Evaluating anti-Toxoplasma gondii activity of new serie of phenylsemicarbazone and phenylthiosemicarbazones in vitro

Article abstract of DOI:10.1007/s00044-012-0347-9

While Toxoplasma gondii is able to infect and replicate within all eukaryotic cells, tachyzoites are the infective form of T. gondii that invades all eukaryotic cells leading to tissue rupture, the main features of toxoplasmosis. The present study evaluates the activity of (benzaldehyde)-4- phenyl-3-thiosemicarbazone and (benzaldehyde)-(4 or 1)-phenylsemicarbazone against intracellular T. gondii. The nine new compounds were incubated in infected Vero cells at concentrations of 0.01, 0.1, 0.5, and 1.0 mM and evaluated for three main effects: cytotoxicity, infection, and number of intracellular parasites. The cytotoxicity test showed a pattern by analyzing the substituent arylhydrazone, where trihydroxy Compounds 4-9 were cytotoxic at concentrations of 0.5 and 1.0 mM. The results highlight Compound 8, which reduced the number of intracellular parasites by 82 % in a concentration of 0.01 mM and showed a LD50 of 0.3 mM in cell culture. These different biological actions are due to changes in the molecular structure and type of radical present in each compound. All compounds tested were more efficient than the control drug sulfadizine.

Full text of DOI:10.1007/s00044-012-0347-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3574–3580
DOI 10.1007/s00044-012-0347-9
ORIGINAL RESEARCH
Evaluating anti-Toxoplasma gondii activity of new serie
of phenylsemicarbazone and phenylthiosemicarbazones in vitro
•
Marco Antonio G. B. Gomes Laıs P. Carvalho Barbara S. Rocha
•
•
ˆ
Rodrigo R. Oliveira Edesio J. T. de Melo Edmilson J. Maria
´
•
•
´
Received: 21 June 2012 / Accepted: 7 November 2012 / Published online: 27 November 2012
Ó Springer Science+Business Media New York 2012
Abstract While Toxoplasma gondii is able to infect and
replicate within all eukaryotic cells, tachyzoites are the
infective form of T. gondii that invades all eukaryotic cells
leading to tissue rupture, the main features of toxoplasmosis.
The present study evaluates the activity of (benzaldehyde)-
4-phenyl-3-thiosemicarbazone and (benzaldehyde)-(4 or 1)-
phenylsemicarbazone against intracellular T. gondii. The
nine new compounds were incubated in infected Vero cells
at concentrations of 0.01, 0.1, 0.5, and 1.0 mM and eval-
uated for three main effects: cytotoxicity, infection, and
number of intracellular parasites. The cytotoxicity test
showed a pattern by analyzing the substituent arylhydraz-
one, where trihydroxy Compounds 4–9 were cytotoxic at
concentrations of 0.5 and 1.0 mM. The results highlight
Compound 8, which reduced the number of intracellular
parasites by 82 % in a concentration of 0.01 mM and
showed a LD50 of 0.3 mM in cell culture. These different
biological actions are due to changes in the molecular
structure and type of radical present in each compound. All
compounds tested were more efficient than the control drug
sulfadizine.
Keywords Anti-Toxoplasma gondii Á
Thiosemicarbazones Á Semicarbazones Á Schiff base Á
Anti-parasitic
Introduction
Toxoplasma gondii, the agent of Toxoplasmosis disease,
belongs to Apicomplexan Phylum which also includes
others pathogens of medical and veterinary importance,
such as the Plasmodium, Cryptosporidium, Sarcocystis,
Eimeria, Babesia, and Neospora species (Levine et al.,
1980). Toxoplasmosis affects millions of people worldwide
and is the main cause of death in patients with AIDS (Luft
and Remington, 1992). While chemotherapeutic treatment
involves the combination of sulfonamides with mainly
pyremithamine (or clindamycin) and folinic acid (Bosch-
Driessen et al., 2002), there is a inefficient elimination of
the intracellular parasites, up to 40 % of treated patients
develop side effects such as the suppression of hemato-
poiesis, teratogenic effect during pregnancy, and severe
skin reaction (James et al., 1996; Hill and Dubey, 2002;
Dannemann et al., 1991; Wong and Remington, 1993). As
consequence, the treatment must be changed or suspended.
(Bosch-Driessen et al., 2002). Toxoplasma infects all
nucleated cells of vertebrates, representing an attractive
model for the study of cellular biology of the host cell-
parasite interaction (Kim and Weiss, 2004). Therefore, it
can offer a useful model for the treatment of other diseases
such as Chagas disease and Leishmaniasis (Melo and
Beiral, 2003).
M. A. G. B. Gomes (&) Á B. S. Rocha Á
R. R. Oliveira Á E. J. Maria
´
ˆ
´
Laboratorio de Ciencias Quımica, Universidade Estadual do
Norte Fluminense, Av. Alberto Lamego,
2000, Horto, Campos dos Goytacazes,
RJ CEP 28015620, Brazil
e-mail: mabgomes@uenf.br
Thiosemicarbazones and semicarbazones were shown to
be useful as anti-parasitic, anti-bacterial, anti-microbial
´
agents (Melo and Souza, 2000; Tenorio et al., 2005;
L. P. Carvalho Á E. J. T. de Melo
´
Laboratorio de Biologia Celular, Universidade Estadual do Norte
Fluminense, Av. Alberto Lamego, 2000, Horto, Campos dos
Goytacazes, RJ CEP 28015620, Brazil
Carvalho and Melo, 2006). Considering this results, our
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Med Chem Res (2013) 22:3574–3580
3575
group developed new compounds belonging to the semi-
carbazone class to examine their anti-Toxoplasmic actions
and to facilitate the development of more efficient drugs
that do not cause severe side effects. The new analogues
were synthesized from thiosemicarbazone and semicarba-
zone substituted at the arylhydrazone moiety and phenyl
group at different positions as shown in Scheme 1.
and 160 ppm in 13C for the compounds with phenyl in
position 1 (3, 6, 9). In summary, all the synthesized com-
pounds exhibited spectral data consistent with the
structures.
Anti-Toxoplasma gondii action
The host cells were initially infected for 24 h with tach-
yzoites of T. gondii. Thereafter the majority of cells
showed proliferating parasites, thus confirming infection.
At this point, the cultures were treated with the compounds.
Quantitative analyses were performed to observe the
anti-T. gondii action of the drugs.
Results and discussion
Chemistry
All compounds synthesized (1–9) were obtained through
precipitation by cooling the reaction medium, showing
solid form with colors ranging from yellow to brown and
yielded an average of 65.41 %.
As expected, untreated cells had a large percentage of
infection, while treated cells showed decrease rates where
the parasites were eliminated (Table 1; Fig. 1). The cyto-
toxic effect including morphological alteration or cell death
and intracellular parasites elimination was considered.
The compounds 1, 2, and 3 (with 2,6-dimethoxy-4-
hydroxy substitution) showed reduced or no toxicity to host
cells until 0.1 mM.
The compounds 3, 6, and 9 have 1-phenylsemicarbazide
15 as a starting material showed the lowest yield. This is
because the amide group is less reactive than the 4-phe-
nylsemicarbazide 14 and 4-phenyl-3-thiosemicarbazide 13
amine portion. The compounds were initially elucidated by
infrared spectroscopy (IR) on a SHIMADZU, model IR
AFFINITY, with the following major shifts observed in the
IR spectra: 1602.91–1666.57 cm-1 due to C=N, which is
the link that represents the formation of the Schiff base,
altering the signals of the starting products, about 1,700 cm
-1 due to C=O of aldehyde. The elucidation was confirmed
However, only the compound 1 exhibited powerful
toxicity from 0.5 (50 %) to 1.0 mM whereupon it reached
100 % of cell lethality. Compounds 4, 5, and 6 (with tri-
hydroxy), with 2,3,4-trihydroxy substituted and 7, 8, and 9
(with 2,4,5-trihydroxy substitution) showed powerful tox-
icity after 0.1 mM treatment where compounds reached
100 %.
1
by nuclear magnetic resonance (NMR) H and 13C on a
There was no cytotoxic effect for any compound at
0.01 mM. Compound 6 exhibited the greatest reduction of
infection, (50 %), but still demonstrated severe toxic
effects at all other concentrations as well.
Varian model Mercury VX 300, where the absence of the
signal for the hydrogen on the carbonyl of aldehydes
starting in 9.50–10.00 ppm was readily apparent. The
confirmation was also made by the identification of Schiff
Although the infection parameters and the number of
parasites (Table 2) were similar. In case of both the
infection and number of intracellular parasites parameters,
the quantification was done considering an infected cell all
1
base formation signaling CH=N, with 8.13 ppm in H and
139 ppm in 13C approximately for the compounds with
1
phenyl at position 4 (1, 2, 4, 5, 7, 8) and 5.84 ppm in H
Scheme 1 Synthetic Scheme
S
S
N
H2
N
NH
NH
N
H
N
H
R1: 2,6-dimethoxy-4hidroxy (1)
R2: 2,3,4-tridroxy (4)
R
R
R3: 2,4,5-tridroxy (7)
O
O
O
+
N
H2
N
NH
NH
N
H
N
H
R1: 2,6-dimethoxy-4hidroxy (2)
R2: 2,3,4-tridroxy (5)
R3: 2,4,5-tridroxy (8)
R
R1: 2,6-dimethoxy-4hidroxy
R2: 2,3,4-tridroxy
R3: 2,4,5-tridroxy
O
NH
N
NH
H
N
H2
N
N
H
R1: 2,6-dimethoxy-4hidroxy (3)
R2: 2,3,4-tridroxy (6)
O
R
R3: 2,4,5-tridroxy (9)
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Med Chem Res (2013) 22:3574–3580
Table 1 Citotoxicity on Vero cells and LD50 values for cell culture and parasites of compounds (1–9)
Compound
Untreated
Control
Treated (mM)
0.01
LD50 of cell culture
LD50 of Parasites
0.1
0.5
1.0
1
583 49
661 47
551 26
492 39
586 36
527 34
624 29
541 15
601 31
420 13
545 37
740 56
538 32
648 50
627 40
791 56
708 37
742 41
600 30
410 13
697 93
755 61
497 33
748 59
669 41
0
234 28
0
0.7
[1
[1
0.4
0.2
2
787 53
837 73
0.5
3
626 43
635 61
0.6
4
0
0
0.03
0.2
5
0
0
0.3
6
0
0
0.07
0.3
0.02
0.08
0.006
0.3
7
695 42
473 23
597 36
515 10
0
0
8
0
0
0.3
9
0
0
0.4
Sulfadiazine
ND
500 15
[10
6
ND not determined
Fig. 1 Optical microscopy of
biological test. N nuclei, PV
parasitophorous vacuole
of them containing tachyzoites, and about the second
parameter, only the parasites morphologically featured as
‘‘half-moon’’ were considered. The different effects
between the compounds, considering the parameters above,
are due to the efficiency of anti-parasitic action of each
analogue as a result of it’s molecular conformation.
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Table 2 Effect of compounds (1–9) on cultures of Vero cells infected and intracelullar multiplication of T. gondii
Compound
Untreated
Control
Treated (mM)
Untreated
Control
Treated (mM)
0.01
0.01
0.1
0.5
1.0
0.1
0.5
1.0
0
1
2
3
4
5
6
7
8
9
75
77
80
65
79
78
78
72
77
3
5
3
4
4
4
3
1
3
75
74
71
46
76
39
59
57
61
4
4
4
2
3
2
2
2
2
35
69
78
37
49
0
4
3
3
3
2
28
45
72
0
1
2
3
0
2,759 136
2,692 98
2,762 127
2,107 152
2,869 163
2,754 141
2,654 106
3,028 234
2,633 114
1,772 86
2,402 165
2,225 163
1,310 69
2,460 114
1,559 115
1,966 101
528 34
1,029 69
2,193 167
2,190 87
825 65
1,770 68
0
776 76
28
37
0
2
4
1,278 77
766 65
1,945 105
241 28
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
42
28
37
2
1
3
0
0
1,216 101
263 18
1,206 77
0
0
0
0
1,761 74
´
Compound 3 induced the most drastic reduction in the
number of parasites at 1.0 mM, followed by the compound
8 at 0.1 mM.
reductase (Tenorio et al., 2005). This class of compound is
capable to bind heavy metal (Hutchinson, 1985), (Casas
et al., 2000), (Chen et al., 2004) which changes their
pharmacological activity (Hancock et al., 2011).
The nine new drugs proved more effective than the other
derivatives for displaying a higher reduction rate at lower
concentrations.
At lowest concentration, the derivative compounds of
methoxy group did not show considerable results, and at
1.0 mM this was the only group which did not show
cytotoxic effects. At 0.01 mM, the semicarbazones ana-
logues were more effective while at 0.1 mM, the thio-
semicarbazones showed better results.
Analyzing the position of the phenyl group, the radicals
can be placed in the arylhydrazone position and the results
of the isomers compared. No linearity was observed, as in
each concentration different compounds stand out, as
exemplified with compound 6 at 0.01 mM with the phenyl
group at position 1 and the compound 8 at 0.1 mM with
phenyl group at position 4.
Experimental section
Chemistry
The nine new compounds (1–9) were synthesized through
nucleophilic addition as described by (Aquino et al., 2008),
were reacted 0.0029 mol of the semicarbazide compounds
(13, 14, and 15) in a 10 mL solution EtOH/ H2O (2:1),
were added 0.0029 mol of benzaldehydes (10, 11, and 12)
and 0.05 mL of acetic acid. The mixture was stirred under
reflux for 1 h, then chilled in an ice bath. The precipitate
was filtered under vacuum and washed with chilled
ethanol.
Considering LD50, Compound 8 exhibited the best
results because the concentration needed to reach parasite
mortality is much lower than the lethal dose for the cell
(Table 1), suggesting it can be a candidate for in vivo
assays. Compound 8 performance is followed by Com-
pound 4 and Compound 7.
This study was performed out due to a need of new
drugs that could improve toxoplasmosis treatment because
current chemotherapy is extremely toxic. Anti-proliferative
drugs are able to arrest the cell cycle inhibiting the syn-
thesis of DNA, preventing their replications. The deriva-
tives of thiosemicarbazones were chosen because of their
wide range of pharmacological properties, including the
anti-proliferative, anti-tumor (Chen et al., 2004), anti-viral
(Kesel, 2011), anti-fungal (Qin et al., 2012), anti-bacterial
(Turan-Zitouni et al., 2002), and anti-parasitic effects,
such as anti-trypanosomal (Rodrigues et al., 2010) and
anti-malarial (Oliveira et al., 2008). Their activities are
related mainly with the inhibition of ribonucleotide
Compound (1): 2.6-dimethoxy-4-hidroxybenzaldehyde-4-
phenyl-3-thiosemicarbazone
Solid with yellow color, and 76.61 % of yield; MP:
127.5 °C; IR (t cm-1 KBr): 1602.91 (C=N); 1H NMR
(DMSO-d6, 300 MHz/ppm): d 11.59 (s, 1H, NH), 10.04
(s, 1H, NH–Ar), 9.62 (s, 1H, OH), 8.40 (s, 1H, CH=N),
7.74-7.72 (d, 2H, J = 6.0 Hz, Ar–H), 7.39-7.33 (t, 2H,
J = 9.0, 6.0 Hz, Ar H), 7.18-7.13 (t, 1H, J = 9.0, 6.0 Hz,
Ar–H), 6.15 (s, 1H, Ar–H), 3.79 (s, 6H, OCH3), 13C NMR
(DMSO-d6, 75.4 MHz/ppm):d 174.04 (C=S), 161.07
(Car-OH), 160.36 (Car-OCH3), 138.78 (CH=N), 138.72
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(Car–NH), 128.31 (Car), 124.37 (Car), 122.57 (Car) 102.01
(Car), 92.32 (Car), 55.79 (CH3).
(DMSO-d6, 300 MHz/ppm): d 10.30 (s, 1H, OH), 9.60 (s,
1H, NH), 9.31 (s, 1H, OH), 8.76 (s, 1H, OH), 8.33 (s, 1H,
NH–Ar), 8.13 (s, 1H, CH=N), 7.60-7.57 (d, 2H,
J = 9.0 Hz, Ar–H), 7.30-7.25 (t, 2H, J = 6.0, 9.0 Hz,
Ar–H), 7.08-7.05 (d, 1H, Ar–H), 7.01-6.96 (t, 1H,
J = 6.0, 9.0 Hz, Ar–H), 6.39-6.36 (d, 1H, J = 9.0 Hz,
Ar–H), 13C NMR (DMSO-d6, 75.4 MHz/ppm): d 152.69
(C=O), 147.75 (C-OH), 146.16 (Car-OH), 141.64 (Car-
OH), 139.24 (CH=N), 132.68 (Car-NH), 128.38 (Car),
122.07 (Car), 119.24 (Car), 118.54 (Car), 112.41 (Car),
107.52 (Car).
Compound (2): 2.6-dimethoxy-4-hidroxybenzaldehyde-4-
phenylsemicarbazone
Solid with yellow color, and 69.72 % of yield; MP:
186.4 °C; IR (t cm-1 KBr): 1655.00 (C=N); 1H NMR
(DMSO-d6, 300 MHz/ppm): d 10.36 (s, 1H, OH), 9.90 (s,
1H, NH), 8.49 (s, 1H, NH–Ar), 8.13 (s, 1H, CH=N), 7.53-
7.50 (d, 2H, J = 9.0 Hz, Ar–H), 7.32-7.27 (t, 2H,
J = 9.0, 6.0 Hz, Ar–H), 7.02-6.97 (t, 1H, J = 6.0,
9.0 Hz, Ar–H), 6.15 (s, 1H, Ar–H), 3.80 (s, 6H, OCH3),
13C NMR (DMSO-d6, 75.4 MHz/ppm): d 160.37 (Car-
OCH3), 159.87 (Car-OH), 152.94 (C=O), 138.98 (CH=N),
136.26 (Car-NH), 128.90 (Car), 122.17 (Car), 118.31
(Car), 102.40 (Car), 92.23 (Car), 58.81 (CH3).
Compound (6): 2.3.4-trihidroxybenzaldehyde-1-
phenylsemicarbazone
Solid with yellow color, and 33.76 % of yield; MP:
137.2 °C; IR (t cm-1 KBr): 1660.78 (C=N); 1H NMR
(DMSO-d6, 300 MHz/ppm): d 7.66 (s, 1H, NH), 7.49 (s,
1H, NH-Ar), 7.18-7.12 (t, 2H, J = 9.0, 6.0 Hz, Ar–H),
7.11-7.08 (d, 1H, J = 9.0 Hz, Ar–H), 6.73.11-6.71 (d,
1H, J = 6.0 Hz, Ar–H), 6.50-6.47 (d, 1H, J = 6.0 Hz,
Ar–H), 5.85 (s, 1H, CH=N), 13C NMR (DMSO-d6,
75.4 MHz/ppm): d 193.14 (C=O), 160.01 (CH=N), 153.25
(Car-OH), 150.89 (Car-OH), 149.44 (Car-OH), 132.16
(Car-NH), 128.57 (Car), 123.84 (Car), 118.52(Car),
115.41 (Car), 112.10 (Car), 108.35 (Car).
Compound (3): 2.6-dimethoxy-4-hidroxybenzaldehyde-1-
phenylsemicarbazone
Solid with yellow color, and 52.63 % of yield; MP:
152.9 °C; IR (t cm-1 KBr): 1664.64 (C=N); 1H NMR
(DMSO-d6, 300 MHz/ppm): d 7.65 (s, 1H, NH), 7.49 (s,
1H, NH–Ar), 7.17-7.12 (t, 2H, J = 9.0, 6.0 Hz, Ar–H),
6.72-6.70 (d, 2H, J = 6.0 Hz, Ar–H), 6.09 (s, 2H, Ar–H),
5.84 (s, 1H, CH=N), 3.76 (CH3), 13C NMR (DMSO-d6,
75.4 MHz/ppm): d 185.12 (C=O), 164.88 (Car-CH3),
163.58 (Car-OCH3), 159.98 (CH=N), 149.44 (Car-NH),
128.56 (Car), 118.49 (Car), 112.09 (Car), 106.94 (Car),
91.82 (Car), 55.64 (CH3).
Compound (7): 2.4.5-trihidroxybenzaldehyde-4-phenyl-3-
thiosemicarbazone
Solid with yellow color, and 63.38 % of yield; MP:
127.7 °C; IR (t cm-1 KBr): 1633.76 (C=N); 1H NMR
(DMSO-d6, 300 MHz/ppm): d 11.47 (s, 1H, NH), 9.87 (s,
1H, NH-Ar), 9.81 (s, 1H, OH), 8.34 (s, 1H, CH=N), 7.62-
7.59 (d, 2H, J = 9.0 Hz, Ar–H), 7.37-7.32 (t, 2H,
J = 9.0, 6.0 Hz, Ar–H), 7.29 (s, 1H, Ar–H), 7.19-7.14 (t,
1H, J=6.0, 9.0 Hz, Ar–H), 6.98 (s, 1H, Ar–H), 13C NMR
(DMSO-d6, 75.4 MHz/ppm): d 174.84 (C=S), 151.49
(C-OH), 149.80 (C-OH), 142.09 (C-OH), 139.16
(CH=N), 138.64 (Car-NH), 129.73 (Car), 128.40 (Car),
125.34 (Car), 113.32 (Car), 111.05 (Car), 103.75 (Car).
Compound (4): 2.3.4-trihidroxybenzaldehyde-4-phenyl-3-
thiosemicarbazone
Solid with yellow color, and 67.36 % of yield; MP:
206.2 °C; IR (t cm-1 KBr): 1631.85 (C=N); 1H NMR
(DMSO-d6, 300 MHz/ppm): d 10.50 (s, 1H, NH), 9.86 (s,
1H, NH-Ar), 9.81 (s, 1H, OH), 8.34 (s, 1H, CH=N), 7.59-
7.57 (d, 2H, J = 6.0 Hz, Ar–H), 7.35-7.30 (t, 2H,
J = 6.0, 9.0 Hz, Ar–H), 7.17-7.13 (t, 1H, J = 6.0,
9.0 Hz, Ar–H), 7.09-7.06 (d, 1H, J = 9.0 Hz, Ar–H),
6.93-6.90 (d, 1H, J = 9.0 Hz, Ar–H), 13C NMR
(DMSO-d6, 75.4 MHz/ppm): d 175.24 (C=S), 150.07
(C-OH), 148.39 (C-OH), 146.63 (C–OH), 139.16
(CH=N), 132.70 (Car–NH), 129.24 (Car), 127.90 (Car),
124.97 (Car), 111.12 (Car), 110.66 (Car), 107.72 (Car).
Compound (8): 2.4.5-trihidroxybenzaldehyde-4-
phenylsemicarbazone
Solid with brown color, and 81.66 % of yield; MP:
162.0 °C; IR (t cm-1 KBr): 1666.57 (C=N); 1H NMR
(DMSO-d6, 300 MHz/ppm): d 10.24 (s, 1H, OH), 9.55 (s,
1H, NH), 9.35 (s, 1H, OH), 9.29 (s, 1H, OH), 8.67 (s, 1H,
NH–Ar), 8.09 (s, 1H, CH=N), 7.90 (s, 1H, Ar–H), 7.29-
7.23 (t, 2H, J = 9.0, 9.0 Hz, Ar–H), 7.14 (s, 1H, Ar–H),
7.00-6.95 (t, 1H, J = 9.0, 6.0 Hz, Ar–H), 6.34-6.32 (d,
1H, J = 6.0 Hz, Ar–H), 13C NMR (DMSO-d6,
Compound (5): 2.3.4-trihidroxybenzaldehyde-4-
phenylsemicarbazone
Solid with yellow color, and 60.51 % of yield; MP:
185.2 °C; IR (t cm-1 KBr): 1639.56 (C=N); 1H NMR
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3579
75.4 MHz/ppm): d 153.60 (C=O), 151.02 (C-OH), 149.16
(Car-OH), 140.94 (Car-OH), 139.24 (CH=N), 129.28
(Car-NH), 129.18 (Car), 122.87 (Car), 120.02 (Car),
119.20 (Car), 113.73 (Car), 111.57 (Car).
and characterized based on their physical, analytical, and
spectral data.
The new synthesized analogues had their anti-T. gondii
activity analyzed in vitro. All compounds showed activity
in all concentrations tested, reducing the percentage of
infected cell and number of intracellular parasites, mainly
at 0.01 and 1.0 mM, respectively. These news compounds
were more effective than the reference drugs Sulfadiazine.
Changing on molecular conformations and modifica-
tions on the ring at arylhydrazone moiety altered the
activity of the drugs. These results indicate that the new
class of drugs representing an interesting model for
development of new medicines against toxoplasmosis and
others parasitic diseases.
Compound (9): 2.4.5-trihidroxybenzaldehyde-1-
phenylsemicarbazone
Solid with brown color, and 54.91 % of yield; MP:
137.5 °C; IR (t cm-1 KBr): 1620.27 (C=N); 1H NMR
(DMSO-d6, 300 MHz/ppm): d 7.68 (s, 1H, NH), 7.50 (s,
1H, NH-Ar), 7.18-7.12 (t, 2H, J = 9.0, 6.0 Hz, Ar–H),
6.98 (s, 1H, Ar–H), 6.73-6.71 (d, 1H, J = 6.0 Hz, Ar–H),
6.36 (s, 1H, Ar–H), 5.87 (s, 1H, CH=N), 13C NMR
(DMSO-d6, 75.4 MHz/ppm): d 190.36 (C=O), 160.10
(CH=N), 156.57 (Car-OH), 154.60 (Car-OH), 149.44
(Car-OH), 139.06 (Car-NH), 128.60 (Car), 118.58 (Car),
114.49 (Car), 112.14(Car), 103.00 (Car).
Acknowledgements The authors are grateful for the support
´
received from the Cordenac¸a˜o de Aperfeic¸oamento de Pessoal Nıvel
Superior (Capes), the Fundac¸a˜o de Amparo aa Pesquisa do Estado do
``
Rio de Janeiro Carlos Chagas Filho (Faperj) and the english reviewer
John Marr Ditty.
Biological assays
Vero cells (kidney fibroblasts of the African monkey) were
plated with DMEM 1152 Sigma supplemented with 5 % of
serum fetal bovine on 24-well tissue plates containing a sterile
round coverslip and maintained at 37 °C , 24 h. Next, the cells
were infected with tachyzoites of T. gondii, RH strain (5:1) at
37 °C, for other 24 h. The infected culture was incubated with
the compounds at concentrations 0.01, 0.1, 0.5, and 1 mM
during 24 h and then, processed for light microscopy.
The concentrations were selected within range of
0.01–1.0 mM, in accord with the preliminary experiments
and previous studies carried out by (Carvalho et al., 2010).
All compounds were dissolved in DMSO in final con-
centration of 1.5 % v/v, that was not toxic for both host cell
and parasites.
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