Synthesis and Pharmacophore Modeling of Naphthoquinone Derivatives with Cytotoxic Activity
in Human Promyelocytic Leukemia HL-60 Cell Line
Elisa Pe ´rez-Sacau,†,‡Raquel G. Dı ´az-Pen ˜ate,‡,§Ana Este ´vez-Braun,*,†,‡Angel G. Ravelo,*,†,‡Jose M. Garcı ´a-Castellano,‡,§
Leonardo Pardo,|and Mercedes Campillo*,|
Instituto UniVersitario de Bio-Orga ´nica “Antonio Gonza ´lez”, UniVersidad de La Laguna, AVda. Astrofı ´sico Fco. Sa ´nchez 2, 38206 La Laguna,
Tenerife, Spain, Instituto Canario de InVestigaciones del Ca ´ncer, Unidad de InVestigacio ´n, Hospital de Gran Canaria “Dr. Negrı ´n”,
Las Palmas de Gran Canaria, and Laboratori de Medicina Computacional, Unitat de Bioestadı ´stica, Facultat de Medicina,
UniVersitat Auto `noma de Barcelona, Barcelona, Spain
ReceiVed July 20, 2006
Catalyst/HypoGen pharmacophore modeling approach and three-dimensional quantitative structure-activity
relationship (3D-QSAR)/comparative molecular similarity indices analysis (CoMSIA) methods have been
successfully applied to explain the cytotoxic activity of a set of 51 natural and synthesized naphthoquinone
derivatives tested in human promyelocytic leukemia HL-60 cell line. The computational models have
facilitated the identification of structural elements of the ligands that are key for antitumoral properties. The
four most salient features of the highly active ?-cycled-pyran-1,2-naphthoquinones [0.1 µM < IC50< 0.6
µM] are the hydrogen-bond interactions of the carbonyl groups at C-1 (HBA1) and C-2 (HBA2), the hydrogen-
bond interaction of the oxygen atom of the pyran ring (HBA3), and the interaction of methyl groups (HYD)
at the pyran ring with a hydrophobic area at the receptor. The moderately active 1,4-naphthoquinone
derivatives accurately fulfill only three of these features. The results of our study provide a valuable tool in
designing new and more potent cytotoxic analogues.
Cancer is, in the developed world, the second most common
cause of death after cardiovascular diseases.1Mass screening
programs of natural products by the National Cancer Institute
have identified the quinone moiety as an important pharma-
cophoric element for cytotoxic activity.2Lapachol 1 and several
natural related 1,4- and 1,2-naphthoquinones are associated with
numerous biological activities3like antibacterial,4fungicidal,4
antimalarial,5trypanocidal,6and antitumoral.7On the basis of
the biological and structural properties, 1,2- and 1,4-naphtho-
quinones are considered privileged structures in medicinal
chemistry.8This term describes selected structural types like
polycyclic heteroatomic systems, capable of orienting varied
substituent patterns in a well-defined three-dimensional space
and binding to multiple, unrelated classes of protein receptors
as high-affinity ligands.
The most important lapachol derivative with antitumoral
activity is ?-lapachone (3,4-dihydro-2,2-dimethyl-2H-naphtho-
[1,2-b]pyran-5,6-dione) 15. This compound induces cell death
in human cancer cells9without causing direct damage to DNA,10
showing low effect on nontumoral proliferative cells.11?-Lapa-
chone also has potent antitumor activity in xenografted human
cancer models with low host toxicity.9a
Although the ?-lapachone cytotoxic action has been known
for more than 20 years,12the detailed mechanism of action has
yet to be investigated. The first proposed biochemical target
for ?-lapachone was DNA topoisomerase I,13being the unique
drug that binds to the enzyme and inhibits its catalytic activity
without stabilizing the DNA-enzyme complex. The enzyme
NAD(P)H:quinone oxidoreductase (NQO1, Xip3, E.C. 220.127.116.11),
a ubiquitous flavoprotein found in most eukaryotes, is also key
for the action of ?-lapachone.9bNQO1 catalyzes a two-electron
reduction of quinones, like ?-lapachone or menadione, using
either NADH or NADPH as electron donor. Reduction of
?-lapachone by NQO1 increases its cytotoxic action, including
growth inhibition and apoptotic proteolysis. NQO1 is overex-
pressed in breast, colon, lung, and prostate tumors,14making
?-lapachone or/and its derivatives a potential therapeutic agent
for these kinds of tumors.15However, ?-lapachone elicits
different responses in cells with similar expression levels of
NQO1, such as NCM460 and SW480, or it elicits apoptosis in
cells lacking NQO1, such as HL-60 and MDAMB-468.9bIn
the human cell line HL60, derived from a promyelocytic
leukemia, ?-lapachone induces apoptosis by a reactive oxygen
species (ROS) and JNK-dependent pathway16and in a p53-
independent manner.17These findings suggest that the antitumor
activity of ?-lapachone is induced by different mechanisms of
action depending on the tumor cell type.
Presently, the structural requirements of quinone derivatives
inducing apoptosis are unknown, hampering the rational devel-
opment of new specific compounds. A pharmacophore definition
is known as the first essential step toward understanding the
interaction between a ligand and its target, and it is clearly
established as a successful computational tool for rational drug
design.18In the present work, with the aim of gaining insight
into the molecular determinants of action of this type of
compound, we have developed a pharmacophore model using
the Catalyst program (Catalyst, version 4.10; Molecular Simula-
tions Inc.: San Diego, CA, 2005) as a systematic and efficient
procedure. In addition, we have performed complementary three-
dimensional quantitative structure-activity relationship (3D-
QSAR) analysis19based on comparative molecular similarity
indices analysis (CoMSIA) studies.20These methodologies were
applied to a set of 51 bioactive naphthoquinones (NQ) tested
* To whom correspondence should be addressed: phone + 34 922
318576 (A.E.-B., A.G.R.), + 34 935 812348 (M.C.); fax + 34 922 318571
(A.E.-B., A.G.R.), + 34 935 812344 (M.C.); e-mail email@example.com
(A.E.-B.), firstname.lastname@example.org (A.G.R.), email@example.com (M.C.).
†Universidad de La Laguna.
‡Instituto Canario de Investigaciones del Ca ´ncer (http://www.icic.es).
§Hospital de Gran Canaria “Dr. Negrı ´n”.
|Universitat Autonoma de Barcelona.
J. Med. Chem. 2007, 50, 696-706
10.1021/jm060849b CCC: $37.00© 2007 American Chemical Society
Published on Web 01/24/2007
in a cytotoxicity assay on HL-60 cells to provide important
information on the active site of the target protein where these
NQ elicit their functions.
Chemistry. Most of the derivatives were obtained from
lapachol 1 and lawsone 53, which are bioactive naphthoquinones
isolated from plants of the Bignonaceae family. They are also
easily available commercial products.
(A) Synthesis of 1,4-Naphthoquinone Derivatives (Scheme
1). Compounds 2-4 were obtained from lapachol 1 by employ-
ing a variety of acylating agents of different electrophilicity and
lipophilicity in the presence of 2,6-lutidine as base. Compound
5 was achieved by treatment of 1 with the voluminous tert-
butyldimethylsilyl chloride, with triethylamine as base. Com-
pound 6, with a shorter side chain, was synthesized by Hooker’s
oxidation5bof 1. Derivatives 8-11 were also synthesized in
order to study the role of the side chain in their cytotoxic
activity. They were obtained by modifications of the exocyclic
double bond of derivative 2. Thus, compound 2 was treated
with m-CPBA to obtain the corresponding (()-epoxy derivative
8 in 60% yield, which, under treatment with HClO4in catalytic
amounts, afforded diol 9 in 96% yield. In addition, the reaction
of 2 with N-bromosuccinimide (NBS) intBuOH/H2O (1/1)
yielded compound 10 (94%) and the dibromo derivative 11
(3%). Hydrogenation of 1 with H2/Pd afforded compounds 12
and 13, the first one exhibiting a partial reduction of the A ring.
Treatment of 13 with methoxymethylchloride (MOMCl) pro-
duced compound 14, which contains a methoxymethyl ether
group at position C-2.
(B) Synthesis of Prenyl Pyran and Furan Naphthoquinones
(Scheme 2). Treatment of 1 with diluted H2SO4 produces a
tertiary carbocation that is intramolecularly trapped by the
hydroxyl group located on carbon C-2 or by the oxygen located
at C-4, yielding ?-lapachone 15 (angular tricyclic derivative)
and R-lapachone 36 (linear tricyclic derivative), respectively.21
Cyclization of 1 with m-chloroperoxybenzoic acid (m-CPBA),
via an epoxide intermediate, rendered the furan and pyran
naphthoquinones 18, 30, 32, and 52 in their racemic forms.
Reaction of 1 with 2 equiv of ammonium cerium(IV) nitrate
[(NH4)2Ce(NO3)6] yielded the linear furan naphthoquinones 33
and 34. Treatment of 1 with NBS in CH2Cl2 afforded com-
pounds 19, 20, 22, and 31. Compound 19 was also obtained in
higher yield (93%) from 2 and Br2/CH2Cl2. This reaction also
afforded the ?-furan isomer 21 as a minor compound.
The (() ester derivatives 23-27 were synthesized from the
racemic alcohol 52 by employing a variety of acylating agents
such as acetyl, isopropyl, lauroyl, p-bromobenzoyl, and p-
cyanobenzoyl chlorides. The stereoisomers 28 and 29 were
obtained from the esterification of 52 with (R)-R-methoxyphe-
nylacetic acid in the presence of DCC (dicyclohexylcarbodi-
imide) and catalytic amounts of DMAP (dimethylaminopyri-
dine). The absolute configuration of these compounds was
established by following the method described by Riguera and
co-workers.22The enantiomeric alcohols 16 and 17 were
obtained by mild basic hydrolysis of the corresponding esters
28 and 29 with NaHCO3/MeOH at rt.
Reaction of compound 15 with hydroxylamine hydrochloride
(NH2OH‚HCl) provided the oxime 39 in a regioselective form.
Compound 40 was obtained from 1, under treatment with NH2-
OH‚HCl and posterior cyclization with m-CPBA. The formation
of the nitro derivative 40 can be due to the formation of a
nitroso-phenol intermediate and a posterior oxidation with
(C) Synthesis of Non-prenyl Pyran and Furan Naphthoquino-
nes (Scheme 3). Compound 35 was obtained by treatment of
lawsone 53 with ceric ammonium nitrate (CAN) and styrene in
aqueous acetonitrile at 0 °C, via [3 + 2]-type cycloaddition.
The Knoevenagel condensation of lawsone 53 and paraform-
aldehyde (CH2O)n leads to a quinone methide intermediate,
which undergoes hetero Diels-Alder reaction with styrene or
ethyl vinyl ether as dienophiles, yielding in a one-pot reaction
the pyran naphthoquinones 37 or 38, respectively. When
2-methylfuran was used as dienophile, compound 7 was formed
instead of the expected pyran naphthoquinone derivative. The
formation of 7 is the result of an electrophilic substitution on
the furan ring, where the quinone methide intermediate acts as
(D) Synthesis of 1,4-Diazaphenanthrene Compounds and 9-
and 10-Membered Macrolactones (Scheme 4). 1,4-Diaza-
Scheme 1. Synthesis of 1,4-Naphthoquinone Derivativesa
aReagents and conditions: (i) KMnO4, NaOH (1%), 0 °C-rt. (ii) H2, dry THF, Pd/C, rt, 10 days. (iii) Dry CH2Cl2, 0 °C, lutidine, RCl. (iv) CH2Cl2,
0 °C, m-CPBA. (v) THF/H2O, HClO4, 0 °C. (vi)tBuOH/H2O, NBS, rt. (vii) Dry CH2Cl2, 0 °C,iPr2EtN, MOMCl.
Naphthoquinone DeriVatiVes with Cytotoxic ActiVityJournal of Medicinal Chemistry, 2007, Vol. 50, No. 4 697
phenanthrene derivatives 41-49 were synthesized by condensa-
tion of lapachol 1 and the o-quinones 15, 18, 19, 23, and 52
with 1,2-ethylendiamine or trans-1,2-diaminecyclohexane.23The
treatment of compounds 46 and 49 with ozone produced a
selective oxidative cleavage of the enol double bond shared by
rings B and C, leading to the corresponding macrolactones 50
Scheme 2. Synthesis of Prenyl Furan and Pyran Naphthoquinonesa
aReagents and conditions: (i) CH2Cl2, rt, NBS. (ii) CH3CN/H2O, 0 °C, CAN. (iii) Dry CH2Cl2, 0 °C, lutidine, AcCl. (iv) Dry CH2Cl2, Br2, rt. (v) CH2Cl2,
0 °C, m-CPBA. (vi) H2SO4, 0 °C-rt. (vii) Dry CH2Cl2, rt, DCC, DMAP, (R)-(-)-MPA. (ix) NaHCO3(0.1 M), rt, MeOH. (x) Dry CH2Cl2, 0 °C, Py RCOCl.
(xi) NH2OH‚HCl, NaOH 5%, 2 h.
Scheme 3. Synthesis of Non-prenyl Pyran and Furan
aReagents and conditions: (i) Dioxane, (CH2O)n, reflux, styrene. (ii)
CAN, CH3CN/H2O (3/1), 0 °C-rt, CH2dCHR. (iii) CH3CN/H2O (3/1),
CAN, 0 °C-rt, 2-methylfuran.
Scheme 4. Synthesis of Diazaphenanthrene Deriviatives and
aReagents and conditions: (i) 1,2-Ethylenediamine or trans-1,2-diamine
cyclohexane, molecular sieves 4 Å, toluene, ∆, 24 h-2 days. (ii) (a) O3,
-78 °C, dry CH2Cl2, 10-20 min; (b) Me2S.
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 Pe ´rez-Sacau et al.
Biological Assays. Cytotoxic assays were performed on
human promyelocytic leukemia HL-60 cell lines by use of the
MTT assay24(see Experimental Section). The concentrations
inducing a 50% inhibition of cell growth (IC50) in µΜ are
reported in Table 1 for p-1,4-naphthoquinones (opened deriva-
tives 1-14); in Table 2 for o-1,2-furan and pyran naphtho-
quinones (?-cycled derivatives 15-29); in Table 3 for p-1,4-
furan and pyran naphthoquinones (R-cycled derivatives 30-
38); and in Table 4 for nitrogenous derivatives and 1,4-
diazaphenanthrene derivatives (compounds 39-51). Compounds
1-51 were classified by their activity as highly active (IC50<
1 µM, +++), moderately active (1 µM < IC50< 10 µM, ++),
or inactive (10 µM > IC50, +). The most cytotoxic derivatives
belong to the o-naphthoquinone series (Table 2) with IC50values
in the 0.1-0.7 µM range, with the only exception being
compound 18 (IC50 ) 1.97 µM). Of the other series of
compounds, only the bromohydrine derivative 10 shows high
activity (IC50) 0.52 µM).
Three-Dimensional Pharmacophore Model for Naphtho-
quinone Derivatives. We have developed a three-dimensional
pharmacophore model for compounds 1-51 (Tables 1-4) with
the Catalyst software. The fixed, null, and configuration costs
are 133, 825, and 10 bits, respectively. The difference of 692
bits between fixed and null costs is a sign of highly predictive
hypotheses. The average linkage cluster method of Catalyst
rendes two groups of hypotheses. The hypothesis with the best
statistics (total cost of 220 bits) is employed throughout this
paper. The statistical significance of this hypothesis was assessed
by the Fisher method as implemented in the CatScramble
module. The IC50values were scrambled randomly 49 times,
and new hypotheses were generated. None of the outcome
hypotheses had a cost lower than the reported hypothesis (results
not shown). Thus, there is at least 98% probability that this
hypothesis represents true correlation in the data.25Figure 1
shows the structural features of the pharmacophore model
consisting of a hydrophobic region (HYD), three hydrogen-
Table 1. p-1,4-Naphthoquinone Derivatives (Bicyclic Compounds)
IC50( SD (mM)
exptl est Catalystest CoMSIA
3.18 ( 1.1
4.51 ( 0.8
26.26 ( 1.9
8.06 ( 3.1
11.44 ( 0.6
27.74 ( 4.4
68.25 ( 1.3
1.89 ( 0.5
48.12 ( 16.3
0.52 ( 0.4
2.23 ( 1.5
34.60 ( 7.1
62.13 ( 2.9
53.91 ( 1.6
aCompounds used to test the CoMSIA model.
Table 2. Pyran- and Furan-1,2-Naphthoquinone Derivatives (?-Cycled, Angular Tricyclic Compounds)
IC50( SD (µM)
exptlest Catalystest CoMSIA
0.27 ( 0.04
0.57 ( 0.02
0.55 ( 0.1
1.97 ( 0.3
0.13 ( 0.02
0.20 ( 0.1
0.70 ( 0.2
0.12 ( 0.02
0.68 ( 0.3
0.24 ( 0.04
0.26 ( 0.1
0.45 ( 0.2
0.13 ( 0.1
0.11 ( 0.01
0.26 ( 0.04
aCompounds used to test the CoMSIA model.
Naphthoquinone DeriVatiVes with Cytotoxic ActiVityJournal of Medicinal Chemistry, 2007, Vol. 50, No. 4 699
bond-acceptor groups (HBA1, HBA2 and HBA3), and six
excluded volumes. The theoretically predicted IC50values are
also listed in Tables 1-4. The good predictive power of this
model is indicated by the high correlation coefficient between
experimentally and theoretically predicted IC50 values (r )
0.937). In fact, only one +++ compound is not correctly
predicted in the ++ category (compound 10); only one ++
compound is incorrectly classified in the + category (compound
10); and all but four + compounds (3, 5, 36, and 38) were
predicted correctly. All compounds that are highly active (+++)
fit the HYD, HBA1, HBA2, and HBA3 pharmacophoric features
with the exception of compound 10.
Three-Dimensional QSAR/CoMSIA Model for Naphtho-
quinone Derivatives. Three-dimensional QSAR/CoMSIA analy-
sis was performed on naphthoquinones 1-51 (Tables 1-4).
Randomly chosen compounds 12, 20, 32, and 45 were not
included in the training set in order to test the derived CoMSIA
model predictiveness. The inactive compounds 42, 50, and 51
were not included in the CoMSIA model, because their residual
values were greater than two standard deviations. The experi-
mentally determined IC50values were related to the independent
variables by the PLS methodology (see Experimental Section).
Table 5 shows the statistical properties of the model. From a
statistical viewpoint, the value of the obtained cross-validated
correlation coefficient q2(0.627) reveals that the model is a
useful tool for predicting the biological activity. The correlation
coefficient between theoretically predicted (see Tables 1-4) and
experimentally determined IC50values is 0.933. As a further
test of robustness, the CoMSIA model was applied to the
excluded ligands 12, 20, 32, and 45. Clearly the theoretically
predicted values for these compounds (marked in Tables 1-4)
are in agreement with the experimentally determined ones. The
relative contributions of the CoMSIA models for the electro-
static, steric, hydrogen-bond-donor and -acceptor, hydrophobic,
and solvation terms are also shown in Table 5. Figure 2
illustrates the CoMSIA (i) steric, (ii) electrostatic, (iii) hydro-
phobic, (iv) hydrogen-bond-acceptor and (v) hydrogen-bond-
donor fields on the receptor maps (the color code of the maps
is described in the caption).
Table 3. Pyran- (n ) 1) and Furan- (n ) 0) 1,4-Naphthoquinone Derivatives (R-Cycled, Linear Tricyclic Compounds)
IC50( SD (µM)
1.35 ( 0.1
1.36 ( 0.4
1.85 ( 0.4
1.24 ( 0.1
1.46 ( 0.6
2.65 ( 0.2
13.46 ( 2.4
22.91 ( 3.6
29.66 ( 0.1
est Catalystest CoMSIA
aCompounds used to test the CoMSIA model.
Table 4. Nitrogenous Derivatives
IC50( SD (mM)
R, X, Y
X ) NOH
Y ) O
X ) NO2
Y ) OH
exptl est CoMSIA
11Me3.20 ( 0.32.21.5 4.6
HH46.97 ( 12.0)
15.53 ( 1.1
13.39 ( 5.7
24.16 ( 9.2
31.88 ( 10.9
23.68 ( 7.0
28.25 ( 5.3
24.10 ( 4.5
44.08 ( 9.6
47.10 ( 7.9
aCompounds used to test the CoMSIA model.
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4Pe ´rez-Sacau et al.
Pyran-1,2-Daphthoquinone Derivatives (?-Cycled, Angu-
lar Tricyclic Compounds). All pyran-1,2-naphthoquinones
(n ) 1) are highly active (+++) compounds (see Table 2).
These compounds share all the chemical features found in the
pharmacophore model. Figure 1A shows compound 15 placed
into the model. Clearly, the carbonyl groups at the C-1 (HBA1)
and C-2 (HBA2) positions, and the oxygen atom of the pyran
ring (HBA3), act as hydrogen-bond acceptors in a hydrogen-
bond interaction with the receptor, whereas the HYD feature is
created in this selected set of ligands by the methyl groups at
the C-13 position of the pyran ring (R1 ) R2 ) Me). The
CoMSIA analysis reproduces similar structural determinants
(Figure 2A). The electrostatic map (Figure 2Aii) and H-bond-
donor map (Figure 2Av) on the receptor show magenta and
orange areas at the upper right side near the carbonyl groups at
C-1 and C-2, which are similar to the HBA1 and HBA2
pharmacophoric elements. In agreement with this hypothesis
that hydrogen-bond-donor groups at the receptor interact with
the two carbonyl groups, the steric map (Figure 2Ai) shows an
unfavorable yellow area at this position where the receptor
would be located. The steric map also contains a favorable green
area similar to the HYD feature (Figure 2Ai, lower right side).
However, the HBA3 pharmacophoric element is not reproduced
in the CoMSIA analysis.
Compound 39 is, among the ligands described in Table 4,
the only one showing moderate activity (++). Remarkably, this
compound has in common with the pyran-1,2-naphthoquinones
the HBA2 carbonyl group, the HBA3 oxygen atom at the pyran
ring, and the HYD methyl groups. However, the HBA1 feature
in 39 is attained by the oxime -CdNOH group rather than the
carbonyl group at the C-1 position of the pyran-1,2-naphtho-
quinones. The longer -CdNOH group is probably not optimal
for interacting with the receptor at this part of the molecule. In
contrast to the carbonyl group, the NOH can also act as
hydrogen-bond donor. The -CdNOH moiety is predicted in
the CoMSIA model to interact with the cyan area at the upper
left side of Figures 2Aivand 3, where H-bond-acceptor groups
of the receptor are located.
The substitution of R3) H in 15 [IC50(15) ) 0.27] by polar
groups such as OH [IC50(16) ) 0.57 and IC50(17) ) 0.55] or
OAc [IC50 (23) ) 0.68] increases the IC50 values; whereas
substitution by Br [IC50(19) ) 0.13 and IC50(20) ) 0.20] or
by a voluminous and polar group (27, 28) decreases the IC50
values. This small dependence on the substituent of R3, ranging
from IC50(28) ) 0.11 to IC50(23) ) 0.68, is not represented
in the pharmacophore model and only partially represented in
the CoMSIA analysis. Steric (Figure 2Ai), electrostatic (Figure
2Aii), and H-bond acceptor (Figure 2Aiv) fields on the receptor
maps show green, red, and cyan areas, respectively, indicating
that the R3substitution modulates the IC50values. The hydrogen-
bond-donor OH group of compounds 16 and 17 would fit the
cyan area where H-bond acceptor groups on the receptor should
be located; however, the IC50increase of compounds 16 and
17 (R3) OH) with respect to compound 15 (R3) H) is not
explained by the CoMSIA model in 1,2-naphthoquinones (?-
cycled). In contrast, the OH substitution in 1,4-naphthoquinones
(R-cycled) decreases the IC50 values in agreement with the
CoMSIA model, as will be discussed later. This different
behavior of 1,2- and 1,4-naphthoquinones having OH substitu-
tions supports the idea of different orientations in the interaction
with the receptor (see next section).
Pyran- (n ) 1) and Furan- (n ) 0) 1,4-Naphthoquinone
Derivatives (R-Cycled, Linear Tricyclic Compounds). None
of the synthesized 1,4-naphthoquinone derivatives shows high
cytotoxic activity, despite the fact that they contain chemical
features similar to the 1,2-naphthoquinones: that is, two
carbonyl moieties, the oxygen atom of the pyran or furan ring,
and two methyl groups (see Table 3). However, these common
elements show different spatial arrangement in the two types
of naphthoquinones. Figure 1B shows superposition of com-
pound 30 into the pharmacophoric model. The carbonyl group
at the C-4 position elicits the HBA2 feature, the oxygen atom
of the pyran ring elicits the HBA3 feature, and the methyl groups
of the pyran ring elicit the HYD feature. In contrast to 1,2-
naphthoquinones, the carbonyl group at the C-1 position of 1,4-
naphthoquinones is not coordinated with any HBA feature,
explaining the fact that this set of compounds is moderately
active (++) or inactive (+) (Table 3).
Figure 2B shows compound 30 placed into the CoMSIA
model. The hydrogen-bond-donor group R3) OH is predicted
to map to the cyan area (Figure 2Biv) where H-bond-acceptor
groups of the receptor are located. This fact explains the 10-
fold decrease of IC50between compounds 36 (R3) H) and 30
(R3) OH). The same effect is not observed in 1,2-naphtho-
quinones (see above).
Figure 1. Pharmacophore model for naphthoquinone derivatives 1-51
created with the HypoGen algorithm as implemented in Catalyst. The
structural features are a hydrophobic region (HYD, cyan), three
hydrogen-bond-acceptor groups (HBA1, HBA2, and HBA3, green), and
six excluded volumes (gray). The HYD feature is drawn as a globe,
whereas HBA features are shown as two globes due to the directional
nature of this chemical function. ?-Cycled-1,2-naphthoquinone 15 (A),
R-cycled-1,4-naphthoquinone 30 (B), and lapachol 1 (C , D) were
mapped onto the pharmacophore model.
Table 5. Statistical Results of Inhibition of Cell Growth (IC50) in the
aLeave-one-out correlation coefficient.bOptimal number of principal
components.cNumber of compounds.dNon-cross-validated correlation
coefficient.ePercentage of contribution.fOn the receptor.
Naphthoquinone DeriVatiVes with Cytotoxic ActiVityJournal of Medicinal Chemistry, 2007, Vol. 50, No. 4 701
1,4-Naphthoquinone Derivatives (Bicyclic Compounds).
1,4-Naphthoquinone derivatives (Table 1) contain two carbonyl
moieties, at positions similar to the carbonyls of the R-cycled
1,4-naphthoquinones, and an oxygenated substituent at C-2,
comparable to the oxygen atom of the pyran or furan ring of
R-cycled 1,4-naphthoquinones. The tricyclic 1,4-naphthoquino-
nes (R-cycled) are more rigid structures than the corresponding
bicyclic 1,4-naphthoquinones (opened), in which the R1and R2
substituents can adopt different conformations and, conse-
quently, achieve different binding modes into the pharmacophore
model. This effect is illustrated in Figure 1C,D for lapachol 1.
Figure 1D shows superposition of lapachol into the pharma-
cophoric model through the C-4 carbonyl group in HBA2, the
R1) OH substituent at C-2 in HBA3, and the isoprenyl chain
in HYD. Notably, the aliphatic chain at C-3 adopts the same
conformation as the pyran ring of R-cycled 1,4-naphthoquinones.
This mode of binding is, thus, similar to R-cycled 1,4-
naphthoquinones (see Figure 1B,D), and it would explain the
moderately active (++) or inactive (+) profile of this set of
compounds (Table 1). Figure 1C shows the other arrangement
of lapachol 1 into the pharmacophore model. In this case, the
C-1 carbonyl group, the hydroxyl group at C-2, and the carbonyl
group at C-4 act as hydrogen-bond acceptors mapping HBA1,
HBA2, and HBA3, respectively, whereas the aliphatic chain at
R2maps the HYD. However, the bicyclic 1,4-naphthoquinones
possess moderate activity or inactivity, in contrast with the high
cytotoxicity of ?-cycled 1,2-naphthoquinones [IC50(1) ) 3.18
vs IC50(15) ) 0.27]. This different behavior can be explained
by considering that the HYD methyl groups of 15 are forced
by the pyran ring to keep their spatial arrangement (see above),
whereas the aliphatic chain of 1 probably changes conformation
to be placed at the HYD feature, causing an energetic penalty.
The electronic nature of the C-2 carbonyl oxygen of 15 and the
OH substituent of 1 is very different. Both functional groups
map the HBA2 feature but in a different way. While the carbon
and oxygen atoms and the lone pairs of CdO are in the same
plane in 15, the carbon, oxygen, and hydrogen atoms and the
lone pairs of the OH group are in a tetrahedral arrangement in
1. Thus, the OH group of 1 cannot have a hydrogen-bond
interaction of the same magnitude as the CdO group of 15.
The replacement of the double bond of the isoprenyl chain
[IC50 (2) ) 4.51] by other groups has varied effects on the
cytotoxicity. Compound 10, with R2) CH2CHBrC(OH)(CH3)2,
presents a cytotoxic activity increase of 8.7-fold with respect
to compound 2 [IC50 (10) ) 0.52]. Figure 3 shows this
compound in orange into the hydrogen-bond-acceptor map of
the CoMSIA model. Clearly, the OH group maps the cyan area
at the lower right side of the figure, where H-bond-acceptor
groups at the receptor are located, explaining the significant
decrease in IC50of this compound. The CoMSIA model also
predicts an unfavorable purple area, which explains the inactivity
of 9 [IC50(9) ) 48.1], which presents two hydroxyl groups on
the isoprenyl chain [R2 ) CH2CH(OH)C(OH)(CH3)2]. The
hydroxyl group at C-2 maps onto this unfavorable area.
Nitrogenous Derivatives. The nitrogenous derivatives (40-
51, Table 4) are inactive compounds. Clearly, compounds 41-
51 occupy the yellow contour area in the steric map of Figure
1, near the C-1 and C-2 carbons, showing that occupancy of
this area by the heterocyclic ring of these compounds is
detrimental for cytotoxic activity.
In conclusion, our study has identified in detail the structural
elements of naphthoquinone derivatives that are keys for
cytotoxic activity in the HL-60 cell line. The independent
generation of a HypoGen pharmacophore model and a 3D-
Figure 2. Steric (i), electrostatic (ii), hydrophobic (iii), hydrogen-bond-acceptor (iv), and hydrogen-bond-donor (v) maps for the IC50CoMSIA
model. ?-Cycled-1,2-naphthoquinone 15 (A) and R-cycled-1,4-naphthoquinone 30 (B) are shown as reference structures. The color code of the
maps is as follows: (i) green or yellow areas depict zones of the space where occupancy by the ligands decreases or increases IC50, respectively;
(ii) areas where a high electron density provided by the ligand decreases or increases IC50are shown in red or blue, respectively; (iii) gray areas
define regions of space where hydrophobic groups are predicted to increase IC50; (iv) areas where H-bond acceptors on the receptor are predicted
to decrease or increase IC50are shown in cyan or purple, respectively; and (v) orange or magenta contours show areas where H-bond donor zones
on the receptor are predicted to increase or decrease IC50.
Figure 3. Hydrogen-bond acceptor on the receptor map for the IC50
CoMSIA model with compounds 10 (yellow) and 39 (green) shown as
reference structures. The color code for the contour maps is as described
for Figure 2.
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4Pe ´rez-Sacau et al.
QSAR/CoMSIA model, using the alignment obtained with the
former, has been shown to be a valuable tool for analysis. Highly
active compounds, such as ?-cycled pyran-1,2-naphthoquinones
[0.1 µM < IC50 < 0.57 µM], contain four pharmacophoric
features: three hydrogen-bond-acceptor groups and a hydro-
phobic region. These results provide the tools for the design
and synthesis of new ligands with high predetermined activities.
General. IR spectra were obtained on a Bruker IFS 55 spectro-
photometer. One-dimensional (1D) NMR spectra (1H and13C NMR)
were recorded on a Bruker AMX-300 spectrometer. Two-
dimensional NMR spectra were registered on Bruker AMX-400
spectrometer. Chemical shifts (δ) are expressed in parts per million
(ppm), and coupling constants (J) are given in hertz. Mass spectra
[electron ionization mass spectrometry (EI-MS) and high-resolution
(HR) EI-MS] were analyzed on Micromass Autospec. TLC 1500/
LS 25 Schleicher and Schuell foils were used for thin-layer
chromatography, while silica gel (0.2-0.63 mm) and Sephadex LH-
20 were used for column chromatography. Lapachol 1, ?-lapachone
15, and lawsone 53 were used as starting material to synthesize
the training set of naphthoquinones. Lapachol 1 was isolated by
extraction of the powdered wood of Tabebuia impetiginosa (Big-
noniaceae) with a cold solution of sodium carbonate 1%. ?-Lapa-
chone 15 was obtained by intramolecular cyclization of lapachol
using concentrated sulfuric acid.7cLawsone 53 (2-hydroxy-1,4-
naphthoquinone) was purchased from Sigma-Aldrich Co. Com-
pounds 2-10, 16-18, and 30-51 were obtained following the same
procedure described in refs 3, 5b, 7c, 21, and 23. Compounds 19-
23 were obtained in higher yield than previously reported methods
by following the procedure described below. The procedures for
the preparation of derivatives 11-14 and 23-29 are also described
below. Spectroscopic data of the new compounds 11, 14 and 24-
29, whose structures were rigorously elucidated and have not been
previously reported in the literature, are also included.
thalenyl Acetate (11). Compound 2 (262 mg) was treated with
1.1 equiv of NBS intBuOH/H2O (1/1). When the starting material
was consumed, the solvent was removed to half of its volume, the
crude material was extracted with ether, and the combined organic
extracts were dried over MgSO4. The mixture was purified on a
Sephadex LH-20 column (eluted with a mixture of hexanes/MeOH/
CHCl32/1/1) to yield 330 mg (94%) of 10 and 11 mg (3%) of 11.
Compound 10 showed spectroscopic data in agreement with those
reported previously in the literature.1H NMR (CDCl3, 300 MHz)
δ 8.12 (m, 2H), 7.76 (m, 2H), 3.60 (dd, J ) 10.1, 2.6 Hz, 1H),
3.04 (dd, J ) 12.9, 2.7 Hz, 1H), 2.81 (dd, J ) 12.9, 10.1 Hz, 1H),
2.41 (s, 3H), 1.86 (s, 3H), 1.85 (s, 3H).13C NMR (CDCl3, 75 MHz)
δ 185.4 (s), 177.8 (s), 168.1 (s), 152.5 (s), 136.5 (s), 134.2 (d),
134.1 (d), 131.9 (s), 130.9 (s), 126.8 × 2 (d), 77.8 (d), 73.1 (s),
30.4 (t), 30.0 (q), 28.6 (q), 20.5 (q). EI-MS m/z 444 (M++ 2, 27),
442 (M+, 70), 361 (M+- Br, 58), 154 (100). HR-EI-MS m/z
441.9409 [(M+); calcd for C17H16O4Br2441.9415]. IR (CHCl3) νmax
(cm-1) 3527, 2970, 1777, 1677, 1595, 1430, 1370, 1340, 1296,
1173, 1068, 1015, 947, 878, 793, 733 cm-1.
Hydrogenation of Lapachol (1) To Obtain Derivatives 12 and
13. Compound 1 (183 mg) was dissolved in 10 mL of dry
tetrahydrofuran (THF). Catalytic amounts of Pd supported on carbon
were added and a stream of H2was passed through the mixture for
10 days. Then the crude product was filtered and the solvent was
eliminated under vacuum. The residue was chromatographed by
preparative TLC (eluted with a mixture of hexanes/acetate 10%),
yielding 50 mg (27%) of 12 and 94 mg (51%) of 13. Both showed
spectroscopic data identical to those reported previously in the
2-Isopentyl-3-(methoxymethoxy)naphthoquinone (14). Com-
pound 13 (40 mg) was treated with 1.6 equiv of methoxymethyl-
chloride (MOMCl) and 2 equiv of ethylisopropylamine (iPr2EtN)
in 4 mL of dry CH2Cl2at room temperature (rt) for 30 min. The
mixture was washed with H2O and the combined organic phases
were dried over MgSO4. After evaporation of the solvent, the
residue was chromatographed on a Sephadex LH-20 column (eluted
with a mixture of hexanes/MeOH/CHCl32/1/1), yielding 47 mg
(100%) of 14.1H NMR (CDCl3, 300 MHz) δ 8.05 (m, 2H), 7.69
(m, 2H), 5.43 (s, 2H), 3.57 (s, 3H), 2.64 (m, 2H), 1.63 (hept, J )
6.6 Hz, 1H), 1.39 (m, 2H), 0.96 (d, J ) 6.6 Hz, 6H).13C NMR
(CDCl3, 75 MHz) δ 185.2 (s), 181.4 (s), 155.3 (s), 137.3 (s), 133.8
(d), 133.2 (d), 132.0 (s), 131.3 (s), 126.2 × 2 (d), 98.4 (t), 57.6
(q), 37.8 (t), 28.6 (d), 22.4 × 2 (q), 22.1 (t). EI-MS m/z 288 (M+,
0.5), 257 (M+- OMe, 0.4), 244 (M+- MOM, 37), 188 (100).
HR-EI-MS m/z 288.1371 [(M+); calcd for C17H20O4288.1362].
Reaction of 1 with NBS To Obtain Derivatives 19, 20, 22,
and 31. Compound 1 (2.25 g) was treated with 2 equiv of NBS in
CH2Cl2for 24 h. Then the solvent was removed under vacuum
and the residue was purified by flash chromatography on silica gel,
eluted with hexanes/EtOAc 3/2, to yield 875 mg (29%) of 19, 752
mg (25%) of 12-bromo-?-dehydrolapachone 20, 773 mg (25%) of
22, 51 mg (2%) of 31, and 6 mg (<1%) of the 12-bromo-R-
dehydrolapachone isomer. All of them showed spectroscopic data
identical to those reported previously in the literature.27
Reaction of 2 with Br2 To Obtain Derivatives 19 and 21.
Compound 2 (184 mg, 0.65 mmol) was treated with 1.0 equiv of
Br2in 10 mL of dry CH2Cl2at rt for 10 min. The solvent was
removed under vacuum and the crude product was purified by flash
chromatography (eluted with hexanes/EtOAc 40%) to yield 171
mg of compound 19 (93%) and 11 mg of compound 21 (5%), which
showed spectroscopic data identical to those reported.27
General Procedure for Preparation of the (() Ester Deriva-
tives 23-27. The racemic alcohol 52 was treated with 1.5 equiv
of the corresponding acyl chloride and 2 equiv of pyridine, in dry
CH2Cl2 at 0 °C. When the starting material was consumed (as
monitored by TLC), the reaction mixture was left to warm to rt,
the solvent was removed under reduced pressure, and the crude
product was chromatographed on silica gel with mixtures of hexane/
EtOAc as eluent. Compound 23 showed spectroscopic data identical
to those reported previously in the literature.28
chromen-3-yl 2-Methylpropanoate (24). Following the procedure
described above, 103 mg (0.39 mmol) of (()-52 was treated with
65 µL (1.5 equiv) of isobutyryl chloride and 65 µL (2 equiv) of
pyridine, in 6 mL of CH2Cl2at 0 °C for 18 h. The crude was purified
by flash chromathography with 5-20% hexanes/EtOAc, to yield
129 mg (100%) of 24.1H NMR (CDCl3, 300 MHz) δ 8.08 (d, J )
7.6 Hz, 1H), 7.83 (d, J ) 7.8 Hz, 1H), 7.67 (t, J ) 7.7 Hz, 1H),
7.54 (t, J ) 7.7 Hz, 1H), 5.12 (t, J ) 4.8 Hz, 1H), 2.83 (dd, J )
18.1, 5.0 Hz, 1H), 2.63 (dd, J ) 18.1, 4.6 Hz, 1H), 2.54 (qq, J )
7.0, 7.0 Hz, 1H), 1.47 (s, 3H), 1.44 (s, 3H), 1.47 (t, J ) 7.0 Hz,
6H).13C NMR (CDCl3, 75 MHz) δ 179.3 (s), 178.5 (s), 176.0 (s),
161.1 (s), 134.9 (d), 131.9 (s), 131.0 (d), 130.1 (s), 128.8 (d), 124.7
(d), 110.2 (s), 79.8 (d), 68.8 (d), 34.0 (d), 25.0 (q), 22.9 (q), 22.6
(t), 19.0 (q), 18.8 (q). EI-MS m/z 329 (M+, 2), 258 (M+- COPri,
2), 258 (M+- COPri- H2O, 100), 212 (240 - CO, 60), 71 (COPri,
46). HR-EI-MS m/z 329.1401 [(M++ 1); calcd for C19H21O5
chromen-3-yl Laurate (25). Following the general procedure
described above, 140 mg (0.54 mmol) of (()-52 was treated with
190 µL (1.5 equiv) of lauroyl chloride and 90 µL (2 equiv) of
pyridine, in 8 mL of CH2Cl2at 0 °C for 6 h. The crude product
was purified by flash chromathography, eluted with hexanes/EtOAc
9/1, to yield 208 mg (87%) of 25.1H NMR (CDCl3, 300 MHz) δ
8.03 (d, J ) 7.6 Hz, 1H), 7.81 (d, J ) 7.8 Hz, 1H), 7.64 (t, J ) 7.7
Hz, 1H), 7.50 (t, J ) 7.6 Hz, 1H), 5.11 (m, 1H), 2.78 (dd, J )
18.2, 4.9 Hz, 1H), 2.63 (dd, J ) 18.2, 4.3 Hz, 1H), 2.30 (m, 6H),
1.56 (m, 6H), 1.45 (s, 3H), 1.41 (s, 3H), 0.83 (m, 11H).13C NMR
(CDCl3, 75 MHz) δ 179.2 (s), 178.4 (s), 162.1 (s), 134.8 (d), 131.9
(s), 130.9 (d), 130.1 (s), 128.7 (d), 124.2 (d), 110.1 (s), 79.7 (s),
68.8 (d), 34.2 (t), 34.0 (t), 31.8 (t), 29.5 (t), 29.4 (t), 29.3 (t), 29.2
(t), 29.1 (t), 29.0 (t), 24.9 (s), 24.8 (q), 24.7 (t), 23.0 (t), 22.6 (q),
14.0 (q). EI-MS m/z 440 (M+, 7), 240 [M+- OCO(CH2)10CH3,
100]. HR-EI-MS m/z 440.2542 [(M+); calcd for C27H36O5440.2563].
Naphthoquinone DeriVatiVes with Cytotoxic ActiVityJournal of Medicinal Chemistry, 2007, Vol. 50, No. 4 703
chromen-3-yl 4-Bromobenzoate (26). (()-52 (133 mg, 0.51 mmol)
was treated with 170 mg (1.5 equiv) of p-bromobenzoyl chloride
and 80 µL (2 equiv) of pyridine, in 15 mL of CH2Cl2at 0 °C. The
crude product was purified by flash chromathography, eluted with
hexanes/EtOAc 9/1, to yield 12 mg (5%) of 26.1H NMR (CDCl3,
300 MHz) δ 8.03 (m, 2H), 7.99 (d, J ) 7.8 Hz, 2H), 7.83 (d, J )
7.8 Hz, 1H), 7.68 (m, 2H), 7.50 (m, H), 5.40 (m, 1H), 2.95 (dd,
J ) 18.3, 4.7 Hz, 1H), 2.85 (dd, J ) 18.3, 4.0 Hz, 1H), 1.55 (s,
3H), 1.52 (s, 3H).13C NMR (CDCl3, 75 MHz) δ 179.3 (s), 178.2
(s), 163.1 (s), 150.9 (s), 134.1 (d), 132.2 × 2 (d), 132.1 (s), 131.9
× 2 (d), 130.1 (s), 129.0 (d), 125.9 (d), 124.3 (d), 127.7 (s), 127.0
(s), 109.8 (s), 79.1 (s), 70.6 (d), 24.9 (q), 23.5 (q), 22.7 (t). EI-MS
m/z 442 (M++ 2, 8), 440 (M+, 8), 257 [M+- COp(Br)Ph, 61],
183 [COp(Br)Ph, 100]. HR-EI-MS m/z 440.0278 [(M+); calcd for
chromen-3-yl 4-Cyanobenzoate (27). (()-52 (130 mg, 0.50 mmol)
in 10 mL of CH2Cl2 was treated with 170 mg (2 equiv) of
p-cyanobenzoyl chloride and 100 µL (2.5 equiv) of pyridine, at 0
°C for 24 h. The crude product was purified by flash chromathog-
raphy, with 5-40% hexanes/EtOAc, to yield 51 mg (35%) of 27.
1H NMR (CDCl3, 300 MHz) δ 8.11 (d, J ) 7.6 Hz, 1H), 8.06 (d,
J ) 7.7 Hz, 2H), 7.89 (d, J ) 7.6 Hz, 1H), 7.68 (m, 3H), 7.58 (m,
1H), 5.40 (m, 1H), 2.95 (dd, J ) 18.4, 4.8 Hz, 1H), 2.85 (dd, J )
18.4, 4.0 Hz, 1H), 1.57 (s, 3H), 1.51 (s, 3H).13C NMR (CDCl3, 75
MHz) δ 179.1 (s), 178.5 (s), 163.9 (s), 161.1 (s), 135.0 (d), 133.2
(s), 132.3 × 2 (d), 131.8 (s), 131.2 (d), 130.6 (s), 130.2 × 2 (d),
129.0 (d), 124.3 (d), 117.7 (s), 116.9 (s), 109.8 (s), 79.1 (s), 70.6
(d), 24.9 (q), 23.5 (q), 22.7 (t). EI-MS m/z 387 (M+, 1), 258 [M+
- COp(CN)Ph, 1], 130 [COp(CN)Ph], 94 (100). HR-EI-MS m/z
387.3401 [(M+); calcd for C23H17O5N 387.3854].
Resolution of 3-Hydroxy-2,2-dimethyl-3,4-dihydro-2H-benzo-
[h]chromene-5,6-dione (52): Preparation of Esters 28 and 29.
(()-52 (149 mg, 0.58 mmol) in 6 mL of dry CH2Cl2was treated
with 192 mg (2 equiv) of (R)-R-methoxyphenylacetic acid, at rt
for 10 h in the presence of dicyclohexylcarbodiimide (2 equiv) and
dimethylaminopyridine (DMAP) in catalytic amounts. When the
starting material was consumed, the solvent was removed under
vacuum and the crude product was purified by flash chromathog-
raphy (silica gel, eluted with mixtures of hexanes/EtOAc increasing
the polarity from 5% to 40%) to yield 105 mg (45%) of the RS
diastereomer 28 and 117 mg (50%) of the RR diastereomer 29.
Both esters were hydrolyzed with NaHCO3(0.1 M) in methanol at
rt, to yield enantiomeric alcohols 16 and 17 in 36% and 58% yield,
chromen-3-yl (2R)-Methoxy(phenyl)ethanoate (28). [R]D25(CHCl3)
) -7.1H NMR (CDCl3, 300 MHz) δ 8.08 (d, J ) 7.6 Hz, 1H),
7.76 (d, J ) 7.7 Hz, 1H), 7.67 (t, J ) 7.6 Hz, 1H), 7.55 (t, J ) 7.7
Hz, 1H), 7.37 (m, 2H), 7.28 (m, 3H), 5.10 (t, J ) 4.2 Hz, 1H),
4.74 (s, 1H), 3.40 (s, 3H), 2.82 (dd, J ) 18.1, 4.9 Hz, 1H), 2.67
(dd, J ) 18.1, 4.7 Hz, 1H), 1.22 (s, 3H), 1.06 (s, 3H).13C NMR
(CDCl3, 75 MHz) δ 179.2 (s), 178.4 (s), 169.6 (s), 161.0 (s), 136.0
(s), 134.8 (d), 131.9 (s), 131.0 (d), 130.1 (s), 128.9 (d), 128.8 (d),
128.6 × 2 (d), 126.8 × 2 (d), 124.2 (d), 109.8 (s), 82.3 (d), 79.6
(s), 69.8 (d), 57.4 (q), 24.7 (q), 22.6 (t), 22.3 (q). EI-MS m/z 240
(M+- MPA, 28), 225 (240 - Me, 9), 121 (MPA - CO2, 100). IR
(CHCl3) νmax(cm-1): 2924, 2853, 1752, 1654, 1608, 1573, 1455,
1395, 1114, 1027, 754.
chromen-3-yl (2R)-Methoxy(phenyl)ethanoate (29). [R]D25(CHCl3)
) -412.1H NMR (CDCl3, 300 MHz) δ 8.05 (d, J ) 7.6 Hz, 1H),
7.78 (d, J ) 7.7 Hz, 1H), 7.66 (t, J ) 7.6 Hz, 1H), 7.55 (t, J ) 7.7
Hz, 1H), 7.24 (m, 2H), 7.09 (m, 3H), 5.04 (t, J ) 4.2 Hz, 1H),
4.73 (s, 1H), 3.38 (s, 3H), 2.57 (dd, J ) 18.3, 4.5 Hz, 1H), 2.41
(dd, J ) 18.3, 3.9 Hz, 1H), 1.46 (s, 3H), 1.34 (s, 3H).13C NMR
(CDCl3, 75 MHz) δ 179.2 (s), 178.4 (s), 169.6 (s), 161.0 (s), 136.0
(s), 134.8 (d), 131.9 (s), 131.0 (d), 130.1 (s), 128.9 (d), 128.8 (d),
128.6 × 2 (d), 127.2 × 2 (d), 124.2 (d), 109.8 (s), 82.3 (d), 79.6
(s), 69.8 (d), 57.4 (q), 24.7 (q), 22.6 (t), 22.3 (q). HR-EI-MS m/z
407.1501 [(M++ 1); calcd for C24H23O6407.1495].
chromene-5,6-dione (16). Hydrolysis of 28 was carried out with
NaHCO3 (0.1 M) in methanol at rt for 2 h. The residue was
extracted several times with CH2Cl2. The combined organic extracts
were washed with water, dried over MgSO4and then purified by
flash chromathography on silica gel (eluted with mixtures of
hexanes/AcOEt 30%), to yield 13 mg (36%) of 16 ([R]D25(CHCl3)
) +47.2) which showed spectroscopic data identical to those
reported in the literature.
chromene-5,6-dione (17). Hydrolysis of 29 was carried out
following the procedure described before to obtain 21 mg (58%)
of 17, which showed spectroscopic data identical to those for 16
except the optical rotation ([R]D25(CHCl3) ) -49.0).
Cell Culture. The human promyelocytic leukemia HL-60 cell
line established by Gallagher et al.29was cultured in suspension in
RPMI-1640 medium (Invitrogen) supplemented with 10% heat-
inactivated fetal bovine serum, penicillin (10 000 units/mL), and
streptomycin in a humidified atmosphere of 95% air and 5% CO2
at 37 °C. Cells were maintained at a density <1 × 106cells/mL.
Cells were resuspended in fresh medium 24 h before each treatment
to ensure exponential growth. Stock solutions (100 or 50 mM) of
lapachol derivatives were made in dimethyl sulfoxide (DMSO),
aliquoted, and stored at -80 °C. Further dilutions were made in
culture medium prior to use. In all experiments, the final concentra-
tion of DMSO did not exceed 0.5%, a concentration that is not
toxic to the cells.
Assay for Cytotoxicity. Cytotoxic assays were performed by
the MTT procedure.30Cells (1 × 104/well) were exposed to different
concentrations of the compounds in 96-well plates for 72 h at
37 °C. Controls and samples were always treated with the same
concentrations of vehicle (DMSO). Surviving cells were detected
on the basis of their ability to metabolize 3-[4,5-dimethylthiazol-
2-yl]2,5-diphenyltetrazolium bromide (MTT) into formazan crystals.
Optical density at 560 nm was used as a measure of cell viability.
The MTT dye reduction assay measures mitochondrial respiratory
function and can detect earlier than dye-exclusion methods. Cell
survival (%) ) (mean absorbance in treated wells/mean absorbance
in control wells) × 100. Concentrations inducing 50% inhibition
of cell growth (IC50) were determined graphically for each
experiment by use of the curve-fitting routine of Prism 2.0
(GraphPad) and the equation derived by De Lean et al.31
Pharmacophore Model. The pharmacophore model was gener-
ated with the HypoGen module of Catalyst 4.10. Compounds 1-51
were built de novo with standard options within the 2D/3D editor
sketcher of the program. In cases where the chirality of the active
form was not known, all possible stereoisomers were generated
and considered. The BEST conformational analysis procedure was
applied. The number of conformers was limited to a maximum of
250, with a 20 kcal/mol energy threshold above the calculated global
minimum. The experimentally determined IC50 values of com-
pounds 1-51 span about 2-3 orders of magnitude from IC50
(7) ) 68.2 µM to IC50(28) ) 0.1 µM. Therefore, the inactivity
spread, uncertainty, and spacing parameters were changed from the
default value of 3.0 to 1.5, 1.6, and 1.8, respectively, as proposed
by Accelrys for training sets with narrower activity span than usual.
The hydrogen-bond-acceptor (HBA) and -donor (HBD), hydro-
phobic (HYD), and aromatic ring (AR) chemical features were
considered for hypothesis generation and up to 10 excluded
volumes. Hypothesis selection was done by a cost analysis
procedure (represented in bit units) based on three terms: weight
cost (increases in a Gaussian form as the feature weight deviates
from an idealized value of 2.0); error cost (penalizes the deviation
between the estimated activities of the training set and their
experimentally determined values); and configuration cost (penalizes
the complexity of the hypothesis, should not exceed a maximum
value of 18). The error cost contributes the most in determining
the overall cost of a hypothesis. In addition, the costs of the ideal
hypothesis, the simplest possible hypothesis that fits the data with
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4Pe ´rez-Sacau et al.
minimal cost (fixed cost), and the null hypothesis in which the error
cost is high (null cost) are computed by the HypoGen module. These
fixed and null costs represent the minimum and maximum energy
cost values, respectively. Statistically significant hypotheses possess
total costs close to the fixed cost and far away from the null cost
Three-Dimensional QSAR/CoMSIA Model. A critical step in
CoMSIA is to select a proper alignment rule. Naphthoquinone
analogues were oriented in space by aligning each compound to
the pharmacophoric hypothesis obtained with the Catalyst software
(see above). The QSAR table consists of IC50as dependent variable
and the electrostatic, steric, hydrophobic, hydrogen-donor, and
hydrogen-acceptor fields and solvation energy as independent
variables. The atom-centered atomic charges used in CoMSIA to
evaluate the electrostatic contributions were computed from the
molecular electrostatic potential33by use of the 6-31G* basis set.
Solvation free energies (∆Gsolv) of 1-51 were calculated with the
PM3--SR5.42R procedure within the AMSOL 6.7.2 program.34
The potential fields were calculated at each lattice intersection of
a regularly spaced grid of 2 Å. An sp3carbon atom with a van der
Waals radius of 1.52 Å carrying a charge of +1.0 served as a probe
atom to calculate the fields with an attenuation factor of 0.3.35Partial
least-squares (PLS) analysis36was used to derive linear equations
from the resulting matrices. Leave-one-out (LOO) cross-validation
was employed to select the number of principal components and
to calculate the cross-validated statistics. The final CoMSIA model
was generated with non-cross-validation and the number of
components suggested by the LOO validation run. The 3D-QSAR/
CoMSIA study was carried out with the QSAR module of the
SYBYL 7.0 program37with default parameters.
Acknowledgment. This work was supported by MCYT
(SAF2003-04200-CO2-02), CICYT (SAF2004-07103-C02-02),
and Generalitat de Catalunya (SGR2005-00390). E.P.-S. thanks
Gobierno Auto ´nomo de Canarias for the short-stay fellowship
and ICIC for the postdoctoral fellowship.
(1) WHO Cancer, http://www.who.int/hpr/NPH/docs/gs_cancer.pdf, http://
(2) (a) Driscoll, J. S.; Hazard, G. F.; Wood, H. B.; Goldin, A. Structure-
antitumor activity relationships among quinone derivatives. Cancer
Chemother. Rep. Part 2 1974, 4, 1-362. (b) Liu, K. C.; Li, J.; Sakya,
S. Synthetic approaches to the 2003 new drugs. Mini-ReV. Med.
Chem. 2004, 4, 1105-1125.
(3) Ravelo, A.; Este ´vez-Braun, A.; Cha ´vez, H.; Pe ´rez-Sacau, E.; Mesa-
Siverio, D. Recent studies on natural products as anticancer agents.
Curr. Top. Med. Chem. 2004, 4, 241-65.
(4) Guiraud, P.; Steiman, R.; Campos-Takaki, G. M.; Seigle-Murandi,
F.; Simeon de Buochberg, M. S. Comparison of antibacterial and
antifungal activities of lapachol and ?-lapachone. Planta Med. 1994,
(5) (a) de Andrade-Neto, V. F.; Goulart, M. O. F.; da Silva Filho, J. F.;
da Silva, M. J.; Pinto, M. C. F. R.; Pinto, A. V.; Zalis, M. G.;
Carvalho, L. H.; Krettli, A. U. Antimalarial activity of phenazines
from lapachol, ?-lapachone and its derivatives against Plasmodium
falciparum in vitro and Plasmodium berghei in vivo. Bioorg. Med.
Chem. Lett. 2004, 14, 1145-49. (b) Pe ´rez-Sacau, E.; Este ´vez-Braun,
A.; Ravelo, A. G.; Gutie ´rrez, D.; Gime ´nez, A. Antiplasmodial activity
of naphthoquinones related to lapachol and ?-lapachone. Chem.
BiodiVersity 2005, 2, 264-274.
(6) (a) Goulart, M. O.; Zani, C. I.; Tonholo, J.; Freitas de Abreu, F. C.;
Oliveira, A. B.; Raaslan, D. S.; Starling, S.; Chiari, E. Trypanocidal
activity and redox potential of heterocyclic- and 2-hydroxy-naph-
thoquinones. Bioorg. Med. Chem. Lett. 1997, 7, 2043-2048. (b)
Ferreira, V.; Jorqueira, A.; Souza, A.; da Silva, M.; de Souza, M.;
Gouvea, R.; Rodrigues, C.; Pinto, A.; Castro, H.; Santos, D.; Araujo,
H.; Bourguignon, S. Trypanocidal agents with low cytotoxicity to
mammalian cell line: A comparison of the theoretical and biological
features of lapachone derivatives. Bioorg. Med. Chem. 2006, 14,
(7) (a) Fujiwara, A.; Mori, T.; Iida, A.; Ueda, S.; Nomura, T.; Tokuda,
H.; Nishino, H. Antitumor-Promoting Naphthoquinones from Catalpa
oVata. J. Nat. Prod. 1998, 61, 629-632. (b) Krishnan, P.; Bastow,
K. F. Novel mechanisms of DNA topoisomerase II inhibition by
pyranonaphthoquinone derivativesseleutherin, R-lapachone, and
?-lapachone. Biochem. Pharmacol. 2000, 60, 1367-1379. (c) Pe ´rez-
Sacau, E.; Este ´vez-Braun, A.; Ravelo, A. G.; Ferro, E.; Tokuda, H.;
Mukainaka, T.; Nishino, H. Inhibitory effects of lapachol derivatives
on Epstein-Barr virus activation. Bioorg. Med. Chem. 2003, 11, 483-
488. (d) Suzuki, M.; Amano, M.; Choi, J.; Park, H.; Williams, B.;
Ono, K.; Song, C. Synergistic effects of radiation and ?-lapachone
in DU-145 human prostate cancer cells in vitro. Radiat. Res. 2006,
165, 525-531. (e) Lee, J.; Choi, D.; Chung, H.; Seo, H.; Woo, H.;
Choi, B.; Choi, Y. Beta-lapachone induces growth inhibition and
apoptosis in bladder cancer cells by modulation of Bcl-2 family and
activation of caspases. Exp. Oncol. 2006, 28, 30-35. (f) Woo, H.;
Park, K.; Rhu, C.; Lee, W.; Choi, B.; Kim, G.; Park, Y.; Choi, Y.
?-Lapachone, a quinone isolated from Tabebuia aVellanedae, induces
apoptosis in HepG2 hepatoma cell line through induction of Bax
and activation of caspase. J. Med. Food 2006, 9, 161-168.
(8) (a) Evans, B. E.; Rittle, K. E.; Bock, M. G.; Di Pardo, R. M.;
Freindinger, R. M.; Whitter, W. L.; Lundell, G. F.; Veber, D. F.;
Anderson, P. S.; Chang, R. S. L.; Lotte, V. J.; Cerino, D. J.; Chen,
T. B.; Kling, P. J.; Kimkel, K. A.; Springer, J. P.; Hirshfield, J.
Methods for drug discovery: development of potent, selective, orally
effective cholecystokinin antagonists. J. Med. Chem. 1988, 31, 2235-
2246. (b) Mason, J. S.; Morize, I.; Menard, P. R.; Cheney, D. L.;
Hulme, C.; Labandiniere, R. F. New 4-Point Pharmacophore Method
for Molecular Similarity and Diversity Applications: Overview of
the Method and Applications, Including a Novel Approach to the
Design of Combinatorial Libraries Containing Privileged Substruc-
tures. J. Med. Chem. 1999, 42, 3251-3264.
(9) (a) Li, C. J.; Li, Y. Z.; Pinto, A. V.; Pardee, A. B. Potent inhibition
of tumor survival in vivo by ?-lapachone plus taxol: combining drugs
imposes different artificial checkpoints. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 13369-13374. (b) Pink, J. J.; Planchon, S. M.; Tagliarino,
C.; Varnes, M. E.; Siegel, D.; Boothman, D. A. NAD(P)H: quinone
oxidoreductase activity is the principal determinant of ?-lapachone
cytotoxicity. J. Biol. Chem. 2000, 275, 5416-5424.
(10) Pardee, A. B.; Li, Y. Z.; Li, C. J. Cancer therapy with ?-lapachone.
Curr. Cancer Drug Targets 2002, 2, 227-242.
(11) Li, Y.; Sun, X.; LaMont, J. T.; Pardee, A. B.; Li, C. J. Selective
killing of cancer cells by ?-lapachone: Direct checkpoint activation
as a strategy against cancer. Proc. Natl. Acad. Sci. U.S.A. 2003, 100
(12) Boorstein, R. J.; Pardee, A. B. Coordinate inhibition of DNA synthesis
and thymidylate synthase activity following DNA damage and repair.
Biochem. Biophys. Res. Commun. 1983, 117 (1), 30-36.
(13) Li, C. J.; Averboukh, L.; Pardee, A. B. ?-Lapachone, a novel DNA
topoisomerase I inhibitor with a mode of action different from
camptothecin. J. Biol. Chem. 1993, 268 (30), 22463-22468.
(14) (a) Malkinson, A. M.; Siegel, D.; Forrest, G. L.; Gazdar, A. F.; Oie,
H. K.; Chan, D. C.; Bunn, P. A.; Mabry, M.; Dykes, D. J.; Harrison,
S. D.; et al. Elevated DT-diaphorase activity and messenger RNA
content in human non-small cell lung carcinoma: relationship to the
response of lung tumor xenografts to mitomycin Cl. Cancer Res.
1992, 52 (17), 4752-7. (b) Marin, A.; Lopez de Cerain, A.; Hamilton,
E.; Lewis, A. D.; Martı ´nez-Penuela, J. M.; Idoate, M. A.; Bello, J.
DT-diaphorase and cytochrome B5 reductase in human lung and
breast tumours. Br. J. Cancer. 1997, 76 (7), 923-9. (c) Ough, M.;
Lewis, A.; Bey, E. A.; Hinkhouse, M. M.; Ritchie, J. M.; Boothman,
D. A.; Oberley, L. W.; Cullen, J. J. Efficacy of ?-lapachone in
pancreatic cancer treatment: exploiting the novel, therapeutic target
NQO1. Cancer Biol. Ther. 2005, 4 (1), 95-102.
(15) Ashwell, M.; Chiang, J. L.; Tandon, M.; Liu, Y.; Lapierre, J.; Jiang,
Z. Pharmaceutical compositions of beta-lapachone and beta-lapachone
analogs with improved tumor targeting potential. U.S. Pat. Appl. Publ.
Patent 2006034796, 2006.
(16) Shiah, S. G.; Chuang, S. E.; Chau, Y. P.; Shen, S. C.; Kuo, M. L.
Activation of c-Jun NH2-terminal kinase and subsequent CPP32/
Yama during topoisomerase inhibitor ?-Lapachone-induced apoptosis
through an oxidation-dependent pathway. Cancer Res. 1999, 59 (2),
(17) (a) Planchon, S. M.; Wuerzberger, S.; Frydman, B.; Witiak, D. T.;
Hutson, P.; Church, D. R.; Wilding, G.; Boothman, D. A. ?-Lapa-
chone-mediated apoptosis in human promyelocytic leukemia (HL-
60) and human prostate cancer cells: a p53-independent response.
Cancer Res. 1995, 55 (17), 3706-11. (b) Planchon, S. M.; Pink, J.
J.; Tagliarino, C.; Bornmann, William, G.; Varnes, M. E.; Boothman,
D. A. ?-Lapachone-induced apoptosis in human prostate cancer
cells: involvement of NQO1/xip3. Exp. Cell Res. 2001, 267 (1), 95-
(18) (a) Kurogi, Y.; Gu ¨ner, O. F. Pharmacophore Modeling and Three-
Dimensional Database Searching for Drug Design Using Catalyst.
Curr. Med. Chem. 2001, 8, 1035-1055. (b) Gu ¨ner, O.; Clement,
O.; Kurogi, Y. Pharmacophore Modeling and Three-Dimensional
Database Searching for Drug Design Using Catalyst: Recent
Advances. Curr. Med. Chem. 2004, 11, 2991-3005.
Naphthoquinone DeriVatiVes with Cytotoxic ActiVityJournal of Medicinal Chemistry, 2007, Vol. 50, No. 4 705
(19) Green, S. M.; Marshall, G. R. 3D-QSAR: A current perspective. Download full-text
Trends Pharm. Sci. 1995, 16, 285-291.
(20) Klebe, G.; Abraham, U.; Mietzner, T. Molecular Similarity Indices
in a Comparative Analysis (CoMSIA) of drug molecules to correlate
and predict their biological activity. J. Med. Chem. 1994, 37, 4130-
(21) Ravelo, A.; Este ´vez-Braun, A.; Pe ´rez-Sacau, E. The chemistry and
biology of lapachol and related natural products R and ? lapachones.
Studies in Natural Products Chemistry, Atta-Ur-Rahman, Ed.;
Elsevier Science Publishers: Amsterdam, 2003, 29 (part J), 719-
760 and references herein.
(22) Seco, J. M.; Quı ´noa, E.; Riguera, R. The Assignment of Absolute
Configuration by NMR. Chem. ReV. 2004, 104, 17-117 and
references cited therein.
(23) Pe ´rez-Sacau, E.; Soto, J; Este ´vez-Braun, A.; Ravelo, A. G. Synthesis
of 9 and 10-membered macrolactones by selective ozonolysis of 1,4-
diazaphenanthrene derivatives. Tetrahedron 2005, 61, 437-445.
(24) Mosmann, T. Rapid colorimetric assay for cellular growth and
survival:application to proliferation and cytotoxicity assays.
J. Immunol. Methods 1983, 65, 55-63.
(25) Fischer, R. The Principle of Experimentation, Illustrated by a Psycho-
Physical Experiment. In The Design of Experiments, 8th ed.; Hafner
Publishing Co.: New York, 1966; Chapt. II.
(26) Silva, M. S.; Camara, C. A.; Barbosa, T. P.; Soares, A. Z.; da Cunha,
L. C.; Pinto, A. C.; Vargas, M. D. Molluscicidal activity of synthetic
lapachol amino and hydrogenated derivatives. Bioorg Med. Chem.
2005, 13 (1), 193-196.
(27) (a) Gupta, R. B.; Khanna, R. N. Bromination with N-bromosuccin-
imide: Part II. An unusual dibromination of ?-lapachone to 3′,4′-
dibromo-?-lapachone. Indian J. Chem., Sect. B 1980, 19B (1), 13-
16. (b) Gupta, R. B.; Khanna, R. N. Bromination with
N-bromosuccinimide: Part III. Formation of 3′-bromo-?-lapachone,
dehydro-iso-?-lapachone and 4′-bromo-iso-?-lapachone from lapa-
chol. Indian J. Chem., Sect B 1980, 19B (1), 17-19. (c) Kapoor,
N. K.; Gupta, R. B.; Khanna, R. N. Reaction of lapachol with lead
tetraacetate and N-bromosuccinimide. Indian J. Chem., Sect B 1981,
20B (6), 508-509.
(28) Sun, J. S.; Geiser, A. H.; Frydman, B. A preparative synthesis of
lapachol and related naphthoquinones. Tetrahedron Lett. 1998, 39
(29) Gallagher, R.; Collins, S.; Trujillo, J.; McCredie, K.; Ahearn, M.;
Tsai, S.; Metzgar, R.; Aulakh, G.; Ting, R.; Ruscetti, F.; Gallo, R.
Characterization of the continuous, differentiating myeloid cell line
(HL-60) from a patient with acute promyelocytic leukemia. Blood
1979, 54 (3), 713-33.
(30) Mosmann, T. Rapid colorimetric assay for cellular growth and
survival: application to proliferation and cytotoxicity assays.
J. Inmunol. Methods. 1983, 65, 55-63.
(31) De Lean, A.; Munson, P. J.; Rodbard, D. Simultaneous analysis of
families of sigmoidal curves: application to bioassay, radioligand
assay, and physiological dose-response curves. Am. J. Physiol. 1978,
(32) Li, H.; Sutter, J.; Hoffmann, R. Int. UniV. Line: La Jolla 2000, 174-
(33) Singh, U. C.; Kollman, P. A. An approach to computing electrostatic
charges for molecules. J. Comput. Chem. 1984, 5, 129-145.
(34) Hawkins, G. D.; Giesen, D. J.; Lynch, G. C.; Chambers, C. C.; Rossi,
I.; Storer, J. W.; Li, J.; Zhu, T.; Winget, P.; Rinaldi, D.; Liotard,
D. A.; Cramer, C. J.; Truhlar, D. G. AMSOL 6.7.2; University of
Minnesota, Minneapolis, MN 55455--0431.
(35) Bo ¨hm, M.; Stu ¨rzebecher, J.; Klebe, G. Three-dimensional quantitative
structure-activity relationship analyses using comparative molecular
field analysis and comparative molecular similarity indices analysis
to elucidate selectivity differences of inhibitors binding to trypsin,
thrombin, and factor Xa. J. Med. Chem. 1999, 42, 458-477.
(36) (a) Dunn, W. J. I.; Wold, S.; Edlund, U.; Hellberg, S.; Gasteiger, J.
Multivariate structure-activity relationships between data from a
battery of biological tests and an ensemble of structure descriptors:
The PLS method. Quant. Struct.-Act. Relat. 1984, 3, 131-137. (b)
Wold, S.; Ruhe, A.; Wold, H.; Dunn, W. J. I. The covariance problem
in linear regression. The partial least squares (PLS) approach to
generalizad inverses. SIAM J. Sci. Stat. Comput. 1984, 5, 735-743.
(c) Wold, S.; Albano, C.; Dunn, W. J. I.; Edlund, U.; Esbenson, W.;
Geladi, P.; Hellberg, S.; Johansson, E.; Lindberg, W.; Sjo ¨stro ¨m, M.
Multivariate data analysis in chemistry. In Chemometrics: Math-
ematics and Statistics in Chemistry; Kowalsky, B. R., Ed.; Reidel:
Dordrecht, The Netherlands, 1984; pp 17-95. (d) Cramer, R. D.;
Bunce, J. D.; Patterson, D. E. Crossvalidation, bootstrapping and
partial least squares compared with multiple regression in conven-
tional QSAR studies. Quant. Struct.-Act. Relat. 1988, 7, 18-25.
(37) SYBYL 7.0; Tripos Inc., 1699 South Hanley Rd., St. Louis, MO
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4Pe ´rez-Sacau et al.