New nitrogen compounds coupled to phenolic units with antioxidant and antifungal activities: Synthesis and structure-activity relationship

Article abstract of DOI:10.3390/molecules23102530

A selection of 1-amino-2-arylidenamine-1,2-(dicyano)ethenes 3 was synthesized and cyclized to 2-aryl-4,5-dicyano-1H-imidazoles 4 upon reflux in ethyl acetate/acetonitrile, in the presence of manganese dioxide. These compounds were tested for their antioxi

Full text of DOI:10.3390/molecules23102530

molecules
Article
New Nitrogen Compounds Coupled to Phenolic
Units with Antioxidant and Antifungal Activities:
Synthesis and Structure–Activity Relationship
Ana Bettencourt 1, Marián Castro 2 , João Silva 3 , Francisco Fernandes 1, Olga Coutinho 3,
M. João Sousa 3, M. Fernanda Proença 1,* and Filipe Areias 1,3,4,
*
1
Department of Chemistry, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal;
abete@quimica.uminho.pt (A.B.); francisco.fernandes@upmc.fr (F.F.)
Department of Pharmacology, Center for Research in Molecular Medicine and Chronic Diseases (CiMUS),
Universidade de Santiago de Compostela, Avenida de Barcelona 22, 15782 Santiago de Compostela, Spain;
marian.castro@usc.es
Department of Biology, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal;
jp.silva@gmail.com (J.S.); olgapc@bio.uminho.pt (O.C.); mjsousa@bio.uminho.pt (M.J.S.)
School of Chemical Science and Engineering, Yachay Tech University, Yachay City of Knowledge,
100650 Urcuqui, Ecuador
2
3
4
*
Correspondence: fproenca@quimica.uminho.pt (M.F.P.); fareias@yachaytech.edu.ec (F.A.);
Tel: +35-8995293765 (F.A.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Josef Jampilek
Received: 9 September 2018; Accepted: 2 October 2018; Published: 3 October 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A selection of 1-amino-2-arylidenamine-1,2-(dicyano)ethenes
3
was synthesized and
cyclized to 2-aryl-4,5-dicyano-1H-imidazoles
4 upon reflux in ethyl acetate/acetonitrile, in the
presence of manganese dioxide. These compounds were tested for their antioxidant capacity by
cyclic voltammetry, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and deoxyribose degradation
assays. The minimum inhibitory concentration of all compounds was evaluated against two yeast
species, Saccharomyces cerevisiae and Candida albicans. Their toxicity was tested in mammal fibroblasts.
Among the synthesised compounds, two presented dual antioxidant/antifungal activity without
toxic effects in fibroblasts. The new compounds synthesized in this work are potential biochemical
tools and/or therapeutic drugs.
Keywords: Imidazoles; phenolic compounds; antifungal activity; MIC; antioxidant capacity; structure-
activity relationship
1. Introduction
The synthesis of novel antioxidants is an important area for the pharmaceutical, cosmetic, and food
industries [1,2]. Indeed, antioxidants have matchless properties in the conservation of many market
products, and an important function in the prevention of many degenerative pathologies, such as,
cancer, cardiovascular diseases, arthritis, arteriosclerosis, and the normal process of aging [3,4].
The antioxidant capacity of a compound is associated with its ability to act as a scavenger of
free radicals [
antioxidant, because iron and copper ions can initiate the production of hydroxyl radicals by the
Fenton reaction [ ]. Phenolic compounds, such as quercetin and gallic acid, are typical examples of
5]. The chelation capacity of metal ions is another important property of an efficient
6
potent antioxidants [7].
The resistance of pathogenic fungi to actual drugs on the market supports the need for the
synthesis of novel antifungal agents [8], presenting selective toxicity to fungi without toxic effects
to other organisms [ ]. Nevertheless, commercial antifungals do not present this selective toxicity.
9
Molecules 2018, 23, 2530; doi:10.3390/molecules23102530
www.mdpi.com/journal/molecules

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Additionally, the actual antifungal agents are highly persistent in the environment, and they present
a threat to living beings in general [10]. Nitrogen heterocycles, like fluconazole and miconazole,
are examples of systemic antifungals with a wide spectrum of action on fungi [11].
In this study, the imidazole ring, present in many antifungal agents, was coupled with the
phenolic unit, which is known to confer antioxidant capacity, to create new nitrogen heterocycles.
The compounds synthesized were assayed for their antioxidant and antifungal activities, and the
results allowed for an understanding of a relationship between chemical structure and activity (SAR).
2. Results
2.1. Chemistry
The products and reactions reported in this section are summarized in Table 1. The 1,2-(dicyano)
ethenes 3a
by the reaction of diaminomaleonitrilo
acid as catalyst. The products 3a
–
3e, used as starting materials for the imidazole scaffolds studied in this work, were prepared
with phenolic aldehydes 2a 2e, in ethanol and using sulphuric
3e were isolated at 81–97% yield after approximately 10 minutes at
1
–
–
room temperature.
The cyclization of the 1,2-(dicyano)ethenes 3a–3e to the corresponding imidazoles 4a–4d was
performed under reflux in tetrahydrofuran (THF), using an excess of MnO2. The products were
isolated in 50–70% yield after 8 hr to 5 days. All attempts to prepare compound 4e from 3e, with either
acid or base catalysis, varying the solvent and using nitrogen atmosphere, resulted in the formation of
dark materials, possibly as the result of extensive oxidation.
Table 1. Experimental conditions for the synthesis of target compounds 3 and 4.
Compound R1
R2
R3
R4
Reaction Conditions
Yield (%)
3a
3b
3c
3d
3e
4a
4b
4c
OH
H
H
H
H
OH
H
H
H
OH
H
H
H
OH
H
H
H
H
1 + 2a (1.0 equiv.), EtOH, H2SO4, r.t., 10 min
1 + 2b (1.0 equiv.), EtOH, H2SO4, r.t., 10 min
1 + 2c (1.0 equiv.), EtOH, H2SO4, r.t., 10 min
1 + 2d (1.0 equiv.), EtOH, H2SO4, r.t., 10 min
1 + 2e (1.0 equiv.), EtOH, H2SO4, r.t., 10 min
3a, ethyl acetate/acetonitrile, MnO2, reflux, 14 h
3b, ethyl acetate/acetonitrile, MnO2, reflux, 5 days
3c, ethyl acetate/acetonitrile, MnO2, reflux, 8 days
3d, ethyl acetate/acetonitrile, MnO2, reflux, 24 h
97
82
87
81
92
50
70
63
55
OH OH
OH OH OH
H
OH
H
H
H
OH
H
H
H
H
4d
H
OH OH
r.t. (room temperature).
2.2. Antioxidant Capacity
The antioxidant capacity of 1,2-(dicyano)ethenes 3a
–
3e and imidazoles 4a–4d was assessed
through a step-by-step cascade using three different experimental procedures. Initially, we determined
the anodic peak potential (Ep) of all compounds by the cyclic voltammetry technique because this
potential represents the ability of compounds to donate their electrons, and it can be used as a parameter
of the scavenging activity [12]. Subsequently, we determined and confirmed the scavenging activity
by the DPPH assay for the compounds that presented the lowest potentials of each chemical series
(3e and 4d) [13]. Finally, we determined the antioxidant capacity by the deoxyribose degradation

Molecules 2018, 23, 2530
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assay for the two previous compounds, because this assay can indicate both the scavenging and the
chelating activities of these compounds [14]. The results obtained were compared with the reference
antioxidant trolox.
Previous work showed that compounds selected by their electrochemical behavior are likely
to act as radical scavengers [12]. In this study, cyclic voltammetry was used to test the anodic peak
potential of the synthesized compounds. The anodic peak potential (Ep) and the half peak potential
(Ep/2) were used to measure the easiness of oxidation, and consequently the antioxidant capacity of
the compounds. The obtained peak and half peak potentials are presented in Table 2. The oxidation
peak potentials of the tested compounds vary from 0.113 V up to 0.896 vs. saturated calomel electrode
(SCE), depending on the number of hydroxyl groups in the aromatic ring. Published literature
corroborates our current observation, stating that compounds with two or more hydroxyl groups have
lower anodic peak potentials and higher antioxidant capacities than monosubstituted phenols [15].
The voltammograms obtained for the oxidation of 1,2-(dicyano)ethenes 3a
3-hydroxy, and 4-hydroxy substituents in the aromatic ring presented values for the peak potential that
were equal to or higher than 0.476 V. The 1,2-(dicyano)ethenes 3d 3e, with the 3,4-di-hydroxyphenyl
–3c with the 2-hydroxy,
–
and 3,4,5-tri-hydroxyphenyl substituents presented lower peak potentials, varying from 0.113 to
0.248 V. These differences in electrochemical behavior indicate that the number of hydroxyl groups of
the phenolic unit is responsible for the easiness of oxidation of the compounds.
Table 2. Data from cyclic voltammetry, DPPH and deoxyribose degradation assays for the target
compounds.
% Inhibition of
deoxyribose degradation
Ep/2 (mV vs. ECS)
Compound
Ep (mV vs. ECS)
DPPH IC50 (µM)
3a
3b
3c
3d
3e
4a
4b
4c
688
740
476
248
113
783
896
620
254
649
-
361
185
40
758
712
534
166
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
3.7 ± 0.7
62.1 ± 2.3
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
4d
12.0 ± 1.0
59.0 ± 3.5
Trolox
173
107
9.0 ± 0.2
23.4 ± 2.6
Ep (Anodic peak potential); Ep/2 (half peak potential); SCE (saturated calomel electrode); DPPH (2,2-diphenyl-1-
picrylhydrazile radical); IC50 (50% inhibitory concentration); n.d. (not determined).
The peak potentials of compounds 3d
the peak potential of trolox (Ep = 0.173 V), suggesting that these compounds have a high scavenging
capacity. Indeed, the peak potentials of the previous compounds (3d 3e) were compared with the peak
–3e were of the same order of magnitude or even lower than
–
potential for trolox, a standard antioxidant, as these values represented the tendency of the compounds
to give up their electrons. In the literature, antioxidant/prooxidant activity has been associated
with the oxidation potential, and compounds with a low peak potential (lower than 0.450 mV) were
identified as antioxidants [12,15].
For each compound series, the scavenging activity of the synthesized molecules with the lowest
peak potential (3e and 4d) was assessed by the DPPH radical assay. Compound 3e (3.7
±
0.7
µM)
presented an IC50 value that was lower than trolox (9.0
an efficient radical scavenger.
±
0.2 µM), establishing that this compound is
The imidazole 4d, substituted with the 3,4-di-hydroxyphenyl moiety, presented a peak potential
value of 0.254 V, which was the lowest of this series (Table 2). The IC50 value obtained for this
imidazole in the DPPH radical assay, 12.0
scavenging activity as consequence of cyclization. Despite this decrease in activity, imidazole 4d could
±
1.0 µM, showed that there was a slight decrease in the
be considered to be a scavenger that is almost as potent as trolox.

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To finalize the antioxidant study, the most potent scavengers of each chemical series were
assayed on the deoxyribose degradation assay. The compounds were assayed at their IC50
concentration, as determined by the DPPH assay. This experimental approach permitted us to estimate
the antioxidant capacity of the synthesized compounds at equipotent scavenging concentrations.
The compounds 3e and 4d inhibited the deoxyribose degradation by approximately 60%, whereas
trolox inhibited deoxyribose degradation only by 23.4
±
2.6%. The higher antioxidant capacity of the
compounds assessed, substituted with 3,4-di-hydroxyphenyl or 3,4,5-tri-hydroxyphenyl moieties,
would be a consequence of the combination of their scavenging activity and of their capacity
to chelate iron ions. These results agree with the literature reporting that phenolic compounds
with 3,4-di-hydroxyphenyl or 3,4,5-tri-hydroxyphenyl moieties, such as quercetin and gallic acid,
respectively, have potent scavenging activities and chelating capacities for iron, and both contribute
to their high antioxidant activities [16–19]. Our hypothesis was valid because the deoxyribose was
oxidized with hydroxyl radicals (HO ) produced through the ascorbate–iron pair by the Fenton
reaction [14]. These results are important, as many pathological diseases are associated with an increase
of free iron concentration, as a consequence of a disruption of iron homeostasis [20]. Additionally,
free iron can react with ascorbate that exists at high concentration inside the cells, generating HO
by the Fenton reaction [21]. In summary, compounds 3e and 4d present excellent properties as
antioxidant agents.
2.3. Antifungal Activity
The antifungal activity of the synthesized compounds was tested on two yeast species by
the method of liquid microdilution, and compared with the reference antifungals fluconazole and
miconazole. We chose the Saccharomyces cerevisiae as the model yeast in this study, because it is not
pathogenic at normal conditions, but it can provoke serious infections in persons with a depressed
immune system, such as patients with human immunodeficiency virus infection and acquired immune
deficiency syndrome (HIV/AIDS) [22]. We also tested the activity of the compounds on Candida albicans
because it is an opportunistic yeast that is frequently found in systemic infections [23]. Therefore,
there is a high interest in the development of new antifungals with activity on these two yeasts.
Minimum inhibitory concentration (MIC) values, defined as the lowest concentration of
compound able to cause 80% inhibition of cellular growth, were assessed for all compounds.
Miconazole presented MIC values on Saccharomyces cerevisiae and Candida albicans of 100.0 and
0.78 µM, respectively, while MIC values for fluconazole were 50.0 and 1.56 µM, respectively (Table 3).
These MIC values exhibited showed that miconazole is more potent than fluconazole on Candida
albicans, while fluconazole is more potent than miconazole on Saccharomyces cerevisiae. In addition,
Candida albicans is more sensitive than Saccharomyces cerevisiae to these two antifungal agents.
In the assessment of the antifungal activity of 1,2-(dicyano)ethenes 3a
compound 3e, with a 3,4,5-tri-hydroxyphenyl moiety, achieved 80% inhibition of the growth of
Saccharomyces cerevisiae at a concentration of 50 M (Table 3). Therefore, 3e showed antifungal activity
that was equipotent to fluconazole, and more potent than miconazole. On Candida albicans, 3e presented
a MIC of 100 M, having weaker activity than miconazole and fluconazole. The highest antifungal
–3e, we observed that
µ
µ
activity of 3e compared to the other compounds of this series seemed to be related with the lowest
anodic peak potential and the highest antioxidant capacity of this compound. Therefore, we confirmed
the presence of a synergistic effect between the phenolic moiety (3,4,5-tri-hydroxyphenyl) and the
rest of the molecule common to all 1,2-(dicyano)ethenes. The other compounds of the series did not
achieve the MIC on the yeast species considered in our study at the highest concentration evaluated in
each case, due to their limited solubility in aqueous solution (Table 3).

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Table 3. Values of minimum inhibitory concentration (MIC) on yeast and the viability of fibroblasts at
the indicated concentrations of the compounds.
Compound
Antifungal Activity MIC (µM)
Cellular Viability (%)
Saccharomyces cerevisiae
Candida albicans
Fibroblasts
3a
3b
3c
3d
3e
4a
4b
4c
>200.0 *
>200.0 *
>400.0 *
>400.0*
50.0
>400.0 *
>400.0 *
>400.0 *
400.0
>200.0 *
>200.0 *
>400.0 *
>400.0 *
100.0
>400.0 *
>400.0 *
>400.0 *
600.0
n.d.
n.d.
n.d.
n.d.
97.3 ± 1.9
n.d.
n.d.
n.d.
4d
94.8 ± 3.5
Miconazole
Fluconazole
100.0
50
0.78
1.56
n.d.
n.d.
n.d. (not determined). * Not achieved at highest concentration tested.
In the evaluation of antifungal activity of imidazoles 4a–4d, we observed that 4d with the
3,4-di-hydroxyphenyl unit had MIC values of 400 and 600 M on Saccharomyces cerevisiae and Candida
µ
albicans, respectively, showing weaker activity than fluconazole and miconazole. The highest antifungal
activity of 4d appeared to be also related with its highest antioxidant capacity in this series. There was
also an increase of antifungal activity with the cyclization, because the 1,2-(dicyano)ethene 3d, which is
the respective linear precursor of imidazole 4d, did not present any antifungal activity.
These results agree with our initial hypothesis, because there is a synergistic effect when the
phenolic moiety is coupled to the nitrogen heterocycle, which was based on previous published
works demonstrating that the antifungal activity increases when a phenolic antioxidant is added to a
solution of antifungal agent [24]. Nevertheless, market antifungal agents do not incorporate a phenolic
moiety, but they are coupled to aromatic groups substituted with chlorine or fluorine atoms [25].
Therefore, these market antifungal agents have low solubility in water and adverse consequences to
the environment [10].
As previously explained, an ideal antifungal must be highly toxic for fungi and not present any
toxicity for other living beings [26]. Therefore, we studied the toxicity of 3e and 4d on fibroblasts using
the lactate dehydrogenase (LDH) release method to obtain toxicological information in mammalian
cells [27]. We observed that these two compounds were not toxic for fibroblasts at concentrations
that presented antifungal activity, because there was no significant release of LDH to the extracellular
media (Table 3). The results showed that 3e and 4d presented antifungal activity at concentrations
that did not result in toxicity for fibroblasts. In addition, the two compounds have the advantage of
being substituted with phenolic units, which form hydrogen bonds with water molecules, and as a
result, they have more solubility in the aqueous medium than compounds substituted with chlorine
and fluorine atoms as the market antifungal agents.
3. Materials and Methods
3.1. Chemistry
Chemicals and solvents were of analytical grade and they were purchased from market sources
and used without purification.
All compounds were fully characterized by spectroscopic data and compared with the data
available in the literature. The IR spectra were recorded on a Bomem MB 104 instrument (ABB Bomem
Inc., Quebec, Canada) using sodium chloride plates and Nujol mulls. The NMR spectra were recorded
on a Varian Unity Plus (1H: 300 MHz and 13C: 75 MHz) or on a Bruker Avance 3400 (1H: 400 MHz
and 13C: 100 MHz) (Bruker Corporation, Billerica, MA, USA), and deuterated DMSO was used as

Molecules 2018, 23, 2530
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solvent. The chemical shifts were expressed in parts per million (ppm), and the coupling constants,
J, were reported in Hertz (Hz). The peak patterns were explained as follows: s, singlet; d, doublet;
t, triplet; m, multiplet. The reactions were monitored by thin layer chromatography (TLC) using
aluminium plates pre-coated with silica gel 60 F254 (Merck) and mixtures of dichloromethane/ethanol
(9/1) and/or chloroform/ethyl acetate/ acetic acid (16/8/1) as an eluent. The melting points were
determined on a Stuart SMP3 melting point apparatus.
3.1.1. General Procedure for the Synthesis of the 1-amino-2-arylidenamine-1,2-(dicyano)ethenes
(3a–3e)
One drop of concentrated sulphuric acid (0.01 mL) was added to a suspension of
diaminomaleonitrile 1 and benzaldehyde 2 (1 molar equivalent) in ethanol. The reaction was stirred
for 10 minutes at room temperature. The yellow solid was filtered, washed with cold diethyl ether,
and identified as the title compound 3.
1-Amino-1,2-dicyano-2-(20-hydroxybenzylidenamine) ethene (3a). 1 (0.65 g, 6.0 mmol), 2-hydroxibenzaldehyde
2a (0.65 mL, 6.08 mmol), ethanol (10 mL). Yellow solid identified as 3a (1.24 g, 97%). Mp 251–254 ◦C.
IR (NaCl): 3465, 3404, 3343, 3191, 2244, 2207, 1626, 1615, 1608, 1562, 1496, 1279, 1230, 1213, 1157, 1118.
1H-NMR (DMSO-d6, 300 MHz) 6.88 (t, 1H, J = 7.8 Hz, H05); 6.92 (dd, 1H, J1 = 8.1 Hz, J2 = 0.6 Hz, H03),
δ
7.32 (dt, 1H, J1 = 7.8 Hz, J2 = 1.8 Hz, H04), 8.03 (dd, 1H, J1 = 7.8 Hz, J2 = 1.8 Hz, H06), 8.58 (s, 1H, H4),
7.84 (s, 2H, NH2), 10.42 (s, 1H, OH0). 13C-NMR (DMSO-d6, 300 MHz) 158,2 (C02), 152.8 (C4), 133.2 (C04),
δ
128.9 (C06), 126.1 (C1), 121.2 (C01), 119.4 (C05), 116.4 (C03), 114.6 (CN), 113.4 (CN), 103.4 (C2). Anal. Calcd.
for C11H8N4O: C, 62.3; H, 3.8; N, 26.4. Found: C, 62.3; H, 3.8; N, 26.1.
1-Amino-1,2-dicyano-2-(3´-hydroxibenzilidenamine) ethene (3b). 1 (0.65 g, 6.0 mmol), 3-hydroxybenzaldehyde
◦
2b (0.73 g, 6.0 mmol), ethanol (8 mL). Yellow solid identified as 3b (1.04 g, 82%). Mp 231–233 C. IR (NaCl):
1
3401, 3379, 3299, 3205, 2244, 2205, 1625, 1611, 1587, 1561, 1491, 1396, 1288, 1225, 1215, 1150. H-NMR
(DMSO-d6, 300 MHz)
7.37 (t, 1H, J = 2.1, H02), 7.43 (d, 1H, J = 8.1 Hz, H06), 7.88 (s, 2H, NH2), 8.16 (s, 1H, H4), 9.64 (s, 1H, OH0).
δ
6.90 (ddd, 1H, J1 = 8.1 Hz, J2 = 2.4, J3 = 0.9 Hz, H04), 7.25 (t, J = 8.1 Hz,1H, H05),
13C-NMR (DMSO-d6, 300 MHz)
δ
157.7 (C03), 155.4 (C4), 136.8 (C01), 129.7 (C05), 126.8 (C1), 120.3 (C06),
118.8 (C04), 115.3 (C02), 114.5 (CN), 113.8 (CN), 102.7 (C2). Anal. Calcd. for C11H8N4O: C, 62.3; H, 3.8; N,
26.4. Found: C, 62.3; H, 3.9; N, 26.3.
1-Amino-1,2-dicyano-2-(4´-hydroxybenzilidenamine) ethene (3c). 1 (0.65 g, 6.0 mmol), 4-hydroxybenzaldehyde
◦
2c (0.73 g, 6.0 mmol), ethanol (10 mL). Yellow solid identified as 3c (1.11 g, 87%). Mp 226–228 C. IR (NaCl):
3455, 3410, 3322, 3272, 3184, 2231, 2207, 1625, 1610, 1589, 1567, 1512, 1306, 1264, 1235, 1207, 1162, 1112.
1H-NMR (DMSO-d6, 300 MHz) 6.82 (d, 2H, J = 8.7 Hz, H03+5), 7.65(s, 2H, NH2), 7.86 (d, 2H, J = 8.7 Hz,
δ
H02+6), 8.14 (s, 1H, H4), 10.21 (s, 1H, OH0). 13C-NMR (DMSO-d6, 300 MHz)
δ
161.0 (C0p), 155.1 (C4),
131.2 (C0o), 126.9 (C01), 125.5 (C1), 115.7 (C0m), 114.7 (CN), 114.0 (CN), 103.4 (C2). Anal. Calcd. for
C11H8N4O.1,1H2O: C, 57.0; H, 4.4; N, 24.2. Found: C, 57.2; H, 4.4; N, 24.0.
1-Amine-1,2-dicyano-2-(3
´
,4´
-dihydroxybenzilidenamine) ethene (3d).
1 (0.32 g, 3.0 mmol), 3,4-
dihydroxybenzaldehyde 2d (0.41 g, 3.0 mmol), ethanol (1.5 mL). Yellow solid identified as 3d (0.55 g,
◦
81%). Mp 219–222 C. IR (NaCl): 3465, 3349, 3297, 3169, 2230, 2201, 1603, 1590, 1565, 1528, 1340,
1
1299, 1264, 1193, 1167, 11002. H-NMR (DMSO-d6, 300 MHz)
δ
6.81 (d, 1H, J = 8.1, H05), 7.29 (dd, 1H,
0
J1 =08.1 Hz, J2 = 2.0 Hz, H 6), 7.40 (d, 1H, J = 2.0 Hz, H 2), 7.60 (s, 2H, NH2), 8.06 (s, 1H, H4), 9.15 (s, 1H,
0
0 0 0
OH ), 9.80 (s, 1H, OH ). 13C-NMR (DMSO-d6, 300 MHz)
δ 155.6 (C4), 149.7 (C 4), 145.7 (C 3), 127.4 (C 1),
125.3 (C1), 122.8 (C06), 115.5 (C02), 115.5 (C05), 114.8 (CN), 114.0 (CN), 103.6 (C2). Anal. Calcd. for
C11H8N4O2.1,1H2O: C, 53,5; H, 4.1; N, 22.7. Found: C, 53.5; H, 4.1; N, 22.7.
1-Amino-1,2-dicyano-2-(3´,4´,5´-trihydroxybenzilidenamine)ethene (3e). 1 (0.22 g, 2.0 mmol), 3,4,5-
trihydroxybenzaldehyde 2e (0.34 g, 2.0 mmol), ethanol (1 mL). Yellow solid identified as 3e (0.45 g,
92%). Mp > 300 ◦C (dec.). IR (NaCl): 3473, 3445, 3359, 3256, 3182, 2242, 2205, 1606, 1591, 1561, 1529,
1
1401, 1338, 1311, 1231, 1190, 1177, 1136. H-NMR (DMSO-d6, 300 MHz)
δ
6.93 (s, 2H, H02+6), 7.56 (s,

Molecules 2018, 23, 2530
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0
0
δ 156.1 (C4), 146.1 (C m),
2H, NH2), 7.98 (s, 1H, H4), 9.09 (s, >2H; OH ). 13C-NMR (DMSO-d6, 300 MHz)
138.0 (C0p), 126.1 (C01), 125.1 (C1), 114.8 (CN), 114.1 (CN), 108.7 (C0o), 103.7 (C2). Anal. Calcd. for
C11H8N4O3.0,1H2O: C, 53.7; H, 3.4; N, 22.8. Found: C, 53.7; H, 3.5; N, 22.6.
3.1.2. General Procedure for the Synthesis of the 2-aryl-4,5-dicyano-1H-imidazoles (4a–4d)
Manganese dioxide was added to a solution of
was kept under reflux until TLC indicated the complete consumption of reagent
3
in ethyl acetate/acetonitrile, and the mixture
. The manganese
3
dioxide suspension was filtered using fiberglass paper, and the solvent was partially removed under
reduced pressure. The yellow solid was filtered, washed with cold dichloromethane, and identified as
the title compound 4.
4,5-Dicyano-2-(2´-hydroxyphenyl)-1H-imidazole (4a). 3a (0.30 g, 1.42 mmol), manganese dioxide (6.16 g),
ethyl acetate/acetonitrile◦(50/5 mL) and reflux for 14 hours. The yellow solid was identified as 4a
(0.15 g, 50%). Mp > 300 C (dec.). IR (NaCl): 3441, 3335, 3240, 2250, 2237, 1675, 1607, 1551, 1521,
1473, 1403, 1351, 1308, 1293, 1264, 1226, 1154, 1111. 1H-NMR (DMSO-d6, 300 MHz)
δ
6.94 (t, 1H,
J = 7.8 Hz, H05), 7.02 (d, 1H, J = 7.8 Hz, H03), 7.34 (dt, 1H, J1 = 7.8 Hz, J2 = 1.5 Hz, H04), 7.94 (dd, 1H,
0 0
J1 = 7.8 Hz, δ 155.4
J2 = 1.5 Hz, H 6), 10.00–13.00 (s, >1H, NH + OH ). 13C-NMR (DMSO-d6, 300 MHz)
(C02), 149.3 (C2), 132.1 (C04), 128.7 (C06), 119.7 (C05), 116.5 (C03), 113.8 (C01), 111.5 (CN), not visible
(C4/C5). Anal. Calcd. for C11H6N4O.0,9H2O: C, 58.3; H, 3.5; N, 24.7. Found: C, 58.6; H, 3.6; N, 24.4.
4,5-Diciano-2-(3´-hidroxifenil)-1H-imidazole (4b). 3b (0.30 g, 1.42 mmol), manganese dioxide (6.16 g) ethyl
acetate/acetonitrile (50/5 mL) and reflux for five days. The yellow solid was identified as 4b (0.21 g,
70%). Mp 264–267 ◦C. IR (NaCl): 3471, 3369, 3223, 2248, 2242, 1664, 1612, 1590, 1567, 1529, 1490, 1409,
1
1392, 1328, 1287, 1229, 1207, 1107. H NMR (DMSO-d6, 300 MHz) 0
δ
6.91 (ddd, 1H, J1 =07.8 Hz, J2 2.4
0
0
Hz, J3 = 1.2 Hz, H 4), 7.33 (t, 1H, J = 7.8 Hz, H 5), 7.35-7.50 (m, 2H, H 2+6), 9.86 (s, 1H, OH ), 12.00–16.00
(s, <1H, NH). 13C-NMR (DMSO-d6, 300 MHz) 157.9 (C03), 151.1 (C2), 130.4 (C05), 128.8 (C01), 118.0
δ
(C04), 117.0 (C06), 113.0 (C02), 111.4 (CN), not visible (C4/C5). Anal. Calcd. for C11H6N4O.0,6H2O: C,
59.6; H, 3.3; N, 25.3. Found: C, 59.8; H, 3.5; N, 25.0.
4,5-Dicyano-2-(4´-hydroxyphenyl)-1H-imidazole (4c). 3c (0.30 g, 1.42 mmol), manganese dioxide (6.16 g),
ethyl acetate/acetonitrile ◦(30/5 mL) and reflux for eight days. The yellow solid was identified as 4c
(0.19 g, 63%). Mp 279–281 C. IR (NaCl): 3435, 3387, 3148, 2242, 2233, 1648, 1613, 1593, 1492, 1403, 1311,
1
1281, 1241, 1177, 1123. H-NMR (DMSO-d6, 300 MHz)
δ
6.89 (d, 2H, J = 9.0 Hz, H’3+5), 7.80 (d, 2H,
159.9
J = 9.0 Hz, H’2+6), 10.16 (s, 1H, OH’), 12.00–16.00 (s, <1H, NH). 13C-NMR (DMSO-d6, 300 MHz)
δ
(C0p), 151.5 (C2), 128.2 (C0o), 118.6 (C01), 116.0 (C0m), 111.6 (CN), not visible (C4/C5). Anal. Calcd. for
C11H6N4O.1,9H2O: C, 54.1; H, 4.0; N, 22.9. Found: C, 54.0; H, 4.2; N, 22.8.
4,5-Dicyano-2-(3´,4´-dihydroxyphenyl)-1H-imidazole (4d). 3d (0.25 g, 1.10 mmol), manganese dioxide
(2.38 g), ethyl acetate/acetonitrile (50/10 mL) and reflux for 24 hours. The yellow solid was identified
as 4d (0.14 g, 55%). Mp > 300 ◦C (dec.). IR (NaCl): 3484, 3366, 3183, 2245, 2236, 1656, 1620, 1605, 1543,
1
1506, 1397, 1323, 1289, 1256, 1227, 1183, 1118. H-NMR (DMSO-d6, 300 MHz)
δ 6.84 (d, 1H, J = 8.1 Hz,
H05), 7.28 (dd, 1H, J1 = 8.1 Hz, J2 = 2.1 Hz, H06), 7.38 (d, 1H, J = 2.1 Hz, H02), 9.38 (s, 1H, OH0), 9.63 (s,
1H, OH0) and the NH signal is not visible on spectrum. 13C-NMR (DMSO-d6, 300 MHz)
δ 151.8 (C2),
148.3 (C04), 145.7 (C03), 119.1 (C01), 118.3 (C06), 116.0 (C05), 113.7 (C02), 111.7 (CN), not visible (C4/C5).
Anal. Calcd. for C11H6N4O2.1,2H2O: C, 53.2; H, 3.4; N, 22.6. Found: C, 53.2; H, 3.4; N, 22.5.
3.2. Antioxidant Capacity
3.2.1. Cyclic Voltammetry Technique
Cyclic voltammograms were obtained in a conventional three-electrode cell. The working
electrode was a glassy carbon electrode; a saturated calomel electrode (SCE) and a platinum wire were

Molecules 2018, 23, 2530
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used as reference electrode and counter electrode, respectively. Cyclic voltammograms were recorded
at a scan rate of 50 mV s-1 and all solutions were purged with argon before assays.
Solutions of the test compounds (1.0 mM) in a 1:1 (v/v) water/ethanol solution containing 0.14 M
KCl and phosphate buffer (1.8 mM KH2PO4 and 10.1 mM Na2HPO4) were prepared, and the pH of
the solutions was adjusted to pH = 7.4.
The experiments were done using Autolab PGSTAT 30 running with GPES (General Purpose
Electrochemical System) version 4.6 software PG (Ecochimie, Utrecht, Netherlands).
3.2.2. DPPH Radical Method
The DPPH• scavenging activity of the compounds was measured as already described [13].
A 0.002% ethanolic DPPH solution was used in the assays, and trolox was used as reference antioxidant.
The IC50 value was determined for the compounds with the lowest oxidation potential from each
chemical series.
3.2.3. Deoxyribose Degradation Method
Antioxidant effects of compounds on hydroxyl radicals (HO ) were determined as described
previously [14]. Reaction mixtures contained deoxyribose, KH2PO4–KOH buffer, ascorbic acid,
FeCl3, H2O2, and synthesized compounds (assessed at IC50 concentration obtained in DPPH assay).
After one hour of incubation at 37 ◦C, the colour appeared by adding 1 mL of thiobarbituric acid and
1 mL of trichloroacetic acid. The reaction mixture was then heated in boiling water for 15 min, and the
absorbance of the resulting solutions was measured at 532 nm.
3.3. Determination of Antifungal Activity
MIC values were evaluated by the method of liquid microdilution described in the standard
document CLSI M27-3A [28,29]. The antifungal activity of the synthesized compounds was evaluated
for the yeasts Saccharomyces cerevisiae PYCC 4072 and Candida albicans PYCC 3436T. For all tests,
a culture of yeasts was prepared in agar tubes with medium YEPD and agar to guaranty purity and
an excellent growth. An inoculum was transferred to a culture medium RPMI-1640, with 0.165 M
of buffer 4-morpholinepropanesulfonic acid (Merck, Darmstadt, Germany), pH = 7.0. An aliquot of
0.1 mL of this cellular suspension was added to 0.1 mL of solution with the synthesized compound in
microplate wells, with the final density of the inoculum being 2.25
×
103 cells/mL. The inoculated
microplaques were incubated for 48 hours at temperatures of 30 and 37 ◦C for Saccharomyces cerevisiae
and Candida albicans, respectively. The MIC values were then measured at 640 nm. Fluconazole and
miconazole (Pfizer Inc., New York, NY, USA) were used as the reference controls.
3.4. Analysis of Toxicity in Mammalian Fibroblast
Cellular viability was determined by the lactate dehydrogenase (LDH) method, as already
described [27]. The fibroblast cell line L929 (European Collection of Animal Cell Cultures) was used
in the tests. Cells were incubated with the compounds and a small volume of medium was collected
for the determination of extracellular LDH. The cells were after lysed to obtain the intracellular LDH.
The enzyme activity was evaluated at 340 nm. The LDH released to the extracellular medium was
described as a percentage of the total LDH activity.
4. Conclusions
Compounds 3e and 4d substituted with 3,4-di-hydroxyphenyl or 3,4,5-tri-hydroxyphenyl units
presented high antioxidant activities, as determined by cyclic voltammetry, DPPH , and deoxyribose
degradation assays. These two compounds also displayed antifungal activity against the yeasts
Saccharomyces cerevisiae and Candida albicans at concentrations not toxic to fibroblasts.

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The cyclization of 1,2-(dicyano)ethene 3d producing imidazole 4d, increased the antifungal activity.
In addition, we observed a synergistic effect between the nitrogen heterocycle and the phenolic unit,
combined in the same molecule. In this work, we describe the synthesis of compounds 3e and 4d with
dual antioxidant/antifungal activity involving two original scaffolds.
Author Contributions: Conceptualization, M.F.P. and F.A.; Funding acquisition, M.F.P.; Investigation, J.S.,
F.F. and F.A.; Methodology, A.B., M.J.S. and M.F.P.; Supervision, A.B, O.C., M.J.S. and F.A.; Data curation,
M.C.; Writing—original draft, A.B., M.C., M.J.S, M.F.P. and F.A.
Funding: This research was funded by the Portuguese Fundação para a Ciência e Tecnologia (PPCDT/QUI/59356).
Filipe Areias gratefully acknowledges a post-PhD grant from the Portuguese FCT (SFRH/BPD/26106).
Conflicts of Interest: The authors declare no conflict of interest.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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