Conjugation of N-acylhydrazone and 1,2,4-oxadiazole leads to the identification of active antimalarial agents

Article abstract of DOI:10.1016/j.bmc.2016.09.013

Malaria, caused by several Plasmodium species, is the major life-threatening parasitic infection worldwide. Due to the parasite resistance to quinoline based drugs, the search for antimalarial agents is necessary. Here, we report the structural design, sy

Full text of DOI:10.1016/j.bmc.2016.09.013

Accepted Manuscript
Conjugation of N-acylhydrazone and 1,2,4-oxadiazole leads to the identification
of active antimalarial agents
José Maurício dos Santos Filho, Diogo Manoel Alves de Queiroz e Silva, Taís
Soares Macedo, Helena Mariana Pitangueira Teixeira, Diogo Rodrigo
Magalhaes Moreira, Soura Challal, Jean-Luc Wolfender, Emerson Ferreira
Queiroz, Milena Botelho Pereira Soares
PII:
S0968-0896(16)30700-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.09.013
BMC 13266
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 July 2016
4 September 2016
7 September 2016
Please cite this article as: Filho, J.M.d., Silva, D.M.A., Macedo, T.S., Teixeira, H.M.P., Moreira, D.R.M., Challal,
S., Wolfender, J-L., Queiroz, E.F., Soares, M.B.P., Conjugation of N-acylhydrazone and 1,2,4-oxadiazole leads to
the identification of active antimalarial agents, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/
10.1016/j.bmc.2016.09.013
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Conjugation of N-acylhydrazone and 1,2,4-oxadiazole leads to the identification of
active antimalarial agents
José Maurício dos Santos Filho,1* Diogo Manoel Alves de Queiroz e Silva,1 Taís Soares Macedo,2
Helena Mariana Pitangueira Teixeira,2 Diogo Rodrigo Magalhaes Moreira,2 Soura Challal,3 Jean-Luc
Wolfender,3 Emerson Ferreira Queiroz,3 Milena Botelho Pereira Soares,2,4
1 Laboratory of Design and Synthesis Appied to Medicinal Chemistry-SintMed®, Centro de Tecnologia e
Geociências, Universidade Federal de Pernambuco, CEP 50740-521, Recife, PE, Brazil.
2 Centro de Pesquisas Gonçalo Moniz, Fundação Oswaldo Cruz, CEP 40296-710, Salvador, BA, Brazil.
3 School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, 30 quai Ernest-
Ansermet, CH-1211 Geneva 4, Switzerland
4 Centro de Biotecnologia e Terapia Celular, Hospital São Rafael, CEP 41253-190, Salvador, BA, Brazil.
*To whom correspondence should be addressed: phone: +55 81 21267288; fax: + 55 81 21267278. E-mail address:
mauricio_santosfilho@yahoo.com.br (J.M.S.F.).

ABSTRACT
Malaria, caused by several Plasmodium species, is the major life-threatening parasitic infection worldwide.
Due to the parasite resistance to quinoline based drugs, the search for antimalarial agents is necessary. Here,
we report the structural design, synthesis and antiparasitic evaluation of two novel series of 1,2,4-
oxadiazoles in conjugation to N-acylhydrazones, both groups recognized as privileged structures, as well as
the studies on the antimalarial activity of 16 previous described analogues. By varying substituents attached
to the phenyl ring, it was possible to retain, enhance or increase the antiparasitic activity in comparison to
the nonsubstituted derivatives. Replacement of substituted aryl rings by ferrocenyl and cinnamyl moieties
attached in the N-acylhydrazone ablated the antiparasitic response, evidencing the structural features
associated with the activity. Active compounds exhibited in vitro potency similar to mefloquine, but not all
inhibited –hematin formation. Additionally, the active compounds displayed low cytotoxicity in HepG2
cells and did not cause hemolysis in uninfected erythrocytes. In P. berghei-infected mice, the compounds
reduced parasitemia but exhibited limited efficacy in increasing mice survival when compared to
chloroquine, suggesting that pharmacological improvement is still necessary.
Keywords: Malaria; Plasmodium falciparum; drug design; N-acylhydrazones; 1,2,4-oxadiazoles.

1. INTRODUCTION
Malaria represents a major threat to the public human health worldwide, with over 230 million
clinical cases and about 660,000 deaths annually. In comparison to the last decade, the mortality is
decreasing, however, malaria remains widespread in endemic areas.1 Current treatment relies on quinolines
and artemisinin combinations; even so, there are significant problems of drug resistance.2,3 It is thus
important to identify drug candidates based on new chemotypes. To address this need, phenotype-based
antimalarial screening has been employed to identify active compounds chemically distinct from artemisinin
and quinolines. For instance, this approach has led to the discovery of the ozonide compounds arterolane
and OZ439, the spiroindolone KAE609, the naphthoquinone decoquinate as well as the thiazole
albitiazolium.4-8 These compounds are considered to be drug candidates and are likely to enter into clinical
trials, strengthening the concept that phenotype-based screening is a suitable approach for antimalarial drug
discovery.
Oxadiazoles are a class of heterocyclic compounds useful for the development of anti-infectious
agents.9-12 The most notable example is the class of the 1,2,5-oxadiazole-2-oxides, collectively known as
furoxans, which have a broad spectrum of antiparasitic activity, including antimalarial property (Figure 1).
Furoxans exert antiparasitic activity in part by releasing endogenous nitric oxide, which is an antimicrobial
molecule. Interestingly, furoxan derivatives without the N-oxide group, which is essential to confer a nitric
oxide release, also exhibit antiparasitic properties. This suggests that furoxan has a multi-target mechanism
of action, therefore oxadiazoles are considered potential antiparasitic compounds.12,13
Figure 1: General representation of 1,2,5-oxadiazole-2-oxide (1) and 1,2,4-oxadiazoles (2).

Previously, N-acylhydrazones derived from 3-aryl-1,2,4-oxadiazoles were identified as antiparasitic
agents against Trypanosoma cruzi, displaying low toxicity in both in vitro and in vivo experiments as well as
high efficacy in in vivo models of T. cruzi-infection.14,15 In this line, N-acylhydrazones derived from 7-
chloroquinoline and 1,3-diiminoisoindoline exhibited strong antimalarial activities.16,17 Based on these
findings, here we investigated the antimalarial activity for N-acylhydrazones derived from 3-aryl-1,2,4-
oxadiazoles (Figure 2). First, a series of known N-acylhydrazones bearing a 4-hydroxy-3-methoxyphenyl
(vanillinyl) portion SintMed (1-8), planned and synthesized on the basis of molecular hybridization
concepts, was investigated. Since the N-acylhydrazone-1,2,4-oxadiazole moiety has been identified as
14,15
important to disclose antiparasitic activity,
its structural association with the vanillinyl portion was
exploited as a promising strategy to increase the molecular recognition by the biological system. Vanillin
itself and its derivatives are largely described as biological active compounds18, reinforcing the grounds of
this strategy. The second series, also previously described in our group, has arisen from the exploitation of
the isosteric relation between the vanillinyl group and the 1,3-benzodioxole-5-phenyl (piperonyl) moiety
SintMed (9-16), also a portion of natural occurrence.
In addition, sixteen new N-acylhydrazone-1,2,4-oxadiazoles were prepared for the first time and
evaluated for their antimalarial activities. Following the defined strategy, the first unexampled series
incorporates a cinnamyl moiety attached to the N-acylhydrazone, which yielded the compounds SintMed
(17-24). This molecular modification was investigated due to the important role played by the cinnamyl
group present in molecules with demonstrated antimalarial activity.19a-b Another novel series of N-
acylhydrazone-1,2,4-oxadiazoles includes a ferrocenyl substitution, a non-natural occurring and unique
group, leading to SintMed (25-32). Ferrocenyl has been for long time associated to bioactive molecules,
promoting meaningful biological improvements, as demonstrated for ferroquine, a ferrocenyl analogue of
chloroquine, which was not only active against chloroquine sensitive Plasmodium parasites, but also against
resistant strains.20a-b Therefore, association of the N-acylhydrazone-1,2,4-oxadiazole structure with each of
the selected groups can represent a great possibility to disclose new potent active antimalarials agents,
whose remarkable property is the structural simplicity and low cost of production.

Figure 2: Structure of the N-acylhydrazone-1,2,4-oxadiazoles analogues SintMed (1-32) evaluated as
antimalarial compounds.
In vitro screening of compounds SintMed (1-32) led us to identify some of them having a potent
activity and selectivity against the blood-stage P. falciparum, as well as to acquire a better understanding of
structure-activity relationships implied in the modification of the N-acylhydrazone-1,2,4-oxadiazole basic
structure used in our group as a fundamental model in drug design.
2 RESULTS AND DISCUSSION
2.1 Chemistry

The synthesis of compounds SintMed (17-32), depicted in Scheme 1, is based on a method
developed by our group.15 Briefly, commercially available aryl nitriles were converted into 4-substituted
arylamidoximes by refluxing during 4 hours in the presence of hydroxylamine with good yields, which
constitutes our primary start material. Under inert atmosphere and dry conditions, arylamidoximes react with
monomethyl oxalyl chloride, leading to methyl 3-(4-arylsubstituted)-1,2,4-oxadiazole carboxylates, which
were converted into the corresponding 1,2,4-oxadiazole hydrazides under mild condition.
Scheme 1: Synthetic route for N-acylhydrazone-1,2,4-oxadiazole compounds.
Although the condensation of hydrazides with vanillin and piperonal, respectively, under sulphuric
acid catalysis at room temperature leads easily to the series SintMed (1-8) and SintMed (9-16) as
previously reported, this same condition did not work for synthesizing ferrocenyl derivatives SintMed (25-
32). In order to overcome this inconvenience, the reaction between the hydrazides and
ferrocenecarboxyaldehyde was carried out in the presence of cerium (III) chloride heptahydrate (CeCl3·
7H2O) as a catalyst with isolated average yields of 90 %, following a method recently developed in our
laboratory.21

13
Compounds SintMed (25-32) were analyzed by usual 1H NMR, and C NMR. Signal assignments
for the ferrocene group was assisted by DEPT, HSQC and HMBC experiments, in addition to comparing the
findings with related literature.22,23 1H NMR analysis of crude ferrocenyl compounds SintMed (25-32)
revealed that only the E-isomer was formed, in agreement with previously reported outcomes for other
compounds synthesized using this method.21 Due to the mild condition and low reaction time, cerium (III)
chloride catalysis has proved to be highly stereoselective, leading exclusively to the thermodynamically
more stable E-isomer. Considering that isomeric purity is important for pharmacological evaluation, the new
series of cinnamyl derivatives SintMed (17-24) as well as compounds SintMed (1-16) were also
1
synthesized in the presence of CeCl3-catalyst and yielded E-isomer only. Taking the H NMR spectra of
representative compounds SintMed23 and SintMed31, the stereoselectivity of the CeCl3-method can be
easily corroborated (Figure 3).
Figure 3: 1H NMR spectra of compounds SintMed23 and SintMed31 as pure E-isomers.
This outcome was analyzed by observing the signal of the iminic proton (–N=CH–), which
ressonates at δ ≈ 8.50-8.30 ppm. In previous reports, it was determined that this value is characteristic for E-
isomers, while Z-isomers usually show signals at δ ≈ 7.90-8.00 ppm.15,21 The presence of E/Z-isomers can
also be established by observing the signal of the amide group (–CONH–), which splits up in two singlets
when both isomers are formed. In all cases discussed in this work, only one signal for each of these groups

was observed, including the known compounds SintMed (1-16), which were previously described with
signals of Z-isomers.15
2.2. Pharmacological evaluation
Compounds SintMed (1-32) were initially screened at 10 μg/mL against the chloroquine-resistant
W2 strain of blood-stage P. falciparum (Table 1). Compounds which have inhibited more than 60 %
parasite growth at this concentration were considered active and had their IC50 values determined.
Mefloquine (Mfq) was used as the reference antimalarial drug in these assays.
First, the parasite grown inhibited by compounds containing a 4-hydroxy-3-methoxyphenyl moiety
SintMed (1-8) was investigated. The unsubstituted compound at phenyl ring SintMed1 was inactive, while
the attachment of a methyl at position 4 of phenyl SintMed2 produced an active antimalarial compound. In
contrast, attaching fluoro SintMed3, chlorine SintMed4 or methoxy SintMed7 substituents did not produce
active compounds. But attaching a bromo SintMed5 or a nitro group SintMed6 increased the activity of
both compounds. In comparison to the unsubstituted compound SintMed1, the attachment of a hydroxyl
group SintMed8 disclosed a compound on the edge of the activity parameter. The series bearing the 1,3-
benzodioxole moiety SintMed (9-16) was investigated, based on its close isosteric relation with the previous
series SintMed (1-8). Except for the compound SintMed16, substituted with a hydroxy group, and
possessing expressive activity, all other members of this series were inactive. Instead of a withdraw, this
outcome gives some key clues about the nature of the structural requirements necessary to enhance the
antimalarial activity of analogue molecules. The presence of a hydroxyl group linked to a phenyl ring at the
N-acylhydrazone-1,2,4-oxadiazole skeleton seems to be essential to the emergence of active antimalarial
compounds.
Table 1. Initial anti-P. falciparum screening against chloroquine-resistant W2 strain.
Compound
R
% inhibition[a]
Compound
R
% inhibition[a]

O
N
O
N
N
N
OH
N
N
H
O
H
R
H
55.4
61.1
24.7
37.0
Br
71.1
68.7
42.2
58.8
SintMed1
SintMed2
SintMed3
SintMed4
SintMed5
SintMed6
SintMed7
SintMed8
CH3
F
NO2
OCH3
OH
Cl
O
N
O
O
N
N
H
O
H
R
H
27.5
21.9
31.6
53.0
Br
45.3
33.9
27.5
71.9
SintMed9
SintMed10
SintMed11
SintMed12
SintMed13
SintMed14
SintMed15
SintMed16
CH3
F
NO2
OCH3
OH
Cl
O
N
O
N
N
H
H
R
H
51.0
52.7
34.3
28.8
Br
58.8
25.7
26.1
39.1
SintMed17
SintMed18
SintMed19
SintMed20
SintMed21
SintMed22
SintMed23
SintMed24
CH3
F
NO2
OCH3
OH
Cl

O
N
O
N
Fe
N
N
H
H
R
H
3.3
35.2
22.7
0
Br
29.8
0
SintMed25
SintMed26
SintMed27
SintMed28
Mfq
SintMed29
SintMed30
SintMed31
CH3
F
NO2
OCH3
OH
-
3.0
43.8
-
Cl
-
SintMed32,
100
-
[a] % of parasite inhibition using the concentration of 10 µg/mL for each compound. Determined 72 h after incubation of W2 strain
P. falciparum (erythrocytic stage) with the respective compounds. Mfq = mefloquine.
This assumption has found support in the results for the series containing cinnamyl SintMed (17-24)
and ferrocenyl SintMed (25-32) moieties, both inactive against P. falciparum. These preliminary
observations narrow the structural possibilities that can be pursued in the search for new antimalarials based
on the N-acylhydrazone-1,2,4-oxadiazole chemotype.
The five active compounds were submitted to the evaluation of their in vitro potency against P.
falciparum, and the standard drug mefloquine was used as a reference parameter, as shown in Table 2.
Mefloquine exhibited an IC50 value of 0.04±0.01 μM. In comparison to mefloquine, vanillinyl moiety-
bearing derivatives of N-acylhydrazone-1,2,4-oxadiazole SintMed2 (4-mehylsubstituted), SintMed5 (4-
bromosubstituted) and SintMed6 (4-nitrosubstituted) displayed potent anti-P. falciparum activities. While
compound SintMed5 was found to be almost as potent as mefloquine with IC50 of 0.07±0.12 μM, the other
two analogues SintMed2 and SintMed6 were less potent, but still exhibited expressive activities: 0.12±0.16
μM and 0.20±0.16 μM, respectively. Replacement by 4-hydroxyl SintMed8 decreased potency of the final
compound. This revealed the importance of a specific substituent located in the phenyl ring attached to the
1,2,4-oxadiazole group, suggesting that lipophilic groups can be more successful in further planning of
antimalarial compounds based on this structural features. In Table 2, the lipophilicity parameters (logP) for

the five active compounds are given, evidencing that the more potent compounds present higher lipophilicity
in the studied group. The only one active compound derived from the 1,3-benzodioxole series SintMed (9-
16), the 4-hydroxylphenyl substituted analogue SintMed16, displayed potency similar to SintMed8.
Table 2. In vitro potency, cytotoxicity, selectivity and logP.
Compounds
P. falciparum IC50 ±S.D. (µM)a
0.12±0.16
HepG2 CC50 ±S.D. (µM)b
46.6±0.5
SIc
388
241
74
-
logPd
3.00
3.36
2.50
2.07
2.62
-
SintMed2
SintMed5
SintMed6
SintMed8
SintMed16
Mfq
0.07±0.12
16.9±1.9
0.20±0.16
14.8±0.8
1.9±0.8
> 200
1.5±1.09
> 200
-
0.04±0.01
11.8 ± 0.3
295
c
a Determined 72 h after incubation with the drugs. b Determined 72 h after incubation with the drugs. SI was determined by the
ratio CC50/IC50. IC50 and CC50 values were determined from two independent experiments using each concentration in triplicate.
IC50 = inhibitory concentration to 50 %. CC50 = cytotoxic concentration to 50 %. S.D. = standard deviation. Mfq = Mefloquine.
dDetermined using the online version of the Molinspiration Cheminformatics software (http://www.molinspiration.com/).
Next, the cytotoxicity against mammalian HepG2 cells was tested and expressed as CC50 values, as
well as the selectivity index (SI), both results described in Table 2. Comparing to mefloquine, which
displayed CC50 value of 11.8 ± 0.3 μM, none of the studied compounds was more cytotoxic. Compounds
SintMed8 and SintMed16 were several folds less cytotoxic than mefloquine, while the other compounds
were similarly cytotoxic. Mefloquine had an SI of 295, and compounds SintMed2 (SI = 388) and SintMed5
(SI = 241) revealed a similar selectivity, reinforcing the evidences that lipophilic groups are important for
the antimalarial activity, especially when the SI of 74 for SintMed6 is taken into account. The nitro group
possesses clearly hydrophilic properties, affecting the activity and selectivity of SintMed6. Although SI for
SintMed8 and SintMed16 could not be precisely determined due to lack cytotoxicity against HepG2 cells,
it is estimated that they are selective antiparasitic agents.

In addition to the cytotoxicity in HepG2 cells, the percentage of hemolysis in uninfected erythrocytes
was determined. As can be seen in Table 3, except for compound SintMed5, which caused hemolysis in the
tested concentrations, none of the compounds did it. Again, the nature of the bromine atom attached to the
molecule plays an important role for its biological response. The ability of active compounds to inhibit the
hemin polymerization into β-hematin was studied. In this assay (Table 3), chloroquine displayed an IC50 of
0.84 ± 0.30 mM. Compounds SintMed2, SintMed5 and SintMed16 inhibited β-hematin in the tested
concentrations with IC50 similar to chloroquine, while compounds SintMed6 and SintMed8 did not. The
CQ index indicates the potency of each compound in comparison with chloroquine.
Table 3. Hemolytic property, β-hematin formation inhibition and CQ-index.
Compounds
SintMed2
SintMed5
SintMed6
SintMed8
SintMed16
CQ
Hemolysis CC50 ±S.D. (µg/mL)a
β-hematin IC50 ±S.D. (mM)
CQ indexb
>100
33.0±0.1
>100
0.6±0.2
0.9±0.03
> 2.0
1.3
0.8
-
>100
> 2.0
-
>100
0.8±0.5
0.8±0.3
1.0
-
>100
aHemolysis was assayed in uninfected human erythrocytes (type O+) after 1h of incubation with compounds, in triplicate.
bDetermined by the ratio IC50 CQ/IC50 test compound. CQ = Chloroquine.
Finally, it was examined the ability of the most potent antimalarial derivatives, compounds
SintMed2, SintMed5 and SintMed6, to decrease parasitemia and increase survival in P. berghei-infected
mice. After 1 h of infection with NK65 strain, mice were treated orally by gavage or intraperitoneal injection
once a day during 4 consecutive days. As can be seen in Figure 4, untreated infected group shows
parasitemia, which usually peaks at 6 to 8 days post infection. By comparison with the untreated group,
chloroquine given orally at 50 mg/kg/day decreases parasitemia (p<0.001) and provides 100 % animal
survival (p<0.05) after 30 days of infection. Under oral treatment with compounds SintMed2 and SintMed5

at 100 mg/kg/day, no parasitemia reduction was detected, and death was observed in less than 30 days
(Figure 4). Even the intraperitoneal administration of compounds SintMed2 and SintMed5 at 100
mg/kg/day did not lead to better outcomes (data not shown).
Vehicle
SintMed 2 (100 mg/kg)
10
SintMed 5 (100 mg/kg)
Chloroquine (50 mg/kg)
8
6
4
2
0
3
4
5
6
7
8
days after infection
100
80
60
40
20
0
Vehicle
(SintMed2)
(SintMed5)
CQ
0
5
10
15
20
25
30
days after infection
Figure 4. (Top) Blood parasitemia and (Bottom) % survival of P. berghei-infected mice. Swiss mice
(n=5/group) were infected with NK65 strain and 1 h after, treated once a day for four consecutive days.
Vehicle group received 10% DMSO in saline. Values represent the means ± SEM of five mice per group in
one experiment of two replicates performed. CQ = chloroquine. i.p. = intraperitoneal injection. p.o. = oral
(gavage). Top: vehicle vs CQ (one-way ANOVA, p<0.001). Bottom: vehicle vs CQ (Mantel-Cox test,
p<0.05).

In contrast, intraperitoneal treatment with compound SintMed6 dosed at 50 mg/kg/day and 25
mg/kg/day decreased parasitemia (p<0.001) in both cases, consisting in a very important experimental result
(Figure 5). Unfortunately, mice survival under such conditions was not so successful, although an
improvement could be registered, reinforcing our initial assumptions about the importance of the 1,2,4-
oxadiazole-N-acylhydrazone backbone structure as a pattern for the design and development of potential
new antiparasitics of high efficacy.
100
80
Vehicle i.p.
60
SintMed6 25 mg/kg i.p.
40
20
0
SintMed6 50 mg/kg i.p.
Chloroquine 50 mg/kg
3
4
5
6
7
8
days after infection
100
80
60
40
20
0
Vehicle i.p.
SintMed6 25mg/kg i.p.
SintMed6 50mg/kg i.p.
Chloroquine 50mg/kg
0
5
10
15
20
25
30
days after infection
Figure 5. (Top) Blood parasitemia and (Bottom) % survival of P. berghei-infected mice. Swiss mice
(n=5/group) were infected with NK65 strain and 1 h after, treated once a day for four consecutive days.
Vehicle group received 10% DMSO in saline. Values represent the means ± SEM of five mice per group in
one experiment of two replicates performed. CQ = chloroquine. i.p. = intraperitoneal injection. p.o. = oral
(gavage). Top: vehicle vs CQ (one-way ANOVA, p<0.001), vehicle vs (SintMed6) 25 mg/kg (one-way

ANOVA, p<0.01), vehicle vs (SintMed6) 50 mg/kg (one-way ANOVA, p<0.001). Bottom: vehicle vs CQ
(Mantel-Cox test, p<0.05).
3. CONCLUSION
Here, new antimalarial compounds were identified on the basis of structural design by employing
hybridization between 1,2,4-oxadiazole, N-acylhydrazone and natural or synthetic occurring relevant
biological groups. Antimalarial activity was observed to be quite dependent on the vanillinyl moiety
attached to the iminic structure, whose biological response for the active derivatives seems to be dependent
on the lipophilicity of the substituents linked to the 4-position of the phenyl ring attached to the oxadiazole
portion. This structural design successfully led to the identification of compounds SintMed2, SintMed5 and
SintMed6 as potent and selective anti-P. falciparum agents, as similarly observed in mefloquine-treated
parasites. Mechanistically, these compounds did show general inhibitory properties for -hematin formation,
suggesting that they present a mechanism of action similar to that of quinoline based drugs, which are well-
known -hematin inhibitors. Therefore, the collected data suggest that the molecular hybridization is a
promising line of research for structural modification targeting antiparasitic agents based on 1,2,4-
oxadiazole-N-acylhydrazone association.
4. EXPERIMENTAL SECTION
4.1 Chemistry: All solvents and reactants were purchased from Sigma-Aldrich, Fluka, Vetec and Acros
Chemicals, and were used without further purification. Reactions’ progresses were monitored by thin layer
chromatography (TLC), performed onto glass-backed plates of silica gel 60 F254 with gypsum, and all
compounds were detected by ultraviolet light (254 nm). Melting points were determined with a capillary
apparatus Gehaka PF 1500 Farma and are uncorrected. NMR spectra were recorded at 400 MHz for
hydrogen and 100 MHz for carbon, using a Varian UNMRS 400 spectrometer, or at 300 MHz for hydrogen
and 75 MHz for carbon nuclei, using a Varian Unitplus 300 NMR. Analyses were determined in DMSO-d6

with chemical shift values (δ) in parts per million (ppm) and coupling constants (J) in Hertz (Hz), and
1
measured at 25 °C. H and 13C assignments were assisted by 2D experiments, such as DEPT full edit,
HMBC and HSQC. IR spectra were recorded on a Tensor27 FTIR spectrometer from Bruker with the
samples being analyzed as KBr pellets. High resolution mass spectrometry (HRMS) analysis of the pure
compounds was performed with an UHPLC-ESI-HRMS on a Micromass LCT Premier time-of-flight mass
spectrometer from Waters with an electrospray ionization (ESI) interface (Waters, Milford, MA, USA)
equipped with an electrospray interface and coupled to an Acquity UHPLC system (Waters, Milford MA,
USA). UHPLC-ESI-HRMS analysis was performed using the following parameters: ESI used capillary
voltage 2800 V, cone voltage 40 V, MCP detector voltage 2400 V, source temperature 120°C, desolvation
temperature 300°C, cone gas flow 20 L/h, and desolvation gas flow 800 L/h. Detection was performed in
positive ion mode (PI) with a m/z range of 100–1300 Da and a scan time of 0.5 s in the W-mode. The MS
was calibrated using sodium formate, and leucine encephalin (Sigma-Aldrich, Steinheim, Germany) was
used as an internal reference at 2μg/mL and infused through a Lock Spray™ probe at a flow rate of 10
μL/min with the help of a second LC pump. The separation was performed on an Acquity BEH C18 UPLC
column (1.7 µm, 50 ×2.1 mm i.d.; Waters, Milford, MA, USA) using a linear gradient, solvent system: A)
0.1% formic acid–water, B) 0.1% formic acid–acetonitrile; gradient: 5–36% B in 1 min, then 36% B to 36%
B in 2 min, 36% B to 95% B in 1.5 min; flow rate 1.1 mL/min. The temperature was set to 40°C. The
injected volume was kept constant (1 µL; solution at the concentration of 0.5 mg/mL). Elemental analyses
were performed in a Perkin Elmer 2400 Series II elemental analyser.
4.1.2. Preparation of compounds SintMed (1-32): to a stirred suspension of 1 mmol of appropriate
hydrazide and 1 mmol of aldehyde in 10 mL of ethanol 10 mol% cerium (III) chloride heptahydrate was
added and the reaction mixture was stirred at 40 °C during 10-30 minutes. Reaction’s completion was
monitored by TLC, using ethyl acetate as eluent system. Once concluded, the heating was put away, and 20
mL of water was added to the medium. After standing at refrigerator, vacuum filtration was carried out, and
1
the solid was washed with cold water/ethanol 1:1 followed by cold water. H NMR analysis of all crude

products confirmed their purity. Recrystallization from dioxane/water mixture afforded the pure products for
biological purposes. Yields, melting points, spectroscopic and spectrometric data are listed below for each
new compound.
4.1.2.1.
3-Phenyl-N'-[(1E,2E)-3-phenylprop-2-en-1-ylideno]-1,2,4-oxadiazol-5-ylcarbohydrazide
SintMed17: Yield: 90 %; Mp 167.6-170-2 °C (from dioxane/H2O); IR (KBr, νmax cm-1): 3266 (NH), 3090
1
(Ar CH), 1700 (C=O), 1625 (C=N, imine), 1529 (C=N, heterocyclic); H NMR (300 MHz, DMSO-d6) δ,
ppm: 12.7 (s, 1H, CONH), 8.42 (d, 1H, 3J = 8.1 Hz, N=CH), 8.10 (d, 2H, 3J = 7.8 Hz, Ph H-2,6), 7.67-7.59
3
3
(m, 5H, Ph H-3,4,5 and Cinnamyl H-2,6), 7.42 (d, 1H, J = 6.6 Hz, CH=CHPh), 7.37 (t, 2H, J = 4.8 Hz,
13
Cinnamyl H-3,5), 7.11 (d, 2H, HC=CHPh and Cinnamyl H-4); C NMR (75 MHz, DMSO-d6) δ, ppm:
168.7 (1C, C=O), 168.1 (1C, Oxadiazole C-3), 153.5 (1C, C=NH), 149.4 (1C, Oxadiazole C-5), 141.3 (1C,
CH=CHPh), 135.6 (1C, Cinnamyl C-1), 132.0 (1C, Ph C-4), 129.4 (2C, Ph C-3,5), 129.2 (1C, CH=CHAr),
128.8 (2C, Cinnamyl C-3,5), 127.3 (2C, Cinnamyl C-2,6), 127.2 (2C, Ph C-2,6), 125.4 (1C, Ph C-1), 125.1
(1C, Cinnamyl C-4); UHPLC-TOF-MS for C18H14N4O2 calcd (found)/Error: 318.1117 (317.1089, [M-H]-
)/15.8 ppm.
4.1.2.2.
3-(4-Tolyl)-N'-[(1E,2E)-3-phenylprop-2-en-1-ylideno]-1,2,4-oxadiazol-5-ylcarbohydrazide
SintMed18: Yield: 94 %; Mp 197.1-199.7 °C (from dioxane/H2O); IR (KBr, νmax cm-1): 3232 (NH), 3058
1
(Ar CH), 1684 (C=O), 1622 (C=N, imine), 1581 (C=N, heterocyclic); H NMR (300 MHz, DMSO-d6) δ,
ppm: 12.7 (s, 1H, CONH), 8.41 (d, 1H, 3J = 6.0 Hz, N=CH), 7.98 (d, 2H, 3J = 5.7 Hz, Tolyl H-2,6), 7.65 (d,
2H, Cinnamyl H-2,6), 7.43-7.40 (m, 5H, Tolyl H-3,5, CH=CHPh and Cinnamyl H-3,5), 7.13 (d, 2H,
13
CH=CHAr and Cinnamyl H-4), 2.39 (s, 3H, CH3); C NMR (75 MHz, DMSO-d6) δ, ppm: 168.5 (1C,
C=O), 168.1 (1C, Oxadiazole C-3), 153.4 (1C, C=NH), 149.4 (1C, Oxadiazole C-5), 142.1 (1C, Tolyl C-4),
141.3 (1C, CH=CHPh), 135.6 (1C, Cinnamyl C-1), 129.9 (2C, Tolyl C-3,5), 129.2 (CH=CHPh), 128.8 (2C,
Cinnamyl C-3,5), 127.3 (2C, Cinnamyl C-2,6), 127.1 (2C, Tolyl C-2,6), 125.1 (1C, Cinnamyl C-4), 122.7
(1C, Tolyl C-1), 21.1 (1C, CH3); UHPLC-TOF-MS for C19H16N4O2 calcd (found)/Error: 332.1273
(331.1189, [M-H]-)/1.8 ppm.

4.1.2.3. 3-(4-Fluorophenyl)-N'-[(1E,2E)-3-phenylprop-2-en-1-ylideno]-1,2,4-oxadiazol-5-ylcarbohydrazide
SintMed19: Yield: 94 %; Mp 214.4-215.8 °C (from dioxane/H2O); IR (KBr, νmax cm-1): 3256 (NH), 3058
1
(Ar CH), 1685 (C=O), 1607 (C=N, imine), 1584 (C=N, heterocyclic); H NMR (400 MHz, DMSO-d6) δ,
ppm: 12.7 (s, 1H, CONH), 8.41 (d, 1H, 3J = 8.0 Hz, N=CH), 8.13 (dd, 2H, 3JFH = 3.2 Hz, 3JHH = 8.8 Hz, 4-
3
FPh H-2,6), 7.68 (d, 2H, J = 7.2 Hz, Cinnamyl H-2,6), 7.49-7.30 (m, 5H, 4-FPh H-3,5, CH=CHPh and
13
Cinnamyl H-3,5), 7.18-7.06 (m, 2H, CH=CHPh and Cinnamyl H-4); C NMR (100 MHz, DMSO-d6) δ,
1
ppm: 168.5 (1C, C=O), 167.3 (1C, Oxadiazole C-3), 164.2 (1C, d, JFC = 249.4 Hz, 4FPh H-4), 153.5 (1C,
C=NH), 149.4 (1C, Oxadiazole C-5), 141.3 (1C, CH=CHPh), 135.6 (1C, Cinnamyl C-1), 129.8 (2C, d, 3JFC
= 9.3 Hz, 4-FPh H-2,6), 129.2 (1C, CH=CHPh), 128.8 (2C, Cinnamyl C-3,5), 127.3 (2C, Cinnamyl C-2,6),
125.1 (1C, Cinnamyl C-4), 122.0 (1C, d, 4JFC = 3.1 Hz, 4-FPh H-1), 116.6 (2C, d, 2JFC = 21.7 Hz, 4FPh H-
3,5); UHPLC-TOF-MS for C18H13N4O2F calcd (found)/Error: 336.1022 (335.0986, [M-H-])/12.5 ppm.
4.1.2.4. 3-(4-Chlorophenyl)-N'-[(1E,2E)-3-phenylprop-2-en-1-ylideno]-1,2,4-oxadiazol-5-ylcarbohydrazide
SintMed20: Yield: 91 %; Mp 214.7-216.6 °C (from dioxane/H2O); IR (KBr, νmax cm-1): 3226 (NH), 3055
1
(Ar CH), 1688 (C=O), 1619 (C=N, imine), 1589 (C=N, heterocyclic); H NMR (300 MHz, DMSO-d6) δ,
ppm: 12.7 (s, 1H, CONH), 8.40 (d, 1H, 3J = 8.1 Hz, N=CH), 8.09 (d, 2H, 3J = 8.7 Hz, 4ClPh H-2,6), 7.69 (d,
3
3
3
2H, J = 5.1 Hz, 4-ClPh H-3,5), 7.65 (d, 2H, J = 8.7 Hz, Cinnamyl H-2,6), 7.42 (d, 1H, J = 6.6 Hz,
13
CH=CHPh), 7.36 (t, 2H, 3J = 4.2 Hz, Cinnamyl H-3,5), 7.11 (2xs, 2H, CH=CHPh and Cinnamyl H-4); C
NMR (75 MHz, DMSO-d6) δ, ppm: 168.8 (1C, C=O), 167.3 (1C, Oxadiazole C-3), 153.5 (1C, C=NH),
149.3 (1C, Oxadiazole C-5), 141.3 (1C, CH=CHPh), 136.8 (1C, 4-ClPh C-4), 135.6 (1C, Cinnamyl C-1),
129.6 (2C, 4-ClPh C-3,5), 129.3 (2C, 4-ClPh C-2,6), 129.1 (1C, HC=CHPh), 129.0 (2C, Cinnamyl C-3,5),
127.4 (2C, Cinnamyl C-2,6), 125.1 (1C, Cinnamyl C-4), 124.3 (1C, 4-ClPh C-1); UHPLC-TOF-MS for
C18H13ClN4O2 calcd (found)/Error: 352.0727 (351.0622, [M-H-])/7.7 ppm.
4.1.2.5. 3-(4-Bromophenyl)-N'-[(1E,2E)-3-phenylprop-2-en-1-ylideno]-1,2,4-oxadiazol-5-ylcarbohydrazide
SintMed21: Yield: 89 %; Mp 195.6-198.3 °C (from dioxane/H2O); IR (KBr, νmax cm-1): 3426 (NH), 3055

1
(Ar CH), 1689 (C=O), 1624 (C=N, imine), 1543 (C=N, heterocyclic); H NMR (400 MHz, DMSO-d6) δ,
ppm: 12.7 (s, 1H, CONH), 8.41 (d, 1H, 3J = 8.0 Hz, N=CH), 8.02 (d, 2H, 3J = 8.4 Hz, 4-BrPh H-2,6), 7.84
3
3
3
(d, 2H, J = 8.4 Hz, 4-BrPh H-3,5), 7.66 (d, 2H, J = 7.2 Hz, Cinnamyl H-2,6), 7.42 (d, 1H, J = 7.2 Hz,
3
CH=CHAr), 7.37 (t, 2H, J = 7.6 Hz, Cinnamyl H-3,5), 7.17-7.06 (m, 2H, CH=CHPh and Cinnamyl H-4);
13C NMR (100 MHz, DMSO-d6) δ, ppm: 168.8 (1C, C=O), 167.4 (1C, Oxadiazole C-3), 153.6 (1C, C=NH),
149.3 (1C, Oxadiazole C-5), 141.3 (1C, CH=CHPh), 135.6 (1C, Cinnamyl C-1), 132.5 (2C, 4BrPh C-3,5),
129.3 (2C, 4-BrPh C-2,6), 129.2 (1C, CH=CHPh), 128.8 (2C, Cinnamyl C-3,5), 127.4 (2C, Cinnamyl C-
2,6), 125.7 (1C, 4-BrPh C-4), 125.1 (1C, Cinnamyl C-4), 124.7 (1C, 4-BrPh C-1); UHPLC-TOF-MS for
C18H13BrN4O2 calcd (found)/Error: 396.0222 (395.0160, [M-H-])/4.1 ppm.
4.1.2.6. 3-(4-Nitrophenyl)-N'-[(1E,2E)-3-phenylprop-2-en-1-ylideno]-1,2,4-oxadiazol-5-ylcarbohydrazide
SintMed22: Yield: 91 %; Mp 235.5-238.1 °C (from dioxane/H2O); IR (KBr, νmax cm-1): 3210 (NH), 3075
1
(Ar CH), 1708 (C=O), 1620 (C=N, imine), 1569 (C=N, heterocyclic); H NMR (300 MHz, DMSO-d6) δ,
ppm: 12.8 (s, 1H, CONH), 8.44 (d, 2H, 3J = 8.7 Hz, 4-NO2Ph H-3,5), 8.40 (d, 1H, 3J = 7.8 Hz, N=CH), 8.32
3
3
3
(d, 2H, J = 9.0 Hz, 4-NO2Ph H-2,6), 7.67 (d, 2H, J = 7.2 Hz, Cinnamyl H-2,6), 7.41 (d, 1H, J = 7.2 Hz,
3
13
CH=CHPh), 7.36 (t, 2H, J = 6.0 Hz, Cinnamyl H-3,5), 7.12 (m, 2H, CH=CHPh and Cinnamyl H-4); C
NMR (75 MHz, DMSO-d6) δ, ppm: 169.1 (1C, C=O), 166.8 (1C, Oxadiazole C-3), 153.6 (1C, C=NH),
149.4 (1C, 4-NO2Ph C-4), 149.1 (1C, Oxadiazole C-5), 141.3 (1C, HC=CHPh), 135.5 (1C, Cinnamyl C-1),
131.2 (1C, 4-NO2Ph C-1), 129.3 (1C, HC=CHAr), 128.8 (2C, Cinnamyl C-3,5), 128.6 (2C, 4-NO2Ph C-2,6),
127.3 (4C, Cinnamyl C-2,6), 125.0 (1C, Cinnamyl C-4), 124.6 (2C, 4-NO2Ph C-3,5); UHPLC-TOF-MS for
C18H13N5O4: [M-H]- 363.0968 calcd (found)/Error: (362.0908, [M-H-])/5.2 ppm.
4.1.2.7.
3-(4-Methoxyphenyl)-N'-[(1E,2E)-3-phenylprop-2-en-1-ylideno]-1,2,4-oxadiazol-5-
ylcarbohydrazide SintMed23: Yield: 96 %; Mp 185.8-188.2 °C (from dioxane/H2O); IR (KBr, νmax cm-1):
3203 (NH), 3055 (Ar CH), 1698 (C=O), 1618 (C=N, imine), 1597 (C=N, heterocyclic); 1H NMR (300 MHz,
3
3
DMSO-d6) δ, ppm: 12.7 (s, 1H, CONH), 8.41 (d, 1H, J = 8.4 Hz, N=CH), 8.03 (d, 2H, J = 8.7 Hz, 4-
3
3
MeOPh H-2,6), 7.65 (d, 2H, J = 6.9 Hz, Cinnamyl H-2,6), 7.41 (d, 1H, J = 7.2 Hz, CH=CHPh), 7.36 (t,

2H, 3J = 3.6 Hz, Cinnamyl H-3,5), 7.16 (d, 1H, 3J = 6.9 Hz, CH=CHPh), 7.14-7.10 (m, 3H, 4-MeOPh H-3,5
13
and Cinnamyl H-4), 3.85 (s, 3H, OCH3); C NMR (75 MHz, DMSO-d6) δ, ppm: 168.4 (1C, C=O), 167.8
(1C, Oxadiazole C-3), 162.0 (1C, 4-MeOPh C-4), 153.5 (1C, C=NH), 149.5 (1C, Oxadiazole C-5), 141.2
(1C, HC=CHPh), 135.6 (1C, Cinnamyl C-1), 129.2 (1C, HC=CHPh), 129.0 (2C, 4-MeOPh C-2,6), 128.8
(2C, Cinnamyl C-3,5), 127.3 (2C, Cinnamyl C-2,6), 125.1 (1C, Cinnamyl C-4), 117.6 (1C, 4-MeOPh C-1),
114.8 (2C, 4-MeOPh C-3,5), 55.4 (C, OCH3); UHPLC-TOF-MS for C19H16N4O3 calcd (found)/Error:
348.1223 (347.1107, [M-H]-)/10.7 ppm.
4.1.2.8.
3-(4-Hydroxyphenyl)-N'-[(1E,2E)-3-phenylprop-2-en-1-ylideno]-1,2,4-oxadiazol-5-
ylcarbohydrazide SintMed24: Yield: 92 %; Mp 170.3-171.7 °C (from dioxane/H2O); IR (KBr, νmax cm-1):
1
3426 (OH), 3260 (NH), 3054 (Ar CH), 1690 (C=O), 1624 (C=N, imine), 1531 (C=N, heterocyclic); H
3
NMR (300 MHz, DMSO-d6) δ, ppm: 12.7 (s, 1H, CONH), 10.3 (s, 1H, OH), 8.41 (d, 1H, J = 7.5 Hz,
N=CH), 7.93 (d, 2H, 3J = 9.0 Hz, 4-OHPh H-2,6), 7.65 (d, 2H, 3J = 7.2 Hz, cinnamyl H-2,6), 7.41 (d, 1H, 3J
3
= 6.3 Hz, HC=CHPh), 7.36 (t, 2H, 3J = 6.3 Hz, Cinnamyl H-3,5), 7.11 (d, 2H, J = 7.8 Hz, HC=CHPh and
3
13
Cinnamyl H-4), 6.97 (d, 2H, J = 8.7 Hz, 4-OHPh H-3,5); C NMR (75 MHz, DMSO-d6) δ, ppm: 168.3
(1C, C=O), 168.1 (1C, Oxadiazole C-3), 160.8 (1C, 4-OHPh C-4), 153.5 (1C, C=NH), 149.6 (1C,
Oxadiazole C-5), 141.3 (1C, HC=CHPh), 135.6 (1C, Cinnamyl C-1), 129.3 (1C, HC=CHPh), 129.1 (2C, 4-
OHPh C-2,6), 128.9 (2C, Cinnamyl C-3,5), 127.4 (2C, Cinnamyl C-2,6), 125.1 (1C, Cinnamyl C-4), 116.2
(2C, 4-OHPh C-3,5), 116.1 (1C, 4-OHPh C-1); LCMS for C18H14N4O3 calcd (found)/Error: 317.1021
(317;1007, [(M-H2O)+H]+)/12.7 ppm.
4.1.2.9. 3-Phenyl-N’-(E-ferrocenylmethylidene)-1,2,4-oxadiazol-5-ylcarbohydrazide SintMed25: Yield: 92
%; Mp 209.9-211.6 °C (from dioxane/H2O); IR (KBr, νmax cm-1): 3240 (NH), 3064 (Ar CH), 1711 (C=O),
1
1601 (C=N, imine), 1556 (C=N, heterocyclic); H NMR (300 MHz, DMSO-d6) δ, ppm: 12.5 (s, 1H,
3
CONH), 8.47 (s, 1H, N=CH), 8.10 (d, 2H, J = 7.2 Hz, Ar H-2,6), 7.65-7.63 (m, 3H, Ar H-3,4,5), 4.71 (s,
13
2H, C=NHFc H-2,5), 4.52 (s, 2H, C=NHFc H-3,4), 4.27 (s, 5H, Fc); C NMR (75 MHz, DMSO-d6) δ,
ppm: 168.9 (1C, C=O), 168.1 (1C, Oxadiazole C-3), 153.5 (1C, C=NH), 148.8 (1C, Oxadiazole C-5), 132.0

(1C, Ar C-4), 129.4 (2C, Ar C-3,5), 127.2 (1C, Ar C-1), 125.5 (2C, Ar C-2,6), 77.7 (1C, C=NHFc C-1),
70.8 (2C, C=NHFc C-3,4), 69.1 (5C, Fc), 68.0 (2C, C=NHFc C-2,5); UHPLC-TOF-MS for C20H16FeN4O2
calcd (found)/Error: 400.0623 (401.0713, [M+H+])/3.0 ppm.
4.1.2.10. 3-(4-Tolyl)-N’-(E-ferrocenylmethylidene)-1,2,4-oxadiazol-5-ylcarbohydrazide SintMed26: Yield:
91 %; Mp 220.0-222.2 °C (from dioxane/H2O); IR (KBr, νmax cm-1): 3182 (NH), 3100, 3029 (Ar CH), 1682
(C=O), 1598 (C=N, imine), 1547 (C=N, heterocyclic); 1H NMR (300 MHz, DMSO-d6) δ, ppm: 12.4 (s, 1H,
CONH), 8.46 (s, 1H, N=CH), 7.99 (d, 2H, 3J = 7.5 Hz, Ar H-2,6), 7.43 (d, 2H, 3J = 7.8 Hz, Ar H-3,5), 4.71
13
(s, 2H, C=NHFc H-2,5), 4.52 (s, 2H, C=NHFc H-3,4), 4.27 (s, 5H, Fc), 2.41 (s, 3H, CH3); C NMR (75
MHz, DMSO-d6) δ, ppm: 168.7 (1C, C=O), 168.0 (1C, Oxadiazole C-3), 153.5 (1C, C=NH), 148.8 (1C,
Oxadiazole C-5), 142.0 (1C, Ar C-4), 129.8 (2C, Ar C-3,5), 127.1 (1C, Ar C-1), 122.6 (2C, Ar C-2,6), 77.7
(1C, C=NHFc C-1), 70.7 (2C, C=NHFc C-3,4), 69.1 (5C, Fc), 67.9 (2C, C=NHFc C-2,5), 21.0 (1C, CH3);
UHPLC-TOF-MS for C21H18FeN4O2 calcd (found)/Error: 414.0779 (415.0830, [M+H+])/6.5 ppm.
4.1.2.11.
3-(4-Fluorophenyl)-N’-(E-ferrocenylmethylidene)-1,2,4-oxadiazol-5-ylcarbohydrazide
SintMed27: Yield: 94 %; Mp 209.4-211.5 °C (from dioxane/H2O); IR (KBr, νmax cm-1): 3179 (NH), 3100,
3048 (Ar CH), 1681 (C=O), 1607 (C=N, imine), 1550 (C=N, heterocyclic); 1H NMR (300 MHz, DMSO-d6)
δ, ppm: 12.5 (s, 1H, CONH), 8.46 (s, 1H, N=CH), 8.16 (dd, 2H, 4JF,H = 3.0 Hz, 3J = 5.4 Hz, Ar H-2,6), 7.48
(t, 2H, 3J = 8.7 Hz, Ar H-3,5), 4.71 (s, 2H, C=NHFc H-2,5), 4.52 (s, 2H, C=NHFc H-3,4), 4.27 (s, 5H, Fc);
13C NMR (75 MHz, DMSO-d6) δ, ppm: 169.0 (1C, C=O), 167.3 (1C, Oxadiazole C-3), 164.2 (1C, d, 1JFC
=
249.5 Hz, Ar C-4), 153.5 (1C, C=NH), 148.7 (1C, Oxadiazole C-5), 129.8 (2C, d, 3JFC = 9.15 Hz, Ar H-2,6),
122.1 (1C, Ar C-1), 116.6 (2C, d, 2JFC = 22.3 Hz, Ar C-3,5), 77.7 (1C, C=NHFc C-1), 70.8 (2C, C=NHFc
C-3,4), 69.1 (5C, Fc), 68.0 (2C, C=NHFc C-2,5); UHPLC-TOF-MS for C20H15FeN4O2F calcd (found):
418.0528 (419.0622, [M+H+])/3.6 ppm.
4.1.2.12.
3-(4-Chlorophenyl)-N’-(E-ferrocenylmethylidene)-1,2,4-oxadiazol-5-ylcarbohydrazide
SintMed28: Yield: 97 %; Mp 217.5 °C (from dioxane/H2O); IR (KBr, νmax cm-1): 3181 (NH), 3105, 3029

1
(Ar CH), 1676 (C=O), 1600 (C=N, imine), 1560 (C=N, heterocyclic); H NMR (300 MHz, DMSO-d6) δ,
3
3
ppm: 12.5 (s, 1H, CONH), 8.46 (s, 1H, N=CH), 8.11 (d, 2H, J = 8.4 Hz, Ar H-2,6), 7.71 (d, 2H, J = 8.4
13
Hz, Ar H-3,5), 4.71 (s, 2H, C=NHFc H-2,5), 4.52 (s, 2H, C=NHFc H-3,4), 4.27 (s, 5H, Fc); C NMR (75
MHz, DMSO-d6) δ, ppm: 169.1 (1C, C=O), 167.3 (1C, Oxadiazole C-3), 153.6 (1C, C=NH), 148.7 (1C,
Oxadiazole C-5), 136.8 (1C, Ar C-4), 129.6 (2C, Ar C-2,6), 129.0 (1C, Ar C-1), 124.4 (2C, Ar C-3,5), 77.7
(1C, C=NHFc C-1), 70.8 (2C, C=NHFc C-3,4), 69.1 (5C, Fc), 68.1 (2C, C=NHFc C-2,5); UHPLC-TOF-
MS for C20H15FeN4ClO2 calcd (found)/Error: 434.0233 (435.0313, [M+H+])/0.2 ppm.
4.1.2.13.
3-(4-Bromophenyl)-N’-(E-ferrocenylmethylidene)-1,2,4-oxadiazol-5-ylcarbohydrazide
SintMed29: Yield: 95 %; Mp 216.0 °C (from dioxane/H2O); IR (KBr, νmax cm-1): 3180 (NH), 3099 (Ar
1
CH), 1676 (C=O), 1598 (C=N, imine), 1548 (C=N, heterocyclic); H NMR (300 MHz, DMSO-d6) δ, ppm:
12.5 (s, 1H, CONH), 8.46 (s, 1H, N=CH), 8.03 (d, 2H, 3J = 8.1 Hz, Ar H-2,6), 7.85 (d, 2H, 3J = 7.8 Hz, Ar
13
H-3,5), 4.71 (s, 2H, C=NHFc H-2,5), 4.52 (s, 2H, C=NHFc H-3,4), 4.26 (s, 5H, Fc); C NMR (75 MHz,
DMSO-d6) δ, ppm: 169.1 (1C, C=O), 167.4 (1C, Oxadiazole C-3), 153.6 (1C, C=NH), 148.6 (1C,
Oxadiazole C-5), 132.5 (2C, Ar-C3,5), 129.1 (Ar-C2,6), 125.6 (Ar-C4), 124.7 (Ar-C1), 77.7 (Fc-C1 to
imine), 70.8 (Fc-C3,4 to imine), 69.1 (Fc), 68.0 (Fc-C2,5 to imino); UHPLC-TOF-MS for C20H15FeN4BrO2
calcd (found)/Error: 477.9728 (478.9811, [M+H+])/1.0 ppm.
4.1.2.14. 3-(4-Nitrophenyl)-N’-(E-ferrocenylmethylidene)-1,2,4-oxadiazol-5-ylcarbohydrazide SintMed30:
Yield: 92 %; Mp 232.4-234.6 °C (from dioxane/H2O); IR (KBr, νmax cm-1): 3181 (NH), 3099 (Ar CH), 1685
(C=O), 1597 (C=N, imine), 1547 (C=N, heterocyclic); 1H NMR (300 MHz, DMSO-d6) δ, ppm: 12.5 (s, 1H,
CONH), 8.49 (d, 2H, 3J = 9.0 Hz, Ar H-3,5), 8.48 (s, 1H, N=CH), 8.37 (d, 2H, 3J = 9.0 Hz, Ar H-2,6), 4.72
(t, 2H, 3J = 1.5 Hz, C=NHFc H-2,5), 4.53 (t, 2H, 3J = 1.5 Hz, C=NHFc H-3,4), 4.28 (s, 5H, Fc); 13C NMR
(75 MHz, DMSO-d6) δ, ppm: 169.4 (1C, C=O), 166.7 (1C, Oxadiazole C-3), 153.7 (1C, C=NH), 149.4 (1C,
Ar C-4), 148.5 (1C, Oxadiazole C-5), 131.2 (1C, Ar C-1), 128.6 (2C, Ar C-2,6), 124.5 (2C, Ar C-3,5), 77.6
(1C, C=NHFc C-1), 70.8 (2C, C=NHFc C-3,4), 69.1 (5C, Fc), 68.0 (2C, C=NHFc C-2,5); UHPLC-TOF-
MS for C20H15FeN5O4 calcd (found)/Error: 445.0473 (446.0538, [M+H+])/3.1 ppm.

4.1.2.15.
3-(4-Methoxyphenyl)-N’-(E-ferrocenylmethylidene)-1,2,4-oxadiazol-5-ylcarbohydrazide
SintMed31: Yield: 93 %; Mp 197.7-199.0 °C (from dioxane/H2O); IR (KBr, νmax cm-1): 3181 (NH), 3100
1
(Ar CH), 1682 (C=O), 1611 (C=N, imine), 1550 (C=N, heterocyclic); H NMR (300 MHz, DMSO-d6) δ,
3
3
ppm: 12.5 (s, 1H, CONH), 8.46 (s, 1H, N=CH), 8.05 (d, 2H, J = 9.0 Hz, Ar H-2,6), 7.17 (d, 2H, J = 8.7
Hz, Ar H-3,5), 4.71 (t, 2H, 3J = 1.5 Hz, C=NHFc H-2,5), 4.53 (t, 2H, 3J = 1.5 Hz, C=NHFc H-3,4), 4.27 (s,
5H, Fc), 3.86 (s, 3H, OCH3); 13C NMR (75 MHz, DMSO-d6) δ, ppm: 168.7 (1C, C=O), 167.8 (1C,
Oxadiazole C-3), 162.1 (1C, Ar C-4), 153.5 (1C, C=NH), 148.8 (1C, Oxadiazole C5), 128.9 (2C, Ar C-2,6),
117.7 (1C, Ar C-1), 114.8 (2C, Ar C-3,5), 77.8 (1C, C=NHFc C-1), 70.8 (2C, C=NHFc C-3,4), 69.1 (5C,
Fc), 68.0 (2C, C=NHFc C-2,5), 55.5 (1C, OCH3); UHPLC-TOF-MS for C21H18FeN4O3 calcd (found)/Error:
430.0728 (431.0822, [M+H+])/3.5 ppm.
4.1.2.16.
3-(4-Hydroxyphenyl)-N’-(E-ferrocenylmethylidene)-1,2,4-oxadiazol-5-ylcarbohydrazide
SintMed32: Yield: 83 %; Mp 221.3-223.0 °C (from dioxane/H2O); IR (KBr, νmax cm-1): 3313 (br OH), 3180
1
(NH), 3100 (Ar CH), 1702 (C=O), 1611 (C=N, imine), 1564 (C=N, heterocyclic); H NMR (300 MHz,
DMSO-d6) δ, ppm: 12.4 (s, 1H, CONH), 10.2 (s, 1H, OH), 8.44 (s, 1H, N=CH), 7.93 (d, 2H, 3J = 6.4 Hz, Ar
3
H-2,6), 6.97 (d, 2H, J = 6.0 Hz, Ar H-3,5), 4.70 (s, 2H, C=NHFc H-2,5), 4.51 (s, 2H, C=NHFc H-3,4),
13
4.27 (s, 5H, Fc); C NMR (75 MHz, DMSO-d6) δ, ppm: 168.4 (1C, C=O), 167.9 (1C, Oxadiazole C-3),
160.7 (1C, Ar C-4), 153.4 (1C, C=NH), 148.9 (1C, Oxadiazole C-5), 129.0 (2C, Ar C-2,6), 116.1 (1C, Ar C-
1), 116.1 (2C, Ar C-3,5), 77.8 (1C, C=NHFc C-1), 70.8 (2C, C=NHFc C-3,4), 69.1 (5C, Fc), 68.0 (2C,
C=NHFc C-2,5); UHPLC-TOF-MS for C20H16FeN4O3 calcd (found)/Error: 416.0572 (417.0664,
[M+H+])/3.4 ppm.
4.2 Pharmacological evaluation
4.2.1 Cytotoxicity assay: HepG2 cells in RPMI-1640 medium without phenol red (Sigma Chemical Co.,
MO, USA) plus 10% fetal bovine serum (FBS; Cultilab, Campinas, SP, Brazil) and 50 µg mL-1 of

gentamycin (Novafarma, Anápolis, GO, Brazil) were seeded in 96-well plates (104 cells/well in 100 μL of
medium). After 24h, the compounds, diluted in RPMI-1640 medium without phenol red, were added (100
μL) in five concentrations, in triplicate, and the plates incubated for 72h at 37°C and 5% CO2. After this
period, 20 % of AlamarBlue (Invitrogen, Carlsbad, CA) was added to each well and the plate incubated for
24 h. The absorbance at 570 and 600 nm was measured according to manufacturer’s instructions. Gentian
Violet (Synth, São Paulo, Brazil) was used as a cytotoxicity control. Controls with untreated cells were read
in parallel and triplicates are read in the same plate. The CC50 values were calculated using a non-linear
regression curve fit in the Graph Pad Prism version 5.03 (Graph Pad Software, San Diego, CA).
4.2.2 Anti-P. falciparum activity: W2 strain P. falciparum was maintained in continuous culture of human
erythrocytes (blood group O+) using the RPMI 1640 medium supplemented with 10% of human plasma.
Parasites grown at 1 % parasitemia and 2.5% hematocrit were distributed into 96-wells culture plate and
incubated with the compounds (previously diluted with 4% DMSO and culture medium). After 24 h of
incubation, [3H]-hypoxanthine was added, the plate incubated again for 24 h, cooled for 24 h and then
parasites were harvested using a cell harvester to quantify the [3H]-hypoxanthine incorporation in a β-
radiation counter. Inhibition of parasite growth was evaluated by comparison with [3H]-hypoxanthine uptake
in treated versus untreated parasite. Each compound was initially tested at concentration of 10 µg/mL in
triplicate and the IC50 values were calculated in triplicate concentration using at least five concentrations.
Mefloquine (Mfq) was used as a standard drug.
4.2.3 Hemolysis assay: The hemolytic activity of the compounds was assayed in fresh human erythrocytes
(type O+).23 Cells were washed three times with phosphate buffered saline, and 100 μL of this suspension
(1% hematocrit) was distributed into each well to a 96-well plate. After, 100 μL of each compound (N-
acylhydrazone), previously dissolved in phosphate buffered saline, were added in triplicate to the plate and
incubated for 1 h. Saponin (Sigma-Aldrich, USA) was used as a hemolytic drug at 1 % v/v. After
incubation, samples were centrifuged (1500 rpm for 10 min.) and 100 μL of each supernatant was

transferred to another microtiter plate. Released haemoglobin was monitored by measuring the absorbance
at 540 nm in a spectrophotometer. The percentage of hemolysis was calculated as previously described.
4.2.4 Inhibition of -hematin formation: 50 μL of a freshly prepared solution of haemin (0.5 mg/mL)
dissolved in 0.2 M NaOH was mixed to a 75 μL of 3 M sodium acetate, 25 μL of 17.4 M acetic acid and 50
μL of the tested compounds (N-acylhydrazones). All N-acylhydrazones were tested at 2.5 mM. After 24 h of
incubation at 37°C, the resulting solution was spun down for 15 min at 3500 rpm, the supernatant was
discarded off and the pellet washed with 200 μL of DMSO. This step was repeated once and after a final
wash with water, the pellet was dissolved in 0.1 M NaOH (150 μL). Chloroquine (CQ) was included in each
experiment as positive control. The absorption at 405 nm was read using a spectrophotometer. Results are
expressed as percentage of inhibition of hematin formation in comparison to the negative control (without
compound). Experiments were carried out at least twice using compound concentrations in triplicate.
4.2.5 Antimalarial tests against Plasmodium berghei in mice: Adult Swiss mice (4-6 weeks) were
inoculated by intraperitoneal route with 106 infected red blood cells with NK65 strain P. berghei. The
infected mice were randomly divided into groups of n= 5 and subsequently treated with compounds
SintMed2, SintMed5 or SintMed6 diluted in DMSO 20 % given once a day for four consecutive days by
oral gavage or intraperitoneal. Two control groups were used in parallel, one treated with Chloroquine (50
mg/kg), standard drug, and one with the vehicle (DMSO 20% in saline solution). Blood smears from mice
tails were prepared on days 4, 5, 6, 7 and 8 post-infection and then methanol-fixed, stained with fast
panoptic (Laborclin, Pinhais, Brazil) and examined by optical microscopy. The parasitemia was evaluated
and the percent inhibition of parasite growth calculated in relation to the untreated control group (considered
100% growth) using the following equation: [(C - T)/C] x 100; where C is the parasitemia where C is the
parasitemia in the control group and T is the parasitemia in the treated group.
ACKNOWLEDGMENTS

This work received support from FAPESB. The authors are grateful to Mrs. Eliete de Fátima V. B. N. da
Silva, Mrs. Abene Silva Ribeiro and the Analytical Centre of Fundamental Chemistry Department,
Universidade Federal de Pernambuco, for the NMR experiments.
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Graphical abstract