6-Aminocoumarin-naphthoquinone conjugates: Design, synthesis, photophysical and electrochemical properties and DFT calculations

Article abstract of DOI:10.5935/0103-5053.20130279

Four novel 6-aminocoumarin-naphthoquinone conjugates were synthesized and their photophysical and electrochemical properties, investigated. 2-Chloro-3-(2-oxo-2H-chromen-6-ylamino)-1,4-naphthoquinone 1 did not present appreciable fluorescence in solution i

Full text of DOI:10.5935/0103-5053.20130279

http://dx.doi.org/10.5935/0103-5053.20130279
J. Braz. Chem. Soc., Vol. 25, No. 1, 133-142, 2014.
Printed in Brazil - ©2014 Sociedade Brasileira de Química
0103 - 5053 $6.00+0.00
Article
A
6-Aminocoumarin-Naphthoquinone Conjugates: Design, Synthesis, Photophysical
and Electrochemical Properties and DFT Calculations
Fabio S. Miranda,* Célia M. Ronconi, Mikaelly O. B. Sousa,
Gleiciani Q. Silveira and Maria D. Vargas*
Instituto de Química, Universidade Federal Fluminense,
Campus do Valonguinho, Centro, 24020-141 Niterói-RJ, Brazil
Quatro novos conjugados do tipo 6-aminocumarina-naftoquinona foram sintetizados e suas
propriedades fotofísicas e eletroquímicas, investigadas. O composto 2-cloro-3-(2-oxo-2H-cromen-
6-ilamino)-1,4-naftoquinona 1 não apresentou fluorescência apreciável, em comparação com a
6-aminocumarina, 6-AC.Visando entender as razões da extinção da fluorescência neste composto,
duas estratégias foram imaginadas. Primeiramente, o composto 1 foi metilado no nitrogênio para
remover a interação eletrostática intramolecular N-H…O=C que mantém as duas unidades fixas.
Entretanto, as propriedades de emissão do produto 2 não se mostraram significantemente diferentes
das do precursor 1. Como os cálculos usando a teoria do funcional da densidade dependente do
tempo (TD-DFT) dos compostos 1 e 2 indicaram que a supressão da fluorescência relaciona-se
ao caráter aceptor no anel naftoquinônico, a segunda estratégia envolveu a substituição do átomo
de cloro na posição 2 do núcleo naftoquinônico por diferentes grupos doadores de elétrons
(compostos 3-5). Novamente não houve mudanças apreciáveis nas propriedades de emissão. Para
explicar estes resultados foram feitos cálculos TD-DFT dos estados fundamental (S0) e excitado
(S1) de todas as moléculas em solução, os quais indicaram que o grupo fluorescente (6-AC) doa
elétrons para o LUMO da naftoquinona, resultando em uma transferência de elétron fotoinduzida
oxidativa (oxidative-PET).
Four novel 6-aminocoumarin-naphthoquinone conjugates were synthesized and their
photophysical and electrochemical properties, investigated. 2-Chloro-3-(2-oxo-2H-chromen-6-
ylamino)-1,4-naphthoquinone 1 did not present appreciable fluorescence in solution in comparison
with 6-aminocoumarin, 6-AC. In order to understand the reasons for the fluorescence quenching
in this compound, two strategies were attempted. Firstly, compound 1 was N-methylated to
remove the intramolecular N-H…O=C electrostatic interaction that maintained the two units
fixed, but the emission properties of the product 2 were not significantly different from those of 1.
Time-dependent density functional theory (TD-DFT) calculations of compounds 1 and 2 indicate
that the fluorescence quenching is related to the electron acceptor character of the naphthoquinone
ring. The second strategy, therefore, involved the substitution of the chlorine atom in position 2 of
the naphthoquinone nucleus for different electron donor groups (compounds 3-5), but again the
emission properties did not change significantly. To explain these experimental findings, TD-DFT
calculations of the ground (S0) and excited (S1) states of all molecules in solution were carried out.
The results suggest that the energy states in these conjugates are such that the fluorescent group
(6-AC) donates electrons to the naphthoquinone LUMO resulting in an oxidative photoinduced
electron transfer (oxidative-PET).
Keywords: aminonaphthoquinone, aminocoumarin, TD-DFT calculations, fluorescence
Introduction
molluscicidal,5,6 antiparasitic,7 antimicrobial,8 pesticide9
and herbicidal,10 among others.11 These properties have
been related to the ability of quinones to accept one or two
electrons with formation of the respective radical-anion
or dianion. Judicious choice of the substituents either on
position 3 of the quinone ring or on the nitrogen atom
2-Amino-1,4-naphthoquinones are known for
their various biological activities, e.g., antitumor,1-4
*e-mail: miranda@vm.uff.br, mdvargascp@gmail.com

134
6-Aminocoumarin-Naphthoquinone Conjugates
J. Braz. Chem. Soc.
allows modulation of the redox,12,13 acid-base, solubility
and pharmacological properties.14,15
Results and discussion
In recent years the use of fluorescent probes attached
to biologically active molecules has attracted great
interest in the field of medical and biological research.16,17
Labelling agents allow a better understanding of drug
accumulation and interaction with the target, e.g., tumor
cell, by monitoring the fluorescence signals.18 Several
organic dyes have been used as fluorescent probes, e.g.,
fluorescein, coumarin, rhodamine,19 cyanine, and Alexa
dyes.20 Among those, coumarins are interesting not only
because of their fluorescence, but also because of their
pharmacological activities.21 6-Aminocoumarin (6-AC) - a
molecule containing a donor amine group and an acceptor
lactone ring - is a probe of choice because it can be easily
prepared and coupled to biologically active molecules
through nucleophilic substitution reactions. In addition
6-AC fluorescence quantum yield and fluorescence
decay time can be modulated by changing the solvent
polarity.22,23
Syntheses of the compounds
The synthetic routes to compounds 1-5 are outlined in
Scheme 1. Compound 1 was obtained from the reaction of
6-AC with DCN, which has been used as the starting material
for the synthesis of a number of N-, S- and O-substituted
derivatives.9,15,27-30 N-methylation of 1 following procedure
previously described31 yielded compound 2 in good yields.
Further substitution of a chloride group with propanethiol,
thiophenol or 4-chlorothiophenol in the presence of Et3N
gave compounds 3, 4 and 5, respectively in 64-82%
yields. Novel compounds 2-5 had their purity confirmed
by elemental analyses and melting point measurements,
1
and were characterized by H and 13C nuclear magnetic
resonance (NMR) and Fourier transform infrared (FTIR)
spectroscopy (see Figures S1-S18 in the SI section).
The X-ray molecular structure of 1 has been reported
previously.32
In the present study we have investigated the
possibility of incorporating the 6-AC fluorescent dye
into a 1,4-naphthoquinone nucleus by reaction with
2,3-dichloro-1,4-naphthoquinone (DCN). Only a few
examples of molecules containing both coumarin and
1,4-naphthoquinone nuclei have been described in the
literature, mostly from natural sources.24-27 Our hypothesis
was that this conjugate would be fluorescent enough to
allow fluorescence microscopy studies in the course of
our further biological activity investigations. Instead,
however, quenching was observed. In order to understand
this process the photophysical and electrochemical
properties of a series of 2-aminocoumarin-naphthoquinone
conjugates (see Scheme 1) have been studied and
interpreted with the help of density functional theory
(DFT) calculations.
Photophysical properties
The normalized UV-Vis absorption and emission
spectra of all aminocoumarin-napthoquinone conjugates
in CH3CN are shown in Figure 1.
The UV-Vis spectra of compounds 1-5 exhibit four
absorption bands: a high energy band around 250 nm, which
is intense only in the spectra of compounds 2 and 5, a very
intense band in the 263-281 nm region and two broad low
intensity bands in the 333-350 nm region (shoulder) and
in the visible region between 468 and 531 nm (Table 1).
The lowest energy band assigned to intramolecular
charge transfer is the most influenced by the nature of the
substituent, both on the nitrogen atom (CH3 vs. H) and on
the naphthoquinone ring (Cl vs. SR). Electron releasing
Scheme 1. The synthetic routes to compounds 1-3. Reactions and conditions: a) DMF, 60-70 °C, 72 h; b) K2CO3/CH3I, DMF, RT; c) thiol/Et3N, DMF (3)
or MeOH (4, 5), 60-70 °C.

Vol. 25, No. 1, 2014
Miranda et al.
135
Figure 1. Normalized absorption (A and B) and emission (C and D) spectra of aminocoumarin-napthoquinone conjugates 1-5 in CH3CN.
groups on the nitrogen (CH3) and on the quinone ring (SR)
shift this band to higher wavelengths. This red shift is more
pronounced upon methylation of the nitrogen atom (2 vs. 1)
than upon thiolation on the naphthoquinone ring (3-5 vs. 1).
The spectra of the three thiolate compounds 3-5 are very
similar, except for the high energy band, as expected.
Excitation of the lowest energy absorption band, λmax(3)
of all compounds results in a very weak emission around
600 nm. Compound 1 has an emission maximum at 538 nm,
between the values found for the 6-AC (λem = 516 nm) and
2-amino-3-chloro-1,4-naphthoquinone 6 (λem = 592 nm)
emissions (Figure 2). Furthermore, the data gathered in
Table 1 evidence that substitution of a hydrogen atom for
a naphthoquinonyl group in 6-AC (compounds 1-5) results
in strong quenching of the aminocoumarin fluorescence
(Φ = 0.25)23. Approximately the same Stokes shift values
are observed for all compounds suggesting that their excited
state geometries are similar.33
Electrochemical properties
The electrochemical behavior of compounds 1-5 was
investigated by cyclic voltammetry experiments which
were carried out in argon-purged CH3CN/Bu4NClO4,
o
at 25 C (see Figure 3 and Figures S19-S24 in the SI
section). The cyclic voltammograms have been obtained
in the potential range of –2.0 to +1.5 V vs. FcH/FcH+ in
four scan rates (50, 100, 250 and 500 mV). The reduction
potentials, LUMO level, HOMO level and optical gap are
given in Table 1. The LUMO level was determined using
Table 1. Optical properties, electrochemical data, calculated HOMO, LUMO and gapopt of compounds 1-5
E1/2 / mVb (DEp / mV)
a
e
Compound
λ
max(3) / nm log ε3
λ
em / nm Dλ / eV
F
HOMO / eVc LUMO / eVd gapopt / eV
Wave I
Wave II
Wave III
–
1
2
3
4
5
468
538
495
503
499
3.66
3.59
3.54
3.39
3.15
538
637
575
588
574
0.35
0.36
0.34
0.35
0.32
0.0001 –1000 (77) –1374 (93)
–6.25
–6.00
–5.98
–5.99
–5.99
–3.80
–3.90
–3.72
–3.76
–3.76
2.45
2.10
2.26
2.24
2.23
0.0001 –905 (96) –1320 (94) 878 (86)
0.0003 –1084 (74) –1422 (100)
0.0003 –1045 (123) –1420 (104)
0.0001 –1036 (102) –1407 (95)
–
–
–
b
aStokes shift; data from voltammetric experiments in a cathodic scan, obtained at a scan rate of 100 mV s–1 in CH3CN + 0.1 mol L−1 n-Bu4NClO4, at
25 °C; potential values are reported vs. ferrocene/ferrocenium (FcH/FcH+); cHOMO = LUMOelec. – gapopt.; dcalculated from the reversible first reduction
process (wave I); eoptical gap estimated from the optical absorption edge. The inflection point in the first derivatives of the absorption spectrum was used.

136
6-Aminocoumarin-Naphthoquinone Conjugates
J. Braz. Chem. Soc.
the semiquinone radical Q•− (Ic/Ia) and the second one
is associated to the reduction of the semiquinone to the
quinone dianion (IIc/IIa), Q2−. Substitution of the chloro
electron attracting group on the naphthoquinone ring in
1 for thiolate groups in 3-5 produces a sizeable cathodic
shift for the two one-electron reduction processes, i.e., the
presence of these electron donor groups makes the reduction
of compounds 3-5 more difficult than reduction of 1. The
magnitude of the observed cathodic shift with respect to
that of 1 for the first wave is roughly equal to 84 (3), 45 (4)
and 36 (5) mV at 100 mV s-1. The presence of the methyl
group bonded to the nitrogen atom in 2 produces an anodic
shift for the two one-electron steps of about 95 (wave I)
and 50 mV (wave II), i.e., naphthoquinone 2 is more easily
reduced than 1, possibly due to the structural changes
caused by the presence of the methyl group (see discussion
below). Furthermore, the oxidation process (IIIa) at positive
potential observed in the voltammograms of compounds 1
and 3-5 associated to the aminocoumarin nucleus is quasi
reversible in the voltammogram of compound 2. Thus, the
methyl group on the nitrogen atom makes the oxidation
product of 2 more stable than the oxidation products of
the other compounds containing the NH group, which
can undergo a number of side reactions.40 No correlation
is observed between the nature of the substituent and the
oxidation process IIIa in the CVs of compounds 1 and 3-5
(see Figures S19-S24 in the SI section).
Figure 2. Normalized absorption and emission spectra of 6-AC and
napthoquinone 6 in CH3CN.
the equation ELUMO = –4.79 – Ered (wave I, vs. FcH/FcH+).34-39
The HOMO energy levels of all compounds were
calculated by subtracting the optical gap. As observed
for other 2-(R-phenyl)amine)-1,4-naphthoquinones13 the
voltammograms in CH3CN show two successive one-
electron reduction processes which generate two separate
cathodic waves.
According to the data, both processes are quasi
reversible (Figures S19-S24 in the SI section). The first
process is attributed to the reduction of the quinone to
Theoretical calculations
In order to understand the spectroscopic and
electrochemical properties of the aminocoumarin-
naphthoquinone conjugates 1-5 calculations were carried
out using DFT at the B3LYP/6-31+G(d,p) level (see
Tables S1-S4 in the SI section).41,42 The geometry of
compound 1 was fully optimized starting from the CIF
file obtained from the X-ray diffraction experiment.32 The
geometries of the other compounds were based on this
structure. To evaluate the individual contributions of the
molecules that represent the fragments composing these
conjugates, calculations of 6-AC and 2-amino-3-chloro-
1,4-naphthoquinone 6 were also carried out.
Geometries of the ground (S0) and excited (S1) state
structures: The ground state (S0) structures of compounds 1
and3-5(seeTable2forthenumberingoftheatomsandvalues
of selected structural parameters) contain a N1–H…O1
intramolecular electrostatic interaction.32 They exhibit
similar C3–N1–C11 angles, however small changes are
noted in the values of the dihedral C2–C3–N1–C11 and
C16–C11–N1–C3 angles as a function of R1. The presence
Figure 3. Cyclic voltammograms at 100 mV s-1 of 1, 2 and 3.

Vol. 25, No. 1, 2014
Miranda et al.
137
Table 2. Selected angles for compounds 1-5 [o] in the ground (S0) and excited (S1) states
a
C2–C3–N1–C11
C16–C11–N1–C3
C3–N1–C11
β
Compound
S0
S1
S0
S1
S0
S1
S0
1(X-ray)32
31.9 (30.8)
52.2
92.8
91.0
99.2
100.4
99.0
31.2 (29.6)
26.6
3.0
1.8
4.9
4.7
4.0
130.5 (129.2)
122.6
125.6
121.3
125.7
125.7
125.7
10.0
1.5
2
3
4
5
36.3
30.4
129.2
12.8
11.5
11.2
32.8
36.4
129.5
32.8
37.2
129.5
aPyramidalization angle β of amine: angle between the N–H or N–CH3 bond and the C3–N–C11 plane.
of the propylthiolate group in compound 3 leads to larger
C2–C3–N1–C11 and smaller C16–C11–N1–C3 dihedral
angles in comparison to compounds 4 and 5.
relation to those observed for compound 1. A comparative
analysis of the bond distances (see Tables S1-S4 in the SI
section) in these structures confirms the conjugation of the
two fragments (coumaryl and naphthoquinonyl) through the
nitrogen heteroatom in 1 as discussed above, as well as the
donor acceptor character (push-pull) of 6-AC.
Analysis of the Mulliken charges (see Tables S5
and S6 in the SI section) for compounds 1-4 shows that
naphthoquinone oxygens O1 and O2 are more negative in
the S1 than in the S0 structures whereas coumaryl oxygens
O3 and O4 are less negative in the S1 than in the S0
structures, thus indicating that the naphthoquinone nucleus
is a better electron acceptor than the coumaryl lactone.
Differently, compound 2 which contains a methyl group
on N1 does not exhibit the N1–H…O1 intramolecular
electrostatic interaction and therefore the C2–C3–N1–C11
dihedral angle is much larger in this molecule than in the
other compounds (Table 2) with a consequent decrease
in the C16–C11–N1–C3 dihedral angle. Furthermore, the
C3‑N1–C11 angle is smaller in 2 (122 o) than in compounds
1 and 3-5 (approximately 130 o). This may be associated to
the larger degree of pyramidalization (β angle, see Table 2)
on the nitrogen atom in the latter compounds compared to
compound 2, possibly due to the N1–H…O1 electrostatic
interaction in 1 and 3-5. In all cases short C3–N1 bond
distances (1.361 – 1.391Å, see Table S1 in the SI section) are
observed, indicating in all cases partial double bond character
and therefore conjugation between the two fragments.
The excited state (S1) structures of compounds 1, 3-5 do
not exhibit the electrostatic N1–H…O1 interaction present
in the S0 structures. Elongation of the C3–N1 bond distance
in compound 1 in the S1 structure (from 1.359 Å in the S0
structure to 1.432 Å) and decrease of the C11–N1 bond
distance (1.416Å to 1.343Å from S0 to S1, respectively, see
Table S1 in the SI section) are noted. Same trend is observed
for compounds 2, 3-5. Importantly, analysis of the dihedral
C2–C3–N1–C11 angles in all compounds (Table 2) reveals
that in the S1 structures the naphthoquinone and coumarin
rings are practically perpendicular to each other, suggesting
that the electronic transition is a twisted intramolecular
charge transfer (TICT).22
Ground state molecular orbital analysis: The contour
plots of the frontier orbitals (HOMOs and LUMOs) and
their corresponding energy levels and gaps are shown in
Figure 4. The HOMO of 1 is located over both moieties,
mostly on the coumarin (except on C17 and C19, see figure
in Table 2 for the numbering), and to a lesser extent on the
quinone (on O2–C1–C2–C3–N1). A smaller contribution
of the quinone fragment to the HOMO of 2 is observed
in comparison with 1, 3-5. Substitution of the Cl group
in 1 for thiolate groups in 3-5 results in the participation
of this group to the HOMO. The aromatic thiolate
groups have a significant contribution to the HOMO of
compounds 4 and 5. The LUMOs of all compounds have
higher amplitude on the quinone ring (e1g type symmetry)
with some contribution of the nitrogen atom.
The calculated ground state energy gaps (2 < 3-5 < 1)
are compatible with the red shifts (gapopt) observed in the
absorption spectra (Table 2) on going from compound 1
to compound 2 and from compound 1 to compounds 3-5.
The S0 and S1 structures of 6-AC and naphthoquinone
6 were also calculated and the differences are discussed in

138
6-Aminocoumarin-Naphthoquinone Conjugates
J. Braz. Chem. Soc.
Figure 4. Frontier orbitals [contour values plotted at 0.03 (e/Bohr3)1/2] and energy diagrams for compounds 1-5.
The LUMO energies of all compounds show a linear
correlation with their first reduction process (see Table
1 and Figure 3). Compound 2 with the most negative
theoretical LUMO (–3.65 eV) is also the most easily
reduced (E1/2 = –0.905 V), whereas compound 3, with
the least negative LUMO, is the hardest to reduce. Both
the shape and energy of the LUMO confirm that the
naphthoquinone nucleus is the reduction electroactive site
of the conjugates 1-5.
Finally, the contour plots of the frontier orbitals (HOMOs
and LUMOs) and the corresponding energy levels and gaps
of 6-AC and compounds 6 and 1 are gathered in Figure 5.
These results show that the HOMO energy of 1 is halfway
between the HOMO energies of 6-AC and 6. However, the
LUMO of 6, about 1.26 eV more stable than that of 6-AC,
dominates both the LUMO shape and energy of 1.
To assign the absorption bands observed in the UV-Vis
spectra, the molecular orbitals of the compounds were
calculated in CH3CN using the PCM model. The theoretical
absorption spectra (see Figures S25-S31 in the SI section)
show a systematic underestimation of the excitation
energies for 6-AC, 6 and 1-5 as previously observed
for other systems.43-47 The largest differences have been
observed for the charge-transfer bands of the conjugates.
In spite of this limitation, the important features of the
spectra have been correctly simulated. The HOMO−LUMO
*
transitions for all compounds are π → π in nature.
To understand the fluorescence quenching of the
conjugates the frontier orbitals in the singlet excited
state conformations (S1) have been calculated and are
displayed in Figure 6. The S1 geometries of the molecules
are very different from those in the S0 state. The geometry
change is related to the increase of the torsion angle
between the naphthoquinone and aminocoumarin
fragments (Table 2). As a consequence the HOMO is
localized on the aminocoumarin and the LUMO on the
naphthoquinone moiety in the excited state. It is evident
from calculated HOMO-LUMO energies (Figure 6) that
all 6-aminocoumarin-naphthoquinone conjugates behave
as push-pull systems and that fluorescence quenching for
all compounds may be related to the stronger π-electron
acceptor character of the naphthoquinone compared to that
of the coumaryl fragment. The naphthoquinone LUMO is
of lower energy than that of 6-AC (see Figure S35 in the
SI section) in both the ground and excited states. Thus the
energy states are such that the fluorescent group (6-AC)
Figure 5. Molecular orbital diagrams for 6-AC, aminonaphthoquinone 6
and aminocoumarin-naphthoquinone conjugate 1 [contour values plotted
at 0.03 (e/Bohr3)1/2].

Vol. 25, No. 1, 2014
Miranda et al.
139
Figure 6. Frontier orbitals in the excited state conformations (S1) [contour values plotted at 0.03 (e/Bohr3)1/2] and energy diagrams for compounds 1-5.
donates electrons to the naphthoquinone LUMO resulting
in an oxidative photoinduced electron transfer (oxidative-
PET).48
the two moieties through the imine like nitrogen which
allow the oxidative-PET. It was found that the LUMO
orbital is located on the naphthoquinone and controls
both electrochemical and photophysical properties of all
conjugates. It would therefore be interesting, in future
studies, to introduce a saturated spacer to avoid quenching
of the coumarin fluorescence.
Conclusions
In this study the fluorescent dye 6-AC was attached
to 1,4-naphthoquinone derivatives aiming at fluorescent
molecules. However the fluorescence of 6-AC was quenched
in the 6-aminocoumarin-naphthoquinone conjugates 1-5.
To explain these results, experimental and theoretical
investigations of the photophysical and electrochemical
properties of conjugates 1-5, 6-AC and 2-amino-3-chloro-
1,4-naphthoquinone 6 were carried out in CH3CN. The
strong π-electron acceptor character of the naphthoquinone
moieties was confirmed by electrochemical measurements
which showed a reduction potential around −1.0 V.
Substitution on either N (H for CH3) or on the quinone
ring (Cl for thiols) did not result in significant changes
(wave I, D = 0.1 V) on the electrochemical properties
of the conjugates. TD-DFT calculations including solvent
effects using the PCM model revealed that the S0 structures
of compounds 1, 3-5 exhibit an intramolecular electrostatic
N1–H…O1 interaction which does not exist in the S1
structures. An analysis of the S1 structures suggests that
the electronic transition is a twisted intramolecular charge
transfer (TICT). The lowest energy absorption band in
the spectra of all conjugates has been assigned to π−π*
charge transfer from aminocumaryl to naphthoquinone.
The emission of all conjugates significantly decreased
(Φ = 10−4) compared to 6-AC (Φ = 0.25). This observation
has been explained in terms of the strong electron acceptor
nature of the naphthoquinone moiety and conjugation of
Experimental section
Materials and instruments
All starting materials were used without previous
treatment and solvents, as received from commercial
suppliers (except for triethylamine and N,N-dimethyl-
formamide, which were previously distilled). Thin-layer
chromatography (TLC) was performed on aluminum sheets
coated with silica gel 60F-254 (Sorbent). The plates were
inspected by UV light (λ = 254 nm). Silica gel 60 Merck
(0.063-0.200 mm) was used for column chromatography.
6-Aminocumarin was synthesized according to the
literature.49
1H NMR and 13C NMR spectra were recorded on a
Varian Unit Plus (300 MHz or 500 MHz) spectrometer
in CD3Cl, CD3CN or DMSO-d6. Chemical shifts (d) are
reported in parts per million (ppm) relative to the signal
of residual solvent. The hydrogen signals were attributed
1
1
through coupling constant values and H × H – COSY
experiments. Elemental analyses were performed using a
Perkin-Elmer CHN 2400 microanalyzer (CentralAnalítica
- Instituto de Química, Universidade de São Paulo,
Brazil). Melting points were obtained on a Digital Melting
Point IA9100 (ThermoFisher Scientific-USA) and are

140
6-Aminocoumarin-Naphthoquinone Conjugates
J. Braz. Chem. Soc.
uncorrected. Transmission infrared spectra were recorded
on an FT-IR Varian 660 spectrophotometer, equipped with
a Pike ATR Miracle accessory (diamond/ZnSe crystal,
resolution: 4 cm-1). Cyclic voltammograms were obtained
with a BAS Epsilon potentiostat-galvanostat system at
room temperature, using n-Bu4NClO4 (0.1 mol L-1) as the
supporting electrolyte in CH3CN (spectroscopic grade)
solutions of the compounds (at 1.0 × 10-3 mol L-1). The
electrochemical cell was a conventional one with three
electrodes: Ag/Ag+ was used as the quasi reference
electrode, a platinum wire as the auxiliary electrode and
glassy carbon as the working electrode. The ferrocene/
ferrocenium (FcH/FcH+) couple was added at the end of
each experiment as internal standard to the bulk solution;
the data are thus reported versus the FcH/FcH+ couple
as recommended by IUPAC.50 Pure argon was bubbled
through the electrolytic solution to remove oxygen in all
experiments. UV-vis absorption spectra were recorded on
a Cary 50 (Varian) spectrophotometer using spectroscopic
grade CH3CN. The values of wavelength are expressed
in nanometers (nm) and epsilons (ε) in their logarithmic
form. Luminescence spectra were obtained in a Varian
Cary Eclipse fluorescence spectrophotometer. Fluorescence
quantum yields were determined using a quinine sulfate
(QB) solution in 0.5 mol L-1 H2SO4 (Φ = 0.546)51 as
reference. The quantum yield was determined using the
following equation:52
that these geometries represent genuine minimum energy
points (with no negative eigenvalue) on the potential energy
surface. Based on the B3LYP/6-31+G(d,p) optimized
geometries, TD-DFT approaches were used to calculate
vertical excitations with linear responses to verify the
effects of these excitations on the absorption spectra
of the examined compounds. The polarized continuum
(overlapping spheres) solvation model (PCM) was used
to ensure that solvent effects (CH3CN) were incorporated
into all of the aforementioned calculations. The PCM
computations used the UFF radii and all of the standard
specifications of the Gaussian package. The TD results were
analysed with the GaussSum 2.2 software.56 The absorption
spectra were fitted with a Gaussian function with a FWHM
of 3000 cm-1.
Synthesis
2-chloro-3-(2-oxo-2H-chromen-6-ylamino)naphthalene-
1,4-dione 1: described elsewhere, see Supplementary
Information.32
2-chloro-3-(methyl(2-oxo-2H-chromen-6-yl)amino)
naphthalene-1,4-dione 2: N-methylation of compound 1 was
carried out as previously described for other 2-(R-phenyl)
amino-1,4-naphthoquinones,31 from 351.7 mg, 1 mmol of
1. After reaction completion the volatiles were removed
under reduced pressure. The crude product was purified by
column chromatography on silica gel (eluent CH2Cl2), giving
a purple solid. Yield: 237.8 mg, 65%; m.p. 196 °C; calcd.
for C20H12ClNO4: C, 65.67; H, 3.31; N, 3.83%; found: C,
65.01; H, 3.43; N, 3.79%; 1H NMR (DMSO-d6, 500 MHz):
d 8.10 (d, 1H, J 6.8 Hz), 8.00 (d, 1H, J 6.8 Hz), 7.93 (d, 1H,
J 9.6 Hz), 7.91-7.87 (m, 2H), 7.32 (t, 1H, J 1.6 Hz), 7.26 (d,
2H, J 1.6 Hz), 6.45 (d, 1H, J 9.6 Hz), 3.38 (s, 3H); 13C NMR-
APT (DMSO-d6, 75 MHz): d180.44, 178.06, 160.67, 149.10,
148.28, 143.28, 143.05, 136.13, 134.41, 134.16, 131.50,
131.44, 127.39, 127.16, 121.83, 119.29, 117.62, 117.42,
114.78, 39.95; IR (KBr; νmax/cm-1): 3080 (C–Harom.), 2923
(C–Haliph.), 1718 (C=Oester), 1677 (C=Oquin.), 1639 (C=Oquin.),
1543 (C=C); UV-Vis [CH3CN; λmax/nm (log ε)]: 263 (4.72),
350 (3.94), 536 (3.59).
Φem,S = Φem,R(fR/fS)(IS/IR)(nS/nR)
(1)
where Φem,s represents the fluorescence quantum yield of
the sample; fS and fR are the absorption factor (f = 1 – 10–Abs
,
whereAbs = absorbance) for the sample and the reference,
respectively, at the excitation wavelength; IS and IR denote
the integrated areas of the corrected emission spectra for
the sample and the reference, respectively; and nS and nR are
the refractive indices of the sample and reference solvents,
respectively.
Calculations
DFT calculations were performed with the Gaussian
09 (Rev. B.01) software package.53 The geometry of
compound 1 was fully optimized using the geometry
obtained in the solid state measurements.32 Compounds
2-5 did not provide single crystal and their geometry
optimizations were carried out based on the geometry of
compound 1. For the geometry optimization calculation
the B3LYP functional together with the 6-31+G(d,p) basis
set was employed.54,55 Harmonic frequency calculations
were performed for the optimized geometries and revealed
2-(2-oxo-2H-chromen-6-ylamino)-3-(propylthio)
naphthalene-1,4-dione 3: 1-Propanethiol (87 μL, 0.96
mmol) was added to a solution of 1 (170 mg, 0.48 mmol)
in DMF (20 mL) in the presence of triethylamine (66.4 μL,
0.48 mmol). The mixture was stirred at 60-70°C for 24 h.
After removal of the solvents under reduced pressure, the
crude product was purified through recrystallization in
cyclohexane, resulting in a purple solid. Yield: 25.3 mg,

Vol. 25, No. 1, 2014
Miranda et al.
141
64%; m.p. 134 °C; calcd. for C22H17NO4S: C, 67.50; H,
4.38; N, 3.58%; found: C, 67.69; H, 4.51; N, 3.45%;
1H NMR (500 MHz, CD3CN): d 8.12 (t, 2H, J 7.7 Hz),
7.88-7.83 (m, 2H), 7.79 (dt, 1H, J 7.7, 1.3 Hz), 7.39 (dd, 2H,
J 8.8, 2.5 Hz), 7.34 (d, 1H, J 8.8 Hz), 7.32 (d, 1H, J 2.5 Hz),
6.45 (d, 1H, J 9.6 Hz), 2.58 (t, 2H, J 7.2 Hz), 1.44-1.35
Acknowledgements
FAPERJ: PRONEX (grant no. E-26/171.512.2010),
Pensa Rio (grant no. E-26/110.692/2012), JCNE (grant no.
E-26/103.213/ 2011) CNE (grant no. E-26/103.084/2011).
CNPq: M.D.V. and C.M.R. are recipients of research
fellowships and M.O.S. of a PIBIC fellowship. The authors
thank LMQC and Prof J. Walkimar de M. Carneiro and
Jusiane M. Costa for their help with the DFT calculations.
13
(m, 2H), 0.84 (t, 3H, J 7.2 Hz); C NMR-APT (CDCl3,
75 MHz): d 180.8, 180.0, 160.4, 150.7, 144.5, 142.8, 134.7,
134.6, 133.2, 132.8, 130.3, 126.8, 126.6, 126.0, 120.4,
118.7, 118.3, 117.3, 116.9, 45.7, 35.8, 8.50; IR (KBr;
ν
max/cm-1): 3329 (N–H), 3079 (C–Harom.), 2957 (C–Haliph.),
References
1721 (C=Oester), 1658 (C=Oquin.), 1639 (C=Oquin.), 1560
(C=C); UV-Vis [CH3CN; λmax/nm (log ε)]: 281 (4.56), 348
(3.73), 500 (3.54).
1. Hallak, M.; Win, T.; Shpilberg, O.; Bittner, S.; Granot, Y.;
Levy, I.; Nathan, I.; Brit. J. Haematol. 2009, 147, 459.
2. Esteves-Souza, A.; Figueiredo, D. V.; Esteves, A.; Camara,
C. A.; Vargas, M. D.; Pinto, A. C.; Braz. J. Med. Biol. Res.
2007, 40, 1399.
3-(2-oxo-2H-chromen-6-ylamino)-3-(arythiol)
naphthalene-1,4-dione 4and (2-oxo-2H-chromen-6-ylamino)-
3-(4-chloroarythiol) naphthalene-1,4-dione 5: Compounds 4
and 5 were synthesized as described in the literature28 for
analogous compounds 351 mg (1 mmol) of 1 and 1 mmol of
the respective aryl thiols in absolute MeOH (20 mL), under
reflux, in the presence of triethylamine (2.5 mmol). After
8 h, the resulting solution was concentrated under vacuum
and purified by column chromatography on silica gel using
EtOAc/hexane. 4: yield: 317 mg, 77%; m.p. 240 °C; calcd.
for C24H15NO4S: C, 69.72; H, 3.66; N, 3.39%.; found: C,
67.88; H, 3.52; N, 3.30%; 1H NMR (DMSO-d6, 500 MHz):
d 8.20 (d, 1H, J 7.6 Hz), 8.13 (d, 1H, J 7.6 Hz), 7.99 (t, 1H,
J 7.4 Hz), 7.95 (d, 1H, J 7.4 Hz), 7.91 (d, 1H, J 9.5 Hz),
7.25-7.23 (m, 1H), 7.19-7.16 (m, 5H), 6.86 (d, 1H, J 7.3 Hz),
6.85 (d, 1H, J 5.9 Hz), 6.54 (d, 1H, J 9.5 Hz); IR (KBr;
3. Esteves-Souza, A.; Lúcio, K. A.; Cunha, A. S.; Pinto, A. C.;
Lima, E. L. S.; Camara, C. A.; Vargas, M. D.; Gattass, C. R.;
Onc. Rep. 2008, 20, 225.
4. Francisco, A. I.; Casellato, A.; Neves, A. P.; Carneiro, J. W.
D; Vargas, M. D.; Visentin, L. C.; Magalhães, A.; Camara, C.
A.; Pessoa, C; Costa-Lotufo, L. V.; Marinho, J. D. B.; Moraes,
M. O.; J. Braz. Chem. Soc. 2010, 21, 169.
5. Barbosa, T. P.; Camara, C. A.; Silva, T. M. S.; Martins, R. M.;
Pinto,A. C.;Vargas, M. D.; Bioorg. Med. Chem. 2005, 13, 6464.
6. Silva, T. M. S.; Camara, C. A.; Barbosa, T. P.; Soares, A. Z.;
Cunha, L. C.; Pinto, A. C.; Vargas, M. D.; Bioorg. Med. Chem.
2005, 13, 193.
7. Kapadia, G. J.;Azuine, M.A.; Balasubramanian,V.; Sridhar, R.;
Pharmacol. Res. 2001, 43, 363.
ν
max/cm-1): 3306 (N–H), 3080 (C–Harom.), 1733 (C=Oester),
8. Ryu, C.-K.; Kim, D.-H.; Arch. Pharm. Res. 1992, 15, 263.
9. Clark, N. G.; Pest. Sci. 1985, 16, 23.
1678 (C=Oquin.), 1664 (C=Oquin.), 1551 (C=C); UV-Vis
[CH3CN; λmax/nm (log ε)]: 284 (4.47), 505 (3.39). 5: yield:
365 mg, 82%; m.p. 210 °C; calcd. for C24H14ClNO4S: C,
64.36; H, 3.15; N, 3.13%.; found: C, 62.58; H, 3.24; N,
10. Koura, S.; Takasuka, K.; Katsuyama, N.; Ogino, C.; Sato, Y.;
Wakabayashi, K.; Biosc. Biotech. Biochem. 1994, 58, 1210.
11. Silveira, G. Q.; Ronconi, C. M.;Vargas, M. D.; San Gil, R.A. S.;
Magalhães, A.; J. Braz. Chem. Soc. 2011, 22, 961.
12. Glezer, V.; Stradins, J.; Friemanis, J.; Baider, L.; Electrochim.
Acta 1983, 28, 87.
1
3.03%; H NMR (DMSO-d6, 500 MHz): d 8.20 (d, 1H,
J 7.6 Hz), 8.13 (d, 1H, J 7.6 Hz), 7.99 (t, 1H, J 7.4 Hz),
7.96-7.92 (m, 2H), 7.26 (d, 1H, J 9.5 Hz), 7.22-7.19 (m,
4H), 6.88 (d, 2H, J 8.6 Hz), 6.56 (d, 1H, J 9.5 Hz); IR (KBr;
13. Aguilar-Martínez, M.; Cuevas, G.; Jiménez-Estrada, M.;
González, I.; Lotina-Hennsen, B.; Macías-Ruvalcaba, N.;
J. Org. Chem. 1999, 64, 3684.
ν
max/cm-1): 3267 (N–H), 3079 (C–Harom.), 1742 (C=Oester),
1663 (C=Oquin.), 1627 (C=Oquin.), 1537 (C=C); UV-Vis
[CH3CN; λmax/nm (log ε)]: 282 (4.29), 510 (3.15).
14. Koyama, J.; Morita, I.; Kobayashi, N.; Osakai, T.; Hotta, H.;
Takayasu, J.; Nishino, H.; Tokuda, H.; Cancer Lett. 2003, 201,
25.
Supplementary information
15. Brun, M. P.; Braud, E.;Angotti, D.; Mondésert, O.; Quaranta, M.;
Montes, M.; Miteva, M.; Gres, N.; Ducommun, B.; Garbay, C.;
Bioorg. Med. Chem. 2005, 13, 4871.
Supplementary Information associated with the paper
contains FTIR, 1H, 13C NMR spectra, cyclic voltammograms
of the compounds and computational details. These data
are available free of charge at http://jbcs.sbq.org.br as a
PDF file.
16. Katritzky, A. R.; Narindoshvili, T.; Org. Biomol. Chem. 2009,
7, 627.
17. Krueger, A. T.; Imperiali, B.; Chembiochem. 2013, 14, 788.

142
6-Aminocoumarin-Naphthoquinone Conjugates
J. Braz. Chem. Soc.
18. Wang, F.; Tan, W. B.; Zhang, Y.; Fan, X.; Wang, M.;
42. Ditchfield, R.; Hehre, W. J.; Pople, J. A.; J. Chem. Phys. 1971,
54, 724.
Nanotechnology 2006, 17, R1.
19. Silveira, G. Q.;Vargas, M. D.; Ronconi, C. M.; J. Chem. Mater.
2011, 11, 6034.
43. Matos, C. R. M. O.; Miranda, F. S.; Carneiro, J. W. de M.;
Pinheiro, C. B.; Ronconi, C. M.; Phys. Chem. Chem. Phys.
2013, 15, 13013.
20. Johnson, I. D. In Handbook of Biological Confocal Microscopy,
3rd ed.; Pawley, J., ed.; Springer: New York, 2006.
21. Riveiro, M. E.; De Kimpe, N.; Moglioni, A.; Vazquez, R.;
Monczor, F.; Shayo, C.; Davio, C.; Curr. Med. Chem. 2010,
17, 1325.
44. Grimme, S.; Parac, M.; ChemPhysChem 2003, 4, 292.
45. Drew, A.; Head-Gordon, M. J.; Am. Chem. Soc. 2004, 126,
4007.
46. Tozer, D. J.; Handy, N. C.; Phys. Chem. Chem. Phys. 2000, 2,
2117.
22. Krystkowiak, E.; Dobek, K.; Maciejewski, A.; Photochem.
Photobiol. Sci. 2013, 12, 446.
47. Pastore, M.; Fantacci, S.; DeAngelis, F. J.; Phys. Chem. C 2013,
117, 3685.
23. Krystkowiak, E.; Dobek, K.; Burdzińskib, G.; Maciejewski,A.;
Photochem. Photobiol. Sci. 2012, 11, 1322.
48. Loudet, A.; Burgess, K.; Chem. Rev. 2007, 107, 4891.
49. Morgan, G. T.; Gore, F. M.; J. Chem. Soc. Trans. 1904, 85,
1230.
24. Rózsa, Z.; Mester, I.; Reisch, R.; Szendrei, K.; Planta Med.
1989, 55, 68.
25. Ito, C.; Ono, T.; Tanaka, E.; Takemura, Y.; Nakata, T.;
Uchida, H.; Ju-ichi, M.; Omura, M.; Furukawa, H.; Chem.
Pharm. Bull. 1993, 41, 205.
50. Gritzner, G.; Kuta, J.; Pure Appl. Chem. 1984, 56, 461.
51. Adams, M. J.; Highfield, J. G.; Kirkbright, G. F.; Anal. Chem.
1977, 49, 1850.
26. Ishikawa, T.; Kotake, K.; Ishii, H.; Chem. Pharm. Bull. 1995,
43, 1039.
52. Birks, J. B.; Photophysics of Aromatic Molecules, 1st ed.; Wiley-
Interscience: London, 1970.
27. Ibis, C.; Deniz, N. G.; J. Chem. Sci. 2012, 124, 657.
28. Tandon,V. K.; Maurya, H. T.; Mishra, N. N.; Shukla, P. K.; Eur.
J. Med. Chem. 2009, 44, 3130.
53. Gaussian 09, Revision B.01, Frisch, M. J.; Trucks, G. W.;
Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman,
J. R.; Scalmani, G.; Barone,V.; Mennucci, B.; Petersson, G.A.;
Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov,
A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;
Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.;
Montgomery Jr., J. A.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.;
Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.;
Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.;
Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.;
Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.;
Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.;
Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski,
J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth,
G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels,
A. D.; Farkas, Ö.; Foresman, J. B.; Ortiz, J. V.; Cioslowski J.;
Fox, D. J. Gaussian, Inc., Wallingford CT, 2009.
54. Becke, A. D.; J. Chem. Phys. 1993, 98, 5648.
55. Ditchfield, R.; Hehre, W. J.; Pople, J. A.; J. Chem. Phys. 1971,
54, 724.
29. Tandon,V. K.; Maurya, H. T.; Tripathi,A.; ShivaKeshava, G. B.;
Shukla, P. K.; Srivastava, P.; Panda, D.; Eur. J. Med. Chem.
2009, 44, 1086.
30. Valente, C.; Moreira, R.; Guedes, R. C.; Iley, J.; Jaffarc, M.;
Douglas, K. T.; Bioorg. Med. Chem. 2007, 15, 5340.
31. Lisboa, C. S.; Santos, V. G.; Vaz, B. G.; Lucas, N. C.; Eberlin,
M. N.; Garden, S. J.; J. Org. Chem. 2011, 6, 5264.
32. Sousa, M. O. B.; Silveira, G. Q.; Gomez, J. A. G.; Acta Cryst.
2013, E69, o1317.
33. Anslyn, E. V.; Dougherty, D. A.; Modern Physical Organic
Chemistry; University Science Books: Sausalito, CA, 2006.
34. Liu, Y.; Liu, M. S.; Jen, A. K. -Y.; Acta Polym. 1999, 50, 105.
35. Chang, S. C.; Weaver, M. J.; J. Phys. Chem. 1991, 95, 5391.
36. Stuve, E. M.; Krasnopoler, A.; Sauer, D. E.; Surf. Sci. 1995,
335, 177.
37. Miller, L. L.; Nordbloman, G. D.; Maybda, E.A.; J. Org. Chem.
1972, 37, 916.
38. Pösch, P.; Thelakkat, M.; Schmidt, H. W.; Synth. Met. 1999,
102, 1110.
56. O’Boyle, N. M.; Tenderholt; A. L.; Langner, K. M.; J. Comp.
Chem. 2008, 29, 839.
39. Pommerehne, J.; Vestweber, H.; Guss, W.; Mahrt, R. F.;
Bassler, H.; Porsch, M.; Daub, J.; Adv. Mater. 1995, 7, 551.
40. Turovska, M.; Stradins, J.; Freimanis, J.; Strazdins, I.; Logins, J.;
Dregeris, J.; J. Electroanal. Chem. 1996, 414, 221.
41. Becke, A. D.; J. Chem. Phys. 1993, 98, 5648.
Submitted: September 9, 2013
Published online: November 26, 2013