t,
CdO) ppm. EI-MS m/z (%) 505 (M+, 52), 503 (100), 488 (M+
-
CH3, 15). EI-HRMS 505.9381 [(M+) calcd para C22H16O4Br2
505.9374]. IR (CHCl3) νmax 2984, 1681, 1646, 1608, 1575, 1446,
1394, 1360, 1337, 1306, 1267, 1212, 1181, 1129, 988, 862, 755,
3
3
3JH,H ) 8.5 Hz, 2H), 8.05 (d, JH,H ) 7.1 Hz, 1H), 8.19 (d, JH,H
) 8.6 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 25.5 (CH3), 26.4
(CH), 26.6 (CH3), 38.9 (CH), 63.7 (CH2), 78.5 (C), 116.2 (Carom),
118.2 (CHarom), 120.1 (Carom), 123.0 (CHarom), 123.5 (CHarom), 126.2
(CHarom), 128.2 (CHarom), 128.9 (Carom), 129.0 (CHarom), 130.3
(Carom), 132.1 (Carom), 133.8 (CHarom), 133.9 (Carom), 134.2 (CHarom),
151.5 (Carom), 153.8 (Carom), 179.5 (C, CdO), 182.8 (C, CdO) ppm.
EI-MS m/z (%) 396 (M+, 100), 381 (M+ - Me, 14). EI-HRMS
396.1354 [(M+), calcd for C26 H20O4 396.1362]. IR (CHCl3) νmax
) 2922, 1730, 1622, 1594, 1568, 1512, 1474, 1374, 1349, 1328,
723, 668, 584 cm-1
.
Synthesis of 5-Bromo-2-(3-methylbut-2-enyloxy)benzaldehyde
(6). An amount of 301.53 mg (1.5 mmol) of 2-hydroxy-5-
bromobenzaldehyde was added to a solution of 1-bromo-3-
methylbut-2-ene (3 mmol, 0.345 mL) and Cs2CO3 (1.8 mmol, 586.7
mg) in dry THF (30 mL). The mixture was heated under reflux for
18 h under nitrogen atmosphere. Then the mixture was filtered and
the solvent removed under vacuum. The residue was treated with
an aqueous solution of 5% NaOH and extracted three times with
Et2O. The organic layers were dried over MgSO4. After removal
of solvent the crude was purified by flash chromatography (silica
gel, 9.5:0.5, hexanes/EtOAc) to provide 281.4 mg (70%) of
1300, 1262, 1203, 1131, 1008, 943, 805, 746, 720, 672, 470 cm-1
.
Data for 4b. It was isolated as an amorphous orange solid. Rf
(hexanes/EtOAc, 7:3) ) 0.45. 1H NMR (300 MHz, CDCl3) δ 1.62
(s, 3H), 1.65 (s, 3H), 2.30 (m, 1H,), 4.15 (t, 3JH,H ) 11.1 Hz, 1H),
3
3
4.43 (m, 1H), 4.79 (d, JH,H ) 4.0 Hz, 1H), 6.97 (d, JH,H ) 8.8

6770 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21
Jime´nez-Alonso et al.
5-bromo-2-(3-methylbut-2-enyloxy)benzaldehyde. 1H NMR
addition of 5 µL of stock solution (5% SDS, 25% ficoll, and 0.05%
bromphenol blue) followed by treatment with 0.25 mg/mL pro-
teinase K (Roche) at 55 °C for 30 min to eliminate the protein.
Samples were resolved by electrophoresis on a 1% (w/v) agarose
gel containing 0.5 µg/mL ethidium bromide in TAE buffer (100
mM Tris-acetate and 2 mM Na2EDTA, pH 8.3). DNA bands were
visualized by UV and photographed and documented with Quan-
tityOne (BioRad).
For quantitation of the substrate (kDNA) and products of the
TopoII reaction (Nck, SC, and catenanes), the numerical values of
intensity profiles of the different lanes were obtained after
background substraction and exported to Microsoft Excel. For each
lane, the overall intensity was used to obtain the fraction of intensity
for the different bands (kDNA, Nck, and SC). Here, we measured
hTopoIIR activity based on the final products (nicked plus super-
coiled minicircles). Positive control (1% DMSO, +hTopoIIR) and
negative control (1% DMSO, -hTopoIIR) were used to set up the
limit values (1 and 0, respectively).
TopoII-Mediated DNA Relaxation Assay. The TopoII drug kit
from TopoGEN (Columbus, OH) was used. The 20 µL reaction
mixture contained 250 ng of pRYG plasmid DNA and 0.5 mM
ATP in the assay buffer [10 mM Tris-HCl, 50 mM KCl, 50 mM
NaCl, 0.1 mM EDTA, 5 mM MgCl2, and 2.5% (v/v) glycerol, pH
8.0]. Chemical compounds were at 100 µM in 1% DMSO. The
reaction components were added as follows: assay buffer, DNA,
chemical compound from DMSO stock or just DMSO, and finally
1-2 units of hTopoIIR. The reaction mixture was incubated at 37
°C for 30 min, and the reaction was quenched with 1% (w/v) SDS
and 25 mM Na2EDTA. The mixture was treated with 0.25 mg/mL
proteinase K (Roche) at 55 °C for 30 min to digest the protein.
pRYG DNA topoisomers were resolved in 1% (w/v) agarose gel
electrophoresis in 1× TBE buffer (89 mM Tris-borate and 2 mM
Na2EDTA, pH 8) without ethidium bromide. The photograph was
taken as above after staining with ethidium bromide.
Quantitation of the products was done as above only that the
SC form was considered as substrate of the reaction and the sum
of all topoisomers (and nicked DNA) as products.
Stabilization of the Cleavage Complex. The same TopoII drug
kit was used as above, but this time an amount of 4-8 units of
hTopoIIR was added 1-3 min before the compound addition.
Electrophoresis was run in TAE buffer with 0.5 µg/mL ethidium
bromide.
DNA Intercalation Assay. Negatively supercoiled pBSKS plas-
mid was prepared using a plasmid mega kit (Qiagen) as described
by the manufacturer. An amount of 250 ng of pBSKS was then
incubated for 10 min at 37 °C with 100 µM of the analyzed drugs
in DMSO 1% (v/v) and loaded in a 1% (w/v) agarose gel.
Electrophoresis was carried out in 1× TBE buffer without ethidium
bromide (0.5 V/cm).
Molecular Docking. The crystal structure of hTopoIIR was
retrieved from the Protein Data Bank (PDB entry 1ZXM).62 The
hydrogen atoms of the protein were added using the software
package ADT Tools. Protein atom types and potentials were
assigned according to the Amber 4.0 force field with Kollman
united-atom charges,63 and the magnesium ion was assigned a
charge of +0.9. The initial structures of naphthoquinones were
optimized using Gaussian 03 at level HF/3-21G with RESP charges.
The minimized geometry was used to prepare the ligands for their
use in docking program AutoDock 3.0.5 (http://www.scripps.edu/
mb/olson/doc/autodock/) following the standard procedure. The
three-dimensional grid with 60 × 60 × 60 points and a spacing of
0.375 Å was created by the AutoGrid algorithm to evaluate the
binding energies between the ligands and the proteins. Default
docking parameters were used except number of generations,
population size, and docking runs, which are fixed at 27 000, 100,
and 25, respectively. The Lamarckian genetic algorithm63 was
applied to analyze protein-ligand interactions. The docked struc-
tures of the ligands were generated after a reasonable number of
evaluations. Figures for best scoring dockings were generated by
PyMOL.64
(CDCl3, 300 MHz) δ 1.69 (s, 3H, CH3), 1.73 (s, 3H, CH3), 4.53
3
3
(d, JH,H ) 7.7 Hz, 2H), 6.82 (d, JH,H ) 8.9 Hz, 1H), 7.49 (dd,
3JH,H ) 8.8, 4JH,H ) 2.6 Hz, 1H), 7.7 (d, 3JH,H ) 2.5 Hz, 1H), 10.3
(s, 1H) ppm. 13C NMR (CDCl3, 75 MHz) δ 14.6 (CH3), 18.7 (CH3),
66.2 (CH2), 113.6 (C), 115.4 (CH), 118.9 (CH), 126.6 (C), 131.0
(CH), 138.4 (CH), 139.5 (C), 160.5 (C), 187.5 (CH, CHO).
Reaction of 2-Hydroxy-1,4-naphthoquinone with 5-Bromo-2-
(3-methylbut-2-enyloxy)benzaldehyde. Following the general pro-
cedure described above, an amount of 50 mg (0.26 mmol) of
2-hydroxy-1,4-naphthoquinone in 10 mL of EtOH was treated with
212.2 mg (0.79 mmol) of 5-bromo-2-(3-methylbut-2-enyloxy)ben-
zaldehyde and 3.0 mg of EDDA. The reaction mixture was refluxed
for 28 min and cooled to room temperature and the solvent removed
under vacuum. The residue was purified by flash chromatography
using hexanes/EtOAc (4:1) to yield 39.5 mg (35.7%) of 6a and
40.5 mg (36.6%) of 6b. Following the procedure B described above,
the reaction mixture was irradiated for 3 min at a preselected
temperature of 130 °C, with an irradiation power of 110 W. The
crude was purified by preparative TLC using hexanes/EtOAc (7:3)
to provide 23.9 mg (39.2%) of 6a and 17.3 mg (28.4%) of 6b.
Data for 6a. It was isolated as an amorphous yellow solid. Rf
(Hex/EtOAct 7:3) ) 0.52. 1H NMR (300 MHz, CDCl3) δ 1.27 (s,
3H, CH3), 1.63 (s, 3H, CH3), 2.18 (m, 1H), 4.36 (dd, 2JH,H ) 12.1,
3
3JH,H ) 3.8 Hz, 1H), 4.47 (m, 2H), 6.63 (d, JH,H ) 8.6 Hz, 1H),
3
4
7.19 (dd, JH,H ) 8.5, JH,H ) 1.6 Hz, 1H), 7.35 (s, 1H), 7.71 (m,
2H), 8.13 (m, 2H,) ppm. 13C NMR (75 MHz, CDCl3) δ 23.6 (CH3),
27.3 (CH3), 29.0 (CH), 37.6 (CH), 64.4 (CH2), 88.5 (C), 112.7
(Carom), 117.7 (CHarom), 120.2 (Carom), 123.0 (Carom), 126.1 (CHarom),
126.3 (CHarom), 130.6 (Carom), 130.9 (CHarom), 132.0 (Carom), 132.3
(CHarom), 132.9 (CHarom), 134.0 (CHarom), 152.8 (Carom), 154.0
(Carom), 179.4 (C, CdO), 184.7 (C, CdO) ppm. EI-MS m/z (%)
426 (M+, 100), 408 (M+ - H2O, 18). EI-HRMS 426.0266 (calcd
for C22H17O4Br (M+) 426.0290). IR (CHCl3) νmax 2922, 2359, 1680,
1645, 1608, 1575, 1480, 1361, 1337, 1255, 1211, 1186, 1227, 1082,
988, 954, 815, 769, 723, 469 cm-1
.
Data for 6b. It was isolated as an amorphous orange solid. Rf
(Hex/EtOAc, 7:3) ) 0.32. 1H NMR (300 MHz, CDCl3) δ 1.27 (s,
3H, CH3), 1.65 (s, 3H, CH3), 2.21 (m, 1H), 4.34-4.45 (m, 3H),
3
3
4
6.60 (d, JH,H ) 8.6 Hz, 1H), 7.11 (dd, JH,H ) 8.8, JH,H ) 1.6
3
3
Hz, 1H), 7.25 (s, 1H), 7.51 (t, JH,H ) 7.5 Hz, 1H), 7.66 (t, JH,H
) 5.4 Hz, 1H), 7.82 (d, 3JH,H ) 7.41 Hz, 1H), 8.03 (d, 3JH,H ) 6.8
Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 23.8 (CH3), 27.8 (CH3),
28.5 (CH), 37.5 (CH), 64.5 (CH2), 81.8 (C), 112.8 (Carom), 113.9
(Carom), 117.6 (CHarom), 123.6 (Carom), 124.7 (CHarom), 128.3
(CHarom), 130.1 (Carom), 130.7 (CHarom), 131.1 (CHarom), 131.8
(Carom), 131.9 (CHarom), 134.8 (CHarom), 152.6 (Carom), 162.1 (Carom),
179.1 (C, CdO), 179.2 (C, CdO). EI MS m/z (%) 426 (M+, 100),
409 (M+ - H2O, 27), 354 (21), 327 (19). EI HRMS 426.0288
[(M+) calcd for C22H17O4Br 426.0290]. IR (CHCl3) νmax 2924,
1643, 1595, 1564, 1481, 1369, 1253, 1126, 1090, 816, 755, 543
cm-1
.
Biology. Biological Assay. The solvent for all the stocks of the
chemical agents employed was dimethyl sulfoxide (DMSO),
Molecular Biology grade (DNase and RNase-free), from Sigma-
Aldrich. Ellipticine and etoposide were purchased from Sigma-
Aldrich and stored at 10 mM in DMSO stock at -20 °C.
Synthesized pyranonaphthoquinones were also stored in DMSO as
a 10 mM stock at -20 °C until their use.
TopoII-Mediated DNA Decatenation Assay. The TopoII assay
kit from TopoGEN (Columbus, OH) was used. Purified hTopoIIR
was also purchased from the same vendor. DNA decatenation assays
were performed according to the manufacturer’s instructions and
earlier procedures61 with some minor modifications. The assay was
performed in a total reaction volume of 20 µL containing 50 mM
Tris-HCl (pH 8.0), 120 mM KCl, 10 mM MgCl2, 0.5 mM
dithiothreitol, 0.5 mM ATP, 30 µg/mL bovine serum albumin, and
150 ng of kDNA. All chemical compounds were then added to a
final concentration of 100 µM in 1% DMSO. Reactions were
initiated by addition of 2-4 units of hTopoIIR, and mixtures were
incubated for 30 min at 37 °C. The reaction was terminated by the

Pyranonaphthoquinones as Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 21 6771
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Acknowledgment. This work has been partly funded by the
Spanish MEC (Project SAF 2006-06720) and ICIC (Instituto
Canario de Investigacio´n del Ca´ncer) to A.E.-B. and by FIS
(Project PI06/1211) to F.M. Ramon y Cajal program (Spanish
Ministry of Science & Technology) contributes to the financial
support of F.M. S.J.-A. thanks the MEC for a predoctoral
fellowship. The authors also thank to B. Delgado and V.
Gonza´lez for granting access to the HLC apparatus.
(10) Lindsey, R. H. J.; Bromberg, K. D.; Felix, C. A.; Osheroff, N. 1,4-
Benzoquinone is a topoisomerase II poison. Biochemistry 2004, 43,
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Note Added after ASAP Publication. This manuscript was
released ASAP on September 25, 2008, with two references missing
from ref 51. The updated version was posted on October 17, 2008.
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Supporting Information Available: 1H NMR and13C NMR
spectra, purity determinations for all pyranonaphthoquinones
synthesized, and figures showing docking models of lapachones
and 2a. This material is available free of charge via the Internet at
http://pubs.acs.org.
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