Synthesis, in vitro antifungal activity and molecular modeling studies of new Mannich bases derived from lawsone

Article abstract of DOI:10.5935/0103-5053.20160104

Hydroxynaphthoquinones such as lawsone (2-hydroxy-1,4-naphthoquinone) have proven to be effective antifungal agents. These compounds were tested for antifungal activity against yeast standard and clinical strains by the broth microdilution method. Among the synthetic lawsone derivatives, 2-hydroxy-3-((2-hydroxyphenyl)(pyrrolidin-1-yl)methyl)naphthalene-1,4-dione, 2-hydroxy-3-(((4-nitrophenyl)amino)(phenyl)methyl)naphthalene-1,4-dione and 2-hydroxy-3-((2-hydroxyphenyl)((4-nitrophenyl)amino)methyl)naphthalene-1,4-dione showed high activity against Candida albicans ATCC 10231, with minimal inhibitory concentrations (MICs) and minimal fungicidal concentrations (MFCs) ranging from 20 to 330 and from 80 to 330 μg mL^-1, respectively. Moreover, they also showed a mechanism of action on exogenous ergosterol. Therapeutic concentrations (CC₅₀) of 2-hydroxy-3-((2-hydroxyphenyl)(pyrrolidin-1-yl)methyl) naphthalene-1,4-dione, 2-hydroxy-3-(((4-nitrophenyl)amino)(phenyl)methyl)naphthalene-1,4-dione and 2-hydroxy-3-((2-hydroxyphenyl)((4-nitrophenyl)amino)methyl)naphthalene-1,4-dione were 52.81, 52.58 and 85.94 μg mL^-1, respectively, which can be considered moderate or low. In addition, docking studies showed that these compounds had similar binding energy to standard ketoconazole, which are recognized as the molecular target by van der Waals interactions. Furthermore, they are under Lipinski's rule of 5 with a druglikeness score better than ketoconazole and nystatin. These findings suggest that 2-hydroxy-3-((2-hydroxyphenyl)(pyrrolidin-1-yl)methyl) naphthalene-1,4-dione, 2-hydroxy-3-(((4-nitrophenyl)amino)(phenyl)methyl)naphthalene-1,4-dione and 2-hydroxy-3-((2-hydroxyphenyl)((4-nitrophenyl)amino)methyl)naphthalene-1,4-dione have potential as leading compounds against human fungal infections.

Full text of DOI:10.5935/0103-5053.20160104

http://dx.doi.org/10.5935/0103-5053.20160104
J. Braz. Chem. Soc., Vol. 27, No. 11, 2127-2140, 2016.
Printed in Brazil - ©2016 Sociedade Brasileira de Química
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Article
Synthesis, in vitro Antifungal Activity and Molecular Modeling Studies of New
Mannich Bases Derived from Lawsone
João F. Allochio Filho,a Larissa L. Roldi,a Maicon Delarmelina,a Rodolfo G. Fiorot,a
Jessica T. Andrade,b Álan A. Aleixo,b Rafaella S. Carvalho,b Marcelo G. F. Araújo,b
Jaqueline M. S. Ferreira,b Alex G. Taranto,c Wanderson Romãod and Sandro J. Greco*,a
aLaboratório de Síntese Orgânica & Medicinal, Departamento de Química, Universidade Federal
do Espírito Santo, Avenida Ferrari, 514, Goiabeiras, 29075-910 Vitória-ES, Brazil
bLaboratório de Microbiologia and cLaboratório de Química Farmacêutica Medicinal,
Universidade Federal de São João Del-Rei, Campus Centro Oeste, Rua Sebastião Gonçalves
Coelho, 400, Chanadour, 35501-296 Divinópolis-MG, Brazil
dLaboratório de Petroleômica, Departamento de Química, Universidade Federal do Espírito Santo,
Avenida Ferrari, 514, Goiabeiras, 29075-910 Vitória-ES, Brazil
Hydroxynaphthoquinones such as lawsone (2-hydroxy-1,4-naphthoquinone) have proven to
be effective antifungal agents. These compounds were tested for antifungal activity against yeast
standard and clinical strains by the broth microdilution method. Among the synthetic lawsone
derivatives, 2-hydroxy-3-((2-hydroxyphenyl)(pyrrolidin-1-yl)methyl)naphthalene-1,4-dione,
2-hydroxy-3-(((4-nitrophenyl)amino)(phenyl)methyl)naphthalene-1,4-dione and 2-hydroxy-3-
((2-hydroxyphenyl)((4-nitrophenyl)amino)methyl)naphthalene-1,4-dione showed high activity
against Candida albicans ATCC 10231, with minimal inhibitory concentrations (MICs) and
minimal fungicidal concentrations (MFCs) ranging from 20 to 330 and from 80 to 330 μg mL-1,
respectively. Moreover, they also showed a mechanism of action on exogenous ergosterol.
Therapeutic concentrations (CC50) of 2-hydroxy-3-((2-hydroxyphenyl)(pyrrolidin-1-yl)methyl)
naphthalene-1,4-dione, 2-hydroxy-3-(((4-nitrophenyl)amino)(phenyl)methyl)naphthalene-1,4-
dione and 2-hydroxy-3-((2-hydroxyphenyl)((4-nitrophenyl)amino)methyl)naphthalene-1,4-dione
were 52.81, 52.58 and 85.94 μg mL-1, respectively, which can be considered moderate or low. In
addition, docking studies showed that these compounds had similar binding energy to standard
ketoconazole, which are recognized as the molecular target by van der Waals interactions.
Furthermore, they are under Lipinski’s rule of 5 with a druglikeness score better than ketoconazole
and nystatin. These findings suggest that 2-hydroxy-3-((2-hydroxyphenyl)(pyrrolidin-1-yl)methyl)
naphthalene-1,4-dione, 2-hydroxy-3-(((4-nitrophenyl)amino)(phenyl)methyl)naphthalene-1,4-
dione and 2-hydroxy-3-((2-hydroxyphenyl)((4-nitrophenyl)amino)methyl)naphthalene-1,4-dione
have potential as leading compounds against human fungal infections.
Keywords: lawsone, Mannich bases, antifungal activity, docking
Introduction
quinone, lawsone can be used to perform Mannich reaction3
and the resulting Mannich bases show particular interest
because of their biological activities.4
Naphthoquinone derivatives possess continuous
interest as potential therapeutic agents. Among them,
hydroxynaphthoquinones such as lawsone (2-hydroxy-1,4-
naphthoquinone), have proven to be effective due to their
chemical and pharmacological properties.1 The antifungal
activity of certain naphthoquinone and lawsone derivatives
has been reported from in vitro studies (Scheme 1).2 As a
Candida infections have always posed a heavy burden
on public health. The incidence of candidiasis has been
increasing, which may be attributable to the growing
numbers of immunocompromised patients.5,6 In addition,
vulvovaginal candidiasis (VVC) is a significant problem
affecting 75% of all women at least once during their
lifetime, and Candida albicans is the causative agent in
more than 90% of these infections.7 However, besides
*e-mail: sandro.greco@ufes.br

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Synthesis, in vitro Antifungal Activity and Molecular Modeling Studies of New Mannich Bases
J. Braz. Chem. Soc.
Scheme 1. Naphthoquinones with antifungal activity.
C. albicans species, an increased incidence has been
observed of invasive candidiasis caused by non-albicans
Candida species, such as Candida glabrata, Candida
tropicalis, Candida krusei or Candida parapsilosis.8 Many
problems remain to be solved for most of the antifungal
drugs available. Mechanisms of resistance to azole drugs
have been described for C. albicans.9 Thus, it has become
necessary to develop new drugs capable of effectively
combating these types of infection.
molecular interactions between these compounds and the
molecular target. These data suggest a lead optimization
process to search for a second generation of antifungal
lawsone-based drugs.
Experimental
General information
In this work, we evaluated the antifungal activity of
synthetic derivative compounds of lawsone, which we
synthesized for the first time, and tested these against
several clinically important microorganisms. This way,
we searched for thirteen new enolamines 4-8, 10-14, 16,
17 and 23, which contain 2-phenyl-2H-1,2,3-triazole or
substituted and non-substituted phenyl groups, themselves
synthesized using a multicomponent Mannich reaction
from lawsone.3 These compounds were also tested against
baby hamster kidney (BHK) cells to study toxicity effects.
Moreover, molecular modeling tools were used to highlight
properties used in drug design,10 for example druglikeness.
In addition, docking studies11-13 described the main
All solvents and reagents were commercially purchased
and were used without any purification.
Melting points were determined using Fisatom 430D
equipment. Infrared (IR) spectra were recorded on a
FTLA 2000-102-ABB BOMEM Fourier transform (FT)
IR spectrophotometer with KBr technique and on a Perkin
Elmer Spectrum 400 FTIR spectrometer model using
attenuated total reflectance (ATR) technique. The 1H and 13C
nuclear magnetic resonance (NMR) spectra were obtained
on aVarianVNMRS spectrometer model 400 (400 MHz for
1H NMR) using CDCl3 or dimethyl sulfoxide (DMSO)-d6
as solvents and tetramethylsilane (TMS) as the internal
standard.

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Allochio Filho et al.
2129
Mass spectra (MS) were recorded on ultra-high
resolution and accuracy mass spectrometer (model
9.4 T Solarix, Bruker Daltonics), operated in both
ionization modes: positive and negative electrospray
ionization Fourier transform ion cyclotron resonance
mass spectrometry (ESI(+) and ESI(–)-FT-ICR MS,
respectively). FT-ICR MS spectra were acquired with
resolving power of m / Dm50% ca. 500000, in which Dm50%
is the full peak width at half-maximum peak height of m/z
400 and a mass accuracy of less than 1 ppm. It provides
an unambiguous molecular formula assignment for singly
charged molecular ions such as [M – H]+ or [M + H]– and
double bound equivalents (DBE) values.
ν
max / cm-1 3343, 3064, 2983, 2874, 1680, 1587, 1531, 1272;
1H NMR (400 MHz, DMSO-d6) d 1.93 (m, 4H, CH2), 3.15
(br s, 4H, NCH2), 5.48 (s, 1H, CH), 7.25 (m, 1H, Ar–H),
7.32 (m, 2H, Ar–H), 7.54 (td, 1H, J 7.5, 1.0 Hz, Naph–H),
7.67 (m, 3H, 2Ar–H and 1H, Naph–H), 7.78 (dd, 1H, J 7.5,
1.0 Hz, Naph–H), 7.89 (dd, 1H, J 7.5, 1.0 Hz, Naph–H),
10.48 (br s, 1H, OH); 13C NMR (101 MHz, DMSO-d6)
d 23.7, 53.9, 67.6, 113.6, 125.5, 125.7, 128.3, 128.4,
128.8, 131.2, 131.9, 134.2, 135.1, 139.1, 170.6, 178.2,
184.9; ESI(+)-FT-ICR-MS calcd. for C21H20NO3+ [M + H]+:
334.14377; found: 334.13985 (DBE = 13); [2M + H]+:
667.2803; found: 667.2790; [2M + Na]+: 689.2622; found:
689.2609.
Synthesis of lawsone derivatives 4-8, 10-14, 17 and 23 -
2-Hydroxy-3-(naphthalen-1-yl(pyrrolidin-1-yl)methyl)
naphthalene-1,4-dione (6)
general procedure
Compound 6 was obtained as an orange solid (38%);
m.p. decomposition at 185 oC; IR (ATR) νmax / cm-1 3337,
3042, 2868, 1678, 1583, 1532, 1275; 1H NMR (400 MHz,
DMSO-d6) d 1.92 (m, 4H, CH2), 3.29 (m, 4H, NCH2), 6.37
(s, 1H, CH), 7.51 (m, 4H, 3Ar-H and 1H, Naph–H), 7.66
(td, 1H, J 7.4, 1.0 Hz, Naph–H), 7.76 (dd, 1H, J 7.4, 1.0 Hz,
Naph–H), 7.84 (d, 1H, J 8.3 Hz,Ar–H), 7.89 (m, 2H, 1Ar–H
and 1H, Naph–H), 8.25 (d, 1H, J 7.4 Hz,Ar–H), 8.59 (d, 1H,
J 8.3 Hz,Ar–H), 10.28 (br s, 1H, OH); 13C NMR (101 MHz,
DMSO-d6) d 23.8, 54.1, 63.3, 112.9, 124.6, 125.5, 125.6,
125.7, 126.1, 126.7, 128.4, 128.9, 131.2, 131.3, 131.9,
133.5, 134.2, 134.5, 135.3, 171.1, 178.4, 184.9; ESI(+)-
FT-ICR-MS calcd. for C25H23NO3+ [M + H]+: 384.15550;
found: 384.15982 (DBE = 16).
A mixture of the aldehyde (1.2 eq) and amine (1.1 eq)
in ethanol was stirred during a few minutes. After this
time, lawsone (1.0 eq) was added to the solution and the
reaction was stirred for 24 h at room temperature in the
dark. The solid formed was filtered under vacuum and
washed with cold ethanol. Recrystallization with ethanol
or ethyl acetate:hexane 1:3 was performed for the impure
compounds. Experimental protocols were initially tested
as described in previous reports.3,14,15
2-Hydroxy-3-((2-hydroxyphenyl)(pyrrolidin-1-yl)methyl)
naphthalene-1,4-dione (4)
Compound 4 was obtained as a red crystalline solid
(77%); m.p. decomposition at 146 oC; IR (ATR) νmax / cm-1
3595, 3088, 2977, 2884, 1685, 1579, 1505, 1271; 1H NMR
(400 MHz, DMSO-d6) d 1.95 (br s, 4H, CH2), 3.18 (br s,
4H, NCH2), 5.82 (s, 1H, CH), 6.78 (td, 1H, J 8.0, 1.0 Hz,
Ar–H), 6.82 (dd, 1H, J 8.0, 1.0 Hz, Ar–H), 7.10 (td, 1H,
J 8.0, 1.0 Hz, Ar–H), 7.60 (td, 1H, J 8.0, 1.0 Hz, Ar–H),
7.67 (td, 1H, J 7.5, 1.0 Hz, Naph–H*), 7.72 (td, 1H, J 7.5,
1.0 Hz, Naph–H), 7.83 (dd, 1H, J 7.5, 1.0 Hz, Naph–H),
7.92 (dd, 1H, J 7.5, 1.0 Hz, Naph–H), 10.36 (sl, 2H, OH);
13C NMR (101 MHz, DMSO-d6) d 22.0, 22.5, 43.8, 52.0,
59.8, 112.5, 115.2, 118.2, 123.4, 123.9, 124.2, 27.9, 128.1,
129.9, 130.2, 132.7, 133.3, 153.8, 169.6, 177.7, 183.0;
2-Hydroxy-3-((4-nitrophenyl)(pyrrolidin-1-yl)methyl)
naphthalene-1,4-dione (7)
Compound 7 was obtained as an orange solid (46%);
m.p. 186-188 oC; IR (ATR) νmax / cm-1 3059, 2863, 1686,
1586, 1532, 1343, 1275; 1H NMR (400 MHz, DMSO-d6)
d 1.94 (m, 4H, CH2), 3.20 (br s, 4H, NCH2), 5.71 (s,
1H, CH), 7.55 (td, 1H, J 7.5, 1.0 Hz, Naph–H), 7.68 (td,
1H, J 7.5, 1.0 Hz, Naph–H), 7.78 (d, 1H, J 7.5, 1.0 Hz,
Naph–H), 7.90 (d, 3H, J 8.7 Hz, 2Ar–H and 1H, Naph–H),
8.20 (d, 2H, J 8.7 Hz, Ar–H), 10.46 (br s, 1H, OH);
13C NMR (101 MHz, DMSO-d6) d 23.8, 54.2, 66.6, 112.4,
124.0, 125.6, 125.9, 129.1, 131.4, 131.9, 134.2, 135.1,
146.0, 146.6, 147.2, 170.8, 178.1, 184.6; ESI(–)-FT-ICR-
MS calcd. for C21H19N2O5– [M – H]–: 377.11430; found:
377.11413 (DBE = 14).
+
ESI(+)-FT-ICR-MS calcd. for C21H20NO4 [M + H]+:
350.13868; found: 350.13476 (DBE = 13); [2M + Na]+:
721.2520; found: 721.2528. *Naph–H: naphthoquinone
hydrogens.
2-Hydroxy-3-(phenyl(pyrrolidin-1-yl)methyl)naphthalene-
1,4-dione (5)
2-Hydroxy-3-((4-methoxyphenyl)(pyrrolidin-1-yl)methyl)
naphthalene-1,4-dione (8)
Compound 5 was obtained as a red crystalline solid
(32%); m.p. 177-179 C with decomposition; IR (ATR)
Compound 8 was obtained as an orange solid (44%);
m.p. 181-183 oC; IR (ATR) νmax / cm-1 3068, 2954, 2835,
o

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Synthesis, in vitro Antifungal Activity and Molecular Modeling Studies of New Mannich Bases
J. Braz. Chem. Soc.
1
1681, 1586, 1538, 1252, 1233; H NMR (400 MHz,
DMSO-d6) d 1.92 (br s, 4H, CH2), 3.10 (br s, 4H, NCH2),
3.70 (s, 3H, OCH3), 5.40 (s, 1H, CH), 6.86 (d, 2H,
J 8.7 Hz, Ar–H), 7.56 (m, 3H, 2Ar–H and 1H, Naph–H),
7.67 (t, 1H, J 7.4 Hz, Naph–H), 7.78 (d, 1H, J 7.4 Hz,
Naph–H), 7.89 (d, 1H, J 7.4 Hz, Naph–H), 10.52 (br s,
1H, OH); 13C NMR (101 MHz, DMSO-d6) d 23.6, 53.6,
55.5, 67.2, 113.9, 114.1, 125.5, 125.7, 129.9, 131.2, 131.9,
134.1, 135.1, 159.4, 170.5, 178.2, 184.9; ESI(+)-FT-ICR-
3349, 3062, 1687, 1640, 1592, 1537, 1320, 1240; 1H NMR
(400 MHz, DMSO-d6) d 6.29 (d, 1H, J 9.4 Hz, CH), 7.07
(br s, 2H, Ar–H), 7.32 (m, 1H, Ar–H), 7.50 (m, 2H, 1Ar–H
and 1H, Naph–H), 7.56 (d, 1H, J 7.5 Hz, Naph–H), 7.73 (d,
1H, J 9.4 Hz, NH), 7.94 (m, 2H, 1Ar–H and 1H, Naph–H),
8.06 (m, 1H, Ar–H), 8.09 (d, 2H, J 9.0 Hz, 1Ar–H and
1H, Naph–H), 8.16 (m, 1H, Ar–H); 13C NMR (101 MHz,
DMSO-d6) d 41.9, 112.0, 117.7, 118.9, 122.4, 126.4, 126.5,
126.7, 129.6, 130.2, 131.0, 131.8, 134.6, 135.4, 136.8,
149.1, 151.4, 152.9, 178.5, 183.3; ESI(–)-FT-ICR-MS
calcd. for C23H13N2O5– [M – 2H – OH]–: 397.08300; found:
397.08265 (DBE = 17); [M – H – OH + Cl]–: 433.05967;
found: 433.05932 (DBE = 17).
MS calcd. for C22H22NO4 [M + H]+: 364.15432; found:
364.15041 (DBE = 13); [2M + Na]+: 749.28347; found:
749.28334 (DBE = 25).
+
2-Hydroxy-3-((4-nitrophenyl)((4-nitrophenyl)amino)methyl)
naphthalene-1,4-dione (10)
2-Hydroxy-3-(naphthalen-1-yl((4-nitrophenyl)amino)methyl)
naphthalene-1,4-dione (13)
Compound 10 was obtained as an yellow solid (82%);
m.p. 134-136 oC; IR (KBr) νmax / cm-1 3446, 3328, 3199,
1675, 1593, 1573, 1507, 1344, 1299; 1H NMR (400 MHz,
DMSO-d6) d 6.37 (s, 1H, CH), 6.93 (d, 2H, J 8.7 Hz,Ar–H),
7.81 (d, 2H, J 8.7 Hz, Ar–H), 7.93 (td, 1H, J 7.5, 1.5 Hz,
Naph–H), 7.98 (td, 1H, J 7.5, 1.5 Hz, Naph–H), 8.10 (m,
4H, 2Ar–H and 2H, Naph–H), 8.31 (d, 2H, J 8.7 Hz,Ar–H);
13C NMR (101 MHz, DMSO-d6) d 49.5, 111.2, 119.2,
122.2, 123.1, 124.8, 124.9, 125.2, 126.6, 129.0, 129.4,
130.5, 132.3, 133.7, 135.5, 145.3, 147.0, 152.3, 154.5,
155.9, 179.9, 182.1, 191.2; ESI(–)-FT-ICR-MS calcd.
for C23H14N3O7– [M – H]–: 444.08372; found: 444.08358
(DBE = 18).
Compound 13 was obtained as an orange solid (90%);
m.p. 165-167 oC with decomposition; IR (ATR) νmax / cm-1
3641, 3407, 3396, 3362, 1678, 1590, 1503, 1308, 1276;
1H NMR (400 MHz, CDCl3) d 5.81 (d, 1H, J 9.3 Hz, CH);
6.67 (dd, 2H, J 7.2, 2.0 Hz, Ar–H), 6.94 (d, 1H, J 9.3 Hz,
NH), 7.43 (t, 1H, J 7.7 Hz,Ar–H), 7.52 (td, 1H, J 7.0, 1.0 Hz,
Naph–H), 7.56 (td, 1H, J 7.0, 1.0 Hz, Naph–H), 7.68 (d,
1H, J 7.4 Hz, Ar–H), 7.71 (td, 1H, J 7.7, 1.0 Hz, Ar–H),
7.79 (td, 1H, J 7.7, 1.0 Hz, Ar–H), 7.83 (d, 1H, J 8.3 Hz,
Ar–H), 7.89 (dd, 1H, J 7.7, 1.0 Hz, Ar–H), 8.06 (dd, 2H,
J 7.2, 2.0 Hz, Ar–H), 8.09 (dd, 1H, J 7.7, 1.0 Hz, Naph–H),
8.12 (dd, 1H, J 7.7, 1.0 Hz, Naph–H), 8.20 (d, 1H, J 8.3 Hz,
Ar–H); 13C NMR (101 MHz, CDCl3) d 49.5, 111.8, 113.3,
120.6, 123.1, 125.2, 125.3, 125.9, 126.5, 126.5, 126.9,
127.3, 129.1, 129.2, 131.2, 132.5, 133.2, 133.5, 134.0, 135.6,
138.7, 151.9, 153.6, 181.0, 184.2; ESI(‑)-FT-ICR-MS calcd.
for C27H17N2O5– [M – H]–: 449.11430; found: 449.11367
(DBE = 20); [M – C6H5N2O2 + OH]–: 329.08193; found:
329.08162 (DBE = 18).
2-Hydroxy-3-(((4-nitrophenyl)amino)(phenyl)methyl)
naphthalene-1,4-dione (11)
Compound 11 was obtained as an pumpkin solid (93%);
m.p. 187-189 oC with decomposition; IR (ATR) νmax / cm-1
3351, 3339, 1656, 1641, 1598, 1585, 1327, 1264; 1H NMR
(400 MHz, DMSO-d6) d 6.09 (s, 1H, CH), 6.55 (d, 1H,
J 9.3 Hz, NH), 6.74 (d, 2H, J 8.5 Hz, Ar–H), 7.19 (m, 1H,
Ar–H), 7.28 (m, 2H, Ar–H), 7.41 (m, 2H, Ar–H), 7.75 (td,
1H, J 7.5, 1.5 Hz, Naph–H), 7.80 (td, 1H, J 7.5, 1.5 Hz,
Naph–H), 7.93 (m, 4H, 2Ar–H and 2H, Naph–H); 13C NMR
(101 MHz, DMSO-d6) d 51.4, 112.8, 121.5, 126.3,
126.3, 126.5, 126.8, 127.1, 127.4, 128.7, 129.6, 129.9,
130.5, 132.1, 133.9, 135.2, 136.7, 140.1, 154.0, 156.1,
156.9, 181.5, 183.9, 193.7; ESI(–)-FT-ICR-MS calcd. for
C23H15N2O5– [M – H]–: 399.09865; found: 399.09808 (DBE
= 20); [M – C6H5N2O2 + C10H5O3]–: 435.08741; found:
435.08684 (DBE = 17).
2-Hydroxy-3-((4-methoxyphenyl)((4-nitrophenyl)amino)
methyl)naphthalene-1,4-dione (14)
Compound 14 was obtained as an yellow solid (68%);
m.p. 222-224 oC with decomposition; IR (ATR) νmax / cm-1
3394, 3245, 1666, 1636, 1593, 1362, 1260, 1237; 1H NMR
(400 MHz, DMSO-d6) d 3.65 (s, 3H, OCH3), 5.88 (s, 1H,
CH), 6.71 (m, 2H,Ar–H), 7.09 (m, 2H,Ar–H), 7.73 (td, 2H,
J 7.4, 1.6 Hz, 1Ar–H and 1H, Naph–H), 7.77 (td, 2H, J 7.5,
1.6 Hz, 1Ar–H and 1H, Naph–H), 7.87 (m, 2H, 1Ar–H
and 1H, Naph–H), 7.93 (m, 2H, 1Ar–H and 1H, Naph–H);
13C NMR (101 MHz, DMSO-d6) d 37.4, 55.3, 113.4, 123.9,
125.9, 126.4, 129.6, 130.2, 132.6, 133.0, 133.5, 135.1,
156.6, 157.6, 181.7, 184.0; ESI(–)-FT-ICR-MS calcd. for
C28H17O7– [M – C6H5N2O2 + C10H5O3]–: 465.09798; found:
465.09757 (DBE = 20).
2-Hydroxy-3-((2-hydroxyphenyl)((4-nitrophenyl)amino)
methyl)naphthalene-1,4-dione (12)
Compound 12 was obtained as an orange solid (69%);
m.p. 237-239 oC with decomposition; IR (ATR) νmax / cm-1

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2-((2-Phenyl-2H-1,2,3-triazol-4-yl)(pyrrolidin-1-yl)methyl)-
3-((1,3,4-trihydroxynaphthalen-2-yl)oxy)naphthalene-1,4-
dione (17)
to give the Mannich adduct 16 as an orange solid (66%),
m.p. 178-180 oC with decomposition. IR (ATR) νmax / cm-1
3353, 3062, 2955, 1684, 1583, 1530, 1326, 1285; 1H NMR
(400 MHz, DMSO-d6) d 1.94 (br s, 4H, CH2), 3.29 (m, 4H,
NCH2), 5.90 (s, 1H, CH), 7.40 (t, 1H, J 7.4 Hz, Bt–H), 7.56
(m, 3H, 2Bt–H and 2H, Naph–H), 7.71 (t, 1H, J 7.1 Hz,
Naph–H), 7.83 (d, 1H, J 7.1 Hz, Naph–H), 7.96 (m, 3H,
Compound 17 was obtained as an orange solid after
recrystallization from ethyl acetate:hexane 1:3 (36%); m.p.
o
above 200 C with decomposition; IR (KBr) νmax / cm-1
3494, 3067, 2976, 2777, 1675, 1597, 1571, 1359, 1278;
1H NMR (400 MHz, CDCl3) d 1.73 (t, 4H, J 6.3 Hz, CH2),
3.19 (br s, 4H, NCH2), 6.90 (s, 1H, CH), 7.16 (t, 1H,
J 7.2 Hz, Bt–H*), 7.29 (m, 2H, Bt–H), 7.47 (t, 2H, J 7.6 Hz,
1Ar–H and 1H, Naph–H), 7.55 (s, 1H, Bt–H), 7.61 (t, 2H,
J 7.7 Hz, 1Ar–H and 1H, Naph–H), 7.81 (d, 2H, J 7.6 Hz,
1Ar–H and 1H, Naph–H), 7.85 (d, 2H, J 8.1 Hz, 1Ar–H
and 1H, Naph–H), 8.12 (d, 2H, J 7.7 Hz, Bt–H); 13C NMR
(101 MHz, CDCl3) d 24.0, 26.8, 45.8, 118.2, 122.4, 125.5,
126.6, 126.8, 129.0, 130.8, 131.9, 133.3, 134.1, 135.1,
138.8, 139.8, 149.5, 162.8, 183.5, 185.2; ESI(+)-FT-ICR-
MS calcd. for C33H26N4O6 [M]+: 574.1852; found: 574.1847
(DBE = 23); [M + H]+: 575.1886; found 575.1925. *Bt–H:
benzotriazole hydrogens.
13
2Bt–H and 1H, Naph–H), 8.11 (s, 1H, Bt–H); C NMR
(101 MHz, DMSO-d6) d 23.6, 53.8, 58.5, 110.7, 118.8,
125.7, 125.9, 128.2, 130.2, 131.4, 132.1, 134.2, 135.2,
136.2, 139.4, 147.5, 171.5, 178.4, 184.7; ESI(–)-FT-ICR-
MS calcd. for C23H19N4O3– [M – H]–: 399.1463; found:
399.1462 (DBE = 16); [M – C4H8N + OH]–: 346.0833;
found: 346.0833 (DBE = 15).
Antimicrobial assay
Microorganism targets
The antimicrobial activity was evaluated using the
following microorganisms from theAmerican Type Culture
Collection (ATCC): C. albicansATCC 10231, C. glabrata
ATCC 2001 and C. krusei ATCC 34135. In addition,
five C. albicans clinical strains given by the Biocentro
Laboratory Ltd., in Divinópolis-MG, Brazil, were employed:
CA1, CA2, CA3, CA4 and CA5.All the fungal strains were
maintained on Sabouraud dextrose agar (SDA) (Oxoid).
The macromorphology and micromorphology
identifications of clinical origin strains were previously
confirmed: (i) growth in broth and SDA; (ii) evidence
of germ tube; (iii) chlamydospores formation test; and
(iv) biochemical tests.16
2-(Morpholino(2-phenyl-2H-1,2,3-triazol-4-yl)methyl)-3-
((1,3,4-trihydroxynaphthalen-2-yl)oxy)naphthalene-1,4-
dione (23)
Compound 23 was obtained as an orange solid after
recrystallization from ethyl acetate:hexane 1:3 (56%);
m.p. decomposition at 160 oC; IR (ATR) νmax / cm-1 3500,
3066, 2866, 2486, 1675, 1598, 1571, 1361, 1279; 1H NMR
(400 MHz, CDCl3) d 3.26 (m, 4H, NCH2), 3.68 (m, 4H,
OCH2), 6.84 (s, 1H, CH), 7.15 (t, 1H, J 7.7 Hz, Bt–H),
7.26 (m, 2H, Bt–H), 7.37 (td, 2H, J 7.6, 1.0 Hz, 1Ar–H
and 1H, Naph–H), 7.48 (td, 2H, J 7.5, 1.0 Hz, 1Ar–H and
1H, Naph–H), 7.60 (s, 1H, Bt–H), 7.73 (d, 2H, J 7.6 Hz,
1Ar–H and 1H, Naph–H), 7.84 (d, 2H, J 7.7 Hz, 1Ar–H
and 1H, Naph–H), 7.98 (d, 2H, J 7.5 Hz, Bt–H); 13C NMR
(101MHz, CDCl3) d 26.8, 44.1, 63.7, 118.3, 122.1, 125.7,
126.6, 126.7, 129.0, 130.7, 132.0, 133.1, 134.0, 135.1,
139.7, 149.3, 183.7, 184.8; ESI(+)-MS-FT-ICR calcd. for
C33H26N4O7 [M]+: 590.1802; found 590.1796 (DBE = 23);
[M + H]+: 591.1835; found 591.1874.
Culture media and inocula
Sabouraud dextrose broth (SDB) (HiMedia) was
prepared according to the Clinical and Laboratory
Standards Institute (CLSI) document M27-A217 for
minimal inhibitory concentration (MIC) fungal assays.
The fungal cultures of the Candida species were freshly
grown at 35 ºC and the inoculum suspensions were
prepared by the spectrophotometric method according to
the CLSI document M27-A318 with a final concentration of
1.5 1.0 × 103 cells mL-1 for susceptibility tests.Yeast cells
in the exponential phase were collected aseptically with a
sterile loop and resuspended in a tube containing 10 mL
of sterile saline; the inoculum of the Candida strains used
in the experiments was standardized to match 0.5 on the
McFarland scale (106 colony forming units (CFU) mL-1)
and confirmed by spectrophotometry (optical density
(OD) 0.08-0.1 at 625 nm). The suspension was produced
by making a 1:50 dilution followed by a 1:20 dilution in
SDB. The suspensions were prepared according to the CLSI
Synthesis of the lawsone derivative 2-hydroxy-3-((2-phenyl-
2H-1,2,3-triazol-4-yl)(pyrrolidin-1-yl)methyl)naphthalene-
1,4-dione (16)
A mixture of 1.2 eq of 2-phenyl-2H-1,2,3-triazole-4-
carbaldehyde 15 and 1.1 eq of pyrrolidine 3 in ethanol
o
(10 mL) was stirred overnight with at 50 C. After this
time, 1.0 eq of lawsone 1 was added and the mixture was
allowed to react for 24 h in the dark. After the completion
of the reaction, the solid was isolated by filtration, washed
with cold water and cold ethanol and dried under vacuum

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Synthesis, in vitro Antifungal Activity and Molecular Modeling Studies of New Mannich Bases
J. Braz. Chem. Soc.
document M27-A3 to obtain a final inoculum size suitable
for each strain.18
Analysis of cell viability
The BHK cells were cultured in 96-well plates until 90%
of confluence was reached. The plates were pre-incubated in
a humidified 5% CO2/95% air atmosphere at 37 ºC for 24 h
to allow the cells to adapt prior to the addition of the test
compounds. All the compounds were dissolved in DMSO
prior to dilution. The cytotoxicity was determined over a
concentration ranging from 9 to 1080 μg mL-1. All the cell
cultures were incubated in a 5% CO2/95% air atmosphere
at 37 ºC for 48 h. The cell viability was estimated by
measuring the rate of the mitochondrial reduction of a
yellow tetrazolium salt, 2-(3,5-diphenyltetrazol-2-ium-
2-yl)-4,5-dimethyl-1,3-thiazole bromide (MTT) (Sigma-
Aldrich), to insoluble purple formazan crystals.21 After
incubation of the cultures with the test compounds, the
MTT solution (0.028 mL at 2 mg mL-1) was added to each
well and the plates were incubated for 3 h at 37 ºC. At the
end of this incubation period, the supernatant was removed
and 0.1 mL of DMSO were added. The plate was then
placed under medium agitation for 7 min, the absorbance
was read at 540 nm in an enzyme linked immunosorbent
assay (ELISA) spectrophotometer (Powder Wave XS2,
Biotec) to determine the concentration that killed 50%
of cells (CC50).22 The cytotoxicity was calculated after
comparing with the control (treated with 0.1% DMSO).
The CC50 values were obtained by regression analysis of the
percentages for the different concentrations of test material.
GraphPad Prism 5.0 software23 was used for non-linear
regression calculations.24 The CC50 values represent the
average of three independent experiments.
Susceptibility test
Broth microdilution testing was performed according
the guidelines presented in the CLSI documents M27-A3
for fungi.18 The susceptibility to antimicrobial agents was
determined by the microbroth dilution method and was
performed in sterile flat-bottomed 96-well microplates
(Difco). The compounds were dissolved in DMSO after
the addition of SDB. Subsequently, serial dilutions were
prepared using the corresponding media as the diluent,
maintaining a constant volume of 0.1 mL in each well.
The compounds were tested at ten concentrations ranging
from 1.5-1500 μg mL-1.
For growth and sterility control, the media was used
without the addition of the test compounds. As a control
for the toxicity of the solvent, a culture was inoculated with
DMSO. The antifungal ketoconazole (Sigma-Aldrich®;
initial concentration 100 μg mL-1) was used as a positive
antifungal control.
After the plates were prepared, the inoculate of each
strain was added and the plates were incubated at 37 ºC
for 48 h. Each test was performed in triplicate. The
endpoints were determined visually by comparison of
the samples with the drug-free control well. The MIC
was detected following the addition of 0.05 mL 2.0%
triphenyltetrazolium chloride solution (TTC) (Sigma-
Aldrich®). The growth of yeast was visualized by changes
to a red color. The MIC was defined as the lowest
compound concentration at which the well was optically
clear, and this value was expressed in μg mL-1.
Molecular modeling studies
The minimal fungicidal concentration (MFC) of
compounds that were showing MIC was determined by
plating up 10 μL of the MIC by technique spread plate
in SDA (Himedia).19 The plates were incubated at 35 ºC
for 48 h. The MFC was considered to be the lowest
concentration of the tested agent capable of preventing the
growth of any yeast colony in SDA. The experiments were
performed in triplicate.
Initially, the most active compounds determined in
the biological assay step (compounds 4, 11 and 12) and
ketoconazole were built and adjusted for the protonation
state at pH 7.4 using MarvinSketch software.25 These
structures were refined by parametric method 7 (PM7)26
implemented in MOPAC 2012.27 Next, the ligands and
molecular target (under protein data bank code 3OZW28)
were prepared using the Autodock tools standard protocol
and submitted toAutodockVina.29 A grid box was generated
with 24, 20 and 20 Å for x, y, z respectively, centered on
the ligand with –17.68, 36.32, –32.46Å. The flavin-adenine
dinucleotide (FAD) was retained at the binding site. The
crystallographic ketoconazole was redocked against 3OZW
to validate the methodology, following the docking of
ligands. All docking simulations were performed with the
‘exhaustiveness’ term set to 20 to achieve more accurate
results. The results were depicted by Discovery Studio 4.1.30
In addition, the physicochemical properties (molecular
Mechanism of action on exogenous ergosterol
The MICs were determined using C. albicans ATCC
10231, C. glabrataATCC 2001 and C. krusei ATCC 34135
by the standard broth microdilution procedure described
above. Duplicate plates were prepared: one contained
the test compounds and exogenous ergosterol (Sigma;
200 μg mL-1) and the other contained the compounds
alone. The MICs were determined after 48 h.20 Nystatin
(Sigma; initial concentration 100 μg mL-1) was used as
the positive control.

Vol. 27, No. 11, 2016
Allochio Filho et al.
2133
weight, partition coefficient (log P), hydrogen acceptors
and donors groups and druglikeness) were calculated using
DataWarrior software.31
yields were obtained in the reaction with p-nitroaniline,
probably due to increased stability of the imine formed as
intermediate. The increased stability shifts the chemical
equilibrium towards its formation thereby increasing the
concentration of electrophile in the reaction medium.
Aiming to exploit the profile of 1,2,3-triazoles as
antifungals32 the aldehyde 2-phenyl-2H-1,2,3-triazole 15
was prepared in three steps from D-(+)-glucose, as
previously reported.33
Results and Discussion
Synthesis of lawsone derivatives
In order to develop the Mannich reaction, and
especially to broaden its scope, we conducted a series
of experiments to optimize the conditions for the
reaction between lawsone 1, the salicylaldehyde 2 and
pyrrolidine 3 to form the corresponding Mannich adduct 4
(Scheme 2). The best results were obtained using a slightly
modified procedure, which used 10 mol% of excess
amine in order to increase the nucleophilic character of
lawsone by deprotonating the hydroxyl and an excess of
20 mol% aldehyde to shift the equilibrium towards the
incipient imine or iminium intermediates.3,14,15 Several
solvents were tested and the best results were obtained
with ethanol to prepare compound 4 in good yields as a
pure red solid after filtration. This protocol allowed to
reach the Mannich adducts 5-8 and 10-14 in moderate
to high yields using pyrrolidine 3 and p-nitroaniline 9,
respectively and various aromatic aldehydes. The higher
To couple the triazole group and naphthoquinone,
we then performed a multicomponent Mannich reaction
between lawsone 1, aldehyde 15 and pyrrolidine 3 in ethanol
at room temperature. However, rather than obtaining the
corresponding Mannich adduct 16, compound 17 was
obtained in this reaction (Scheme 3) as a pure orange solid in
36% yield after recrystallization from ethyl acetate:hexane
1:3. IR of the compound 17 showed a band at 1278 cm-1
likely due to the C–O–C ether bond. Additionally, the
1
integration of signals in the H NMR spectrum revealed
additionally four hydrogen atoms (7.82 ppm, d, 2H,
J 7.3 Hz, H18/H21 and 7.48 ppm, t, 2H, J 7.3 Hz, H19/H20)
in the aromatic region. The compound was also
characterized by ESI(+)-FT-ICR MS, which provided an
unambiguous molecular formula of C33H26N4O6, giving an
ion [C33H26N4O6 + H]+ with m/z of 575.1925.
Scheme 2. Multicomponent Mannich reaction in lawsone.

2134
Synthesis, in vitro Antifungal Activity and Molecular Modeling Studies of New Mannich Bases
J. Braz. Chem. Soc.
Scheme 3. Synthesis of compound 17.
Compound 17 was likely formed via Michael addition
between the alkoxide derived 20, obtained from the
deprotonation of the hydroxyl of the Mannich adduct 16
by pyrrolidine and the lawsone 1 remaining in the reaction
medium (Scheme 4). Compound 16 was formed in low
concentration, probably due to low concentration of the
iminium ion 18 in the reaction medium. The formation of 17
from 21 is thermodynamically favored due to the formation
of the aromatic ring indicated with arrow in enolization
step of the intermediate 21. This reaction was repeated
using p-nitroaniline and morpholine but also in this case
the compound 23 was obtained in 56% yield as an orange
solid after recrystallization from ethyl acetate:hexane 1:3,
as a result from the Michael addition between the alkoxide
of the Mannich adduct 22 and lawsone (Scheme 4).
strains. Compound 4 (MIC = 20 μg mL-1) showed the highest
inhibitory effect growth of C. albicans when compared to
ketoconazole (MIC = 62.5 μg mL-1), the standard antifungal
agent utilized in this assay. The compounds 4, 11 and 12
showed fungicidal activity against C. albicansATCC 10231,
which was not observed for the antifungal ketoconazole.
The MIC value of compound 4 obtained against this yeast
was lower than the MFC value, which suggests that this
compound is fungistatic at a lower concentration and
fungicidal at a slightly higher concentration.
It is worth mentioning that C. glabrata ATCC 2001
displayed sensitivity towards the compounds 4 and 12 that
showed fungistatic activity. In addition, C. krusei ATCC
34135 was susceptible to the compound 12 that showed
MIC and MFC of 330 μg mL-1. All the yeasts tested were
resistant to compounds 5, 6, 7, 8, 14 and 23.
Regarding clinical strains, the compound 4 showed
the best fungistatic activity against C. albicans clinical
strains CA1, CA2, CA4 and CA5 with MICs ranging from
40-670 μg mL-1. The compound 10 showed fungistatic
activity against CA1 with an MIC of 170 μg mL-1. The
other compounds showed less activity.
In this study, the compounds 4, 11 and 12 had the
greatest activity against C. albicans strains; therefore,
their effect on membrane ergosterol was determined using
the exogenous ergosterol assay. Compound 4 showed
binding action on membrane ergosterol, since the MIC
was enhanced 4 fold against C. albicans ATCC 10231,
when performed in the presence of exogenous ergosterol
(MIC = 80 μg mL-1) indicating their binding to ergosterol
(Table 2).
For compound 23, the presence of four aromatic
1
hydrogen atoms in the H NMR spectrum was also
observed by integrating the signals. Additionally, an ion
[C33H26N4O7 + H]+ with an m/z of 591.18733 was detected
in the ESI(+)-FT-ICR mass spectrum.
Thus, the Mannich adduct 16 containing the triazole
group was obtained by a slight experimental modification
(Scheme 5). For this, the 2-phenyl-2H-1,2,3-triazole-4-
carbaldehyde 15 and pyrrolidine 3 in ethanolic solution were
allowed to react overnight with a constant temperature of
50 oC. Then, lawsone 1 was added to the reaction and after
24 h the Mannich adduct 16 was obtained in 66% yield.
Antimicrobial assay
The synthetic compounds were tested against three
Candida strains and five clinical isolates. The best antifungal
activities were observed against C. albicans ATCC 10231
(Table 1). The compounds 4, 11 and 12 showed the greatest
activity, with MICs ranging from 20 to 330 μg mL-1 and
MFCs ranging from 80 to 330 μg mL-1 for the C. albicans
The cytotoxicity assay was performed with the
compounds 4, 11 and 12, which showed best MIC values
against C. albicans ATCC 10231. The CC50 obtained for
those compounds were 52.81, 52.58 and 85.94 μg mL-1,
respectively.

Vol. 27, No. 11, 2016
Allochio Filho et al.
2135
Scheme 4. Proposed mechanism for the formation of compounds 17 and 23.
Molecular modeling studies
was evaluated by redocking34 of ketoconazole, which
yielded a root mean square deviation (RMSD) of 2.29 Å
(Figure 1a). Even though this value deviates 0.29Å from the
ideal value (2.0Å), as can be seen in Figure 1a, it could keep
the moieties in the similar order of crystallographic ligand.
Next, the compounds 4, 11 and 12 were docked into the
In order to initiate a lead optimization process, docking
studies (Figure 1) were performed between the most
active lawsone compounds and the molecular target of
ketoconazole (3OZW).28 Initially, the docking methodology

2136
Synthesis, in vitro Antifungal Activity and Molecular Modeling Studies of New Mannich Bases
J. Braz. Chem. Soc.
Scheme 5. Synthesis of compound 16.
Table 1. Antifungal activity (μg mL-1) of 2-hydroxy-1,4-naphthoquinone derivatives against Candida spp. strains
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. glabrata
C. krusei
ATCC 10231
ATCC 1417
ATCC 1083
ATCC 1203
ATCC 1252
ATCC 11763
ATCC 2001
ATCC 34135
Concentration / (μg mL-1)
MIC
20
–
MFC
80
–
MIC
670
–
MFC
MIC
40
–
MFC
MIC
670
–
MFC
MIC
–
MFC
MIC
170
–
MFC
MIC
170
–
MFC
MIC
670
–
MFC
4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
170
–
–
–
–
–
5
–
6
–
–
–
–
–
–
–
–
–
–
–
–
–
7
–
–
–
–
–
–
–
–
–
–
–
–
–
8
–
–
–
–
–
–
–
–
–
–
–
–
–
10
11
12
13
14
16
17
23
–
–
170
–
350
660
330
–
–
–
–
700
–
–
700
–
–
–
–
330
85
–
330
85
–
660
670
–
–
–
–
–
–
–
670
350
–
330
–
670
–
–
–
330
–
–
330
–
330
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
730
–
–
–
730
–
730
–
–
–
730
–
–
730
670
–
–
–
–
–
670
–
–
–
–
670
–
–
–
–
–
–
–
–
–
–
–
–
–
MIC: minimum inhibitory concentration; MFC: minimum fungicidal concentration; –: no activity.
Table 2. Mechanism of action on membrane ergosterol against three Candida spp. strains
C. albicans
C. glabrata
C. krusei
ATCC 10231
ATCC 2001
ATCC 34135
MIC without
ergosterol / (μg mL-1) ergosterol / (μg mL-1) ergosterol / (μg mL-1) ergosterol / (μg mL-1) ergosterol / (μg mL-1) ergosterol / (μg mL-1)
MIC with
MIC without
MIC with
MIC without
MIC with
4
80
330
85
20
330
85
170
N
170
N
N
N
N
N
11
12
330
31.25
330
3.9
330
31.25
330
3.9
Nystatin
31.25
3.9
MIC: minimum inhibitory concentration; N: not performed.
binding site following a similar conformation (Figure 1b).
The intermolecular interactions between the most active
compound 4 and target are illustrated by Figures 1c and 1d.
As can be observed, the pyrrolidine moiety is able to bind to
the heme group by a cation-pi, whereas the naphthoquinone
and phenyl moieties perform van der Waals interactions.
Further, the binding site cavity was studied by hydrophobic
(Figure 1e) and hydrogen bond (Figure 1f) surfaces. In
general, the binding site showed a high hydrophobic
character (Figure 1e). However, close to the phenyl moiety,
the binding site can recognize a hydrogen bond acceptor
and donor. In other words, nonpolar moieties and hydrogen
acceptor and donor moieties in the phenyl ring can improve
the biological activity.

Vol. 27, No. 11, 2016
Allochio Filho et al.
2137
Figure 1. Docking results between lawsone and 3OZW. (a) Redocking of ketoconazole; (b) cluster formed by compounds 4, 11 and 12; (c) 3D compound
4-3OZW intermolecular interactions; (d) 2D intermolecular interactions diagram between compound 4 and 3OZW; (e) hydrophobic; and (f) hydrogen
bond surfaces of binding site. The hydrogen atoms were omitted for a better visualization.
Finally, tests on the physicochemical properties
were carried out to evaluate the most promising ligand35
among compounds 4, 11 and 12 to reach the market
(Table 3). According to the rule of 5 (Ro5) proposed by
Lipinski et al.,36 the lawsone derivatives 4, 11 and 12 do not
violate any rule. They showed molecular weights less than
500, the number of hydrogen bond donors and acceptors
less than 5 and 10, respectively, and log P less than 5. In
addition, the binding energy obtained from docking studies
is similar to that of ketoconazole. On the other hand,
ketoconazole and nystatin violated one and two Ro5 rules,
respectively. Moreover, druglikeness37 shows that these
compounds contain fragments present in commercial drugs,
of which 80% have positive druglikeness values close to
3. These findings suggest that the compounds 4, 11 and 12
are more promising drugs than ketoconazole and nystatin.
Conclusions
In this study, we determined the MICs and MFCs of
thirteen new naphthoquinones derivatives 4-8, 10-14, 16,
17 and 23 obtained by multicomponent Mannich reaction

2138
Synthesis, in vitro Antifungal Activity and Molecular Modeling Studies of New Mannich Bases
J. Braz. Chem. Soc.
Table 3. Physicochemical properties for the bioactive lawsone derivatives
Binding energy /
(kcal mol-1)
Molecular weight /
(g mol-1)
Compound
log P
H-acceptors
H-donors
Druglikeness
4
–10.2
–11.7
–11.6
–10.5
–
334.39
383.40
399.40
533.45
926.10
0.81
3.40
3.05
3.12
0.40
4
5
2
2
0.00
1.80
1.80
6.58
–4.30
11
12
6
3
Ketoconazole
Nystatin
8
1
18
12
log P: partition coefficient.
containing 2-phenyl-2H-1,2,3-triazole or substituted and
non-substituted phenyl groups against Candida strains and
clinical isolates.Among them, compounds 4, 11 and 12 were
the most active. These compounds showed fungistatic and
fungicidal activity against strains of C. albicans ATCC 10231.
This finding indicates that they are strong candidates for
clinical use, since the complete elimination of the pathogen
is the safest option.38 Moreover, the results indicate that these
compounds were more active against C. albicans ATCC
10231, which shows greater sensitivity than C. glabrata and
C. krusei. It is noteworthy that C. albicans is the causative
agent in 90% of fungal infections such as VVC.39 Besides,
C. albicans and C. tropicalis strains are responsible for a
number of major diseases as well as providing recent cases
of resistance to the current main antifungals.40
In addition, the effect on the membrane ergosterol was
determined for compounds 4, 11 and 12. The evaluation of
this mechanism of action is important due to the ergosterol
molecule, or enzymes used in its biosynthesis, being
important targets for the action of azole antifungals.41
Hence, the antifungal activity of the compound 4 against
C. albicans ATCC 10231 can be attributed to its action
on membrane ergosterol. The antifungal activity of the
compounds 11 and 12 against C. albicans ATCC 10231
can be attributed probably to the reactive oxygen species
(ROS) generation, because these compounds do not
have the triazole group and because of the presence of
naphthoquinone core with recognized pharmacological
action via formation of ROS.1,42
The most active compounds showed similar binding
energy with ketoconazole, suggesting their mechanism
of action. In general, the cavity of the binding site has a
hydrophobic character, addressing for lipophilic moieties
in phenyl ring. Furthermore, the compounds 4, 11 and 12
are under Ro5 with a druglikeness score better than that
for ketoconazole and nystatin. These findings suggest a
therapeutic alternative against the increased resistance of
Candida spp. to azole-based antifungals.40
Supplementary Information
1
Supplementary information (spectra for IR, H and
13C NMR and FT-ICR MS of the synthesized compounds)
is available free of charge at http://jbcs.sbq.org.br as
PDF file.
Acknowledgments
The authors thank FAPES (process 54684722/2012),
CNPq (process 449984/2014-1) and FAPEMIG
(APQ-00557-14) for financial support.
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on the cell strain tested.43

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Submitted: February 5, 2016
Published online: April 6, 2016