Electronic and cytotoxic properties of 2-amino-naphtho[2,3-b]furan- 4,9-diones

Article abstract of DOI:10.1021/jo102233j

The electronic properties of a new set of cytotoxic 2-amino-naphtho[2,3-b] furan-4,9-dione derivatives (1-8) are evaluated. The electron delocalization of these compounds is described by means of their redox potentials and solvatochromic properties. The l

Full text of DOI:10.1021/jo102233j

ARTICLE
pubs.acs.org/joc
Electronic and Cytotoxic Properties of 2-Amino-naphtho[2,3-b]furan-
4,9-diones
Sandra Jimꢀenez-Alonso,†,‡ Judith Guasch,§ Ana Estꢀevez-Braun,*,†,‡ Imma Ratera,§ Jaume Veciana,*,§ and
Angel G. Ravelo*,†,‡
†Instituto Universitario de Bio-Orgꢀanica “Antonio Gonzꢀalez”, Universidad de La Laguna, Avda. Astrofísico Fco. Sꢀanchez, 38206 La
Laguna, Tenerife, Spain
‡Instituto Canario de Investigaciones del Cꢀancer (ICIC)
§Department of Molecular Nanoscience and Organic Materials, Institut de Ciꢁencia de Materials de Barcelona (CSIC), Universitat
Autꢁonoma de Barcelona and CIBER de Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN), 08193 Bellaterra, Barcelona,
Spain
S Supporting Information
b
ABSTRACT: The electronic properties of a new set of cytotoxic
2-amino-naphtho[2,3-b]furan-4,9-dione derivatives (1-8) are
evaluated. The electron delocalization of these compounds is
described by means of their redox potentials and solvatochromic
properties. The large solvatochromism of their intramolecular
electron transfer band is analyzed using the linear solvation
energy relationship method. In addition, this method deter-
mined the importance of the molecular environment, quantifying the interactions that compounds (1-8) establish with their
surrounding media, with the capacity of acting as hydrogen-bond acceptors (HBA) and hydrogen-bond donors (HBD) and the
dipolarity/polarizability being the most significant ones. As a result, a relationship between the electronic and the cytotoxic
properties of these compounds is proposed.
uinones are organic compounds of major importance in
biological systems and industrial applications as dyes or
Q
drugs.1 Compounds with quinone cores are common in
nature and play important physiological roles in animals
and plants.2 They are used in anticancer,3 antibacterial,4 and
antimalarial5 drugs and as fungicides.6 On the basis of their
numerous biological activities and structural properties, this
scaffold is considered a privileged structure in medicinal
chemistry.7,8 Quinones are oxidants and electrophiles. Since
a nucleophilic addition to a quinone represents a formal two-
electron reduction, these properties are interrelated.9 Hence,
they play a major role as bioreductive drugs, oxidative stress
enhancers, and redox catalysts.10 Two major mechanisms of
cytotoxicity have been proposed: stimulation of oxidative
stress and alkylation of cellular nucleophiles, which encom-
pass a large range of biomolecules.11 Quinones undergo
reversible oxidation-reduction processes (Figure 1) gener-
ating reactive oxygen species (ROS) such as superoxide
Figure 1. Redox cycling process of quinone.
reactive oxygen species, which can cause additional damage to
the DNA.14
Another transformation is mainly found in the cytoplasm of
tissues, is catalyzed by the enzyme NAD(P)H:quinone oxidor-
eductase, and involves a two-electron reduction of the quinone
function producing hydroquinone using either NADH or
NADPH as electron donor. This intermediate may then be
inactivated by a subsequent glucuronidation and/or sulfation
or by the conversion of the hydroquinone into an alkylating
intermediate, the quinone methide. Two-electron reduction is
(O2 -) and hydrogen peroxide (H2O2), causing oxidative
3
stress and inducing DNA damage to some essential
proteins.12
Under aerobic conditions, i.e., in tumors with sufficient blood
supply, a one-electron reduction predominates, resulting in free-
radical intermediates that can react with molecular oxygen and
ensuing superoxide production.13 The superoxide anion radical is
further metabolized to hydrogen peroxide (H2O2) and other
Received: November 16, 2010
Published: February 15, 2011
r
2011 American Chemical Society
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three-component reaction following a modified methodology de-
scribed by Temouri et al.18 Thus, the reaction of 2-hydroxynaphto-
quinone with various aldehydes and aromatic or aliphatic
isocyanides, under refluxing in toluene, afforded the corresponding
2-amino-naphtho[2,3-b]furan-4,9-dione (Figure 3). The synthesis
of linear naphtho[2,3-b]furan-4,9-dione derivatives can be rationa-
lized by the initial formation of a conjugated electron-deficient
enone (A) through a Knoevenagel condensation of the 2-hydroxy-
1,4-naphthoquinone and an aldehyde. The next step of this
mechanism could involve a [4 þ 1] cycloaddition reaction of the
electron-deficient heterodiene moiety of the adduct (A) with the
isocyanide to afford an iminolactone intermediate (B). The sub-
sequent isomerization of iminolactone (B) leads to the formation of
the corresponding naphthofuran derivative. The use of EDDA as a
catalyst accelerated the process and afforded the desired products in
higher yields and shorter times.
The structures of the new 2-amino-naphtho[2,3-b]furan-4,9-
diones (1-8) and the corresponding reaction yields are detailed in
Table 1. Good yields were obtained when aliphatic or aromatic
aldehydes were used. The worst yield (57%) resulted from the
combination of the hindered tert-butyl isocyanide with benzaldehyde.
An intense solvatochromic effect was detected during the
manipulation of the obtained compounds with different solvents,
and all products were isolated as amorphous deep blue solids. As
an example, single crystals of 3 were grown by slow evaporation
from acetone and used for their X-ray crystal determination.
Weak H-bonds were found between the amino proton and the
carbonyl groups of neighboring quinones leading to a distance of
2.44 Å between molecules (Figure 4).
Figure 2. Structure of 2-amino-naphtho[2,3-b]furan-4,9-dione deriva-
tives.
generally known as the detoxification step by reducing
the quinone into the stable hydroquinone, which is readily
excreted after conjugation. Such a pattern is believed to
predominate under anaerobic conditions or inside anaerobic loci
within a cell.
However, to obtain more information about the mechanisms
of cytotoxicity is it interesting to study not only the electronic
properties of the bioactive quinones but also their molecular
environment. Changes of the intermolecular interactions
(hydrogen-bond ability, dipolar moment, etc.) are clearly capable
of affecting the structures, reactivities, biological activities, equi-
libria, reaction rate constants, and a host of other aspects that are
of central interest to chemistry and biology.15 Electrochemical
and spectroscopic measurements are well suited for characteriz-
ing the molecules in their local environment.16 Thus, specific
shifts in the redox potentials and in the absorption spectra reflect
the extent of stabilization that the molecular ground and excited
states experience due to their own nature as well as their
solvent-solute interactions.17
Our research group is especially interested in antitumoral
compounds based on quinone cores fused to heterocyclic
rings.5,8c-8f In this sense, we have recently published the design
and synthesis of novel pyranonaphthoquinones through an
intramolecular domino Knoevenagel hetero-Diels-Alder reac-
tion. The obtained adducts resulted to be effective catalytic
inhibitors of topoisomerase II.8d
With the aim of obtaining new bioactive quinones, we
synthesized a set of new 2-amino-naphtho[2,3-b]furan-4,9-dione
derivatives (Figure 2). These compounds were tested for cyto-
toxicity against several human solid tumor cell lines (MCF7,
MCF7/BUS, and SK-Br-3). Moreover, the electronic properties
of these aromatic donor-acceptor derivatives were analyzed by
means of their redox potentials and solvatochromic properties.
This study revealed that the higher the electron delocalization
within the molecule, the higher the cytotoxicity against the tested
human solid tumor cell lines. In addition, thanks to the linear
solvation energy relationship method applied to determine the
solvatochromism of these compounds, we were able to identify the
interactions that the molecules establish with their environment.
To determine the potential cytotoxicity of these derivatives
according to antecedents of different naphthoquinones,19 com-
pounds 1-8 were tested against the following tumoral cell lines:
MCF7, MCF7/BUS, and SK-Br-3 (Table 2).
Compounds 4, 6, and 8 exhibit the highest cytotoxicities.
Compound 4 shows selectivity toward the hormone-dependent
cell line MCF7/BUS with a GI50 value of 9.2 μM. Compounds
6 and 8 present good cytotoxicities against MCF7, MCF7/BUS,
and SK-Br-3 tumor cell lines. The best GI50 was achieved
by compound 6 against the SK-Br-3 cell line, with GI50 = 1.6 μM.
Electrochemistry. The electrochemistry of a plethora of
quinones has been widely studied.13 However, there are only
few reports on electrochemical studies of heterocyclic
naphthoquinones.9c Cyclic voltammograms of naphthoquinones
in organic aprotic solvents present two typical waves correspond-
ing to two sequential reversible or quasi-reversible one-electron
transfer processes (Figure 5). The first wave is related to the
redox couple quinone (Q)/semiquinone anion radical (Q -),
3
whereas the second one is related to the semiquinone anion
radical (Q -)/quinone dianion (Q2-).20
3
’ RESULTS AND DISCUSSION
In our case, cyclic voltammograms of the 2-amino-naphtho-
[2,3-b]furan-4,9-dione derivatives were performed in DMF
using tetrabutylammonium hexafluorophosphate (HFPTBA)
Synthesis and Cytotoxic Activity. 2-Amino-naphtho[2,3-
b]furan-4,9-diones (1-8) were synthesized using a one-pot
Figure 3. Synthesis of compounds (1-8) through a one-pot three-component reaction.
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Figure 4. Crystal structure and packing of compound 3.
Table 1. Summary of the Synthesis of 2-Amino-naphtho[2,3-
b]furan-4,9-dione Derivatives (1-8)
Table 2. Cytotoxic Activity of Compounds 1-8 against a
Representative Panel of Human Breast Cancer Cell Linesa
compound
MCF7
MCF7/BUS
SK-Br-3
1
2
3
4
5
6
7
8
18.8 ((0.07)
29.8 ((0.4)
29.9 ((1.3)
22.2 ((1.2)
82.0 ((2.5)
5.9 ((0.4)
12.1 ((0.6)
15.2 ((0.4)
17.9 ((0.4)
9.22 ((0.4)
95.2 ((0.9)
2.8 ((0.5)
10.2 ((0.3)
14.6 ((0.5)
24.5 ((1.5)
22.9 ((2.0)
87.8 ((2.4)
1.6 ((0.4)
14.1 ((0.3)
5.8 ((0.6)
19.4 ((0.4)
7.6 ((0.4)
17.4 ((0.6)
3.9 ((0.07)
a Expressed as GI50 values given in μM and determined as means of two
to five experiments; standard deviations are given in parentheses.
atmosphere by saturation with argon during several minutes at room
temperature. As expected, these molecules exhibit two waves, the
Q/Q - reversible and the Q -/Q2- quasi-reversible (Figure 6).
3
3
The residual water present in the solvent might catalyze the
disproportionation of the radical anion or be reduced by the
electrogenerated dianion (Q2-),21 explaining the quasi-reversi-
ble character of the second reduction process. It could also be
explained as being due to the ion-pairing stabilization of the
quinone dianion (Q2-) by the tetrabutylammonium cation.22
Taking into account the nature of the two waves, the discussion
refers only to the reversible wave I (Table 3).
The presence of an aromatic moiety in the structure of the
2-amino-naphtho[2,3-b]furan-4,9-dione derivatives favors
the delocalization of their electrons. Moreover, as reported
for aromatic dianions,23 charges dispersed over a large
molecular framework are usually less affected or unaffected
by counterions. Consequently, compounds 5, 6, 7, and 8
present higher redox potentials than the rest and therefore,
I
their reductions are facilitated (E1/2 > -1,300 V). It is
interesting to highlight that compounds 5 and 6, which
exhibit the best electron delocalization due to the presence
of an aromatic group directly attached to the carbon C-3 of
the furan ring, have the highest reduction potentials. Another
crucial factor to take into account for compound 6 is its extra
stabilization caused by the formation of an intramolecular
seven-membered ring between the 20-H (furan numbering)
and one oxygen of the quinone structure (Figure 7).
as supporting electrolyte under different scan rates (50-125
mV s-1). The obtained potential values are referred to Fc/Fcþ, as
recommended by IUPAC, and all studies were carried out in an inert
This O-H interaction reduces the electron density on the
oxygen, increases the electrophilic nature of the aromatic system,
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Figure 8. Neutral and zwitterionic resonance formulas of 2-amino-
naphtho[2,3-b]furan-4,9-dione derivatives (the neutral form dominates
in the electronic ground state, and the dipolar zwitterionic form
dominates in the CT excited state).
Figure 5. Scheme of the redox processes of the 2-amino-naphtho[2,3-
b]furan-4,9-dione derivatives.
grams. The expected ones can be obtained after degasification with
argon, which demonstrates the reversibility of the involved process and
the stability of these compounds after reacting with oxygen.
According to these results, the 2-amino-naphtho[2,3-b]furan-
4,9-dione derivatives with the higher electron delocalizations
present higher reduction potentials and higher cytotoxic activ-
ities against the studied cell lines, as already observed in other
similar systems.25 High reduction potentials facilitate the semi-
quinone formation and therefore the initial bioreduction step
described in many cytotoxic mechanisms of action of different
naphthoquinones. Moreover, the influence of the oxygen ob-
served in the cyclic voltammograms supports the importance of
this factor as previously reported,26 as well as the mechanism of
action related to reactive oxygen species. Consequently, although
caution must be taken in interpreting these results, it is reason-
able to think that a plausible mechanism of action of these
compounds involves an initial bioreduction process followed by
the generation of reactive oxygen species. Moreover, 2-amino-
naphtho[2,3-b]furan-4,9-dione derivatives with E1/2I > -1,250 V
are very susceptible to show cytotoxic activity against the MCF7,
MCF7/BUS, and SK-Br-3 cell lines. However, even though the
trend between the redox potentials and the cytotoxicities of these
compounds is clear, it was not possible to establish an unequi-
vocal lineal relationship between these parameters pointing out
the importance of other factors such as steric, liphophilic, etc. of
in vitro cytotoxicity. Compound 5, with an aromatic substituent,
is the only product that having a high redox potential shows a low
cytotoxic activity. This fact indicates that compound 5 possibly
acts through a different mechanism or presents other properties
that are significant for its cytotoxicty.
Intramolecular Electron Transfer Process and Its Solvent
Dependence. It is known that the molecular environment and
the different intermolecular interactions are very important for
bioactive compounds. They affect parameters such as diffusion,
solubility, and membrane permeability, among others.27 To
study the molecular environment, spectroscopic measurements
were used. The solvatochromic shifts related to the intramolecular
electron transfer (IET) process (Figure 8) were studied using an
empirical method called linear solvation energy relationship (LSER),
developed by Kamlet, Taft, and co-workers.28 This method not only
determines the media effect in terms of specific solvent-solute
interactions, such as possible solvation spheres that surround the
molecules and affect their capacity of bonding with the substrates, but
also gives an alternative approach to the cyclic voltammetry technique
to quantify the electronic properties of such molecules. Therefore, the
LSER method is extremely suitable to analyze the electronic proper-
ties of the 2-amino-naphtho[2,3-b]furan-4,9-diones to relate them to
their cytotoxic activity.
-
Figure 6. Cyclic voltammogram of 3 (wave I: Q/Q -; wave II: Q
/
.
3
3
Q2-). [Q] = 1.0 mM in DMF/HFPTBA (0.1 M); 25 °C; v = 0.1 V s-1
Table 3. Voltammetric Parameters of 1-8 vs Fc/Fcþa
wave I
wave II
EaI (V)
EcI (V)
ΔEI (V)
E1/2I (V)
Ia/IcI
EaII (V)
1
2
3
4
5
6
7
8
-1.2977
-1.3017
-1.2779
-1.2606
-1.2523
-1.2018
-1.2432
-1.2339
-1.3903
-1.3780
-1.3742
-1.3891
-1.3379
-1.3030
-1.3635
-1.3621
0.0926
0.0764
0.0963
0.1285
0.0856
0.1012
0.1202
0.1282
-1.3440
-1.3398
-1.3260
-1.3248
-1.2951
-1.2524
-1.3033
-1.2980
0.86
0.80
0.98
0.90
0.96
0.71
0.55
0.62
0.4134
0.3987
0.4246
0.4392
0.4610
0.4660
0.4767
0.4070
a [Q] = 1.0 mM in DMF/HFPTBA 0.1 M; 25 °C; ν = 0.1 V s-1
.
Figure 7. H-bonding interaction between the furan ring and the
carbonyl group in compound 6.
and stabilizes the semiquinone anion radical formed during the
intramolecular electron transfer process (IET) that these com-
pounds present (Figure 8).13,24 Obviously, this interaction can
also take place in the other compounds but is favored in compound 6
thanks to the electron-withdrawing oxygen element of the furan ring.
Compounds without aromatic groups directly bonded to the furano-
naphthoquinone structure present lower redox potentials all of them
being E1/2I <-1,300 V. Finally, it is worth to mention that the presence
of atmospheric oxygen in the sample modifies the cyclic voltammo-
Marcus theory for donor-acceptor (DA) systems makes it
possible to correlate the energy of the optical absorption due to a
charge transfer (CT) (Eopt or hνmax) with the free energy
changes that the molecule experiences during the charge separa-
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Figure 9. (Top) Solvatochromism of compound 3 in different solvents. (Bottom) Charge-transfer bands of compound 3 in 17 different solvents.
tion and recombination resulting from the internal bond length
alterations (or vibrational changes), λv, and the outer solvent
reorganizations, λo. For asymmetric compounds, an additional
term, ΔG°, named redox or energetic asymmetry, describing the
energy difference between the charge-separated DþA- state and
the neutral DA ground state is added, so that the total energy
balance is expressed as follows:29
Eopt ¼ hνmax ¼ λν þ λo þ ΔG°
ð1Þ
The solvent dependence of the optical energy of the CT band
enters into this formula in two different ways: one is by the outer
solvent reorganization energy, but it also plays a role through the
redox asymmetry term, while the internal (vibrational) reorga-
nization energy is supposed not to vary with the solvent.30 In this
part, the solvent dependence of spectroscopic data of our set of
2-amino-naphtho[2,3-b]furan-4,9-dione derivatives (1-8) is
described. This will corroborate the cyclic voltammetry results
in terms of the electron delocalization. Moreover, it will describe
the interactions of such compounds with their surrounding
media, an important factor for their cytotoxic activity.
Figure 10. Relationship between the observed IET band ν3 (cm-1) for
compound 3 and the relative permittivity εr of the solvents.
All compounds exhibit a broad absorption band in the visible
region of their spectra that presents an intense solvatochromism (Δλ
≈ 80 nm) going from orange to blue when moving from hexane to
ethanol (Figure 9 and Table S1 in Supporting Information).
These bands correspond to the excitation of a neutral DA
ground state to the charge separated state DþA-, indicative of an
intramolecular electron transfer process of amino-substituted
quinonoid π-π* structures.31 Looking how the νi (cm-1) of the
IET band varies with the solvent polarity measured by their
relative permittivity (εr) (Figure 10), a clear deviation from
linearity is detected. Therefore, it is corroborated that the solvent
cannot be considered as a structureless continuum and that
specific solvent/solute interactions must be playing an important
role. To take into account such specific interactions and explain
in more detail the solvatochromic behavior that these com-
pounds exhibit, a linear solvation energy relationship was used.
The LSER treatment is a powerful tool for the study of the
principal intermolecular interactions that control a specific
physicochemical process in solution. The LSER assumes that
the changes in the free energy associated with a physicochemical
process are the sum of different independent contributions
generated from the different solute/solvent interactions. In this
case, it was confirmed that there are three parameters to be
considered: R, β, and π*.28 The nonspecific parameter π*
measures the attractive effect of dipole/dipole and dipole/
induced dipole interactions between the solute and the solvent
molecules, i.e., it is a measure of the dipolarity/polarizability. The
solvatochromic specific parameter β is a quantitative empirical
measure of the ability of a solvent to act as hydrogen-bond
acceptor (HBA)15 or electron donor toward a standard solute, i.
e., it is a measure of the basicity of the solvent. By contrast, the
specific empirical parameter R quantitatively measures the ability
of a solvent to act as a hydrogen-bond donor (HBD)15 or
electron-pair acceptor toward a standard solute, i.e., it is a
measure of the acidity of the solvent.29 Therefore, the generalized
LSER equation that describes the IET energy associated with the
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Table 4. LSER Values Obtained by a Multivariable Linear
Regression Fit for Compound 3a
coeff
std error std coeff tolerance
t
p
ν°3
a3
b3
s3
20.543
-1.064
-1.679
-1.602
0.056
0.126
0.106
0.087
0
365.938 <0.0004
-8.446 <0.0004
-15.915 <0.0004
-18.402 <0.0004
-0.272
-0.541
-0.547
0.721
0.647
0.847
a Standardized coefficients and distribution values (t) indicate the
goodness of this method (p); whereas the tolerance is an indication of
the orthogonality among a3, b3, and s3. R = 0.995. Standard error of
estimate = 0.095
CT absorption for a donor-acceptor dyad in any solvent media
adopts the form of eq 2:
ꢀ
νi ¼ ν°i þ aiR þ biβ þ siπ
ð2Þ
Figure 11. IET energies, calculated using the LSER eq 3, versus the
experimentally observed values.
where the coefficients ai, bi, si, and the constant ν°i are char-
acteristic of each studied compound i independent of the nature
of the solvent. These coefficients are indicative of the sensitivity
of the IET process exhibited by the compound i toward the
variation of each solvent property (R, β, and π*). Such coeffi-
cients must have negative (or positive) signs according to the
nature of each term, and the independent term ν°i (in cm-1) is
the energy of the absorption expected for the IET process in the
absence of a solvent, i.e., in a vacuum.29 The energies of the CT
bands were measured for compound 3 at room temperature in 17
different solvents, which were chosen as representative of the 11
groups in which the most common laboratory solvents are
classified.32 By fitting the experimental data ν3 (cm-1) obtained
in different solvents with the known R, β, and π* parameters for
each solvent to the eq 2 through a multivariable linear regression,
the coefficients ai, bi, si and the independent term ν°i correspond-
ing to the IET process were calculated for compound 3. By
means of statistical analysis, those coefficients with a low
significance level were removed from the calculated model.
The LSER treatment of the experimental spectroscopic data of
compound 3 demonstrates the presence of specific H-bonding
interactions between this compound and the solvent along with
nonspecific dipolarity/polarizability interactions that contribute
significantly to the solvent-induced IET process. The final LSER
model obtained is described in Table 4 and summarized by eq 3:
Figure 12. Scheme of different solvent interactions with the 2-amino-
naphtho[2,3-b]furan-4,9-dione derivatives.
hydrogen-bond formation between these compounds and the
solvent enhances the stability of the whole system. (b) The
excited state of 3 and the corresponding ones for the other
compounds 1-8 present larger dipole moments and greater
polarizabilities than the corresponding electronic ground states.
Since the π* values measure the dipolarity/polarizability of the
solvents, the negative sign of s3 indicates a greater stabilization of
the excited state by means of larger dipole/dipole and dispersion
interactions with increasing π* values, as compared to the
electronic ground state.
To determine the solvent effect on the IET band of the rest of our
family of 2-amino-naphtho[2,3-b]furan-4,9-diones, derivatives 1-8
were studied. Taking into account the important specific and
nonspecific parameters R, β, and π*, three representative solvents
were selected to describe the solvent influence on the IET of these
2-amino-naphtho[2,3-b]furan-4,9-diones: toluene as a nonpolar one,
acetonitrile as a polar one, and ethanol as polar and protic one.
The following spectroscopic data were obtained for each
compound (Table 5): (a) In general terms, the solvent tendency
previously described for compound 3 is maintained in each
derivative. Therefore, an increase of any parameter R, β, or π*
causes a decrease in the energy and a red shift of the IET band,
which can be explained as an increase of the stability of these
molecules in the surrounding media. (b) Derivatives substituted
with aromatic functions like 5, 6, 7, and 8 present less solvato-
chromism, which is indicative of being more stable than the other
ones due to their capability of delocalizing their electrons and
stabilizing the charge separated state as it was observed in the
cyclic voltammetry experiments. Moreover, those compounds
that contain aromatic groups at the C-3 of the furan ring (5 and
ν3 ¼ 20:543ð ( 0:056Þ - 1:064ð ( 0:126ÞR -
ꢀ
1:679ð ( 0:106Þβ - 1:602ð ( 0:087Þπ
ð3Þ
Figure 11 shows the good agreement achieved between the
calculated IET energy from the LSER model obtained for each
solvent and the experimental data for compound 3.
From the analysis of the LSER model, the following informa-
tion can be extracted: (a) The IET energy for compound 3 is very
sensitive to changes of the HBD or HBA abilities of the
surrounding media. Thus, the negative values obtained for the
coefficients a3 and b3 result in a decrease in the energy (red shift)
of the IET band with increasing solvent hydrogen-bond donor
(R) or acceptor abilities (β). The decrease of energy with the
increasing of R can be explained in terms of the quinone structure
due to the capability of its carbonyl groups to interact with HBD
solvents. Similarly, the HBA solvents are able to interact with the
2-amino-naphtho[2,3-b]furan-4,9-dione derivatives through
their amino function (Figure 12) leading to a decrease in energy
when β increases. Therefore, it can be concluded that the
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Table 5. Solvatochromism of the IET Band of Several 2-Amino-naphtho[2,3-b]furan-4,9-dione Derivatives in Toluene (tol),
Acetonitrile (AN), and Ethanol (OH)
λtola (nm)
λANa (nm)
λOHa (nm)
εtolb (cm-1 M-1
)
εANb (cm-1 M-1
)
εOHb (cm-1 M-1
)
Δλtol/OHc (nm)
1
2
3
4
5
6
7
8
521
523
508
511
524
531
514
509
550
553
541
542
541
549
538
538
577
580
568
569
556
568
565
564
4780
4076
4033
4170
5427
4908
4263
1475
5744
5184
4714
5331
5784
5407
4834
1708
7605
7327
4903
6700
6230
6162
5259
2443
56
57
60
58
32
37
51
55
a λmax of a given compound in solvent i. b εmax of a given compound in solvent i. c Δλi/j = λmax,j - λmax,i being i, j solvents.
6) are even more stable than those that have aromatic functions
in the amino position (7 and 8) due to their capability of a better
delocalization of their electrons through the quinoid structure.
(c) The dependence of the solvent polarity with the molar
absorption coefficients presents the same tendency. Therefore,
the highest ε values were obtained in ethanol according to other
systems previously described in the literature.29
using Pt as working and counter electrodes. The reference electrode was Ag/
AgCl. However, the potential values obtained are referred to Fc/Fcþ as
recommended by IUPAC. The concentrations of the samples were 1 mM in
DMF using tetrabutylammonium hexafluorophosphate (HFPTBA) as sup-
porting electrolyte (0.1 M). Different scan rates (50-125 mV s-1) were
used. All studies were carried out in an inert atmosphere by saturation with
argon during several minutes at room temperature. Each absorption spectrum
reported is the median of 3 spectra whose extinction coefficients did not differ
significantly. Each spectrum was obtained from an independent solution of
the corresponding studied compound. The spectra were performed at room
temperature.
’ CONCLUSIONS
In this paper we present a new set of cytotoxic 2-amino-
naphtho[2,3-b]furan-4,9-dione derivatives (1-8). A relationship
between the electronic properties of these compounds and their
cytotoxicity against several human tumor cell lines (MCF7,
MCF7/BUS, and SK-Br-3) is established: the more delocalized
the electronic system, the greater the cytotoxic activity. The
electron delocalization is quantitatively evaluated by means of
reduction potentials and, in a more novel approach, by the
solvatochromism of the IET band through a LSER treatment.
Hence, high electron delocalizations imply high redox potentials
and small solvatochromic effects. In fact, we can affirm that
compounds with E(1/2)1 > -1,250 V and Δλtol/OH < 40 nm are
extremely susceptible to present cytotoxic activity against the
studied tumor cell lines. This observed trend can be easily
explained taking into account the reduction potential. Thus, a
high reduction potential facilitates semiquinone formation and
therefore the bioreduction step that is supposed to initiate the
cytotoxic activity of such compounds. Moreover, an influence of
the atmospheric oxygen is demonstrated that suggests that the
mechanism of action of our compounds involves reactive oxygen
species. Moreover, the LSER treatment allowed the determina-
tion of the principal interactions that these compounds 1-8
present with their surrounding media, the capacity of acting as
electron donors and acceptors and the dipolarity/polarizability
being the most important. These interactions not only play a role
from the electronic point of view but also from the structural one.
General Procedure for the Synthesis of 2-Amino-
naphtho[2,3-b]furan-4,9-dione Derivatives. The synthesis of
2-amino-naphtho[2,3-b]furan-4,9-dione derivatives was made following
a modified methodology described by Teimouri, et al.18 2-Hydroxy-1,4-
naphthoquinone (100.0 mg, 0.547 mmol) in 10 mL of toluene was
treated with 1.2 equiv of aldehyde, 1.2 equiv of isocyanide, and catalytic
amounts of EDDA (ethylenediamino diacetate, 5% mmol). The reaction
mixture was heated under reflux and checked by TLC until disappear-
ance of the starting naphthoquinone. Then, the reaction mixture was
cooled, and the toluene was removed under reduced pressure. The crude
was purified by silica gel column chromatography with Hex/EtOAc as a
mixture of solvents.
2-Cyclohexylamino-3-ethylnaphtho[2,3-b]furan-4,9-dione (1). Follo-
wing the general procedure described above, 100.0 mg (0.574 mmol) of
2-hydroxy-1,4-naphthoquinone in 10 mL of toluene was treated with 1.2
equiv of propionaldehyde (0.689 mmol, 0.049 mL), 1.2 equiv of cyclohexyl
isocyanide (0.689 mmol, 0.085 mL), and 0.1 equiv of EDDA (0.057 mmol,
10.3 mg). The reaction mixture was refluxed for 30 min. The solvent was
removed under vacuum, and the crude was purified by flash chromatography
(silica gel, Hex/EtOAc 9:1) to provide 1 (144.3 mg, 77%) as an amorphous
blue solid. IR (neat) νmax 2931, 2856, 1642, 1587, 1540, 1489, 1453, 1350,
1285, 1257, 1220, 1153, 1114, 1082, 1030, 996, 966, 891, 847, 812, 755, 698
cm-1; 1H NMR (300 MHz; CDCl3) δ 1.11 (3 H, t, J = 7.4 Hz), 1.18-1.42
(5H, m), 1.60(1H, m), 1.71(2H, m), 2.02(2H, m), 2.59(2H, m), 3.74(1
H, m), 4.84 (1 H, d, J = 8.2 Hz), 7.53 (1 H, t, J= 7.3 Hz), 7.62 (1 H, t, J = 7.3
Hz), 7.99 (1 H, d, J = 7.3 Hz), 8.09 (1 H, d, J =7.4 Hz); 13C NMR (75 MHz;
CDCl3) δ13.6 (CH3), 15.5 (CH2), 24.5 (2 ꢁ CH2), 25.1 (CH2), 33.7 (2ꢁ
CH2), 52.2 (CH), 101.0 (C), 125.7 (CH), 125.8 (CH), 131.5 (CH), 132.4
(C), 132.6 (C), 133.4 (CH), 133.7 (C), 142.0 (C), 159.3 (C), 167.6 (C),
182.8 (C); EIMS m/z 323 [Mþ] (43), 241 (100), 226 (39), 55 (15);
HREIMS 323.1521(calcd for C20H21NO3, 323.1516).
’ EXPERIMENTAL SECTION
General Methods. All solvents and reagents were purified by
standard techniques reported (Perrin, D. D.; Amarego, W. L. F. In
Purification of Laboratory Chemicals, 3rd ed.; Pergamon: Oxford, 1988 )
or used as supplied from commercial sources when appropriate. Reac-
tions were monitored by TLC (on silica gel POLYGRAM SIL G/UV254
foils). Precoated TLC plates SIL G-100 UV254 were used for preparative
TLC purification. 1H NMR spectra were recorded in CDCl3 at 300 and 400
MHz. All compounds were named using ACD40 Name-Pro program, which
is based on IUPAC rules. Cyclic voltammetry experiments were performed
2-Cyclohexylamino-3-hexyl-naphtho[2,3-b]furan-4,9-dione (2). Follo-
wing the general procedure described above 100.0 mg of 2-hydroxy-1,4-
naphthoquinone (0.574 mmol) in toluene (10 mL) was treated with 1.2
equiv of heptaldehyde (0.689 mmol, 0.096 mL), 1.2 equiv of cyclohexyl
isocyanide (0.689 mmol, 0.085 mL), and 0.1 equiv of EDDA (0.057 mmol,
10.3 mg). The mixture was heated under reflux for 30 min under nitrogen
atmosphere. Then, the solution was cooled at room temperature, and the
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The Journal of Organic Chemistry
ARTICLE
solvent was removed under reduced pressure. The crude was purified by flash
chromatography (silica gel, Hex/EtOAc 9:1) to provide 2(205.4 mg, 94%) as
an amorphous blue solid. IR (neat) νmax 2926, 1725, 1662, 1641, 1587, 1537,
1465, 1447, 1085, 875 cm-1; 1H NMR (300 MHz; CDCl3) δ 0.80 (3 H, s),
1.23-1.30 (8 H, m), 1.40-1.45(2H, m), 1.53(2H, m), 1.64(2H, m), 1.76
(2H, m), 2.25(2H, m), 2.55(2H, t,J=7.4 Hz), 3.79 (1 H, m), 4.33 (1 H, d,
J=7.8Hz), 7.70(1H, t, J= 7.2 Hz), 7.77 (1 H, t, J=7.1 Hz), 8.12 (1H, d, J=
7.2 Hz), 8.16 (1H, d, J = 7.3 Hz); 13C NMR (75 MHz; CDCl3) δ 13.8 (c),
22.2(t), 22.4(t), 24.5(2t), 25.1 (t), 28.8 (t), 29.1 (t), 31.4 (t), 33.8 (2t),52.1
(d), 99.5 (s), 125.8 (d ꢁ 2), 131.5 (d), 132.5 (s), 132.7 (s), 133.4 (d),
133.6(s), 142.4 (s), 159.3 (s), 168.1 (s), 182.5 (s); m/z(EI):379(Mþ, 100),
308 (20), 297 (31), 226 (80), 55 (21); HREIMS 379.2147 (calcd for
C24H29NO3, 379.2129).
2-tert-Butylamino-3-ethyl-naphtho[2,3-b]furan-4,9-dione (3). Follo-
wing the general procedure described above, 100.0 mg (0.574 mmol) of
2-hydroxy-1,4-naphthoquinone in 10 mL of toluene was treated with 1.2
equiv of propionaldehyde (0.689 mmol, 0.049 mL), 1.2 equiv of tert-butyl
isocyanide (0.689 mmol, 0.078 mL), and 0.1 equiv of EDDA (0.057 mmol,
10.3 mg). The mixture was refluxed for 30 min under nitrogen atmosphere.
Then, the solution was cooled to room temperature, and the solvent was
removed under reduced pressure. The crude was purified by flash chroma-
tography (silica gel, Hex/EtOAc 9:1) to provide 3 (154.3 mg, 90%) as an
amorphous blue solid. IR (neat) νmax 2930, 1709, 1641, 1584, 1538, 1464,
1369, 1281, 1207, 1083, 874, 755, 702 cm-1;1H NMR (300 MHz; CDCl3) δ
1.10 (3 H, t, J = 7.5 Hz), 1.42 (9 H, s), 2.57 (2 H, c), 4.74 (1 H, s), 7.53 (1 H,
t, J= 7.4 Hz), 7.59 (1 H, t, J=7.4 Hz), 7.96 (1 H, d, J= 7.5 Hz), 8.07 (1 H, d, J
= 7.5 Hz); 13C NMR (75 MHz; CDCl3) δ 13.6 (3 ꢁ CH3), 15.7 (CH2),
29.8(CH3), 53.6(C), 103.3(C), 125.7(CH), 125.8(CH), 131.5(C), 131.6
(CH), 132.6 (C), 133.3 (CH), 133.5 (C), 143.1 (C), 159.3 (C), 168.1 (C),
182.6 (C); EIMS m/z 297 [Mþ] (15), 241 (100), 55 (12); HREIMS
297.1365 (calcd for C18H19NO3 297.1357).
2-tert-Butylamino-3-ethyl-naphtho[2,3-b]furan-4,9-dione (4). 2-
Hydroxy-1,4-naphthoquinone (100.0 mg, 0.574 mmol) in 10 mL of
toluene was treated with 1.2 equiv of heptaldehyde (0.689 mmol, 0.096
mL), 1.2 equiv of tert-butyl isocyanide (0.689 mmol, 0.078 mL), and 0.1
equiv of EDDA (0.057 mmol, 10.3 mg). The reaction mixture was
refluxed for 30 min under nitrogen atmosphere. The solvent was
removed under vacuum, and the crude was purified by flash chroma-
tography (silica gel, Hex/EtOAc 9:1) to provide 4 (159.4 mg, 79%) as an
amorphous blue solid. IR (neat) νmax 2859, 1641, 1583, 1539, 1463,
1369, 1273, 1258, 1083, 933, 756 cm-1; 1H NMR (300 MHz; CDCl3) δ
0.77 (3 H, s), 1.20 (6 H, m), 1.42 (6 H, s), 1.47 (5 H, m), 2.53 (2 H, t, J =
7.4 Hz), 4.82 (1 H, s), 7.53 (1 H, t, J = 7.4 Hz), 7.59 (1 H, t, J = 7.4 Hz),
7.96 (1 H, d, J = 7.5 Hz), 8.06 (1 H, d, J = 7.5 Hz); 13C NMR (75 MHz;
CDCl3) δ 13.17 (CH3), 22.3 (2 ꢁ CH2), 28.8 (CH2), 29.1 (CH2), 29.8
(CH3 ꢁ 3), 31.4 (CH2), 53.5 (C), 101.8 (C), 125.7 (CH), 125.8 (CH),
131.5 (CH), 131.7 (C), 132.7 (C), 133.3 (CH), 133.5 (C), 143.0 (C),
159.7 (C), 167.9 (C), 182.6 (C); EIMS m/z 353 [Mþ] (24), 297 (96),
226 (100); HREIMS 353.1991 (calcd for C22H27NO3 353.1996).
2-tert-Butylamino-3-phenyl-naphtho[2,3-b]furan-4,9-dione (5). Fol-
lowing the general procedure described above, 100.0 mg (0.574 mmol) of
2-hydroxy-1,4-naphthoquinone in 10 mL of toluene was treated with 1.2
equiv of benzaldehyde (0.689 mmol, 0.070 mL), 1.2 equiv of tert-butyl
isocyanide (0.689 mmol, 0.078 mL), and 0.1 equiv of EDDA (0.057 mmol,
10.3 mg). The reaction mixture was refluxed for 30 min under nitrogen
atmosphere. The solvent was removed under vacuum, and the crude was
purified by flash chromatography (silica gel, Hex/EtOAc 9:1) to provide 5
(114.2 mg, 57%) as an amorphous blue solid. IR (neat) νmax 2971, 1709,
1671, 1640, 1580, 1538, 1508, 1460, 1345, 1250, 1202, 1169, 1083, 973, 918,
805, 752, 696 cm-1; 1H NMR (300 MHz; CDCl3) δ 1.48 (9 H, s), 5.02 (1
H, m), 7.31(1H, m), 7.39(4H, m), 7.46 (1H, t, J=6.5), 7.58(1H, t, J=6.4
Hz), 7.98 (1 H, d, J = 7.4 Hz), 8.11 (1 H, d, J= 7.5 Hz); 13C NMR(75 MHz;
CDCl3) δ 29.7 (3 ꢁ CH3), 53.7 (C), 100.0 (C), 125.7 (CH), 126.1 (CH),
127.4 (CH), 128.5 (CH ꢁ 2), 129.1 (CH ꢁ 2), 129.9 (C), 130.1 (C), 131.9
(CH), 132.9 (C), 133.0 (C), 133.3 (CH), 143.6 (C), 159.2 (C), 168.9 (C),
181.4 (C); EIMS m/z 345 [Mþ] (17), 289 (100), 272 (8), 233 (10), 204
(7); HREIMS 345.1365 (calcd for C22H19NO3 345.1376).
2-tert-Butylamino-3-furan-3-yl-naphtho[2,3-b]furan-4,9-dione (6). Fol-
lowing the general procedure described above, 100.0 mg (0.574 mmol) of
2-hydroxy-1,4-naphthoquinone in 10 mL of toluene was treated with 1.2 equiv
of 3-furaldehyde (0.689 mmol, 0.060 mL), 1.2 equiv of tert-butyl isocyanide
(0.689 mmol, 0.078 mL), and 0.1 equiv of EDDA (0.057 mmol, 10.3 mg).
The reaction mixture was refluxed for 30 min under nitrogen atmosphere. The
solvent was removed under vacuum, and the crude was purified by flash
chromatography (silica gel, Hex/EtOAc 9:1) to provide 6 185.6 mg (96%) as
an amorphous blue solid. IR (neat) νmax 2974, 1647, 1595, 1541, 1458, 1370,
1260, 1204, 1160, 1099, 1048, 995, 935, 908, 871, 790, 756, 727, 696, 644 cm-
1; 1H NMR (300 MHz; CDCl3) δ 1.51 (9H, s), 4.87 (1H, s), 6.73 (1H, s), 7
53 (1H, s), 7.59 (1H, t, J= 7.4 Hz), 7.67 (1H. t, J= 7.4 Hz), 7.82 (1H, s), 8.03
(1H d, J = 7.4 Hz), 8.13 (1H, d, J= 7.5 Hz); 13C NMR (75 MHz; CDCl3) δ
29.7(CH3 ꢁ 3), 53.8 (C), 91.5 (C), 110.4 (CH), 114.5 (C), 125.7 (CH),
126.1 (CH), 130.0 (C), 131.9 (CH), 132.8 (C), 133.0 (C), 133.4 (CH),
140.7 (CH), 143.0(CH), 143.7 (C), 159.0(C), 168.9 (C), 181.6 (C); EIMS
m/z 335 [Mþ] (20), 279 (100), 251 (18), 223 (12), 139 (88); HREIMS
335.1158 (calcd for C20H17NO4 335.1161).
2-Benzylamino-3-ethyl-naphtho[2,3-b]furan-4,9-dione (7). 2-Hy-
droxy-1,4-naphthoquinone (100.0 mg, 0.574 mmol) in 10 mL of toluene
was treated with 1.2 equiv of propionaldehyde (0.689 mmol, 0.5 mL),
1.2 equiv of benzyl isocyanide (0.689 mmol, 0.078 mL), and 0.1 equiv of
EDDA (0.057 mmol, 10.3 mg). The reaction mixture was refluxed for 30
min under nitrogen atmosphere. The solvent was removed under
vacuum, and the crude was purified by flash chromatography (silica
gel, Hex/EtOAc 9:1) to provide 7 (170.1 mg, 89%) as an amorphous
blue solid. IR (neat) νmax 1709, 1643, 1591, 1543, 1493, 1450, 1354,
1285, 1220, 1126, 1083, 1026, 994, 961, 754, 697 cm-1; 1H NMR (300
MHz; CDCl3) δ 1.16 (3 H, t, J = 7.4 Hz), 2.63 (2 H, c), 4.62 (2 H, d, J =
5.9 Hz), 5.19 (1 H, t, J = 5.3 Hz), 7.26-7.31 (5 H, m), 7.57 (1 H, t, J = 7.2
Hz), 7.64 (1 H, t, J = 7.3 Hz), 8.01 (1 H, d, J = 7.4 Hz), 8.09 (1 H, d, J =
7.4 Hz); 13C NMR (75 MHz; CDCl3) δ 13.7(CH3), 15.6 (CH2), 47.3
(CH2), 100.8 (C), 125.8 (CH), 125.9 (CH), 127.5 (2 ꢁ CH), 127.7
(CH), 128.3 (C), 128.6 (CH ꢁ 2), 131.7 (CH), 132.3 (C), 132.7 (C),
133.4 (CH), 137.3 (C), 142.5 (C), 158.9 (C), 168.5 (C), 182.7 (C);
EIMS m/z 331 [Mþ] (45), 240 (10), 213 (8), 91 (100); HREIMS
331.1208 (calcd for C21H17NO3 331.1211).
2-Benzylamino-3-cyclohexyl-naphtho[2,3-b]furan-4,9-dione (8). Fol-
lowing the general procedure described above, 100.0 mg (0.574 mmol) of
2-hydroxy-1,4-naphthoquinone in 10 mL of toluene was treated with 1.2
equiv of cyclohexanecarbaldehyde (0.689 mmol, 0.083 mL), 1.2 equiv of
benzyl isocyanide (0.689 mmol, 0.084 mL), and 0.1 equiv of EDDA (0.057
mmol, 10.3 mg). The reaction mixture was refluxed for 30 min under nitrogen
atmosphere. The solvent was removed under vacuum, and the crude was
purified by flash chromatography (silica gel, Hex/EtOAc 9:1) to provide 8
(216.5 mg, 97%) as an amorphous blue solid. IR (neat) νmax 2925, 2853,
1725, 1641, 1587, 1534, 1447, 1345, 1212, 1083, 874, 789, 754, 698 cm-1;1H
NMR (300 MHz; CDCl3) δ 1.20-1.28 (2 H, m), 1.65-1.76 (8 H, m), 1.98
(1 H, m), 4.05 (2 H, s), 5.69 (1 H, s), 7.14-7.25 (5 H, m), 7.59 (1 H, t, J =
7.4 Hz), 7.65 (1 H, t, J=7.2Hz),8.04(1H,d,J= 7.1 Hz), 8.11 (1 H, d, J=7.2
Hz); 13C NMR (75 MHz; CDCl3) δ 25.4 (CH3), 26.5 (2 ꢁ CH2), 30.75 (2
ꢁ CH2), 33.58 (CH), 47.1 (CH2), 104.7 (C), 125.5 (CH), 126.0 (CH),
127.3 (CH ꢁ 2), 127.4 (CH), 128.4 (2 ꢁ CH), 131.7 (CH), 132.4 (C),
132.8 (C), 133.1 (C), 133.3 (CH), 137.5 (C), 142.7 (C), 158.9 (C), 168.3
(C), 182.3 (C); EIMS m/z 385 (Mþ, 67), 91 (100); HREIMS 385.1678
(calcd for C25H23NO3 385.1675).
Biological Assays. All starting materials were commercially avail-
able research-grade chemicals and were used without further purification.
RPMI 1640 medium was purchased from Flow Laboratories (Irvine, U.K.),
fetal calf serum (FCS) was from Gibco (Grand Island, NY), trichloroacetic
acid (TCA) and glutamine were from Merck (Darmstadt, Germany), and
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The Journal of Organic Chemistry
ARTICLE
penicillin G, streptomycin, dimethyl sulfoxide (DMSO), and sulforhodamine
B (SRB) were from Sigma (St. Louis, MO).
Iniciativa Ingenio 2010, Consolider Program, CIBER Actions
and financed by the Instituto de Salud Carlos III with assistance
from the European Regional Development Fund). J.G. thanks
CSIC for a predoctoral grant. J.G. realized part of this work in the
framework of the “Material Science PhD Program” of the
Autonomous University of Barcelona (UAB). S.J.-A. thanks
MICINN-Spain for a predoctoral grant. We also thank
CEAMED (Dr. Rubꢀen Pꢀerez-Machín) for carrying out the
cytotoxic assays and the ICIC for financial support.
Cells, Culture, and Plating. The human solid tumor cell lines
MCF- 7 and SK-Br-3 were obtained from the American Type Culture
Collection (ATCC, Rockville, MD), and MCF-7BUS cells were kindly
provided by Dr. Nicolꢀas Olea Serrano from the University of Granada
(Spain). Cells were maintained in 25 cm2 culture flasks in RPMI 1640
medium supplemented with 5% heat-inactivated fetal calf serum and 2
mM L-glutamine in a 37 °C, 5% CO2, 95% humidified air incubator.
Exponentially growing cells were trypsinized and resuspended in anti-
biotic-containing medium (100 units penicillin G and 0.1 mg of
streptomycin per mL). Single cell suspensions displaying >97% viability
by trypan blue dye exclusion were subsequently counted. After counting,
dilutions were made to give the appropriate cell densities for inoculation
onto 96-well microtiter plates. Cells were inoculated in a volume of 100
μL per well at densities of 12,000 (MCF-7 and MCF-7/BUS) and
15,000 (SK-Br-3) cells per well, based on their doubling times.
Chemosensitivity Testing. Chemosensitivity tests were per-
formed using the SRB assay of the U.S. NCI with slight modifications.
Briefly, pure compounds were initially dissolved in DMSO at 400 times
the desired final maximum test concentration. Control cells were
exposed to an equivalent concentration of DMSO (0.25% v/v, negative
control). Each agent was tested in triplicate at different dilutions in the
range 1-100 μM. The test compound treatment was started on day 1
after plating. Drug incubation times were 48 h, after which time cells
were precipitated with 25 μL of ice-cold 50% (w/v) trichloroacetic acid
and fixed for 60 min at 4 °C. Then, the SRB assay was performed. The
optical density (OD) of each well was measured at 492 nm, using a
PowerWave XS Absorbance microplate reader (BioTek). Values were
corrected for background OD from wells containing only medium. The
percentage growth (PG) was calculated with respect to untreated
control cells (C) at each of the drug concentration levels based on the
difference in OD at the start (T0) and end of drug exposure (T),
according to the NCI protocol. Therefore, if T was greater than or equal
to T0 the calculation was 100 ꢁ [(T - T0)/(C - T0)]. If T was less than
T0, denoting cell death, the calculation was 100 ꢁ [(T - T0)/T0]. The
effect was defined as percentage of growth, where 50% growth inhibition
(GI50), total growth inhibition (TGI), and 50% cell killing (LC50)
represent the concentration at which PG is þ50, 0, and -50, respec-
tively. With these calculations, a PG value of 0 corresponded to the
amount of cells present at the start of drug exposure, while negative PG
values denoted net cell kill.
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’ ASSOCIATED CONTENT
Supporting Information. 1H NMR and 13C NMR spec-
S
b
tra of compounds 1-8, crystallographic data of compound 3 in
CIF format, and spectral data for the IET band of 3 in different
solvents. This material is available free of charge via the Internet
at http://pubs.acs.org.
’ AUTHOR INFORMATION
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Corresponding Author
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’ ACKNOWLEDGMENT
We gratefully acknowledge the financial support from MI-
CINN-Spain Projects “Eje C-Consolider” CTQ2006-06333/
BQU and CTQ2010-19501, SAF 2009-13296-CO2-01, AGAUR
grant 2009SGR-00516 from Generalitat de Catalunya, CIBER de
Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN is an
initiative funded by the VI National R&D&i Plan 2008-2011,
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