Synthesis, antibacterial and antifungal activities of naphthoquinone derivatives: a structure–activity relationship study

Article abstract of DOI:10.1007/s00044-016-1550-x

The synthesis of 1,4-naphthoquinone derivatives is of great interest since these compounds exhibit strong activity as antimalarial, antibacterial, antifungal and anticancer agents. A series of 50 naphthoquinone derivatives was synthesized and evaluated for antibacterial and antifungal activity against Escherichia coli, Pseudomonas aeruginosa, Enterococcus faecalis, Staphylococcus aureus, Candida krusei, Candida parapsilosis and Cryptococcus neoformans using the broth microdilution method. The Candida species were the most susceptible microorganisms. Halogen derivatives of 1,4-naphthoquinone presented strong activity, e.g., 2-bromo-5-hydroxy-1,4-naphthoquinone, which exhibited inhibition at an MIC of 16?μg/mL in S. aureus, and 2-chloro-5,8-dihydroxy-1,4-naphthoquinone, with an MIC of 2?μg/mL in C. krusei. These compounds showed higher activity against fungi, but the antibacterial activities were very low. The study of structure–activity relationships is very important in the search for new antimicrobial drugs due to the limited therapeutic arsenal.

Full text of DOI:10.1007/s00044-016-1550-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2016) 25:1274–1285
DOI 10.1007/s00044-016-1550-x
ORIGINAL RESEARCH
Synthesis, antibacterial and antifungal activities
of naphthoquinone derivatives: a structure–activity
relationship study
1
2
2
2
•
Juan M. S a´ nchez-Calvo
•
Gara R. Barbero
•
Guillermo Guerrero-V a´ squez
•
Alexandra G. Dur a´ n
3
4
2
2
Mariola Mac ´ı as
•
Manuel A. Rodr ´ı guez-Iglesias
•
Jos e´ M. G. Molinillo
•
Francisco A. Mac ´ı as
Received: 17 November 2015 / Accepted: 26 February 2016 / Published online: 12 April 2016
Ó Springer Science+Business Media New York 2016
Abstract The synthesis of 1,4-naphthoquinone deriva-
tives is of great interest since these compounds exhibit
strong activity as antimalarial, antibacterial, antifungal and
anticancer agents. A series of 50 naphthoquinone deriva-
tives was synthesized and evaluated for antibacterial and
antifungal activity against Escherichia coli, Pseudomonas
aeruginosa, Enterococcus faecalis, Staphylococcus aureus,
Candida krusei, Candida parapsilosis and Cryptococcus
neoformans using the broth microdilution method. The
Candida species were the most susceptible microorgan-
isms. Halogen derivatives of 1,4-naphthoquinone presented
strong activity, e.g., 2-bromo-5-hydroxy-1,4-naphtho-
quinone, which exhibited inhibition at an MIC of 16 lg/
mL in S. aureus, and 2-chloro-5,8-dihydroxy-1,4-naph-
thoquinone, with an MIC of 2 lg/mL in C. krusei. These
compounds showed higher activity against fungi, but the
antibacterial activities were very low. The study of
structure–activity relationships is very important in the
search for new antimicrobial drugs due to the limited
therapeutic arsenal.
Keywords Naphthoquinone Á Antifungal Á Antibacterial Á
Minimum inhibitory concentration Á
Structure–activity relationship
Introduction
Naphthoquinones are natural pigments that are widely
distributed in plants, fungi and some animals (Premalatha
et al., 2012; Zhou et al., 2012; Yang et al., 2013). Naph-
thoquinones are structurally characterized by the presence
of two carbonyl groups at the 1,4-positions and, less fre-
quently, at the 1,2- or 1,3-positions of the naphthalene,
1
&
&
Manuel A. Rodr ´ı guez-Iglesias
manuel.rodrigueziglesias@uca.es
A´ rea de Gesti o´ n Sanitaria Norte de C a´ diz, Hospital de Jerez,
Unidad de Gesti o´ n Cl ´ı nica de Enfermedades Infecciosas y
Microbiolog ´ı a, Ronda de circunvalaci o´ n s/n, 11407 Jerez,
C a´ diz, Spain
Jos e´ M. G. Molinillo
chema.gonzalez@uca.es
2
Grupo de Alelopat ´ı a, Departamento de Qu ´ı mica Org a´ nica,
Juan M. S a´ nchez-Calvo
juma27581@hotmail.com
Instituto de Biomol e´ culas (INBIO), Facultad de Ciencias,
Universidad de C a´ diz, Campus de Excelencia Internacional
Agroalimentario (ceiA3), C/Rep u´ blica Saharaui, n 7, 11510
Puerto Real, C a´ diz, Spain
Gara R. Barbero
gariak14@hotmail.com
3
Guillermo Guerrero-V a´ squez
guillermo.guerrero@univ-nantes.fr
Departamento de Anatomıa Patologica, Biologıa Celular,
´
´
´
Histologıa, Historia de la Ciencia, Medicina Legal y Forense
y Toxicolog ´ı a, Facultad de Medicina, Plaza Fragela, 9, 11003
C a´ diz, Spain
´
Alexandra G. Dur a´ n
alexandra.garcia@uca.es
4
Unidad de Gesti o´ n Cl ´ı nica de Microbiolog ´ı a, Instituto de
Biomol e´ culas (INBIO), Hospital Universitario Puerta del
Mar, Avenida Ana de Viya, 21, 11009 C a´ diz, Spain
Mariola Mac ´ı as
mariola.macias@uca.es
Francisco A. Mac ´ı as
famacias@uca.es
1
23

Med Chem Res (2016) 25:1274–1285
1275
which has ring (A) and ring (B) (Fig. 1). These com-
pounds, when they occur naturally, have hydroxyl and/or
methyl groups as substituents (Glazunov and Berdyshev,
antibacterial and antifungal activities of a number of
naphthoquinone derivatives by these methods. Some
compounds were commercially obtained, and other ones
were synthesized in our laboratory to perform a structure–
activity relationship study. The compounds are numbered
in order to facilitate the discussion of their bioactivity
rather than by their chemical nature.
2
012). They are privileged structures in medicinal chem-
istry due to their characteristics, structural properties and
biological activities on prokaryotic and eukaryotic cells. In
most cases, the biological activity of naphthoquinones is
related to their redox and acid–base properties, both of
which can be modulated synthetically.
The synthesis of novel derivatives of 1,4-naphtho-
quinone is of particular interest since these compounds
exhibit strong action as antimalarial, antibacterial, anti-
fungal and anticancer agents (Koyama, 2010).
Results and discussion
Fifty compounds were screened for activity against bacte-
ria and yeast; 45 of these generated by parallel synthesis,
and 5 purchased for comparison purposes.
The mechanism of action of naphthoquinones could be
due to their properties as oxidizing or dehydrogenation
agents, in a similar way to hydrogen peroxide and super-
oxide radicals (Tran et al., 2004b; Pinto and de Castro,
Chemistry
2
009). This behavior is related to the ability of quinones to
accept one or two electrons to form highly reactive radical
anion intermediates, which are responsible for the oxida-
tive stress observed in the cells (Valderrama et al., 2008).
However, several other mechanisms have been attributed to
quinonoid compounds, including DNA intercalation,
alkylation, induction of DNA strand breaks or the inhibi-
tion of special proteins or enzymes such as topoisomerases
Most of the naphthoquinones were prepared from 47,
which can easily be obtained from 38 by reduction with
tin(II)chloride (Guerrero-V a´ squez et al., 2013). Com-
pounds 39, 40 and 41 were also prepared from 38 by
acetylation and methylation. Similarly, 37 was prepared
using chlorohydroquinone and maleic anhydride rather
than dimethoxybenzene and dichloromaleic anhydride
(Bekaert et al., 1986). Compound 25 was obtained when
the same reaction was carried out using 2-bromo-1,4-
dimethoxybenzene (Scheme 1).
(
Plyta et al., 1998).
Some naphthoquinones, such as juglone (5-hydroxy-1,4-
naphthoquinone), lawsone (2-hydroxy-1,4-naphthoquinone)
and plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone),
are present in plants, and their antibacterial effects on vari-
ous species of aerobic and anaerobic organisms have been
demonstrated (Lim et al., 2006; Sakunphueak and
Panichayupakaranant, 2012; Bhattacharya et al., 2013).
Other naphthoquinones, such as toxins derived from
naphthazarin (5,8-dihydroxy-1,4-naphthoquinone), which
are produced by Fusarium solani, can attack plants, fungi
and bacteria (Rohnert et al., 1998). Alkannin, its enan-
tiomer shikonin and its derivatives are active against Gram-
positive and Gram-negative bacteria such as Staphylococ-
cus aureus, Enterococcus faecalis/faecium, Escherichia
coli and Pseudomonas aeruginosa (Shen et al., 2002; Al-
Mussawi, 2010). Some compounds show strong activity
against fungi such as Candida sp. and filamentous fungi
Oxidation of 47 with potassium superoxide produced
compound 48 in good yield (92 %) (Lewis and Paul, 1977),
whereas air oxidation in the presence of a solution of
sodium hydroxide yielded 1 (93 %) (Lewis and Paul,
1977). Compound 1 was brominated with Br in acetic acid
2
to produce 43 and 44, and acetylation of 43 afforded 46
(Horowska et al., 1988; Takeya et al., 1999; Tandon et al.,
2005). Compound 42 was prepared by following the same
strategy as for the preparation of 43 (Horowska et al.,
1988; Takeya et al., 1999; Tandon et al., 2005).
Permethylation of 47 with dimethyl sulfate and potas-
sium hydride gave 33 (Kawasaki et al., 1988). This com-
pound was used to produce 32 by bromination with NBS
(Bloomer and Zheng, 1998), 34 by formylation with POCl3
in chloroform and 3 by oxidation with ceric ammonium
nitrate (CAN) (Scheme 2) (Terada et al., 1987; Kawasaki
et al., 1988). Bromination of 3 produced 45 (Huot and
Brassard, 1974). Compounds 16 and 17 were obtained by
similar strategies employing bromochloromethane and
potassium carbonate for the formation of the methylene-
dioxy derivatives and NBS for the bromination (Dallaeker
et al., 1983).
(
Pawar et al., 2014). These activities depend on the posi-
tion(s) of the substituents in the naphthoquinone ring
(
Figs. 1, 2).
In most studies, antibacterial and antifungal activities
have been studied with not standardized methods. Best
methods to achieve these activities are based by broth
dilution antibacterial and antifungal susceptibility testing
of bacteria that grow aerobically and yeasts. These methods
are used in all microbiology laboratories around of the
world. For this, the aim of our study was to test the
Compounds 20, 34, 35 and 36 were obtained by the
methodology described for the synthesis of the potent
phytotoxic agent naphthotectone (Guerrero-V a´ squez et al.,
123

1
276
Med Chem Res (2016) 25:1274–1285
O
O
O
OH
O
O
OMe O
OH
O
H C
3
8
1
4
O
O
O
9
O
O
O
(CH2)2
2
3
O
O
O
7
6
B
A
1
0
5
OMe O
OH
O
1
2
3
4
5
6
O
O
O
O
O
H C
H C
H C
H C
H C
3
3
3
3
3
O
O
O
O
O
O
O
O
O
O
O
O
(
CH2)3
(CH )
(CH )
(CH )
(CH )
2 4
2 5
2 6
2 7
O
O
O
O
O
7
8
9
10
11
O
O
O
H C
H C
3
3
H C
3
O
O
O
H C
O
O
3
O
O
O
O
O
(CH2)14
O
(CH2)12
(
CH )
(
CH2)8
2 10
O
O
O
O
O
1
2
13
14
15
OH
O
O
O
O
OMe O
OMe
OMe
OMe OMe
OMe
OMe
O
OMe
HO
Br
O
OMe
O
O
OMe OMe
OMe
2
1
1
9
20
1
8
HO
HO
O
OMe O
OMe
OH OMe
O
OMe
O
OH
O
O
O
Br
OMe
OMe
OMe
OMe
22
23
24
25
Fig. 1 Natural and synthetic naphthoquinones and derivatives tested (1–25)
2
013). Compounds 21–24 were obtained as described for
Compounds 2, 29, 39, 49 and 50 were obtained
from Sigma-Aldrich
the synthesis of speciosins G and P (Guerrero-V a´ squez
et al., 2014). Acyl derivatives of juglone were obtained
using standard conditions; acetylation was carried out
using acetic anhydride and pyridine, and the other 5-acetyl-
juglone derivatives 5–15 were prepared with 4-dimethy-
laminopyridine (DMAP) as base in reactions with the
corresponding acid chloride.
1
13
All derivatives were characterized by H and C nuclear
magnetic resonance (NMR), infrared spectroscopy and
high-resolution mass spectrometry (HRMS). The spectro-
scopic data for the compounds are consistent with the
assigned structures (see ‘‘Experimental’’ section).
1
23

Med Chem Res (2016) 25:1274–1285
1277
O
O
O
O
O
O
OH
OH
OMe OMe
OMe
O
Br
O
OH
OH
O
OMe OMe
2
6
27
28
29
30
31
32
OH
O
OMe OMe
OMe OMe O
OMe OMe
O
OMe OMe OH
Cl
H
OH
OH
O
OMe OMe
OMe OMe
34
OMe OMe
OMe OMe
3
3
35
36
37
OH
OH
O
OAc O
OMe O
OMe O
O
O
O
Cl
Cl
Cl
Cl Cl
Br
Br
Cl
Cl Cl
OH
O
OAc O
39
OMe O
OMe O
OMe O
41
OH
OH
O
43
3
8
40
42
OH
O
OAc O
O
O
OH
O
O
O
O
O
O
Br
Br
Br
Br
OH
OH
OH
O
OMe O
OAc O
OH
OH
4
4
45
46
47
48
49
50
Fig. 2 Naphthoquinone derivatives tested (26–50)
OH
O
OCH3
OH
OH
O
OH
O
O
O
i NaOH 5M, 60ºC, 90min
ii HCl 20%
i AlCl3, NaCl,
ii H2O, HClcc,
SnCl2, HCl 4M,
140 °C,
Cl
Cl
Cl
Cl
O
93%
145-185°C;
80%
O
OH
OH
O
O
OCH3
O
OH
Br
47
1
38
OAc
O
OH
O
Br
Ac2O, H2SO4, 35°C
4%
OH
9
KO2, 18-Crown-6, DMSO (dry)
2%
9
OAc
46
O
OH
O
O
Br2/AcOH, reflux
43
OH
O
4
8
OH
Br
Br
OH
O
44
Scheme 1 General procedure for the preparation of naphthoquinone derivatives
123

1
278
Med Chem Res (2016) 25:1274–1285
OMe OMe
OMe OMe
Br
NBS, CH Cl2
2
4
4%
3
2
OMe OMe
OMe OMe
OH
O
O
Me SO , KH, DMF
CHO
2
4
POCl , DMF, CHCl3
3
0
to 40 °C
9
4%
9
5%
OMe OMe
OMe OMe
OH
4
7
33
34
O
O
OMe
OMe
OMe
O
i Br , CHCl , -5°C Br
2
3
ii Et N, 0°C
CAN/H O, CH CN, 0°C
3
2
3
9
9%
97%
OMe
O
45
3
Scheme 2 Procedure for the preparation of methoxylated derivatives of naphthoquinones
Biological evaluation
susceptible fungus in the study, with an MIC value of 2 lg/
mL. This behavior is significant because C. krusei pos-
sesses an intrinsic resistance to many triazole antifungal
drugs, especially fluconazole, which is the main drug used
in antifungal therapy (Yadav et al., 2012).
All of the compounds were screened by the broth
microdilution method for their antibacterial activity against
two Gram-positive bacteria, E. faecalis and S. aureus, and
two Gram-negative bacteria, E. coli and P. aeruginosa. The
compounds were also evaluated for their in vitro antifungal
activity against Candida krusei, Candida parapsilosis and
Cryptococcus neoformans. We did not test Candida albi-
cans because this one has an antifungal susceptibility
profile very similar to C. parapsilosis. The lowest con-
centrations of the compounds that prevented visible growth
are listed in Table 1.
SAR for naphthoquinone derivatives
In order to carry out a structure–activity relationship study,
a number of naphthoquinone derivatives and related com-
pounds were prepared and tested. These compounds com-
prise
a variety of functionalization and structural
modifications that allowed us to establish structural
requirements for activity.
It was determined that the solvent did not have
antibacterial or antifungal activities against any of the test
microorganisms. Gentamicin, doxycycline and ampho-
tericin B were used as standard drugs, and these were also
tested under similar conditions for comparison as positive
controls. The minimum inhibitory concentrations (MIC) of
the synthesized compounds against highly inhibited
organisms are listed in Table 1.
Influence of hydroxyl and methoxy groups at the C5
and/or C8 positions
Several authors have reported that the presence of hydroxyl
groups in the B ring has a significant effect on activity,
particularly in compounds with hydroxyl groups at the C5
and C8 positions. In these compounds, the antimicrobial
and antifungal activities were twice as high as in those
compounds with only one hydroxyl group (Tran et al.,
2004b; Yakubovskaya et al., 2009a; Hughes et al., 2011).
In our study, naphthazarin (1) showed antimicrobial
activity against E. coli, E. faecalis and S. aureus with MIC
values between 64 and 128 lg/mL. In yeasts, the MIC
values were 32–128 lg/mL. Nevertheless, juglone (2)
Some of the tested compounds were very active against
Candida sp., showed moderate antifungal activity against
C. neoformans and were inactive or less active against all
other test organisms. P. aeruginosa was the least suscep-
tible microorganism as only 6 compounds (4, 6, 7, 37, 38
and 42) exhibited activity at CMIC 64 lg/mL. Only
compound 42 showed moderate antibacterial activity
against S. aureus at MIC 16 lg/mL. C. krusei was the most
1
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Med Chem Res (2016) 25:1274–1285
1279
Table 1 Antimicrobial activity (MIC profiles) of the synthesized compounds
Compounds
Antibacterial activity
E. coli P. aeruginosa
Minimum inhibitory concentration (MIC) (lg/mL)
Antifungal activity
E. faecalis
S. aureus
C. krusei
C. parapsilosis
C. neoformans
1
2
3
4
5
6
7
8
9
1
1
1
3
3
3
4
4
4
4
4
4
4
4
4
5
64
–
–
128
–
128
–
32
32
4
64
128
8
128
–
–
–
–
–
–
16
32
32
16
64
256
512
256
–
128
–
128
–
256
–
64
–
16
8
16
16
8
64
128
256
–
64
256
–
128
–
32
256
–
4
16
64
256
64
128
256
2
64
128
256
128
128
256
4
–
–
–
–
0
1
2
7
8
9
0
1
2
3
4
5
6
7
8
0
–
–
–
–
–
–
–
–
–
–
–
–
–
64
64
64
–
128
128
–
128
64
128
256
128
128
64
–
32
128
32
256
64
16
64
64
256
–
8
8
8
4
4
8
16
64
–
–
8
8
–
–
256
8
–
128
64
64
256
–
128
–
8
8
8
8
16
8
–
4
8
–
256
–
64
8
128
8
128
16
8
–
–
–
–
–
4
16
–
–
–
64
128
–
64
64
0,5
–
–
–
128
0,5
–
16
–
64
–
16
–
Gentamicin
Doxycycline
Amphotericin B
Control
1
–
–
–
4
–
–
–
–
–
–
2
2
4
–
–
–
–
–
–
showed antifungal activity against C. krusei and C. parap-
silosis with MICs of 32 and 128 lg/mL, respectively, while
it was inactive against C. neoformans and all bacteria
tested. Antifungal and antibacterial activities have been
reported previously in these compounds. For example,
naphthazarin was active against E. coli and S. aureus
et al., 2012; Sreelatha et al., 2014a). However, juglone did
exhibit very good activity against the filamentous fungi
Aspergillus niger, Paecilomyces variotii, Trametes versi-
color and Gloeophyllum trabeum (Yang et al., 2009).
These results are consistent with ours, although the activity
against filamentous fungi was not tested in this work.
Methoxylation at the C5 and/or C8 positions converted 1
to 5,8-dimethoxy-1,4-naphthoquinone (3), which was
inactive against bacteria but very active against yeasts. In
these microorganisms, compound 3 had an MIC B 16 lg/
mL, i.e., eight times lower than that of 1. This structural
change is very important, since it demonstrates that the
presence of hydroxyl groups in the naphthoquinone ring
improves the cytotoxicity on normal cells, whereas the
permethylated products of naphthazarin and its derivatives
showed slightly lower cytotoxicity in this respect (Zhou
(
Yakubovskaya et al., 2009b) and other fungi such as
Saccharomyces carlsbergensis (Yakubovskaya et al.,
009b), while Tandon et al. (2004) did not find in vitro
2
antifungal activity against C. albicans, C. neoformans,
Sporothrix schenckii, Trichophyton mentagrophytes, Mi-
crosporum canis and Aspergillus fumigatus. Juglone is one
of the most widely studied naphthoquinones. In some
studies, this compound did not demonstrate good antimi-
crobial activity and it was also inactive against the bacteria
P. aeruginosa, S. aureus and the fungus C. albicans (Pawar
123

1
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Med Chem Res (2016) 25:1274–1285
et al., 2011). This compound was more active than com-
pounds 1 and 2 against the yeasts tested.
modulated by the presence of 1,4-substituents (Tran et al.,
2004a). The presence of two carbonyl groups at the C1 and
C4 positions is essential for antimicrobial activity, since
the loss of both groups makes the molecule inactive
Hydrophilicity versus lipophilicity
(Verma and Hansch, 2004). Moreover, the presence of two
The level of activity may be influenced by excessive
hydrophilicity or lipophilicity, as in compounds that con-
tain substituents with 10 or more carbon atoms (Riffel
et al., 2002). Among the series of compounds tested, from
free keto groups at the C1 and C4 positions leads to a
greater inhibitory activity than at the C1 and C2 positions
(Tran et al., 2004a). In our study, it was found that all
compounds that lack the carbonyl groups at these positions
were inactive on the seven microorganisms tested, i.e.,
compounds 32, 33, 34, 35 and 36. The addition or removal
of other substituents in these inactive compounds did not
improve their antimicrobial activity.
5
-acetyl-juglone (4) to 5-palmitoyl-juglone (15), the
structures progressively changed by an increase by 1 car-
bon atom at the C5 position. This increase in the number of
carbon atoms resulted in the loss of antimicrobial activity.
In bacteria, 5-heptanoyl-juglone (9), with 7 carbons at the
C5 position, was the first compound that was inactive. In
yeast, 5-lauroyl-juglone (13) was inactive and this com-
pound has 12 carbons in the same position. 5-Butanoyl-
juglone (6) was the most active against bacteria and fungi.
The activity of these compounds could be influenced by the
partition coefficient or log P, which is a ratio of concen-
trations of non-ionized compounds between two solutions.
The log P value is used in the study of quantitative struc-
ture–property relationships as a measure of lipophilicity
Halogen-substituted 1,4-naphthoquinones
The addition of halogen groups in the structure of naph-
thoquinone can produce a marked improvement in the
antimicrobial and antifungal activity. The activity of these
compounds has been explained as a short circuiting of the
cell electron transfer normally executed by quinones
(Holmes et al., 1964). The compounds with halogen groups
in the nucleus had the lowest MIC values of all compounds
in our study.
(
Leo et al., 1971). Log P values for the series of com-
pounds tested were between 1.51 (4) and 7.16 (15). MIC
values in the range 4–32 mg/L for yeasts correspond to log
P values between 1.51 (4) and 2.26 (6). Log P values [3
were associated with inactive compounds and low activity
compounds such as 7–15. This trend is consistent with
other compounds in this study, where log P values of \1,
e.g., compound 49 (0.99), and close to or [3, e.g., com-
pounds 32–36, were inactive against all microorganisms
tested.
As far as the chloro-1,4-naphthoquinone derivatives are
concerned, comparison of compound 1, which does not
have substituents, with 2-chloro-5, 8-dihydroxy-1,4-naph-
thoquinone (37) showed increase in antimicrobial and
antifungal activity by a factor of four against S. aureus and
by a factor of sixteen against all yeasts tested. However, the
presence
in
2,3-dichloro-5,8-dihydroxy-1,4-naphtho-
quinone (38) of two chloro-substituents in the A ring led to
a decrease in the antimicrobial and antifungal activity,
except on C. neoformans, which gave the lowest MIC
value (B4 lg/mL) in our study for this microorganism.
These compounds also showed activity against P. aerugi-
nosa, albeit very low in this case (MIC 128 lg/mL).
Methoxylation at C5 and C8, as in the change from 2,3-
dichloro-5,8-dimethoxy-1,4-naphthoquinone (40) to com-
pound 38, did not improve the activity. In general,
methoxylation led to lower antimicrobial activity, although
the activity against Candida remained the same. Chlorina-
tion at positions C6 and C7, as in 6,7-dichloro-5,8-dime-
thoxy-1,4-naphthoquinone (41), led to the loss of activity.
Ethoxylation at the C5 and C8 positions, as in 2,3-dichloro-
5,8-diacethoxy-1,4-naphthoquinone (39), improved the
activity against S. aureus and C. krusei, but the activity
against C. neoformans was four times lower. This finding
demonstrates that ethoxylation improved the activities more
markedly than methoxylation, although the most active
compound against Candida sp. in the series tested was the
monochloro derivative with hydroxyl groups at C5 and C8
(37) (MIC 2 lg/mL).
Number of rings
Number of rings can affect antibacterial and antifungal
activities. So, structures with more than two rings did not
show any antimicrobial activity, including methylene-
dioxy-naphthalene (16), 2-bromo-methylenedioxy-naph-
thalene (17) and 1,4,8-trimethoxy-3-methyl-anthraquinone
(
18). This finding is supported by the results reported by
Tran et al. (2004a) who found that addition of other rings
led to a marked loss of activity. They also showed that
compounds without a quinone structure were less active, as
is the case with our compounds (from 19 to 31), where
structures differed from the 1,4-naphthoquinone core.
Influence of carbonyl groups at the C1 and C4
positions
Previous studies showed that the biological activity of 1,4-
naphthoquinone and its derivatives are affected and/or
1
23

Med Chem Res (2016) 25:1274–1285
1281
Regarding bromo 1,4-naphthoquinone derivatives, the
presence of a bromo substituent at the C2 position, as in
inactive against Gram-negative bacteria and yeasts but
more active against Gram-positive bacteria. This activity
was very poor in general. These results are consistent with
those obtained in a study carried out by Yakubovskaya
et al. (2009b) who found significantly higher MIC values
([100 lg/mL) in E. coli and S. aureus. This behavior could
occur because this hydroxyl derivative exhibited low
cytotoxicity. The low activity of hydroxyl derivatives could
be connected with deprotonation of this group under the
conditions of the biological experiment or when it acts in
cells (Pelageev et al., 2014).
2
-bromo-5,8-dihydroxy-1,4-naphthoquinone (43), improved
the activity against the Gram-positive bacteria and yeasts
tested, albeit to a lesser extent than that observed for the
monochloro derivative. The removal of the hydroxyl group at
C8, as in 2-bromo-5-hydroxy-1,4-naphthoquinone (42),
improved the activity against P. aeruginosa, S. aureus and C.
neoformans, although the activities against other microor-
ganisms assessed were similar. This compound showed the
best antimicrobial activity against S. aureus of all compounds
studied (MIC 16 lg/mL).
Methoxylation of monobromo derivatives at the C5 and
C8 positions, as in 2-bromo-5,8-dimethoxy-1,4-naphtho-
quinone (45), markedly reduced the activity against all
microorganisms studied and gave rise to considerably
higher MIC values (64 lg/mL) compared to compound 43.
However, ethoxylation of this compound gave 2-bromo-
The absence of hydroxyl groups at the C5 and C8
positions in lawsone (49), compared to 48, make this
compound inactive against all microorganisms tested.
Several authors have found similar results on using dif-
ferent methods for the evaluation of antibacterial and
antifungal activities of lawsone, namely the disk diffusion
(Tekin et al., 2015) and broth microdilution methods
(Sreelatha et al., 2014b). This behavior could be attributed
to the chemical structure of lawsone, which is a hydrox-
yquinone that can exist as a 1,2-diketone or a 1,4-quinone.
Structural modification of the phenolic hydroxyl group
could result in a reduction in activity, as observed in our
study (Sreelatha et al., 2014b).
5
,8-diacethoxy-1,4-naphthoquinone (46), which was inac-
tive against bacteria but showed the same activity against
yeast.
Dihalogenation at C2 and C3, as in 2,3-dibromo-5,8-
dihydroxy-1,4-naphthoquinone (44), led to a slight increase
in the antifungal activity but not in the antimicrobial
activity.
Replacement of the hydroxyl group at the C2 position of
49 by a methyl group gave 2-methyl-1,4-naphthalenedione
(or menadione, 50), which has a low activity against bac-
teria but moderate activity against yeast. Compound 50
showed antimicrobial activity, whereas compound 49 was
completely inactive. This is because menadione inhibited
the growth of Candida species by stimulating the produc-
tion of radical oxygen species (Ueno et al., 2008).
In general, halogenated derivatives showed activity
against all microorganisms assessed and they were more
active against Candida sp., especially against C. krusei,
with MIC values between 2 and 8 lg/mL (Ambrogi et al.,
1
970).
Ambrogi et al. observed that chloro derivatives showed
stronger activity than bromo derivatives. Similar behavior
was found in our study, and comparison of compounds 37
and 38 with 42 and 43 shows that the chloro derivatives are
slightly more active against most of the microorganisms
assessed. It has been demonstrated in several studies that
the presence of a Cl group in ring A is essential for anti-
fungal activity (Tandon et al., 2009; Tran et al., 2009).
In summary, chloro derivatives were more active than
bromo derivatives, followed by compounds with methyl
and hydroxyl groups, respectively.
Of these compounds, only 47 had a moderate activity
against yeasts. The antibacterial activity was very low.
Experimental
General
All reagents (obtained from Aldrich Chemical Co.) and
solvents (HPLC grade) were used without further purifi-
cation. NMR spectra were recorded at room temperature on
Other modifications in the ring
of 1,4-naphthoquinone
1
Agilent Inova 500- and 400-MHz spectrometers. The H
1
and C chemical shifts are referenced to the CDCl solvent
3
3
Finally, we decided to study other modifications in structure
of 1,4-naphthoquinone. First, for comparison, the lack of
the double bond in the A ring, as compound 47, enhanced
antifungal activity with MIC B 16 lg/mL, compared to 1,
although 47 was inactive against all bacteria tested.
at d 7.25 and d 77.0 ppm. Melting points were taken on a
H
C
Kofler hot stage apparatus and are uncorrected. General IR
spectra (KBr) were recorded on a PerkinElmer FTIR
Spectrum 1000 spectrophotometer. High-resolution mass
spectra (HRMS) were obtained on a WATERS SYNAPT
G2 mass spectrometer (70 eV). Reactions were monitored
by thin-layer chromatography (TLC) on silica gel (F245
Merck plates).
Addition of a hydroxyl group at C2 of the 1,4-naph-
thoquinone moiety, as in 2,5,8-trihydroxy-1,4-naphtho-
quinone (48), compared to 1, led to a compound that was
123

1
282
Med Chem Res (2016) 25:1274–1285
Procedure for the synthesis of compound 4
J = 1.2, 8.0 Hz, H-6), 6.91 (1H, d, J = 10.3 Hz, H-3),
6
.82 (1H, d, J = 10.3 Hz, H-2), 2.73 (2H, t, J = 7.5 Hz,
0 0
Compound 4 was prepared by acetylation of juglone
according to a literature procedure with modifications
H-2 ), 1.80 (2H, tt, J = 7.5, 7.5 Hz, H-3 ), 1.48 (2H, tt,
13
J = 7.5, 7.4 Hz, H-4 ), 0.98 (3H, t, J = 7.4 Hz, H-5 );
0
0
C
(
Greco et al., 2010). Juglone (50 mg, 1 eq.) was dissolved
in the minimum amount of pyridine. Acetic anhydride
271 lL, 10 eq.) was added dropwise to the solution of
NMR (CDCl , 100 MHz): d = 184.3 (C, C-4), 183.7 (C,
C-1), 172.2 (C, C-1 ), 149.7 (C, C-5), 140.0 (CH, C-2),
3
0
(
137.4 (CH, C-3), 134.9 (CH, C-7), 133.6 (C, C-8a), 129.9
juglone, and the reaction mixture was stirred at room
temperature and monitored by TLC. Saturated aqueous
copper sulfate was added, and liquid–liquid extraction was
carried out to remove the pyridine. The organic layer was
dried over anhydrous Na SO and evaporated under
(CH, C-6), 125.0 (CH, C-8), 123.5 (C, C-4a), 34.0 (CH2,
0
0
0
0
C-2 ), 26.6 (CH , C-3 ), 22.4 (CH , C-4 ), 13.9 (CH , C-5 );
2
2
3
?
HRESIMS m/z (pos) 259.0936 C H O [M ? H]
5 15 4
1
(calcd.: 259.0970).
-O-Heptanoyloxy-1,4-naphthoquinone (9) This com-
pound was obtained as a brown oil in 56 % yield; IR (KBr)
2
4
5
reduced pressure. The crude product was purified by col-
umn chromatography using 10 % AcOEt/hexane as eluent
to give 5-acetyl-1,4-naphthoquinone (40 % yield).
-
1 1
mmax 3079, 2929, 1768, 1667, 1596, 1135 cm ; H NMR
CDCl , 400 MHz): d = 8.03 (1H, dd, J = 1.2, 7.8 Hz,
(
3
H-8), 7.75 (1H, dd, J = 8.0, 7.8 Hz, H-7), 7.36 (1H, dd,
J = 1.2, 8.1 Hz, H-6), 6.92 (1H, d, J = 10.3 Hz, H-3),
Procedure for the synthesis of compounds 5–15
6
.83 (1H, d, J = 10.3 Hz, H-2), 2.72 (2H, t, J = 7.5 Hz,
0 0
These derivatives were obtained according to the method-
ology previously described by Mathew et al. (2010) with
some modifications. These acylation reactions were per-
formed in dichloromethane with juglone (50 mg, 1 eq.) as
the starting material. 4-Dimethylaminopyridine (68.4 lL,
H-2 ), 1.80 (2H, tt, J = 7.5, 7.5 Hz, H-3 ), 1.48–1.31 (6H,
13
m, H-4 , H-5 , H-6 ), 0.90 (3H, t, J = 7.1 Hz, H-7 );
0
0
0
0
C
NMR (CDCl , 100 MHz): d = 184.2 (C, C-4), 183.2 (C,
C-1), 172.1 (C, C-1 ), 149.6 (C, C-5), 139.9 (CH, C-2),
3
0
137.3 (CH, C-3), 134.8 (CH, C-7), 133.5 (C, C-8a), 129.8
3
eq.) and the corresponding acyl chloride (125 lL, 5 eq.)
(CH, C-6), 124.9 (CH, C-8), 123.3 (C, C-4a), 34.2 (CH2,
0
0
0
0
were added at 0 °C. The reaction mixture was stirred at room
temperature and monitored by TLC. After completion of the
reaction, the organic phase was washed with brine, dried
over anhydrous Na SO and concentrated under vacuum.
C-2 ), 31.5 (CH , C-5 ), 28.8 (CH , C-4 ), 24.4 (CH , C-3 ),
2
2
2
0
0
22.5 (CH , C-6 ), 14.0 (CH , C-7 ); HRESIMS m/z (pos)
2
3
?
287.1320 C H O [M ? H] (calcd.: 287.1283).
1
7 19 4
2
4
5
-O-Octanoyloxy-1,4-naphthoquinone (10) This com-
pound was obtained as a brown oil in 49 % yield; IR (KBr)
The resulting mixture was purified by column chromatog-
raphy using 100 % chloroform as eluent to obtain com-
pounds 5–15. New compounds are characterized.
-
1 1
mmax 3038, 2924, 1768, 1668, 1596 cm ; H NMR (CDCl3,
00 MHz): d = 8.03 (1H, dd, J = 1.2, 7.8 Hz, H-8), 7.75
(1H, dd, J = 8.1, 7.8 Hz, H-7), 7.36 (1H, dd, J = 1.2,
4
5-O-Butanoyloxy-1,4-naphthoquinone (6) This com-
pound was obtained as a yellow solid in 78 % yield; mp
8.1 Hz, H-6), 6.92 (1H, d, J = 10.3 Hz, H-3), 6.83 (1H, d,
0
7
2–75 °C; IR (KBr) m
138 cm ; H NMR (CDCl , 400 MHz): d = 8.02 (1H,
3078, 2963, 1753, 1664, 1594,
J = 10.3 Hz, H-2), 2.72 (2H, t, J = 7.5 Hz, H-2 ), 1.80 (2H,
0 0 0
max
-
1
1
1
tt, J = 7.5, 7.5 Hz, H-3 ), 1.48–1.24 (8H, m, H-4 , H-5 ,
3
0
0
0
13
H-6 , H-7 ), 0.89 (3H, t, J = 6.9 Hz, H-8 ); C NMR
dd, J = 1.2, 7.8 Hz, H-8), 7.74 (1H, dd, J = 8.0, 7.8 Hz,
H-7), 7.36 (1H, dd, J = 1.2, 8.0 Hz, H-6), 6.92 (1H, d,
(CDCl , 100 MHz): d = 184.2 (C, C-4), 183.6 (C, C-1),
172.1 (C, C-1 ), 149.6 (C, C-5), 139.9 (CH, C-2), 137.3 (CH,
3
0
J = 10.3 Hz, H-3), 6.82 (1H, d, J = 10.3 Hz, H-2), 2.71
0
(
2H, t, J = 7.5 Hz, H-2 ), 1.84 (2H, qt, J = 7.5, 7.4 Hz,
C-3), 134.7 (CH, C-7), 133.5 (C, C-8a), 129.8 (CH, C-6),
0
0
0
13
H-3 ), 1.08 (3H, t, J = 7.4 Hz, H-4 ); C NMR (CDCl3,
124.9 (CH, C-8), 123.4 (C, C-4a), 34.2 (CH , C-2 ), 31.7
2
0
0
0
1
00 MHz): d = 184.0 (C, C-4), 183.4 (C, C-1), 171.7 (C,
(CH , C-6 ), 30.9 (CH , C-4 ), 29.0 (CH , C-5 ), 24.4 (CH ,
2
2
2
2
0
0
0
0
C-1 ), 149.4 (C, C-5), 139.7 (CH, C-2), 137.1 (CH, C-3),
C-3 ), 22.6 (CH , C-7 ), 14.1 (CH , C-8 ); HRESIMS m/z
2
3
?
1
34.6 (CH, C-7), 133.3 (C, C-8a), 129.6 (CH, C-6), 124.7
0
(pos) 301.1422 C H O [M ? H] (calcd.: 301.1440).
18 21 4
(
CH, C-8), 123.2 (C, C-4a), 35.8 (CH , C-2 ), 17.8 (CH ,
2
2
5
-O-Nonanoyloxy-1,4-naphthoquinone (11) This com-
pound was obtained as a yellow solid in 64 % yield; mp
6–61 °C; IR (KBr) m 3079, 2926, 1767, 1667, 1595,
0
0
C-3 ), 13.5 (CH , C-4 ); HRESIMS m/z (pos) 245.0788
3
?
C H O [M ? H] (calcd.: 245.0814).
1
4 13 4
5
max
-
1 1
5-O-Pentanoyloxy-1,4-naphthoquinone (7) This com-
pound was obtained as a brown oil in 90 % yield; IR (KBr)
1133 cm ; H NMR (CDCl , 400 MHz): d = 8.02 (1H,
3
dd, J = 1.3, 7.8 Hz, H-8), 7.73 (1H, dd, J = 8.0, 7.8 Hz,
H-7), 7.36 (1H, dd, J = 1.3, 8.0 Hz, H-6), 6.91 (1H, d,
J = 10.3 Hz, H-3), 6.82 (1H, d, J = 10.3 Hz, H-2), 2.72
-
1 1
mmax 3079, 2957, 1762, 1669, 1594, 1138 cm ; H NMR
CDCl , 400 MHz): d = 8.02 (1H, dd, J = 1.2, 7.8 Hz,
H-8), 7.73 (1H, dd, J = 8.0, 7.8 Hz, H-7), 7.36 (1H, dd,
(
3
0
(2H, t, J = 7.5 Hz, H-2 ), 1.80 (2H, tt, J = 7.5, 7.5 Hz,
1
23

Med Chem Res (2016) 25:1274–1285
1283
0
0
0
13
0
0
0
H-3 ), 1.48–1.24 (10 H, m, H-4 , H-5 , H-6 , H-7 , H-8 ),
2.42 mmol) followed by AlCl (644 mg, 4.83 mmol) in
3
0
0
.87 (3H, t, J = 6.9 Hz, H-9 );
C NMR (CDCl3,
small portions over 2 h 30 min. The reaction mixture was
stirred at 0 °C and warmed slowly to room temperature and
then stirred for 72 h. The reaction mixture was poured into
1
00 MHz): d = 184.2 (C, C-4), 183.6 (C, C-1), 172.1 (C,
0
C-1 ), 149.6 (C, C-5), 139.9 (CH, C-2), 137.2 (CH, C-3),
1
34.7 (CH, C-7), 133.5 (C, C-8a), 129.8 (CH, C-6), 124.8
0
ice water, and then, aqueous NaHCO 5 % was added until
3
(
CH, C-8), 123.3 (C, C-4a), 34.2 (CH , C-2 ), 31.8 (CH ,
2
the solid had dissolved. The resulting mixture was concen-
trated under reduced pressure to remove dichloroethane. The
aqueous solution was acidified to pH 1 by addition of 5 %
HCl. The mixture was stirred for 15 min and extracted with
ethyl acetate (3 9 20 mL), and the organic extracts were
combined and washed with water and brine and dried over
anhydrous Na SO . The crude material was purified by silica
2
0
0
0
C-7 ), 29.23 (CH , C-4 ), 29.13 (CH , C-6 ), 29.11 (CH ,
2
2
2
0
0
0
0
C-5 ), 24.4 (CH , C-3 ), 22.6 (CH , C-8 ), 14.1 (CH , C-9 );
3
2
2
?
HRESIMS m/z (pos) 315.1586 C H O [M ? H]
19 23 4
(
calcd.: 315.1596).
5-O-Decanoyloxy-1,4-naphthoquinone (12) This com-
pound was obtained as a brown solid in 84 % yield; mp
2
4
gel column chromatography, eluting with hexane/ethyl
acetate (1:1) with 0.5 % acetic acid to afford 25 as a yellow
solid (348.4 mg).
4
4–49 °C; IR (KBr) m
3076, 2924, 1766, 1667, 1594,
max
-
1 1
1
137 cm ; H NMR (CDCl , 400 MHz): d = 8.02 (1H,
3
dd, J = 1.3, 7.9 Hz, H-8), 7.73 (1H, dd, J = 7.9, 7.9 Hz,
0
0
0
H-7), 7.36 (1H, dd, J = 1.3, 7.9 Hz, H-6), 6.91 (1H, d,
4 -(4-bromo-2,5-dimethoxyphenyl)-4 -oxobut-2 -enoic acid
(25) This compound was obtained as a yellow solid in
48 % yield; mp 153–154 °C; IR (KBr) mmax 3082, 3013,
2944, 1712, 1658, 1595, 1488, 1388, 1289, 1269,
J = 10.3 Hz, H-3), 6.82 (1H, d, J = 10.3 Hz, H-2), 2.72
0
(
2H, t, J = 7.7 Hz, H-2 ), 1.80 (2H, tt, J = 7.7, 7.7 Hz,
0
0
0
0
0
0
H-3 ), 1.40–1.24 (12 H, m, H-4 , H-5 , H-6 , H-7 , H-8 ,
13
H-9 ), 0.87 (3H, t, J = 6.7 Hz, H-10 ); C NMR (CDCl3,
0
0
-1 1
1215 cm ; H NMR (Pyridine-d , 400 MHz): d = 8.23
´
(1H, d, J = 15.6, H-3), 7.47 (1H, s, H-6), 7.46 (1H, s, H-3),
´
7.31 (1H, d, J = 15.6, H-2), 5.72 (1H, s, COOH), 3.75 (3H,
5
1
00 MHz): d = 184.2 (C, C-4), 183.6 (C, C-1), 172.1 (C,
0
C-1 ), 149.6 (C, C-5), 139.9 (CH, C-2), 137.2 (CH, C-3),
1
3
1
34.7 (CH, C-7), 133.5 (C, C-8a), 129.8 (CH, C-6), 124.8
0
s, C-2–O–CH ), 3.74 (3H, s, C-5–O–CH ); C NMR
3 3
0
(
CH, C-8), 123.3 (C, C-4a), 34.2 (CH , C-2 ), 31.8 (CH ,
2
(Pyridine-d , 100 MHz): d = 191.2 (C, C-4 ), 169.0 (C,
2
5
0
0
0
0
0
C-8 ), 29.41 (CH , C-6 ), 29.28 (CH , C-7 ), 29.24 (CH ,
2
C-1 ), 154.0 (C, C-5), 153.0 (C, C-2), 140.8 (CH, C-3 ),
0
2
2
0
0
0
C-4 ), 29.13 (CH , C-5 ), 24.4 (CH , C-3 ), 22.6 (CH ,
2
133.4 (CH, C-2 ), 128.2 (C, C-4), 118.9 CH, C-6), 118.7
2
2
0
0
C-9 ), 14.1 (CH , C-10 ); HRESIMS m/z (pos) 329.1787
3
(C, C-1), 113.9 (CH–C-3), 57.2 (CH , C-2–O–CH ), 57.1
3
3
?
C H O [M ? H] (calcd.: 329.1753).
2
0
25
4
(CH , C-5–O–CH ); HRESIMS m/z (pos) 314.9861,
3 3
?
16.9842 C H O Br [M] (calcd.: 314.9864).
3
12 12 5
5
-O-Palmitoyljuglone (15) This compound was obtained
as a yellow solid in 49 % yield; mp 66–69 °C; IR (KBr) m
max
-
1 1
3
076, 2921, 1760, 1664, 1590, 1140 cm ; H NMR (CDCl ,
3
Biological evaluation
4
00 MHz): d = 8.03 (1H, dd, J = 1.1, 7.8 Hz, H-8), 7.74
1H, dd, J = 8.0, 7.8 Hz, H-7), 7.36 (1H, dd, J = 1.1, 8.0 Hz,
(
The compounds were subjected to an evaluation of their
level activity using the broth microdilution method to
estimate the minimal inhibitory concentration (MIC)
according to the recommendations of (CLSI 2008; PA,
2009).
H-6), 6.92 (1H, d, J = 10.3 Hz, H-3), 6.83 (1H, d,
0
J = 10.3 Hz, H-2), 2.72 (2H, t, J = 7.6 Hz, H-2 ), 1.81 (2H,
0
0
0
0
0
0
m, H-3 ), 1.48–1.25 (24 H, m, H-4 , H-5 , H-6 , H-7 , H-8 ,
13
0
0
0
0
H-9 , H-10 , H-11 ), 0.87 (3H, t, J = 6.4 Hz, H-12 );
C
NMR (CDCl , 100 MHz): d = 184.2 (C, C-4), 183.2 (C,
C-1), 172.1 (C, C-1 ), 149.6 (C, C-5), 139.9 (CH, C-2), 137.2
The microorganisms assessed were E. coli ATCC
25922, P. aeruginosa ATCC 27853, E. faecalis ATCC
29212, S. aureus, C. krusei ATCC 6258, C. parapsilosis
ATCC 22019 and C. neoformans.
3
0
(
CH, C-3), 134.7 (CH, C-7), 133.5 (C, C-8a), 129.8 (CH,
0
C-6), 124.8 (CH, C-8), 123.3 (C, C-4a), 34.2 (CH , C-2 ), 31.9
2
0
0
0
(
(
(
CH , C-10 ), 29.58 (CH , C-6 ), 29.56 (CH , C-8 ), 29.4
2
A series of twofold dilutions from 1024 to 2 lg/mL
(dissolved in DMSO up to 2 % final DMSO concentration)
were prepared in a 96-well sterile microplate.
2
2
0
0
0
CH , C-7 ), 29.24 (CH , C-4 ), 29.31 (CH , C-5 ), 29.28
2
2
2
0
0
0
CH , C-9 ), 29.14 (CH , C-4 ), 24.4 (CH , C-3 ), 22.6 (CH ,
2
2
2
2
0
0
C-11 ), 14.1 (CH , C-12 ); HRESIMS m/z (pos) 357.2024
3
For bacteria In each well was introduced 50 lL of the
dilution compound in Muller Hinton broth. Subsequently,
?
C H O [M ? H] (calcd.: 357.2060).
2
2 29 4
6
50 lL of an inoculum containing 5 9 10 CFU was added
Procedure for the synthesis of compound 25
to each well. Gentamicin and doxycycline were used as the
antibacterial references. The microplate was incubated at
37 °C for 24 h.
To a stirred solution of commercially available 1-bromo-2,5-
dimethoxybenzene (500 mg, 2.3 mmol) in dichloroethane
3
For yeasts 100 lL of an inoculum containing 1 9 10 –
3
5 9 10 CFU was added to each well. In each well was
(
15 mL) at 0 °C was added maleic anhydride (237 mg,
123

1
284
Med Chem Res (2016) 25:1274–1285
introduced 100 lL of the dilution compound in RPMI 1640
with L-glutamine and without sodium bicarbonate (Sigma-
Aldrich). Amphotericin B was used as the antifungal ref-
erence. The microplate was incubated at 37 °C for 48 h in
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Several known and new naphthoquinone derivatives have
been tested for antibacterial and antifungal activity. In
bacteria, compound 42 displayed the highest activity
against S. aureus at MIC 16 lg/mL. Compound 37 was the
most active against Candida spp. with MIC of 2–4 lg/mL,
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decrease in the activity until they became inactive. The two
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additional rings in the structure or the lack of either ring
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groups at the C-5 and/or C-8 positions is critical for
antibacterial activity. The replacement of these groups with
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