In vitro anti-toxoplasma gondii and antimicrobial activity of amides derived from cinnamic acid

Article abstract of DOI:10.3390/molecules23040774

Most cinnamic acids, their esters, amides, aldehydes, and alcohols present several therapeutic actions through anti-inflammatory, antitumor, and inhibitory activity against a great variety of microorganisms. In this work, eight amines derived from cinnami

Full text of DOI:10.3390/molecules23040774

molecules
Article
In Vitro Anti-Toxoplasma gondii and Antimicrobial
Activity of Amides Derived from Cinnamic Acid
Graziela Rangel Silveira 1,*, Karoline Azerêdo Campelo 1, Gleice Rangel Silveira Lima 1,
Lais Pessanha Carvalho 2, Solange Silva Samarão 3, Olney Vieira-da-Motta 3, Leda Mathias 1,
Carlos Roberto Ribeiro Matos 1, Ivo José Curcino Vieira 1, Edesio José Tenório de Melo 2 and
Edmilson José Maria 1
1
Laboratório de Ciências Químicas, Centro de Ciências e Tecnologia, Universidade Estadual do Norte
Fluminense-Darcy Ribeiro, Av. Alberto Lamego, 2000—Parque Califórnia, 28013-602 Campos dos
Goytacazes/RJ, Brazil; karolcampelo16@yahoo.com.br (K.A.C.); gleice.sil@hotmail.com (G.R.S.L.);
leddam8@gmail.com (L.M.); matos@uenf.br (C.R.R.M.); curcino@uenf.br (I.J.C.V.);
edmilson_maria@yahoo.com.br (E.J.M.)
2
Laboratório de Biologia Celular e Tecidual, Centro de Biociências e Biotecnologia, Universidade Estadual do
Norte Fluminense-Darcy Ribeiro, Av. Alberto Lamego, 2000—Parque Califórnia, 28013-602 Campos dos
Goytacazes/RJ, Brazil; lais_pessanha@hotmail.com (L.P.C.); ejtm1202@gmail.com (E.J.T.M.)
Laboratório de Sanidade Animal, Centro de Ciências e Tecnologias Agropecuárias, Universidade Estadual
3
do Norte Fluminense-Darcy Ribeiro, Av. Alberto Lamego, 2000—Parque Califórnia, 28013-602 Campos dos
Goytacazes/RJ, Brazil; solangesamarao@gmail.com (S.S.S.); olney.motta@gmail.com (O.V.-d.-M.)
*
Correspondence: graziela_sil@hotmail.com; Tel.: +55-022-2728-6167
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 1 March 2018; Accepted: 23 March 2018; Published: 28 March 2018
Abstract: Most cinnamic acids, their esters, amides, aldehydes, and alcohols present several
therapeutic actions through anti-inflammatory, antitumor, and inhibitory activity against a great
variety of microorganisms. In this work, eight amines derived from cinnamic acid were synthesized
and tested against host cells infected with Toxoplasma gondii and the bacteria Escherichia coli,
Pseudomonas aeruginosa, Staphylococcus epidermidis, and three strains of Staphylococcus aureus.
Compounds 3 and 4 showed the best result against intracellular T. gondii, presenting antiparasitic
activity at low concentrations (0.38 and 0.77 mM). The antibacterial activity of these compounds was
also evaluated by the agar microdilution method, and amides 2 and 5 had a minimum inhibitory
concentration of 250 µ
g mL−1 against two strains of S. aureus (ATCC 25923 and bovine strain LSA 88).
These also showed synergistic action along with a variety of antibiotics, demonstrating that amines
derived from cinnamic acid have potential as pharmacological agents.
Keywords: synthetic amides; antiparasitic; antibiotic; acrylamides
1. Introduction
The cinnamic acids belong to a group of (C6-C3) substances derived from the secondary metabolism
of plants. The term “cinnamic” refers to Cinnamomum verum (Sin. C. zeylanicum), commonly known
as the “cinnamon tree”. From this plant, “cinnamon” is obtained by drying the central part of the
bark [1,2].
Most of the cinnamic acids, their esters, amides, aldehydes, and alcohols present several
therapeutic actions like anti-inflammatory [
and inhibitory activity against a broad range of microorganisms [
is still poorly known [ ]. Several researchers tried to produce alternative compounds obtained from
3,
4], antitumor [
5], antimalarial [
6,7], antitrypanosomal [8],
2,9–11]. The mechanism of action
2
derivates of this acid to potentiate its therapeutic properties.
Molecules 2018, 23, 774; doi:10.3390/molecules23040774
www.mdpi.com/journal/molecules

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Despite the broad range of biologic activities described in the derivates of the cinnamic
acid, there has been no literature record until now on its effect against the protozoan parasite
Toxoplasma gondii, the causative agent of toxoplasmosis [12
estimated to be infected by this disease [12 14]. The most used treatment for this disease is the joint
use of sulfadiazine and pyrimethamine [15]. This combination of drugs shall be used as a reference for
other therapeutic protocols [13 15 16] to reduce the collateral effects associated with the treatment [16].
,13]. One-third of the global population is
,
,
,
Several studies using thiosemicarbazones and thiazolidines aimed to reduce the toxic effects of the
treatment [17–19].
Studies on the amides derived from cinnamic acid described inhibitory activity against several
main bacteria such as Escherichia coli and Staphylococcus spp. [2]. The development of alternative
antimicrobials against pathogenic bacteria is of great interest to the pharmaceutical industry [20].
With the aim of verifying the antimicrobial and antiparasitic activity, we synthesized eight amides
derived from the cinnamic acid (Figure 1).
Figure 1. Chemical structure of compounds 1–8.
2. Results and Discussion
2.1. Chemistry
The amides were synthesized by two different methods: coupling of the derivates of
the cinnamic acids with phenylethylamines substituted by dicyclohexylcarbodiimine (DCC) as
a coupling agent (to obtain amides
1–5 and 8) and nucleophilic substitution reaction using

Molecules 2018, 23, 774
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p-nitrophenylethylamine, p-bromophenylethylamine, and trans-p-nitrocynnamoil chloride (to obtain
amines 6 and 7). Among these reactions, those with acid chloride displayed higher yield.
The products of the reactions were solid at room temperature. These were analyzed using
1
a nuclear magnetic resonance spectrometer. In all compounds, a quartet signal in the H-NMR
spectrum was visible between 3.64–3.74 ppm, associated to the methylene adjacent to the nitrogen
atom of the amide. In the 13C-NMR spectrum, signals relating to carbonyl groups of the amides
were present between 164–172 ppm. The confirmation of these groups was also found in the HMBC
spectrum by the correlation at two and three bonds between the carbonyl carbon and the adjacent
hydrogens. The adsorptions in the infrared spectrum in the ranges of 1639–1657 and 3277–3347 cm−1
are characteristic of the C=O and N-H stretch, respectively. To verify the functional groups and to
assess the purity of the obtained products and their molecular weight we used gas chromatography
coupled to the mass spectrophotometer (GC-MS).
In this way, all the synthesized compounds showed spectral data consistent with the
respective structures.
Of the eight synthesized compounds, five are unpublished as of yet (2, 4, 6–8).
2.2. Assay for Anti-Toxoplasma gondii Activity
We focused the tests against T. gondii on the amides 1, 3–7. For this purpose, they were incubated
for 24 h in cultures of LCC-MK2 cells infected and non-infected with T. gondii.
The cellular toxicity test was performed to assess the concentration of the synthesized amides
causing neither toxic effects nor cell death in LCC-MK2 cells. The presence of morphological alterations,
degenerations or cell death induced due to the treatment with the different compounds defined the
presence of cellular toxicity. In general, increased cellular toxicity is directly proportional to the
increase in concentration, as displayed in Figure 2. The figure highlights that, as compared to the
control (Figure 2a), increasing the concentration (Figure 2b–d) caused nuclear morphological changes
and the progressive increase of toxicity with the reduction of cell numbers.
Figure 2. Toxicity in uninfected LLC-MK2 cells. The black arrow indicates nuclei with physiological
appearance. White arrows indicate nuclear morphological changes indicative of toxicity. (
In ( ) a gradual increase of cytotoxicity is visible, associated with significant nuclear disorders and
a decrease of the cellular number.
a) Control case;
b–d

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Table 1 displays the results of the toxicity test in uninfected cells. Data show that compounds
exhibited no toxicity at 2.92 mM and are, therefore, the least toxic. Compound had a similar behavior
at 1.69 mM. Compounds and showed no toxicity at concentrations of 0.35 and 0.17 mM, respectively.
6 and
7
5
3
4
Table 1. Cytotoxicity of the compounds 1 and 3–7 in LLC-MK2 uninfected cells.
Cytotoxicity (Uninfected Culture) a
Concentration (mM)
Drug
Untreated
226 ± 09
196 ± 12
196 ± 12
226 ± 09
206 ± 10
206 ± 10
0.17
-
-
0.35
1.69
2.92
0
0
Compound 1
Compound 3
Compound 4
Compound 5
Compound 6
Compound 7
229 ± 13
112 ± 14
194 ± 11
-
0
200 ± 21
-
-
-
-
0
-
-
-
217 ± 30
71 ± 10
209 ± 12
209 ± 17
-
-
a
Values means ± SD.
Compounds
concentration, these compounds caused 100% of cellular death. No substance caused significant
damages at levels equal or lower than 0.17 mM. The 1.69 mM concentration of compound caused the
caused cellular
1, 3, and 4 displayed higher cellular damages at the concentration of 2.92 mM. At this
1
reduction of 50% of the LLC-MK2 cells. The 2.92 mM concentration of compound
damages in 69% of the cells.
5
To define the dose of each compound which does not cause toxicity in uninfected cells, we tested
cells infected with T. gondii at concentrations lower than the toxic dose established. After the infection
of the LLC-MK2 cells with the parasite, we quantified the parasites based on the presence of tachyzoites.
Infection of the T. gondii parasite was visible in untreated (control) cells. After treatment with the
compounds, the number of infected host cells was reduced due to the elimination of the intracellular
parasites (Figure 3).
Figure 3. Optical microscopy of the biological test showing infected LLC-MK2 cells. The black arrows point
to the cell nucleus; the white arrows indicate the parasitophorous vacuole. (a) Control case. Reduction of
infection with the elimination of the parasite in the cells treated with (b) compound 1 and (c) 7.
Table 2 displays the effect of compounds 1, 3–7 on LLC-MK2 cells and intracellular parasites.
The number of cells in the control tests and the treatments are similar, suggesting no cytotoxicity of the
tested compounds at the concentration used in the experiment.

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Table 2. Effect of compounds
1
and
3–7
on cultures of LLC-MK2 cells infected and multiplication of T. gondii.
Cytotoxicity (Infected Culture) a
Drugs
Concentration (mM) Uninfected Cells
Infected Cells
Total of Cells
111 ± 12
153 ± 10
159 ± 25
170 ± 8
159 ± 25
184 ± 34
111 ± 12
126 ± 4
116 ± 10
120 ± 17
116 ± 10
132 ± 12
Parasites
179 ± 29
166 ± 15
254 ± 38
127 ± 32
254 ± 38
134 ± 32
179 ± 29
82 ± 19
174 ± 16
83 ± 23
174 ± 16
98 ± 12
Control
Compound
1
Control
Compound
3
Control
Compound
4
Control
Compound
5
Control
Compound
6
Control
Compound
7
0
0.18
0
83 ± 8
129 ± 6
108 ± 19
147 ± 3
108 ± 19
149 ± 24
83 ± 8
114 ± 3
79 ± 7
89 ± 12
79 ± 7
104 ± 9
a
28 ± 4
24 ± 4
51 ± 6
23 ± 5
51 ± 6
35 ± 10
28 ± 4
12 ± 1
37 ± 3
0.077
0
0.038
0
0.16
0
1.33
0
31 ± 5
37 ± 3
28 ± 3
1.46
Values means ± SD.
During the incubation, tachyzoites displayed morphologic disorganization before being
eliminated. These results corroborate with similar experiences using thiosemicarbazones and
thiazolidines [17–19].
Compound
parasite. Compounds
This concentration corresponds to 22.01 and 56.3
T. gondii among the compounds we tested. Compounds
5
required a 0.16 mM concentration to be effective against the intracellular
and eliminated 50% of the parasites at 0.077 and 0.38 mM, respectively.
g mL−1, which are the lowest effective doses against
and showed better effects at concentrations
3
4
µ
6
7
of 1.33 and 1.46 mM, respectively (Table 2), although they had low cytotoxicity.
These results align with similar experiments performed by other researchers which studied the
anti-toxoplasmic activity of thiosemicarbazone and thiazolidine analogues. Their results showed,
the active concentrations of these drugs varied between 0.1 mM and 1.5 mM and cytotoxicity was
between 0.5 and 10 mM [17–19]. Regarding the activity of the compounds used in this study,
compounds and displayed better results at lower concentrations. This data suggests that the
3
4
substitution of chlorine and nitro (NO2) in the para position in the ethylphenylamine portion promotes
a significative action on the parasite.
The analysis of both cytotoxicity and antitoxoplasmic activity shows that the presence of an active
electron scavenger group (NO2) in the para position of the cinnamoyl portion (as in compounds
) are characteristic of the least cytotoxic compounds. Similarly, these compounds display the highest
concentration for the induction of parasite mortality.
6 and
7
All the compounds had antiparasitic activity at concentrations lower than the standard drugs
hydroxyurea (DL50 4 mM) [18] and sulfadiazine (DL50 3 mM) [19]. The high concentration of these
drugs suggests the possibility of toxic effects.
2.3. Assay for Antimicrobial Activity
All synthesized compounds were tested for their antimicrobial activity by the broth microdilution
method according to the Standards Institute and Laboratory [21]. Minimum inhibitory concentration
(MIC) values were the lowest concentration that presented growth between 30 and 300 colonies.
Compounds
25923 and LSA 88, respectively (Table 2). Most of the compounds displayed some activity against
the tested strains at concentrations higher than 250 were effective against
g mL−1. Compounds
2 and 5 showed MICs of 250 µ
g mL−1 against strains of Staphylococcus aureus ATCC
µ
1–5

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Pseudomonas aeruginosa ATCC 15442. Compound
2
was effective against Staphylococcus aureus LSA
88 (clinical strain isolated from bovine mastitis) and Staphylococcus aureus ATCC 33591. Compounds
6
and
7
were effective against Escherichia coli ATCC 25922; compounds
1
,
6
and
7 were effective against
Staphylococcus epidermidis ATCC 12228−1. Compound
8 displayed no inhibitory activity on the tested
microorganisms (Table 3).
Table 3. Minimum inhibitory concentration (MIC) of compounds 1–8 against different bacterial strains.
−1 a
Minimum Inhibitory Concentration (MIC) in µg mL
Compound
ATCC 15442
LSA 88
ATCC 33591 ATCC 25923 ATCC 25922 ATCC 12228
1
2
3
4
5
6
7
8
>250
>250
>250
>250
>250
−
−
>250
−
−
>250
−
−
250
−
−
−
−
>250
−
−
−
−
−
−
−
250
−
−
−
−
−
−
−
>250
>250
−
>250
>250
−
−
−
−
−
−
−
−
−
a
The maximum concentration tested was 250
(concentration 10
g mL−1). Tests were performed in triplicate. - No inhibition at the concentration tested. >250 indicates
that there was sensitivity, but the concentration should be higher than 250
g mL−1 for a count of fewer than 300 colonies.
µg
mL−1
.
Gentamicin was used as control antibiotic
µ
µ
−1
)
Georgiev et al. [22] synthesized cinnamoylamides effective against S. aureus strains (62.5–500
as observed in the present study.
µg mL
Table 4 displays the antibiograms of the bacteria treated with the compounds
2
and
5.
The comparison between control bacteria and those treated with compound
to the antibiotics displays larger inhibition halos of the strain ATCC 25923 (p < 0.01) for all the
antibiotics that have been tested together with compound . The increase of halo size of the compounds
demonstrates that this product has synergic actions with these antibiotics,
2 after the exposition
2
treated with compound
boosting their effects.
2
Table 4. Antibiogram (average halo diameter expressed in mm) of strains of S. aureus treated with
amides derived from cinnamic acid and submitted to 12 different antibiotics. Assessed by agar gel
diffusion method [23].
Strains
ATCC 25923
Control
LSA 88
Antibiotic
2
Control
5
VAN
AMP
SUT
CLI
CFX
ERI
AMO
PEN
GEN
AMC
OXA
TET
16.74Bg
27.05Ba
25.00Bbc
21.40Be
23.70Bcd
20.87Be
24.95Bbc
25.69Bab
21.37Be
26.81Ba
19.02Bf
23.98Ad
36.99Aa
37.00Aa
17.50Ae
33.17Aab
28.05Bc
26.76Ac
28.09Bc
17.45Ai
34.64Ab
29.68Ade
27.17Ag
28.90Aef
27.99Afg
31.36Bc
36.21Aa
26.99Ag
35.62Aab
21.32Ah
30.91Acd
34.42Aab
34.86Aab
32.42Aabc
32.88Aab
33.40Aab
30.24Abc
35.52Aab
26.61Acd
33.33Aab
25.73Bc
33.98Aab
36.11Aa
26.72Ac
35.06Aab
21.31Ad
32.49Ab
22.40Bde
The averages followed by the same small letter in the column (antibiotics) and a capital letter on the line
(treatment with amines derived from cinnamic acid) do not differ from each other. Tukey test, 1 and 5% probability.
VAN: Vancomycin; AMP: Ampicillin; SUT: Sulfazotrim; CLI: Clindamycin; CFX: Cefalexin; ERI: Erythromycin;
AMO: Amoxicillin; PEN: Penicillin G; GEN: Gentamicin; AMC: Amoxicillin/ Clavulanic acid; OXA: Oxacillin;
TET: Tetracycline.

Molecules 2018, 23, 774
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Compound
5
had a significant synergism (p < 0.05) on the effect of Sulfazotrim (SUT),
Cefalexin (CFX), and Erythromycin (ERI) against the S. aureus bovine clinical sample LSA 88. On the
contrary, the same compound had an antagonistic effect toward AMO, reducing its inhibition
5
halo (p < 0.05). Carlos et al. [20] also described the antagonistic effect between the control and the
Rauvolfia grandiflora extracts with the antibiotics ampicillin, oxacillin, and cephalosporin.
Comparing the results among drugs, AMC and AMP displayed more significant activity in the
control treatment for the ATCC 25923 strain. With the treatment of compound 2, SUT activity is
potentiated, producing a broader inhibition halo together with AMP. The LSA 88 strain displayed
higher sensitivity toward penicillin G (PEN). This result remained after treatment with compound 5.
The ATCC 25923 strain remained less sensitive to VAN and OXA even after the treatments,
although an increase in the inhibition zone was confirmed (p < 0.01).
The results suggest the differences in the inhibition halos of S. aureus treated with compounds
and 5 increased the sensibility of strains ATCC 25923 and LSA 88 to different antibiotics.
2
3. Materials and Methods
3.1. General Information
All the chemical reagents used in this experiments were from Sigma Aldrich (St. Louis, MI, USA).
Nuclear magnetic resonance spectra (NMR 1H, 13C, HMBC e HSQC) were obtained using BRUKER
Avance III equipment (125 and 500 MHz) in deuterated chloroform (CDCl3) measured as parts per
million (ppm) and coupling constants (J) in Hz. Infrared spectra were obtained using an IR-affinity
spectrometer/Shimadzu (Tokyo, Japan) using KBr pellets. Purity and atomic mass of the compounds
were confirmed by gas chromatography coupled with mass spectrometry (GC/MS), model 5975C
Inert XL EI/CI/MS Agilent Technologies. All spectra are available in Supplementary Materials
Figure S1–S48.
3.2. General Synthetic Procedure
The eight compounds were synthesized by the condensation reaction of carboxylic acids with
phenylethylamines by activation of the acid portion; six compounds (1, 2, 3, 4, 5 and 8) were produced
using N,N-dicyclohexylcarbodiimide (DCC-Sigma Aldrich, St. Louis, MI, USA) as the coupling agent.
For compounds 6 and 7, the corresponding acid chloride was used (Figure 1).
3.2.1. General Synthetic Procedure for Compounds 1–5 and 8
The addition of 0.2 mmol of cinnamic acid together with equimolar amounts of DCC and their
amines in dichloromethane is done at 0 ◦C and stirred for 48 h under argon atmosphere. The reaction
was monitored by thin layer chromatography and purified through a silica gel chromatographic
column, eluting hexane and ethyl acetate (1:1) [9,24].
N-[2-(4-Methoxyphenyl)ethyl]-3-phenyl-acrylamide (1). Yellow amorphous solid; Yield: 39.9%; MP:
◦
1
137.4–137.9 C; IR (υ υ
cm−1 KBr): 1624 (N-H), 3312 (N-H), 1655 cm−1 (C=O); NMR H (CDCl3,
500 MHz): δH 7.64 (d, J = 15.6 Hz, 1H, C=CH), 7.51–7.48 (m, 2H, Ar-H), 7.39–7.35 (m, 3H, Ar-H),
7.18–7.14 (m, 2H, Ar-H), 6.90–6.86 (m, 2H, Ar-H), 6.36 (d, J = 15.6 Hz, 1H, C=CH), 3.64 (q, J = 6.7 Hz, 2H,
-CH2), 2.85 (t, J = 6.7 Hz, 2H, -CH2), 2.81 (s, 3H, OCH3); 13C-NMR (CDCl3, 125 MHz) δC 165.9 (C=O),
158.3 (C), 140.0 (CH), 134.8 (C), 130.8 (C), 129.8 (C), 129.7 (C), 128.8 (C), 127.8 (C), 120.7 (CH), 114.1 (C),
55.2 (OCH3), 41.0 (CH2), 34.8 (CH2); MS (EI), m/z = 281.0.
3-Phenyl-N-(2-m-tolylethyl)-acrylamide (
IR (
cm−1 KBr): 1618 (N-H), 3277 (N-H), 1657 (C=O) cm−1; 1H-NMR (CDCl3, 500 MHz): δH 7.64
2
). Yellow amorphous solid; Yield: 49.4%; MP: 126.0–126.4 ◦C;
υ
υ
(d, J = 15.3 Hz, 1H, C=CH), 7.52–7.48 (m, 2H, Ar-H), 7.40–7.35 (m, 3H, Ar-H), 7.07 (m, 1H, Ar-H),
7.10–7.03 (m, 3H, Ar-H), 6.36 (d, J = 15.3 Hz, 1H, C=CH), 3.68 (q, J = 6.8 Hz, 2H, -CH2), 2.88 (t, J = 6.8 Hz,
2H, -CH2), 2.38 (s, 3H, CH3); 13C-NMR (CDCl3, 125 MHz) δC 165.9 (C=O), 141.0 (CH), 138.9 (C),

Molecules 2018, 23, 774
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138.5 (C), 134.9 (C), 129.7 (C), 129.7 (C), 128.8 (C), 128.6 (C), 127.8 (C), 127.3 (C), 125.8 (C), 120.7 (CH),
40.9 (CH2), 35.6 (CH2), 21.6 (CH3); MS (EI), m/z = 265.1.
N-[2-(4-Chlo◦rophenyl)ethyl]-3-phenyl-acrylamide (
3
). White amorphous solid; Yield: 63.8%; MP:
1
163.4–164.0 C; IR (
υ
cm−1 KBr):
υ
1620 (N-H), 1651 (C=O) 3300 (N-H) cm−1; H-NMR (CDCl3,
500 MHz): δH 7.64 (d, J = 15.6 Hz, 1H, C=CH), 7.52–7.49 (m, 2H, Ar-H), 7.39–7.36 (m, 3H, Ar-H),
7.32–7.29 (m, 2H, Ar-H), 7.19–7.16 (m, 2H, Ar-H), 6.35 (d, J = 15.6 Hz, 1H, C=CH), 3.65 (q, J = 6.7 Hz,
2H, -CH2), 2.89 (t, J = 6.7 Hz, 2H, -CH2); 13C-NMR (CDCl3, 125 MHz) δC 165.9 (C=O), 141.3 (CH),
137.3 (C), 134.7 (C), 132.4 (C), 130.2 (C), 129.8 (C), 128.8 (C), 127.8 (C), 40.7 (CH2), 35.0 (CH2); MS (EI),
m/z = 285.1.
◦
N-[2-(4-Nitrophenyl)ethyl]-3-phenyl-acrylamide (
4
). Yellow amorphous solid; Yield: 41.2%; MP: 152.9–153.0 C;
IR (υ
cm−1 KBr):
υ
1612 (N-H), 1651 (C=O) 3312 (N-H) cm−1; 1H-NMR (CDCl3, 500 MHz): δH 8.20–8.15
(m, 2H, Ar-H), 7.66 (d, J = 15.3 Hz, 1H, C=CH), 7.52–7.49 (m, 2H, Ar-H), 7.42–7.36 (m, 5H, Ar-H),
6.39 (d, J = 15.3 Hz, 1H, C=CH), 3.71 (q, J = 7.0 Hz, 2H, -CH2), 3.04 (t, J = 7.0 Hz, 2H, -CH2); 13C-NMR
(CDCl3, 125 MHz) δC 166.1 (C=O), 146.8 (C), 146.8 (C), 141.6 (CH), 134.6 (C), 129.9 (C), 128.9 (C),
127.8 (C), 123.9 (C), 120.2 (CH), 40.5 (CH2), 35.7 (CH2); MS (EI), m/z = 296.1.
◦
N-[2-(3,4-Dimethoxyphenyl)ethyl]-3-phenyl-acrylamide (
5
). Crystalline solid; Yield: 32.5%; MP: 110.8–111.0 C;
1
IR (υ
cm−1 KBr):
υ
1622 (N-H), 1655 (C=O) 3347 (N-H) cm−1; H RMN (CDCl3, 500 MHz): δH 7.64
(d, J = 15.6 Hz, 1H, C=CH), 7.52–7.47 (m, 2H, Ar-H), 7.39–7.34 (m, 3H, Ar-H), 6.80–6.74 (m, 2H, Ar-H),
6.84 (m, 1H, Ar-H), 6.36 (d, J = 15.6 Hz, 1H, C=CH), 3.88 (s, 6H, OCH3), 3.65 (q, J = 6.8 Hz, 2H, -CH2), 2.86
(t, J = 6.8 Hz, 2H, -CH2); 13C-NMR (CDCl3, 125 MHz) δC 165.9 (C=O), 149.1 (C), 147.8 (C), 141.1 (CH),
131.4 (C), 129.7 (C), 128.8 (C), 127.9 (C), 120.7 (C), 120.6 (CH), 112.0 (C), 111.4 (C), 56.0 (OCH3), 55.9
(OCH3), 41.0 (CH2), 35.2 (CH2); MS (EI), m/z = 311.1.
3-(4-Bromophenyl)-N-[2-(4-chlorophenyl)ethyl]-propionamide (
130.0–131.1 ◦C; IR ( cm−1 KBr):
8
). Crystalline solid; Yield: 64.0%; MP:
1
υ
υ
1639 (C=O), 3290 (N-H est) cm−1; H RMN (CDCl3, 500 MHz):
δ
7.44–7.39 (m, 2H, Ar-H), 7.28–7.25 (m, 2H, Ar-H), 7.09–7.06 (m, 2H, Ar-H), 7.04–6.99 (m, 2H, Ar-H),
3.47 (q, J = 6.9 Hz, 2H, -CH2), 2.92 (t, J = 7.5 Hz, 2H, -CH2), 2.73 (t, J = 6.9 Hz, 2H, -CH2), 4.01 (t, J = 7.5 Hz,
2H, -CH2); 13C-NMR (CDCl3, 125 MHz): δC 171.6 (C=O), 139.8 (C), 137.2 (C), 132.4 (C), 131.6 (C),
130.2 (C), 130.0 (C), 128.1 (C), 120.1 (C), 40.5 (CH2), 38.2 (CH2), 35.0 (CH2), 31.0 (CH2); MS (EI),
m/z = 367.0.
3.2.2. Synthetic Procedure for Compounds 6 and 7
The addition of 0.2 mmol of trans-p-nitrocinnamoyl chloride together with diisopropylamine with
equimolar amounts of their phenylethylamines in dichloromethane [4,9
] was done at 0 ◦C and stirred
for 48 h under argon atmosphere. The reaction was monitored by thin layer chromatography and
purified through a separation funnel (dichloromethane to water).
N-[2-(4-Bromophenyl)ethyl]-3-(4-nitrophenyl)-acrylamide (6). Yellow crystalline solid; Yield: 89.6%; MP:
◦
1
146.0–147.0 C; IR (υ υ
cm−1 KBr): 1618 (N-H), 1651 (C=O) 3292 (N-H) cm−1; H-NMR (CDCl3,
500 MHz): δH 8.25–8.22 (m, 2H, Ar-H), 7.66–7.63 (m, 2H, Ar-H), 7.68 (d, J = 15.7 Hz, 1H, C=CH),
7.48–7.45 (m, 2H, Ar-H), 7.13–7.10 (m, 2H, Ar-H), 6.47 (d, J = 15.7 Hz, 1H, C=CH), 3.67 (q, J = 6.8 Hz, 2H,
-CH2), 2.88 (t, J = 6.8 Hz, 2H, -CH2); 13C-NMR (CDCl3, 125 MHz) δC 164.9 (C=O), 148.2 (C), 141.2 (C),
138.7 (CH), 137.6 (C), 131.8 (C), 130.5 (C), 128.4 (C), 124.5 (CH), 124.2 (C), 40.8 (CH2), 35.0 (CH2); MS
(EI), m/z = 374.0.
3-(4-Nitrophenyl)-N-[2-(4-nitrophenyl)ethyl]-acrylamide (7). Red amorphous solid; Yield: 82.3%; MP:
◦
1
149.6–151.2 C; IR (υ υ
cm−1 KBr): 1620 (N-H), 1651 (C=O) 3294 (N-H) cm−1; H-NMR (CDCl3,
500 MHz): δH 8.26–8.18 (m, 4H, Ar-H), 7.70 (d, J = 15.4 Hz, 1H, C=CH), 7.67–7.59 (m, 2H, Ar-H),
7.44–7.40 (m, 2H, Ar-H), 6.48 (d, J = 15.4 Hz, 1H, C=CH), 3.74 (q, J = 7.0 Hz, 2H, -CH2), 3.06 (t, J = 7.0 Hz,

Molecules 2018, 23, 774
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2H, -CH2); 13C-NMR (CDCl3, 125 MHz) δC 164.9 (C=O), 148.3 (C), 146.9 (C), 146.5 (C), 140.9 (C), 139.1
(CH), 129.7 (C), 128.4 (C), 124.2 (C), 123.9 (C), 123.9 (CH), 40.6 (CH2), 35.6 (CH2); MS (EI), m/z = 341.1.
3.3. Anti-Toxoplasma Gondii Activity
Cells of LLC-MK2 (monkey kidney fibroblasts) were seeded on culture plates with 24 sumps
with RPMJ 1640 (Sigma Aldrich, USA) supplemented with 5% fetal bovine serum for 24 h at 37 ◦C,
as described by Liesen et al. [18]. These cultures were treated with different concentrations of selected
amides (ranging from 0.19 to 2.92 mM) to verify cytotoxicity. The dose which did not show cellular
toxicity was determined by observation of morphological alterations when compared to the control.
Subsequently, for the anti-T. gondii activity, test cells were infected with tachyzoites (1:5) and
◦
incubated at 37 C for 24 h. After confirmation of infection, the cultures were treated with the cinnamic
acid derivatives at different concentrations (Table 2) for 24 h.
After treatment, the cultures were washed with phosphate buffer solution (PBS), fixed using
4% paraformaldehyde, and stained with GIEMSA solution. Subsequently, cultures were analyzed by
optical microscopy (AXIOPLAN/Zeis, Jena, Germany). The number of infected and uninfected cells,
as well as intracellular parasites, were quantified, and quantitative analyses were performed to verify
anti-toxoplasma activity.
The data presented were performed in triplicate.
3.4. Antimicrobial Activity
The biofilm forming bacterial strains of S. aureus (ATCC 33591), E. coli (ATCC 25922), P. aeruginosa
(ATCC 15442) and those strains which did not form biofilm—S. aureus (ATCC 25923), bovine strain
S. aureus (LSA88 SEC+/SED+/TSST-1+) [25], and S. epidermidis (ATCC 12228)—were obtained from
the bacterial collection of the Animal Health Laboratory (Laboratório de Sanidade Animal—LSA,
Universidade Estadual do Norte Fluminense – Darcy Ribeiro, Campos dos Goytacazes, Brazil).
The in vitro antibacterial activity of the amides was investigated using the broth dilution method.
Mueller Hinton agar (Acumedia, Lansing, MI, USA) was used as the bacterial growth medium.
Stock solutions of the compounds were prepared in dimethyl sulfoxide (DMSO—Sigma Aldrich,
St. Louis, MI, USA) and serial dilutions. The maximum concentration tested was 250 µg mL−1
.
Through the photometric reading (Densimat—bioMérrieux, Marcy-l’Étoile, France) the inoculum
−1
containing 1
×
108 CFU mL corresponding to 0.5 McFarland was obtained from the culture broth in
the log phase of growth and diluted to 1
10 µg mL−1 concentration were conducted simultaneously.
×
106 CFU mL−1. Tests with gentamicin (control antibiotic) at
Subsequently, a cell culture plate was used for MIC trials and solutions of serially diluted chemical
compounds (0.1 mL) were seeded into◦each sample of each bacterial strain (0.1 mL). These were
incubated in a bacteriological oven at 37 C for 24 h. The MIC of each compound tested was defined as
the lowest concentration that presented growth between 30 and 300 colonies.
After determining the MIC value of each compound, a synergy test was performed to verify
the potentiation of the action of the conventional antibiotics together with the products for S. aureus
strains (ATCC 25923 and LSA 88). For the antimicrobial activity pattern, the disc diffusion method was
used on Mueller Hinton agar, according to the guidelines of the National Committee of Clinical
Laboratory Standards [23]. The disks contained the following drugs: penicillin G (PEN, 10UI),
oxacillin (OXA, 1
ampicillin (AMP, 10
erythromycin (ERI, 15
(VAN, 30 µg) (Laborclin, Pinhais, Brazil).
µ
g), amoxicillin (AMO, 10
g), cefalexin (CFX, 30 g), sulfazotrim (SUT, 25
g), gentamicin (GEN, 10 g), tetracycline (VAN, 30
µ
g), amoxicillin/clavulanic acid (AMC, 20/10
µ
g),
g),
µ
µ
µg), clindamycin (CLI, 2
µ
µ
µ
µg), and vancomycin
The inhibition halos formed around each disc were measured with a digital caliper
(Mitutoyo/CD-600CSX-B) and values submitted to Tukey’s t-test [26] were 1 and 5%. All trials were
performed in triplicate.

Molecules 2018, 23, 774
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4. Conclusions
Eight amides derived from cinnamic acid were synthesized and characterized based on their
physical, analytical, and spectrometric data. All amides tested showed anti-T. gondii activity,
reducing the number of infected cells and intracellular parasites. Compound
the best results. Compounds , and showed weak activity against different strains of
bacteria at a concentration higher than 250 and demonstrated activity
g mL−1. Compounds
against two strains of S. aureus (ATCC 25923 and LSA 88) at a concentration of 250
g mL−1. The same
3 and 4 displayed
1,
2,
3,
4,
5,
6
7
µ
2
5
µ
compounds showed a synergic effect with different antibiotics. These results indicate that these classes
of analogs are potential pharmacological agents.
Supplementary Materials: MS and IV spectra, 1H-, 13C, HMBC, HSQC, and HMBC spectral data are available as
supplementary materials online.
Acknowledgments: The authors are very grateful for the financial support from the “Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior” (CAPES).
Author Contributions: G.R.S., K.A.C., and G.R.S.L. prepared the amides. G.R.S., K.A.C. and S.S.S. performed
the antimicrobial experiments. O.V.-d.-M. developed the antimicrobial study. L.P.C. performed the anti-T.gondii
experiments. E.J.T.dM. developed the anti-T. gondii study. L.M., C.R.R.M. and I.J.C.V. were responsible for the
analysis of these data and E.J.M. planned and coordinated the study.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of all the compounds are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).