Trisubstituted barbiturates and thiobarbiturates: Synthesis and biological evaluation as xanthine oxidase inhibitors, antioxidants, antibacterial and anti-proliferative agents

Article abstract of DOI:10.1016/j.ejmech.2017.11.070

Barbituric and thiobarbituric acid derivatives have become progressively attractive to medicinal chemists due to their wide range of biological activities. Herein, different series of 1,3,5-trisubstituted barbiturates and thiobarbiturates were prepared in moderate to excellent yields and their activity as xanthine oxidase inhibitors, antioxidants, antibacterial agents and as anti-proliferative compounds was evaluated in vitro. Interesting bioactive barbiturates were found namely, 1,3-dimethyl-5-[1-(2-phenylhydrazinyl)ethylidene]pyrimidine-2,4,6(1H,3H,5H)-trione (6c) and 1,3-dimethyl-5-[1-[2-(4-nitrophenyl)hydrazinyl]ethylidene]pyrimidine-2,4,6(1H,3H,5H)-trione (6e), which showed concomitant xanthine oxidase inhibitory effect (IC₅₀ values of 24.3 and 27.9 μM, respectively), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (IC₅₀ values of 18.8 and 23.8 μM, respectively). In addition, 5-[1-(2-phenylhydrazinyl)ethylidene]pyrimidine-2,4,6(1H,3H,5H)-trione (6d) also revealed DPPH radical scavenger effect, with an IC₅₀ value of 20.4 μM. Moreover, relevant cytotoxicity against MCF-7 cells (IC₅₀ = 13.3 μM) was observed with 5-[[(2-chloro-4-nitrophenyl)amino]methylene]-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (7d). Finally, different 5-hydrazinylethylidenepyrimidines revealed antibacterial activity against Acinetobacter baumannii (MIC values between 12.5 and 25.0 μM) which paves the way for developing new treatments for infections caused by this Gram-negative coccobacillus bacterium, known to be an opportunistic pathogen in humans with high relevance in multidrug-resistant nosocomial infections. The most promising bioactive barbiturates were studied in silico with emphasis on compliance with the Lipinski's rule of five as well as several pharmacokinetics and toxicity parameters.

Full text of DOI:10.1016/j.ejmech.2017.11.070

European Journal of Medicinal Chemistry 143 (2018) 829e842
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Trisubstituted barbiturates and thiobarbiturates: Synthesis and
biological evaluation as xanthine oxidase inhibitors, antioxidants,
antibacterial and anti-proliferative agents
,
b
a
,
,
Joana Figueiredo a , Joao L. Serrano b, Eunice Cavalheiro a b, Leena Keurulainen c,
~
^
Jari Yli-Kauhaluoma , Vania M. Moreira d, Susana Ferreira a, Fernanda C. Domingues a
,
c
c
,
, b
,
e,
, b
Samuel Silvestre a *, Paulo Almeida a
a
~
CICSeUBI e Health Sciences Research Centre, University of Beira Interior, 6200-506 Covilha, Portugal
b
~
Department of Chemistry, University of Beira Interior, 6201-001 Covilha, Portugal
c Drug Research Program, Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, Viikinkaari 5 E (PO Box 56), FI-00014, University of
Helsinki, Helsinki, Finland
d Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 161 Cathedral Street, Glasgow G4 0RE, UK
e CNC e Center for Neuroscience and Cell Biology, University of Coimbra, Rua Larga, 3004-517 Coimbra, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
Barbituric and thiobarbituric acid derivatives have become progressively attractive to medicinal chemists
due to their wide range of biological activities. Herein, different series of 1,3,5-trisubstituted barbiturates
and thiobarbiturates were prepared in moderate to excellent yields and their activity as xanthine oxidase
inhibitors, antioxidants, antibacterial agents and as anti-proliferative compounds was evaluated in vitro.
Interesting bioactive barbiturates were found namely, 1,3-dimethyl-5-[1-(2-phenylhydrazinyl)ethyl-
idene]pyrimidine-2,4,6(1H,3H,5H)-trione (6c) and 1,3-dimethyl-5-[1-[2-(4-nitrophenyl)hydrazinyl]eth-
ylidene]pyrimidine-2,4,6(1H,3H,5H)-trione (6e), which showed concomitant xanthine oxidase inhibitory
Received 25 September 2017
Received in revised form
21 November 2017
Accepted 25 November 2017
Available online 27 November 2017
Keywords:
Barbiturates
Xanthine oxidase inhibition
Antioxidant
Antibacterial
effect (IC50 values of 24.3 and 27.9
scavenging activity (IC50 values of 18.8 and 23.8
ethylidene]pyrimidine-2,4,6(1H,3H,5H)-trione (6d) also revealed DPPH radical scavenger effect, with an
IC50 value of 20.4 M) was observed
m
M, respectively), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical
m
M, respectively). In addition, 5-[1-(2-phenylhydrazinyl)
m
M. Moreover, relevant cytotoxicity against MCF-7 cells (IC50 ¼ 13.3
m
Cytotoxicity
with 5-[[(2-chloro-4-nitrophenyl)amino]methylene]-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (7d).
Finally, different 5-hydrazinylethylidenepyrimidines revealed antibacterial activity against Acinetobacter
baumannii (MIC values between 12.5 and 25.0 mM) which paves the way for developing new treatments
for infections caused by this Gram-negative coccobacillus bacterium, known to be an opportunistic
pathogen in humans with high relevance in multidrug-resistant nosocomial infections. The most
promising bioactive barbiturates were studied in silico with emphasis on compliance with the Lipinski's
rule of five as well as several pharmacokinetics and toxicity parameters.
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
has become increasingly attractive to medicinal chemists due to the
discovery that they bear a wide range of biological activities. More
Barbituric acid (BA) is a relevant pyrimidine derivative that was
synthesized for the first time in 1864 by cyclization of urea and
malonic acid [1]. Until the commercialization of benzodiazepines in
the 1960s, barbiturates were almost the only group of drugs used as
sedative-hypnotics [2,3]. More recently, this class of compounds
specifically, several of these compounds are enzyme inhibitors
[collagenase-3, matrix metalloproteinases (MMPs) [4], xanthine
oxidase (XO) [5] and methionine aminopeptidase-1] as well as
antibacterial, anticancer, antiangiogenic, immunomodulatory,
antifungal and antioxidant agents [6e9]. These activities are
~
* Corresponding author. CICSeUBIeHealth Sciences Research Centre, University of Beira Interior, 6200-506 Covilha, Portugal.
E-mail address: samuel@fcsaude.ubi.pt (S. Silvestre).
https://doi.org/10.1016/j.ejmech.2017.11.070
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

830
J. Figueiredo et al. / European Journal of Medicinal Chemistry 143 (2018) 829e842
associated to several structural changes at positions N1, N3, C2 and
C5, the latter being the most studied position. Within these struc-
tural modifications, various cyclization reactions at C5 and C6 of BA,
thiobarbituric acid (TBA) and their derivatives (BADs and TBADs,
respectively) [10e19] as well as at C5 with spiro formation [20e24]
have been described. Interestingly, some of these transformations
involve a multicomponent reaction (MCR) [11,14,16]. However, in
most cases, the cyclization path is preceded by the formation of
substituted methyl [10,18], arylidene [12,15,19e24], or methylene
[13,17,19] derivatives at C5 as key isolated or potential precursors of
these modified pyrimidines.
Among the most important C5 functionalized BAs and TBAs
are the 5-benzylidene or 5-methylene derivatives, usually pro-
duced by their reaction with benzaldehydes or triethyl ortho-
formate, respectively. Out of these, a large variety of 5-arylidene
BADs and TBADs have been biologically evaluated and several
compounds of this class was found to possess XO inhibitory effect
[5] and antimicrobial activity against a number of Gram-positive
and Gram-negative bacteria, as well as against fungal strains
[25]. Methylene BADs and TBADs are most often used as in-
termediates for the synthesis of other derivatives [26,27]. Within
these, hydrazone BADs have the ability to inhibit fungal growth
2. Results and discussion
2.1. Chemistry
Thirty-five BADs and TBADs were synthesized as shown in
Scheme 1, in moderate to excellent yields, i.e. 41e97% (Experi-
mental section and Table S1). The majority of 5-substituted BAD
and TBAD families described herein are known precursors of fused
heterocycles reported in the literature [10,12,13,15,17e24]. From
this set of compounds and to the best of our knowledge, two 5-
phenylcyanomethylpyrimidines
hydrazinylethylidenepyrimidine
(4a-b),
(6a),
one
seven
5-
5-
phenylaminomethylenepyrimidines (7a-g) and six 1-(pyrimidine-
5-ylidene)ureas and thioureas (8a-c, e-g) are described for the first
time. All starting materials were commercially obtained, except 1,3-
diphenylthiobarbituric acid (1a) which was synthesized from
phenylisothiocyanate, aniline, malonic acid and acetyl chloride, in
two steps [35,36]. Almost all prepared 5-substituted BADs and
TBADs were easily isolated in a high degree of purity by simple
filtration, as confirmed by nuclear magnetic resonance (NMR)
spectra. In general, the BADs yields were higher than their congener
TBADs.
in the
m
M range [28]. In addition, through the introduction of
A full spectroscopic characterization of all 5-substituted BADs
and TBADs 2e8 was made, including 1H and 13C NMR shift as-
signments, which were established whenever possible with the aid
of distortionless enhancement by polarization transfer (DEPT),
heteronuclear multiple quantum coherence (HMQC) and hetero-
nuclear multiple bond correlation (HMBC) experiments. The most
representative 1H and 13C NMR chemical shifts of 5-substituted
BADs and TBADs 2e8 are presented in Tables S2 and S3, corre-
sponding to the structural moiety depicted in bold, in Scheme 1.
5-Benzylidenepyrimidines 2a-e were synthesized by a Knoe-
venagel condensation reaction in excellent yields (90e97%), based
on the method reported by Deb et al. [37] in which the reaction
temperature was changed from 70 ꢀC to reflux. Then, the C-5
double bond of 5-benzylidenepyrimidine 2e was reduced at room
temperature by NaBH4 in ethanol according to the method
described by Yan et al. [38] to give compound 3, in 41% yield. The
change of the yellow arylidene color to white, due to a conjugation
extension reduction, together with the absence of the 1H NMR
singlet at 8.25 ppm (2e unsaturated methine CH) and the obser-
vation of a triplet and doublet signals corresponding to the methine
and methylene in adjacent carbons at 3.82 and 3.19 ppm, respec-
tively, observed in the 1H NMR spectra of 3, are the most important
evidences of a successful reduction reaction. Additionally, the
cyanomethyl derivatives 4a-b were obtained, in 67e77% yield, from
the respective 5-(4-bromobenzylidene)pyrimidines followed by
the addition of sodium cyanide [39]. Once again the reduction was
evidenced by the change in color of the solid to white and by
observation of deshielded methylene CH 1H NMR singlet signals at
5.24e5.35 ppm for compounds 4a-b.
The 5-acetylpyrimidines 5a-c were synthesized using the pro-
cess described by Jursic et al. [40] in good to very good yields
(45e83%). Then, their hydrazine derivatives 6a-g were easily pre-
pared after reaction with hydrazine and different phenyl-
hydrazines, in refluxing methanol, using the method described by
the same authors [40], also in good to very good yields (50e92%).
The 5-phenylaminomethylenepyrimidines 7a-j and 1-(pyr-
imidin-5-ylidene)ureas/thioureas 8a-g were obtained in moderate
to very good yields (52e98%) by reaction of triethyl orthoformate
with different anilines and urea or thiourea, respectively, in
refluxing butan-1-ol, by a MCR based on the method described by
Rauf et al. [27]. In this procedure, the solvent was exchanged from
butan-2-ol to butan-1-ol, which allowed the increase of the reflux
temperature, resulting in higher yields of products. As
urea, thiourea, guanidine, hydrazine or hydroxylamino groups to
BADs and TBADs, new bioactive compounds can be obtained
[13,17,29].
XO is an important and versatile molybdo-flavoprotein
responsible for the oxidation of purine substrates, catalyzing the
conversion of hypoxanthine into xanthine and xanthine into uric
acid, with subsequent reduction of oxygen, forming reactive oxygen
species (ROS) which include the superoxide anion radical. An in-
crease in this enzyme's activity, as well as in its metabolites levels
(uric acid and ROS), are associated with many pathological condi-
tions, including gout and oxidative damage of tissues [30e32].
Therefore, the discovery of new XO inhibitors, preferably with
antioxidant capacity, is an important goal in medicinal chemistry
[33].
Infections caused by microorganisms, especially those involving
multidrug-resistant strains, place public health at serious risk.
Acinetobacter baumannii is a bacterial species increasingly associ-
ated with nosocomial infections. Risk factors for an infection with
this bacterium include prolonged hospitalization and recent sur-
gical procedures. In this context, the identification of new anti-
bacterial agents against A. baumannii, with new mechanisms of
action, is an object of major efforts by the scientific community
[34].
Taken together, the basic BA and TBA scaffolds may play a
critical role in both XO inhibitory and promoting antibacterial
effects. Therefore, in search for new active BADs and TBADs with
these activities, herein we describe the synthesis and in vitro
biological evaluation of 35 (16 new) 1,3,5-trisubstituted barbi-
turates and thiobarbiturates where position 5 was conveniently
substituted with methylene, benzylidene and methyl derivatives.
All BADs and TBADs were evaluated as XO inhibitors and their
antioxidant activity was determined using the 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical scavenging method. In addition,
their antibacterial activity against several Gram-positive and
Gram-negative strains and cytotoxicity towards normal human
dermal fibroblasts (NHDF) and a mammalian cancer cell line
(MCF-7) were assessed. Structure-activity relationships were also
discussed and relevant pharmacokinetic and toxicity properties
were calculated in silico to evaluate their potential interest for
in vivo applications and/or in the development of compounds
with improved pharmacological properties.

J. Figueiredo et al. / European Journal of Medicinal Chemistry 143 (2018) 829e842
831
Scheme 1. Chemical synthesis of BADs and TBADs 2e8.
representative
examples,
the
yields
of
5-
NMR signal between 11.10 and 13.57 ppm correlated to the 5-C
signal in HMBC experiments, which makes unequivocal evidence
of the exclusive existence of tautomer A in DMSO-d6.
phenylaminomethylenepyrimidines 7h and 7j were changed from
82 and 72% to 97 and 84%, respectively, when the reaction tem-
perature increased.
Detailed 1H and 13C NMR analysis suggested that the enamine
tautomer form A was present in all pyrimidine derivatives 6e8, in
DMSO-d6. The imine form (hydrazone) B appears to be absent
2.2. Xanthine oxidase inhibitory activity
The evaluation of the XO inhibitory activity of BADs and TBADs
2e8 was made using a spectrophotometric method, under aerobic
conditions, with xanthine as the substrate, and following uric acid
(Fig. 1),
contrary
to
what
was
reported
for
5-
hydrazinylethylidenepyrimidines [28]. Specifically, the NH 1H

832
J. Figueiredo et al. / European Journal of Medicinal Chemistry 143 (2018) 829e842
Table 1
In vitro IC50 values for XO inhibition and DPPH radical scavenging activity of com-
pounds 2c, 2d, 6c-e and respective references allopurinol and Trolox.
Compound
XO (
m
M)a
DPPH (
m
M)a
20 min
60 min
2c
2d
6c
6d
26.1
31.5
24.3
ndb
27.9
3.2
ndb
ndb
ndb
ndb
22.6
23.9
23.9
ndb
18.8
20.4
23.8
ndb
6e
Allopurinol
Trolox
ndb
33.8
35.9
a
IC50 values represent as mean of three determinations.
nd: not determined.
b
Fig. 1. Enamine A and imine B tautomeric forms.
2.3. Antioxidant activity as DPPH radical scavenger
formation by measuring the absorbance at 295 nm, as described in
the literature [41,42]. The commercial drug allopurinol was used as
the positive control. The study started with a screening at 30 mM
concentration (Table S4) and after this preliminary evaluation,
concentration-response studies were performed for the BADs and
TBADs which presented an inhibitory effect of 40% or higher, in
order to determine their half maximal inhibitory concentration
(IC50) values.
The DPPH radical scavenging activity of BADs and TBADs 2e8
was evaluated using a spectrophotometric method by measuring
the absorbance of this radical at 517 nm, based on the available
literature [41,42] and using Trolox as the reference compound. A
screening was first performed at the concentration of 30 mM, after
20 and 60 min of incubation (Table S4). IC50 values were further
determined for BADs and TBADs which presented a DPPH radical
scavenging activity higher than 50% (Table 1).
Most of the tested compounds presented no appreciable DPPH
radical scavenging capacity (Table S4). However, 5-
hydrazinylethylidenepyrimidines 6c-e were clear exceptions
In general, the majority of BADs and TBADs was able to inhibit
XO activity (Table S4), with the most active compounds being the 5-
benzylidenepyrimidines
hydrazinylethylidenepyrimidines 6c and 6e (Fig. 2), with an IC50
of 26.1, 31.5, 24.3 and 27.9 M, respectively (Table 1). For the pos-
itive control (allopurinol) and under the assay conditions
employed, an IC50 value of 3.17 M was calculated, which is in
2c
and
2d
and
the
5-
(Fig. 3) and the determined IC50 values were 22.6, 23.9 and 23.9
respectively, at 20 min, and 18.8, 20.4 and 23.8 M, at 60 min
(Table 1). Particularly, under these experimental conditions, the
radical scavenging ability of these 5-
mM,
m
m
m
agreement with the reported values [43]. Through the analysis of
all these results, some important structural features for the XO
inhibition could be inferred. Generally, BADs showed better XO
inhibitory effect in comparison to their congeners TBADs, and
bulkier substituents at C5 and smaller groups at N1 and N3 (Scheme
1) also seemed to favour this activity. Moreover, the 5-
hydrazinylethylidenepyrimidine 6c, substituted with a methyl
group at N1 and N3, clearly presented better activity than its hy-
drogenated analogue 6d. Additionally, the presence of a 2,4-dinitro
system (6f) seemed to reduce this activity when compared to the
congeners 5-hydrazinylphenyl 6c and 4-nitrophenyl 6e (Fig. 2).
hydrazinylethylidenepyrimidines was superior to the one
observed with Trolox, whose calculated IC50 values (33.8 and
35.9 mM after 20 and 60 min, respectively) are in agreement with
the literature [44]. Concerning the structural features relevant for
this activity, the hydrazinyl derivatives globally presented higher
DPPH radical scavenging capacity when compared with most of the
other compounds. More specifically, the aromatic 5-
hydrazinylethylidenepyrimidines 6c-e can be relevant starting
points to develop improved antioxidant compounds acting as
radical scavengers. In addition, it is interesting to note that the 5-
Fig. 2. In vitro XO inhibitory activity of compounds 2c, 2d, 6c-f and allopurinol. Results are expressed as average values standard error of the mean (SEM). A p < 0.05 versus
negative control in the statistical significance analysis (Student's t-test) was observed for all compounds except for 6d.

J. Figueiredo et al. / European Journal of Medicinal Chemistry 143 (2018) 829e842
833
Fig. 3. In vitro DPPH radical scavenging activity for 5-hydrazinylethylidenepyrimidines 6a-g, 1-(pyrimidin-5-ylidene)ureas 8d, 8f and Trolox (positive control). Results are expressed
as average values SEM. A p < 0.05 versus negative control in the statistical significance analysis (Student's t-test) was observed for all compounds.
hydrazinylphenyl 6c-d and 5-hydrazinyl-(4-nitrophenyl) 6e de-
rivatives had better activity than their hydrogenated 6a-b or 2,4-
dinitrophenyl 6f-g counterparts. In this context, it is worth
mentioning that the antioxidant action of other different organic
hydrazines has already been described [45e47]. Finally, the 1-
(pyrimidin-5-ylidene)ureas 8d and 8f exhibited a moderate radical
scavenging activity (Fig. 3).
Taken together, it is important to note the relevant XO inhibition
and the high DPPH radical scavenging activity of the 5-
hydrazinylethylidenepyrimidines 6c and 6e. As the new XO in-
hibitors should have a remarkable radical scavenging capacity,
these results seem promising [43].
The emergence of infections and outbreaks associated with
strains of A. baumannii resistant to antibiotics, or even extensively
drug-resistant and pandrug-resistant, has highlighted the lack of
available drugs to treat infections by this bacterium. In fact,
carbapenem-resistant A. baumannii is marked as one of the critical
pathogens in the World Health Organization priority list for
research and development of new antibiotics [51]. Therefore, the
activity of compounds 6c-e against A. baumannii makes them
promising new starting points for the development of antibacterial
agents against this high-profile pathogen.
2.5. Cytotoxicity studies in human cells
2.4. Antibacterial activity
Considering the relevant results observed in the XO inhibition,
DPPH radical scavenging and antibacterial studies, the cytotoxicity
of BADs and TBADs 2e8 was evaluated in normal human dermal
fibroblasts (NHDF). Additionally, a mammalian breast cancer cell
line (MCF-7) was included in this study to evaluate the anti-
proliferative potential and selectivity of these compounds. The
The antibacterial potential of all synthesized BADs and TBADs
2e8 was studied in Bacillus cereus, Staphylococcus aureus, Escher-
ichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmo-
nella enterica subsp. enterica serovar Typhimurium and
Acinetobacter baumannii strains, using tetracycline as the reference
compound (Table S5).
Regarding the antibacterial activity of the compounds under
study, most of the assayed BADs and TBADs did not show activity
against the tested Gram-positive and Gram-negative bacterial
strains, with minimum inhibitory concentrations (MIC) higher than
well-established
3-(4,5-dimethylthiazolyl-2)-2,5-
diphenyltetrazolium bromide (MTT) cell proliferation assay [52]
was used in which the soluble yellow MTT is converted into pur-
ple and insoluble formazan crystals by active mitochondrial lactate
dehydrogenases of living cells [53]. Initially, both cell lines were
exposed to BADs and TBADs 2e8 at the concentration of 30 mM,
200
m
M. These results are in agreement with the previous reports
during a period of 72 h. Untreated cells were used as the negative
control and cells treated with 5-fluorouracil (5-FU) as the positive
control.
referring to several 5-functionalized BADs and TBADs which were
inactive against different strains of bacteria and fungi [28,48e50].
However, pyrimidines 6c-d (MIC 25.0
mM) and 6e (MIC 12.5
mM)
The results (Table S4) showed that most of the compounds were
exhibited a relevant and selective antibacterial activity against
A. baumannii. In fact, the presence of a p-nitrophenyl moiety bound
to the hydrazine group of 5-hydrazinylethylidenepyrimidines
(Scheme 1) seems to play an important role, since an increase in
this antibacterial activity was observed in relation to the unsub-
stituted phenyl analogs 6c-d. In addition, the presence of more than
one nitro group in the phenyl group (2,4-dinitrophenyl derivatives
not markedly cytotoxic for NHDF cells at 30
IC50 of 5-hydrazinylethylidenepyrimidine 6c was determined as a
representative example of pyrimidines 6c-e, and a value of 82.0
was obtained. Considering the potential future use of this type of
compounds in the treatment of A. baumannii infections, this can be
an interesting result as it demonstrates a possible selectivity of
these agents for bacterial versus normal human cells.
mM. Additionally, the
mM
6f-g)
decreased
the
activity
of
these
5-
On the other hand, a more marked reduction of MCF-7 cells
proliferation was observed (Fig. 4 and Table S4). The phenyl-
aminomethylpyrimidines 7 seemed to have higher cytotoxicity
than the other groups of compounds. Specifically, it is clear that the
hydrazinylethylidenepyrimidines. Hence, a suitable aromatic sub-
stituent at R2 of these pyrimidines can favour this activity.
Furthermore, it is important to note that compound 6d, with
unsubstituted pyrimidinone nitrogens, had antibacterial activity
against A. baumannii but was devoid of XO inhibitory effects.
5-hydrazinylethylidenepyrimidine
6d
and
the
5-
phenylaminomethylenepyrimidines 7d-e,g-h have
a
relevant

834
J. Figueiredo et al. / European Journal of Medicinal Chemistry 143 (2018) 829e842
Fig. 4. In vitro cytotoxic effect of compounds 6c, 6d, 7a-j and 5-fluorouracil (5-FU) in normal human dermal fibroblasts (NHDF) and in mammalian breast cancer cells (MCF-7).
Results are expressed as average values SEM. A p < 0.05 versus negative control in the statistical significance analysis (Student's t-test) was observed for all compounds.
anti-proliferative effect at 30
m
M. In this last group, the presence of
pharmacokinetic interactions involving this protein is expected. A
similar situation should occur in the case of the excretory protein
organic cation transporter 2 (OCT2). In the distribution field, a
generic tendency for a low probability for these compounds to
access the central nervous system was estimated. Concerning
metabolism, in which the cytochrome P450 isoforms have a key
role, only the 5-phenylaminomethylenepyrimidine 7d was pre-
dicted to be a potential substrate of the important isoform CYP3A4.
In general, it is expected that pyrimidines 2c,d, 6c-e and 7d should
accomplish most of the pharmacokinetic and toxicity criteria
studied, however, the estimated maximum tolerated dose in
humans and potential genotoxicity (Ames toxicity) for several
compounds (Table S7) appeared to be the potential major prob-
lems. In fact, a mutagenic potential for the nitroaromatic de-
rivatives 2c-d, 6e and 7d is expected and therefore this point should
be experimentally studied and the use of non-mutagenic bio-
isosteres should be considered.
a chloro or a carboxyl group at position 2 of the aromatic ring
(Scheme 1) seems to improve this activity when compared to the
compound containing the nitro (7f) group in the same position. For
the 5-phenylaminomethylenepyrimidine 7d, the most cytotoxic
compound in MCF-7 cells at 30 mM, the IC50 value was 13.3 mM.
Considering its selectivity for cancer cells versus NHDFs, this
structure can be a future starting point to develop this class of
compounds as potential novel anticancer agents.
2.6. In silico studies
Poor pharmacokinetics and/or unacceptable side effects are the
main reasons why the majority of drugs evaluated in clinical trials
do not reach the market [54]. Thus, computational approaches to
predict the absorption, distribution, metabolism, excretion and
toxicity (ADMET) may be a decisive tool to minimize these risks.
The pkCSM online software (available from http://bleoberis.bioc.
cam.ac.uk/pkcsm/prediction) is a recent freely accessible web
platform that allows rapid evaluation of pharmacokinetic and
toxicity properties and was herein used for the in silico evaluation
of the drug-likeness, in terms of the Lipinski's rule of five (Table S6),
and to predict ADMET properties (Table S7) for the six most
promising pyrimidines (2c,d, 6c-e and 7d) tested in vitro.
Four parameters are taken into consideration in the Lipinski's
rule of five, including an n-octanol-water partition coefficient (log
P) no higher than 5, a molecular weight (MW) lower than 500 Da,
no more than 10 hydrogen bond acceptors (n-HBA) and no more
than 5 hydrogen bond donors (n-HBD) [54]. Generally, an orally
active drug has no more than one violation [55]. According to
Table S6, all these pyrimidines are compatible with the parameters
of the Lipinski's rule of five, suggesting that these compounds have
favourable properties to accomplish the drug-likeness criteria.
The pkCSM online software was also used to predict pharma-
cokinetic and toxicity properties of pyrimidines 2c-d, 6c-e and 7d
(Table S7). The pyrimidines 2c,d, 6c-e and 7d entirely fulfil the
Lipinski's rule of five and a good intestinal absorption was pre-
dicted. However, for compounds 2d and 6d-e a poor Caco-2
permeability was estimated. In addition, despite the fact that py-
rimidines 6e and 7d can be potential P-glycoprotein substrates, it
was predicted that all studied compounds should not be potential
3. Conclusion
A series of 1,3-disubstituted barbiturates and thiobarbiturates
functionalized at the position 5 with benzylidene, benzyl, phenyl-
cyanomethyl,
acetyl,
hydrazinylethylidene,
phenyl-
hydrazinylethylidene, phenylaminomethylene, ylideneureas and
the corresponding thioureas were synthesized, in moderate to
excellent yields. In addition, the activity of these compounds as
xanthine oxidase inhibitors, antioxidants as well as antibacterial
and anti-proliferative agents was also evaluated. Some of the syn-
thesized barbiturates were found to have potential for health-
related
applications,
namely
1,3-dimethyl-5-[1-(2-
phenylhydrazinyl)ethylidene]pyrimidine-2,4,6(1H,3H,5H)-trione
(6c) and 1,3-dimethyl-5-[1-[2-(4-nitrophenyl)hydrazinyl]ethyl-
idene]pyrimidine-2,4,6(1H,3H,5H)-trione (6e) which showed
moderate inhibition of xanthine oxidase (IC50 values of 24.3 and
27.9
picrylhydrazyl scavenging activity (IC50 values of 18.8 and
23.8 M respectively). Aditionally, 5-[1-(2-phenylhydrazinyl)eth-
mM, respectively), and potent radical 2,2-diphenyl-1-
m
ylidene]pyrimidine-2,4,6(1H,3H,5H)-trione (6d) was also high-
lighted as a relevant starting point for developing new radical
scavengers. Interestingly, a selective antibacterial effect of 5-
hydrazinylethylidenepyrimidines 6c-e against Acinetobacter bau-
P-glycoprotein I/II inhibitors. Therefore,
a
low probability of
mannii (12.5e25.0 mM MIC values) was observed with concomitant

J. Figueiredo et al. / European Journal of Medicinal Chemistry 143 (2018) 829e842
835
low cytotoxicity in normal human dermal fibroblasts. On the other
4.1.2. 1,3-Diphenylthiobarbituric acid (1a) [36]
hand, compound 5-[[(2-chloro-4-nitrophenyl)amino]methylene]-
A stirred solution of 1,3-diphenylthiourea (1.00 mmol; 228 mg),
malonic acid (1.30 mmol; 135 mg) and acetyl chloride (3.00 mmol;
2-thioxodihydropyrimidine-4,6(1H,5H)-dione (7d) presented
relevant and selective anti-proliferative effect against MCF-7 cells
(IC50 ¼ 13.3 M). It should be noted that the presence of a conju-
a
235 mg; 214 m
L) was heated at 60 ꢀC for 30 min. The solid product
m
obtained was ground into finer powder, filtered, washed with water
gated carbon-carbon double bond in some of the studied barbituric
and thiobarbituric acid derivatives may affect the observed bio-
logical activity due to possibility of the compounds to act as
Michael acceptors [56e58]. According to in silico studies, all
selected pyrimidines 2c, 2d, 6c-e and 7d fulfil the Lipinski's rule of
five as well as most of the pharmacokinetic and toxicity criteria
determined for the different ADMET parameters. Therefore, a novel
compound class with members bearing xanthine oxidase inhibitory
activity, antioxidant effects as well as anti-proliferative action and
the ability to inhibit the growth of Acinetobacter baumannii was
revealed. However, future studies should be performed to improve
not only the potency and selectivity of this family of compounds but
also its pharmacokinetics.
and recrystallized from acetic acid; Yield 95%; needle-like yellow
crystals; mp 252e253 ꢀC (lit [36] 258e259 ꢀC); IR ymax (cmꢁ1
)
3053, 2895, 1727, 1707, 1594, 1490, 1454, 1381, 1338, 1260, 1212,
1165, 1169, 1037, 1003, 927, 747, 696, 687, 667; 1H NMR (400 MHz,
CDCl3)
d
(ppm) 7.55 (m, 6H, ArCH), 7.21 (d, J ¼ 7.3 Hz, 4H, 60 and 20-
ArCH), 4.10 (s, 2H, 5-CH2); 13C NMR (101 MHz, CDCl3)
d (ppm)
181.64 (2-CS), 163.32 (4 and 5-CO), 138.75 (ArC), 129.68 (ArCH),
129.20 (ArCH), 128.64 (ArCH), 41.23 (5-CH2).
4.1.3. 5-Benzylidenepyrimidines 2a-e [37]
A stirred mixture of compound 1a-d (1.00 mmol) and benzal-
dehyde (1.00 mmol) in water (5 mL) was refluxed for 2 h. After
cooling, the formed product was filtered, washed with water,
ethanol and diethyl ether to give the following 5-
benzylidenepyrimidines:
4. Experimental section
4 . 1. 3 . 1. 5 - ( 2 - N i t r o b e n z y l i d e n e ) - 1, 3 - d i p h e n y l - 2 -
4.1. Chemistry
thioxodihydropyrimidine-4,6(1H,5H)-dione
(2a). From
1,3-
diphenylthiobarbituric acid (1a) and 2-nitrobenzaldehyde; Yield
90%; pale orange solid. mp 235 ꢀC dec. (lit [60] 232 ꢀC); IR ymax
(cmꢁ1) 3032, 1717, 1691, 1624, 1607, 1591, 1520, 1486, 1353, 1325,
1265, 1189, 1156, 788, 781, 752, 722, 692; 1H NMR (400 MHz,
All reagents were purchased from commercial suppliers and
used without further purification. Reactions were monitored by
thin-layer chromatography (TLC) on Merck-Nagel 60 G/UV254
(0.2 mm) plates which were visualized by ultra-violet (UV) detec-
tion. Melting points were recorded on a Büchi B-540 melting point
apparatus and are uncorrected. Infrared (IR) spectra were obtained
with Thermo Fisher Scientific Nicolet iS10: smart iTR infrared
spectrophotometer equipped with a diamond ATR crystal using
OMNIC 8.2 software. The sample spectra were collected at room
temperature in the 4000-400 cmꢁ1 range by averaging 32 scans at a
DMSO-d6)
d
(ppm) 8.80 (s, 1H, 5-CCH), 8.19 (d, J ¼ 8.2 Hz, 1H), 7.71
(t, J ¼ 7.5 Hz, 1H), 7.59 (t, J ¼ 7.9 Hz, 2H), 7.45 (t, J ¼ 7.6 Hz, 2H),
7.40e7.28 (m, 6H), 7.20 (d, J ¼ 7.7 Hz, 2H); 13C NMR (101 MHz,
DMSO-d6)
d (ppm) 181.43 (2-CS), 160.28 (CO), 158.81 (CO), 155.03
(5-CCH), 146.20 (20-ArC), 140.02 (ArC), 139.72 (ArC), 134.04 (50-
ArCH), 131.76 (10-ArC), 130.40 (40-ArCH), 130.16 (60-ArCH), 129.05
(ArCH),128.88 (ArCH),128.85 (ArCH),128.83 (ArCH),128.28 (ArCH),
128.10 (ArCH), 124.12 (30-ArCH), 121.30 (5-C).
spectral resolution of
4 .
cmꢁ1 1H- (400.13 MHz) and 13C-
(100.62 MHz) NMR spectra were performed on a Brüker Avance III
400 MHz spectrometer and were processed with the software
MestReNova 11.0.3 (trial). Chemical shifts were referenced to the
residual solvent signal (DMSO-d6: dH ¼ 2.50 and dC ¼ 39.52, or
4.1.3.2. 5-(2-Nitrobenzylidene)-2-thioxodihydropyrimidine-
4,6(1H,5H)-dione (2b). From thiobarbituric acid (1b) and 2-
nitrobenzaldehyde; Yield 90%; pale yellow solid; mp 239e241 ꢀC
(lit [61] 246e250 ꢀC); IR ymax (cmꢁ1) 3255, 3156, 2876, 1718, 1692,
1626, 1540, 1513, 1439, 1351, 1288, 1203, 1141, 785, 759, 732; 1H
CDCl3: dH ¼ 7.26 and dC ¼ 77.16). The chemical shift (
d) values are
given in parts per million (ppm), and the coupling constants (J) are
given in Hertz (Hz). The multiplicity of the signals is reported as s
(singlet), d (doublet), dd (doublet of doublets), dt (doublet of trip-
lets), t (triplet), td (triplet of doublets) and m (multiplet). High
resolution mass spectrometry (ESI-HRMS) was carried out on a
Microanalysis QSTAR XL spectrometer by the microanalysis service
(Salamanca, Spain). The 1H NMR and 13C NMR spectra of all com-
pounds are presented on Supplementary Material.
NMR (400 MHz, DMSO-d6)
d (ppm) 12.56 (s, 1H, NH), 12.33 (s, 1H,
NH), 8.63 (s, 1H, 5-CCH), 8.24 (dd, J ¼ 8.3 and 1.0 Hz, 1H, 30-ArCH),
7.80 (td, J ¼ 7.6 and 1.1 Hz, 1H, 50-ArCH), 7.72e7.66 (m, 1H, 40-ArCH),
7.62 (dt, J ¼ 7.7 and 1.2 Hz,1H, 60-ArCH); 13C NMR (101 MHz, DMSO-
d6) d (ppm) 179.05 (2-CS), 160.63 (CO), 159.18 (CO), 153.31 (5-CCH),
146.28 (20-ArC), 133.77 (50-ArCH), 131.58 (10-ArC), 130.49 (40-ArCH),
130.39 (60-ArCH), 124.08 (30-ArCH), 120.61 (5-C).
4.1.3.3. 1,3-Dimethyl-5-(2-nitrobenzylidene)pyrimidine-
2,4,6(1H,3H,5H)-trione (2c). From 1,3-dimethylbarbituric acid (1c)
and 2-nitrobenzaldehyde; Yield 97%; white solid; mp 158e159 ꢀC
(lit [62] 159e161 ꢀC); IR ymax (cmꢁ1) 3028, 2950, 1663, 1603, 1518,
1457, 1415, 1377, 1342, 1323, 1161, 1092, 1057, 787, 754, 737, 692; 1H
4.1.1. 1,3-Diphenylthiourea [35]
To a stirred solution of phenylisothiocyanate (1.00 mmol;
135 mg) in dichloromethane (0.5 mL) at room temperature, was
added dropwise a solution of aniline (1.00 mmol; 93 mg) in
dichloromethane (0.5 mL). The reaction was followed by TLC
(dichloromethane) and completed in 3 h. The resulting suspension
was filtered and washed with diethyl ether; Yield 95%; white
crystals; mp 142e144 ꢀC (lit [59].140e142 ꢀC); IR ymax (cmꢁ1) 3202,
3053, 1598, 1526, 1491, 1449, 1342, 1313, 1288, 1242, 1171, 1069,
1021, 1003, 933, 756, 694, 642, 629, 610; 1H NMR (400 MHz, DMSO-
NMR (400 MHz, DMSO-d6)
d
(ppm) 8.71 (s, 1H, 5-CCH), 8.26 (dd, J ¼
8.3 and 1.2 Hz, 1H, 30-ArCH), 7.80 (td, J ¼ 7.6 and 1.3 Hz, 1H, 50-
ArCH), 7.69 (t, J ¼ 7.7 Hz, 1H, 40-ArCH), 7.53 (d, J ¼ 7.8 Hz, 1H, 60-
ArCH), 3.25 (s, 3H, NCH3), 3.06 (s, 3H, NCH3); 13C NMR (101 MHz,
DMSO-d6)
d (ppm) 161.20 (CO), 159.96 (CO), 153.65 (5-CCH), 151.10
(2-CO), 146.15 (20-ArC), 133.90 (50-ArCH), 132.01 (10-ArC), 130.09
(40-ArCH), 130.02 (60-ArCH), 124.06 (30-ArCH), 120.14 (5-C), 28.44
(N-CH3), 27.82 (N-CH3).
d6)
d
(ppm) 9.79 (s, 2H, 2 ꢂ NH), 7.48 (d, J ¼ 7.4 Hz, 4H, 20 and 60-
ArCH), 7.33 (t, J ¼ 8.2 and 7.6 Hz, 4H,30 and 50-ArCH), 7.12 (t, J ¼
7.4 Hz, 2H, 40-ArCH); 13C NMR (101 MHz, DMSO-d6)
d (ppm) 179.63
(2-CS), 139.46 (10-ArC), 128.45 (ArCH), 124.44 (40-ArCH), 123.66
(ArCH).
4.1.3.4. 5-(2-Nitrobenzylidene)pyrimidine-2,4,6(1H,3H,5H)-trione
(2d). From barbituric acid (1d) and 2-nitrobenzaldehyde; Yield

836
J. Figueiredo et al. / European Journal of Medicinal Chemistry 143 (2018) 829e842
97%; white solid; mp 274e275 ꢀC (lit [61] 274e276 ꢀC); IR ymax
(cmꢁ1) 3230, 3070, 2849, 1739, 1676, 1597, 1516, 1434, 1368, 1341,
1312, 1218, 848, 791, 803, 735, 710; 1H NMR (400 MHz, DMSO-d6)
120.26,119.59, 86.08 (5-C), 30.03 (5-CCH); ESI-HRMS Calcd for [M þ
Na]þ C12H7N3O2Na2SBr 381.9232, found 381.9223.
d
(ppm) 11.49 (s, 1H, NH), 11.24 (s, 1H, NH), 8.60 (s, 1H, 5-CCH), 8.23
4.1.5.2. Sodium 5-[(4-bromophenyl) (cyano)methyl]-1,3-dimethyl-
(d, J ¼ 7.9 Hz, 1H, 30-ArCH), 7.79 (t, J ¼ 7.4 Hz, 1H, 50-ArCH), 7.68 (t,
2,6-dioxo-1,2,3,6-tetrahydropyrimidine-4-olate
(4b). From
1,3-
J ¼ 7.7 Hz, 1H, 40-ArCH), 7.57 (d, J ¼ 7.7 Hz, 1H, 60-ArCH); 13C NMR
dimethylbarbituric acid (1c); Yield 77%; white solid; mp 273 ꢀC
dec.; IR ymax (cmꢁ1) 3445, 2951, 2247 (CN), 1668 (C¼O), 1583 (C¼C),
(101 MHz, DMSO-d6)
d (ppm) 162.38 (CO), 161.20 (CO), 152.45 (5-
CCH), 150.26 (2-CO), 146.28 (20-ArC), 133.76 (50-ArCH), 131.72 (10-
ArC), 130.41 (40-ArCH), 130.16 (60-ArCH), 124.06 (30-ArCH), 120.54
(5-C).
1442, 1317, 1010, 776, 1H NMR (400 MHz, DMSO-d6)
d
7.45 (d, J ¼
8.5 Hz, 2H, 2 ꢂ ArCH), 7.31 (d, J ¼ 8.2 Hz, 2H, 2 ꢂ ArCH), 5.35 (s, 1H,
5-CCH), 3.04 (s, 6H, 2 ꢂ NCH3); 13C NMR (101 MHz, DMSO-d6)
d
161.10 (4 and 6-C),152.75 (2-CO),138.46,130.82 (2 ꢂ ArCH),129.17
4.1.3.5. 5-(4-Methoxybenzylidene)pyrimidine-2,4,6(1H,3H,5H)-tri-
one (2e). From barbituric acid (1d) and 4-methoxybenzaldehyde;
Yield 92%; yellow solid; mp 286e289 ꢀC (lit [37] 297e300 ꢀC); IR
ymax (cmꢁ1) 3046, 2838, 1745, 1698, 1651, 1601, 1567, 1532, 1509,
1444, 1431, 1393, 1355, 1316, 1266, 1215, 1179, 1117, 1041, 1001, 834,
(2 ꢂ ArCH), 120.85, 119.37, 82.03 (5-C), 31.56 (5-CCH), 26.97
(2 ꢂ NCH3); ESI-HRMS Calcd for [M þ Na]þ C14H11N3O3Na2Br
393.9774, found 393.9760.
4.1.6. 5-Acetylpyrimidines (5a-c) [40]
790, 752, 691, 631; 1H NMR (400 MHz, DMSO-d6)
d
(ppm) 11.29 (s,
A solution of compound 1a or 1c-d (1.00 mmol) and concen-
trated sulfuric acid (0.100 mmol) was refluxed in acetic anhydride
(1.00 mmol) for 1 h. The reaction was followed by TLC (dichloro-
methane/5% methanol). The reaction mixture was concentrated to
half of the initial volume and poured onto ice. The formed solid was
isolated, washed with hot water, dried and recrystallized from
ethanol/water (2:1) to give the following 5-acetylpyrimidines:
1H, NH), 11.17 (s, 1H, NH), 8.37 (d, J ¼ 8.9 Hz, 2H, 20 and 60-ArCH),
8.25 (s, 1H, 5-CCH), 7.06 (d, J ¼ 9.2 Hz, 2H, 30 and 50-ArCH), 3.87 (s,
3H, CH3); 13C NMR (101 MHz, DMSO-d6)
d (ppm) 163.91 (CO),
163.44 (CO), 162.18 (40-ArCOCH3), 154.94 (5-CCH), 150.20 (2-CO),
137.49 (20 and 60-ArCH), 125.16 (10-ArC), 115.56 (5-C), 113.95 (30 and
50-ArCH), 55.70 (O-CH3).
4.1.4. 5-(4-Methoxybenzyl)pyrimidine-2,4,6(1H,3H,5H)-trione (3)
[38]
4.1.6.1. 5-Acetyl-1,3-diphenyl-2-thioxodihydropyrimidine-
4,6(1H,5H)-dione (5a). From 1,3-diphenylthiobarbituric acid (1a);
Yield 65%; brown needle crystals; mp 256e257 ꢀC (lit [36]
258e259 ꢀC). IR ymax (cmꢁ1) 3051, 3014, 1701, 1635, 1595, 1557,
1488, 1452, 1414, 1364, 1320, 1271, 1240, 1187, 1161, 1094, 1004, 967,
914, 828, 781, 746, 730, 639, 652, 612; 1H NMR (400 MHz, DMSO-d6)
To a stirred solution of 5-(4-methoxybenzylidene)pyrimidine-
2,4,6(1H,3H,5H)-trione (2e) (1.00 mmol) in ethanol (30 mL), were
added little portions of sodium borohydride (3.00 mmol). The re-
action was followed by TLC (ethyl acetate) and completed after 4 h
at room temperature. The solvent was evaporated to dryness, and
water (20 mL) was added with the formation of a suspension,
which was acidified with 1.00 M hydrochloric acid until pH 5. The
formed solid was filtered and recrystallized from methanol. Yield
41%; white solid; mp 194e195 ꢀC (lit. [63] 205e210 ꢀC); IR ymax
(cmꢁ1) 3224, 2966, 2929, 2835, 1743, 1669, 1608, 1566, 1504, 1421,
1390, 1324, 1299, 1273, 1249, 1175, 1113, 1093, 1033, 835, 797, 779,
d
(ppm) 7.48 (t, J ¼ 7.4 Hz, 4H, 30 and 50-ArCH), 7.39 (t, J ¼ 7.7 Hz, 2H,
40-ArCH), 7.30 (d, J ¼ 7.7 Hz, 4H, 20 and 60-ArCH), 2.66 (s, 3H, 5-
CCCH3); 13C NMR (101 MHz, DMSO-d6)
d (ppm) 196.90 (5-CCCH3),
179.97 (2-CS), 139.68 (10-ArC), 129.54 (20 and 60-ArCH), 129.32 (30
and 50-ArCH), 128.79 (40-ArCH), 98.72 (5-C), 25.06 (5-CCCH3).
4.1.6.2. 5-Acetyl-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione
(5b). From 1,3-dimethylbarbituric acid (1c); Yield 45%; golden
solid; mp 91e92 ꢀC (lit [64] 96.5e98.5 ꢀC); IR ymax (cmꢁ1) 3011,
2963, 1721 (C¼O), 1656 (C¼O), 1557, 1494, 1455, 1364, 1337, 1274,
1221, 1164, 1017, 988, 876, 787, 754, 676; 1H NMR (400 MHz, DMSO-
751, 688, 668; 1H NMR (400 MHz, DMSO-d6)
d (ppm) 11.15 (s, 2H,
NH), 6.99 (d, J ¼ 8.4 Hz, 2H, 20 and 60-ArCH), 6.81 (d, J ¼ 8.5 Hz, 2H,
30 and 50-ArCH), 3.82 (t, J ¼ 4.8 Hz, 1H, 5-CH), 3.70 (s, 3H, CH3), 3.19
(d, J ¼ 4.8 Hz, 2H, 5-CCH2); 13C NMR (101 MHz, DMSO-d6)
d (ppm)
170.04 (4 and 6-CO), 158.05 (40-ArC), 150.56 (2-CO), 130.03 (10-ArC),
128.93 (20 and 60-ArCH), 113.71 (30 and 50-ArCH), 54,94 (O-CH3),
49,57 (5-CH), 32,83 (5-CHCH2).
d6)
d
(ppm) 3.18 (s, 6H, 2 ꢂ NCH3), 2.63 (s, 3H, 5-CCCH3); 13C NMR
(101 MHz, DMSO-d6)
d (ppm) 194.96 (5-CCCH3), 150.44 (2-CO),
96.36 (5-C), 28.06 (2 ꢂ N-CH3), 24.55 (5-CCCH3); ESI-HRMS Calcd
for [M þ H]þ C8H11N2O4 199.0713, found 199.0726.
4.1.5. 5-Phenylcyanomethylpyrimidines 4a-b [39]
A stirred suspension of compound 1b-c (1.00 mmol) and 4-
bromobenzaldehyde (1.00 mmol) in water (5 mL) was refluxed
for 2 h. After cooling, sodium cyanide (1.00 mmol) was added and
the resulting mixture was stirred at 70 ꢀC. The reaction was fol-
lowed by TLC (dichloromethane/methanol 20%) and completed
after 1 h. The reaction mixture was evaporated to dryness and the
obtained solid was dissolved in an equimixture of methanol and
ethyl acetate. The product formed after partial evaporation of the
solvent was filtered and washed with diethyl ether to give the
following 5-phenylcyanomethylpyrimidines:
4.1.6.3. 5-Acetylpyrimidine-2,4,6(1H,3H,5H)-trione (5c). From bar-
bituric acid (1d); Yield 83%; brown solid; mp 305e307 ꢀC (lit [65]
296e300 ꢀC); IR ymax (cmꢁ1) 3280, 3202, 3115, 3029, 2776, 1779,
1732, 1688, 1625, 1515, 1463, 1380, 1249, 1207, 1114, 1061, 1028, 966,
800, 741, 650; 1H NMR (400 MHz DMSO-d6)
d
(ppm) 11.78 (s, 1H,
NH), 11.04 (s, 1H, NH), 2.58 (s, 3H, 5-CCCH3); 13C NMR (101 MHz,
DMSO-d6) (ppm) 195.35 (5-CCCH3), 149.52 (2-CO), 95.89 (5-C),
d
24.29 (5-CCCH3).
4.1.7. 5-Hydrazinylethylidenepyrimidines (6a-g) [40]
A stirred suspension of 5-acetylpyrimidine 5a-c (1.00 mmol)
and the appropriate hydrazine (1.00 mmol) in methanol (20 mL)
was refluxed overnight. The formed solid was filtrated, washed
with hot water and methanol to give the following 5-
hydrazinylethylidenepyrimidines:
4.1.5.1. Sodium 5-[(4-bromophenyl) (cyano)methyl]-6-oxo-2-thioxo-
1,2,3,6-tetrahydropyrimidine-4-olate (4a). From thiobarbituric acid
(1b); Yield 67%; white solid; mp 310 ꢀC dec.; IR ymax (cmꢁ1) 3092,
2881, 2245, 1592, 1525, 1485, 1418, 1300, 1173, 1010, 789; 1H NMR
(400 MHz, DMSO-d6)
8.1 Hz, 2H, 2 ꢂ ArCH), 7.31 (d, J ¼ 7.9 Hz, 2 ꢂ ArCH, 2H), 5.24 (s, 1H,
5-CCH); 13C NMR (101 MHz, DMSO-d6)
(ppm) 173.82 (2-CS),
161.41 (4 and 6-C), 137.61, 130.91 (2 ꢂ ArCH), 129.17 (2 ꢂ ArCH),
d
(ppm) 10.72 (s, 2H, 2 ꢂ NH), 7.47 (d, J ¼
4 .1. 7 .1. 5 - ( 1 - H y d ra z i n yl e t hyl i d e n e ) - 1, 3 - d i p h e nyl - 2 -
thioxodihydropyrimidine-4,6(1H,5H)-dione (6a). From 5-acetyl-1,3-
diphenyl-2-thioxodihydropyrimidine-4,6(1H,5H)-dione (5a) and
d

J. Figueiredo et al. / European Journal of Medicinal Chemistry 143 (2018) 829e842
837
hydrazine hydrate; Yield 88%; brown solid; mp 242e243 ꢀC; IR ymax
(cmꢁ1) 3331, 3246, 3047, 1662, 1620, 1593, 1552, 1489, 1454, 1410,
1363, 1355, 1311, 1298, 1284, 1175, 1122, 1066, 1021, 977, 817, 800,
and 60-ArCH), 89.98 (5-C), 28.03 (2 ꢂ N-CH3), 16.77 (5-CCCH3); ESI-
HRMS Calcd for [M þ H]þ C14H16N5O5 334.1146, found 334.1148.
772, 752, 720, 692, 607; 1H NMR (400 MHz, DMSO-d6)
d
(ppm)
13.35 (s, 1H, 5-CCNH), 7.44e7.20 (m, 12H, ArCH and NH2), 2.61 (s,
3H, 5-CCCH3); 13C NMR (101 MHz, DMSO-d6)
(ppm) 178.47 (2-CS),
4.1.7.6. 5-[1-[2-(2,4-Dinitrophenyl)hydrazinyl]ethylidene]-1,3-
dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (6f). From 5-acetyl-1,3-
d
dimethylpyrimidine-2,4,6(1H,3H,5H)-trione
(5b)
and
2,4-
166.18 (5-CCCH3), 162.03 (4 and 6-CO), 140.66 (100-ArC), 129.22 (200
and 600-ArCH), 128.63 (300 and 500-ArCH), 127.48 (400-ArCH), 89.19 (5-
C), 15.91 (5-CCCH3); ESI-HRMS Calcd for [M þ H]þ C18H17N4O2S
353.1067, found 353.1069.
dinitrophenylhydrazine; Yield 83%; mp 245e247 ꢀC; IR ymax
(cmꢁ1) 3362, 3093, 2952, 1705, 1650, 1615, 1594, 1558, 1538, 1509,
1455, 1360, 1332, 1315, 1275, 1237, 1207, 1157, 1142, 1114, 1060, 972,
923, 850, 817, 754, 744, 715, 651, 620; 1H NMR (400 MHz, DMSO-d6)
d
(ppm) 13.25 (s, 1H, 5-CCNH), 10.64 (s, 1H, 10-ArCNH), 8.89 (d, J ¼
4.1.7.2. 5-(1-Hydrazinylethylidene)pyrimidine-2,4,6(1H,3H,5H)-tri-
one (6b). From 5-acetylpyrimidine-2,4,6(1H,3H,5H)-trione (5c)
and hydrazine hydrate; Yield 86%; yellow solid; mp 300 ꢀC dec.; IR
ymax (cmꢁ1) 3344, 3246, 3004, 1639, 1647, 1609, 1573, 1435, 1374,
1346, 1262, 1242, 1144, 1093, 1037, 977, 923, 872, 797, 781, 755, 672;
2.7 Hz, 1H, 30-ArCH), 8.38 (dd, J ¼ 9.4 and 2.7 Hz, 1H, 50-ArCH), 7.17
(d, J ¼ 9.5 Hz, 1H, 60-ArCH), 3.20 (s, 6H, 2 ꢂ NCH3), 2.65 (s, 3H, 5-
CCCH3); 13C NMR (101 MHz, DMSO-d6)
d (ppm) 176.01 (5-CCCH3),
150.59 (2-CO), 146.98 (10-ArC), 137.80 (40-ArC), 130.38 (20-ArC),
130.37 (50-ArCH), 122.88 (30-ArCH), 115.37 (60-ArCH), 90.30 (5-C),
27.65 (2 ꢂ N-CH3), 16.73 (5-CCCH3); ESI-HRMS Calcd for [M þ Na]þ
C12H10N6O7Na 373.0503, found 373.0498.
1H NMR (400 MHz, DMSO-d6)
d (ppm) 13.19 (s, 1H, 5-CCNH), 10.34
(s, 2H, 2 ꢂ NH), 5.55 (s, 2H, NH2), 2,61 (s, 3H, 5-CCCH3); 13C NMR
(101 MHz, DMSO-d6)
d (ppm) 168.35 (5-CCCH3), 165.64 (4 and 6-
CO), 149.87 (2-CO), 86.07 (5-C), 15.19 (5-CCCH3); ESI-HRMS Calcd
for [M þ Na]þ C6H8N4O3Na 207.0489, found 207.0493.
4.1.7.7. 5-[1-[2-(2,4-Dinitrophenyl)hydrazinyl]ethylidene]pyrimi-
dine-2,4,6(1H,3H,5H)-trione
(6g). From
5-acetylpyrimidine-
4.1.7.3. 1,3-Dimethyl-5-(1-(2-phenylhydrazinyl)ethylidene)pyrimi-
2,4,6(1H,3H,5H)-trione (5c) and 2,4-dinitrophenylhydrazine; Yield
87%; mp 342 ꢀC dec.; IR ymax (cmꢁ1) 3325, 3231, 3094, 3033, 2797,
1710,1682,1634,1616,1591,1505,1455,1334,1313,1272,1234,1175,
1143, 1127, 1059, 1035, 925, 912, 805, 765, 740, 714, 661, 634; 1H
dine-2,4,6(1H,3H,5H)-trione
(6c). From
5-acetyl-1,3-
dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (5b) and phenyl-
hydrazine; Yield 50%; white needle-like crystals; mp 182e184 ꢀC;
IR ymax (cmꢁ1) 3250, 3111, 2997, 2950, 1704, 1672, 1600, 1561, 1459,
1415, 1374, 1355, 1306, 1252, 1214, 1157, 1123, 1053, 1026, 971, 816,
NMR (400 MHz, DMSO-d6)
d (ppm) 13.22 (s, 1H, 5-CCNH), 10.95 (s,
1H, NH),10.67 (s,1H, NH),10.57 (s,1H,10-ArCNH), 8.89 (d, J ¼ 2.6 Hz,
1H, 30-ArCH), 8.38 (dd, J ¼ 9.5 and 2.6 Hz, 1H, 50-ArCH), 7.20 (d, J ¼
9.5 Hz, 1H, 60-ArCH), 2.62 (s, 3H, 5-CCCH3); 13C NMR (101 MHz,
736, 749, 690, 641; 1H NMR (400 MHz, DMSO-d6)
d (ppm) 13.36 (s,
1H, 5-CCNH), 8.63 (s, 1H, 10-ArCNH), 7.27 (t, J ¼ 7.8 Hz, 2H, 30 and 50-
ArCH), 6.89 (t, J ¼ 7.4 Hz, 1H, 40-ArCH), 6.77 (d, J ¼ 7.4 Hz, 2H, 20 and
60-ArCH), 3.17 (s, 6H, 2 ꢂ NCH3), 2.70 (s, 3H, 5-CCCH3); 13C NMR
DMSO-d6) d
(ppm) 175.82 (5-CCCH3), 149.63 (2-CO), 147.10 (10-ArC),
137.79 (40-ArC), 130.89 (20-ArC), 130.42 (50-ArC), 122.89 (30-ArCH),
115.45 (60-ArCH), 89.99 (5-C), 16.08 (5-CCCH3); ESI-HRMS Calcd for
[M þ H]þ C14H15N6O7 379.0997, found 379.0992.
(101 MHz, DMSO-d6)
d (ppm) 174.83 (5-CCCH3) 150.66 (2-CO),
146.43 (10-ArC), 129.39 (30 and 50-ArCH), 120.71 (40-ArCH), 112.76
(20 and 60-ArCH), 88.57 (5-C), 27.50 (2 x NCH3), 16.28 (5-CCCH3);
ESI-HRMS Calcd for [M
289.1302.
þ
H]þ C14H17N4O3 289.1295, found
4.1.8. 5-Phenylaminomethylenepyrimidines (7a-j)
Obtained from the corresponding compounds 1a-c and anilines
by the reaction conditions A or B adapted from the literature [27].
All products were isolated by hot filtration and washed with hot
ethanol and diethyl ether to give the following 5-
phenylaminomethylenepyrimidines.
Reaction conditions A: A stirred mixture of compound 1b-c
(1.00 mmol) and triethyl orthoformate (1.00 mmol) was refluxed in
butan-1-ol (10 mL). o-Phenylenediamine (1.00 mmol) was added
after approximately 1 h, when the color of the reaction mixture
changed to red. The resulting mixture was refluxed for 3 h.
Reaction conditions B: A stirred mixture of compound 1a-
d (1.00 mmol), appropriate aniline (1.00 mmol) and triethyl
orthoformate (1 mL) was refluxed in butan-1-ol (10 mL) for 4 h.
4.1.7.4. 5-[1-(2-Phenylhydrazinyl)ethylidene]pyrimidine-
2,4,6(1H,3H,5H)-trione (6d). From 5-acetylpyrimidine-
2,4,6(1H,3H,5H)-trione (5c) and phenylhydrazine; Yield 92%; dark
white solid; mp 307e310 ꢀC; IR ymax (cmꢁ1) 3267, 3173, 3108, 3064,
3010, 2828, 1712, 1662, 1628, 1601, 1567, 1498, 1458, 1429, 1407,
1376, 1359, 1306, 1255, 1241, 1181, 1152, 1048, 832, 799, 772, 746,
691, 651; 1H NMR (400 MHz, DMSO-d6)
d (ppm) 13.26 (s, 1H, 5-
CCNH), 10.65 (s, 2H, 2 ꢂ NH), 8.54 (s, 1H, 10-ArCNH), 7.26 (t, J ¼
8.0 Hz, 2H, 30 and 50-ArCH), 6.89 (t, J ¼ 7.8 Hz, 1H, 40-ArCH), 6.76 (d,
J ¼ 7.3 Hz, 2H, 20 and 60-ArCH), 2.67 (s, 3H, 5-CCCH3); 13C NMR
(101 MHz, DMSO-d6)
d (ppm) 174.87 (5-CCCH3), 149.72 (2-CO),
146.59 (10-ArC), 129.39 (30 and 50-ArCH), 120.66 (40-ArCH), 112.75
(20 and 60-ArCH), 88.20 (5-C), 15.69 (5-CCCH3); ESI-HRMS Calcd for
[M þ H]þ C12H13N4O3 261.0982, found 261.0982.
4 .1. 8 .1. 5 - [ [ ( 2 - A m i n o p h e n y l ) a m i n o ] m e t h y l e n e ] - 2 -
thioxodihydropyrimidine-4,6(1H,5H)-dione
(7a). From
thio-
4.1.7.5. 1,3-Dimethyl-5-[1-[2-(4-nitrophenyl)hydrazinyl]ethylidene]
barbituric acid (1b) by reaction conditions A; Yield 81%; dark yel-
low solid; mp 247e249 ꢀC; IR ymax (cmꢁ1) 3429, 3342, 3098, 2998,
2894, 1688, 1616, 1587, 1538, 1471, 1330, 1309, 1162, 1008, 868, 776;
pyrimidine-2,4,6(1H,3H,5H)-trione
dimethylpyrimidine-2,4,6(1H,3H,5H)-trione
(6e). From
(5b)
5-acetyl-1,3-
and 4-
nitrophenylhydrazine; Yield 67%; brick-colored solid; mp
228e232 ꢀC; IR ymax (cmꢁ1) 3327, 3254, 2948, 2602, 2432, 2160,
1706, 1612, 1524, 1498, 1455, 1416, 1376, 1357, 1321, 1299, 1270,
1202, 1176, 1104, 971, 836, 797, 794, 690, 658; 1H NMR (400 MHz,
1H NMR (400 MHz, DMSO-d6)
d (ppm) 12.14 (s, 1H, NH), 11.99 (s, 1H,
NH), 11.89 (d, J ¼ 10.8 Hz, 1H, 10-Ar-NH), 8.43 (d, J ¼ 10.8 Hz, 1H,5-
CCH), 7.35 (d, J ¼ 7.8 Hz, 1H, 60-ArCH), 7.05 (t, J ¼ 7.6 Hz, 1H, 40-
ArCH), 6.87 (d, J ¼ 7.7 Hz, 1H, 30-ArCH), 6.73 (t, J ¼ 7.5 Hz, 1H, 50-
ArCH), 5.20 (s, 2H, 10-Ar-NH2); 13C NMR (101 MHz, DMSO-d6)
DMSO-d6)
d
(ppm) 13.29 (s, 1H, 5-CCNH), 9.76 (s, 1H, 10-ArCNH),
8.16 (d, J ¼ 9.8 Hz, 2H, 30 and 50-ArCH), 6.85 (d, J ¼ 9.1 Hz, 2H, 20 and
d (ppm) 177.70 (2-CS), 164.03 (CO), 161.64 (CO), 153.24 (5-CCH),
60-ArCH), 3.19 (s, 6H, 2 ꢂ NCH3), 2.66 (s, 3H, 5-CCCH3); 13C NMR
140.40 (20-ArC), 127.71 (40-ArCH), 126.02 (10-ArC), 119.71 (60-ArCH),
118.37 (50-ArCH), 117.62 (30-ArCH), 93.81 (5-C); ESI-HRMS Calcd for
[M þ Na]þ C11H10N4O2NaS 285.0417, found 285.0407.
(101 MHz, DMSO-d6)
d
(ppm) 175.81 (10-ArC), 152.80 (5-CCCH3),
151.07 (2-CO), 140.09 (40-ArC), 126.51 (30 and 50-ArCH), 111.82 (20

838
J. Figueiredo et al. / European Journal of Medicinal Chemistry 143 (2018) 829e842
4.1.8.2. 5-[[(2-Chloro-5-nitrophenyl)amino]methyleno]-2-
thioxodihydropyrimidine-4,6(1H,5H)-dione (7b). From thio-
barbituric acid (1b) and 2-chloro-5-nitroaniline by reaction con-
ditions B; Yield 97%; dark white solid; mp 348e349 ꢀC; IR ymax
(cmꢁ1) 3074, 2891, 1686, 1620, 1592, 1576, 1517, 1458, 1345, 1317,
1301, 1161, 1053, 1003, 848, 795, 740; 1H NMR (400 MHz, DMSO-d6)
8.09 (d, J ¼ 8.2 Hz, 1H, ArCH), 7.86 (td, J ¼ 7.9 and 1.1 Hz, 1H, ArCH),
7.47 (td, J ¼ 7.8 and 1.0 Hz,1H, ArCH); 13C NMR (101 MHz, DMSO-d6)
d (ppm) 178.10 (2-CS), 163.57 (CO), 161.35 (CO), 151.86 (5-CCH),
138.03, 136.23, 133.94, 126.20, 125.98, 119.60, 96.75 (5-C); ESI-
HRMS Calcd for [M þ H]þ C11H9N4O4S 293.0339, found 293.0336.
d
(ppm) 12.57 (d, J ¼ 13.1 Hz, 1H, 10-Ar-NH), 12.37 (s, 1H, NH), 12.20
4.1.8.7. 2-[[(4,6-Dioxo-1,3-diphenyl-2-thioxotetrahydropyrimidin-
(s, 1H, NH), 8.91 (d, J ¼ 13.0 Hz, 1H, 5-CCH), 8.76 (d, J ¼ 2.6 Hz, 1H,
5(2H)-ylidene)methyl]amino]benzoic
acid
(7g). From
1,3-
60-CH), 8.06 (dd, J ¼ 8.8 and 2.6 Hz, 1H, 40-CH), 7.89 (d, J ¼ 8.8 Hz,
diphenylthiobarbituric acid (1a) and 2-aminobenzoic acid by re-
1H, 30-CH); 13C NMR (101 MHz, DMSO-d6)
d (ppm) 178.04 (2-CS),
action conditions B; Yield 83%; yellow solid; mp 322e324 ꢀC; IR
164.56 (CO), 161.06 (CO), 152.92, 147.29, 136.10, 131.07, 129.64,
ymax (cmꢁ1) 3400 to 2700 (broad), 3061, 1689, 1660, 1644, 1607,
ꢀ
120.96, 113.70, 96.17 (5-C). ESI-HRMS Calcd for [M
C11H8ClN4O4S 326.9949, found 326.9947.
þ
H]þ
1569, 1439, 1324, 1290, 1259, 1208, 1147, 1070, 753, 723, 691; 1H
NMR (400 MHz, DMSO-d6) d
(ppm) 13.82 (s, 1H, 20-Ar-COOH), 13.57
(d, J ¼ 14.2 Hz, 1H, 10-Ar-NH), 8.83 (d, J ¼ 14.1 Hz, 1H, 5-CCH), 8.03
(dd, J ¼ 7.9 and 1.8 Hz, 1H, ArCH), 7.88 (d, J ¼ 8.4 Hz, 1H, ArCH), 7.73
(td, J ¼ 7.6 and 1.2 Hz, 1H, ArCH), 7.52e7.43 (m, 4H, Ar-CH),
7.42e7.31 (m, 5H, ArCH), 7.29 (d, 2H, J ¼ 7.2 Hz, ArCH); 13C NMR
4.1.8.3. 5-[[(2-Chloro-5-nitrophenyl)amino]methyleno]-1,3-
dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (7c). From 1,3-
dimethylbarbituric acid (1c) and 2-chloro-5-nitroaniline by reac-
tion conditions B; Yield 92%; white solid; mp 257e259 ꢀC; IR ymax
(cmꢁ1) 3068, 2956, 1731, 1631, 1599, 1573, 1528, 1498, 1466, 1419,
1344, 1293, 1204, 1086, 1053, 1000, 882, 825, 740; 1H NMR
(101 MHz, DMSO-d6)
d
(ppm) 180.74 (2-CS), 167.30 (20-Ar-COOH),
161.92 (CO), 161.05 (CO), 152.23 (5-CCH), 140.30, 139.66, 139.33,
134.58, 131.68, 129.27, 129.09, 128.90, 128.84, 128.00, 127.88, 125.81,
(400 MHz, DMSO-d6)
d
(ppm) 12.52 (s, 1H, 10-Ar-NH), 8.93 (s, 1H, 5-
119.30, 117.50, 96.12 (5-C); ESI-HRMS Calcd for [M
C24H17N3O4NaS 466.0832, found 466.0823.
þ
Na]þ
CCH), 8.74 (d, J ¼ 2.4 Hz, 1H, 60-Ar-CH), 8.06 (dd, J ¼ 8.8 and 2.4 Hz,
1H, 40-Ar-CH), 7.89 (d, J ¼ 8.8 Hz, 1H, 30-Ar-CH), 3.23 (s, 6H,
2 ꢂ NCH3); 13C NMR (101 MHz, DMSO-d6)
d
(ppm) 164.67 (CO),
4.1.8.8. 2-[[(4,6-Dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)
methyl]amino]benzoic acid (7h). From thiobarbituric acid (1b) and
2-aminobenzoic acid by reaction conditions B; Yield 97%; dark
161.38 (CO), 152.26, 151.14 (2-CO), 147.31, 136.26, 131.02, 129.46,
120.59, 113.26, 95.00 (5-C), 27.66 (NCH3), 27.15 (NCH3); ESI-HRMS
Calcd for [M þ H]þ C13H12ClN4O5 339.0490, found 339.0485.
white solid; mp 365 ꢀC dec. (lit [27] 340e344 ꢀC); IR ymax (cmꢁ1
)
3142 to 2511 (broad), 3142, 3041, 2966, 2892, 1676, 1602, 1581, 1527,
4.1.8.4. 5-[[(2-Chloro-4-nitrophenyl)amino]methyleno]-2-
1457, 1437, 1348, 1321, 1288, 1251, 1158, 1008, 858, 757; 1H NMR
thioxodihydropyrimidine-4,6(1H,5H)-dione
barbituric acid (1b) and 2-chloro-4-nitroaniline by reaction con-
ditions B; Yield 91%; yellow solid; mp 365 ꢀC dec.; IR ymax (cmꢁ1
3332, 3053, 2880, 1698, 1650, 1575, 1506, 1457, 1347, 1302, 1288,
(7d). From
thio-
(400 MHz, DMSO-d6) d
(ppm) 13.86 (s, 1H, 20-Ar-COOH), 13.53 (d,
J ¼ 14.0 Hz, 1H, 10-Ar-NH), 12.14 (s, 1H, NH), 12.05 (s, 1H, NH), 8.67
(d, J ¼ 14.0 Hz, 1H, 5-CCH), 8.03 (d, J ¼ 7.7 Hz, 1H, ArCH), 7.86 (d, J ¼
8.4 Hz,1H, ArCH), 7.70 (t, J ¼ 7.5 Hz, 1H, ArCH), 7.35 (t, J ¼ 7.6 Hz, 1H,
)
1267, 1139, 815, 744, 731; 1H NMR (400 MHz, DMSO-d6)
d
(ppm)
ArCH); 13C NMR (101 MHz, DMSO-d6)
d (ppm) 178.00 (2-CS), 167.61
12.63 (d, J ¼ 12.9 Hz, 1H, 10-Ar-NH), 12.44 (s, 1H, NH), 12.27 (s, 1H,
NH), 8.83 (d, J ¼ 12.9 Hz, 1H, 5-CCH), 8.48 (d, J ¼ 2.2 Hz, 1H, 30-
ArCH), 8.25 (dd, J ¼ 9.2 and 1.9 Hz, 1H, 50-ArCH), 8.19 (d, J ¼ 9.2 Hz,
(20-Ar-COOH), 162.92 (CO), 161.78 (CO), 150.92 (5-CCH), 139.59,
134.56, 131.67, 125.50, 118.91, 117.30, 95.48 (5-C).
1H, 60-ArCH); 13C NMR (101 MHz, DMSO-d6)
d
(ppm) 178.13 (2-CS),
4.1.8.9. 2-[[(1,3-Dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-
164.48 (CO), 160.99 (CO), 151.47 (5-CCH), 144.11, 140.41, 125.45,
124.03, 123.08, 117.96, 97.31 (5-C); ESI-HRMS Calcd for [M þ H]þ
C11H8ClN4O4S 326.9949, found 326.9947.
ylidene)methyl]amino]benzoic
acid
(7i). From
1,3-
dimethylbarbituric acid (1c) and 2-aminobenzoic acid by reaction
conditions B; Yield 77%; pale yellow needle crystals; mp
314e315 ꢀC (lit [66] 210 ꢀC); IR ymax (cmꢁ1) 3300 to 2600 (broad),
3083,1715,1644,1602,1577, 1450, 1355,1315, 1197, 1142, 1072, 1013,
4 .1. 8 . 5 . 5 - [ [ ( 2 - C h l o r o p h e n y l ) a m i n o ] m e t h y l e n o ] - 2 -
thioxodihydropyrimidine-4,6(1H,5H)-dione
(7e). From
thio-
869, 789, 752; 1H NMR (400 MHz, DMSO-d6)
d (ppm) 13.60 (s, 1H,
barbituric acid (1b) and 2-chloroaniline by reaction conditions B;
20-Ar-COOH), 13.46 (d, J ¼ 14.1 Hz, 1H, 10-Ar-NH), 8.67 (d, J ¼
13.9 Hz,1H, 5-CCH), 8.01 (d, J ¼ 7.7 Hz, 2H, ArCH), 7.79 (d, J ¼ 8.4 Hz,
1H, ArCH), 7.69 (t, J ¼ 7.7 Hz,1H, ArCH), 7.32 (t, J ¼ 7.5 Hz,1H, ArCH),
3.18 (d, J ¼ 3.1 Hz, 6H, 2 ꢂ NCH3); 13C NMR (101 MHz, DMSO-d6)
Yield 93%; pale yellow solid; mp 357e358 ꢀC; IR ymax (cmꢁ1) 3106,
ꢀ
3008, 2894, 2562, 1680, 1621, 1599, 1588, 1571, 1536, 1496, 1442,
1316, 1285, 1157, 1053, 1000, 854, 802, 767; 1H NMR (400 MHz,
DMSO-d6)
d
(ppm) 12.53 (d, J ¼ 13.4 Hz, 1H, 10-Ar-NH), 12.31 (s, 1H,
d
(ppm) 167.58 (20-Ar-COOH), 163.04 (CO), 162.06 (CO), 151.41 (5-
NH), 12.14 (s, 1H, NH), 8.74 (d, J ¼ 13.4 Hz, 1H, 5-CCH), 7.93 (d, J ¼
8.1 Hz, 1H, ArCH), 7.62 (dd, J ¼ 8.0 and 1.1 Hz, 1H, ArCH), 7.45 (t, J ¼
7.7 Hz, 1H, ArCH), 7.29 (td, J ¼ 7.7 and 1.1 Hz, 1H, ArCH); 13C NMR
CCH), 150.40 (2-CO), 139.81, 134.53, 131.66, 125.09, 118.69, 116.83,
94.19 (5-C), 27.64 (NCH3), 27.03 (NCH3).
(101 MHz, DMSO-d6)
d
(ppm) 177.93 (2-CS), 164.49 (CO), 161.26
4.1.8.10. 2-[[(2,4,6-Trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]
amino]benzoic acid (7j). From barbituric acid (1d) and 2-
aminobenzoic acid by reaction conditions B; Yield 84%; white
solid; mp 365 ꢀC dec. (lit [27] 310e314 ꢀC); IR ymax (cmꢁ1) 3163 to
2514 (broad), 3136, 3084, 2815, 1729, 1673, 1614, 1593, 1573, 1503,
1458, 1439, 1420, 1350, 1324, 1290, 1255, 941, 856, 795, 758; 1H
(CO), 151.86 (5-CCH), 134.81, 130.00, 128.82, 127.22, 123.15, 118.18,
95.27 (5-C); ESI-HRMS Calcd for [M þ H]þ C11H9ClN3O2S 282.0098,
found 282.0095.
4 .1. 8 . 6 . 5 - [ [ ( 2 - N i t r o p h e n y l ) a m i n o ] m e t h y l e n e ] - 2 -
thioxodihydropyrimidine-4,6(1H,5H)-dione
(7f). From
thio-
NMR (400 MHz, DMSO-d6) d
(ppm) 13.75 (s, 1H, 20-Ar-COOH), 13.38
barbituric acid (1b) and 2-nitroaniline by reaction conditions B;
Yield 98%; yellow solid; mp 330 ꢀC dec.; IR ymax (cmꢁ1) 3121, 3062,
2899, 1684, 1633, 1587, 1567, 1532, 1511, 1436, 1337, 1299, 1261, 1151,
(d, J ¼ 13.9 Hz, 1H, 10-Ar-NH), 10.96 (s, 1H, NH), 10.88 (s, 1H, NH),
8.62 (d, J ¼ 13.8 Hz, 1H, 5-CCH), 8.02 (d, J ¼ 7.6 Hz, 1H, ArCH), 7.81
(d, J ¼ 8.4 Hz, 1H, ArCH), 7.69 (t, J ¼ 7.9 Hz, 1H, ArCH), 7.32 (t, J ¼
991, 856, 790, 740; 1H NMR (400 MHz, DMSO-d6)
d
(ppm) 13.33 (d,
7.6 Hz,1H, ArCH); 13C NMR (101 MHz, DMSO-d6) (ppm) 167.69 (20-
d
J ¼ 13.3 Hz, 1H, 10-Ar-NH), 12.32 (s, 1H, NH), 12.18 (s, 1H, NH), 8.72
(d, J ¼ 13.3 Hz, 1H, 5-CCH), 8.26 (dd, J ¼ 8.4 and 1.0 Hz, 1H, ArCH),
Ar-COOH), 165.08 (CO), 163.72 (CO), 150.71 (5-CCH), 149.85 (2-CO),
139.97, 134.57, 131.69, 124.97, 118.57, 116.87, 94.26 (5-C).

J. Figueiredo et al. / European Journal of Medicinal Chemistry 143 (2018) 829e842
839
4.1.9. 1-[(Pyrimidin-5-ylidene)methyl]ureas 8a-g
4.1.9.6. 1-[(1,3-Dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-yli-
dene)methyl]thiourea (8f). From 1,3-dimethylbarbituric acid (1c)
and thiourea; Yield 89%; yellow solid; mp 267e270 ꢀC; IR ymax
(cmꢁ1) 3290, 3145, 1716, 1635, 1589, 1469, 1429, 1338, 1230, 1186,
A stirred mixture of compound 1a-d (1.00 mmol), urea/thiourea
(1.00 mmol) and triethyl orthoformate (1.50 mmol) was refluxed in
butan-1-ol (10 mL) for 4 h. The formed product was filtered and
washed with diethyl ether to give the following 1-(pyrimidin-5-
ylidene)ureas:
1086, 798, 754; 1H NMR (400 MHz, DMSO-d6)
d
(ppm) 11.95 (d, J ¼
12.4 Hz, 1H, 5-CCHNH), 9.59 (s, 1H, NH2), 9.48 (s, 1H, NH2), 9.19 (d,
J ¼ 12.4 Hz, 1H, 5-CCH), 3.19 (d, J ¼ 3.3 Hz, 6H, 2 ꢂ N-CH3); 13C NMR
(101 MHz, DMSO-d6) d (ppm) 181.46 (NHCNH2), 163.30 (CO), 161.70
4.1.9.1. 1-[(4,6-Dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)
methyl]urea (8a). From thiobarbituric acid (1b) and urea; Yield
77%; reddish solid; mp 298e300 ꢀC; IR ymax (cmꢁ1) 3406, 3147,
2889, 1730, 1693, 1643, 1529, 1375, 1259, 1149, 815, 757; 1H NMR
(CO),152.35 (5-CCH),151.14 (2-CO), 95.78 (5-C), 27.76 (N-CH3), 27.17
(N-CH3); ESI-HRMS Calcd for [M þ H]þ C8H11N4O3S 243.0552,
found 243.0551.
(400 MHz, DMSO-d6)
d (ppm) 12.19 (s, 1H, NH), 12.13 (1H, s, NH),
4.1.9.7. 1-[(2,4,6-Trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]
thiourea (8g). From barbituric acid (1d) and thiourea; Yield: 86%;
light-brown solid; mp 375 ꢀC dec.; IR ymax (cmꢁ1) 3280, 3217, 3145,
3045, 2808, 1733, 1701, 1645, 1571, 1438, 1407, 1247, 1024, 783; 1H
11.24 (d, J ¼ 13.2 Hz, 1H, 5-CCHNH), 8.54 (d, J ¼ 13.1 Hz, 1H, 5-CCH),
7.88 (s, 1H, NH2), 7.56 (s, 1H, NH2); 13C NMR (101 MHz, DMSO-d6)
d
(ppm) 178.46 (2-CS), 163.01 (CO), 161.73 (CO), 152.03 (NHCNH2),
151.03 (5-CCH), 96.12 (5-C); ESI-HRMS Calcd for [M þ H]þ
NMR (400 MHz, DMSO-d6)
d
(ppm) 11.84 (d, J ¼ 12.3 Hz, 1H, 5-
C6H7N4O3S 215.0239, found 215.0237.
CCHNH), 11.15 (s, 1H, NH), 11.03 (s, 1H, NH), 9.53 (s, 1H, NH2), 9.45
(s, 1H, NH2), 9.08 (d, J ¼ 12.3 Hz, 1H, 5-CCH); 13C NMR (101 MHz,
4.1.9.2. 1-[(1,3-Dimethyl-2,4,6-trioxotetrahydropyrimidin-5(2H)-yli-
dene)methyl]urea (8b). From 1,3-dimethylbarbituric acid (1c) and
urea; Yield 52%; yellow solid; mp 260e261 ꢀC; IR ymax (cmꢁ1) 3371,
3278, 3197, 1704, 1666, 1635, 1560, 1406, 1371, 1255, 1085; 1H NMR
DMSO-d6)
d (ppm) 181.52 (NHCNH2), 165.17 (CO), 163.22 (CO),
151.54 (5-CCH), 150.40 (2-CO), 96.96 (5-C); ESI-HRMS Calcd for
[M þ H]þ C6H7N4O3S 215.0239, found 215.0238.
(400 MHz, DMSO-d6)
d
(ppm) 11.23 (d, J ¼ 13.1 Hz, 1H, 5-CCHNH),
4.2. In vitro activity
8.61 (d, J ¼ 13.1 Hz, 1H, 5-CCH), 7.82 (s, 1H, NH2), 7.47 (s, 1H, NH2),
3.17 (d, J ¼ 3.7 Hz, 6H, 2 ꢂ N-CH3); 13C NMR (101 MHz, DMSO-d6)
4.2.1. Xanthine oxidase inhibitory activity
d
(ppm) 163.24 (CO), 161.95 (CO), 152.17 (NHCNH2), 151.27 (5-CCH),
4.2.1.1. Preparation of sample solutions. All assayed compounds
were dissolved in DMSO in a concentration of 10 mM for the initial
screening and 50 mM for the concentration-response studies. A
10 mM xanthine stock solution was prepared in a 25 mM aqueous
NaOH solution. All stock solutions were stored at 4 ꢀC and the
diluted solutions were prepared in 50 mM dihydrogen phosphate
buffer (pH 7.4) before each experiment. The concentrations in each
150.67 (2-CO), 94.90 (5-C), 27.66 (N-CH3), 27.02 (N-CH3); ESI-HRMS
Calcd for [M þ H]þ C8H11N4O4 227.0780, found 227.0780.
4.1.9.3. 1-[(2,4,6-Trioxotetrahydropyrimidin-5(2H)-ylidene)methyl]
urea (8c). From barbituric acid (1d) and urea; Yield 71%; light-
brown solid; mp 248e250 ꢀC; IR ymax (cmꢁ1) 3411, 3172, 1733,
1703, 1629, 1569, 1384, 1305, 1272, 792; 1H NMR (400 MHz, DMSO-
well were 30
50 and 100
m
M for the initial screening as well as 0.1, 1, 7.5, 15, 30,
d6)
10.97 (s, 1H, NH), 8.51 (d, J ¼ 13.1 Hz, 1H, 5-CCH), 7.83 (s, 1H, NH2),
7.46 (s, 1H, NH2); 13C NMR (101 MHz, DMSO-d6)
(ppm) 165.22
d
(ppm) 11.10 (d, J ¼ 13.1 Hz, 1H, 5-CCHNH), 11.06 (s, 1H, NH),
mM for the concentration-response studies. The final
concentration of DMSO in each well was 1%, and no significant
interference with the enzyme activity was observed (data not
shown).
d
(CO), 163.57 (CO), 152.33 (NHCNH2), 150.61 (5-CCH), 149.91 (2-CO),
95.08 (5-C); ESI-HRMS Calcd for (Mþ þ Na) C6H6N4O4Na 221.0281,
found 221.0282.
4.2.1.2. Experimental procedure. The xanthine oxidase (XO) activity
was spectrophotometrically determined by quantifying the uric
acid formation from xanthine adapting the methods of Zhao et al.
4.1.9.4. 1-[(4,6-Dioxo-1,3-diphenyl-2-thioxotetrahydropyrimidin-
5(2H)-ylidene)methyl]thiourea
(8d). From
1,3-
[41] and Gupta et al. [68]. For each essay, 50 mL of test solution and
diphenylthiobarbituric acid (1a) and thiourea; Yield 85%; orange
solid; mp 259e261 ꢀC (lit 263e264 ꢀC [67]); IR ymax (cmꢁ1) 3442,
3263, 3172, 3051,1685, 1654, 1591,1487,1402,1321, 1217, 1190, 1153,
50 mL of a XO bovine serum suspension (0.1 U/mL) were added to
each well of a 96-well plate followed by a pre-incubation at 37 ꢀC
for 5 min. The enzymatic reaction started after the addition of
823, 688; 1H NMR (400 MHz, DMSO-d6)
d
(ppm) 12.13 (d, J ¼
150
m
L of xanthine (0.42 mM) and further incubation at 37 ꢀC
11.7 Hz, 1H, 5-CCHNH), 9.72 (s, 1H, NH2), 9.44 (s, 1H, NH2), 9.28 (d,
during 10 min. The absorbances were measured at 295 nm and at
every minute, with 20 s of a constant and automatic slow stirring
before each reading. Dihydrogen phosphate buffer (50 mM, pH 7.4)
was used as negative control and allopurinol as a positive control.
In order to discount the absorbance of each compound at this
J ¼ 11.7 Hz, 1H, 5-CCH), 7.49e7.27 (m, 10H, ArCH); 13C NMR
(101 MHz, DMSO-d6)
d (ppm) 181.16 (NHCNH2), 180.79 (2-CS),
162.01 (CO),160.72 (CO), 153.88 (5-CCH), 139.94 (ArC), 139.29 (ArC),
128.96 (2 ꢂ ArCH), 128.91 (2 ꢂ ArCH), 128.80 (2 ꢂ ArCH), 128.75
(2 ꢂ ArCH), 128.00 (ArCH), 127.94 (ArCH), 98.49 (5-C); ESI-HRMS
Calcd for [M þ H]þ C18H15N4O2S2 383.0636, found 383.0635.
wavelength, a blank sample containing 50
mL of test solution and
200 L of buffer was performed. Three independent experiments,
m
each one in triplicate, were performed. The percentage of enzyme
inhibition for each compound was calculated according to the
following formula:
4.1.9.5. 1-[(4,6-Dioxo-2-thioxotetrahydropyrimidin-5(2H)-ylidene)
methyl]thiourea (8e). From thiobarbituric acid (1b) and thiourea;
Yield 74%; red solid; mp 252 ꢀC dec.; IR ymax (cmꢁ1) 3284, 3136,
1695, 1664, 1574, 1506, 1413, 1259, 1143, 1012, 997, 804; 1H NMR
% of inhibition ¼ [1 - (Abssample e Absblank of sample) / Absnegative
control] ꢂ 100
(400 MHz, DMSO-d6)
d (ppm) 12.32 (s, 1H, NH), 12.23 (s, 1H, NH),
11,94 (d, J ¼ 13.1 Hz, 1H, 5-CCHNH), 9.70 (s, 1H, NH2), 9.55 (s, 1H,
NH2), 9.11 (d, J ¼ 13.1 Hz,1H, 5-CCH); 13C NMR (101 MHz, DMSO-d6)
The direct breakdown of uric acid by the synthetized BADs and
TBADs 2e8 was spectrophotometrically studied in experimental
conditions similar to the established in enzymatic inhibition assays.
d
(ppm) 181.31 (NHCNH2), 178.48 (2-CS), 163.05 (CO), 161.50 (CO),
152.57 (5-CCH), 97.95 (5-C); ESI-HRMS Calcd for [M þ H]þ
C6H7N4O2S2 231.0010, found 231.0013.
For each assay, 50 mL of test solution (60 mM) and 50 mL of uric acid

840
J. Figueiredo et al. / European Journal of Medicinal Chemistry 143 (2018) 829e842
(200
m
M) were added to each well of a 96-well plate. The absor-
also tetracycline was used as reference. The MIC was defined as the
lowest concentration at which no growth was visually observed.
Each test was carried out at least three independent times and
modal MIC values were selected.
bances were measured at 295 nm after further incubation at 37 ꢀC
during 10 min. In order to discount the absorbance of each com-
pound at this wavelength, a blank sample containing 50
mL of test
solution and 50 L of buffer was performed. Three independent
m
experiments, each one in triplicate, were performed. No significant
interference with uric acid levels was observed for any compound
under study.
4.2.4. Cytotoxicity in human cell lines
4.2.4.1. Preparation of sample solutions. All the tested compounds
were dissolved in DMSO in a concentration of 10 mM and stored at
4.2.2. Radical scavenging activity
4
ꢀC. From this solution, several solutions of the compounds in
4.2.2.1. Preparation of sample solutions. All assayed compounds
were dissolved in DMSO in a 10 mM concentration and stored at
4
study were prepared by their adequate dilutions in the complete
culture medium before each experiment. The maximum DMSO
concentration in the studies was 1% (v/v), a concentration with no
significant effect on cell proliferation (data not shown).
ꢀC. The diluted solutions were prepared in 99.5% ethanol before
each experiment. The concentrations in each well were 30
m
M for
the initial screening as well as 1, 7.5, 15, 30, 50 and 100
concentration-response studies.
mM for the
4.2.4.2. Cells cultures. The cells used in this study were human
breast cancer cells (MCF-7) and normal human dermal fibroblasts
(NHDF) and were obtained from American Type Culture Collection
(ATCC). These cells were maintained at 37 ꢀC in a humidified at-
mosphere containing 5% CO2. MCF-7 cells were cultured in high-
glucose Dulbecco's modified Eagle's medium (DMEM) supple-
mented with 10% fetal bovine serum (FBS) and 1% antibiotic/anti-
mycotic (10,000 units/mL penicillin, 10 mg/mL streptomycin and
4.2.2.2. Experimental procedure. The radical scavenging activity
was spectrophotometrically determined by the quantification of
the DPPH reduction extension by the compounds under study,
according to Zhao et al. [41] and Nicolaides et al. [42]. The reaction
mixture contained 100 mL of the test solution and 100 mL of the
DPPH solution (0.2 mM). The capacity of each compound to reduce
DPPH was followed by measuring the absorbance at 517 nm after
20 and 60 min. The solutions remained in the absence of light be-
tween measurements. Ethanol was used as the negative control and
Trolox as the positive control. To discount the absorbance of each
compound at 517 nm, for each test compound a blank was per-
formed where DPPH was replaced by ethanol. Three independent
experiments were performed, each one in triplicate. The reduction
capacity of DPPH was calculated according to the following
formula:
25
mg/mL amphotericin B). The RPMI medium supplemented with
10% fetal bovine serum (FBS),
L
-glutamine (2 mM), HEPES (10 mM),
sodium pyruvate (1 mM) and 1% antibiotic/antimycotic was used to
culture NHDF cells. The cells used in the experiments were used in
passages 26 to 31 (MCF-7) and 6 to 11 (NHDF).
4.2.4.3. MTT assay. The cell proliferation was evaluated by quanti-
fying the extent of the reduction of 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) according to a previously
described procedure [52]. Cells were seeded in 96-well plates
(2 ꢂ 104 cells/mL) in the culture medium and after 48 h of adher-
ence they were treated with the different compounds and con-
centrations under study for 72 h. Untreated cells were used as
negative control and 5-FU as positive control. At the end of incu-
bation, the medium was removed and replaced with fresh culture
medium and MTT solution [5 mg/mL in phosphate buffer saline
(NaCl 137 mM, KCl 2.7 mM, Na2HPO4 10 mM and KH2PO4 1.8 mM),
pH 7.4] and further incubated at 37 ꢀC during 4 h. Then, the
medium-containing MTT was removed and formazan crystals were
dissolved with DMSO followed by absorbance readings at 570 nm.
The cytotoxicity was expressed as the relative cell proliferation in
percentage in comparison with the negative control cells.
% Reduction ¼ [1 - (Abssample - Abssample blank / Absnegative
control)] ꢂ 100
4.2.3. Antibacterial activity
4.2.3.1. Sample solutions, bacteria, stock and growth conditions.
All the tested compounds were dissolved in DMSO in a concen-
tration of 10 mM, stored at 4 ꢀC, and diluted with culture medium
before assays.
The following strains were used in the study: Bacillus cereus
ATCC11778, Staphylococcus aureus ATCC 25923, Escherichia coli
ATCC 25922, Klebsiella pneumoniae ATCC 13883, Pseudomonas aer-
uginosa ATCC 27853, Salmonella enterica subsp. enterica serovar
Typhimurium ATCC 13311 and Acinetobacter baumannii LMG 1025.
All bacterial cultures were stocked at ꢁ80 ꢀC in medium with 20%
glycerol. The bacteria were sub-cultured on Müeller Hinton Agar
and incubated overnight at 37 ꢀC prior to each antimicrobial assay.
4.2.5. Statistics
The results are expressed as mean values standard error of the
mean (SEM) and are representative of least two independent ex-
periments. The difference between groups was considered statis-
tically significant at p < 0.05 (Student's t-test). The IC50 values were
calculated by sigmoidal fitting analysis considering a 95% confi-
dence interval.
4.2.3.2. Determination of minimum inhibitory concentration (MIC).
The antibacterial activity of the test compounds was studied by
measurement of MIC by broth microdilution method according
with CLSI guidelines (M7-A6). The compounds were two-fold
serially diluted in a 96-well plate with Müeller Hinton Broth
4.3. In silico simulations
(MHB) to a volume of 50
mL, to give a final concentration range from
200 to 1.56 M. The maximum DMSO concentration was 2% (v/v).
Bacterial suspensions were prepared from overnight culture using
sterile saline solution, i.e. NaCl 0.85% (w/v), adjusted to 0.5
m
The structures of tested compounds were manually drawn in
ChemBioDraw 13.0 software and the SMILES notation was obtained
for each molecule. To calculate the parameters of Lipinski's rule of
five, pharmacokinetic (absorption, distribution, metabolism and
excretion) and toxicity properties, the SMILES notation in pkCSM
online software (available from http://bleoberis.bioc.cam.ac.uk/
pkcsm/prediction, accessed at 04.04.17) was employed [54].
McFarland and diluted with MHB. Then 50 mL of suspension was
added to each well to a final concentration of about 5 ꢂ 105 colony
forming units/ml, and the plate was incubated at 37 ꢀC for 24 h.
Sterility, solvent and growth controls were included in the assays,

J. Figueiredo et al. / European Journal of Medicinal Chemistry 143 (2018) 829e842
50 (2013) E121eE125.
841
Acknowledgments
[23] Z. Huang, Q. Zhao, G. Chen, H. Wang, W. Lin, L. Xu, H. Liu, J. Wang, D. Shi,
Y. Wang, An efficient synthesis of novel dispirooxindole derivatives via one-
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(2012) 12704e12717.
This work is supported by FEDER funds through the POCI -
COMPETE 2020 - Operational Programme Competitiveness and
Internationalization in Axis I - Strengthening research, technolog-
ical development and innovation (Project POCI-01-0145-FEDER-
007491) and National Funds by FCT - Foundation for Science and
Technology (Project UID/Multi/00709/2013). The authors also
acknowledge the contribution of Dr. Adriana Santos for the revision
of written English.
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