Synthesis using microwave irradiation and antibacterial evaluation of new N,O-acetals and N,S-acetals derived from 2-amino-1,4-naphthoquinones

Article abstract of DOI:10.1016/j.ejmech.2013.01.010

This paper describes a novel series of N,O-acetals and N,S-acetals (7a-o) derived from 2-amino-1,4-naphthoquinones that were synthesized and evaluated as potential antimicrobial agents. These compounds were obtained in good yields using microwave irradiation, and several of them showed promising antibacterial profiles. Three of our biologically active 2-amino-1,4-naphthoquinone N,O-acetals and N,S-acetals tested against hospital bacterial strains were identified as potential lead compounds. Characterization of all compounds was performed using one-dimensional NMR techniques (¹H, ¹³C-APT), IR spectra, elemental analyses and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS).

Full text of DOI:10.1016/j.ejmech.2013.01.010

European Journal of Medicinal Chemistry 63 (2013) 196e201
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis using microwave irradiation and antibacterial evaluation of
new N,O-acetals and N,S-acetals derived from 2-amino-1,4-
naphthoquinones
Alessandro K. Jordão a, Juliana Novais b, Bruno Leal b, Ana C. Escobar a, Helvécio M. dos Santos Júnior c,
,
Helena C. Castro b, Vitor F. Ferreira a
*
a Universidade Federal Fluminense, Departamento de Química Orgânica, Programa de Pós-Graduação em Química, Outeiro de São João Batista, s/nꢀ, Niterói 24020-141, RJ, Brazil
b Universidade Federal Fluminense, PPBI e LABiEMol, GCM, Instituto de Biologia, Outeiro de São João Batista, s/nꢀ, Niterói 24020-141, RJ, Brazil
c Universidade Federal do Rio de Janeiro, Instituto de Química, Departamento de Química Orgânica, 21944-970 Cidade Universitária, RJ, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
This paper describes a novel series of N,O-acetals and N,S-acetals (7aeo) derived from 2-amino-
1,4-naphthoquinones that were synthesized and evaluated as potential antimicrobial agents. These
compounds were obtained in good yields using microwave irradiation, and several of them showed
promising antibacterial profiles. Three of our biologically active 2-amino-1,4-naphthoquinone N,O-ace-
tals and N,S-acetals tested against hospital bacterial strains were identified as potential lead compounds.
Characterization of all compounds was performed using one-dimensional NMR techniques (1H, 13C-APT),
IR spectra, elemental analyses and high-resolution electrospray ionization mass spectrometry (HR-
ESI-MS).
Received 9 October 2012
Received in revised form
7 January 2013
Accepted 8 January 2013
Available online 23 January 2013
Keywords:
Quinones
Microwave chemistry
Biological activity
Ó 2013 Published by Elsevier Masson SAS.
1. Introduction
(2) [15] and antibacterial agents (3) [16]. Recently, our research group
described the synthesis of diethyl 2-[(3-hydroxy-1,4-dioxo-1,4-
Naphthoquinones are important natural substances that are
commonly found throughout different families of plants, fungi and
certain animals [1]. This class of compounds has been studied
extensively for their antitumor [2], molluscicidal [3], antiparasitic
[4], leischmanicidal [5], anti-inflammatory [6], antifungal [7],
antimicrobial [8] and trypanocidal [9] properties. Reports indicate
that the biological activity of these molecules is derived from their
ortho- or para-quinonoid moieties, which accept one or two elec-
trons (redox cycling) to form the corresponding radical anion or
dianion species in situ [10]. The resultant semi-quinone radicals
accelerate intracellular hypoxic conditions by producing superox-
ide anions [11,12]. By this mechanism, quinones may be cytotoxic in
mammalian cells by affecting such enzymes as topoisomerases,
a group of enzymes that are critical for DNA replication [13].
By introducing amino substituents onto the quinone moiety, its
redox properties can be tuned to induce oxidative stress in cells and
to alkylate DNA. Such aminonaphthoquinones are found in impor-
tant bioactive compounds, such as antitumor (1) [14], antimalarial
dihydronaphthalen-2-yl)-hydrazono]-malonate (4), which shows
promising antibacterial activity [17] (Fig. 1).
2. Results and discussion
2.1. Synthesis
The aim of this work was to synthesize a new series of N,O-ac-
etals and N,S-acetals derived from 2-amino-1,4-naphthoquinones
and to evaluate their antimicrobial properties. N,O-Acetals repor-
ted by Barluenga and co-workers [18] were unstable compounds at
room temperature and needed to be stored at ꢁ18 ꢀC to avoid
decomposition. In this study, we describe a methodology that
employs microwave irradiation to obtain N,O-acetals and N,S-ace-
tals in moderate to good yields.
In the first step, 1,4-naphthoquinone (5a) or 2-methyl-1,4-
naphthoquinone (5b) was reacted with sodium azide in acetic acid
to produce 2-amino-1,4-naphthoquinone (6a) [19] and 2-amino-3-
methyl-1,4-naphthoquinone (6b) [20], respectively, according to
a previous report.
Compounds 6a and 6b were converted readily into their cor-
responding aminonaphthoquinone derivatives (7aeo). Treatment
* Corresponding author. Tel.: þ55 21 26292345; fax: þ55 21 26292136.
E-mail address: cegvito@vm.uff.br (V.F. Ferreira).
0223-5234/$ e see front matter Ó 2013 Published by Elsevier Masson SAS.
http://dx.doi.org/10.1016/j.ejmech.2013.01.010

A.K. Jordão et al. / European Journal of Medicinal Chemistry 63 (2013) 196e201
197
Fig. 1. Examples of bioactive aminonaphthoquinones.
with paraformaldehyde in the presence of selected alcohols or
thiols under microwave irradiation afforded the N,O-acetals and
N,S-acetals in moderate yields (Scheme 1). The aim of the use of
heat microwave is that solvents can be applied at higher temper-
atures than usual since an increased pressure limit can be achieved
and also low-absorbing solvents can be used efficiently in micro-
wave protocols without any problems.
N,O-Acetals 7aeg were prepared by the condensation of com-
pound 6a with paraformaldehyde using the corresponding alcohols
simultaneously as both solvent and reactant (route I). Sulfur de-
rivatives N,S-acetals 7heo were obtained by the condensation of
compounds 6a, b with paraformaldehyde and various thiols in
chloroform (route II).
By utilizing this synthetic route, it was possible to prepare 2-
amino-1,4-naphthoquinone N,O-acetals and N,S-acetals 7aeo in
moderate to good yields. The reactions were performed in a sealed
glass vessel. These compounds were characterized using spectro-
scopic methods, such as 1H and 13C NMR.
These synthetic compounds are the first examples of stable N,O-
acetals and N,S-acetals derived from naphthoquinones and are
notably promising for biological activity screenings.
acetals series against several bacterial strains, including a control
strain with susceptibility to several known antibiotics (Staphylo-
coccus ATCC 25923) and seven hospital strains [five gram-positive
strains (Staphylococcus aureus, methicillin-resistant S. aureus e
MRSA, oxacillin-resistant S. aureus e ORSA, and oxacillin-resistant
staphylococci e Staphylococcus epidermidis ORS and Staphylococ-
cus haemolyticus ORS) and two gram-negative strains (Klebsiella
pneumonia and Pseudomonas aeruginosa)] (Tables 1 and 2).
Initially, we screened our N,O-acetal and N,S-acetals series
against all bacterial strains with the disc diffusion method. Inter-
estingly, three compounds (7a, 7b and 7c) showed an active profile
against almost all of the strains tested (Table 1). This antibacterial
spectrum is advantageous as it reveals biological activity against
multi-resistant strains of medical importance [23]. Importantly,
none of 2-amino-1,4-naphthoquinone-N,S-acetals, 7(heo) showed
antibacterial activity.
According to our results, only N,O-acetals 7(aeg) were effective
against the bacteria evaluated herein including gram-positive
(staphylococci) and gram-negative strains. The S. aureus strains
was the most susceptible to the new derivatives, whereas the K.
pneumoniae was the least affected. This in accord to the current
literature that reports more antibacterial lead compounds against
gram-positive than against gram-negative bacteria. This phenome-
non is generally due to the cellular organization of gram-negative
bacteria that includes an outer lipid bilayer membrane. Known an-
tibiotics, such as aminoglycosides, tetracyclines and chlor-
amphenicol, and several newer ones are prevented from entering
into these bacteria due to the presence of enzyme-containing
2.2. Antibacterial activity
Due to the increasing antibiotic resistance of various bacterial
strains, primarily in hospital environments, new alternatives to
conventional antibiotics are needed [21,22]. To that end, we eval-
uated our new 2-amino-1,4-naphthoquinone N,O-acetal and N,S-
Scheme 1. Synthesis of 2-amino-1,4-naphthoquinone N,O-acetals and N,S-acetals 7aeo.

198
A.K. Jordão et al. / European Journal of Medicinal Chemistry 63 (2013) 196e201
Table 1
was increased, leading to the lowest observed MIC values (4e
g/mL, Table 2). However, this substitution negatively affected
the compound’s activity against the multi-resistant bacterial
strains (Table 2).
Antibacterial profile of the active compounds from 2-amino-1,4-naphthoquinone
N,O-acetals and N,S-acetals series against hospital multi-resistant bacterial strains
on disc diffusion assays.
8
m
Bacterial strain
Inhibition growth zone (Halo ¼ mm)a
Finally, changing R2 ¼ methyl (7a) to R2 ¼ propynyl (7c) led to
7a
7b
7c
the highest observed S. aureus MIC values (64 mg/mL), suggesting
adverse steric effects resulting from the large propynyl substituent.
Despite this result, this compound is still effective against P.
aeruginosa.
Importantly, 7a and 7b antimicrobial profile is biologically sig-
nificant (p ꢂ 0.005) and suggests that these derivatives should be
further explored for targeting a future use against S. aureus-related
infections. This feasible use may allow other effective antibiotics,
such as vancomycin, to be saved for using only against exceptional
dangerous multi-resistant strains.
Staphylococcus aureus ATCC 25923 17
15
18
14
17
10
16
0
16
20
16
12
10
18
0
Staphylococcus aureus
24
16
18
14
20
17
0
Staphylococcus aureus MRSA
Staphylococcus aureus ORSA
Staphylococcus epidermidis ORS
Staphylococcus haemolyticus ORS
Klebsiella pneumoniae
Pseudomonas aeruginosa
14
18
a
Ciprofloxacin (Halo ¼ 22 mm) and vancomycin (16e19 mm) were used as
positive controls for gram-negative and gram-positive bacteria, respectively.
Our qualitative antibacterial data also revealed an active profile
for compounds 7a, 7b and 7c against K. pneumoniae or P. aeruginosa,
two gram-negative bacteria. However the MIC assay revealed that
periplasmic space, the peptide glycans layer and the polysaccharides
in the membrane surface mainly associate with the efflux pump
resistance mechanism [24]. These features increase bacterial
resistance to antibiotics and reiterate the need for finding new
molecules that are active against gram-negative strains.
The inhibitory results from some of the biologically active de-
rivatives (24 mm) were greater than those observed for the positive
control, which was vancomycin (16e19 mm). The biological profiles
of the three active derivatives (7aec) correlate with the nature of
their R2 substituents (7a, R2 ¼ methyl, 7b, R2 ¼ ethyl and 7c,
R2 ¼ propynyl), especially when contrasted with the inactive de-
rivatives (7deg, route I and 7hen, route II).
Because the disk diffusion test is a qualitative assay [25,26],
these derivatives were also submitted to a minimal inhibitory
concentration (MIC) determination assay (Table 2). Notably, the
MIC values for the active compounds ranged from 4 to 64 mg/mL
against the gram-positive bacteria, which are close to the reported
values of some commercially available antibiotics.
compound 7c is more active against P. aeruginosa (MIC ¼ 16
than 7b (MIC ¼ 128 g/mL) whereas 7a showed a lower active
profile against K. pneumoniae (MIC ¼ 256 g/mL) (Table 2). Due to
mg/mL)
m
m
the importance of these bacteria worldwide and the current need
for more treatment options, further structural modification in 7c
may allow exploring and/or increasing its antimicrobial properties
for future treatment purpose.
Many studies have shown that some antimicrobials are able to
affect genes involved in the bacterial oxidative stress response by
inducing the production of reactive oxygen species (ROS) [28,29].
The presence of the quinone group in certain derivatives is
described as essential for the inhibitory activity on enzymes
involved in ROS formation. For future perspective, additional
studies to investigate the antibacterial effects of 2-amino-1,4-
naphthoquinone N,O-acetals and their relationship with ROS for-
mation [30] will be performed.
According to the literature, the emergence of resistant Staphy-
lococcus species has increased continually, including the methicillin-
resistant and oxacillin-resistant S. aureus (MRSA and ORSA, respec-
tively) and oxacillin-resistant staphylococci (ORS). These bacterial
strains cause several diseases ranging from soft tissue infections to
life-threatening diseases such as toxic shock syndrome, endocarditis
and necrotizing pneumonia [27].
3. Conclusion
Since their discovery in the twentieth century, antimicrobial
agents have been responsible for significant reduction of worldwide
bacterial infection rates [31,32]. Currently, the urgent worldwide
need for new bacterial outbreak treatment options warrants the
need for further exploration of the new derivatives described herein.
Three of our biologically active 2-amino-1,4-naphthoquinone N,O-
acetals tested against hospital bacterial strains were identified as
potential lead compounds that can be explored to generate new and
more active antibiotics. At the same time, it is necessary to evaluate
these compounds’ biological activity against additional bacterial
strains to assess their ability to treat other threatening bacteria.
Further development of the biological profiles of these compounds
may help to emphasize their potential as lead compounds for
treating a large variety of bacterial infections.
The quantitative assay data confirmed that active derivatives
7aec significantly affect these Staphylococcus resistant strains
with substituent-dependent biological profiles. Derivative 7a
(R2 ¼ methyl) presented similar antibacterial effects against all
staphylococci strains studied (MIC ¼ 16
mg/mL), independent of
the bacteria resistance profile. The only exception was for
S. epidermidis, in which compound 7a presented a more active
profile (MIC ¼ 8 g/mL, Table 2). By changing R2 ¼ methyl (7a) to
m
R2 ¼ ethyl (7b), the activity against non- or less-resistant strains
4. Experimental section
Table 2
Comparison of the minimal inhibitory concentration values of the active 2-amino-
1,4-naphthoquinone N,O-acetals against various bacterial strains.
Melting points were determined with a Fisher-Johns instrument
and are uncorrected. Infrared (IR) spectra were recorded on an ABB
FTLA2000-100 spectrophotometer using KBr pellets. NMR spectra,
unless otherwise stated, were obtained in Me2SO-d6 or CDCl3 with
Strain
MIC (mg/mL)
7a
7b
7c
32
Staphylococcus aureus ATCC 25923
Staphylococcus aureus
16
8
a Varian Unity Plus 300 MHz spectrometer. Chemical shifts (d) are
16
16
16
16
4
32
32
64
32
64
256
32
expressed in ppm and the coupling constants (J) in Hertz. Flash
column chromatography purification was performed on silica gel
from Acros. Reactions were routinely monitored by thin-layer
chromatography (TLC) on silica gel pre-coated F254 plates from
Merck. Microanalyses were performed on a PerkineElmer Model
2400 instrument, and all values were within ꢃ0.4% of the
Staphylococcus aureus MRSA
Staphylococcus aureus ORSA
Staphylococcus haemolyticus ORS
Staphylococcus epidermidis ORS
Klebsiella pneumoniae
8
16
16
256
ꢂ256
ꢂ256
ꢂ256
Pseudomonas aeruginosa
128
16

A.K. Jordão et al. / European Journal of Medicinal Chemistry 63 (2013) 196e201
199
0
~
calculated composition values. High-resolution electrospray ion-
137.0 (C-1 ), 147.2 (C-7), 181.7 (C]O), 183.5 (C]O) ppm; IR (KBr)
n
3340, 1681, 1597, 1570 cmꢁ1; HMRS (ESI) [M þ H]þ. Found: 294.1128.
Calc. for C18H16NO3þ: 294.1125. Anal. Calcd. for C18H15NO3: C, 73.71;
H, 5.15; N, 4.78. Found: C, 73.33; H, 5.47; N, 4.45.
ization mass spectrometry (HR-ESI-MS) was performed in positive
ion mode on a Waters-Micromass Q-Tof Micro instrument. Com-
pounds 7aeo were prepared using an Anton Paar Monowave 300
microwave reactor.
4.1.1.5. 2-(((1-Phenyl-1H-1,2,3-triazol-4-yl)methoxy)methyl)-amino-
4.1. Chemistry
1,4-naphthoquinone 7e. Obtained as a yellow solid (114 mg, 22%);
mp: 145e156 ꢀC; 1H NMR (300 MHz, CDCl3):
d
¼ 3.33 (s, 2H, CH2),
4.1.1. General procedures for the preparation of amino-1,4-
naphthoquinone acetals 7aeg
4.66 (s, 2H, CH2), 5.96 (s, 1H, H-6), 7.45e7.50 (m, 1H, H-40), 7.55e
7.61 (m, 2H, H-30 and H-50), 7.73e7.77 (m, 1H, H-1 or H-4), 7.81e
7.87 (m, 3H, H-20, H-60, H-1 or H-4), 7.98e8.01 (m, 2H, H-2 and
A 10 mL microwave tube was loaded with 6a (1.4 mmol), par-
aformaldehyde (4 mmol), the appropriate alcohol (4.5 mmol) and
were irradiated for 20 min. The internal temperature reached
150 ꢀC. The alcohol excess was evaporated under reduced pressure.
The residual solid product was purified by column chromatography
on silica gel and eluted with an increasing polarity gradient mixture
of hexane and ethyl acetate (9/1). For the product 7e was used 5 mL
of chloroform as solvent.
H-3) ppm; 13C NMR (75 MHz, CDCl3):
d
¼ 59.9 (CH2), 71.9 (CH2),
103.2 (C-6), 119.9 (C-10), 125.2 (C-20 and C-60), 125.8 (C-1 and C-4),
128.5 (C-40), 129.7 (C-30 and C-50), 130.2 (C-4a), 132.4 (C-8b), 134.6
(C-2 and C-3), 136.4 (C-10), 144.7 (C-9), 148.1 (C-7), 181.3 (C]O),
~
182.2 (C]O) ppm; IR (KBr)
n
3340, 1681, 1597, 1570 cmꢁ1; HMRS
(ESI) [M þ H]þ. Found: 361.1297. Calc. for C20H17N4O3þ: 361.1295.
Anal. Calcd. for C20H16N4O3: C, 66.66; H, 4.48; N, 15.55. Found: C,
66.69; H, 4.64; N, 15.24.
4.1.1.1. 2-(Methoxymethyl)-amino-1,4-naphthoquinone (7a). Obtained
as a yellow solid (232 mg, 74%); mp: 115e116 ꢀC; 1H NMR (300 MHz,
4.1.1.6. 2-(Isopropoxymethyl)-amino-1,4-naphthoquinone 7f. Obtained
as a yellow solid (223 mg, 63%); mp: 125e126 ꢀC; 1H NMR (300 MHz,
CDCl3):
7.67 (m, 1H, H-1 or H-4), 7.70e7.76 (m, 1H, H-1 or H-4), 8.05e8.11 (m,
2H, H-2 and H-3) ppm; 13C NMR (75 MHz, CDCl3):
d
¼ 3.35 (s, 3H, CH3), 4.69 (s, 2H, CH2), 6.09 (s, 1H, H-6), 7.61e
CDCl3):
d
¼ 1.20 (d, 6H, J ¼ 6.4, CH3), 3.77 (hep,1H, J ¼ 6.4, CH), 4.75 (s,
d
¼ 55.4 (CH3), 74.5
2H, CH2), 6.07 (s, 1H, H-6), 7.61e7.65 (m, 2H, H-1 or H-4), 7.72e7.74
(CH2), 104.5 (C-6), 126.2 (C-1 and C-4), 130.4 (C-4a), 132.3 (C-2 and C-
(m, 2H, H-1 or H-4), 8.04e8.10 (m, 2H, H-2 and H-3) ppm. 13C NMR
3), 133.0 (C-8b), 134.7 (C-2 and C-3), 147.3 (C-7), 181.7 (C]O), 183.5
(75 MHz, CDCl3):
126.2 (C-1 and C-4), 130.4 (C-4a), 133.1 (C-8b), 134.6 (C-2 and C-3),
d
¼ 22.1 (CH3), 69.2 (CH), 71.1 (CH2), 104.1 (C-6),
(C]O) ppm; IR (KBr):
3341, 1681, 1611, 1571 cmꢁ1; HRMS (ESI)
~
n
[M þ H]þ. Found: 218.0807. Calc. for C12H12NO3þ: 218.0812. Anal.
Calcd. for C12H11NO3: C, 66.35; H, 5.10; N, 6.45. Found: C, 66.04; H,
5.50; N, 6.31.
147.3 (C-7), 181.7 (C]O), 183.5 (C]O) ppm; IR (KBr)
n
3249, 1677,
~
1604, 1573 cmꢁ1; HMRS (ESI) [M þ H]þ. Found: 246.1129. Calc. for
C
14H16NO3þ: 246.1125. Anal. Calcd. for C14H15NO3: C, 68.56; H, 6.16;
N, 5.71. Found: C, 68.32; H, 6.49; N, 5.39.
4.1.1.2. 2-(Ethoxymethyl)-amino-1,4-naphthoquinone 7b. Obtained
as a yellow solid (157 mg, 47%); mp: 134e135 ꢀC; 1H NMR
4.1.1.7. 2-(tert-Butoxymethyl)-amino-1,4-naphthoquinone
(300 MHz, CDCl3):
d
¼ 1.22 (t, 3H, CH3, J ¼ 14.1), 3.54 (q, 2H, J ¼ 14.1,
7g. Obtained as a yellow solid (113 mg, 30%); mp: 104e105 ꢀC;
CH2), 4.73 (s, 1H, CH2), 6.09 (s, 1H, H-6), 7.60e7.66 (m, 1H, H-1 or H-
4), 7.70e7.76 (m, 1H, H-1 or H-4), 8.04e8.10 (m, 2H, H-2 and H-3)
1H NMR (300 MHz, CDCl3):
d
¼ 1.28 (s, 9H), 4.75 (s, 2H, CH2),
6.07 (s, 1H, H-6), 7.61e7.64 (m, 2H, H-1 or H-4), 7.70e7.73 (m, 2H,
ppm; 13C NMR (75 MHz, CDCl3):
d
¼ 14.9 (CH3), 63.4 (CH2), 72.9
H-1 or H-4), 8.03e8.10 (m, 2H, H-2 and H-3) ppm. 13C NMR
(CH2), 104.2 (C-6), 126.2 (C-1 and C-4), 130.4 (C-4a), 132.3 (C-2 and
(75 MHz, CDCl3):
126.2 (C-1 and C-4), 130.4 (C-4a), 133.2 (C-8b), 134.5 (C-2 and C-
d
¼ 27.9 (CH3), 67.1 (C), 74.1 (CH2), 103.5 (C-6),
C-3), 133.0 (C-8b), 134.6 (C-2 and C-3), 147.3 (C-7), 181.7 (C]O),
3273, 1679, 1625, 1542 cmꢁ1; HMRS
3), 147.2 (C-7), 181.7 (C]O), 183.4 (C]O) ppm. IR (KBr)
n
3361,
~
~
183.5 (C]O) ppm; IR (KBr):
n
(ESI) [M þ H]þ. Found: 232.0967. Calc. for C13H14NO3þ: 232.0968.
Anal. Calcd. for C13H13NO3: C, 67.52; H, 5.67; N, 6.06. Found: C,
67.58; H, 6.04; N, 5.76.
1673, 1603, 1572 cmꢁ1; HMRS (ESI) [M þ H]þ. Found: 260.1280.
Calc. for C15H18NO3þ: 260.1281. Anal. Calcd. for C15H17NO3: C,
69.48; H, 6.61; N, 5.40. Found: C, 69.13; H, 6.44; N, 5.57.
4.1.1.3. 2-((Prop-2-ynyloxy)methyl)-amino-1,4-naphthoquinone 7c.
Obtained as a yellow solid (73 mg, 21%; mp: 126e127 ꢀC); 1H NMR
4.1.2. General procedures for the preparation of amino-1,4-
naphthoquinone acetals 7heo
(300 MHz, CDCl3):
d
¼ 2.50 (s, 1H, CH), 4.19 (s, 2H, CH2), 4.87 (s, 1H,
A 10 mL microwave tube was loaded with 6a or 6b (1.4 mmol),
paraformaldehyde (4 mmol), the appropriate thiol (1.5 mmol) and
chloroform (5 mL) and were irradiated for 20 min. The internal
temperature reached 150 ꢀC. The solvent was evaporated under
reduced pressure. The residual solid product was purified by col-
umn chromatography on silica gel and eluted with an increasing
polarity gradient mixture of hexane and ethyl acetate (9/1).
CH2), 6.14 (s, 1H, H-6), 7.61e7.66 (m, 1H, H-1 or H-4), 7.70e7.76 (m,
1H, H-1 or H-4), 8.04e8.10 (m, 2H, H-2 and H-3) ppm; 13C NMR
(75 MHz, CDCl3):
75.5 (C^CH), 104.9 (C-6), 126.2 (C-1 and C-4), 130.4 (C-4a), 132.3
(C-2 or C-3), 132.9 (C-8b), 134.7 (C-2 or C-3), 147.1 (C-7), 181.6 (C]
d
¼ 54.4 (OeCH2eC), 71.4 (CH2), 73.6 (C^CH),
3365, 1677, 1608, 1515 cmꢁ1; HMRS
~
n
O), 183.5 (C]O) ppm; IR (KBr)
(ESI) [M þ H]þ. Found: 242.0812. Calc. for C14H12NO3þ: 242.0812.
Anal. Calcd. for C14H11NO3: C, 69.70; H, 4.60; N, 5.81. Found: C,
69.47; H, 4.49; N, 5.77.
4.1.2.1. 2-(Phenylthiomethyl)-amino-1,4-naphthoquinone
1
7h. Obtained as a yellow solid (404 mg, 95%); mp: 196e197 ꢀC; H
NMR (300 MHz, CDCl3):
d
¼ 4.62 (s, 2H, CH2), 5.88 (s, 1H, H-6), 7.32e
4.1.1.4. 2-(Benzyloxymethyl)-amino-1,4-naphthoquinone 7d. Obtained
as a yellow solid (191 mg, 45%); mp: 149e150 ꢀC; 1H NMR (300 MHz,
7.33 (m, 3H, H-30, H-40 and H-50), 7.44e7.46 (m, 2H, H-20 and H-60),
7.62e7.65 (m, 1H, H-1 or H-4), 7.73e7.76 (m, 1H, H-1 or H-4), 8.03e
8.04 (m, 1H, H-2 or H-3), 8.09e8.11 (m,1H, H-2 or H-3) ppm. 13C NMR
CDCl3):
d
¼ 4.57 (s, 2H), 4.80 (s, 2H), 6.13 (s,1H, H-6), 7.31e7.36 (m, 5H,
H-20, H-30, H-40, H-50 and H-60), 7.62e7.67 (m, 1H, H-1 or H-4), 7.71e
(75 MHz, CDCl3):
d
¼ 48.4 (CH2), 104.1 (C-6), 126.2 (C-1 and C-4),
7.77 (m, 1H, H-1 or H-4), 8.05e8.11 (m, 2H, H-2 and H-3) ppm; 13C
128.3 (C-40), 129.3 (C-30 and C-50), 130.4 (C-4a), 132.9 (C-20 and C-60),
NMR (75 MHz, CDCl3):
d
¼ 69.7 (CH2), 72.4 (CH2), 104.6 (C-6), 126.2
133.2 (C-8b), 134.7 (C-2 and C-3), 146.1 (C-7), 181.5 (C]O), 183.0 (C]
(C-1 and C-4), 127.8 (C-20 and C-60), 128.0 (C-40), 128.5 (C-30 and C-50),
130.4 (C-4a), 132.3 (C-2 and C-3), 133.0 (C-8b), 134.6 (C-2 and C-3),
O) ppm. IR (KBr)
n
3340, 1677, 1624, 1571 cmꢁ1; HMRS (ESI) [M þ H]þ.
~
Found: 296.0738. Calc. for C17H14NO2Sþ: 296.0740. Anal. Calcd. for

200
A.K. Jordão et al. / European Journal of Medicinal Chemistry 63 (2013) 196e201
C17H13NO2S: C, 69.13; H, 4.44; N, 4.74. Found: C, 69.58; H, 4.84; N,
4.45.
7.60e7.66 (m, 1H, H-1 or H-4), 7.71e7.76 (m, 1H, H-1 or H-4), 8.03e
8.06 (m, 1H, H-2 or H-3), 8.10e8.12 (m, 1H, H-2 or H-3) ppm; 13C
NMR (75 MHz, CDCl3) d 15.4 (CH3), 48.9 (CH2), 104.1 (C-6), 126.2 (C-
4.1.2.2. 2-(2-Tolylthiomethyl)-amino-1,4-naphthoquinone 7i. Obtained
as a yellow solid (195 mg, 46%); mp: 179e180 ꢀC; 1H NMR (300 MHz,
1 and C-4), 126.9 (C-20 and C-60 or C-30 and C-50), 128.7 (C-20 and C-
60 or C-30 and C-50), 130.4 (C-4a), 132.2 (C-40), 133.2 (C-8b), 134.7 (C-
2 and C-3), 140.0 (C-10), 146.0 (C-7), 181.5 (C]O), 182.9 (C]O) ppm;
~
Found: 342.0619. Calc. for C18H16NO2S2þ: 342.0617. Anal. Calcd. for
C18H15NO2S2: C, 63.32; H, 4.43; N, 4.10. Found: C, 63.32; H, 4.51; N,
CDCl3):
d
¼ 2.43 (s, 3H, CH3), 4.58 (s, 2H, CH2), 5.89 (s, 1H, H-6), 7.15e
7.17 (m, 1H, H-50), 7.20e7.23 (m, 2H, H-40 and H-60), 7.39e7.41 (m, 1H,
IR (KBr)
n
3252, 1679, 1624, 1571 cmꢁ1; HMRS (ESI) [M þ H]þ.
H-30), 7.62e7.65 (m, 1H, H-1 or H-4), 7.72e7.76 (m, 1H, H-1 or H-4),
8.02e8.04 (m,1H, H-2 or H-3), 8.10e8.12 (m,1H, H-2 or H-3) ppm; 13
C
NMR (75 MHz, CDCl3):
d
¼ 20.8 (CH3), 47.6 (CH2),104.0 (C-6),126.2 (C-
3.75.
40), 126.8 (C-50), 126.8 (C-1 and C-4), 128.5 (C-60), 130.4 (C-4a), 130.7
(C-30),132.2 (C-10),133.4 (C-8b),134.7 (C-2 and C-3),140.7 (C-20),146.2
4.1.2.7. 2-(Propylthiomethyl)-amino-1,4-naphthoquinone
(C-7), 181.5 (C]O), 183.0 (C]O) ppm; IR (KBr)
3351, 1681, 1620,
7n. Obtained as a yellow solid (262 mg, 70%); mp: 140e141 ꢀC; 1H
~
n
1569 cmꢁ1; HMRS (ESI) [M þ H]þ. Found: 310.0911. Calc. for
NMR (300 MHz, CDCl3):
d
¼ 0.98e1.01 (m, 3H, CH3),1.63e1.66 (m, 2H,
C
18H16NO2Sþ: 310.0896. Anal. Calcd. for C18H15NO2S: C, 69.88; H, 4.89;
H-10), 2.58e2.61 (m, 2H, H-9), 4.33 (s, 2H, CH2), 5.87 (s, 1H, H-6),
7.62e7.64 (m, 1H, H-1 or H-4), 7.72e7.75 (m, 1H, H-1 or H-4), 8.05e
N, 4.53. Found: C, 69.89; H, 5.11; N, 4.17.
8.11 (m, 2H, H-2 and H-3) ppm. 13C NMR (75 MHz, CDCl3):
d
¼ 13.4
4.1.2.3. 2-(4-Tolylthiomethyl)-amino-1,4-naphthoquinone 7j. Obtained
as a yellow solid (165 mg, 39%); mp: 185e186 ꢀC; 1H NMR (300 MHz,
(CH3), 22.9 (C-10), 33.5 (C-9), 44.7 (CH2),103.2 (C-6),126.2 (C-1 and C-
4), 130.4 (C-4a), 133.3 (C-8b), 134.7 (C-2 and C-3), 146.6 (C-7), 18ꢁ1.16
~
n
CDCl3):
d
¼ 2.32 (s, 3H, CH3), 4.56 (s, 2H, CH2), 5.87 (s, 1H, H-6), 7.10e
(C]O), 182.9 (C]O) ppm; IR (KBr)
3278, 1678, 1622, 1571 cm
;
7.14 (m, 1H, H-30 and H-50), 7.33e7.37 (m, 1H, H-20 and H-60), 7.62e
7.65 (m, 1H, H-1 or H-4), 7.72e7.76 (m, 1H, H-1 or H-4), 8.03e8.11
HMRS (ESI) [M þ H]þ. Found: 262.0901. Calc. for C14H16NO2Sþ:
262.0896. Anal. Calcd. for C14H15NO2S: C, 64.34; H, 5.79; N, 5.36.
Found: C, 64.57; H, 5.89; N, 5.25.
(m, 2H, H-2 and H-3) ppm; 13C NMR (75 MHz, CDCl3):
d
¼ 21.1
(CH3), 48.8 (CH2), 104.0 (C-6), 129.2 (C-10), 129.8 (C-1 and C-4), 130.4
(C-4a), 130.7 (C-20 and C-60), 132.2 (C-30 and C-50), 133.2 (C-8b), 134.7
(C-2 and C-3), 138.7 (C-40), 146.1 (C-7), 181.5 (C]O), 182.9 (C]O)
~
4.1.2.8. 2-(Phenylthiomethyl)-amino-3-methyl-1,4-naphthoquinone
7o. Obtained as a yellow solid (124 mg, 30%); mp: 104e105 ꢀC; 1H
ppm; IR (KBr)
n
3413, 1680, 1624, 1572 cmꢁ1; HMRS (ESI) [M þ H]þ.
NMR (300 MHz, CDCl3):
d
¼ 2.19 (s, 3H, CH3), 4.94 (s, 2H, CH2),
Found: 310.0911. Calc. for C18H16NO2Sþ: 310.0896. Anal. Calcd. for
7.26e7.28 (m, 3H, H-30, H-40 and H-50), 7.42e7.44 (m, 2H, H-1 or H-
4), 7.61e7.64 (m, 1H, H-1 and H-4), 7.69e7.72 (m, 1H, H-1 and H-4),
7.99e8.01 (m, 1H, H-2 and H-3), 8.09e8.10 (m, 1H, H-2 and H-3)
C18H15NO2S: C, 69.88; H, 4.89; N, 4.53. Found: C, 69.53; H, 5.11; N, 4.18.
4.1.2.4. 2-((4-Fluorophenylthio)methyl)-amino-1,4-naphthoquinone
ppm; 13C NMR (75 MHz, CDCl3):
d
¼ 11.2 (CH3), 51.9 (CH2), 117.1 (C-
7k. Obtained as a yellow solid (247 mg, 61%); mp: 199e200 ꢀC; 1H
6), 126.2 (C-1 and C-4), 128.3 (C-40), 129.2 (C-30 and C-50), 130.3 (C-
NMR (300 MHz, CDCl3):
d
¼ 4.56 (s, 2H, CH2), 5.88 (s, 1H, H-6),
4a), 132.8 (C-10), 132.9 (C-8b), 133.4 (C-20 and C-60), 134.1 (C-2 and
7.01e7.05 (m, 2H, H-30 and H-50), 7.43e7.47 (m, 2H, H-20 and H-60),
7.63e7.66 (m, 1H, H-1 or H-4), 7.73e7.76 (m, 1H, H-1 or H-4), 8.03e
8.05 (m, 1H, H-2 or H-3), 8.10e8.12 (m, 1H, H-2 or H-3) ppm; 13C
C-3), 144.7 (C-7), 181.9 (C]O), 183.5 (C]O). IR (KBr)
3312, 1662,
~
n
1624, 1574 cmꢁ1; HMRS (ESI) [M þ H]þ. Found: 310.0885. Calc. for
C
18H16NO2Sþ: 310.0896. Anal. Calcd. for C18H15NO2S: C, 69.88; H,
NMR (75 MHz, CDCl3):
d
¼ 49.1 (CH2), 104.3 (C-6), 116.6 (C-30 and C-
4.89; N, 4.53. Found: C, 69.84; H, 4.79; N, 4.27.
50, J ¼ 21.5), 126.3 (C-1 and C-4), 127.9 (C-10), 130.4 (C-4a), 133.2 (C-
8b), 134.9 0(C-2 and C-3), 135.9 (C-20 and C-60, J ¼ 8.8), 146.0 (C-7),
4.2. Microbiological assays
~
n
163.1 (C-4 , J ¼ 249.4), 181.5 (C]O), 182.9 (C]O) ppm; IR (KBr)
3253, 1680, 1623, 1572 cmꢁ1; HMRS (ESI) [M þ H]þ. Found:
The assays were performed according to the Clinical and Labo-
ratory Standards Institute (CLSI) [33]. The antimicrobial disc diffusion
assays included gram-positive (Enterococcus faecalis, S. aureus, S.
aureus ORSA, S. aureus MRSA, S. aureus ATCC 25923, S. epidermidis
ORS and S. haemolyticus ORS) and gram-negative (K. pneumoniae
and P. aeruginosa) bacteria. All strains used in this study were from
clinical isolates from the University Antônio Pedro Hospital.
314.0647. Calc. for
C
17H13FNO2Sþ: 314.0646. Anal. Calcd. for
C
17H12FNO2Sþ: C, 65.16; H, 3.86; N, 4.47. Found: C, 65.51; H, 3.37; N,
4.48.
4.1.2.5. 2-((4-Methoxyphenylthio)methyl)-amino-1,4-
naphthoquinone 7l. Obtained as a yellow solid (412 mg, 89%); mp:
140e141 ꢀC; 1H NMR (300 MHz, CDCl3): 3.78 (s, 3H, CH3), 4.50 (s,
2H, CH2), 5.86 (s, 1H, H-6), 6.84 (d, 2H, J ¼ 5.4, H-20 and H-60), 7.39
(d, 2H, J ¼ 5.4, H-30 and H-50), 7.61e7.65 (m, 1H, H-1 or H-4), 7.72e
7.75 (m, 1H, H-1 or H-4), 8.02e8.04 (m, 1H, H-2 or H-3), 8.09e8.11
The strains were grown briefly at 37 ꢀC in MüellereHinton me-
dia, and 3 mL of a stock solution (5 mg/mL) of each derivative in
dimethyl sulfoxide (DMSO) was placed in Whatman disks. The
inocula used in the growth method were those with turbidity that
was equal to a 0.5 McFarland standard. Filter paper disks, 5 mm in
diameter, were placed on top of the plate containing exponentially
growing plated cultures diluted to 1.0 ꢄ 107 colony-forming units
(CFU/mL). These cultures were subsequently incubated for 18e24 h
at 37 ꢀC. Ciprofloxacin and vancomycin were used as positive con-
trols, and DMSO was used as a negative control. The results were
verified by measuring the inhibitory zones surrounding the disk.
Minimum inhibitory concentration (MIC) assays were per-
formed using the broth macrodilution method. After 5 h of bacterial
growth, the culture was diluted to obtain a concentration of
1.0 ꢄ 105 CFU/mL. Next, each compound was added to reach a final
(m, 1H, H-2 or H-3) ppm; 13C NMR (75 MHz, CDCl3)
d 49.4 (CH2),
55.3 (CH3), 103.4 (C-6), 114.9 (C-30 and C-50), 123.1 (C-10), 126.2 (C-1
and C-4), 130.4 (C-4a), 132.1 (C-20 and C-60), 133.2 (C-8b), 134.7 (C-2
and C-3), 146.1 (C-7), 160.3 (C-40), 181.5 (C]O), 182.9 (C]O) ppm;
~
IR (KBr)
n
3258, 1680, 1624, 1571 cmꢁ1; HMRS (ESI) [M þ H]þ.
Found: 326.0853. Calc. for C18H16NO3Sþ: 326.0845. Anal. Calcd. for
C
18H15NO3S: C, 66.44; H, 4.65; N, 4.30. Found: C, 66.61; H, 4.82; N,
3.93.
4.1.2.6. 2-((4-(Methylthio)phenylthio)methyl)-amino-1,4-
naphthoquinone 7m. Obtained as a yellow solid (380 mg, 78%); mp:
176e177 ꢀC; 1H NMR (300 MHz, CDCl3):
d
¼ 2.46 (s, 3H, CH3), 4.57
concentration ranging from 0.5 to 1024 mg/mL. The mixture was
(s, 2H, CH2), 5.87 (s, 1H, H-6), 7.19 (d, 2H, J ¼ 8.4, H-20 and H-60 or H-
incubated at 37 ꢀC for 18e24 h. The MIC was defined as the lowest
30 and H-50), 7.36 (d, 2H, J ¼ 8.4, H-20 and H-60 or H-30 and H-50),
compound concentration preventing visible bacterial growth. All

A.K. Jordão et al. / European Journal of Medicinal Chemistry 63 (2013) 196e201
201
[7] S. Gafner, J.-L. Wolfender, M. Nianga, H. Stoeckli-Evans, K. Hostettmann,
Phytochemistry 42 (1996) 1315e1320.
strains were tested at least three times, and ciprofloxacin
g/mL) were used as
positive controls.
(MIC ¼ 1
m
g/mL) and vancomycin (MIC ¼ 2
m
[8] M. Tabata, M. Tsukada, H. Fukui, Planta Med. 44 (1982) 234e236.
[9] C.N. Pinto, A.P. Dantas, K.C.G. de Moura, F.S. Emery, P.F. Polequevitch,
M.D. Pinto, S.L. de Castro, A.V. Pinto, Arzneim. Forsc. 50 (2000) 1120e1128.
[10] L. Salmon-Chemin, E. Buisine, V. Yardley, S. Kohler, M.-A. Debreu, V. Landry,
C. Sergheraert, S.L. Croft, R.L. Krauth-Siegel, E. Davioud-Charvet, J. Med. Chem.
44 (2001) 548e565.
Acknowledgments
[11] E.V.M. dos Santos, J.W.M. Carneiro, V.F. Ferreira, Bioorg. Med. Chem. 12 (2004)
87e93.
The authors are grateful to FAPERJ-PRONEX, FAPERJ-PAPD,
CAPES-PNPD, FINEP and CNPq for financial support and fellowships
and would like to thank Universidade Federal do Rio de Janeiro,
Departamento de Química Orgânica, LABEM e Laboratório Multi-
usuário de Espectrometria de Massas for the mass spectra.
[12] J.A. Hickman, Curr. Opin. Genet. Dev. 12 (2002) 67e72.
[13] A. Esteves-Souza, D.V. Figueiredo, A. Esteves, C.A. Camara, M.D. Vargas,
A.C. Pinto, A. Echevarria, Braz. J. Med. Biol. Res. 40 (2007) 1399e1402.
[14] J. Benites, J.A. Valderrama, K. Bettega, R.C. Pedrosa, P.B. Calderon, J. Verrax, Eur.
J. Med. Chem. 45 (2010) 6052e6057.
[15] G.J. Kapadia, M.A. Azuine, V. Balasubramanian, R. Sridhar, Pharmacol. Res. 43
(2001) 363e367.
[16] S. Matsuda, K. Adachi, Y. Matsuo, M. Nukina, Y. Shizuri, J. Antibiot. 62 (2009)
519e526.
Appendix A. Supplementary data
[17] C.G.T. Oliveira, F.F. Miranda, V.F. Ferreira, C.C. Freitas, R.F. Rabello,
J.M. Carballido, L.C.D. Corrêa, J. Braz. Chem. Soc. 12 (2001) 339e345.
[18] J. Barluenga, A.M. Bayón, P. Campos, G. Asensio, E. Gonzalez-Nuñez,
Y.J. Molina, J. Chem. Soc. Perkin. Trans. I (1988) 1631e1636.
[19] L.F. Fieser, J.L. Hartwell, J. Am. Chem. Soc. 57 (1935) 1482e1484.
[20] G. Yoshio, T. Yuichi, JP2008303149, 2008.
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.01.010.
References
[21] S. Donadio, S. Maffioli, P. Monciardini, S. Margherita, D. Jabes, Appl. Microbiol.
Biotechnol. 88 (2010) 1261e1267.
[22] C. Walsh, Nat. Rev. Microbiol. 1 (2003) 65e70.
[23] G. Ebrahim, J. Trop. Pediatr. 56 (2010) 141e143.
[24] T. Luangtongkum, T.Y. Morishita, A.B. El-Tayeb, A.J. Ison, Q. Zhang, J. Clin.
Microbiol. 45 (2007) 590e594.
[1] C.F.J. Franco, A.K. Jordão, V.F. Ferreira, A.C. Pinto, M.C.B.V. de Souza,
J.A.L.C. Resende, A.C. Cunha, J. Braz. Chem. Soc. 22 (2011) 187e193.
[2] D.R. Da Rocha, A.C.G. de Souza, J.A.L.C. Resende, W.C. Santos, E.A. dos Santos,
C. Pessoa, M.O. de Moraes, L.V. Costa-Lotufo, R.C. Montenegro, V.F. Ferreira,
Org. Biomol. Chem. 9 (2011) 4315e4322.
[25] M.A.G.S. Silva, E.F. de Mendonça, K.F.M. Rodrigues, C. Bariani, G.B.L. Silva, Rev.
Odontol. Bras. Cent. 15 (2006) 40e42.
[3] A.F. dos santos, P.A.L. Ferraz, A.V. Pinto, M.C.F.R. Pinto, M.O.F. Goulart,
A.E.G. Sant’Ana, J. Int. Parasitol. 30 (2000) 1199e1202.
[26] S.K. Fridkin, J.C. Hageman, M. Morrison, L.T. Sanza, K. Como-Sabetti,
J.A. Jernigan, K. Harriman, L.H. Harrison, R. Lynfield, M.M. Farley, N. Eng. J.
Med. 352 (2005) 1436e1444.
[27] E. Cabiscol, J. Tamarit, J. Ros, Int. Microbiol. 3 (2000) 3e8.
[28] P.L. Páez, M.C. Becerra, I. Albesa, Fundam. Clin. Pharmacol. 24 (2010) 771e776.
[29] M.N. da Silva, V.F. Ferreira, M.C.B.V. de Souza, Quim. Nova 26 (2003) 407e416.
[30] D.J. Payne, M.N. Gwynn, D.J. Holmes, D.L. Pompliano, Nat. Rev. Drug Discov. 6
(2007) 29e40.
[31] J. Fernebro, Drug Resist. Updates 14 (2011) 125e139.
[32] (a) A.R.M. Coates, Y. Hu, Br. J. Pharmacol. 152 (2007) 1147e1154;
(b) B. Ozer, N. Duran, Y. Onlen, L. Savas, J. Antibiot. 64 (2011) 158.
[33] Clinical and Laboratory Standards Institute, Performance Standards for Anti-
microbial Susceptibility Testing; Twenty-Third Informational Supplement,
CLSI document M100-S21. 32 (2012) 1e188.
[4] (a) V.F. Ferreira, A. Jorqueira, A.M.T. Souza, M.N. da Silva, M.C.B.V. de Souza,
R.M. Gouvea, C.R. Rodrigues, A.V. Pinto, H.C. Castro, D.O. Santos, H.P. Araújo,
S.C. Bourguignon, Bioorg. Med. Chem. 14 (2006) 5459e5466;
(b) A. Jorqueira, R.M. Gouvêa, V.F. Ferreira, M.N. da Silva, M.C.B.V. de Souza,
A.A. Zuma, D.F.B. Cavalcanti, H.P. Araújo, D.O. Santos, S.C. Bourguignon, Par-
asitol. Res. 99 (2006) 429e433;
(c) E.N. da Silva Jr., R.F.S. Menna-Barreto, M.C.F.R. Pinto, R.S.F. Silva,
D.V. Teixeira, M.C.B.V. de Souza, C.A. de Simone, S.L. de Castro, V.F. Ferreira,
A.V. Pinto, Eur. J. Med. Chem. 43 (2008) 1774e1780.
[5] M.J. Teixeira, Y.M. de Almeida, J.R. Viana, J.G. Holanda-Filha, T.P. Rodrigues,
J.R.C. Prata Jr., I.C.B. Côelho, V.S. Rao, M.M.L. Pompeu, Phytother. Res. 15 (2001)
44e48.
[6] E.R. de Almeida, A.A. da Silva-Filho, E.R. dos Santos, C.A.C. Lopes,
J. Ethnopharmacol. 29 (1990) 239e241.