Synthesis and structure - Activity relationships of new benzodioxinic lactones as potential anticancer drugs

Article abstract of DOI:10.1021/jm061184g

A set of disubstituted tetracyclic lactones has been synthesized and tested for potential antitumor activity. Several of them possess a noticeable cytotoxicity against L1210 and HT-29 colon cells in vitro. Relationships between chain nature and biological

Full text of DOI:10.1021/jm061184g

294
J. Med. Chem. 2007, 50, 294-307
Synthesis and Structure-Activity Relationships of New Benzodioxinic Lactones as Potential
Anticancer Drugs
Manel Romero,† Pierre Renard,‡ Daniel-Henry Caignard,‡ Ghanen Atassi,‡ Xavier Solans,§ Pere Constans,† Christian Bailly,| and
Maria Dolors Pujol*,†
Laboratori de Qu´ımica Farmace`utica (Unitat Associada al CSIC), Facultat de Farma`cia, UniVersitat de Barcelona, AV. Diagonal 643,
08028-Barcelona, Spain, Les Laboratoires SerVier, 1 rue Carle Hebert, 92415-CourbeVoie Cedex, France, Departament de Cristallografia,
Mineralogia i Dipo`sits Minerals, Facultat de Geologia, UniVersitat de Barcelona, C/ Mart´ı i Franque`s s/n, 08028-Barcelona, Spain, and
INSERM U-524, IRCL, Place de Verdum, 59045 Lille, France
ReceiVed October 11, 2006
A set of disubstituted tetracyclic lactones has been synthesized and tested for potential antitumor activity.
Several of them possess a noticeable cytotoxicity against L1210 and HT-29 colon cells in Vitro. Relationships
between chain nature and biological properties were sought. Lactones with a pentyl or hexyl substituent at
C-11 are the most active ones. The introduction of a functional group at the side chain of C-11 modified the
potency; carboxylic acid and primary amine decreased the cytotoxicity, whereas a cyano group increased
the activity. An extensive structure-activity relationship study of these derivatives, including carbon
homologues and bioisosteres has been performed. The synthesis and cytotoxicity of these compounds are
discussed. Two lactones are recognized as potential lead compounds.
Introduction
great majority of the antitumor compounds, both alone and in
combination with other antitumor agents, is a reason for their
restricted deployment. These problems motivate the research
of new compounds. There has been an enormous amount of
effort directed toward the research of new anticancer agents,
related or not, to known compounds and the elucidation of their
structure-activity relationships.
The presence of polycyclic lactones in a wide variety of
antineoplastic compounds of natural or synthetic origin6 induced
us to introduce this structural motif in a new research line. It is
well documented that compounds possessing a butyrolactone
fused to a polycyclic system, such as podophyllotoxin7 or their
semi-synthetic analogues etoposide and teniposide,8 show an
interesting cytotoxic and antitumor activity.
The relationships between structure and cytotoxicity from
inhibition of topoisomerase II are not yet well established, and
the research of further classes of new antitumor agents remains
of high interest. We have previously studied 1,4-benzodioxino-
isoquinolines designed and synthesized as related analogues to
the ellipticine.9 They are formally derived by substitution of
the indole with a 1,4-benzodioxine subunit. This substitution
leads to analogues that retain a noticeable in Vitro cytotoxic
activity. Structurally, they possess a basic nitrogen atom suitable
for the DNA-intercalation. At a physiological pH, these
compounds are ionized and it is expected as a limitation of their
applicability. The present investigation was undertaken to
determine the effect of introducing a nonbasic lactone instead
of a pyridine. Therefore, we decided to further investigate a
novel tetracyclic system consisting on an 1,4-benzodioxine
skeleton fused with a benzofuranone, to form the new isoben-
zofuro[5,6-b][1,4]benzodioxines depicted in Chart 2. The aim
of the present study was to establish new structure requirements
for these new compounds and determine structural tolerances
for cytotoxic activity in cancer cell cultures.
Anticancer drugs have been classified according to the origin
or the mechanism of action involved as antitumor antibiotic,
antimetabolites, antitubulin agents, topoisomerase inhibitors,
alkylating agents, and miscellaneous agents.1 In recent years,
difficulties in classifying new antitumoral compounds have
arisen. Several of them exhibit multiple mechanisms of action
in tumor cells and many demonstrate a wide range of biochemi-
cal and pharmacological properties. Major progress in chemo-
therapy requires new and useful antitumor agents to eradicate
the entire range of cancer diseases in the world over. New
compounds can be developed by a wide variety of approaches,
ranging from empirical screening to rational design: (a) from
natural products, (b) by screening of synthetic compounds
prepared for other purposes, and (c) by semisynthesis or total
synthesis using new biological concepts and structure-activity
relationships of potentially interesting known compounds.2
It has been established that ellipticine, a drug isolated from
Ochrosia elliptica (Apocinaceae), has powerful antiproliferative
properties. Its chemical structure consists in a tetracyclic skeleton
composed of pyridine fused with the carbazole system (subunits
X and Y).3 Ellipticine and related compounds exert their
cytotoxic and antineoplastic effects by a multimodal mechanism
of biological action (inhibition of DNA-topoisomerase II, DNA-
intercalation, covalent binding to DNA, and redox generation
of cytotoxic free radicals).4 Several quaternary ellipticinium salts
(datelliptium, pazellipticine, and others) were previously reported
to possess high cytotoxic activity.5 Datelliptium and pazellip-
ticine bearing hydrophilic substituents on a pyridine ring which
increase water solubility were pursued as targets for synthesis
(Chart 1).5
These antitumor compounds are clinically effective, albeit
they might cause serious adverse reactions. The toxicity of the
* To whom correspondence should be addressed. Phone: 0034 93
4024534. Fax: 0034 93 4035941. E-mail: mdpujol@ub.es.
† Laboratori de Qu´ımica Farmace`utica, Facultat de Farma`cia, Universitat
de Barcelona.
We focused our attention on the lactone due to its appealing
antitumor activity for epipodophyllotoxins, such as etoposide,
tenoposide,8 and other topoisomerase II inhibitors containing
this moiety. The 1,4-benzodioxine, whose synthesis has been
developed by us, was also chosen as a heterocyclic bioisostere
‡ Laboratoires Servier.
§ Facultat de Geologia, Universitat de Barcelona.
| INSERM U-524, IRCL, Place de Verdum, 59045 Lille, France.
10.1021/jm061184g CCC: $37.00 © 2007 American Chemical Society
Published on Web 12/22/2006

Benzodioxinic Lactones as Anticancer Drugs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 295
Chart 1. Chemical Structures of Ellipticine, Datelliptium, and Etoposide
Chart 2. Design of Compounds 1-19 from Ellipticine
of indole.9 Structure-activity relationship studies and theoretical
determination of relevant parameters to their cytotoxic activity
were very useful in the design and development of new
compounds.
To our knowledge, the isobenzofuro[5,6-b][1,4]benzodioxine
system has not previously been incorporated into an antitumor
structure. Several modifications maintaining the same backbone
were carried out, as those indicated in Chart 2: (a) modification
of the R2 and R3 substituents, (b) modifications on the lactone
ring, and (c) alkylation of the 1,4-benzodioxine ring.
cycloaddition of the diene 48 and the 2(5H)-furanone at 90 °C.
The results shown in the Table 1 indicate that Yb(OTf)3 is the
best catalyst for this cycloaddition reaction. When TiCl4 as a
typical Lewis acid was used in CH2Cl2, the reaction could be
only poorly observed. Moreover, the reaction of cycloaddition
with SiO2 as catalyst did not lead to the desired product. On
the other hand, it was found that BF3‚Et2O catalyzed the Diels-
Alder cycloaddition in lower yield than ytterbium(III) trifluo-
romethanesulfonate.
The one-pot formation of tetracyclic systems was investigated
by using disubstituted dienes and lactone or thiolactone as
dienophiles under the most suitable conditions at 90 °C in the
presence of Lewis acid catalyst. It is noteworthy that a catalytic
amount of Lewis acid is enough to give directly a mixture of
two isomeric tetracyclic lactones (a, b). The diene 60 was
obtained with difficulties in little yield, possibly due to the
interaction of the heteroatom in the side chain with the reagents
used for its preparation. The reaction between diene 60 and
lactone 63 leads to the complex mixture of degradation
compounds (dicarbonyl compound, diol, etc.) instead of the
expected lactones.
The mixture of isomeric compounds proved difficult to
separate by usual methods, but finally, after extensive experi-
mentation, the mixture could be separated by meticulous column
chromatography on silica gel. The structure of the isomer
obtained was judged from 1H NMR and 13C NMR spectra. X-ray
experiments of the lactone 4a allowed us to unambiguously
ascribe structure a to the minor constituent of the mixture and
structure b to the main compound. Subsequent NMR studies
agreed well with the X-ray structure (see Supporting Informa-
tion).
To increase the number of structural changes, compounds
14, 15, 16, and 19 were synthesized by transformation of the
cyano-derivatives 12a and 13a. Thus, base hydrolysis of the
cyano-derivatives (12 and 13a) with 2 N NaOH gave the
corresponding carboxylic acids (14 and 15) in excellent yields.
Based on molecular modeling, best activity was expected for
the amine 16. Treatment of the appropriate nitrile with H2 in
Chemistry
Our initial task was to establish the most efficient method
for performing the synthesis of the tetracyclic lactones (1, 2a-
8a, 2b-8b) from the carboxylic acids 20 and 21. The results
are summarized in Schemes 1-3.
Treatment of 1,4-benzodioxin-6-yl carboxylic acids (20 and
21) with LDA and subsequent addition of the corresponding
aldehyde allowed the preparation of the desired hydroxyacids
(22-34). After the ring closure of the hydroxyacids (22-34)
using PTSA (p-toluenesulfonic acid monohydrate) as catalyst
in toluene at reflux led to the lactones (35-47) in very high
yields.10-11
The intermediate lactones (35-47) were treated with alkyl-
lithium followed by the addition of trimethylsilylchloride at -78
°C to give the furo-derivatives (48-62). These dienes were
relatively stable at low temperatures but unstable at room
temperature.12
Finally, the treatment of the furo[3,4-b][1,4]benzodioxines
(48-62) with the lactone (63) or the thiolactone (64) in acidic
media (Yb(OTf)3, BF3.Et2O, SnCl4, or TiCl4) gives a mixture
of tetracyclic lactones a and b in acceptable yields. Surprisingly,
the expected Diels-Alder mixture of exo-endo adducts was not
detected by reason of the facile cyclodehydration catalyzed by
Lewis acid of the intermediate 1,4-epoxy-derivatives to the
aromatic compounds. Aromatization of similar adducts by
dehydration was generally effected by using catalytic amounts
of acid.13 Several Lewis acids were tested as catalysts in the

296 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Scheme 1. Synthesis of Dienes 48-62
Romero et al.
methanol using PtO2 as catalyst at room temperature afforded
the corresponding amine in quantitative yields. The nucleophilic
treatment of the bromide 18a with methylamine hydrochloride
in basic media afforded the corresponding amine 19 in accept-
able yield (Scheme 3).
This analysis was performed using fully optimized ab initio
geometries, and electron density overlaps as a measure of
similarity. Geometries were found to be almost planar. The
density overlap alignment at the global maximum of the
similarity function is presented in Figure 1. Figures 1 and 2c,d
display the isocontour surface of the largest common pattern
density, see Computational Methods for details and definitions.
On the other hand, Figure 2a,b displays the electron density
isocontours for the two molecules. The complete set of figures
illustrates the extent of similarity, which embraces nearly the
whole molecule but half of ring A. In the figures, the surfaces
were plotted at the isocontour value of 0.03 and 0.032, in a.u.,
for the density and common pattern density, respectively, as
being this value is an approximate equivalent for molecular
accessible surface areas. Albeit being similar on an overall and
structural analysis, the two electronic densities differ in some
aspects. Differences on electronic rearrangements are manifest
on the electrostatic potential maps. There are important differ-
ences in charge distribution among the studied compounds due
to the presence of specific side groups. Figure 3 plots the density
Results and Discussion
Structure-Activity Relationship. Many experimental stud-
ies indicate that the size, shape, and planarity of the ellipticine
and derivatives are important factors for their mechanism of
cytotoxicity and antitumor activity.14 Furthermore, the acid-basic
properties of ellipticine have been estimated. It is known that
the pharmacological activities of drugs depend on their interac-
tion. They are expected to be significantly different for the
charged and uncharged drugs.15
A detailed similarity analysis, Figures 1 to 3, has shown a
remarkable structural coincidence of 1-oxo-3(H)-isobenzofuro-
[5,6-b][1,4]benzodioxine with the ellipticine nucleus. It is
important to point out that only the behavior of the tetracyclic
system was studied without considering the alkyl substituents.

Benzodioxinic Lactones as Anticancer Drugs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 297
Scheme 2. Synthesis of Tetracyclic Lactones by Diels-Alder Cycloaddition
isocontours colored according to the potential values. As
expected, the electronegative N and O atoms exhibit negative
charge. The polarized Nδ- - Hδ+ bond is also reflected in this
particular isosurface because the surface goes nearby the indole
H.
and (d) the nature of polycyclic lactone. First, with respect to
the substituents on ring A (Figure 4), in general, the substitution
of the H for a methoxy group presents a decreasing of activity
(see Table 2, 17a and 17b versus 5a and 5b).
A parallel examination of compounds 1 and 2a-8a, which
have alkyl substituents on ring C, shows that the cytotoxic
activity is best for compound 5a, which possesses a pentyl chain
on the C-11 and a methyl group on the C-4. The introduction
of substituents with less than four C or more than six C at the
C-11 position confers a decrease of cytotoxic activity. Com-
pound 5a, bearing a pentyl group on the C-11, showed an
increase in the potency relative to the one bearing a propyl group
substituent 3a. The butyl homologue 4a was less potent than
5a and more potent than the new analogue compounds bearing
a lower chain 3a, 2a, and 1.
All prepared lactones (compounds 1-19) were assayed in
Vitro for the ability to inhibit cell proliferation L1210 (murine
leukemia) and HT-29 (human colon carcinoma).16 Also, the
action over the phases of cell cycle was considered on the first
cell line (L1210). The results expressed as IC50 values (mean
of at least 4 determinations in triplicate obtained in independent
experiments with variations of <5%) are listed in Table 2. The
lactone 4a was also evaluated against 60 tumor cell lines in the
NCI (National Cancer Institute, MD).17 The cytotoxic activity
of these compounds was compared with the activity of ellipticine
and adriamycin, both known antitumor compounds. Ellipticine
and adriamycin were the chosen patterns for their structural
similarity and for possessing bulky side chains, respectively.
Some of these compounds were found to possess significant
activity at micromolar concentration. For analyzing structure-
activity relationships, four structural components are consid-
ered: (a) the presence of substituents on ring A, (b) the nature
of the ring C substituents, (c) position of the ring C substituents,
It is important to point out that the side chain at C-11 (the
same side of the lactone carbonyl) confers greater activity for
the inhibition of cell proliferation (4a, 5a, and 6a vs 4b, 5b,
and 6b). Thus, in general, compounds with the alkyl chain on
the C-11 (series a) are more active than compounds with this
chain on the C-4 (series b).
The lactones 5a and 6a showed similar cytotoxic activity,

298 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Scheme 3. Preparation of Lactones 14-16 and 19
Romero et al.
Table 1. Lewis Acids Tested as Catalyst in the Diels-Alder Reaction of 48a
b
b
b
b
catalyst
SiO2
0*
MgBr2
5*
BF3‚(CH3CH2)2Ob
SnCl4
15**
TiCl4
16**
Yb(CF3SO3)3
77***
yield (%)
0*
40**
a The symbol * designates a time of reaction ) 12 h; ** designates a time of reaction ) 10 min; and *** designates a time of reaction ) 4 h. b Yields
of isolated products.
Figure 1. Electron density alignment of 4,11-dimethyl-1-oxo-3(H)-
isobenzofuro[5,6-b][1,4]benzodioxine and ellipticine, in yellow and
blue, respectively. The largest FCP(r) isocontour surface is displayed
at 0.001 e2 a-0 6
.
but 6a was slightly more specific in disturbing cell cycle (G2/
M, 8N, 86%, 5 µM) than 5a.
Particularly remarkable was the considerable cytotoxic activ-
ity of 4a and 5a on HT-29 human colon line (IC50 ) 5.8 ×
10-7 M and IC50 ) 1.2 × 10-7 M, respectively) and also the
high selectivity for the G2 phase of the cell cycle (induced the
accumulation of 67% of cells at the G2/M phase by 4a and 80%
by 5a).
On the other hand, the introduction of branched substituents
at the alkyl chain on C-11 decreases the activity by more than
10 times (see 5a vs 10a).
The activity appears closely dependent on the number of
methylenes in the side chain and on the nature of the substituents
of this chain.
Until now, the C-4 and/or C-11 substituents had homologous
alkyl chains, but it is interesting to study that the introduction
of three distinct functions on the side chain is related to obtaining
complementary results. It may be seen that 13a exhibits greater
Figure 2. Electron density isocontour surfaces at 0.03 e a-0 3, and ball
and stick representation of (a) 4,11-dimethyl-1-oxo-3(H)-isobenzofuro-
[5,6-b][1,4]benzodioxine and (b) ellipticine. The corresponding largest
F
CP(r) isocontour is displayed in (c) and (d), respectively, at 0.001 e2
a-0 6
.
activity according to the results of the nonfunctionalized chain
compounds (3-fold more activity than 6a and 5a against
leukemia L1210 and more activity than 6a on HT-29 line cells).
Whereas the introduction of the nitrile group on the side chain
increases the activity (compare 13a and 6a), the introduction
of some hydrophilic function as carboxylic acid (15) or primary

Benzodioxinic Lactones as Anticancer Drugs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 299
group on the aromatic ring A showed more cytotoxicity than
the unsubstituted parent.18
Some of these compounds (4a, 5a, 5b, 6a, and 19) could
block the cell cycle progression of L1210 cells in the G2/M
phase in the same pattern as shown by adriamycin, while
compounds 7a and 13a showed an affect on the 8 N phase.
In conclusion, a series of tetracyclic lactones were synthesized
and evaluated in Vitro against the leukemia L1210 cell line,
colon HT-29 cell line, and also the cycle inhibition studied
(Table 2). The presence of an alkyl chain at position C-11
appears as an important structural requirement to observe potent
cytotoxic activity in this series. The decrease of cytotoxicity
observed by lengthening and shortening the alkyl chain at the
C-11 position of the tetracyclic system reveals that the optimal
length was five C (see Figure 5). The presence of a cyano,
carboxylic acid, or amino group on the side chain has a marked
influence on the activity.
Considering substitutions on the side chain at the C-11, it
appeared, as indicated in Table 2, that weak structural modifica-
tions were responsible for the activity variation and that this
series and the ellipticine series are not comparable in terms of
structure-activity relationships.18
Despite the interesting activity of lactones, the mechanism
of their action at both cellular and molecular level has not yet
been clearly established. Considering that the main accepted
mode of action for ellipticine is based on the interaction with
the enzyme topoisomerase II in terms of stabilization of the
cleaVage complex, which is formed between DNA and topoi-
somerase II during a particular stage of the cell cycle, 4a and
13a were examined for their ability to facilitate the formation
of a cleaVage complex as an indication of the inhibition of
topoisomerase II.19 These compounds did not show inhibition
of topoisomerase II consistent with their cytotoxicity.
Figure 3. Electron density isocontour surface of (a) 4,11-dimethyl-
1-oxo-3(H)-isobenzofuro[5,6-b][1,4]benzodioxine and (b) ellipticine at
0.03 e a-0 3, mapped with the electrostatic potential values.
amine on the C-11 chain (16) produces a 15-fold reduction of
the activity on the colon HT-29 cell line and a 2-fold reduction
on the leukemia L1210 cells, with respect to the unsubstituted
compound 6a.
The cyano alkyl 12a displayed less cytotoxicity than 5a. The
hexyl derivative 6a, enhances the activity due to the addition
of the cyano group (compare 6a and 13a). Comparison of the
analogues 16 and 19 suggested that a secondary amine was more
active than a primary amine.
Generally, these compounds were globally more potent on
the colon HT-29 cell line than on the leukemia L1210 cells
(15-fold in the case of 5a and 4.5-fold in the case of 13a and
19). Compounds 4a-7a, 12a, 13a, 16, and 19 showed selectivity
toward colon HT-29 cell lines, and only 5b, 10a, 11, and 14
showed little selectivity toward leukaemia L1210 cell lines.
Particularly remarkable was the lack of activity obtained for
the thiolactone 11a (9- and 90-fold less activity than the lactone
6a on L1210 and on colon HT-29 cell line, respectively).
Among all of these compounds, only the compound 4a
(NSC: 707690-M/1) was selected by the NCI for testing in Vitro
against 60 cells lines of the NCI anticancer drug screen derived
from eight cancer types (leukemia, non small cell lung cancer,
small cell lung cancer, colon cancer, CNS cancer, melanoma,
ovarian cancer, and renal cancer). The GI50 (GI50 values are
the concentrations corresponding to 50% growth inhibition, and
are averages of at least two determinations) and LC50 (a
concentration at which 50% of the cells were killed in a 96 h
assay) values obtained with selected cell lines are summarized
in Table 3. For the most part, there are no differences between
log GI50 or LC50 values for the cell lines tested, only a small
CNS and colon selectivity at the levels for growth inhibition
was displayed. However, the cytotoxicity remains at the
micromolar level and confirms the results provided by the
Institut de Recherches SerVier.
Compound 9 has two long and flexible side chains at C-4
and C-11 of the tetracyclic lactone, which potentially could
provide hindrance and make difficult the binding with the site
of action. This fact could explain the inactivity of this
compound.
On the other hand, the simplified lactone 35, which bears
important structural modifications on the proposed skeleton of
this series, is inactive. The inactivity may be due to an
insufficient structural similarity to the tetracyclic lactones, such
as 5a. The compounds 13a and 5a were the most promising,
and 6a was of particular interest because of its marked activity
in Vitro against leukemia L1210 and against colon HT-29 cell
line. Compound 13a exhibited potent activity against both tumor
cell lines and, in particular, its cytotoxic effect against HT-29
(IC50 ) 0.12 µM) was greater than that of adriamycin (IC50
0.48 µM) or ellipticine (IC50 ) 14.9 µM).
)
Results from COMPARE searches often provide insight into
the mechanism of action of new compounds.20 COMPARE
analysis of 4a and standard anticancer agent database of the
NCI anticancer drug screen using the determination of Pearson
correlation coefficient found no significant correlation (P > 100)
between the IC50 of the 4a and the IC50 of known topoisomerase
II inhibitors (9-hydroxy-2-methylellipticinium NSC, 264137;
amsacrine NSC, 249992; etoposide NSC, 141540; adriamycin
(doxorubicin) NSC, 123127; mitoxantrone NSC, 301739).17
The molecular target of these compounds is not known, and
the study was continued by making additional biological studies.
The capacity of compounds 4a and 5a to bind to DNA and to
inhibit human DNA topoisomerases I and II21 was investigated.
However, no significant effect was observed compared with the
reference compounds camptothecin or etoposide. These com-
The favorable influence on cytotoxic activity of the presence
of a lipophilic alkyl chain was not mentioned before in antitumor
studies. Instead, the introduction of polar chains that facilitate
the solubility and also the biological interactions are better
known.
The compound 6a induced the accumulation of 86% of L1210
cells in the G2/M, 8 N phase.
In regard to the amino functionalized derivatives, the most
active was the secondary amine 19 (5-fold more potent than
the primary amine 16 on the L1210 cells and 10-fold more
potent on HT-29 cells).
The bulk of the methoxy group could explain the fact that
the substituted compounds (17a and 17b) are inactive. In other
works related to the ellipticine, compounds with a methoxy

300 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Table 2. Cytotoxic Activity of 1-19a
Romero et al.
a Inhibition of L1210 and HT-29 cell proliferation measured by in Vitro biological assay. IC50 values are mean concentrations that inhibit growth by 50%
(IC50 values are the average of at least four determinations in triplicate obtained in independent experiments. Variation between replicates was less than 5%).
b Percentage of L1210 cells arrested in cell cycle phase after 24 h of exposure to the indicated concentration of problem compound. n.t. ) not tested
(considered not interesting due to poor activity on L1210 cell lines).

Benzodioxinic Lactones as Anticancer Drugs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 301
performing new pharmacological investigations to elucidate the
mechanism of action. These biological results corroborate the
observation that a chemical modification of the structure
framework may result in a profound impact in the biological
activity.
In summary, several new synthetic tetracyclic lactones
described in this paper have antitumor activity at the micromolar
level. The cytotoxic activity is quantitatively comparable to the
natural ellipticine, but they show a different mechanism of
action. Likewise, the main structural features influencing the
antitumor activity are different from the ellipticine series.
Among the tested compounds, 4a, 5a, and 13a are considered
promising leads for further structural modifications, perhaps
guided by the valuable information obtainable from detailed
analysis of theoretical studies.
Figure 4. General structure of series a and b of lactones.
The results of this study, we believe, will provide useful
information for the design of novel class of cytotoxic agents.
Experimental Section
Chemistry. Melting points were obtained on a MFB-595010M
Gallenkamp apparatus in open capillary tubes and are uncorrected.
IR spectra were obtained using a FTIR Perkin-Elmer 1600 infrared
spectrophotometer. Only noteworthy IR absorptions are listed (υ
1
expressed in cm-1). H and 13C NMR spectra were recorded on a
Varian Gemini-200 (200 and 50.3 MHz, respectively) or Varian
Gemini-300 (300 and 75.5 MHz) instrument using CDCl3 as solvent
with tetramethylsilane as internal standard or (CD3)2CO. Other 1H
NMR spectra and heterocorrelation 1H-13C (HMQC and HMBC)
experiments were recorded on a Varian VXR-500 (500 MHz). Mass
spectra were recorded on a Hewlett-Packard 5988-A. Column
chromatography was performed with silica gel (E. Merck, 70-
230 mesh). Reactions were monitored by TLC using 0.25 mm silica
gel F-254 (E. Merck). Microanalysis was determined on a Carlo
Erba-1106 analyzer. All reagents were of commercial quality or
were purified before use. Organic solvents were of analytical grade
or were purified by standard procedures. Commercial products were
obtained from Sigma-Aldrich.
Preparation of Lactones (1-13a, 2b-13b, 17a-18a, 17b-
18b) from Diels-Alder Cycloadditions of the Furans (48-62).
General Procedure. 2(5H)-Furanone 63 (1.5 mmols) or 2(5H)-
thiofuranone 64 (1.5 mmols) and a catalytic amount of ytterbium
triflate were added to the corresponding diene (1 mmol) without
solvent at room temperature.
Figure 5. Plots -log10(IC50) versus cancer type (L1210, and HT-29)
for compounds 1, 2a-8a, and 2b-8b.
pounds have no effect on the melting temperature of the DNA
double helix, be it with calf thymus (42% GC) or with the
polynucleotide poly(dAT)2.
DNA sequence recognition was studied by DNase I foot-
printing.22 No marked modification of the absorption spectra
of the compounds was observed upon addition of DNA, and
no specific binding site was detected in the DNase I footprinting
assay, indicating also that these molecules do not appreciably
bind to DNA.
In parallel, a plasmid supercoiled DNA (pKMp27 DNA) was
used to study the relaxation and cleavage of DNA by purified
topoisomerases I and II.23 Here again, we found no effect of
compounds 4a and 5a in these biochemical assays. Moreover,
compounds 4a and 13 were tested using another native
supercoiled DNA (pYRG DNA), and the results were incon-
clusive (no DNA binding).24 These compounds do not stimulate
DNA cleavage by the enzymes and do not interfere with the
poisoning effects of camptothecin on topoisomerase I or
etoposide on topoisomerase II. The effect of no detectable
binding to DNA, the absence of inhibition of either topoi-
somerase I or topoisomerase II, and the negative response to
the DNA relaxation test confirm the results previously obtained
from COMPARE analysis.
The mixture was stirred in a hermetically closed flask on a
preheated bath at 90-100 °C for 4 h. Purification of the crude
product by silica gel column chromatography, using hexane/ethyl
acetate as eluent, allowed us to obtain two positional isomers that
were accurately identified as a or b.
4,11-Dimethyl-1-oxo-3(H)-isobenzofuro[5,6-b][1,4]benzodio-
xine (1). Starting from the diene 48 (523 mg, 2.59 mmol) and the
lactone 63, and following the general procedure described above
for the preparation of lactones, we obtained the lactone 1 (535 mg,
1.99 mmol). The crude product was purified by silica gel column
chromatography, using hexane/ethyl acetate 95/5 as eluent, giving
the lactone as an oil in 77% of yield. Anal. (C16H12O4) C, H.
11-Ethyl-4-methyl-1-oxo-3(H)-isobenzofuro[5,6-b][1,4]benzodio-
xine (2a). Starting from the diene 49 (621 mg, 2.87 mmol) and the
lactone 63, and following the general procedure described above
for the preparation of lactones, we obtained the lactone 2a.
Purification of the crude product by silica gel column chromatog-
raphy, using hexane/ethyl acetate 95/5 as eluent, gave the lactone
as a white solid in 32% of yield. When hexane/ethyl acetate 96/4
mixtures were used as eluent, the isomer 2b was obtained in 44%
yield. For 2a: mp 205-208 °C (hexane/ethyl acetate). Anal.
(C17H14O4) C, H.
These compounds do not correlate with any known compound
in the National Cancer Institute’s data base, suggesting a unique
mechanism of action.
The structure-activity relationships allowed refining the
structural requirements of this series of dioxygenated com-
pounds. Lactone appears essential for cytotoxicity, and its
substitution by a thiolactone prevents us from obtaining the same
activity. On the other hand, if we consider the side chain position
and the nature of its substituents, it appears that the substituent
on the C-11 position leads to an increase of the relative activity
compared with C-4 substitution. Finally, this SAR study
indicated that the best substituent must possess a linear alkyl
chain of 5 C (5a) or a linear alkyl chain with a cyano group in
position 5 (13a). These results strengthen our purpose of
4-Ethyl-11-methyl-1-oxo-3(H)-isobenzofuro[5,6-b][1,4]benzodio-
xine (2b). Compound 2b was obtained as a white solid, 44% yield;
mp 185-188 °C (hexane /ethyl acetate). Anal. (C17H14O4) C, H.

302 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Romero et al.
4-Methyl-11-propyl-1-oxo-3(H)-isobenzofuro[5,6-b]1,4benzodio-
xine (3a). Starting from the diene 50 (681 mg, 2.96 mmol) and the
lactone 63, and following the general procedure described above
for the preparation of lactones, we obtained the lactone 3a.
Purification of the crude product by silica gel column chromatog-
raphy, using hexane/ethyl acetate 96/4 as eluent, gave the lactone
as a white solid in 33% yield. When hexane/ethyl acetate 97/3
mixtures were used as eluent, the isomer 3b (378 mg, 1.27 mmol)
was obtained as a white solid in 45% yield. For 3a: mp 199-202
°C (hexane/ethyl acetate). Anal. (C18H16O4) C, H.
white solid in 44% yield; mp 84-86 °C (hexane/ethyl acetate).
Anal. (C23H26O4) C, H.
11-Methyl-4-octyl-1-oxo-3(H)-isobenzofuro[5,6-b][1,4]benzo-
dioxine (8b). Mp 137-138 °C (hexane/ ethyl acetate). Anal.
(C23H26O4) C, H.
4,11-Dihexyl-1-oxo-3(H)-isobenzofuro[5,6-b][1,4]benzodiox-
ine (9). Starting from the diene 54 (721 mg, 2.11 mmol) and the
lactone 63, and following the general procedure described above
for the preparation of lactones, we obtained the lactone 9.
Purification of the crude product by silica gel column chromatog-
raphy, using hexane/ethyl acetate 99/1 as eluent, gave the lactone
as a white solid in 73% of yield; mp 115-117 °C (hexane/ethyl
11-Methyl-4-propyl-1-oxo-3(H)-isobenzofuro[5,6-b][1,4]ben-
zodioxine (3b). Mp 191-194 °C (hexane /ethyl acetate). Anal.
(C18H16O4) C, H.
acetate). Anal. (C26H32O4) C, H.
11-Butyl-4-methyl-1-oxo-3(H)-isobenzofuro[5,6-b][1,4]benzo-
dioxine (4a). Starting from the diene 51 (695 mg, 2.84 mmol) and
the lactone 63, and following the general procedure described above
for the preparation of lactones, we obtained the lactone 4a.
Purification of the crude product by silica gel column chromatog-
raphy, using hexane/ethyl acetate 97/3 as eluent, gave the lactone
as a white solid in 31% of yield. When the same eluent was used,
the isomer 4b (380 mg, 1.2 mmol) was obtained in 43% yield; mp
122-125 °C (hexane/ethyl acetate). Anal. (C19H18O4) C, H.
4-Butyl-11-methyl-1-oxo-3(H)-isobenzofuro[5,6-b][1,4]benzo-
dioxine (4b). Mp 153-155 °C (hexane /ethyl acetate). Anal.
(C19H18O4) C, H.
4-Methyl-11-pentyl-1-oxo-3(H)-isobenzofuro[5,6-b][1,4]benzo-
dioxine (5a). Starting from the diene 52 (598 mg, 2.32 mmol) and
the lactone 63, and following the general procedure described above
for the preparation of lactones, we obtained the lactone 5a.
Purification of the crude product by silica gel column chromatog-
raphy, using hexane/ethyl acetate 98/2 as eluent, gave the lactone
as a white solid in 28% yield (256 mg, 0.77 mmol). When hexane/
ethyl acetate 99/1 was used as eluent, the isomer 5b was obtained
in 45% yield; mp 108-110 °C (hexane/ethyl acetate). Anal.
(C20H20O4) C, H.
4-Methyl-11-(2,4,4-trimethylpentyl)-1-oxo-3(H)-isobenzofuro-
[5,6-b][1,4]benzodioxine (10a). Starting from the diene 57 (659
mg, 2.01 mmol) and the lactone 63, and following the general
procedure described above for the preparation of lactones, we
obtained the lactone 10a. Purification of the crude product by silica
gel column chromatography, using hexane/ethyl acetate 98/2 as
eluent, gave the lactone as a white solid in 29% of yield. When
hexane/ethyl acetate 99/1 was used, the isomer 10b (356 mg, 0.90
mmol) was obtained as a white solid in 45% yield; mp 182-183
°C (hexane/ethyl acetate). Anal. (C23H26O4) C, H.
11-Methyl-4-(2,4,4-trimethylpentyl)-1-oxo-3(H)-isobenzofuro-
[5,6-b][1,4]benzodioxine (10b). Mp 161-162 °C (hexane/ethyl
acetate). Anal. (C23H26O4) C, H.
11-Hexyl-4-methyl-1-oxo-3(H)-isobenzothiofeno[5,6-b][1,4]-
benzodioxine (11a). Starting from the diene 53 (700 mg, 2.57
mmol) and the thiolactone 64, and following the general procedure
described above for the preparation of lactones, we obtained the
lactone 11a. Purification of the crude product by silica gel column
chromatography, using hexane/ethyl acetate 80/20 as eluent, gave
the lactone as a white solid in 36% of yield. When hexane/ethyl
acetate 70/30 was used, the isomer 11b (382 mg, 1.06 mmol) was
obtained as a white solid in 42% yield; mp 126-129 °C (hexane/
ethyl acetate). Anal. (C21H22O3S) C, H.
11-Methyl-4-pentyl-1-oxo-3(H)-isobenzofuro[5,6-b][1,4]benzo-
dioxine (5b). Mp 145-146 °C (hexane/ethyl acetate). Anal.
(C20H20O4) C, H.
4-Hexyl-11-methyl-1-oxo-3(H)-isobenzothiofeno[5,6-b][1,4]-
benzodioxine (11b). Mp 131-133 °C (hexane/ethyl acetate). Anal.
(C21H22O3S) C, H.
11-Hexyl-4-methyl-1-oxo-3(H)-isobenzofuro[5,6-b][1,4]benzo-
dioxine (6a). Starting from the diene 53 (603 mg, 2.21 mmol) and
the lactone 63, and following the general procedure described above
for the preparation of lactones, we obtained the lactone 6a.
Purification of the crude product by silica gel column chromatog-
raphy, using hexane/ethyl acetate 98/2 as eluent, gave the lactone
as a white solid in 36% of yield. When the same eluent was used,
the isomer 6b (292 mg, 0.86 mmol) was obtained in 39% yield;
mp 131-133 °C (hexane/ethyl acetate). Anal. (C21H22O4) C, H.
4-Hexyl-11-methyl-1-oxo-3(H)-isobenzofuro[5,6-b][1,4]benzo-
dioxine (6b). Mp 149-150 °C (hexane/ ethyl acetate). Anal.
(C21H22O4) C, H.
11-Heptyl-4-methyl-1-oxo-3(H)-isobenzofuro[5,6-b][1,4]benzo-
dioxine (7a). Starting from the diene 55 (627 mg, 2.19 mmol) and
the lactone 63, and following the general procedure described above
for the preparation of lactones, we obtained the lactone 7a.
Purification of the crude product by silica gel column chromatog-
raphy, using hexane/ethyl acetate 98/2 as eluent, gave the lactone
as a white solid in 29% of yield. When the same eluent was used,
the isomer 7b (302 mg, 0.86 mmol) was obtained as a white solid
in 39% yield; mp 103-104 °C (hexane/ethyl acetate). Anal.
(C22H24O4) C, H.
11-(4-Cyanobutyl)-4-methyl-1-oxo-3(H)-isobenzofuro[5,6-b]-
[1,4]benzodioxine (12a). Starting from the diene 58 (458 mg, 1.70
mmol) and the lactone 63, and following the general procedure
described above for the preparation of lactones, we obtained the
lactone 12a. Purification of the crude product by silica gel column
chromatography, using hexane/ethyl acetate 99/1 as eluent, gave
the lactone as a white solid in 30% of yield. When hexane/ethyl
acetate 98/2 was used, the isomer 12b (245 mg, 0.73 mmol) was
obtained as a white solid in 43% yield; mp 169-171 °C (hexane/
ethyl acetate). Anal. (C20H17NO4) C, H, N.
4-(4-Cyanobutyl)-11-methy-1-oxo-3(H)-isobenzofuro[5,6-b]-
[1,4]benzodioxine (12b). Mp 178-181 °C (hexane/ethyl acetate).
Anal. (C20H17NO4) C, H, N.
11-(5-Cyanopentyl)-4-methyl-1-oxo-3(H)-isobenzofuro[5,6-b]-
[1,4]benzodioxine (13a). Starting from the diene 59 (597 mg, 2.11
mmol) and the lactone 63, and following the general procedure
described above for the preparation of lactones, we obtained the
lactone 13a. Purification of the crude product by silica gel column
chromatography, using hexane/ethyl acetate 99/1 as eluent, gave
the lactone as a white solid in 36% of yield. When hexane/ethyl
acetate 98/2 was used, the isomer 13b (318 mg, 0.92 mmol) was
obtained as a white solid in 43% yield; mp 110-113 °C (hexane/
ethyl acetate). Anal. (C21H19NO4) C, H, N.
4-(5-Cyanopentyl)-11-methyl-1-oxo-3(H)-isobenzofuro[5,6-b]-
[1,4]benzodioxine (13b). Mp 137-139 °C (hexane/ethyl acetate).
Anal. (C21H19NO4) C, H, N.
4-Methyl-8-methoxy-11-pentyl-1-oxo-3(H)-isobenzofuro[5,6-
b][1,4]benzodioxine (17a). Starting from the diene 61 (527 mg,
1.83 mmol) and the lactone 63, and following the general procedure
described above for the preparation of lactones, we obtained the
lactone 17a. Purification of the crude product by silica gel column
chromatography, using hexane/ethyl acetate 99/1 as eluent, gave
4-Heptyl-11-methyl-1-oxo-3(H)-isobenzofuro[5,6-b][1,4]benzo-
dioxine (7b). Mp 153-154 °C (hexane/ ethyl acetate). Anal.
(C22H24O4) C, H.
4-Methyl-11-octyl-1-oxo-3(H)-isobenzofuro[5,6-b][1,4]benzo-
dioxine (8a). Starting from the diene 56 (598 mg, 1.99 mmol) and
the lactone 63, and following the general procedure described above
for the preparation of lactones, we obtained the lactone 8a.
Purification of the crude product by silica gel column chromatog-
raphy, using hexane/ethyl acetate 98/2 as eluent, gave the lactone
as a white solid in 27% of yield. When hexane/ethyl acetate 99/1
was used, the isomer 8b (321 mg, 0.88 mmol) was obtained as a

Benzodioxinic Lactones as Anticancer Drugs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 303
the lactone as a white solid in 34% of yield. When hexane/ethyl
acetate 98/2 was used, the isomer 17b (304 mg, 0.82 mmol) was
obtained as a white solid in 45% yield; mp 137-139 °C (hexane/
ethyl acetate). Anal. (C21H22O5) C, H.
11-Methyl-7-methoxy-4-pentyl-1-oxo-3(H)-isobenzofuro[5,6-
b][1,4]benzodioxine (17b). Mp 117-119 °C (hexane/ethyl acetate).
Anal. (C21H22O5) C, H.
pressure. The hydroxyacid was obtained as a white solid that was
further transformed into the corresponding lactone.
3-(1-Hydroxyethyl)-1,4-benzodioxin-2-carboxylic Acid (22).
Following the general procedure applied to the carboxylic acid 20
(1 g, 5.60 mmol), the crude residue was purified to furnish the
hydroxyacid (1.19 g, 5.36 mmol) in a 96% of yield; mp 210-212
°C (methanol). Anal. (C11H10O5) C, H.
4-Methyl-11-(4-bromobutyl)-1-oxo-3(H)-isobenzofuro[5,6-b]-
[1,4]benzodioxine (18a). Starting from the diene 62 (602 mg, 1.86
mmol) and the lactone 63, following the general procedure described
above for the preparation of lactones, we obtained the tetracyclic
compound 18a. Purification of the crude product by silica gel
column chromatography, using hexane/ethyl acetate 90/10 as eluent,
gave the lactone as a white solid in 29% of yield. The isomer 18b
(308 mg, 0.78 mmol) was obtained as a white solid in 43% yield
using hexane/ethyl acetate 88/12 as eluent; mp 146-148 °C
(hexane/dichloromethane). Anal. (C19H17BrO4) C, H.
3-(1-Hydroxypropyl)-1,4-benzodioxin-2-carboxylic Acid (23).
Following the general procedure applied to the carboxylic acid 20
(0.8 g, 4.20 mmol) to furnish the hydroxyacid 23 (0.9 g, 3.86 mmol)
in a 92% yield; mp 151-153 °C (methanol). Anal. (C12H12O5) C,
H.
3-(1-Hydroxybutyl)-1,4-benzodioxin-2-carboxylic Acid (24).
Following the general procedure applied to the acid 20 (0.78 g,
4.40 mmol) furnished the hydroxyacid 24 (1.04 g, 4.16 mmol) in
a 95% yield; mp 152-154 °C (methanol). Anal. (C13H14O5) C, H.
3-(1-Hydroxyhexyl)-1,4-benzodioxin-2-carboxylic Acid (25).
Following the general procedure applied to the acid 20 (0.95 g,
5.33 mmol) furnished the hydroxyacid 25 (1.23 g, 4.42 mmol) in
83% yield; mp 154-157 °C (methanol). Anal. (C15H18O5) C, H.
3-(1-Hydroxyheptyl)-1,4-benzodioxin-2-carboxylic Acid (26).
Following the general procedure applied to the acid 20 (1.01 g,
5.67 mmol) furnished the hydroxyacid 26 (1.35 g, 4.62 mmol) in
82% yield; mp 154-155 °C (methanol). Anal. (C16H20O5) C, H.
11-Methyl-4-(4-bromobutyl)-1-oxo-3(H)-isobenzofuro[5,6-b]-
[1,4]benzodioxine (18b). Mp 160-162 °C (hexane/dichloromethane).
Anal. (C19H17BrO4) C, H.
11-(4-Carboxybutyl)-4-methyl-1-oxo-3(H)-isobenzofuro[5,6-
b][1,4]benzodioxine (14). A solution containing the corresponding
lactone (12a; 50 mg, 0.15 mmol) in 2 N NaOH (40 mL) and
methanol (5 mL) was refluxed for 2 h. After 1 N HCl was slowly
added until pH ) 1, and the suspension was filtered. The residue
was dried to give the lactone 14 as a white solid (48 mg, 0.14
mmol) in a 91% yield; mp 197-200 °C (H2O). Anal. (C20H18O6)
C, H.
3-(1-Hydroxyoctyl)-1,4-benzodioxin-2-carboxylic Acid (27).
Following the general procedure applied to the acid 20 (1.31 g,
7.35 mmol) furnished the hydroxyacid 27 (1.78 g, 5.81 mmol) in
79% yield; mp 150-152 °C (methanol). Anal. (C17H22O5) C, H.
11-(5-Carboxypentyl)-4-methyl-1-oxo-3(H)-isobenzofuro[5,6-
b][1,4]benzodioxine (15). A solution containing the corresponding
lactone (13a; 60 mg, 0.17 mmol) in 2 N NaOH (40 mL) and
methanol (5 mL) was refluxed for 2 h. HCl (1 N) was slowly added
until pH ) 1, and the suspension was filtered. The residue was
dried to give the lactone 15 as a white solid (57 mg, 0.16 mmol)
in a 96% yield; mp 182-185 °C (H2O). Anal. (C21H20O6) C, H.
11-(5-(Aminopentyl)-4-methyl-1-oxo-3(H)-isobenzofuro[5,6-
b][1,4]benzodioxine Hydrochloride (16). To a solution containing
the corresponding lactone (12a; 15 mg, 0.045 mmol) in methanol
(15 mL) was added PtO2 (5 mg) and two drops of 1 N HCl. The
suspension was stirred at room temperature under hydrogen
atmosphere for 12 h. Then the suspension was filtered, and the
solvent was removed under vacuum. The residue obtained was
diluted with water and extracted with dichloromethane. The organic
layers were dried (Na2SO4), filtered, and concentrated under reduced
pressure to give the reduced compound 16 (15 mg, 0.04 mmol) as
a white solid in 88% yield. Anal. (C20H22ClNO4‚H2O) C, H, N.
11-(5-(Aminomethyl)pentyl-4-methyl-1-oxo-3(H)-isobenzofuro-
[5,6-b][1,4]benzodioxine (19). To a solution of the lactone 18a
(20 mg, 0.05 mmol) in DMF (3 mL) were added K2CO3 (22 mg,
0.15 mmol) and methylamine hydrochloride (5 mg, 0.08 mmol).
The resulting mixture was stirred seven days at room temperature.
Every two days, the same amount of K2CO3 and methylamine
hydrochloride was added. The reaction mixture was diluted with
water (50 mL) and was extracted with ether (3 × 12 mL). The
organic layer was dried over Na2SO4, filtered off, and the solvent
removed under vacuum. The residue was purified by silica gel
column chromatography using hexane/ethyl acetate as eluent, giving
the desired amine in 57% yield; mp 114-116 °C (methanol). Anal.
(C20H21NO4) C, H, N.
3-(1-Hydroxynonyl)-1,4-benzodioxin-2-carboxylic acid (28).
Following the general procedure applied to the acid 20 (0.98 g,
5.50 mmol) furnished the hydroxyacid 28 (1.43 g, 4.46 mmol) in
81% yield. Mp 148-150 °C (methanol). Anal. (C18H24O5) C, H.
3-(3,5,5-Trimethyl-1-hydroxyhexyl)-1,4-benzodioxin-2-car-
boxylic Acid (29). Following the general procedure applied to the
acid 20 (0.68 g, 3.82 mmol) furnished the hydroxyacid 29 (1.11 g,
3.19 mmol) in 83% yield; mp 168-170 °C (methanol). Anal.
(C18H24O5) C, H.
3-(5-Bromo-1-hydroxypentyl)-1,4-benzodioxin-2-carboxylic
Acid (30). Following the general procedure applied to the acid 20
(0.87 g, 4.90 mmol) furnished the hydroxyacid 30 (1.49 g, 4.32
mmol) in 89% yield; mp 160-162 °C (methanol). Anal. (C14H15-
BrO5) C, H.
3-(2-Benzyloxy-1-hydroxyethyl)-1,4-benzodioxin-2-carboxy-
lic Acid (31). Following the general procedure applied to the acid
20 (0.97 g, 5.45 mmol) furnished the hydroxyacid 31 (1.59 g, 4.84
mmol) in 89% yield; mp 145-147 °C (methanol). Anal. (C18H16O6)
C, H.
3-(1-Hydroxyhexyl)-6-methoxy-1,4-benzodioxin-2-carboxyl-
ic Acid (32). Following the general procedure applied to the acid
21 (0.97 g, 4.65 mmol) furnished the hydroxyacid 32 as colorless
oil (1.19 g, 3.86 mmol) in 83% yield. Anal. (C16H20O6) C, H.
3-(5-Cyano-1-hydroxypentyl)-1,4-benzodioxin-2-carboxylic Acid
(33). Following the general procedure applied to the acid 20 (0.72
g, 4.04 mmol) furnished the hydroxyacid 33 (0.88 g, 3.10 mmol)
in 76% yield; mp 158-160 °C (methanol). Anal. (C15H15NO5) C,
H, N.
3-(6-Cyano-1-hydroxyhexyl)-1,4-benzodioxin-2-carboxylic Acid
(34). Following the general procedure applied to the acid 30 (0.53
g, 2.96 mmol) furnished the hydroxyacid 34 (0.67 g, 2.21 mmol)
in 75% yield; mp 162-163 °C (methanol). Anal. (C16H17NO5) C,
H, N.
Preparation of Lactones (35-47). Lactonization of Hydroxy-
acids (22-34). General Procedure. To a solution of the carboxylic
acid (1 mmol) in dry toluene (25 mL) under an argon atmosphere
were added PTSA (catalytic amount) and molecular sieves of 4 Å
(100 mg), and the mixture was refluxed for 12 h. Then the
suspension was filtered and washed with ethyl acetate. The crude
product was extracted with ether and washed with 1 N NaOH until
General Procedure for the Preparation of Hydroxyacids (22-
34). ortho-Lithiation of Carboxylic Acids (20-21). To a solution
of the carboxylic acid (1 mmol) in THF cooled at -78 °C was
added dropwise LDA (2.2 mmol; 2 M solution in THF/heptane),
and this mixture was stirred. After 3.5 h, the electrophile (1.2 mmol)
was added dropwise. The resulting mixture was stirred for 12 h,
and then the reaction mixture was allowed to warm to room
temperature. A solution of 0.5 N HCl was added until the pH of
the mixture was acidic. Then the mixture was stirred at room
temperature for 5 min. Finally the reaction mixture was diluted
with water and extracted with ether (4 × 15 mL). The organic layers
were dried (Na2SO4), filtered, and concentrated under reduced

304 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Romero et al.
basic pH. The obtained residue was purified by column chroma-
tography on silica gel eluting with hexane/ethyl acetate mixtures.
3-Methyl-1-oxo-3(H)-furo[3,4-b][1,4]benzodioxine (35). Start-
ing from the hydroxyacid 22 (0.95 g, 4.28 mmol) and following
the general procedure, the lactone 35 (0.78 g, 3.82 mmol) was
obtained as a white solid. The crude product was purified on silica
gel column, eluting with hexane/ethyl acetate (7/3), to furnish the
desired lactone in 89% yield; mp 125-127 °C (hexane/ethyl
acetate). Anal. (C11H8O4) C, H.
3-Ethyl-1-oxo-3(H)-furo[3,4-b][1,4]benzodioxine (36). Starting
from the hydroxyacid 23 (0.7 g, 3.02 mmol) and following the
general procedure, the lactone 36 (0.55 g, 2.57 mmol) was obtained
as a white solid. The crude product was purified on silica gel
column, eluting with hexane/ethyl acetate (7/3), to furnish the
desired lactone in 85% yield. Anal. (C12H10O4) C, H.
3-Propyl-1-oxo-3(H)-furo[3,4-b][1,4]benzodioxine (37). Start-
ing from the hydroxyacid 24 (1.1 g, 4.40 mmol) and following the
general procedure, the lactone 37 (0.88 g, 3.79 mmol) was obtained
as a white solid. The crude product was purified on silica gel
column, eluting with hexane/ethyl acetate (7/3), to furnish the
desired lactone in 86% yield; mp 80-81 °C (hexane/ethyl acetate).
Anal. (C13H12O4) C, H.
3-Pentyl-1-oxo-3(H)-furo[3,4-b][1,4]benzodioxine (38). Starting
from the hydroxyacid 25 (0.63 g, 2.26 mmol) and following the
general procedure, the lactone 38 (0.51 g, 1.96 mmol) was obtained
as a white solid. The crude product was purified on silica gel
column, eluting with hexane/ethyl acetate (7/3), to furnish the
desired lactone in 87% yield; mp 92-93 °C (hexane/ethyl acetate).
Anal. (C15H16O4) C, H.
3-Hexyl-1-oxo-3(H)-furo[3,4-b][1,4]benzodioxine (39). Starting
from the hydroxyacid 26 (0.74 g, 2.52 mmol) and following the
general procedure, the lactone 39 (0.63 g, 2.30 mmol) was obtained
as a white solid. The crude product was purified on a silica gel
column, eluting with hexane/ethyl acetate (8/2), to furnish the
desired lactone in 91% yield; mp 82-83 °C (hexane/ethyl acetate).
Anal. (C16H18O4) C, H.
3-Heptyl-1-oxo-3(H)-furo[3,4-b][1,4]benzodioxine (40). Start-
ing from the hydroxyacid 27 (0.99 g, 3.22 mmol) and following
the general procedure, the lactone 40 (0.79 g, 2.74 mmol) was
obtained as a white solid. The crude product was purified on silica
gel column, eluting with hexane/ethyl acetate (8/2), to furnish the
desired lactone in 85% yield; mp 84-85 °C (hexane/ethyl acetate).
Anal. (C17H20O4) C, H.
6-Methoxy-3-pentyl-1-oxo-3(H)-furo[3,4-b][1,4]benzodiox-
ine (45). Starting from the hydroxyacid 32 (0.92 g, 2.97 mmol)
and following the general procedure, the lactone 45 (0.77 g, 2.65
mmol) was obtained as a yellow oil. The crude product was purified
on silica gel column, eluting with hexane/ethyl acetate (7/3), to
furnish the desired lactone in 89% yield. Anal. (C16
H18O5) C, H.
3-(4-Cyanobutyl)-1-oxo-3(H)-furo[3,4-b][1,4]benzodioxine (46).
Method a. Starting from the hydroxyacid 33 (0.43 g, 1.48 mmol)
and following the general procedure, the lactone 46 (0.32 g, 1.20
mmol) was obtained as a white solid. The crude product was
purified on silica gel column, eluting with hexane/ethyl acetate (7/
3), to furnish the desired lactone in 81% yield; mp 103-105 °C
(hexane/ethyl acetate). Anal. (C15H13NO4) C, H.
Method b. To a solution of the corresponding lactone (110 mg,
0.34 mmol) in DMF (3 mL) was added HMPA (1 mL) and KCN
(37 mg, 0.58 mmol). The obtained mixture was stirred at room
temperature for 12 h. Then the solvent was removed and the crude
of reaction was purified by silica gel column chromatography to
give catechol (32 mg, 0.29 mmol).
3-(5-Cyanopentyl)-1-oxo-3(H)-furo[3,4-b][1,4]benzodioxine (47).
Starting from the hydroxyacid 34 (0.94 g, 3.10 mmol) and following
the general procedure, the lactone 47 (0.77 g, 2.70 mmol) was
obtained as a yellow solid. The crude product was purified on silica
gel column, eluting with hexane/ethyl acetate (7/3), to furnish the
desired lactone in 87% yield; mp 117-118 °C (hexane/ethyl
acetate). Anal. (C16H15NO4) C, H, N.
Synthesis of Dienes (48-60). General Procedure. The lactone
(1 mmol) was dissolved in dry THF (4 mL) under argon, and the
solution was then cooled to -78 °C. A solution of trimethylsilyl
chloride (6 mmol) in THF and the corrresponding alkyllithium (3
mmol) were added with stirring over 5 min, and stirring was
continued until the mixture reached room temperature. A solution
of NH4Cl (5 mL) was added and the organic layer was separated.
The combined organic solutions were dried (Na2SO4), and the
solvent was removed to give the crude product. The product was
purified by column chromatography (silica gel, hexane/ethyl
acetate).
1,3-Dimethylfuro[3,4-b][1,4]benzodioxine (48). Starting from
the lactone 35 (631 mg, 3.09 mmol) and following the general
procedure using methyllithium (1.6 M in ether), the furan 48 (531
mg, 2.63 mmol) was obtained as an oil. The crude product was
purified on silica gel column, eluting with hexane/ethyl acetate (98/
2), to furnish the desired furan in 85% yield. Anal. (C12H10O3) C,
H.
1-Ethyl-3-methylfuro[3,4-b][1,4]benzodioxine (49). Starting
from the lactone 36 (501 mg, 2.34 mmol) and following the general
procedure using methyllithium (1.6 M in ether), the furan 49 (440
mg, 2.06 mmol) was obtained as an oil. The crude product was
purified on silica gel column, eluting with hexane/ethyl acetate (98/
2), to furnish the desired furan in 87% yield. Anal. (C13H12O3) C,
H.
1-Methyl-3-propylfuro[3,4-b][1,4]benzodioxine (50). Starting
from the lactone 37 (750 mg, 3.23 mmol) and following the general
procedure using methyllithium (1.6 M in ether), the furan 50 (625
mg, 2.71 mmol) was obtained as an oil. The crude product was
purified on silica gel column, eluting with hexane/ethyl acetate (98/
2), to furnish the desired furan in 84% yield. Anal. (C14H14O3) C,
H.
1-Butyl-3-methylfuro[3,4-b][1,4]benzodioxine (51). Starting
from the lactone 35 (523 mg, 2.56 mmol) and following the general
procedure using butyllithium (1.6 M in hexane), the furan 51 (425
mg, 1.74 mmol) was obtained as an oil. The crude product was
purified on silica gel column, eluting with hexane/ethyl acetate (98/
2), to furnish the desired furan in 68% yield. Anal. (C15H16O3) C,
H.
3-Octyl-1-oxo-3(H)-furo[3,4-b][1,4]4benzodioxine (41). Starting
from the hydroxyacid 28 (1.13 g, 3.53 mmol) and following the
general procedure, the lactone 41 (0.88 g, 2.91 mmol) was obtained
as a white solid. The crude product was purified on silica gel
column, eluting with hexane/ethyl acetate (8/2), to furnish the
desired lactone in 82% yield; mp 82-83 °C (hexane/ethyl acetate).
Anal. (C18H22O4) C, H.
3-(2,4,4-Trimethylpentyl)-1-oxo-3(H)-furo[3,4-b][1,4]benzo-
dioxine (42). Starting from the hydroxyacid 29 (1.11 g, 3.19 mmol)
and following the general procedure, the lactone 42 (0.88 g, 2.66
mmol) was obtained as a white solid. The crude product was
purified on silica gel column, eluting with hexane/ethyl acetate (8/
2), to furnish the desired lactone in 84% yield; mp 69-70 °C
(hexane/ethyl acetate). Anal. (C18H22O4) C, H.
3-(4-Bromobutyl)-1-oxo-3(H)-furo[3,4-b][1,4]benzodioxine (43).
Starting from the hydroxyacid 30 (0.52 g, 1.52 mmol) and following
the general procedure, the lactone 43 (0.41 g, 1.26 mmol) was
obtained as a white solid. The crude product was purified on silica
gel column, eluting with hexane/ethyl acetate (7/3), to furnish the
desired lactone in 83% yield; mp 82-84 °C (hexane/ethyl acetate).
Anal. (C14H13BrO4) C, H.
3-(Benzyloxymethyl)-1-oxo-3(H)-furo[3,4-b][1,4]benzodiox-
ine (44). Starting from the hydroxyacid 31 (0.82 g, 2.49 mmol)
and following the general procedure, the lactone 44 (0.61 g, 1.97
mmol) was obtained as a white solid. The crude product was
purified on silica gel column, eluting with hexane/ethyl acetate (7/
3), to furnish the desired lactone in 79% yield; mp 95-97 °C
(hexane/ethyl acetate). Anal. (C18H14O5) C, H.
1-Methyl-3-pentylfuro[3,4-b][1,4]benzodioxine (52). Starting
from the lactone 38 (541 mg, 2.08 mmol) and following the general
procedure using methyllithium (1.6 M in ether), the furan 52 (457
mg, 1.77 mmol) was obtained as an oil. The crude product was

Benzodioxinic Lactones as Anticancer Drugs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 305
purified on silica gel column, eluting with hexane/ethyl acetate (99/
1), to furnish the desired furan in 85% yield. Anal. (C16H18O3) C,
H.
1-Hexyl-3-methylfuro[3,4-b][1,4]benzodioxine (53). Starting
from the lactone 39 (480 mg, 1.75 mmol) and following the general
procedure using methyllithium (1.6 M in ether), the furan 53 (396
mg, 1.45 mmol) was obtained as an oil. The crude product was
purified on silica gel column, eluting with hexane/ethyl acetate (99/
1), to furnish the desired furan in 83% yield. Anal. (C17H20O3) C,
H.
1,3-Dihexylfuro[3,4-b][1,4]benzodioxine (54). Starting from the
lactone 39 (637 mg, 2.32 mmol) and following the general
procedure using hexyllithium (2.5 M in hexane), the furan 54 (453
mg, 1.32 mmol) was obtained as an oil. The crude product was
purified on silica gel column, eluting with hexane/ethyl acetate (99/
1), to furnish the desired furan in 87% yield. Anal. (C22H30O3) C,
H.
1-Heptyl-3-methylfuro[3,4-b][1,4]benzodioxine (55). Starting
from the lactone 40 (523 mg, 1.81 mmol) and following the general
procedure using methyllithium (1.6 M in ether), the furan 55 (435
mg, 1.52 mmol) was obtained as an oil. The crude product was
purified on silica gel column, eluting with hexane/ethyl acetate (99/
1), to furnish the desired furan in 84% yield. Anal. (C18H22O3) C,
H.
1-Methyl-3-octylfuro[3,4-b][1,4]benzodioxine (56). Starting
from the lactone 41 (527 mg, 1.74 mmol) and following the general
procedure using methyllithium (1.6 M in ether), the furan 56 (444
mg, 1.49 mmol) was obtained as an oil. The crude product was
purified on silica gel column, eluting with hexane/ethyl acetate (99/
1), to furnish the desired furan in 85% yield. Anal. (C19H24O3) C,
H.
3-Methyl-1-(2,4,4-trimethylpentyl)furo[3,4-b][1,4]benzodiox-
ine (57). Starting from the lactone 42 (625 mg, 1.89 mmol) and
following the general procedure using methyllithium (1.6 M in
ether), the furan 57 (503 mg, 1.53 mmol) was obtained as an oil.
The crude product was purified on silica gel column, eluting with
hexane/ethyl acetate (98/2), to furnish the desired furan in 87%
yield. Anal. (C19H24O3) C, H.
1-(4-Cyanobutyl)-3-methylfuro[3,4-b][1,4]benzodioxine (58).
Starting from the lactone 46 (440 mg, 1.62 mmol) and following
the general procedure using methyllithium (1.6 M in ether), the
furan 58 (353 mg, 1.31 mmol) was obtained as an oil. The crude
product was purified on silica gel column chromatography, eluting
with hexane/ethyl acetate (98/2), to furnish the desired furan in
81% yield. Anal. (C16H15NO3) C, H, N.
1-(5-Cyanopentyl)-3-methylfuro[3,4-b][1,4]benzodioxine (59).
Starting from the lactone 47 (520 mg, 1.82 mmol) and following
the general procedure using methyllithium (1.6 M in ether), the
furan 59 (407 mg, 1.44 mmol) was obtained as an oil. The crude
product was purified on silica gel column, eluting with hexane/
ethyl acetate (98/2), to furnish the desired furan in 79% yield. Anal.
(C17H17NO3) C, H, N.
ethyl acetate (99/1), to furnish the desired furan in 84% yield. Anal.
(C15H15BrO3) C, H.
Biological Experimental
Biological Materials. Cell Culture and Cytotoxicity. L1210
leukemia and HT-29 cells were grown in nutrient medium RPMI
1640 or DMEM (Dulbecco’s modified Eagle’s medium, Sigma,
St. Louis, MO), respectively, supplemented with 2 mM L-glutamine,
200 IU/mL penicillin, 50 µg/mL streptomycin, and 20% heat
inactivated horse serum. They were incubated in a 5% CO2
atmosphere at 37 °C. For the experiments, the drugs were dissolved
in dimethyl sulfoxide (0.5% final) and added to the cells in
exponential phase of growth at an initial concentration of 0.8 ×
105 cells/mL. The cells were counted in quadruplicate after 48 h
for L1210 cells and 96 h for HT-29 cells with Coultronics Coulter
Counter, and results were expressed as the drug concentration that
inhibited cell growth by 50% as compared to the controls (IC50).
The IC50 values were calculated from regression lines obtained from
the probit of the percent cell growth inhibition plotted as a function
of the logarithm of the dose. This experiment was performed four
independents times in triplicate for every compound.
Inhibition of Cellular Proliferation and Cell Cycle Effects.
L1210 leukemia cells were grown in nutrient medium RPMI 1640
supplemented with 2 mM L-glutamine, 200 IU/mL penicilium, 50
µg/mL streptomycin, and 20% heat inactivated horse serum. They
were incubated in a 5% CO2 atmosphere at 37 °C for 21 h with
several drug concentrations. Cells were fixed by ethanol (70% v/v),
then washed, and incubated with PBS containing 100 µM RNAse
and 25 µM/mL propidium iodide for 30 min at room temperature.
For each concentration, 10-4 cells were analyzed on an Epics XL
flow cytomer (Modele Beckman Coulter, French). Results were
expressed as a percentage of cells accumulated in each phase of
the cell cycle and are indicated.
Effect on Topoisomerase I and II. The complete methodology
to measure topoisomerase I and topoisomerase II inhibition by
relaxation assay has been previously described.21
DNA Interaction. Sequence recognition was determined by
DNase I footprinting using a protocol previously described in
detail.22
Formation of Cleavage Complex. Topoisomerase II Inhibi-
tion. Topoisomerase II inhibition activity by 4a and 13a was
measured by quantization of the formation of the cleavage complex
on pYRG plasmid DNA by human topoisomerase II (purified from
placenta).19 The cleavage complex product, linear DNA, was
quantized (relative to ellipticine) by densitometric analyses.24
Computational Methods
The molecular geometries of ellipticine and 4,11-dimethyl-1-
oxo-3(H)-isobenzofuro[5,6-b][1,4]benzodioxine were optimized at
the B3LYP/6-31G(d) level of theory. The B3LYP/6-31G(d) electron
densities at the optimized geometries were fitted to spherical atomic
shell approximation (ASA) expansions25 and were used to perform
the molecular alignment26 of the two structures. Once aligned, the
electron densities were computed at the finer level B3LYP/6-311G
(d,p) to analyze and visualize the charge distributions and electro-
static potentials. The computation of the molecular geometries,
electron densities, and electrostatic potentials was carried out using
the Gaussian 0327 suite of programs. The electron density alignment
was produced with ASASim,26 and the visualization figures were
prepared with VMD28 and OpenDX.29
1-(Benzyloxymethyl)-3-methylfuro[3,4-b][1,4]benzodioxine (60).
Starting from the lactone 44 (535 mg, 1.72 mmol) and following
the general procedure using methyllithium (1.6 M in ether), the
furan 60 (58 mg, 0.19 mmol) was obtained as an oil. The crude
product was purified on silica gel column, eluting with hexane/
ethyl acetate (98/2), to furnish the desired furan in 11% yield. Anal.
(C19H16O4) C, H.
1-Methyl-6-methoxy-3-pentylfuro[3,4-b][1,4]benzodioxine (61).
Starting from the lactone 45 (823 mg, 2.83 mmol) and following
the general procedure using methyllithium (1.6 M in ether), the
furan 61 (677 mg, 2.34 mmol) was obtained as an oil. The crude
mixture was purified on silica gel column, eluting with hexane/
ethyl acetate (98/2), to furnish the desired furan in 83% yield. Anal.
(C17H20O4) C, H.
Figures were arranged to display electron density isocontours at
the aligned positions. A novel visualization technique for the
analysis of similarity, that is, isocontour plots of the product of
densities, was used. Briefly, the common pattern density FCP(r) )
FA(r)FB(r) on an information or similarity theoretic setting gives
the simultaneous probability distribution for the two entities FA(r)
1-Methyl-3-(4-bromobutyl)furo[3,4-b][1,4]benzodioxine (62).
Starting from the lactone 43 (530 mg, 1.63 mmol) and following
the general procedure using methyllithium (1.6 M in ether), the
furan 62 (441 mg, 1.52 mmol) was obtained as an oil. The crude
product was purified on silica gel column, eluting with hexane/
and FB(r). If A and B were two equal and aligned molecules, FCP
-
(r) would possess exactly the same set of isocontour surfaces as
FA(r), with the values shifted and given by the product FA(r)FA(r).
In general, at the global maximum arrangement of the similarity

306 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2
Romero et al.
(11) Bozzo, C.; Pujol, M. D. Study of Preparation of Methylfuro[3,4-b]-
[1,4]benzodioxinones as Intermediates for the Synthesis of Substituted
Polycyclic Systems. Importance of the Acid Used as Catalyst.
Tetrahedron 1999, 55, 11843-11852.
(12) Bozzo, C.; Pujol, M. D. Intermolecular Diels-Alder Cycloadditions
of Isobenzofuran: A Synthesis of Fused Heterocycles. Heterocycl.
Commun. 1996, 2, 163-167.
function, FCP(r) displays the largest common pattern (LCP) for the
two molecules A and B.
Acknowledgment. The authors express their gratitude to the
Laboratoires Servier (France), Ministerio de Ciencia y Tecno-
log´ıa (MCYT, BQU 2002-00148) and to the Generalitat de
Catalunya, Spain (SGR2005-00180), for financial support.
(13) (a) Gribble, G. W.; Saulnier, M. G.; Sibi, M. P.; Obaza-Nutaitis, J.
A. Synthesis and Diels-Alder Reactions of 1,3-Dimethyl-4-(phenyl-
sulfonyl)-4H-furo[3,4-b]indole. A New Annulation Strategy for the
Construction of Ellipticine and Isoellipticine. J. Org. Chem. 1984,
49, 4518-4523. (b) Smith, J. G.; Welankiwar, S. S.; Shantz, B. S.;
Lai, E. H.; Chu, N. G. Synthetic Routes to Derivatives of Polycyclic
Aromatic Hydrocarbons Using Isobenzofurans as Transient Reactive
Intermediates. J. Org. Chem. 1980, 45, 1817-1824. (c) Newman,
M. S.; Cells, J. A. A General Route to 2,3-Diacyl-1,4-dihydro-1,4-
disubstituted-1,4-epoxynaphtahlenes and 1,4-Disubstituted 2,3-Naph-
tahlic Anhydrides. J. Org. Chem. 1973, 38, 3482-3487.
(14) (a) Auclair, C. Multimodal Action of Antitumor Agents on DNA:
The Ellipticine Series. Arch. Biochem. Biophys. 1987, 259, 1-14.
(b) Schwaller, M.-A.; Dodin, G.; Aubard, J. Thermodynamics of
Drug-DNA Interactions: Entropy-Driven Intercalation and Enthalpy-
Driven Outside Binding in the Ellipticine Series. Biopolymers 1996,
50, 510-527. (c) Mauffret, O.; Rene, B.; Convert, O.; Monnot, M.;
Lescot, E.; Fernandjian, S. Drug-DNA Interactions: Spectroscopic
and Footprinting Studies of Site and Sequence Specificity of
Elliptinium. Biopolymers 1991, 31, 1325-1341.
(15) (a) Dodin, G.; Schwaller, M. A.; Aubard, J.; Paoletti, C. Binding of
Ellipticine Base and Elliptinium Cation to Calf-Thymus DNA. A
Thermodynamic and Kinetic Study. Eur. J. Biochem. 1988, 176,
371-376. (b) Aubard, J.; Levi, G.; Pantigny, J.; Marsault, J. P.;
Dodin, G.; Schwaller, M. A. SERS of Ellipticine and Derivatives
Free and Bound to DNA. Spec. Publ.sR. Soc. Chem. 1991, 94, 385-
386. (c) Canals, A.; Purciolas, M.; Aymani, J.; Coll, M. The
Anticancer Agent Ellipticine Unwinds DNA by Intercalative Binding
in an Orientation Parallel to Base Pairs. Acta Crystallogr., Sect. D
2005, 61 (7), 1009-1012. (d) Cirino, J. J. V.; Belletato, P.; Dantas,
S.; Ribeiro, L.; Ferreira, G. R.; dos Santos, H. F. Structure and
Properties of Ellipticines. Quim. NoVa 2005, 28, 30-36.
(16) (a) Bermejo, A.; Andreu, I.; Suvire, F.; Le´once, S.; Caignard, D. H.;
Renard, P.; Pierre, A.; Enriz, R. D.; Cortes, D.; Cabedo, N. Synthesis
and Antitumor Targeting G1 Phase of the Cell Cycle of Benzoyldihy-
droisoquinolines and Related 1-Substituted Isoquinolines. J. Med.
Chem. 2002, 45, 5058-5068. (b) Melliou, E.; Magiatis, P.; Mitaku,
S.; Skaltsounis, A.-L.; Pierre´, A.; Atassi, G.; Renard, P. 2,2-Dimethyl-
2H-anthra[2,3-b]pyran-6,11-diones: A New Class of Cytotoxic
Compounds. Bioorg. Med. Chem. 2001, 9, 607-612. (c) Wu, J. M.;
Shan, F.; Wu, G. S.; Li, Y.; Ding, J.; Xiao, D.; Han, J. X.; Atassi,
G.; Le´once, S.; Caignard, D. H.; Renard, P. Synthesis and Cytotox-
icity of Artemisinin Derivatives Containing Cyanoarylmethyl Group.
Eur. J. Med. Chem. 2001, 36, 469-479. (d) Pierre´, A.; Dunn, T. A.;
Kraus-Berthier, L.; Le´once, S.; Saint-Dizier, D.; Regnier, G.;
Dhainaut, A.; Bizzari, J. P.; Atassi, G. In Vitro and In ViVo
Circumvention of Multidrug Resistance by Servier 9788, a Novel
Triazinoaminopiperidine Derivative. I. InVest. New Drugs 1992, 10,
137-141. (e) Bojarski, C.; Gitter, A. H.; Bendfeldt, K.; Mankertz,
J.; Schmitz, H.; Wagner, S.; Fromm, M.; Schulzke, J. D. Permeability
of Human HT-29/B6 Colonic Epithelium as a Function of Apoptosis.
J. Physiol. 2001, 535, 541-552.
Supporting Information Available: X-ray crystallographic data
of 4a, cytotoxicity of 4a evaluated by the NCI, 1H NMR, 13C NMR,
and elemental analysis of all prepared compounds. This material
is available free of charge via the Internet at http://pubs.acs.org.
References
(1) Schepartz, S. A. Introduction and Historial Background. In Cancer
Chemotherapeutic Agents, 1st edition; Foye, W. O., Ed.; American
Chemical Society: Washington D.C., 1995; pp 1-7.
(2) (a) Doyle, T.-W. In Cancer and Chemotherapy; Crooke, S. T.,
Prestayko, A. W., Eds.; Academic Press: New York, 1980; Vol. 1,
pp 295-305. (b) Carter, S. K.; Bakowski, M. T.; Hellmann, K. In
Chemotherapy of Cancer, 3rd edition; John Wiley & Sons: New
York, 1987; pp 43-52. (c) Slichenmyer, W. J.; Rowinsky, E. K.;
Donehower, R. C.; Kaufmann, S. H. The Current Status of Camp-
tothecin Analogues as Antitumor Agents. J. Natl. Cancer Inst. 1993,
85, 271-278.
(3) (a) Sengupta, S. K. Topoisomerase II inhibitors. In Cancer Chemo-
therapeutic Agents; Foye, W. O., Ed.; American Chemical Society:
Washington, D. C., 1995; pp 246-250. (b) Foye, W. O.; Sengupta,
S. K. Cancer Chemotherapy. In Principles of Medicinal Chemistry;
Foye, W. O., Ed.; American Chemical Society: Washington, D. C.,
1995; chapter 37, pp 822-845. (c) Mosher, C. W.; Greus, O. P.;
Acton, E. M.; Goodman, L. Preparation and Antitumor Activity of
Olivacine and Some Analogs. J. Med. Chem. 1966, 9, 237-241. (d)
Kohn, K. W.; Waring, M. J.; Galubiger, D.; Friedman, C. A.
Intercalative Binding of Ellipticine to DNA. Cancer Res. 1975, 35,
71-76.
(4) (a) Jurayj, J.; Haugwitz, R. D.; Varma, R. K.; Paull, K. D.; Barret,
J. F.; Cushman, M. Design and Synthesis of Ellipticinium Salts and
1,2-Dihydroellipticines with High Selectivities against Human CNS
Cancers In Vitro. J. Med. Chem. 1994, 37, 2190-2197. (b) Sainsbury,
M. Ellipticines. In Chemistry of Antitumor Agents; Wilman, D. E.
V., Ed.; Chapman and Hall: New York, 1990; pp 410-435.
(5) (a) Auclair, C.; Pierre, A.; Voisin, E.; Pepin, O.; Cros, S.; Colas, C.;
Saucier, J. M.; Vershuere, B.; Gros, P.; Paoletti, C. Physicochemical
and Pharmacological Properties of the Antitumor Ellipticine Deriva-
tives 2-(2-Diethylaminoethyl)-9-hydroxyellipticinium Chloride, HCl.
Cancer Res. 1987, 47, 6254-6261. (b) Pierson, V.; Pierre, A.;
Pommier, Y.; Gros, P. Production of the Protein-Associated DNA
Breaks by 10-[Diethylaminopropylamino]-6-methyl-5H-pyrido[3′,4′:
4,5]pyrrolo[2,3-g]isoquinoline in Cultured L1210 Cells and in Isolated
Nuclei: Comparison with Other Topoisomerase II Inhibitors. Cancer
Res. 1988, 48, 1404-1409.
(6) (a) Tormo, J. R.; Estornell, E.; Gallardo, T.; Gonzalez, M. C.; Granell,
S.; Cortes, D.; Zafra-Polo, M. C. Gamma-Lactone-Functionalized
Antitumoral Acetogenins are the Most Potent Inhibitors of Mito-
chondrial Complex I. Bioorg. Med. Chem. Lett. 2001, 11, 681-684.
(b) Quintero, A.; Pelcastre, A.; Solano, J. D. Antitumoral Activity
of New Pyrimidine Derivatives of Sesquiterpene Lactones. J. Pharm.
Pharmaceut. Sci. 1999, 2, 108-112.
(7) (a) Zavala, F.; Guenard, D.; Robin, J.-P.; Brown, E. Structure-
Antitubulin Activity Relationships in Steganacin Congeners and
Analogs. Inhibition of Tubulin Polymerization In Vitro by (()-
Isodeoxypodophyllotoxin. J. Med. Chem. 1980, 23, 546-549. (b)
Beard, A. R.; Drew, M. G. B.; Hilgard, P.; Hudson, B. D.; Mann, J.;
Neidle, S.; Wong, L. F. T. Podophyllotoxin Analogs: Synthesis and
Computer Modeling Investigation of Structure-Activity Relation-
ships. Anticancer Drug Des. 1987, 2, 257-252.
(17) Experimental design and publicly available data (including the results
of this compound reported here) are available at http://dtp.nci.nih.gov/
yacds/default.html.
(18) Le Pecq, J. B.; Dat-Xuong, N.; Gosse, C.; Paoletti, C. A New
Antitumoral Agent: 9-Hydroxyellipticine. Possibility of a Rational
Design of Anticancerous Drugs in the Series of DNA Intercalating
Drugs. Proc. Natl. Acad. Sci. U.S.A. 1974, 71, 5078-5082.
(19) (a) Spitzner, J. R.; Muller, M. T. A. Consensus Sequence for Cleavage
by Vertebrate DNA Topoisomerase II. Nucleic Acids Res. 1988, 16,
5533-5556. (b) Rowe, T. D.; Chen, G. L.; Hsuing, Y. H.; Liu, L.
H. Damage by Antitumor Acridines Mediated by Mammalian DNA
Topoisomerase II. Cancer Res. 1986, 46, 2021-2026.
(20) (a) Paull, K. D.; Lin, C. M.; Malspeis, L.; Hamel, E. Identification
of Novel Antimitotic Agents Acting at the Tubulin Level by
Computer-Assisted Evaluation of Differential Cytotoxicity Data.
Cancer Res. 1992, 52, 3892-3900. (b) Bai, R.; Paull, K. D.; Herald,
C. L.; Malspeis, L.; Petttit, G. R.; Hamel, E. Halichondrin B and
Homohalichondrin B, Marine Natural Products Binding in Vinca
(8) Terada, T.; Fujimoto, K.; Nomura, M.; Yamashita, J.; Wierzba, K.;
Yamazaki, R.; Shibata, J.; Sugimoto, Y.; Yamada, Y. Antitumor
Agents. 3. Synthesis and Biological Activity of 4â-Alkyl Derivatives
Containing Hydroxy, Amino, and Amido Groups of 4′-O-Demethyl-
4-desoxypodophyllotoxin as Antitumor Agents. J. Med. Chem. 1993,
36, 1689-1699.
(9) (a) Pujol, M. D.; Romero, M.; Sa´nchez, I. Synthesis and Biological
Activity of New Class of Dioxigenated Anticancer Agents. Curr.
Med. Chem.: Anti-Cancer Agents 2005, 5, 215-239. (b) Ruiz, N.;
Bouyssou, P.; Maurizis, J. C.; Madelmont, J. C.; Coudert, G.
Synthesis of a Benzodioxinic Analogue of Ellipticine and Evaluation
of its Antitumor Activity. Heterocycl. Commun. 1997, 3, 509-514.
(10) Ruiz, N.; Buon, C.; Pujol, M. D.; Guillaumet, G.; Coudert, C. Diels-
Alder Reactions of Furo[3,4-b]1,4-Benzodioxins: An Efficient
Approach to Substituted Dibenzo[b,e]1,4-dioxins. Synth. Commun.
1996, 26, 2057-2066.

Benzodioxinic Lactones as Anticancer Drugs
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 2 307
Domain of Tubulin: Discovery of Tubulin-Based Mechanism of
Action by Analysis of Differencial Cytotoxicity Data. J. Biol. Chem.
1991, 266, 15882-15889. (c) Schepartz, S. A. Prediction of
Biochemical Mechanism of Action from the In Vitro Antitumor
Screen of the National Cancer Institute. In Cancer Chemotherapeutic
Agents, 1st edition; Foye, W. O., Ed.; American Chemical Society:
Washington D.C., 1995; pp 9-46.
(24) Fosse, P.; Rene, B.; Charra, M.; Paoletti, C.; Saucier, J. M.
Stimulation of Topoisomerase II-Mediated DNA Cleavage by El-
lipticine Derivatives: Structure-Activity Relationships. Mol. Phar-
macol. 1992, 42, 590-595.
(25) Constans, P.; Carbo´, R. Atomic Shell Approximation-Electron-
Density Fitting Algorithm Restricting Coefficients to Positive Values.
J. Chem. Inf. Comput. Sci. 1995, 35, 1046-1053.
(26) Constans, P.; Amat, L.; Carbo´-Dorca, R. Toward a Global Maximiza-
tion of the Molecular Similarity Function: Superposition of Two
Molecules. J. Comput. Chem. 1997, 18, 826-846.
(21) Bailly, C.; Arafa, R. K.; Tanious, F. A.; Laine, W.; Tardy, C.;
Lansiaux, A.; Colson, P.; Boykin, D. W.; Wilson, W. D. Molecular
Determinants for DNA Minor Groove Recognition: Design of a bis-
Guanidinium Derivative of Ethidium that is Highly Selective for AT-
Rich DNA Sequences. Biochemistry 2005, 44, 1941-1952.
(22) Bailly, C.; Kluza, J.; Martin, C.; Ellis, T.; Waring, M. J. DNase I
Footprinting of Small Molecule Binding Sites on DNA. In DNA
Synthesis: Methods and Protocols. In Methods in Mol. Biol.;
Herdewijn, P., Ed.; Humana Press: Totowa, NJ, 2004; Vol. 288, pp
319-342.
(27) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb,
M. A.; Cheeseman, J. R.; Vreven, T.; Kudin, K. N.; et al. Gaussian
03, revision B.05; Gaussian, Inc.: Wallingford, CT, 2003.
(28) Humprey, W.; Dalke, A.; Schulten, K. VMD-Visual Molecular
Dynamics. J. Mol. Graphics 1996, 14, 33-38.
(29) IBM Open Visualization Data Explorer, version 4.2.0, 2005; http://
www.research. ibm.com/dx.
(23) Bailly, C. DNA Relaxation and Cleavage Assays to Study Topoi-
somerase I Inhibitors. Methods Enzymol. 2001, 340, 610-623.
JM061184G