Synthesis, characterization and biological activities of 3-aryl-1,4-naphthoquinones-green palladium-catalysed Suzuki cross coupling

Article abstract of DOI:10.1039/c6nj00872k

Quinones are important scaffolds that are present in a variety of natural products or synthetic bioactive molecules. Arylation is an important strategy for accomplishing structural modifications, leading to new potential candidates for use as drugs. In the present work, palladium-catalysed, ligandless and phosphine-free Suzuki coupling reactions between 2-hydroxy-3-iodo-1,4-naphthoquinone and boronic acids were employed to prepare several 2-hydroxy-3-aryl-1,4-naphthoquinones in aqueous conditions using microwave irradiation or conventional heating. Because of the biological activities of quinones, which are related to their ability to accept electrons to form semiquinones and hydroquinones, the electrochemical behaviour of the synthesized molecules was investigated. The Osiris and Molinspiration Cheminformatics programs, utilizing in silico analyses, imply that these naphthoquinones are candidates for use as drugs which was reinforced by the outcomes of the in vitro antifungal and trypanocidal activity tests. Our in vitro data indicated a MIC value of 8 μg mL^-1 against Candida albicans ATCC 24433 strains, and an EC₅₀ of 0.67 μM with respect to trypanocidal activity against Trypanosoma cruzi epimastigote strains (Y).

Full text of DOI:10.1039/c6nj00872k

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Synthesis, characterization and biological activities of 3-aryl-1,4-
naphthoquinones – Green palladium-catalysed Suzuki cross
couplings
Received 00th January 20xx,
Accepted 00th January 20xx
Aline da R. Louvis,a Nayane A. A. Silva,a Felipe S. Semaan,b Fernando de C. da Silva,a Gabriela
Saramago,c Laís C. S. Velasco de Souza,c Bruno L. A. Ferreira,c Helena C. Castro,c Juliana P. Salles,e
André Luís A. Sousa,d Robson X. Faria,e Vitor F. Ferreira,a* Daniela de L. Martins,a*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Quinones are important scaffolds that are present in a variety of natural occurrings or synthetic bioactive molecules.
Arylation is an important strategy for accomplishing structural modifications, leading to new potential candidates to drugs.
In the present work, palladium-catalysed, ligandless and phosphine-free Suzuki couplings between 2-hydroxy-3-iodo-1,4-
naphthoquinone and boronic acids were employed to prepare several 2-hydroxy-3-aryl-1,4-naphthoquinones in aqueous
conditions using microwave irradiation or conventional heating. Because the biological activities of quinones, which are
related to their ability to accept electrons to form semiquinones and hydroquinones, the electrochemical behaviour of the
synthesized molecules was investigated. The Osiris and Molinspiration Cheminformatic programs, utilized in silico
analyses, imply that these naphthoquinones are candidates inclined to drug which was reinforced by the in vitro antifungal
and trypanocidal activities tests outcomes. Our in vitro data indicated a MIC value of 8 µg/mL against Candida albicans
ATCC 24433 strains, and an EC50 of 0.67 µm with respect to trypanocidal activity against Trypanosoma cruzi epimastigote
strains
(Y).
O
OH
Introduction
Quinones occur widely in nature and play important roles
in the life cycle of cells. Quinone-based compounds display a
variety of pharmacological activities, such as anticancer,
antiviral and anti-neurodegenerative activities, among others.1
Atovaquone (Figure 1) is a potent anti-malarial drug that
inhibits the mitochondrial electron transport chain
(cytochrome bc1 complex) of parasites. The naphthoquinone
moiety in atovaquone interacts with highly conserved
aminoacids residues of cytochrome b by multiple non-polar
interactions. Currently, atovaquone is used for the treatment
and prophylaxis of malaria in association with proguanil
O
1
Cl
Figure 1 – Atovaquone
Fungi are eukaryotic organisms that are found in the
commensal microbiota of healthy humans. They may colonize
mucosal surfaces such as the skin, gastrointestinal tract and
oral cavity. These organisms may present different forms,
including a rounded form, a hyphae form or a dimorphic form.3
Candida spp are fungi of clinical importance that undergo
phenotypic switching from yeast to hyphae forms, becoming
infectious and causing candidiasis. There are several
predisposing factors for the development of candidiasis, such
as AIDS, diabetes, cytotoxic therapy and prolonged antibiotic
therapy.4
hydrochloride (Malarone
Other biologically active molecules were synthesized based
as the lead compound using rational drug design.2
– GlaxoSmithKline, UK).
on
1
Over the last few years, Candida strains have been
recognized as a major cause of opportunistic fungal infections
that occur around the world. They represent a great threat,
especially to immune compromised patients, and cause
significant morbidity and mortality.5 In this regard, Candida
albicans is the most common aetiological agent of candidiasis,
but non-albicans Candida species, such as C. glabrata, C.
krusei, C. parapsilosis and C. tropicalis, are also important in
causing this infection.
a
Departamento de Química Orgânica, Universidade Federal Fluminense, Campus
do Valonguinho, Centro, Niterói 24020-141, Brazil. dlmartins@id.uff.br
b
Departamento de Química Analítica, Universidade Federal Fluminense, Campus
do Valonguinho, Centro, Niterói 24020-141, Brazil.
cLABioMol – Laboratório de Antibióticos, Bioquímica e Modelagem Molecular,
Instituto de Biologia, Universidade Federal Fluminense, Niterói 24210-130, Brazil.
d Laboratory of Biochemistry of Peptides, Oswaldo Cruz Institute - Fiocruz, Brazil.
e
Laboratory of Toxoplasmosis and other Protozooses – Oswaldo Cruz Institute-
Fiocruz, Brazil.
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Currently, there are a few classes of antifungal agents that inhibitors of the sterol biosynthesis. Antifungals such as
are available for the treatment of candidiasis. They include posaconazole or ketoconazole can inhibit the growth of the T.
polyenes, azoles and echinocandins, but none of them are fully cruzi strains.15
effective due to their lack of efficacy or their toxicity. This
Arylation is an interesting approach that can accomplish
scenario worsens because of the emergence and/or increase in structural modifications of quinones and naphthoquinones
fungi resistance against the antifungals that are clinically in with the goal of synthesizing new substances with biological
use. Widespread and non-rational use of these antifungal activities.16 In this regard, palladium-catalysed Suzuki coupling
agents has led to a strong selection of resistant strains.6
between boronic acids and electrophiles is an important C-C
The emergence of antifungal resistance and the side bond-forming tool. Additionally, greener protocols, including
effects of current antifungal therapy make it difficult to the reactions in aqueous medium, have been developed to
treatment of the fungi-caused infections. Therefore, new accomplish the Suzuki couplings.17
treatment options ought to be developed by researching new
antifungal prototypes with effective activities against Candida analogues of atovaquone, but with a simpler structure. This is
strains and with low toxicity. very interesting because although atovaquone is a highly
3-Aryl-2-hydroxy-1,4-naphthoquinones can be considered
A similar scenario is observed in a protozoan infection that active and safe drug against protozoan infections its
affects millions of people and kills 12,000 per year. Chagas production costs have precluded its use in the developing
disease is caused by the protozoan parasite Trypanosoma countries.
cruzi. The transmission of such a disease can occur in different
Arylations of naphthoquinones have already been reported
ways (e.g., via the faeces of bloodsucking bugs known as in the literature by using C-H activation procedures,18
‘kissing bugs’). Within urban areas, it is not only caused by diazonium salts, Lewis acid-catalyzed arylations with aromatic
transfusion of contaminated blood, but also by food aldehydes, arenes or hydrazines, Stille and Suzuki cross-
contaminated with the parasite, as well as by urine or anal coupling reactions.18 Many of the procedures reported fail
secretions from infected marsupials, to name
Additionally, mothers can transmit T. cruzi to their foetus 3 position of the naphthoquinones. Additionally, there are few
during pregnancy.7
reports regarding the arylation of unprotected 2-hydroxy-1,4-
a few. when it comes in to introducing electron-poor groups at the C-
In Latin America, Chagas disease affects mainly poor naphthoquinones. Spyroudis and co-workers19 reported the
communities, where it sets in endemically. However, this Suzuki cross-coupling of phenyliodonium ylides of
neglected disease has been scattered to other continents, and hydroxyquinones with arylboronic acids (0.5 mmol of
more than 6 million people are estimated to be infected.8
The main drugs used in the treatment of Chagas disease DME:H2O, R3P, 5-8 h). Yields were found to be dependent on
are Nifurtimox and Benznidazole (Figure 2). These drugs the phosphine employed, with better yields when P(t-Bu)3 was
iodonium, 1.1 mmol boronic acid, 4% Pd(OAc)2, LiOH,
2
3
are very effective if applied in the acute phase, but not in the used, which was attributed to the minimization of
chronic phase, when the effectiveness decreases. In addition, transylidation reaction with formation of the corresponding
treatments with these drugs are of long duration and have phosphonium ylides. Iodobenzene was formed as side product
severe side effects.9 Similar to the Candidiasis scenario, it is and coupled with the excess of the boronic acids, resulting in
also necessary to discover new trypanocidal agents to replace the formation of Ph-Ar. 3-Aryl-2-hydroxy-1,4-naphthoquinones
the few that are currently available for treating Chagas bearing electron-withdrawing groups (F, CHO or CN) in the
disease.
phenyl ring were not synthesized using the procedure
reported by Spyroudis. 2-Hydroxy-3-iodo-1,4-naphthoquinone
or 2-amino-3-iodo-1,4-naphthoquinone were not employed in
the Suzuki cross-couplings, phosphine-free couplings were not
reported for these substrates, as well as the effect of
microwave irradiation in these reactions was not investigated.
Furthermore, the 3-aryl-2-hydroxy-1,4-napthoquinones were
not evaluated concerning their antifungal or trypanocidal
activities.
Figure 2 – Trypanocidal agents
Several reports in the literature describe the potential of
naphthoquinones as antifungals and trypanocidal agents. Tran
and co-workers demonstrated the antifungal activity of several
halo-1,4-naphthoquinones,10 Yoshizaki and co-workers
reported on the antifungal activity of naphthoquinone
derivatives constituting Lithospermum erythrorhizon11 and
terpenyl-1,4-naphthoquinones were considered active in vitro
against pathogenic yeasts and filamentous fungi.12 In our
research group and in others,13,14 several naphthoquinones
were prepared that displayed promising chemotherapeutic
activity against T. cruzi.
We
prepared
different
3-aryl-2-hydroxy-1,4-
naphthoquinone analogues of atovaquone under aqueous
conditions, employing conventional heating or microwave
irradiation using inexpensive phosphine-free sources of
palladium and inexpensive bases. These molecules were tested
for in vitro antifungal and trypanocidal activities, and an in
silico study was performed to evaluate their potential as
pharmaceuticals, as well as their pharmacokinetic properties
and toxicity.
T. cruzi shows some similarities to fungi in terms of the
sterol lipid biosynthesis, being both susceptible to the
2 | J. Name., 2012, 00, 1-3
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brominated lawsone, because this carbon resonates at 111.11
ppm (lawsone C-3 = 110.9 ppm).
Results and discussion
Different 3-arylnaphthoquinones were prepared via
palladium-catalysed Suzuki couplings between arylboronic
acids and 3-iodo-naphthoquinones in aqueous conditions,
using conventional heating or microwave irradiation.
Synthesis and characterization of 3-halo-1,4-naphthoquinones
Scheme 2 –Preparation of 3-bromo-lawsone
Initially, a morpholine-iodine complex 4 was prepared by
reacting morpholine and iodine20 in an aqueous solution
3-Iodo-1,4-naphthoquinones must be stored protected
from light to prevent the loss of iodine. 2-Amino-3-iodo-1,4-
(Scheme 1). This complex was, then, used in the iodination of
2-amino-1,4-naphthoquinone
naphthoquinone under
iodonaphthoquinones and
and
conditions.
7 were obtained in excellent This compound is resistant to GC/MS (EI) analysis conditions
2-hydroxy-1,4-
naphthoquinone
7 appear to be less prone to iodine loss, as it
basic
The 3-
can be stored for a longer period of time without losing iodine.
6
and the molecular ion (m/z = 299) can be recognized as being
the most prominent signal in the spectrum, followed by a peak
that corresponds to the loss of iodine (m/z = 172). 2-Hydroxy-
yields (82 and 93%, respectively) after acidification with a
concentrated phosphoric acid solution.21 Iodination at
position C-3 was confirmed by the absence of a signal at 6.20
1
3-iodo-1,4-naphthoquinone 6, however, is more prone to the
ppm (DMSO-d6) in an H-NMR spectrum that corresponds to
deiodination process and therefore must be stored carefully.
This compound does not survive the conditions under which
GC/MS analysis is performed, and the molecular ion cannot be
the vinylic CH of lawsone 5a (3-hydroxy-1,4-naphthoquinone).
In lawsone, the C-3 signal in the 13C-NMR spectrum is located
at 110.9 ppm, whereas halogenation of this position produces
a shielding effect, and the C-3 of 2-hydroxy-3-iodo-1,4-
naphthoquinone resonates at 92.7 ppm. The upfield trend in
the chemical shifts caused by bromination or iodination of a
carbon is attributed to an increase in the diamagnetic
shielding, which is due to the introduction of a large number of
electrons by the bromine or iodine atoms. The “shielding
effect” on the α-carbon is known as “the heavy atom effect”.
Bromo-1,4-naphthoquinone can also decompose, but this
process seems to be slower than the iodine loss. Halogenation
of 2-amino-1,4-napthoquinone 5b was confirmed in a similar
manner: a signal for the vinylic CH of the reagent (5.8 ppm)
observed. Iodo-lawsone
chromatographic step.
6 appears to lose iodine in the
Substitution of hydrogen in the C-3 position by halogen
atoms resulted in the appearance of an additional band at
approximately 414 nm in the UV electromagnetic spectrum,
indicating halogenation at this position.23
Synthesis and characterization of 3-aryl-1,4-naphthoquinones
3-Halo-1,4-naphthoquinones were subject to Suzuki
couplings with boronic acids. Perez and co-workers24
employed Pd(OAc)2/K2CO3/H2O to couple 3-iodolawsone with
electron-deficient olefins in a Heck coupling procedure and
obtained coupled naphthoquinones in high yields. Therefore,
we used the conditions described by Perez as the initial
conditions for the Suzuki couplings in the present work, using
1
was absent in the H-NMR spectrum of the product, and the
iodination of C-3 produces a shielding effect: from 102.3 ppm
in 2-amino-1,4-naphthoquinone to 82.2 ppm in 2-amino-3-
iodo-1,4-naphthoquinone (DMSO-d6).
the reaction between 2-hydroxy-3-iodo-1,4-naphthoquinone
6
and phenylboronic acid 9 as a model system. Palladium was
tested from different sources, bases and solvents (Table 1,
Scheme 3).
Scheme 3 – Suzuki coupling with phenylboronic acid
Under basic conditions for the Suzuki couplings, 2-hydroxy-
Scheme 1 – Preparation of iodo-1,4-naphthoquinones 6 and 7
3-iodo-1,4-naphthoquinone
6 (yellow) is soluble in water, as it
Lawsone was also brominated at the C-3 position using forms their lawsonate (red), as well as the reaction product is
in the form of a salt. After acidification of the aqueous reaction
medium, the crude product precipitates from water (crude
yields are shown in Table 1). We analysed the aqueous phase
and observed that the majority of the product precipitates.
bromine in acetonitrile (Scheme 2), and 3-bromo-2-hydroxy-
1,4-naphthoquinone22
was obtained in 77% yield. Although
8
bromine can, in principle, also cause an upfield effect in the
chemical shifts of the Cα signal, this “heavy atom effect” due
to bromination wasn’t observed at the C-3 position of the
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Therefore, extraction of the aqueous phase was not necessary
to improve the recovery of the product.
When the reactions were heated for more than 6-8h, by-
products began to form, and iodo-lawsone was not fully
consumed.
Table 1 – Suzuki coupling with phenylboronic acid
The conditions described in Table 1, entry 1 (5% Pd(OAc)2,
K2CO3, H2O) were considered the best of those that were
evaluated and were applied to the synthesis of other
arylnaphthoquinones for use the coupling of 3-halo-1,4-
naphthoquinones with different arylboronic acids (Scheme 4).
Crude products were recovered easily (60-99 %) from the
acidified solutions. However, low to moderate yields were
obtained, after flash chromatography.
Conditions
Catalyst
Pd(OAc)2
Pd(OAc)2
Pd(OAc)2
PdCl2
Base
K2CO3
K2CO3
Na2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
K2CO3
Solvent Recovery (%)
1
2
3
4
5
6
7
8
9
H2O
EtOH
H2O
H2O
H2O
H2O
H2O
H2O
H2O
85 (53)*
66
69
44
Pd2dba3
84 (54)*
99
PdCl2(PPh3)2
Pd/C
86
Pd(II)/BaSO4
Pd(0)/CaCO3
75
81
Reaction conditions: 0.5 mmol of 2-hydroxy-3-iodo-1,4-naphthoquinone,
0.75 mmol of boronic acid, 5 mol% of the palladium source, 2.5 mmol
K2CO3 and 5 mL of solvent. The reactions were monitored by TLC
analysis, and after 6 h, virtually no more consumption of
6 occurred,
however by-products began to form. Reactions were performed in water
and crude products were analysed by 1H-NMR. *Yield after column
chromatography.
Scheme 4 – Suzuki coupling of 6 with different boronic acids
Microwave irradiation can improve yields and accelerate
many organic reactions. Beneficial effects of the use of
microwave irradiation in cross-coupling reactions have already
been reported in the literature.25 Therefore, we employed the
conditions that were determined to be optimal for coupling
with conventional heating (5% Pd(OAc)2, K2CO3, H2O).
Intensive deiodination occurred when 5% Pd(OAc)2 was
employed, using microwave heating (P = 300 W, T = 120° C, 10
min). Increasing temperatures (150, 200° C) also resulted in
Products were obtained with low crude yields when alcohol
was employed as the solvent (entry 2) and 1H-NMR analysis
indicated a 4% yield of lawsone (product of deiodination). This is
likely due to the insolubility of carbonate in ethanol, thus
producing heterogeneous conditions; whereas both the base
(carbonate) and the substrate are soluble under aqueous basic
conditions.
No
lawsone
was
observed
when
Pd(OAc)2/K2CO3/H2O (entry 1) was employed, whereas 9%
lawsone was obtained using Pd(OAc)2/Na2CO3/H2O (entry 3).
Water was considered the best solvent, and potassium
carbonate the best base. Several catalysts were tested using
H2O/K2CO3.
deiodination of 2-hydroxy-3-iodo-1,4-naphthoquinone
6. At
80° C, a large amount of unreacted iodo compounds were
recovered. The best reaction conditions were: 1%
Pd(OAc)2/100°C/H2O. Therefore, using microwave irradiation,
excellent results were obtained using a lower loading of
palladium (1% irradiation versus 5% conventional) and shorter
reaction times (10 min versus 6 h). Using these conditions,
different boronic acids were employed in cross-coupling
reactions with 3-halo-1,4-naphthoquinones (Table 2). The
products that were obtained were higher in purity than those
obtained using the conventional conditions, and better yields
were obtained using flash chromatography (yields are an
average of duplicates). Moderate to good yields were
obtained, except for that of the reaction with 2-
thiopheneboronic acid (entry 4). Several conditions were
evaluated to improve the yield (increasing temperatures,
catalyst loading, time or different solvents), but in all cases a
low conversion of the iodinated precursor was obtained.
During homogeneous catalysis, palladium dichloride
exhibited the worst performance. TLC indicated a high content
of 6, which was confirmed by NMR analysis. Although a high
recovery of the product from acidified water was accomplished
when Pd2dba3 and PdCl2(PPh3)2 (entries 5 and 6) were employed,
TLC analysis of the crude products revealed
formation of lawsone, and in the case of Pd2dba3, a significant
amount of iodo-lawsone remained. NMR analysis identified
a significant
6
numerous impurities in the aromatic region.
Supported catalysts were also evaluated (entries 7-9). All of
the catalysts that were tested were successful in the Suzuki
coupling reactions, but the reactions appeared to be slower with
Pd/CaCO3 and Pd/BaSO4 (entries 8 and 9), because after 6h, a
significant amount of iodo-lawsone remained. Alternatively,
when Pd/C was used, 6% lawsone was obtained, and some
impurities were detected in the resonance spectra.
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Table 2 – Suzuki reactions under microwave irradiation
f
and 10g) and the boronic acids (3-formylphenylboronic acid
Yield (%)*
and 4-formylphenylboronic acid, respectively). This small
difference made the purification by column chromatography
Compounds
O
difficult.
The
difference
between
10f
and
3-
OH
formylphenylboronic acid, however, was slightly larger than
the difference between 10g and 4-formylphenylboronic acid.
Hence, the isolated yields were higher in all the reactions with
3-formylphenylboronic acid, despite of reaction conditions
(microwave versus conventional heating and Pd(OAc)2 versus
Pd/C).
10a
1
2
71
Ph
O
O
OH
10b
10c
64
78
4
O
F
Because of the apparent greater stability of 3-bromo-2-
hydroxy-1,4-naphthoquinone compared with 2-hydroxy-3-
iodo-1,4-naphthoquinone, couplings with the former were also
studied (Scheme 5). Under conventional heating and
employing the best conditions that were employed in
O
OH
3
4
F
O
O
O
couplings
with
2-hydroxy-3-iodo-1,4-naphthoquinone
OH
(Pd(OAc)2/K2CO3/H2O/6h), a low conversion was observed.
Increasing the time of the reaction to 24 hours did not improve
the conversion rate. Taking into consideration the observation
that microwave irradiation was beneficial to the couplings with
2-hydroxy-3-iodo-1,4-naphthoquinone, we evaluated different
conditions using microwave heating. The best conditions that
led to the greatest conversion of 3-bromo-2-hydroxy-1,4-
10d
S
O
OH
10e
5
6
7
72
S
O
O
O
OH
naphthoquinone were: 5% Pd(OAc)2/H2O/25 min/
(2:1) (Y = 40%).
8: PhB(OH)2
10f
60 (83)**
43 (65)**
O
O
OH
OH
10g
10h
O
O
O
8
9
75
66
43
Scheme 5 – Coupling reactions with
8
O
OMe
The low solubility of 2-amino-3-iodo-1,4-naphthoquinone
affected its conversion to product. Therefore, a mixture 4:1
water/EtOH was employed in the couplings with this
naphthoquinone. The coupling product 11 is partially soluble in
water and could not be recovered in higher yields by filtration
(47% the crude product). The pure product was obtained at
26% yield by extraction of the aqueous acidified phase.
Microwave conditions were also tested in the coupling of 2-
amino-3-iodo-1,4-naphthoquinone with phenylboronic acid.
The best condition obtained using microwave irradiation was:
5% Pd(OAc)2/H2O:EtOH(1:1)/25 min, using 100% excess of
O
OH
10i
OMe
O
O
OH
10
10j
CN
O
11
77
PhB(OH)2, instead of 50% that was used with
column chromatography).
6 (Yield = 54%,
Reaction conditions: (0.5 mmol of 6), 0.75 mmol of boronic acid, 1.0
mol % of the Pd(OAc)2, 2.5 mmol K2CO3, and 5 mL of H2O. *Isolated
yields (flash column chromatography). **Pd/C (1%).
Pd/C is a low cost, interesting, heterogeneous pre-catalyst.
Therefore, in this work, we also tested this catalyst with
Scheme 6 – Coupling reactions with the naphthoquinone
In silico analysis of 3-aryl-1,4-naphthoquinones
7
microwave heating for the reactions of
6
with
formylbenzeneboronic acids to obtain compounds 10f and 10g
(entries 6 and 7). The moderate isolated yields that were
obtained with formylbenzeneboronic acids were due to the
small retention factor (rf) difference between the products (10
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Recently, several research groups have exploited the toxicological risks, bioavailability properties and biological
potential of cheminformatic tools to conduct a rational activity scores for six receptor classes (GPCR ligands, ion
approach to drug design. Currently, these tools are used in channel modulators, kinase inhibitors, nuclear receptor
partnership with experimental tests.26 In view of the interest ligands, protease inhibitors and enzyme inhibitors) were
in the synthesis of new antifungal and trypanocidal drug calculated for all of the compounds and were compared with
candidates and the potential of naphthoquinones, compounds Fluconazole and Benznidazole, which are antifungal and
10a-10k were evaluated in silico using the Osiris Property trypanocidal drugs, respectively.
Explorer27 and Molinspiration Cheminformatic programs28. The
Table 3 – Comparison of the in silico results of the 3-aryl-1,4-naphthoquinones series
cLogP
Sol
M
-
T
-
-
-
-
-
-
-
-
-
-
-
-
-
REf
-
I
-
DL
DS L. R. viol. GCPRL ICM
KI
NRL
PI
EI
10a
10b
10c
10d
10e
10f
10g
10h
10i
2.19 -3.16
-1,12 0.53
-1.79 0.46
-4.76 0.41
0.29 0.66
-1.63 0.5
-5.89 0.26
-8.8 0.24
-1.29 0.30
-2.07 0.46
-10.0 0.38
-2.59 0.43
-1.66 0.33
4.84 0.95
0
0
0
0
0
0
0
0
0
0
0
0
0
-0.26 -0.28 0.15 -0.09 -0.40 0.27
-0.20 -0.29 0.22 -0.01 -0.37 0.25
-0.17 -0.27 0.25 0.04 -0.34 0.26
-0.40 -0.40 -0.01 -0.23 -0.57 0.10
-0.32 -0.28 0.23 -0.15 -0.47 0.26
-0.24 -0.29 0.18 0.12 -0.43 0.23
-0.27 -0.31 0.14 0.07 -0.46 0.20
-0.21 -0.34 0.16 -0.02 -0.34 0.20
-0.22 -0.34 0.19 -0.01 -0.36 0.21
-0.14 -0.27 0.31 0.12 -0.25 0.30
-0.26 -0.37 0.12 -0.07 -0.41 0.19
-0.33 -0.39 -0.49 -0.71 -0.05 -0.02
2.3
-4.04
-
-
-
2.3
-4.04
-
-
-
2.06 -3.74
1.98 -3.63
2.13 -4.05
2.13 -4.05
2.12 -3.75
2.12 -3.75
2.03 -4.50
2.54 -4.07
-
-
-
-
-
-
+
-
-
+
+
-
-
-
+
-
-
-
10j
-
-
-
10k
-
-
-
Benz -0.25 -1.62
Flu -0.31 -1.55
-
+
-
-
-
-
0.04
0.01 -0.09 -0.23 -0.09 0.03
Properties calculated by the Osiris Explorer program: clogP, Sol = solubility, M = mutagenic, T = tumourigenic, Ref = reproductive
effect, I = irritant, DL = druglikeness and DS = drug-score. Properties estimated by the Molinspiration Program: L.R.viol. = number
of violations of Lipinski’s rule. Theoretical inhibitory profile against GPCRL = GPCR ligand, ICM= ion channel modulator, KI =
kinase inhibitor, NRL= nuclear receptor ligand, PI = protease inhibitor, EI = enzyme inhibitor. Flu = Fluconazole, Benz =
Benznidazole
According to our in silico analysis, most of the 3-aryl-1,4-
Our in silico analyses revealed negative values for
naphthoquinones presented a low toxic risk profile regarding druglikeness parameter of these naphthoquinones, which
mutagenic, tumourigenic, irritant and reproductive effects. suggested fragments different from those present in
This low toxicity theoretical profile was as low as, or even commercial pharmaceuticals. The drug-score, which is a global
lower than the antifungal positive control (Fluconazole) or the parameter that is related to the potential of a molecule to be a
trypanocidal positive control (Benznidazole) (Table 3).
drug candidate, indicates that the 3-aryl-1,4-naphthoquiones
Water solubility is an important parameter to be evaluated are good drug candidates. Analysis of the predictions of
for the drug candidates because it is related to their bioactivities, according to the Molinspiration tool, indicated
distribution in the body. Interestingly, suitable values for logs > the inhibition of kinase and other enzymes as latent activities
-4 were observed for the naphthoquinones that were of the compounds under study. In agreement, most of the
synthesized
herein.
Analogously,
lipophilicity
and drugs that interact with enzymes in the body show a clogP
hydrophilicity are important properties that affect the between 2 and 5 and all of the naphthoquinones that were
absorption of a drug in the body. On this subject, the Lipinsk’s evaluated herein presented clogP values in this range.
rule of five29 is used to predict oral bioavailability. According to
Based on the in silico analyses that suggested the potential
our analysis, none of the compounds studied violated Lipinski’s biological profiles for this new series, we performed in vitro
rule of five, which indicates a good theoretical biovailability.
antifungal and antiparasitic tests to search for biological
6 | J. Name., 2012, 00, 1-3
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Journal Name
ARTICLE
activities associated with the arylnaphthoquinones. Among all
Figure 4 – Naphtoquinones-induced injury. Mouse peritoneal
of the compounds that were tested, 10a, 10b, 10d and 10e macrophages were treated with naphtoquinones (100 µM) for
were found to inhibit the growth of Candida strains, whereas
compound 10e exhibited a high trypanocidal activity, which
was superior to that observed with Benznidazole (Tables 4 and
5 and Figures 3-5). These data are in agreement with those of
the active compounds detected in silico that presented drug-
24 h. Cytotoxicity was measured using resazurin reduction to
resorufin as an index of proliferation. Data shown are the
mean SD of four independent experiments. Comparisons:
***, treated vs. non-treated. n.s. – no significant difference
scores higher than 0.4 except for compound 10d, and scores Disk diffusion susceptibility test (DDST)
for kinase inhibitor and enzyme inhibition greater than 0.2.
These experimental observations reinforced our in silico
Among the naphthoquinones that were tested (6, 8, 10a-
predictions, as the in vitro data are in good agreement with k), four exhibited antifungal activity. Compound 10b presented
them. The low theoretical toxicity value was also confirmed by the highest inhibition growth zone against C. albicans ATCC
the in vitro viability studies in mice primary cells that were 24433, as well as the widest spectrum of action of all
exposed to the arylnaphthoquinones. These results supported compounds that were tested. Compounds 10a
, 10b and 10d
additional studies on the spectrum and level of their biological showed a great spectrum of action, being able to inhibit the
activities, which we present in the in vitro assays that follow.
growth of three strains. None of the compounds were active
against the strain C. parapsilosis, ATCC 90028 (data not
shown).
Toxicity assay in mouse cell lines
Naphthoquinone-induced injuries to J774.G8 mouse Table 4 - Comparison of the inhibitory effects (halo = mm) of
macrophage cells derived from a sarcoma (Figure 3), or mouse the active naphthoquinones (10a 10b 10d and 10e) in the
,
,
primary peritoneal macrophages (Figure 4), were measured in DDST against Candida strains of clinical importance
terms of the ability to reduce resazurin. Cell viability decreased
Inhibition growth zone (halo = mm)
significantly when J774.G8 cells were exposed to
naphthoquinone 10g at a concentration of 100 µM for 24 h
Compounds
Control
MO strain
(Fig. 3). However, the concentration used in the testes is 10-
100 times greater than the concentration that was applied in
the subsequent biological assays. None of naphthoquinones
displayed toxicity when tested with wild type primary cells.
10a
0
10b 10d 10e Fluconazole
C. tropicalis
ATCC 750
11
15
11
18
11
0
15
0
14
12
14
13
C. glabrata
ATCC 90030
C. krusei
N=3
09
13
13
150
ns
11
11
0
*
100
ATCC 34135
C. albicans
ATCC 24433
0
50
***
***
0
Determination of the minimum inhibitory concentrations
(DMIC)
Figure 3 – Naphthoquinone-induced injury. J774.G8 cells
The four compounds that were active in the DDST were
submitted to the MIC test to determine the minimum
inhibitory concentration. Compound 10d presented the lowest
treated with naphthoquinones (100 µM) for 24 h. Cytotoxicity
was measured using resazurin reduction to resorufin as an
index of proliferation. Data shown are the mean SD of four
independent experiments. Comparisons: *** or *, treated vs.
non-treated. n.s. – no significant difference
MIC against C. albicans ATCC 2433 MIC = 8 µg/mL) (Table 5).
Most of the compounds had MIC values that were at the same
level of the antifungals most used in clinical practice (0.125 to
32
µg/mL) (listed in: CLINICAL LABORATORY STANDARDS
N=3 - peritoneal macrophages
INSTITUTE - Reference Method for Broth Dilution Antifungal
Susceptibility Testing of Yeasts; Approved Standard - Second
Edition. M27-A2. CLSI, Wayne, PA.2013). This level of activity
suggested their use as food molecules for further exploration
against multiresistant strains.
120
ns
100
80
60
40
***
20
Table
5
–
Comparison of the minimum inhibitory
***
0
concentration (MIC) values for the derivatives that presented
antifungal activities in the DDST
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Journal Name
150
MIC
µg/mL
MO strain
10a
16
10b
16
10d 10e Fluconazole
100
50
0
C. albicans
ATCC 24433
C. krusei
08
256
32
-
-
-
0.75
64
***
>512
-
128
>512
32
ATCC 34135
C. tropicalis
ATCC 750
32
-
03
C.glabrata
ATCC 90030
16
03
Treatments
In vitro antiparasitic activity
Figure 5 - Trypanocidal activity in 24 h (10 µM
naphthoquinones, n=3, Y strain). This graph is representative
of triplicate treatments performed on 4 distinct days
The effect of 3-aryl-1,4-naphthoquinones on in vitro
cultures of epimastigotes of T. cruzi was evaluated in
comparison with Benznidazole. Initially, we treated the cells
150
100
with a fixed dose of 10
µM naphthoquinones (see the
Experimental section and Figure 5) and compared the results
with those obtained with Benznidazole treatment. Treatment
with 100 µM Benznidazole was considered our positive control
(approximately 0-30% survive), whereas untreated cells were
the negative control (approximately 90-100% survive). The
results are shown in Figure 5 as % of trypanocidal activity. The
derivative that reduced the trypanosome viability below 75%
(compared with the positive control) was selected for
additional assays and MTT was used to perform the initial
screening of activity.
***
***
50
0
According to our data the analogues 5a, 6, 10a, 10b, 10c,
Treatments
10d, 10e, 10f, 10g and 11 did not exhibit a response
comparable to Benznidazole after 24 h (Figure 5). After 72 h of
Figure 6- Trypanocidal activity in 72 h (10µM
continuous exposure, only the derivative 10e exhibited
naphthoquinones, n=3, Y strain). This graph is representative
of triplicate treatments performed on 4 distinct days
trypanocidal
activity
(Figure
6).
Therefore,
this
naphthoquinone analogue was tested against epimastigote T.
cruzi strains (Y) using increasing concentrations ranging from 1
nM – 1 mM for 72 h (Figure 7). Interestingly, the EC50 value for
10e to cause trypanocidal activity (0,67 µM) was more potent
than that described for Benznidazole (EC50 = 40 µM).30 Thus,
although the 10e derivative effects were observed only after
72h, the potency that was measured was approximately 59
times greater than that with Benznidazole, indicating that the
development of new derivatives of this class would be
desirable.
150
10e
100
50
0
-10
-8
-6
-4
-2
[10e] M
Figure 7– Analysis of the trypanocidal effect of 10e after 72 h
using increasing concentrations (1nM – 1mM) against Y strains.
This graph is representative of triplicate treatments performed
on 3 distinct days
Electroanalytical studies
Naphthoquinones are known for their biological activities
that are often related to their ability to accept electrons (in
8 | J. Name., 2012, 00, 1-3
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ARTICLE
general one or two), forming semiquinones and A diffusion-controlled mass transport may be considered
hydroquinones.31,32,33 Such interesting electrochemical limiting in this case, because a linear relationship was obtained
behaviour encouraged us to conduct electroanalytical studies. between Ip (µA) and v1/2 (mV/s). From the series of synthesized
Thus, cyclic voltammetry33 was chosen as exploratory compounds, three were exceptional, because during
technique. For the assessment of the electroanalytical voltammetric measurements, solutions containing compounds
behaviour of the synthesized naphthoquinones, voltammetric 5a
measurements were performed using an IVIUM CompactSTAT red (Figure 9) in the surroundings of the working and counter
potentiostat (IviumTechnologies). The synthesized electrodes; such changes were monitored by coupling
naphtoquinones (from 5a 10a-k) were added to a 10 mL elecroanalytical (this time using an ITO transparent electrode
, 10a and 10d exhibited colour changes from colourless to
, 6,
electrochemical cell filled with a supporting electrolyte, and in and fixing the potential into suitable values) and
which a three electrode arrangement was assembled. The spectrophotometric measurements (Red Tide Fiber Optic
electrodes consisted of a platinum wire as a counter electrode, Spectrometer USB-650-UV-VIS). When UV-VIS spectrum was
Ag|AgCl(sat.) as
a
pseudo reference electrode, and
a
obtained by applying a potential when a colour change
occurred on the working electrode, it detected a new band in
commercially available glassy carbon as a working electrode (
φ
= 0.0519 cm2, Metrohm). Initially, solutions prepared by direct the spectrum. According to Singh,23 this is due to a broad local
solubilization of lithium perchlorate or tetrabutylammonium excitation band (L. E.) in the 400-500 nm region which is
perchlorate (0.1 mol/L) in acetonitrile were used as supporting attributable to a n→π* transition of the quinone carbonyls.
electrolytes; however, because noisy and non-reproductive
electroanalytical responses were obtained using the former, all
subsequent studies were performed in tetrabutylammonium
perchlorate solutions. Analytes were assessed by direct
dissolution of suitable amounts in the supporting electrolytes,
reaching a final concentration of 1 mmol/L just before the
electrochemical measurements were conducted. Cyclic
voltammograms were recorded from -2.00 to +1.25 V, at
different scan rates (from 50 to 200 mV/s), and were assessed
against respective blanks. From these experiments, some
issues were raised. It could be noted that the
arylnaphthoquinones exhibited one reduction peak (from -1.33
to -1.24 V) and one oxidation peak (from -1.16 to -1.28 V).32
Complimentary experiments were performed by inverting the
sweep rate, from +1.25 to -2.00 V, which indicated an
independence between the observed peaks, which was then
confirmed by narrowing the potential window (assessing this
time from 0.00 to +2.00 V). Within this new potential window
it was possible to note that the peaks, which appear at more
positive potentials, are independent of the others (Figure 8).
a)
5,0x10-5
0,0
-5,0x10-5
-1,0x10-4
-1,5x10-4
-2,0
-1,5
-1,0
-0,5
0,0
0,5
1,0
E/V vs Ag|AgCl pseudo-ref.
sat
b) 3,00
2,25
1,50
0,75
0,00
5,0x10-5
0,0
UV 10a
UV 10a at E = -1.19 V
O
OH
-5,0x10-5
F
O
10c
-1,0x10-4
-2.0 to +2.0 V
-2.0 to 0.0 V
0.0 to +2.0 V
250 300 350 400 450 500 550 600 650 700 750 800
-1,5x10-4
λ/nm
-2,0 -1,5 -1,0 -0,5
0,0
0,5
1,0
1,5
2,0
Figure 9: a) Cyclic voltammogram of 10a, b) UV-VIS of 10a at
potential of -1.19 V
E/V vs Ag|AgCl pseudo-ref.
sat
Figure 8 - Cyclic voltammogram of 10c: 200 mV/s
Although naphthoquinone activities can, in many cases, be
related to their redox properties, in the present work, it was
not possible to correlate the electrochemical behaviour of the
synthesized arylnaphthoquinones with their biological
Thus the electrochemical studies demonstrated that the
assessed naphthoquinones have quasi-reversible redox peaks.
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Journal Name
activities, as both of bioactive and the inactive compounds and molecular ions are reported in m/z units. An Agilent® 122-
exhibited the same reduction and oxidation values. Chemical 5532 DB-5MS column was used for GC analysis. High-
activity, in this case, may involve another mechanism of action. resolution mass spectra (HRMS) were recorded on a Mass
spectrometer MICROTOF Bruker Daltonics mass spectrometer.
Samples were dissolved in CH3Cl and diluted in MeOH and
could not be ionized in the positive mode.
Conclusions
Arylnaphthoquinones were obtained in moderate to good
yields by conducting palladium-catalysed Suzuki reactions
Biological assays
using conventional heating and microwave irradiation. Suzuki
couplings were performed under aqueous conditions using
inexpensive palladium phosphine-free pre-catalysts. were obtained from America Type Culture Collection and were
Five fungal strains of different species of the genus Candida
used to evaluate the antifungal activity of the
naphthoquinones: C. parapsilosis ATCC 90028, C. albicans
ATCC 24433, C. krusei ATCC 34135, C. glabrata ATCC 90030
and C. tropicallis ATCC 750.
Microwave irradiation resulted in an improvement of the
isolated yields, decreasing reaction times and decreasing
catalyst load (1% with microwave vs. 5% with conventional
heating). Five of the aryl-naphthoquinones synthesized herein
are new. Arylnaphthoquinones were evaluated in silico with
respect to their ADME/Tox and druglikeness properties. The in
silico analysis indicated good biological and toxicological
profiles for the synthesized naphthoquinones, and this was
reinforced by the biological assays. None of the
arylnaphthoquinones displayed toxicity to wild type mouse
Disk diffusion susceptibility test (DDST)
To determine the susceptibility of each of the five Candida
ATCC strains to the compounds used, a suspension of each
strain in 5 mL of saline solution was prepared. Then, an aliquot
of 500 µL was inoculated onto a Sabouraud-Dextrose Agar
plate, and a 6 mm filter paper disk impregnated with the
naphthoquinones diluted in dimethyl sulfoxide (DMSO) (5
mg/mL) was placed on the agar plate. The plate was then
incubated for 24-48 h at a temperature of 35°C. Fungal growth
inhibition zones were measured when the compounds
displayed activity against the Candida strains tested. DMSO
was used as a negative control, and Fluconazole (5 mg/mL) as
a positive control.
primary peritoneal macrophages at a concentration of 100
(24 h). Four of the arylnaphthoquinones that were prepared
10a 10b 10d and 10e) exhibited antifungal activities against
strains of clinical importance (10d, MIC = 8 g/mL, against C.
albicans ATCC 24433). Compound 10d exhibited an
antiparasitic effect against the epimastigote strain (Y) of T.
cruzi, with an EC50 of 0.67 M. Although the trypanocidal
µM
(
,
,
µ
µ
effects were observed only after 72h, this EC50 value is 59
times higher than the reported EC50 value for Benznidazole, a
trypanocidal agent that is used clinically. These results indicate
that arylnaphthoquinones are promising hit compounds whose
structures deserve to be optimized.
Determination of minimum inhibitory concentrations (DMIC
)
The compounds that exhibited antifungal activity in the
DDST were evaluated further to define the lowest
concentrations of the compounds that inhibited visible growth
of the Candida strains (MIC). Serial dilutions of the compounds
were prepared using Sabouraud-Dextrose Broth medium in a
96-well microtiter plate (0.5 µg/mL to 512µg/mL) and 100 µL
inoculums of the Candida strains. The microtiter plate was
incubated for 24-48 h at a temperature of 35°C, and the
minimum inhibitory concentration (MIC) was defined as the
lowest concentration that was capable of inhibiting visible
fungal growth.
Experimental
Materials and measurement
Boronic acids were purchased from Sigma Aldrich Brazil
LTDA and Combi-Blocks. They were used without further
purification. 2-Hydroxy-1,4-naphthoquinone and other
reagents and solvents were purchased from Sigma Aldrich
Brazil LTDA. Column flash chromatography was performed
using silica gel 60 (35-70 µm). Analytical thin-layer
chromatography was conducted on silica gel plates (Silicycle
Ultrapure Silica Gels, F254) and the spots were visualized using
UV light or iodine vapours. Yields refer to isolated yields after
flash chromatography. Melting points were recorded on a
Fisatom 413D apparatus. Infrared spectra were measured
In silico analysis of the toxicity
The compounds 10a-10k were submitted to toxicological
analysis in silico using the Osiris Property Explorer27 and
Molinspiration programs.28 The Osiris program presents a
predictive algorithm that evaluates the potential toxicological
risks of the compounds tested by analysis of the molecular
structure using a database of 3300 drugs. This program
predicts whether the compounds are mutagenic,
tumourigenic, irritants, or have reproductive effects. Other
properties, including solubility, lipophilicity, druglikeness, TPSA
(permeability of the molecule in the plasma membrane) and
molecular weights are also estimated. The Osiris program
indicates the potential toxicological risks using a colour table in
which a green colour represents a low potential toxic risk, a
using KBr pellets on
a Perkin-Elmer model 1420 FT-IR
Spectrophotometer. NMR spectra were recorded on a Varian
VNMRS (300 MHz or 500 MHz) instrument in DMSO-d6 or
CDCl3. The chemical shift data are reported in units of δ (ppm)
downfield from tetramethylsilane, which was used as an
internal standard. Coupling constants (J) are reported in Hertz
units and refer to apparent peak multiplicities. The gas
chromatography/mass spectrometry (GS/MS) analysis was
conducted on an Agilent® 6890 chromatograph coupled to the
Agilent® 5973 mass spectrometer. The fragmentation values
10 | J. Name., 2012, 00, 1-3
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yellow colour represents a medium potential toxic risk, and a
red colour represents a high potential toxic risk.
foetal bovine serum (FBS) (Cultilab - Campinas, Brazil) at 37 °C
in a humidified atmosphere with 5% CO2. On reaching 80-90%
confluence, the cells were detached using a solution of 0.025%
trypsin and 0.4% EDTA (ethylene diamine tetraacetic acid) for
1 minute and then used in flow cytometry experiments.
Violations of the Lipinski’s rule of five29 were determined
using Molinspiration web based software. Important
pharmacokinetic properties in humans (absorption,
distribution, metabolism, excretion and toxicity – ADME/Tox)
are described by this rule. The activity score for GPCR ligands,
ion channel modulators, nuclear receptor ligands, kinase
inhibitors, protease inhibitors and enzyme inhibitors were
calculated using Molinspiration. The results obtained were
compared with the values obtained with standard drugs.
Molecules with positive scores are considered active. Scores
between -5.0 and 0.0 indicate moderate activities and scores
bellow -5.0, are considered inactive.
Resazurin (Alamar Blue) proliferation assay
Reduction of the dye resazurin to resorufin was used to
measure proliferation of the cell in cultures. Cells were seeded
at 1 x 105 trypan blue excluding cells/mL in 96-well microtiter
plates.34 After incubating for 24 h, the DMEM medium was
removed and replaced with fresh medium containing 40 µM
resazurin, followed by an incubation period of 24 h, after
which reduction of resazurin to resorufin was determined by
fluorescence (excitation 530 nm; emission 590 nm) using a M5
microplate fluorometer (Molecular Devices, Florida, U.S.A.).
Appropriate cell free controls were included in the test to
account for potential interactions between the derivatives and
resazurin.
Trypanosomatid cultures
T. cruzi epimastigote Y strains were cultured in LIT medium
(Tryptose liver infusion) supplemented with 10% foetal calf
serum at a temperature of 28◦C, with strong agitation (100
rpm) as reported previously, in the absence or presence of
either naphthoquinone derivatives or Benznidazole. Live
parasites were counted daily using a Neubauer chamber. The
initial parasite concentration was 106 parasites/mL. Either the
drug or vehicle (DMSO) was added after 24h and 72h for each
Synthesis of 3-halo-1,4-naphthoquinones
condition. At least
3 independent experiments were
Iodination of 1,4-naphthoquinones 5a and 5b
performed for each drug and dose, and the 50% effective
concentration (EC50) was determined using Prisma GraphPad
5.0.
The morpholine-iodine complex was synthesized using the
procedure described by Frota and co-workers and then stored
protected from light.21
Trypomastigote viability assay
Morpholine-iodine complex (12.8 g, 37.5 mmol) was added
to a 500 mL Erlenmeyer flask containing 1,4-naphthoquinone
(lawsone 5a or 2-amine-1,4-naphthoquinone 5b 30 mmol),
K2CO3 (12.4g, 90 mmol) and distilled water (300 mL) in small
portions every 15 minutes over a period of two hours. The
reaction mixture was then stirred for at least 30 minutes.
Subsequently, the mixture was cooled using an ice bath and
acidified with concentrated H3PO4. The solution remained
under agitation at room temperature for at least 24 hours,
after which the mixture was filtered using a vacuum, and the
solid obtained was washed with ice-cold water. The solid was
dried at room temperature protected from light and then
stored protected from light and under refrigeration.21
Viability assays were performed using the formazan
formation method, also referred to as the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay, as previously described.33 Briefly, 1x107 trypomastigotes
were incubated in RPMI 1640 culture medium at 37 °C for 24 h
with and without addition of the naphthoquinones at a final
concentration of 10 µM and then incubated for 24 or 72h.
Naphthoquinones with trypanocidal activities were tested at
concentrations range from 1nM
– 1mM. Formation of
formazan was monitored at 570 nm in a multi-well reader
(Spectramax M5, Molecular Devices).
Cell cultures
2-Hydroxy-3-iodonaphthalene-1,4-dione (6) - The
reaction
produced the compound (7.38 g, 82%) as an orange solid, mp
6
Macrophages were obtained from the peritoneal cavities of
4-week-old Swiss Webster mice. After centrifugation, the cells
were counted using a Neubauer chamber, maintained in
serum-free medium for macrophages (Gibco, USA) and
cultured at 37 °C in an atmosphere of 5% CO2 for 24 hours. Our
protocols adhered to the Ethical Principles in Animal
Experimentation adopted by the Brazilian College of Animal
Experimentation and were approved by the Fiocruz Research
Ethics Committee (number L-041/08).
172-173°C (lit. 177°C).21 IR (KBr) ʋmax/cm-1 1620 (C=O), 1670
(C=O), 3161 (OH). δH (500 MHz; DMSO-d6; TMS) 8.06-7.98 (2H,
m), 7.85-7.78 (2H, m). δC (125 MHz; DMSO-d6) 179.5, 177.2,
162.2, 134.4, 133.3, 130.7, 129.6, 126.6, 126.3, 92.7 ppm.
2-Amino-3-iodonaphthalene-1,4-dione.
The
reaction
produced the compound (0.592 g, 91%) as a red-brick solid
7
mp 188-190°C (lit. 201-202°C).21 IR (KBr) ʋmax/cm-1 1271 (C-N),
1579 (N-H), 1621 (C=O), 1671 (C=O), 3346 and 3447 (NH2). δH
(500 MHz; DMSO-d6; J in Hz) 8.12-8.10 (2H, m), 7.91 (1H, td, J =
1.5, 7.5 Hz), 7.88-7.85 (1H, m). δC (125 MHz; DMSO-d6) 177.3,
176.5, 152.5, 134.5, 132.5, 131.5, 129.4, 126.4, 126.3, 82.2
ppm.
The murine macrophage line J774.G8 was used in all
experiments. This strain is derived from the original J774.A1
cell line from the American Type Cell Collection (ATCC,
Rockville, MD). The cells were cultured in Dulbecco's Modified
Eagle's Medium (DMEM)(Sigma, St. Louis, MO) containing 10%
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 11
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DOI: 10.1039/C6NJ00872K
ARTICLE
Journal Name
2-Bromo-3-hydroxynaphthalene-1,4-dione (8) – Lawsone 5a 2-(4-Fluorophenyl)-3-phenylnaphthalene-1,4-dione (10b)
-
(1.16 g, 6.7 mmol) and acetonitrile (100 mL) were added to a The reaction produced the compound 10b as a yellow solid
250 mL flask equipped with a magnetic stirrer. Bromine (0.5 (0.086 g, 0.321 mmol, 64%), mp 185-188°C. IR (KBr) ʋmax/cm-1
mL) was added after solubilization of 5a and the resulting 1643 (C=O), 1665 (C=O), 3330 (OH). δH (500 MHz; DMSO-d6; J
solution was refluxed for 3 hours and then 1.2 mL of in Hz) 8.07 (1H, dd, J = 1.0, 7.5 Hz); 8.03 (1H, dd, J = 1.5, 7.5
cyclohexene was added. Solvents were removed using a rotary Hz); 7.88 (1H, td, J = 1.5, 7.5 Hz); 7.84 (1H, td, J = 1.5, 7.5 Hz);
evaporator. The residue was washed with hexane, solubilized 7.45-7.41 (2H, m); 7.27-7.22 (2H, m). δC (125 MHz; CDCl3)
in ether and treated with charcoal. Subsequently, the solution 183.3, 181.3, 163.0, 159.8, 154.9, 134.6, 133.2, 132.8, 132.0,
was filtered, and the ether was evaporated. The solid that was 129.9, 127.6, 126.0, 125.5, 121.1, 114.4 ppm. MS (EI+) m/z (%):
obtained was dried at room temperature protected from light 268 (M+, 100), 239 (18) 240 (19), 183 (57).
and was then stored protected from light and under
refrigeration. This reaction produced compound
8
(1.306 g, 2-(3-Fluorophenyl)-3-phenylnaphthalene-1,4-dione (10c)
-
77%) as a yellow solid, mp 203-205°C (lit. 201-203°C).22, 35 IR The reaction produced the compound 10c as an orange solid
(KBr) ʋmax/cm-1 3176 (O-H), 1672 (C=O), 1580 and 1454 (C=C), (0.105 g, 0.391 mmol, 78%), mp 163-165°C. IR (KBr) ʋmax/cm-1
3161 (OH). δH (500 MHz; DMSO-d6; J in Hz) 8.03-8.00 (2H, m); 1648 (C=O), 1672 (C=O), 3246 (OH). δH (500 MHz; DMSO-d6; J
7.85-7.80 (2H, m). δC (125 MHz; DMSO-d6) 179.2, 178.6, 158.7, in Hz) 8.07 (1H, dd, J = 1.5, 7.5 Hz); 8.04 (1H, dd, J = 1.0, 7.5
135.1, 134.0, 131.9, 130.2, 126.9, 126.8, 111.1 ppm.
Hz); 7.89 (1H, td, J = 1.5, 7.5 Hz); 7.84 (1H, td, J = 1.5, 7.5 Hz);
7.48-7.43 (1H, m); 7.23 (1H, dd, J = 1.0, 7.5 Hz); 7.20-7.17 (2H,
General procedure for preparing 3-arylnaphthoquinones m).δC (125 MHz; DMSO-d6) 183.4, 181.5, 162.6, 160.7, 155.3,
under conventional heating 10a-k
135.0, 133.9, 133.5, 132.1, 130.1, 129.5, 127.1, 126.3, 125.8,
121.0, 117.6, 114.6 ppm. HRMS (ESI) m/z calc for C16H9FO3 [M-
2-Hydroxy-3-iodo-1,4-naphthoquinone (0.150 g, 0.5 mmol), H]- 267.0457, found 267.0462.
K2CO3 (0.346 g, 2.5 mmol), arylboronic acid (0.75 mmol) and
Pd(OAc)2 (0.005 g, 5 mol%) were added to a 25 mL round- 2-Hydroxy-3-(thiophen-2-yl)naphthalene-1,4-dione (10d)
-
bottom flask equipped with a reflux condenser and a magnetic The reaction produced the compound 10d as a purple solid
stirrer. Then, 5.0 mL of distilled water was added. The reaction (0.046 g, 0.179 mmol, 36%), mp 146-147°C (lit. 134-137°C).16d
mixture was refluxed for 6h. The reaction mixture was IR (KBr) ʋmax/cm-1 1652 (C=O), 3344 (OH). δH (500 MHz; DMSO-
acidified with concentrated H3PO4 (pH
≅
2). The solid that d6; J in Hz) 8.27 (1H, dd, J = 1.0, 4.0 Hz); 8.18 (1H, dd, J = 1.0,
formed was vacuum filtered, dried at room temperature and 7.5 Hz); 8.13 (1H, dd, J = 1.0, 7.5 Hz); 7.98 (1H, td, J = 1.0, 7.5
purified by flash chromatography using silica gel and was Hz); 7.94-7,91 (1H, m); 7.85 (1H, dd, J = 1.0, 5.5 Hz); 7.32-7.30
eluted with an increasing polarity gradient mixture of hexane (1H, m). δC (125 MHz; DMSO-d6) 183.5, 180.5, 153.4, 134.7,
and dichloromethane - 4:1 to 1:1).
133.4, 132.0, 131.9, 130.9, 129.8, 129.7, 126.5, 126.4, 125.5,
115.4 ppm. MS (EI+) m/z (%): 256 (M+, 100), 228 (33), 172 (27),
General procedure for preparing 3-arylnaphthoquinones 171 (64), 76 (17).
under microwave irradiation 10a-k
2-Hydroxy-3-(thiophen-3-yl)naphthalene-1,4-dione (10e)
-
A 10 mL Pyrex tube equipped with a magnetic stirrer was The reaction produced the compound 10e as a red-brick solid
charged with 3-halo-1,4-naphthoquinone (0.5 mmol), K2CO3 (0.092 g, 0.359 mmol, 72%), mp 132-134°C (lit. 125-127°C).16 IR
(0.346 g, 2.5 mmol), arylboronic acid (0.75 mmol) and (KBr) ʋmax/cm-1 1649 (C=O), 3325 (OH). δH (500 MHz; DMSO-
Pd(OAc)2 (0.001 g, 1 mol%). The mixture was irradiated for 10 d6; J in Hz) 8.06-8.03 (2H, m); 7.94-7.93 (1H, m); 7.87 (1H, td, J
min at 100°C. Then, the reaction media were acidified with = 1.5, 7.5 Hz); 7.82 (1H, td, J = 1.5, 7.5 Hz); 7.56-7.53 (2H, m).
H3PO4 (pH
≅
2). The solid that was obtained was purified by δC (125 MHz; DMSO-d6) 183.5, 181.2, 154.4, 134.6, 133.2,
flash column chromatography on silica gel and eluted with an 132.2, 130.8, 129.9, 129.8, 127.9, 126.2, 125.4, 123.7, 117.0
increasing polarity gradient mixture of hexane and ppm. MS (EI+) m/z (%): 256 (M+, 100), 228 (63), 172 (34), 171
dichloromethane.
(95), 76 (28).
2-Hydroxi-3-phenylnaphthalene-1,4-dione (10a)
–
Product 3-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
10a was obtained as a yellow solid (0.089 g, 0.356 mmol, 71%), yl)benzaldehyde (10f) - The reaction produced the compound
mp 145-146°C (lit. 146°C).36 IR (KBr) ʋmax/cm-1 1649 (C=O), 10f as a yellow solid (0.083 g, 0.300 mmol, 60%), mp 221-
1659 (C=O), 3346 (OH). δH (500 MHz; DMSO-d6; J in Hz) 8.19 223°C. IR (KBr) ʋmax/cm-1 1667 (C=O), 1691 (C=O), 3137 (OH).
(1H, dd, J = 1.0, 7.5 Hz); 8.15 (1H, dd, J = 1.0, 7.5 Hz); 8.02-7.99 δH (500 MHz; DMSO-d6; J in Hz) 10.05 (1H, s, CHO); 8.09–8.04
(1H, m), 7.95 (1H, td, J = 1.5, 7.5 Hz); 7,55-7.45 (5H, m, Ph). δC (2H, m); 7.93 (1H, s); 7.91–7.88 (2H, m); 7.86–7.83 (1H, m);
(125 MHz; CDCl3) 183.7, 181.8, 152.2, 135.3, 133.1, 132.8, 7.73 (1H, dt, J = 1.5; 7.5 Hz); 7.66 (1H, t, J = 7.5 Hz). δC (125
130.6, 130.0, 129.3, 128.6, 127.9, 127.3, 126.1, 122.2 ppm. MS MHz; DMSO-d6) 193.0, 183.3, 181.3, 155.4, 136.8, 135.6,
(EI+) m/z (%): 250 (M+, 100), 221 (19) 194 (21), 165 (47).
134.7, 133.3, 132.5, 132.0, 131.6, 130.0, 128.8, 128.3, 126.1,
125.6, 120.8 ppm. HRMS (ESI) m/z calc for C17H10O4 [M-H]-
277.0501,found 277.0510.
12 | J. Name., 2012, 00, 1-3
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Journal Name
4-(3-Hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-
View Article Online
DOI: 10.1039/C6NJ00872K
ARTICLE
2-amino-3-iodo-1,4-naphthoquinone (0.149 g, 0.5 mmol),
yl)benzaldehyde (10g) - The reaction produced the compound K2CO3 (0.346 g, 2.5 mmol), arylboronic acid (0.75 mmol) and
10g as a yellow solid (0.058 g, 0.208 mmol, 42%), mp 217- Pd(OAc)2 (0.005 g, 5 mol%) were added to a 25 mL round-
218°C. IR (KBr) ʋmax/cm-1 1671 (C=O), 1689 (C=O), 3261 (OH). bottom flask equipped with a reflux condenser and a magnetic
δH (500 MHz; DMSO-d6; J in Hz) 10. 05 (1H, s, CHO); 8.07 (1H, stirrer. Then, 4.0 mL of distilled water and 1.0 mL of ethanol
dd, J = 1.0, 7.,5 Hz); 8.04 (1H, dd, J = 1.0, 7.5 Hz); 7.95 (2H, d, J were added. The reaction mixture was refluxed for 6h. The
= 8,0 Hz); 7.89 (1H, td, J = 1.0, 7.5 Hz); 7.84 (1H, td, J = 1.0, 7.5 reaction mixture was then acidified with concentrated H3PO4
Hz); 7.62 (2H, d, J = 8.0 Hz). δC (125 MHz; DMSO-d6) 192.6, (pH
≅ 2). The solid that formed was filtered, dried at room
183.0, 181.2, 155.4, 137.9, 135.0, 134.7, 133.2, 131.9, 131.4, temperature and purified by extraction with ethyl acetate.
129.9, 128.4, 126.0, 125.6, 120.9 ppm. HRMS (ESI) m/z calc for
C17H10O4 [M-H]- 277.0501, found 277.0509.
2-Amino-3-phenylnaphthalene-1,4-dione (11) - The reaction
produced the compound 11 in 24% as a red darkness solid,
2-hydroxy-3-(4-methoxyphenyl)naphthalene-1,4-dione (10h) m.p. 177-178°C (lit. 178-179°C).37 IR (KBr) ʋmax/cm-1 1603
-The reaction produced the compound 10h as a red darkness (C=O), 1675 (C=O), 3343 and 3416 (NH2). δH (500 MHz; DMSO-
solid (0.106 g, 0.378 mmol, 75%), mp 180-182°C (lit. 172- d6; J in Hz) 8.13 (1H,d, J = 7.5 Hz); 8.10 (1H,d, J = 7,0 Hz); 7.95
173°C).18f IR (KBr) ʋmax/cm-1 1609 (C=O), 1635 (C=O), 3366 (1H,td, J = 1.0, 7.5 Hz); 7.86 (1H,td, J = 1.0, 7.5 Hz); 7,57 (2H, t,
(OH). δH (500 MHz; DMSO-d6; J in Hz) 8.07-8.01 (2H, m), 7.90- J = 7.5 Hz); 7.47 (1H, t, J = 7.5 Hz); 7.41-7.39 (2H, m). δC (125
7.79 (2H, m), 7.37-7.32 (2H, m), 7.00-6.95 (2H, m), 3.80 (3H, s, MHz; DMSO-d6) 181.7, 180.2, 146.1, 134.7, 130.4, 128.3,
CH3). δC (125 MHz; DMSO-d6) 184.4, 181.8, 159.2, 154.9, 127.3, 125.7, 125.5, 114.8 ppm.
135.2, 133.8, 132.5, 132.4, 130.3, 126.6, 126.0, 123.7, 122.5,
113.5, 55.6 ppm.
Acknowledgements
2-Hydroxy-3-(3-methoxyphenyl)naphthalene-1,4-dione (10i) -
The reaction produced the compound 10i as a yellow solid
(0.092 g, 0.328 mmol, 66%), mp 126-127°C. IR (KBr) ʋmax/cm-1
1652 (C=O), 1661 (C=O), 3359 (OH). δH (500 MHz; DMSO-d6; J
in Hz) 8.06 (1H,dd, J = 0.5, 7.5 Hz), 8.03 (1H,dd, J = 1.0, 7.5 Hz);
7.88 (1H, td, J = 1.5, 7.5 Hz); 7.83 (1H, td, J = 1.0, 7.5 Hz); 7.34-
7.31 (1H, m); 6.94-6.92 (3H, m); 3.76 (3H, s, CH3). δC (125 MHz;
DMSO-d6) 183.4, 181.4, 158.4, 154.8, 134.6, 133.1, 132.7,
132.0, 129.9, 128.3, 126.0, 125.5, 123.0, 122.1, 116.4, 113.0,
55.0 ppm. HRMS (ESI) m/z calc for C17H12O4 [M-H]- 279.0657,
found 279.0658.
CNP-q/Universal (455739/2014-5), CAPES (for the student
scholarships and researcher fellowships), FAPERJ/APQ-1
(E26/110.724/2012/2), and FAPERJ (Support for Studies of
Neglected and Reemerging Diseases - E-26/010.001535/2014),
and Brazilian foundations for financial support and researcher
fellowships. We thank Dr. Ivano Philippis, responsible for the
Reference Microorganisms Collection (CMRVS) – Fiocruz, for
the donation of the fungi strains. We also thank Dr. Marcos da
Veiga Kalil for allowing the use of his laboratory facilities to
perform some antifungic assays and Professor Elias Barreto for
his kind suggestions regarding written in English.
4-(3-Hydroxy-14-dioxo-1,4-dioxo-1,4-dihydronaphthalen-2-
yl)benzonitrile (10j) -The reaction produced the compound 10j
as a yellow solid (0.059 g, 0.214 mmol, 43%), mp 253-255°C. IR
(KBr) ʋmax/cm-1 1653 (C=O), 1674 (C=O), 2239 (C≡N), 3211
(OH). δH (500 MHz; DMSO-d6; J in Hz) 8.07 (1H, dd, J = 1.5, 7.5
Hz), 8.05-8.03 (1H, m), 7.91-7.83 (4H, m), 7.59 (2H, d, J = 8,0
Hz). δC (125 MHz; DMSO-d6) 182.9, 181.1, 155.5, 136.8, 134.8,
133.3, 131.9, 131.7, 131.2, 130.0, 126.0, 125.6, 120.4, 118.8,
110.0 ppm. HRMS (ESI) m/z calc for C17H9NO3 [M-H]- 274.0504,
found 274.0520.
Notes and references
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Synthesis of 2-amino-3-phenylnaphthalene-1,4-dione using
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DOI: 10.1039/C6NJ00872K
ARTICLE
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O
O
O
O
X
Y
X
Pd(OAc)2
ArB(OH)2
+
K2CO3, H2O
Ar
reflux or
microwave heating
X = NH2, OH
Y = I, Br
Trypanocidal and
antifungal activities
Antifungal and trypanocidal aryl-1,4-naphthoquinones were prepared through aqueous Suzuki protocol with reflux or microwave irradiation.