Characterization of antimicrobial, antioxidant, and leishmanicidal activities of Schiff base derivatives of 4-aminoantipyrine

Article abstract of DOI:10.3390/molecules24152696

Our main interest is the characterization of compounds to support the development of alternatives to currently marketed drugs that are losing effectiveness due to the development of resistance. Schiff bases are promising biologically interesting compounds

Full text of DOI:10.3390/molecules24152696

Article
Characterization of Antimicrobial, Antioxidant, and
Leishmanicidal Activities of Schiff Base Derivatives
of 4-Aminoantipyrine
1,
1,2,†
1
3
Rommy Teran †, Rommel Guevara , Jessica Mora , Lizeth Dobronski ,
3,4
2,5
1,2
6
Olalla Barreiro-Costa , Timo Beske , Jorge Pérez-Barrera , Ramiro Araya-Maturana ,
Patricio Rojas-Silva , Ana Poveda * and Jorge Heredia-Moya
3,4
1,2,
3,4,
*
1
Facultad de Ciencias Químicas, Universidad Central del Ecuador, Quito 170521, Ecuador
Instituto de Investigación en Salud Pública y Zoonosis-CIZ, Universidad Central del Ecuador, Quito
170521, Ecuador
Centro de Investigación Traslacional, Universidad De Las Américas, Quito 170503, Ecuador
Centro de Investigación Biomédica (CENBIO), Facultad de Ciencias de la Salud Eugenio Espejo,
Universidad UTE, Quito 170527, Ecuador
Facultad de Medicina Veterinaria, Universidad Central del Ecuador, Quito 170521, Ecuador.
Instituto de Química de Recursos Naturales, Programa de Investigación Asociativa en Cáncer Gástrico
(PIA-CG), Universidad de Talca, Talca 3460000, Chile
2
3
4
5
6
* Correspondence: apoveda@uce.edu.ec (A.P.); jorgeh.heredia@ute.edu.ec (J.H.-M.).
† These authors contributed equally to this work
Received: 26 March 2019.; Accepted: 08 April 2019.; Published: 24 July 2019
Abstract: Our main interest is the characterization of compounds to support the development of
alternatives to currently marketed drugs that are losing effectiveness due to the development of
resistance. Schiff bases are promising biologically interesting compounds having a wide range of
pharmaceutical properties, including anti-inflammatory, antipyretic, and antimicrobial activities,
among others. In this work, we have synthesized 12 Schiff base derivatives of 4-aminoantipyrine. In
vitro antimicrobial, antioxidant, and cytotoxicity properties are analyzed, as well as in silico
predictive adsorption, distribution, metabolism, and excretion (ADME) and bioactivity scores.
Results identify two potential Schiff bases: one effective against E. faecalis and the other with
antioxidant activity. Both have reasonable ADME scores and provides a scaffold for developing
more effective compounds in the future. Initial studies are usually limited to laboratory in vitro
approaches, and following these initial studies, much research is needed before a drug can reach
the clinic. Nevertheless, these laboratory approaches are mandatory and constitute a first filter to
discriminate among potential drug candidates and chemical compounds that should be discarded.
Keywords: Schiff base synthesis; Leishmania; antimicrobial activity; cytotoxicity
1. Introduction
In the last decades, antibiotic resistances have been described in several microorganisms,
including those having multiresistance phenotypes [1–4]. This is an alarming situation, as noted by
the World Health Organization, and many researchers have focused on the development of new
therapeutic alternatives. Oldfield and Feng have proposed different strategies to develop a new
generation of antibiotics (resistance-resistant antibiotics) that should have the characteristic of acting
against multiple targets simultaneously [5]. Alternatively, other authors have suggested the
development of new antibiotic classes with novel chemical properties not present in existing classes
of antibiotics [6]. In this context, Schiff bases offer an alternative that has not been adequately
explored.
Molecules 2019, 24, 2696; doi:10.3390/molecules24152696
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Molecules 2019, 24, 2696
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Schiff bases have received much attention due to their wide applications in several fields,
ranging from industrial uses such as advanced nanomaterials [7] to chemotherapeutics and new drug
development [8–11]. However, even regarding the chemotherapeutic applications, the reported roles
are very divergent, sometimes with proposed roles that could seem contradictory. For example,
antioxidant activity has been ascribed to Schiff bases, acting as cell-protective agents [12–15], but
antimicrobial [16,17], antitumor [18,19], analgesic [12], anti-inflammatory [9], and antidiabetic
activities [8,18] have also been reported. A detailed review covering the patented therapeutics
applications of Schiff bases has been published by Khan and collaborators [18]. These diversities of
functions could rely on the wide variety of Schiff base compounds that can interfere with biological
activities. The open question is what is the mechanism(s) of action by which they operate. Recent
publications suggest that different groups of Schiff bases can exert actions through different
mechanisms. For example, some compounds have a potential application as antitumor drugs [18],
but different molecular modes of action could explain this property. While some Schiff bases are able
to intercalate into the DNA helix [6], others display DNA cleavage activity [20,21]. It is not possible
to rule out the possibility that in some compounds, both activities are linked. Also, this DNA-
damaging activity could explain antimicrobial activities in some cases.
Other Schiff bases seem to act by inhibiting specific targets. Some of the most prominent
examples are the patented inhibitors specific for β-secretase, an enzyme present in aberrantly high
levels in Alzheimer’s patients [22]; Schiff base copper complexes that target and inhibit reversibly
caspase enzymes involved in apoptosis [23]; or Schiff bases of thiazoles specific for urease, an enzyme
present in bacteria, fungi, and plants [24] (all of them reviewed by Khan and collaborators [18]).
However, the broad range of described effects of these compounds make it reasonable to think
that a more generalized mechanism may be involved. The role of Schiff bases in a generalized process,
the in vivo nonenzymatic glycation of macromolecules, is very interesting. These reactions take place
between a reducing sugar and a macromolecule—DNA, lipid, or protein—and generate advanced
glycation end products (AGEs) [8,25] through a Schiff base intermediary. The accumulation in blood
of AGE products, closely related to oxidative stress, triggers many health complications associated
with diseases such as ageing, diabetes, Alzheimer’s, and cancer, among others [8,25,26]. A class of
Schiff bases has been described as agents with antiglycation activity, being very promising for
prevention of late diabetic complications [27]. This suggests that some Schiff bases can interfere with
AGE production, possibly indicating a generalized mode of action that can explain, at least partially,
the wide roles and activities of these compounds. Protein glycation leads to crosslinking and protein
aggregation, which indeed constitute a source of oxidative species. Some proteins suffering massive
glycation are immunoglobulins, apolipoprotein, fibrinogen, and transferrin, which have been
associated with several disorders [25]. It is possible that the antioxidant activity described for some
Schiff bases [13,18] is related to the prevention of the protein glycation processes, but to our
knowledge, nothing has been published regarding this; however, this correlation has been reported
for some flavonoids inhibiting the formation of both initial and advanced stages of Maillard reaction
in tissue protein sources [28]. Other mechanisms could operate by trapping of carbonyl compounds,
similar to that reported with OPB-9195, a known inhibitor of in vitro formation of AGEs [29].
The diversified Schiff base structures and their different mechanisms of action can explain the
wide variety of biological activities described for these compounds that deserve more attention as
promising candidates for therapies are being more and more delineated. Some of the Schiff bases
reported with interesting bioactivities are those derived from 4-aminoantipyrine, and early reports
have shown that these compounds have also important antioxidant properties [14], as well as
antifungal and antibacterial activity against several microorganisms [30–33]. However, most of these
studies were performed by antibiograms. Here, we report the synthesis of a series of Schiff base
derivatives of 4-aminoantipyrine [14,30–32,34–37] and their evaluation of antifungal, antibacterial,
(inhibitory concentration of 50% of population (IC50), minimum inhibitory concentration (MIC), and
noninhibitory concentration (NIC)), leishmanicidal, and antioxidant activities, which will provide
useful information to explain the potential chemotherapeutic use of these compounds.
2. Results

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2.1. Synthesis of Schiff Base of 4-Aminoantipyrine
The Schiff bases 3 were synthesized following the scheme depicted in Figure 1. Compound 1
was reacted with the respective benzaldehydes 2a–j to obtain 3a–j, while 4 and 5 were obtained by
reaction with furfural (6) and cinnamaldehyde (7), respectively. The reactions were performed using
ethanol as solvent to give good to excellent yields (Table 1). All compounds were characterized, and
1
all the data obtained agreed with the proposed structures. The H NMR spectra for 3a–j show a singlet
between 9.41 and 9.79 ppm corresponding to the azomethine –CH=N proton, while 5 shows a doublet
at 9.40 ppm. These signals are like those reported in the literature, and as expected, the azomethine
proton for all compounds with electron-donating groups are shifted to a higher field relative to 3a,
except for 3b, which shows a signal at 9.70 ppm, possibly due to an intramolecular hydrogen bond
with the hydroxyl group.
O
R4
R3
H
R1
R4
R2
O
2a-j
R3
R2
N
N
N
R1
O
3a-j
H
O
O
O
6
N
NH2
N
N
N
N
O
4
O
1
H
O
7
N
N
N
5
Figure 1. Synthesis of Schiff base derivatives of 4-aminoantipyrine. Depiction of the scheme followed
to synthesize the Schiff bases described in this study, designated as 3a–j, 4, and 5.
Table 1. Description of substituents R1, R2, R3, and R4 and the obtained synthesis yield (%) of Schiff
bases.
Compounds
R1
H
OH
H
H
H
OH
H
H
H
H
R2
H
H
R3
H
H
OMe
OH
OMe
H
R4
H
H
H
H
H
NO2
H
H
H
Yield (%)
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
4
77
94
86
94
93
85
95
95
97
91
86
90
H
OMe
OH
H
NO2
H
OH
OMe
----
----
H
N(Me)2
OH
OH
----
OMe
----
----
----
----
5
----
2.2. Antimicrobial Evaluation
The in vitro antifungal and antimicrobial activities were determined by testing the Schiff bases
against the fungi Saccharomyces cerevisiae, Candida albicans, and Aspergillus niger and the bacteria
Staphylococcus aureus, Enterococcus faecalis (Gram-positive), Pseudomonas aeruginosa, and Escherichia coli

Molecules 2019, 24, 2696
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(Gram-negative). Additionally, the values of minimum inhibitory concentration (MIC), noninhibitory
concentration (NIC), and inhibitory concentration of 50% of population (IC50) were determined as
described by Lambert et al. [38,39].
Since the Schiff bases have a limited solubility in dimethyl sulfoxide (DMSO), first, we tested the
toxicity of DMSO against the selected microorganism. According to published data [40],
concentrations over 2% DMSO are significantly toxic for S. cerevisiae. Drop test assay (Figure 2)
revealed that wild-type cells from the BY4741 strain grow with no major difference at DMSO
concentrations below 2%, but at concentrations of 3% DMSO or higher, wild-type cells begin to show
diminished viability, and the effect is more acute in sod1 cells. Wild-type cells from W303 background
seem to be more resistant, and sensitivity is observed at around 4% DMSO.
Figure 2. DMSO toxicity. Drop test of serial dilution of yeast cell cultures (wild type from genetic
background W303, wild type from genetic background BY4741, and sod1 mutant from genetic
background BY4741) exposed to different DMSO concentrations ranging from 1 to 6%. A control
without DMSO (−DMSO) is included.
More accurate determinations of noninhibitory concentration (NIC) [38] revealed that toxicity
ranges from a minimum of 1.55% DMSO (E. faecalis) to a maximum of 15.86% (E. coli) (Table 2).
Table 2. MIC, IC50, and NIC values determined for DMSO in the indicated microorganism.
A. niger
C. albicans
S. cerevisiae
E. faecalis
E. coli
S. aureus
MIC
DMSO (%)
IC50
19.22 ± 1.3
4.36 ± 2.0
1.78 ± 0.4
14.98 ± 0.6
8.50 ± 0.5
4.94 ± 0.5
71.47 ± 1.7 35.62
± 1.0
20.02 ± 1.3
8.66 ± 0.6
3.68 ± 0.2
13.81 ± 3.5
16.36 ± 0.33
11.95 ± 2.9
32.64 ± 6.9 20.36 ±
5.2 8.52 ± 0.9
5.04 ± 1.1
NIC
Considering the limited solubility of Schiff bases and the DMSO toxicity, we set up our protocol
at a maximal DMSO working concentration of 2%. Values of NIC, MIC, and IC50 are summarized in
Table 3, and all parameters were compared with that of the commercial drugs geneticin (GN) for S.
cerevisiae, voriconazole (VR) for A. niger and C. albicans, and ciprofloxacine (CP) for all bacteria
studied. It is important to note that although in the literature there appear several reports regarding
the activities of some of Schiff bases reported here with these microorganisms [14,30–34,41–48], those
tests were done using different methods of evaluation in different strains, when it was reported.
Overall, A. niger and S. aureus are inhibited by Schiff base 3f; C. albicans by 3b and 3f; S. cerevisiae by
5; E. faecalis by 3a; and E. coli by 3b, 3d, 3f, and 5. Only P. aeruginosa was not sensitive to any of the
assayed compounds. Interestingly, different microorganisms are inhibited by different compounds,
revealing specific activities against specific microorganisms. The specificity is a property required in
drug candidates for applications in pharmacology. Compounds 3c, 3e, 3g, 3h, 3i, 3j, and 4 did not
show any activity against the tested microorganisms, and the activity of 3e, 3f, and 5 had not been
reported previously. Schiff base 3f is the only compound that showed activity with all bacteria, except
E. faecalis, and 3d had the lowest activity (MIC of 10.2 ± 0.1 μg/mL). When the observed MIC
concentration was >200 μg/mL or out of the assay range concentration, the drug was considered
ineffective (reported as NE, no effect).
Next, we evaluated qualitatively if the inhibitory effect is exerted by killing the microorganism
(bactericidal/fungicidal effect) or by preventing growth (bacteriostatic/fungistatic effect). After
incubating cells in the presence of different concentration of Schiff bases for 24 h, cells were plated

Molecules 2019, 24, 2696
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on media free of drug. Results indicated that only compound 5 has a fungicidal mechanism on S.
cerevisiae, since cells are not able to recover after exposure (see Supplementary Figure S16A). The rest
of the Schiff bases assayed showed a bacteriostatic behavior, allowing the growth of microorganism
on drug-free media after 24 h of exposure (Supplementary Figure S16B).
Finally, Schiff bases were evaluated against L. mexicana parasites in promastigote form by (3-
(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results are shown in
Table 3. Some compounds were inactive or too weak (a cutoff value of IC50 > 200 μM was considered),
but others showed leishmanicidal activity to different extents, particularly 5 and 3f, with the latter
being the most powerful. Pentamidine and amphotericin were included as controls.
In conclusion, Schiff bases 3f and, to a lesser extent, 5 show a wide range of activities against
several bacteria. Also, 3a, 3b, and 3d are interesting candidates, with narrow but specific activity.
Compounds 3f and 5 showed the best activity against parasites. So, we will focus our next efforts in
evaluation of these compounds.

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Table 3. Growing inhibitory parameters (MIC, NIC, and IC50, expressed in μg/mL), determined by serial microdilution in 96-well plates, for Schiff bases 3a–j, 4, and 5. The
sensitivity was tested for the microorganism indicated in the first column. Geneticin and voriconazole were used as control against fungi, ciprofloxacin for bacteria, and
amphotericin and pentamidine for leishmania.
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
4
5
GN
CP
VR
AP
PT
MIC
A. niger IC50
NIC
32.6 ± 2.1
10.6 ± 0.8
3.4 ± 0.5
15.6 ± 0.9
10.4 ± 0.6
7.5 ± 1.1
55.9 ± 6.2 179.2 ± 59.5
17.9 ± 1.4 105.1 ± 22.1
5.6 ± 0.9
1.2 ± 0.05
0.2 ± 0.02
0.05 ± 0.01
29.7 ± 3.52
0.9 ± 0.07
0.00 ± 0.00
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
ND
NA
NA
NA
MIC
C. albicans IC50
NIC
MIC
S. cerevisiae IC50
NIC
13.2 ± 1.6
5.8 ± 2.5
3.0 ± 2.2
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
ND
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
76.7 ± 5.7 93.7 ± 0.9
22.8 ± 0.3 59.9 ± 0.9
6.6 ± 0.7
NE
NE
NE
NA
NA
NA
NA
NA
NA
63.7 ± 8.7
44.5 ± 1.7
MIC 32.3 ± 11.7
E. faecalis IC50 12.2 ± 3.3
1.15 ± 0.42
0.49 ± 0.07
0.30 ± 0.07
0.44 ± 0.09
0.10 ± 0.01
0.06 ± 0.02
0.15 ± 0.009
0.07 ± 0.004
0.06 ± 0.003
0.47 ± 0.1
0.94 ± 0.0
ND
NE
NE
NE
NA
NIC
MIC
3.6 ± 0.7
27.7 ± 4.7
9.5 ± 1.5
3.3 ± 1.5
47.7 ± 1.0
15.8 ± 0.1
4.9 ± 0.2
S. aureus IC50
NIC
MIC
E. coli IC50
NIC
NE
NE
NE
NE
NE
NE
NA
NA
NA
NA
49.8 ± 5.0
12.1 ± 0.5
2.8 ± 0.5
10.2 ± 0.0
3.3 ± 0.8
2.3 ± 0.1
22.6 ± 14.1
12.4 ± 6.2
5.1 ± 2.4
NE
NE
NE
MIC
P. aeruginosa IC50
NIC
NE
NE
NE
NE
MIC
*
L. mexicana IC50
NE
NE
5.3 ± 2.8
81.5 ± 12.5
NA
0.18 ± 0.07 4.50 ± 0.46
NIC
*Leishmanicidal activity expressed in μM
MIC: minimum inhibitory concentration; NIC: noninhibitory concentration; IC50: inhibitory concentration of 50% of population. NA: Not applicable; ND: not determined; NE: no
effect. GN: geneticin; CP: ciprofloxacin; VR: voriconazole; AP: amphotericin; PT: pentamidine.

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2.3. Evaluation of Antioxidant/Oxidative Activity
All the synthesized compounds were evaluated for antioxidant activity by the N,N-diphenyl-N'-
picrylhydrazyl (DPPH) assay (Table 4). All compounds evaluated showed lower radical scavenging
activity than the standards ascorbic acid (entry 13), quercetin (entry 14), and caffeic acid (entry 15).
In general, the presence of hydroxyl groups in the para position of the aromatic ring (3d, 3i, and 3j)
confers a better radical scavenging activity compared to those with hydroxyl groups in other
positions or with other electron-donating groups, with the exception of 3h, which contains one
dimethylamino group (entry 8) that also can stabilize the radical by resonance. Compounds 3d, 3h,
3i, and 3j possess the best antioxidant activities, comparable to the standards, but only 3d presents
activity against bacteria (E. coli), while 3f and 5 are effective against leishmania promastigotes.
Table 4. Antioxidant activities of Schiff bases. DPPH scavenging IC50 values (μM), for Schiff bases 3a–
j, 4, and 5. Ascorbic acid, quercetin, and caffeic acid were used as controls for antioxidant activity.
DPPH scavenging activity
Entry
Compound
IC50 (µM)
1
2
3
3a
3b
3c
>200
NA
>200
4
5
3d
3e
28.33 ± 4.35
>200
6
3f
>200
7
8
9
10
11
12
13
14
15
3g
3h
3i
3j
4
>200
129.4 ± 18.7
18.9 ± 2.4
15.7 ± 3.2
>200
5
>200
ascorbic acid
quercetin
caffeic acid
14.5 ± 2.2
7.3 ± 1.0
16.2 ± 2.4
DPPH: N,N-diphenyl-N'-picrylhydrazyl.
Next, we wanted to evaluate the oxidative activity. To do this, drop test assays were performed
with wild-type (W303 and BY4741 backgrounds) and sod1 (BY4741 background) yeast cells in the
presence of 200 μg/mL of the analyzed Schiff bases (Figure 3). The SOD1 gene encodes superoxide
dismutase 1, an enzyme with a major role in detoxifying ROS species generated during metabolism
or because of environmental exposure. sod1 mutant cells are unprotected against oxidative agents,
decreasing survival in their presence. Figure 3A shows the relevant results obtained with drugs 3b
and 5, where sod1 cells are sensitive to these compounds compared to the control (no drug, DMSO),
after 48 h of incubation at 25 °C. Also, wild-type cells are shown as control, unaffected by the Schiff
bases. Figure 3B and Table 5 show the graphic representation of optical density (OD600) of wild and
sod1 cells exposed to increasing concentrations of compounds 3b and 5, and the MIC, IC50, and NIC
values obtained for these compounds, respectively.
These results reveal that 3b and 5 act by an oxidative mechanism, in accordance with the results
obtained in the DPPH assay, which detected no significant antioxidant activity of these compounds.

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Figure 3. Oxidative effect. (A) Drop test of serial dilution of yeast cell cultures (wild type from genetic
background W303, wild type from genetic background BY4741, and sod1 mutant from genetic
background BY4741) exposed to Schiff bases at 200 μg/mL. A control without drug and with 2%
DMSO are included. (B) Graphic representation of optical density (OD600) of wild-type (black line)
and sod1 (grey line) cells (BY4741) exposed to increasing concentrations of compounds 3b and 5.
Table 5. MIC, IC50, and NIC values obtained for wild type and sod1 strains exposed to compounds 3b
and 5.
Wt
sod1
MIC
IC50
NIC
208.2 ± 4.0
192.0 ± 0.6
178.0 ± 2.1
65.2 ± 0.2
22.6 ± 0.6
8.1 ± 0.4
164.1 ± 0.1
100.7 ± 4.2
53.3 ± 0.1
39.4 ± 0.9
19.5 ± 0.8
10.4 ± 1.1
3b
MIC
NIC IC50
5
2.4. Toxicity in Mammalian Cell Lines
The specificity and selectivity for the pathogen target is a key point for any potential drug. To
evaluate these, we determined the cytotoxicity (% of living cells) of mammal macrophage cells
(murine RAW 264.7, ATCC TIB 71) after exposure to Schiff bases; see Table 6. Cytotoxicity was
determined at 400 μM of each Schiff base, except for those marked with an asterisk (*), which were
assayed at 300 μM because of the limited solubility inherent to these compounds. Compounds 3d, 3f,
3h*, 3j, and 5 showed high cytotoxicity with ≤20% of cells surviving, with 3f and 5 being the most
cytotoxic, thus making them useless for pharmacological purposes, at least in this simple formulation
(chemical compound directly in solution). Cytotoxicity data restrain the potential application to 3a
and 3i, the only two compounds producing viability of >25% at 0.1165 and 0.1293 mg/mL,
respectively. A control with saponine was included, giving an IC50 value of 0.1400 mg/mL. 3a and 3i
are effective against E. faecalis and as an antioxidant, respectively. Overall, these results are an early
filter indicating a potential application for compounds 3a and 3i, which deserve to be analyzed more
deeply.

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Table 6. Cytotoxicity of the investigated compounds. Cytotoxicity is expressed as % of live cells after
exposure to 400 μM of Schiff bases (compounds with limited solubility were evaluated at 300 μM).
Equivalent concentrations are expressed in mg/mL units for comparison to the IC50 of saponine (in
mg/mL), used as control.
Entry Compound % live cells µM mg/mL
1
2
3
3a
3b
3c
3d
27.1 ± 3.4 400
19.3 ± 0.2 400
26.1 ± 1.9 400
17.6 ± 0.4 400
0.1165
0.1229
0.1285
0.1349
4
5
6
3e
3f
27.1 ± 0.8 300* 0.1012
4.5 ± 1.4 400 0.1409
7
8
3g
3h
22.1 ± 0.6 300* 0.1009
20.4 ± 1.7 300* 0.1003
9
3i
3j
4
5
34.4 ± 2.4 400
14.2 ± 0.6 400
23.8 ± 3.4 400
0.1293
0.1470
0.1125
0.1270
10
11
12
13
2.7 ± 0.5
400
Saponine**
IC50 (50%) NA 0.1410 ± 0.02
*Compounds with limited solubility were evaluated at 300 μM.
**The saponine used was a commercial mix of different saponines, so the μM cannot be calculated.
2.5. Evaluation of Pharmacokinetic and Drug-Like Properties
To evaluate the drug-likeness of a compound, Lipiniski’s rule of five (ROF) is usually used. This
rule states that good absorption or permeation are more likely when: (a) there are no more than five
hydrogen bond donors (HBD); (b) no more than 10 hydrogen bond acceptors (HBA); (c) formula
weight less than 500; and (d) n-octanol/water partition coefficient (log P) is less than 5. Two or more
violations of ROF suggest the probability of problems in bioavailability [49]. One of the big problems
with the development of new drugs is that they usually fail before reaching the clinics due to poor
pharmacokinetics, and these physicochemical drug descriptors provide a useful tool for evaluating
drug activity. These descriptors were calculated for all the synthesized compounds 3a–j, 4, and 5
using the Osiris DataWarrior software (Table 7) [50]. Additionally, the bioactivity scores of all
compounds were calculated using the Molinspiration cheminformatics software (Table 8), and all
parameters were compared with those of the standard drugs voriconazole (VR) and ciprofloxacin
(CP).
Table 7. Theoretical prediction of adsorption, distribution, metabolism, and excretion (ADME)
properties of Schiff bases 3a–j, 4, and 5, calculated using Osiris DataWarrior software. Voriconazol
and ciprofloxacin were included as controls.
Compou
nd
3a
cLog cLog
MW
HBA HBD nrotb
PSA
M
T
R
I
DL DS
P
S
291.35
307.35
321.38
337.38
337.38
352.35
336.35
334.42
323.35
367.40
281.31
317.39
349.32
331.35
4
5
5
6
6
8
7
5
6
7
5
4
6
6
0
1
0
1
1
1
0
0
2
1
0
0
1
2
3
3
4
4
4
4
4
4
3
5
3
4
5
3
35.91 high
56.14 high
45.14 high
65.37 high
65.37 high
101.96 high
81.73 high
39.15 high
76.37 high
74.60 high
49.05 high
35.91 high
NE
NE
NE
low
NE
NE
NE
high
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
NE
2.16 −3.24 4.25 0.52
1.81 −2.94 4.25 0.53
2.09 −3.26 4.24 0.51
1.74 −2.96 4.24 0.41
1.74 −2.96 4.24 0.52
0.67 −3.40 −0.85 0.33
1.01 −3.70 −0.85 0.32
2.05 −3.28 4.56 0.30
1.47 −2.65 4.25 0.53
1.67 −2.98 4.24 0.50
1.35 −2.92 4.08 0.54
2.18 −3.53 4.28 0.50
1.48 −3.23 4.08 0.84
−1.53 −3.32 2.05 0.82
3b
3c
3d
3e
3f
3g
3h
3i
3j
4
5
VR
CP
76.72
72.88
NE
NE

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MW: molecular weight; HBA: number of hydrogen bond acceptors; HBD: number of hydrogen bond
donors; nrotb: number of rotatable bonds; PSA: polar surface area; M: mutagenicity; T;
tumorigenicity; R: reproductive effect; I: irritant effect; cLogP: logarithm of compound partition
coefficient between n-octanol and water; cLogS: logarithm of compound aqueous solubility; DL:
drug-likeness; DS: drug score. VR: voriconazole; CP: ciprofloxacin. NE: no effect.
Table 8. Predicted bioactivity scores of the synthesized Schiff bases 3a–j, 4, and 5, calculated using
Molinspiration Cheminformatics Osiris software. Voriconazol and ciprofloxacin were included as
controls.
Entry Compound GPCR
ICM
−1.12
−1.14
−1.10
−1.03
−1.03
−1.07
−1.05
−1.01
−1.01
−0.96
−1.26
−0.84
0.17
KI
NRL
−1.08
−0.92
−0.98
−0.90
−0.90
−0.91
−1.03
−0.91
−0.86
−0.86
−1.35
−0.93
−0.22
−0.19
PI
EI
1
2
3
4
5
6
7
8
9
10
11
12
13
14
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
4
5
VRC
CP
−0.90
−0.82
−0.85
−0.79
−0.79
−0.88
−0.93
−0.76
−0.77
−0.75
−1.01
−0.63
0.23
−0.63
−0.58
−0.60
−0.54
−0.54
−0.65
−0.69
−0.51
−0.54
−0.49
−0.96
−0.75
0.14
−1.07
−0.95
−1.00
−0.98
−0.98
−0.96
−1.05
−0.92
−0.95
−0.89
−1.30
−0.96
0.02
−0.53
−0.48
−0.54
−0.47
−0.47
−0.55
−0.59
−0.48
−0.44
−0.41
−0.70
−0.51
0.19
0.12
−0.04
−0.07
−0.20
0.28
GPCR: GPCR ligand; ICM: ion channel modulator; KI: kinase inhibitor; NRL: nuclear receptor ligand;
PI: protease inhibitor; EI: enzyme inhibitor; VR: voriconazole; CP: ciprofloxacin.
Calculations of the physicochemical drug descriptors (see Table 7) show that molecular weights
of all synthesized compounds are below 500; HBD and HBA values are 4–7 and 0–2, respectively;
logP and logS values were below 2.2 and above −3.70, respectively; and all compounds have a low
number of nrotb (3–5); so, a very low conformational flexibility and good oral bioavailability are
expected. According to this, all Schiff bases meet Lipinski´s rule of five. Additionally, all compounds
show permeability to cell membranes, and half of them (3a–c, 3h, 4, and 5) show PSA values from
2
35.91 to 56.14 Å , suggesting good permeability at the blood–brain barrier.
The evaluation of toxicity risk assessment is also important prior to structural drug design, and
many potential drugs fail to reach the clinical stage because of ADME toxicity issues. The toxicity risk
predictor locates fragments within a molecule that constitute a potential toxicity risk for one of four
major toxicity classes: mutagenicity, tumorigenicity, reproductive effect, and irritant effect. The
results are displayed, regarding the category specified, as high, low, or none as risk for a compound
to possess a toxic effect. The result show that all the compounds have a high risk to be mutagenic and
only 3d and 3h show a low and high risk, respectively, to be tumorigenic. In the other categories,
none of the compounds showed effect.
Drug-likeness (DL) of a compound may be defined as a complex balance of various molecular
properties and structural features which determine whether a particular molecule is similar to known
drugs [33]. Considering that most drugs have drug-likeness with a positive value, it is desirable that
new candidate stay in the positive range. Osiris DataWarrior calculations show that all nitro
compounds, 3f, and 3g have negative values, while other compounds have values higher than 4.08,
a value similar to that observed for voriconazole. The drug score index (DS) is a parameter that
combines drug-likeness, cLogP, logS, molecular weight, and toxicity risks in a single descriptor that
is very useful for determining whether a compound can be qualified as a potential drug. According
to established criteria, values closer to 1 indicate a higher probability of a compound being a future
drug candidate. All the evaluated compounds exhibited poor to moderate scores (0.30 to 0.54), 4 being
the compound with the highest value. Finally, the bioactivity scores of all compounds for being a G
protein-coupled receptor (GPCR) ligand, ion channel modulator, kinase inhibitor, nuclear receptor

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ligand, protease inhibitor, and enzyme inhibitor were calculated and are shown in Table 8. A high
score implies a higher probability for a molecule to be biologically active. For many organic molecules,
the prediction is as follows: if the bioactivity score is more than 0.00, it is active; if −0.50 to 0.00,
moderately active; and if less than −0.50, inactive. According to this, all compounds in this study are
predicted to be inactive as GPCR ligands, ion channel modulators, nuclear receptor ligands, and
protease inhibitors, and only 3j shows a moderate activity as a kinase inhibitor, while 3b, 3d, 3e, 3h,
3i, and 3j show similar activity as enzyme inhibitors. Both standard drugs show moderate to high
activity against all bioactivities evaluated. These results suggest that the mechanism of action would
be unspecific and/or generalized.
3. Discussion
In this study, we synthesized a series of Schiff base derivatives of 4-aminoantipyrine and tested
their cytotoxic properties against selected bacteria, fungi, and leishmania parasites, as well their
antioxidant/oxidative activities. Finally, we analyzed their properties regarding their suitability for
therapeutic uses.
Schiff bases are an emerging alternative for disease treatments. In the last decades, many types
of resistance have been described in bacteria, generating the need for new drugs for dealing with the
diseases caused by microbes [3,4]. In addition, the prevalence of cancer and metabolic diseases such
as diabetes are predicted to rapidly grow in the next years [51,52]. For many of these diseases, there
are still no effective therapies. Schiff bases have shown a wide range of properties that make them
promising candidates as alternative drugs, but they have not been adequately characterized. There
have been described—and some patented—Schiff bases that are effective against microorganisms
(bacteria, virus, fungi, parasites) or against cancer, with antiglycation, antioxidant, or anti-
inflammatory activities, among others [15,18]. A major question is how these compounds act against
several targets and display different properties. The most reliable answer is that due to the vast
variety of compounds, there should be many modes of action.
Our results indicate that Schiff bases 3a–b, 3d, 3f, and 5 show antimicrobial activities with
moderate inhibitory values (Table 3). Evaluation of scavenging activity reveals antioxidant properties
for 3d and 3h–j, while compounds 3b, 3f, and 5 possess oxidative activity. Overall, according to the
in silico calculations, Schiff bases present good scores for the parameters evaluated, such as
bioavailability, and have membrane permeability similar to commercial drugs (DS/DL), except 3d
and 3f–h, which have poor scores for DS (<0.5), DL, and risk of tumorigenicity. The main problem
with the low DL score of 3f is the presence of the nitro group in the structure, with is related to the
toxicity of some drugs [53]. Actually, drug discovery pipelines for the ‘neglected diseases’ are now
heavily populated with nitro compounds [54,55], so this compound should not be discarded. Also,
cytotoxicity is significant in mouse macrophages. These analyses limit compounds 3a and 3i to be the
ones with the most promising potential applications (summarized in Figure 4), with some
considerations discussed below. Compounds 3c, 3e, 3g, and 4 do not present any kind of activity and
were discarded from further analysis. It deserves to be discussed that compound 3g is equivalent to
compound 9 previously described by Thirunarayanan and collaborators [30]. The authors describe
microorganism sensitivity by the Kirby–Bauer method with comparison to the commercial drugs
used as controls, namely ciprofloxacin for bacteria and miconazol for fungi. They reported significant
activity against B. subtilis, M. luteus, S. aureus, E. coli, P. aeruginosa, and the fungi A. niger and T. viride.
In our hands, only S. cerevisiae presents a relative sensitivity to Schiff base 3g, but for the rest of the
tested microorganisms, the MIC values obtained were too high (>200 μg/mL). These two techniques
are not directly comparable, since the Kirby–Bauer method is qualitative and MIC is quantitative, but
two possible explanations for these differences are as follows: First, Thirunarayanan and
collaborators did not report the disk potency (μg) used in the assay; and second, the origin of the
strains used in their work is not indicated and apparently are not from the American Type Culture
Collection (ATCC). Together, this could explain the differences obtained with the same compound.
Nevertheless, we should omit compound 3g from next analyses, since we are not able to describe any
significant activity.

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Figure 4. Summarizing diagram highlighting the evaluated Schiff bases. Relevant compounds are in
black/bold track, non-relevant compounds discarded because of associated cytotoxicity are in light
grey/dashed tracks. A grey scale indicates the intensity of cytotoxicity.
Compounds 3b, 3f, and 5 show both antimicrobial and oxidative activity, opening the possibility
that the antimicrobial effect is a consequence of its oxidative activity. Since the effect is specific (3b
for C. albicans and E. coli; 3f for C. albicans, A. niger, S. cerevisiae, E. coli, S. aureus, and L. mexicana; 5 for
S. cerevisiae, E. coli, and L. mexicana), we cannot rule out that these compounds are oxidizing specific
targets. This is consistent with the bioactivity analyses, at least for 3b, indicating that it could be acting
as an enzyme inhibitor (Table 8). However, due to the difference observed in the time-response
experiments, the action mechanism of both compounds could be different. Additionally, 3a and 3d
showed antimicrobial activity against E. faecalis and E. coli, respectively.
The analysis of the structure–activity of the Schiff bases shows that 5 presents an extension in
the imine conjugation and the aromatic ring does not possess any substituent, while 3f has an ortho
hydroxyl group (electron-donating group) and a meta nitro group (electron-withdrawing group), and
it is evident there is a synergistic effect between both substituents, because when only the hydroxyl
group (3b) or only the nitro group (3g) is present, the activity is limited to C. albicans and E. coli in the
first case, and no activity observed in 3g, while no activity against leishmania was observed for either
compound. The hydroxyl group in the ortho position can form an intramolecular hydrogen bond with
the imine nitrogen [56], which, due to the inductive and resonance effect of the nitro group, favors
the keto–enol tautomerism to generate the keto tautomer which could be, in part, responsible for the
enhancement of the observed antimicrobial and leishmanicidal activities.
Toxicity was assayed in RAW cells (Table 6). Results indicate that compounds 3a and 3i are the
least toxic in our system. Although it is true that other systems should be tested, such as primary
macrophages isolated from mice or human macrophages cell lines, the results with RAW cells are a
sensitive indication of toxicity that should be considered before continuing any new experimental
design. Indeed, 3a and 3i are effective against E. faecalis and as antioxidant, respectively. E. faecalis is
an opportunistic pathogen highly associated with resistance development in the last decades. This
bacterial infection is common in hospital environments, presenting such clinical manifestations as
urinary tract infections, bacteremia, and endocarditis, among others [57].
Regarding compounds 3d and 3h–j, their potential application is related to their antioxidant
activity, and thus as cell-protective agents. In accordance with our results, antioxidant activity for
compounds 3i–j has been previously described [14], along with a very low inhibition of RAW cell
viability. These compounds have similar activities as the controls: ascorbic acid, quercetin, or caffeic
acid (Table 4). Nevertheless, potential applications of 3d and 3h are not very promising, as the ADME
test gave poor scores for several of the parameters (Table 7). Neither is 3j a good candidate, as the
percentage of viable RAW cells was compromised in our experiments (Table 6). So, only 3a seems to
be a reliable option for an antioxidant activity approach.
The biggest limitation of these compounds is that all of them seem to present mutagenicity
characteristics, according to our predictive analyses for ADME toxicity issues (Table 7). If so, this

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prediction should be tested experimentally; it would be interesting to exploit these compounds to
identify chemical varieties that keep or improve their useful properties, while diminishing the
associated mutagenicity. Nevertheless, a possible explanation is that compounds 3a, 3b, 3d, 3f, and 5
exert their antimicrobial and/or leishmanicidal activities through a mutagenic mechanism, but this
could not be the main mechanism as none of the other compounds show this activity, despite
reported mutagenicity. Although this is not a desired property for antimicrobial compounds, this
observation opens the question as to whether these compounds could be used in cancer treatment.
It is recognized that most anticancer drugs are potentially hazardous substances, since they are
mutagenic, teratogenic, and/or carcinogenic [58] and as they exert their activities probably due to
these properties. Some of them have been classified as carcinogenic by the International Agency for
Research on Cancer (IARC), and the in silico prediction of their ADME properties show poor scores
in some categories (Supplementary Table S1). For example, cyclophosphamide, a drug used in the
treatment of chronic lymphocytic leukemia, lymphomas, soft tissue and osteogenic sarcomas, and
solid tumors, is in group 1 (carcinogenic to humans) according to IARC, and according to the
calculations, presents a high risk to be mutagenic, tumorigenic, and have reproductive effects.
Considering this, it cannot be ruled out that Schiff bases could have anticancer properties. Indeed, a
review of the literature describes some Schiff bases with associated anticancer activity [32]. However,
more studies should be done to determine if these compounds are able to discriminate between
normal mammalian cells and certain kinds of pathogens or cancer cells [18,19], or if, in contrast, they
are generalized mutagens useless for therapeutics.
The toxicity of drugs can be greatly controlled using nanoformulations (nanoparticles,
nanotubes, liposomes, etc.) [41,42]. These systems enable specific selection of the desired target and
delivery of the drug locally and in a controlled way, reducing the side effects generated by
conventional drug delivery systems that usually require high dosage. So, the choice of the system
will depend on the final purpose of the formulation. Nanomedicine offers new approaches to drug
design, improving the specificity and limiting the undesired toxicity. Major efforts have been
focusing on developing nanocarriers to fight against cancer. Main limitations of chemotherapy are
multidrug resistance and nonspecific tissue targeting, generating serious side effects that
compromise the patient’s health. Nanoformulations of chemotherapeutic agents enhance the
selective delivery, diminishing the toxicity. Despite the associated benefits, only a few
nanoformulations for chemotherapy have been approved by the FDA and are currently marketed.
Nevertheless, many research projects and clinical trials are studying different nanoparticles,
disclosing them as an emerging therapeutic approach [43,52]. Nanoparticles are not only useful in
chemotherapy; many potential applications against infectious microorganisms and brain or
metabolic diseases are emerging [41,44].
The work here presented is a first approach to discriminate among compounds to determine
those which have application potential and those which are useless (have no biological activity or
intrinsic toxicity, etc.). Additional efforts to characterize and improve Schiff base properties and
toxicity should be done by the scientific community to uncover the real uses and applications of these
compounds in therapy as antimicrobial agents or otherwise, such as anti-inflammatory, antipyretic,
antitumor, or antidiabetic agents.
4. Materials and Methods
4.1. General Procedures
All solvents and reagents were from Sigma Aldrich (St. Louis, MO, USA) and used without
further purification. All melting points are uncorrected and were determined on a Büchi Melting
Point M-560 (Büchi Labortechnik AG, Flawil, Switzerland). Infrared spectra (KBr discs) were
recorded on a Perkin Elmer RX1 infrared spectrophotometer (PerkinElmer, Waltham, MA, US); wave
−1
numbers are reported in cm . NMR spectra were recorded at 298 K on a Bruker Advance 500 MHz
spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) equipped with a z-gradient, triple-
1
13
15
resonance ( H, C, N) cryoprobe using CDCl3 as solvent or in a Bruker AVANCE DRX 300

Molecules 2019, 24, 2696
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spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany) using DMSO-d6 as solvent. Chemical
shifts are expressed in ppm with TMS as an internal reference. Accurate mass data were obtained
using a Waters model LCT Premiere time-of-flight (TOF) mass spectrometer (Waltham, MA, US).
Absorbance was measured at different wavelength on a Multi-Mode Microplate Reader Synergy H1
(Biotek, VT, US) or HT BioTek spectrophotometer (Biotek, VT, US). Reactions were monitored by TLC
on pre-coated silica gel aluminum plates, Kieselgel 60 F254 from Merck (Darmstadt,Germany) using
ethyl acetate/hexane mixtures as a solvent and compounds were visualized by UV lamp (UVP,
Upland, CA, US). The reported yields are for the purified material and are not optimized. The IC50
values were calculated in GraphPad Prism 7.02 (GraphPad Software, San Diego, US) The results are
given as a mean ± standard deviation (SD) of experiments done in triplicate.
4.2. Synthesis of Schiff Base Derivatives (3a–j, 4, 5)
The synthesis of the Schiff base derivatives 3a–j was adapted from a procedure reported by Alam
and coworkers [14]. To an ethanol solution (10 mL) of 4-amino-1,5-dimethyl-2-phenylpyrazol-3-one
(1) (1.0 g, 4.92 mmol) was added an ethanol solution (10 mL) of substituted benzaldehyde (4.92 mmol),
and the mixture was refluxed for 6 h. The progress of the reaction was monitored by TLC. The
precipitates formed were collected by filtration, purified by recrystallization with ethanol, and then
dried under vacuum to produce the pure compounds. Spectra analyses can be consulted in the
Supplementary Materials (Figures S1–S15).
4-Benzylideneamino-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3a): yield 77% as white
1
crystals; m.p. 176.8–177.2 °C (Lit [14] 178.3 °C); H-NMR (300 MHz, DMSO-d6) δ 9.59 (s, 1 H), 7.81 (dd,
J = 7.3, 2.1 Hz, 2 H), 7.49–7.57 (m, 2 H), 7.41–7.48 (m, 3 H), 7.34–7.41 (m, 3 H), 3.18 (s, 3 H), 2.46 (s, 3
H).
4-(2-Hydroxybenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3b): yield 94%
1
as yellow crystals; m.p. 201.0-201.5 °C (Lit [34] 199 °C); H-NMR (300 MHz, DMSO-d6) δ 12.94 (s, 1 H),
9.70 (s, 1 H), 7.51–7.59 (m, 2 H), 7.45–7.50 (m, 1 H), 7.36–7.44 (m, 3 H), 7.28–7.35 (m, 1 H), 6.88–6.95
(m, 2 H), 3.21 (s, 3 H), 2.41 (s, 3 H).
4-(4-Methoxybenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3c): yield 86%
1
as white crystals; m.p. 169.8–170.3 °C (Lit [32] 171–173 °C); H-NMR (300 MHz, DMSO-d6) δ 9.52 (s, 1
H), 7.76 (d, J = 8.8 Hz, 2 H), 7.57–7.49 (m, 2 H), 7.41–7.33 (m, 3 H), 7.02 (d, J = 8.8 Hz, 2 H), 3.81 (s, 3
H), 3.15 (s, 3 H), 2.44 (s, 3 H).
4-(4-Hydroxy-3-methoxybenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3d):
1
yield 94% as white crystals; m.p. 210.6–210.9 °C (Lit [32] 208–210 °C); H-NMR (300 MHz, DMSO-d6)
δ 9.46 (s, 1 H), 7.48–7.57 (m, 2 H), 7.32–7.44 (m, 4 H), 7.19 (dd, J = 8.2, 1.7 Hz, 1 H), 6.84 (d, J = 8.2 Hz,
1 H), 3.84 (s, 3 H), 3.14 (s, 3 H), 2.44 (s, 3 H).
4-(3-Hydroxy-4-methoxybenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3e):
1
yield: 93.4% as white crystals; m.p. 246.8–247.2 °C; H-NMR (500 MHz, CDCl3) δ 9.66 (s, 1 H), 7.61 (d,
J = 1.3 Hz, 1 H), 7.48 (dd, J = 8.2, 7.7 Hz, 2 H), 7.42 (dd, J = 8.5, 1.4 Hz, 2 H), 7.32 (tt, J = 7.2, 1.2 Hz, 1
H), 7.29 (dd, J = 8.2, 1.7 Hz, 1 H), 6.89 (d, J = 8.2 Hz, 1 H), 5.71 (br. s, 1 H), 3.94 (s, 3 H), 3.14 (s, 3 H),
13
2.49 (s, 3 H); C NMR (126 MHz, CDCl3) δ 161.0, 156.9, 151.7, 145.8, 134.9, 129.2, 129.0, 126.8, 125.9,
−1
124.3, 122.8, 114.1, 112.1, 110.2, 56.0, 35.9, 10.2; IR (cm ) (KBr discs) 3096.15, 2958.95, 1608.34, 1573.63,
+
1257.36, 1020.16, 776.21; HRMS (TOF ES+) m/z calcd for C19H20N3O3 (M+H) : 338.1505; found: 338.1506.
4-(2-Hydroxy-5-nitrobenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one
(3f):
1
yield 84.6% as yellow crystals; m.p. 204.9–205.6 °C (Lit [36] 205.3–205.9 °C); H-NMR (300 MHz,
DMSO-d6) δ 13.73 (br. s., 1 H), 9.79 (s, 1 H), 8.52–8.58 (m, 1 H), 8.18 (dd, J = 9.0, 2.8 Hz, 1 H), 7.56 (t, J
= 7.7 Hz , 2 H), 7.35–7.46 (m, 3 H), 7.10 (d, J = 9.2 Hz, 1 H), 3.26 (s, 3 H), 2.45 (s, 3 H).
4-(3-Nitrobenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3g): yield 95.4% as
1
light orange crystals; m.p. 219.7–220.1 °C (Lit [30] 218–219 °C); H-NMR (300 MHz, DMSO-d6) δ 9.68
(s, 1 H), 8.59–8.63 (m, 1 H), 8.19–8.28 (m, 2 H), 7.74 (t, J = 8.0 Hz, 1 H), 7.51–7.59 (m, 2 H), 7.35–7.44 (m,
3 H), 3.24 (s, 3 H).
4-(4-Dimethylaminobenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one
yield 94.5% as yellow crystals; m.p. 220.4–221.4 °C (Lit [37] 223–224 °C); H-NMR (300 MHz, DMSO-
(3h):
1

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d6) δ 9.43 (s, 1 H), 7.62 (d, J = 8.8 Hz, 2 H), 7.55–7.48 (m, 2 H), 7.40–7.30 (m, 3 H), 6.75 (d, J = 8.8 Hz, 2
H), 3.11 (s, 3 H), 2.98 (s, 6 H), 2.41 (s, 3 H).
4-(3,4-Dihydroxybenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (3i): yield
1
97.4% as cream powder; m.p. 279.2–280.5 °C (Lit [14] 287.2 °C); H-NMR (300 MHz, DMSO-d6) δ 9.41
(br. s., 1 H), 9.38 (s, 1 H), 9.19 (br. s., 1 H), 7.52 (dd, J = 8.2, 7.4 Hz, 2 H), 7.32–7.38 (m, 3 H), 7.29 (d, J =
1.9 Hz, 1 H), 7.02 (dd, J = 8.1, 1.9 Hz, 1 H), 6.78 (d, J = 8.1 Hz, 1 H), 3.12 (s, 3 H), 2.41 (s, 3 H).
4-(4-Hydroxy-3,5-dimethoxybenzylideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one
1
(3j): yield 90.5% as white crystals; m.p. 259.2–260.7 °C (Lit [14] 258.9 °C); H-NMR (300 MHz, DMSO-
d6) δ 9.46 (s, 1 H), 8.88 (br. s., 1 H), 7.53 (dd, J = 8.5, 7.1 Hz, 2 H), 7.41–7.33 (m, 3 H), 7.10 (s, 2 H), 3.83
(s, 6 H), 3.14 (s, 3 H), 2.45 (s, 3 H).
4-(2-furfurilideneamino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (4): yield 85.6% as dark
1
yellow crystals; m.p. 212.7–213.7 °C (Lit [45] 206 °C); H-NMR (300 MHz, DMSO-d6) δ 9.43 (s, 1 H),
7.84 (d, J = 1.7 Hz, 1 H), 7.53 (dd, J = 8.1, 7.7 Hz, 2 H), 7.41 - 7.33 (m, 3 H), 6.96 (d, J = 3.4 Hz, 1 H), 6.64
(dd, J = 3.4, 1.8 Hz, 1 H), 3.17 (s, 3 H), 2.40 (s, 3 H).
4-Phenylallylideneamino-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one (5); yield 90% as yellow
1
crystals; m.p. 165.5–165.9 °C (Lit [37] 164–165 °C); H-NMR (300 MHz, DMSO-d6) δ 9.40 (d, J = 8.2 Hz,
1 H), 7.64 (dd, J = 8.3, 1.4 Hz, 2 H), 7.57–7.49 (m, 2 H), 7.43–7.29 (m, 6 H), 7.11 (d, J = 16.1 Hz, 1 H), 7.01
(dd, J = 16.1, 8.3 Hz, 1 H), 3.17 (s, 3 H), 2.39 (s, 3 H).
4.3. Evaluation of Antimicrobial Activity
The antimicrobial activities of the synthesized compounds were tested against the Gram-
positive bacteria Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212, the Gram-
negative bacteria Escherichia coli ATCC 25922 and P. aeruginosa ATCC 27853, and the fungal strains
Candida albicans ATCC 10231, Aspergillus niger ATCC 6275, and S. cerevisiae (backgrounds W303 and
BY4741), using the microdilution method [46,47].
The bacterial inoculum was prepared by emulsifying overnight colonies from an agar medium
5
in broth (Mueller–Hinton broth). The suspensions were adjusted to a final organism density of 5 × 10
colony former units (cfu)/mL. The suspensions of C. albicans were made in saline solution and
adjusted to an optical density of 0.155 at 530 nm. The emulsifying of A. niger cultures was done by
pouring 5 mL of saline and 0.1% of polysorbate 80 over the Petri dishes. Then, 100 μL of this
suspension were diluted in 5 mL of saline and adjusted to an optical density of 0.1 at 530 nm. For S.
cerevisiae, fresh exponential YPAD (yeast extract 1%, peptone 2%, adenine 0.004%, dextran 2%)
cultures were adjusted to an optical density of 0.5 at 600 nm, and then diluted to the equivalent of 1
4
−3
× 10 cells/mL (10 dilution). Stock solutions of Schiff bases were prepared by dissolving them in
DMSO at 10 mg/mL. For controls, ciprofloxacin was used for all bacteria tested, voriconazol for A.
niger and C. albicans, and geneticin for S. cerevisiae. The range of concentrations used was empirically
selected for each organisms and the Schiff base. Drug sensitivities were assayed by serial dilutions in
96-well plates as previously described [39,46,47]. A row with media and cells but without drug was
the positive control. The blank was done by preparing a row with media and drugs, but without cells.
Plates were fitted with a tight lid before incubation to prevent desiccation. Plates were incubated at
37 °C in air for 24 h for bacterial cultures and 35 °C for 48 h for fungi. The turbidity was recorded at
600 nm for bacteria and 530 nm for fungi in a plate reader (Synergy H1, Biotek), and calculations of
MIC (minimal inhibitory concentration), NIC (noninhibitory concentration), and IC50 (inhibitory
concentration for 50% of the population) were done with GraphPad Prism [39]. The minimum
bactericidal concentration (MBC) and fungicidal concentration (MFC) were determined by spreading
100 μL of culture from each well onto a plate (Mueller–Hinton agar for bacteria, Sabouraud dextrose
agar for fungi except S. cerevisiae, and YPAD for S. cerevisiae). MBC and MFC values represented the
lowest concentration of a compound that completely inhibited growth.
4.4. Evaluation of Leishmanicidal Activity
Leishmanicidal activities of all Schiff bases were evaluated by measuring promastigote
mitochondrial activity using MTT colorimetric assay as described previously [57]. In this study,

Molecules 2019, 24, 2696
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promastigotes from L. mexicana (M379), donated from the Instituto de Medicina Tropical Alexander
von Humboldt (Universidad Cayetano Heredia, Peru) were cultured at 24 °C in Schneider's
Drosophila Medium supplemented with 20% fetal bovine serum and 100 IU/mL penicillin + 100
mg/mL streptomycin. The medium was renewed every four days. Parasite density was determined
in a Neubauer chamber.
6
Into each well of a 96-well cell culture dish was dispensed 1 × 10 parasites. A stock solution of
each of the Schiff Base was prepared in DMSO, and serial dilutions were added to the parasite
suspension, keeping the solvent concentration at 1%. The final volume was 100 μL for each well and
triplicate conditions were carried out. Pentamidine treatment (100 μg/mL) and untreated parasites
were used as positive control and negative control, respectively. After exposure to the compounds
for 48 h in culture medium, 10 μL of a solution of 5 mg/mL MTT dissolved in PBS was added to each
well. The plate was incubated at 24 °C for 4 h in darkness. Later, the plate was centrifuged at 4400
rpm for 10 min and the culture medium was then aspirated. Next, 100 μL of DMSO were added into
each well to solubilize the formazan and the plate was shaken for 5 min. The colored formazan salt
was measured by recording changes in absorbance at 570 nm using a microplate reader, a BioTek
Synergy HT spectrophotometer. A reference wavelength of 630 nm was used to subtract background.
Optical densities were analyzed as the quantity of formazan is directly proportional to the number
of viable parasites. Data were analyzed with the statistical software GraphPad Prism.
4.5. Cell Viability
RAW 264.7 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) (Gibco,
Invitrogen, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Eurobio) and 100 IU/mL
penicillin + 100 μg/mL streptomycin (Gibco, Invitrogen) at 37 °C in an atmosphere containing 5% CO2.
The medium was renewed once a week. The viability was determined using MTT (thiazolyl blue
tetrazolium bromide) dye assay as described previously for leishmanicidal activity assessment, with
4
some variations. Here, 5 × 10 cells/well in a final volume of 100 μL were deposited into a 96-well
plate in triplicate. Saponin (2 and 4 mg/mL) and untreated cells were used as the positive control and
negative control, respectively. Schiff bases were dissolved in DMSO to obtain different serial
concentrations (0.01–2000 μM). After 48 h exposure to the compounds, 10 μL/well of MTT (5 mg/mL
MTT in PBS) was added, and the plate was incubated at 37 °C for 2 h in darkness. Cells were pelleted
by centrifugation at 4400 rpm for 10 minutes and the media was removed. Next, 100 μL/well of DMSO
were added and the absorbance at 570 nm was recorded. A reference wavelength of 630 nm was used
for background subtraction.
4.6. Drop Test
For the drop test with S. cerevisiae, wild-type strain (background W303), and wild type and sod1
mutant cells (background BY4741) were grown overnight in YPAD media until an optical density of
7
0.5 at 600 nm was reached (equivalent to 1 × 10 cells/mL). Three serial dilutions were prepared and
5
4
3
2
5 μL of each was spotted onto a plate (approximately 1×10 , 1×10 , 1×10 , and 1×10 cells/spot). YPAD
plates were supplemented with 200 μg/mL of each Schiff base, and one plate supplemented with 2%
DMSO and one plate with YPAD without any drug were used as controls. Plates were incubated at
25 °C for 24, 48, and 72 h.
4.7. DPPH Radical Scavenging Assay
The stock solutions of the compounds were prepared by dissolving 3 in DMSO to a concentration
of 4 mg/mL, then diluted with methanol to give a concentration of 400 μg/mL. This was used
immediately. The experimental procedure was adapted from the literature [59]. Briefly, in a 96-well
plate, 100 μL of solution of DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical (0.2 mM in methanol) was
added to 100 μL of methanolic solutions of 3a–j, 4, or 5, prepared as serial two-fold dilutions from
the stock solution. Standards were also prepared in the same concentrations. The mixture was

Molecules 2019, 24, 2696
17 of 21
incubated in the dark at room temperature for 30 min, and the absorbance was read at 515 nm on a
microplate reader, a BioTek Synergy H1 spectrophotometer.
The percentage of DPPH scavenging was then calculated by using the following formula:
(Aꢁꢂꢃꢄꢅꢆꢇꢈꢉꢉꢊ − Aꢁꢂꢃꢄꢅꢆ ꢋꢅꢂꢌꢍ
% DPPH scavenging = 100 ∗ ꢀ
)
ꢑ
(Aꢈꢉꢉꢊ − Aꢁꢎꢅꢏꢆꢌꢐ
)
The antioxidant activity of the compound was expressed as IC50, which is defined as the
concentration that could scavenge 50% of the DPPH free radicals.
4.8. Theoretical Prediction of ADME Properties and Bioactivity Scores
The predictions of ADME toxicity parameters and bioactivity scores were calculated as
described previously [49,50,60,61]. The Osiris DataWarrior version 4.4.4 software on a Windows 10
operating system [50] was used to determine the drug-like properties of the synthesized compounds.
These properties included the molecular polar surface area (PSA), octanol–water partition coefficient
(clogP), aqueous solubility (clogS), number of rotatable bonds (nrotb), number of hydrogen donors
(HBD), number of hydrogen acceptors (HBA), toxicities (mutagenic, tumorigenic, irritant, and
reproductive), drug scores (DS), and drug likeness (DL). The bioactivity scores, including activities
as a GPCR ligand, ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor,
and enzyme inhibitor, were calculated using the Molinspiration Cheminformatics software.
1
Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1. H NMR
1
1
spectrum of compound 3a., Figure S2. H NMR spectrum of compound 3b, Figure S3. H NMR spectrum of
1
1
compound 3c, Figure S4. H NMR spectrum of compound 3d, Figure S5. H NMR spectrum of compound 3e,
Figure S6. 13C NMR spectrum of compound 3e, Figure S7. IR spectrum of compound 3e, Figure S8. HRMS of
compound 3e, Figure S9. 1H NMR spectrum of compound 3f, Figure S10. 1H NMR spectrum of compound 3g,
Figure S11. 1H NMR spectrum of compound 3h, Figure S12. 1H NMR spectrum of compound 3i, Figure S13. 1H
NMR spectrum of compound 3j, Figure S14. 1H NMR spectrum of compound 4, Figure S15. 1H NMR spectrum
of compound 5, Figure S16. Evaluation of biocidal/biostatic effect, Table S1. Theoretical prediction of ADME
properties of some drugs used in chemotherapy.
Author Contributions: Conceptualization, R.T., A.P. and J.H.-M.; Formal analysis, R.G., J.M., L.D., O.B.-C., T.B.,
J.P.-B., R.A.-M., P. R.-S., A. P. and J. H.-M.; Funding acquisition, R.T., A.P. and J.H.-M.; Investigation, R. T., R.G.,
J.M., L.D., O.B.-C., T.B., J.P.-B., R.A.-M. and P. R.-S.; Methodology, R.G., J.M., L.D., O.B.-C., P.R.-S., A.P. and J.H.-
M.; Project administration, R.G. and O.B.-C.; Supervision, R.T., A.P. and J.H.-M.; Writing—original draft, A.P.
and J.H.-M.; Writing—review & editing, R.T.
Funding: This research was supported by grants from the Universidad Central del Ecuador (Dirección General
de Investigación y Proyectos-DGIP, project #6), Universidad UTE, Universidad Las Américas, SENPLADES
(9175.0000.0000.377784 and 91750000.0000.375239, managed by Dirección General de Investigación y Proyectos,
DGIP, from UCE), and Academie de Recherche et d´Enseignement Supérieur (ARES) from Belgium.
Acknowledgments: The authors express their sincere thanks to John R. Lloyd and Robert O’Connor at NIDDK—
NIH for providing, respectively, HRMS and NMR spectra. We thank Rui Oliveira and Björn Johansson from the
Universidade do Minho for yeast strain donation. We thank all the staff from the Instituto de Investigación y
Salud Pública en Zoonosis, CIZ, the Chemical Sciences Faculty from UCE, and the Instituto CENBIO from UTE.
Kenneth L. Kirk (NIDDK—NIH) provided helpful comments.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to
publish the results.
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Sample Availability: Samples of the compounds are available from the authors.
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