Synthesis and in vitro anti-Toxoplasma gondii activity of a new series of aryloxyacetophenone thiosemicarbazones

Article abstract of DOI:10.1007/s11030-019-09986-9

Abstract: A new series of aryloxyacetophenone thiosemicarbazones 4a–q have been synthesized as anti-Toxoplasma gondii agents. All compounds showed significant inhibitory activity against T. gondii-infected cells (IC₅₀ values 1.09–25.19?μg/mL). The 4-fluorophenoxy derivative (4l) was the most potent compound with the highest selectivity toward host cells (SI = 19), being better than standard drug pyrimethamine. SAR study indicated that the concurrence of proper substituents on both aryl ring of phenoxyacetophenone is important for potency and safety profile. Further in vitro experiments with the representative compounds 4l and 4p revealed that these compounds at the concentration of 5?μg/mL can significantly reduce the viability of T. gondii tachyzoites, as well as their infectivity rate and intracellular proliferation, comparable to those of pyrimethamine. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s11030-019-09986-9

Molecular Diversity
https://doi.org/10.1007/s11030-019-09986-9
ORIGINAL ARTICLE
Synthesis and in vitro anti‑Toxoplasma gondii activity of a new series
of aryloxyacetophenone thiosemicarbazones
Mahsa Ansari1 · Mahbobeh Montazeri2,3,4 · Ahmad Daryani2,4 · Kaveh Farshadfar5 · Saeed Emami6
Received: 27 May 2019 / Accepted: 7 August 2019
© Springer Nature Switzerland AG 2019
Abstract
A new series of aryloxyacetophenone thiosemicarbazones 4a–q have been synthesized as anti-Toxoplasma gondii agents.
All compounds showed signifcant inhibitory activity against T. gondii-infected cells (IC50 values 1.09–25.19 μg/mL). The
4-fuorophenoxy derivative (4l) was the most potent compound with the highest selectivity toward host cells (SI=19), being
better than standard drug pyrimethamine. SAR study indicated that the concurrence of proper substituents on both aryl ring
of phenoxyacetophenone is important for potency and safety profle. Further in vitro experiments with the representative
compounds 4l and 4p revealed that these compounds at the concentration of 5 μg/mL can signifcantly reduce the viability of
T. gondii tachyzoites, as well as their infectivity rate and intracellular proliferation, comparable to those of pyrimethamine.
Graphic abstract
Keywords Acetophenone derivatives · Anti-apicomplexan · Thiosemicarbazones · Toxoplasmosis · Toxoplasma gondii
Introduction
Toxoplasma gondii (T. gondii), the ubiquitous parasitic pro-
tozoan belonging to phylum Apicomplexa, is the etiologic
Electronic supplementary material The online version of this
agent of toxoplasmosis. This successful obligate intracel-
article (https://doi.org/10.1007/s11030-019-09986-9) contains
lular parasite can infect all warm-blooded vertebrates such
as humans, livestock, birds and marine mammals [1]. It is
estimated that up to one-third of the world’s population is
infected with T. gondii specifcally in developing countries
supplementary material, which is available to authorized users.
* Saeed Emami
sdemami12@gmail.com; semami@mazums.ac.ir
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

Molecular Diversity
[2]. Although the infection is asymptomatic in most immu-
nocompetent people with normal immunity, signifcant mor-
bidity and mortality may especially occur in congenitally
infected and immunocompromised individuals such as HIV-
positive, cancer or organ transplant human patients [3].
The recommended drugs for treatment of toxoplasmosis
are the combination of pyrimethamine (an inhibitor of dihy-
drofolate reductase) and sulfonamides such as sulfameth-
oxazole or sulfadiazine which inhibit dihydropteroate syn-
thetase in the folate synthesis pathway. In addition, other
drugs such as clarithromycin, azithromycin and atovaquone
can be used for treatment of toxoplasmosis [4, 5]. However,
current chemotherapy for toxoplasmosis is often associated
with serious side efects including hypersensitivity, hema-
tological toxicity, teratogenicity and allergic reactions. Most
of chemotherapeutic agents are only efective in the acute
phase of the disease and cannot eliminate intracellular para-
sites. Therefore, these limitations clearly indicate the need
for newer, safer and more efcacious therapy for toxoplas-
mosis [6].
Thiosemicarbazones were shown to be as potential
chemical leading groups for producing new therapeutic
agents as antitumor, antimicrobial and anti-parasitic agents
[10–13]. Biological activities of thiosemicarbazones
and also avoiding of drug resistance are related to their
abilities to form complex with metal cations, by bonding
through the sulfur and azomethine nitrogen atoms [13, 14].
Previously, Tenório et al. have reported the synthesis
and anti-T. gondii activity of thiosemicarbazones, sub-
stituted at arylhydrazone moiety with nitro group (struc-
ture C, Fig. 1). The results of their studies showed that
thiosemicarbazones were efective against intracellular T.
gondii, while the treated host cells with the thiosemicar-
bazones did not show any morphological alterations after
24 h of drugs treatment [11]. Inspired by these fndings,
our group has made eforts to develop new derivatives of
thiosemicarbazone class to broaden these initial conse-
quences with further details on anti-T. gondii activity to
achieve more efcient drugs. In our previous study, we
have synthesized a series of phenoxyacetophenones (D) as
antimicrobial agents derived from dihydrochalcones [15].
In the current work, the thiosemicarbazone functionality
was introduced in the phenoxyacetophenone framework
(Fig. 1). Thus, we report here, the synthesis and anti-T.
gondii activity of a new series of aryloxyacetophenone
thiosemicarbazones.
Several reports have previously described the efcacy
of hydroxyurea (A) and thiosemicarbazones (B) in vitro, in
eliminating the intracellular parasites of T. gondii, Trypa-
nosoma cruzi and Leishmania amazonensis [7–9] (Fig. 1).
Hydroxyurea (A) and thiosemicarbazones (B) act by arrest-
ing the cell cycle in the G1/S phase and inactivating the
enzyme ribonucleotide reductase, then the DNA synthesis.
However, thiosemicarbazones are known inhibitors of this
enzyme, being approximately 1000 times more potent than
hydroxyurea [7–10].
Fig. 1 Design of target compounds bearing thiosemicarbazone functionality
1 3

Molecular Diversity
calculations with M06-D3, M06-2X, B3LYP-D3 and B97D
methods demonstrated that among two possible geometric
isomers, the (Z)-isomer is 0.4, 0.6, 0.1 and 0.7 kcal/mol,
respectively, more stable than the corresponding (E)-isomer
in term of Gibbs free energy (Fig. 2).
Results and discussion
Chemistry
The target compounds 4a–q were synthesized through the
synthetic route outlined in Scheme 1. Initially, the commer-
cially available acetophenone derivatives 1a–c were bro-
minated by CuBr2 in CHCl3/EtOAc to give corresponding
2-bromoacetophenone derivatives 2a–c. The intermediate
compounds phenoxyacetophenones 3a–k were obtained by
treatment of 2a–c with diferent phenolic derivatives in the
presence of potassium carbonate in DMF at room tempera-
ture. Finally, the reaction of phenoxyacetophenones 3a–k
with thiosemicarbazide or 4-phenylthiosemicarbazide in the
presence of a few drops of HCl in ethanol gave thiosemicar-
bazone derivatives 4a–q in good yields. The structures of
newly synthesized compounds were confrmed by spectro-
scopic data (IR, 1H NMR and MS) and elemental analysis
as interpreted in the Experimental part.
The optimized structure of (Z)-4a displays the intramo-
lecular hydrogen bond between the NH and the oxygen of
phenoxy with distance of 2.09 Å, which stabilizes somewhat
the (Z)-isomer. It is clear that in all cases the nitrogen lone
pair on both sides of C=S moiety can donate electrons to
the π* orbital of C=S, causing N–C bonds to get stronger,
in contrast, carbon–sulfur bond of thiocarbonyl moiety tends
to single bond.
The main conformers of this compound will be obtained
from rotation around C–C bond (see Fig. 3). Torsion angle
of O–C^C–N was chosen to explore its changing efect on
the energy of the molecule and the stability of the compound
in the solution phase. For this purpose, this torsion angle
was scanned in the range of 360° by 5° steps. As depicted in
Fig. 3, the local minimum energy points in the optimization
results indicated that each of the (Z)- and (E)-isomers have
two stable conformers; the (Z)-isomer conformers are at 56°
and 301° torsion angles and the (E)-isomer at 125° and 235°
torsion angles.
Considering the possibility for formation of E/Z-isomers
in the reaction of phenoxyacetophenones 3a–k with thio-
semicarbazides, it should be noted that the exact structure of
thiosemicarbazone derivatives 4a–q is unknown. Although
the formation of E or Z-isomer is controlled kinetically dur-
ing the preparation of compounds, isomerization around the
C═N double bond is also possible via rotation and inversion
mechanisms in the absence or presence of catalysts such as
acids or bases [16, 17].
Besides confgurational and conformational aspects of
compounds, we explored the drug-like properties of all
compounds. Molecular properties were calculated using
Molinspiration web tool (www.molinspiration.com) and
listed in Table S1. As seen, all compounds have acceptable
properties, suggesting no problem in their bioavailability.
Furthermore, the metabolism and toxicity properties of
representative compounds (4l and 4p) were predicted by a
freely accessible web server namely pkCSM (http://struc
ture.bioc.cam.ac.uk/pkcsm) [18]. The obtained results were
presented in Table S2–S5 (Supplementary material).
Computational studies
In the lack of spectroscopic data for distinguishing the E/Z-
isomers, a computational study using density functional the-
ory has been applied for the parent compound 4a. Our DFT
Scheme 1 Synthesis of com-
pounds 4a–q. Reagents and
conditions: a) CuBr2, CHCl3/
EtOAc; b) appropriate phenolic
derivatives, K2CO3, DMF, rt;
c) thiosemicarbazide or phenyl
thiosemicarbazide, HCl, EtOH,
rt
1 3

Molecular Diversity
Fig. 2 Fully optimized struc-
tures of 4a: (Z)-4a (a); (E)-4a
(b)
(a)
(b)
active than 4a. The comparison of unsubstituted acetophenone
derivative 4a with corresponding 4-chloro analog 4n (R1=Cl)
revealed that the introduction of 4-chloro group diminished the
activity against T. gondii-infected cells. Inversely, the same
modifcation in 4-fuorophenoxyacetophenone 4h resulted in
more potent compound 4p. Therefore, it can be concluded that
the efect of substituent on the acetophenone part depends on
the substitution type of phenoxy moiety.
Biological activity
In vitro anti‑toxoplasma activity
The synthesized compounds 4a–q were subjected to assess the
inhibition of T. gondii in vitro. For this purpose, the host cells
(Vero cells) were infected with intracellular parasites (tachy-
zoites) of RH strain, and the efect of test compounds on T.
gondii-infected cells was evaluated. Furthermore, the cytotoxic
efect of compounds was evaluated by MTT assay against unin-
fected Vero cells and selectivity index (SI) was determined for
each compound. The obtained IC50 values were listed in Table 1.
The IC50 values of compounds against T. gondii-infected cells
were in the range of 1.09–25.19 μg/mL. The CC50 values of
all compounds with exception of 4n and 4o against uninfected
host cells were signifcantly higher than their IC50 values. Com-
pounds 4c, 4l and 4p with IC50 values of≤1.38 μg/mL showed
the highest inhibitory activity against infected cells, displaying
also good selectivity index (SI≥10). Notably, compound 4c
was 3-chlorophenoxy derivative and compounds 4l and 4p were
derived from 4-fuorophenoxyacetophenone. Indeed, compound
4l (R1=MeO, R2=4-F and R3=H) showed the highest potency
toward T. gondii-infected cells with more favorable safety for
host cells (IC50 =1.09 μg/mL, SI=19).
Other modifcation in the target compounds was insertion
of phenyl ring on the thiosemicarbazone moiety. While the
N-phenyl-thiosemicarbazones 4g, 4i, 4k, 4m and 4q were
less active than their corresponding simple thiosemicarba-
zones, the N-phenyl analog 4o was found to be more active
than its unsubstituted thiosemicarbazone 4n.
Efect of compounds 4l and 4p on tachyzoites
The most active compounds (4l and 4p) against T. gondii-
infected cells were selected for further evaluation against
tachyzoites. Thus, T. gondii tachyzoites were treated with
compounds 4l and 4p at the concentration of 5 μg/mL for
24 h. Pyrimethamine (at the concentration of 50 μg/mL)
and PBS were used as positive and negative control groups.
As seen in Fig. 4, while the mortality rate of tachyzoites
in PBS group was 4.7%, the 4l- and 4p-treated tachyzoites
showed 75.3 and 72.3% mortality, respectively. Therefore,
the representative compounds 4l and 4p can signifcantly
reduce the viability of T. gondii tachyzoites at the concentra-
tion of 5 μg/mL. It should be noted that the standard drug
pyrimethamine could induced 80.5% mortality at the higher
concentration of 50 μg/mL.
Structurally, the fnal compounds 4a–q were thiosemicar-
bazone of acetophenone, 4-methoxyacetophenone or 4-chlo-
roacetophenone. In the acetophenone analogs 4a–i, the intro-
duction of 3-F or 3-Cl on the phenoxy moiety increased the
activity (compare 4b and 4c with 4a). In contrast, the presence
of 2-MeO, 3-MeO or 4-F on the phenoxy moiety had unfavora-
ble efect on activity; thus, compounds 4d, 4f and 4h were less
1 3

Molecular Diversity
(a)
(b)
Fig. 3 Scan of potential energy surface (rotating the O–C^C–N dihedral angle in steps of 5°) at the B3LYP/6-31G(d) level a (Z)-4a; b (E)-4a
1 3

Molecular Diversity
Table 1 Inhibitory activity
of compounds 4a–q against
Toxoplasma gondii-infected
Vero cells (IC50s) in comparison
with their cytotoxicity against
Vero cells (CC50s)
Compounds
R1
R2
R3
IC50 (μg/mL)
CC50 (μg/mL)
SI
1.3
4a
H
H
H
H
H
H
H
H
H
3-F
3-Cl
2-MeO
3,4,5-(MeO)3
3-MeO
H
4-F
4-F
H
H
H
H
H
H
H
Ph
H
Ph
H
Ph
H
Ph
H
Ph
H
Ph
4.02
2.85
1.38
12.66
15.19
5.51
5.92
13.09
25.19
3.93
7.07
1.09
4.22
22.13
7.29
1.19
8.27
2.63
5.12
14.11
13.83
41.57
36.72
22.77
7.27
51.25
34.13
11.58
15.47
20.73
13.99
19.41
6.38
4b
4.9
10.0
3.3
2.4
4.1
1.2
3.9
1.4
2.9
2.2
19.0
3.3
0.9
0.9
10.9
4.3
3.0
4c
4d
4e
4f
4g
4h
4i
H
4j
MeO
MeO
MeO
MeO
Cl
Cl
Cl
4k
H
4l
4-F
4-F
H
H
4-F
4-F
4m
4n
4o
4p
13.00
35.41
7.99
4q
Cl
Pyrimethamine
IC50: half maximal inhibitory concentration, CC50: half cytotoxic concentration; SI: selectivity index.
SI=CC50/IC50
100
4p or pyrimethamine (5 μg/mL). After fxing and staining,
****
the cells were counted under a light microscope in order to
80
60
40
20
0
****
****
determine the infection index (number of infected cell per
100 examined cells) and T. gondii intracellular proliferation
(total number of tachyzoites per 100 examined cells). Each
experiment was repeated three times; the values were deter-
mined as mean SEM. The obtained data were presented
in Fig. 5 and compared with control group. As observed
)
in Fig. 5, the infection index in the negative control group
PBS
g//mL
g//mL)
g//mL)
µ
µ
µ
(the cells were infected, but not treated) was 71%. Treat-
ment with compounds 4l and 4p at the concentration of 5 μg/
mL resulted in the decrease in infection index to 42.3 and
47.3%, respectively. The infection index for pyrimethamine-
treated cells at the same concentration was 50.7%. On the
other hand, after treatment with 4l, 4p and pyrimethamine,
the total number of tachyzoites per 100 examined cells was
352.7, 408 and 325.3, being signifcantly less than that of
control (663.3). Based on these data, it can be concluded that
compounds 4l and 4p can signifcantly reduce the infectivity
rate and intracellular proliferation of T. gondii, comparable
to those of pyrimethamine.
4l (5
4p (5
Pyrimethamine (50
Fig. 4 The efect of compounds after 24 h on the mortality of T. gon-
dii tachyzoites
Efect of compounds 4l and 4p on the invasion and intra‑
cellular proliferation of T. gondii
The host Vero cells were infected with tachyzoites at a ratio
of 5:1 for 3 h and then treated with the compounds 4l and
1 3

Molecular Diversity
800
80
60
40
20
0
600
400
200
0
*
**
*
**
***
**
p
ne
i
l
4l
4
l
m
4
4p
a
mine
Control
Contro
imeth
metha
yr
P
Pyri
Fig. 5 Infection index (% of infected cells) and parasite proliferation
(total number of tachyzoites per 100 cells) in Vero cells treated with
compounds 4l, 4p and pyrimethamine in concentration of 5 µg/mL,
staining by blue toluidine. Controls: the cells were infected, but not
treated. Data were shown as mean SEM in triplicates. Signifcant
diference compared to control (*P<0.05)
procedure previously reported by us [15]. The atom num-
bering of compounds 4a–q for NMR interpretations was
illustrated in Scheme 1.
Conclusion
We have synthesized a new series of aryloxyacetophenone
thiosemicarbazones with anti-T. gondii activity. The in vitro
assay of compounds against T. gondii-infected cells revealed
that all thiosemicarbazones had signifcant inhibitory activ-
ity, displaying IC50 values between 1.09 and 25.19 μg/mL.
The most active compound namely 2-(2-(4-fuorophenoxy)-
1-(4-methoxyphenyl)ethylidene)hydrazine-1-carbothioamide
(4l) showed also the highest selectivity (SI=19) toward host
cells (Vero cells), being better than standard drug pyrimeth-
amine. SAR study showed that the concurrence of substituents
on both aryl ring of phenoxyacetophenone is important for
potency and safety profles of promising compounds 4l and 4p.
The representative compounds 4l and 4p were further tested
for actions on the tachyzoite viability, invasion and intracel-
lular proliferation. The obtained results indicated that these
compounds at the concentration of 5 μg/mL can signifcantly
reduce the viability of T. gondii tachyzoites, as well as their
infectivity rate and intracellular proliferation, comparable to
those of pyrimethamine. These fndings suggest that these
compounds can be considered as new leads for development
of anti-T. gondii agents.
General method for the preparation of fnal com-
pounds 4a–q
To a solution of phenoxyacetophenone derivative 3 (1 mmol)
in ethanol (4 mL), a few drops of concentrated HCl were
added and stirred at room temperature. Then, thiosemicar-
bazide or 4-phenylthiosemicarbazide (1 mmol) was added
portion-wise and stirring was continued. After consumption
of starting materials (checked by TLC), the reaction mixture
was left in refrigerator overnight. Subsequently, the precipi-
tated solid was separated by fltration and washed with cool
ethanol and dried to give pure compounds 4a–q.
2‑(2‑Phenoxy‑1‑phenylethylidene)hydrazine‑1‑carbothio‑
amide (4a)
White solid; yield: 88%; m.p: 121–123 °C; IR (KBr, cm−1):
3412, 3248, 1599, 1472, 1290, 1212, 1119, 1075, 881,
1
751, 685. H NMR (400 MHz, DMSO-d6): δ 5.33 (s, 2H,
CH2O), 6.96–7.03 (m, 3H, H-2′, H-4′ and H-6′), 7.32 (t, 2H,
J = 7.60 Hz, H-3′ and H-5′), 7.37–7.43 (m, 3H, H-3, H-4
and H-5), 7.89–7.96 (m, 2H, H-2 and H-6), 8.09 (br s, 1H,
NH2), 8.48 (br s, 1H, NH2), 10.76 (br s, 1H, NH). 13C NMR
(100 MHz, DMSO-d6): 179.58, 158.05, 144.66, 136.14,
129.96, 129.76, 128.74, 127.51, 121.82, 115.28, 61.65. MS
(m/z, %) 285 (M+, 5), 268 (7), 195 (10), 180 (32), 178 (100),
133 (69), 105 (36), 103 (100), 94 (38), 91 (29), 77 (97), 66
(22), 60 (25). Anal. Calcd for C15H15N3OS: C, 63.13; H,
5.30; N, 14.73. Found: C, 63.09; H, 5.39; N, 14.70.
Experimental
Chemistry
All solvents and chemical reagents were purchased from
Merck Co. and used without further purifcation. 2-Bro-
moacetophenone derivatives (2a–c) were prepared by using
literature method [19]. The synthesis of the phenoxyace-
tophenones (3a–k) has been carried out according to the
1 3

Molecular Diversity
2‑(2‑(3‑Fluorophenoxy)‑1‑phenylethylidene)hydra‑
zine‑1‑carbothioamide (4b)
3H, OCH3), 3.71 (s, 6H, 2 × OCH3), 5.32 (s, 2H, CH2O),
6.28 (s, 2H, H-2′ and H-6′), 7.37–7.43 (m, 3H, H-3, H-4
and H-5), 7.93–7.99 (m, 2H, H-2 and H-6), 8.12 (br s, 1H,
NH2), 8.51 (br s, 1H, NH2), 10.74 (br s, 1H, NH). MS (m/z,
%) 375 (M+, 2), 358 (7), 284 (6), 233 (6), 193 (9), 184 (80),
178 (100), 159 (6), 141 (41), 124 (53), 103 (83), 77 (54), 51
(16). Anal. Calcd for C18H21N3O4S: C, 57.58; H, 5.64; N,
11.19. Found: C, 57.44; H, 5.60; N, 11.30.
White solid; yield: 84%; m.p: 158–160 °C; IR (KBr, cm−1):
3357, 3262, 3179, 1643, 1604, 1538, 1486, 1384, 1281,
1
1144, 802, 553. H NMR (400 MHz, DMSO-d6): δ 5.33
(s, 2H, CH2O), 6.79–6.86 (m, 2H, H-5′ and H-6′), 6.88 (dt,
1H, J = 11.2 and 2.4 Hz, H-2′), 7.31–7.37 (m, 1H, H-4′),
7.38–7.41 (m, 3H, H-3, H-4 and H-5), 7.90–7.96 (m, 2H,
H-2 and H-6), 8.10 (br s, 1H, NH2), 8.49 (br s, 1H, NH2),
10.85 (br s, 1H, NH). MS (m/z, %) 303 (M+,<1), 133 (17),
112 (100), 103 (27), 92 (15), 83 (37), 76 (18), 73 (7), 64
(15), 51 (6). Anal. Calcd for C15H14FN3OS: C, 59.39; H,
4.65; N, 13.85; Found: C, 59.50; H, 4.57; N, 13.66.
2‑(2‑(3‑Methoxyphenoxy)‑1‑phenylethylidene)hydra‑
zine‑1‑carbothioamide (4f)
White solid; yield: 76%; m.p: 125–127 °C; IR (KBr, cm−1):
3406, 3160, 2954, 1606, 1584, 1494, 1482, 1288, 1279,
1194, 1158, 1066, 807, 758. 1H NMR (400 MHz, DMSO-
d6): δ 3.71 (s, 3H, OCH3), 5.32 (s, 2H, CH2O), 6.50–6.58
(m, 3H, H-2′, H-4′ and H-6′), 7.19 (t, 1H, J=8.00 Hz, H-5′),
7.36–7.42 (m, 3H, H-3, H-4 and H-5), 7.89–7.98 (m, 2H,
H-2 and H-6), 8.10 (br s, 1H, NH2), 8.49 (br s, 1H, NH2),
10.78 (br s, 1H, NH). MS (m/z, %) 315 (M+,<1), 284 (6),
240 (8), 207 (16), 178 (100), 159 (8), 141 (17), 124 (77),
103 (95), 77 (81), 51 (29). Anal. Calcd for C16H17N3O2S: C,
60.93; H, 5.43; N, 13.32. Found: C, 61.09; H, 5.31; N, 13.28.
2‑(2‑(3‑Chlorophenoxy)‑1‑phenylethylidene)hydra‑
zine‑1‑carbothioamide (4c)
Yellow solid; yield: 91%; m.p: 111–113 °C; IR (KBr, cm−1):
1
3123, 3049, 1968, 1872, 1498, 1072, 1049, 681, 572. H
NMR (400 MHz, DMSO-d6): δ 5.33 (s, 2H, CH2O), 6.92
(dd, 1H, J=8.4 and 1.6 Hz, H-6′), 7.05 (dd, 1H, J=7.6 and
1.2 Hz, H-4′), 7.10 (t, 1H, J = 2.0 Hz, H-2′), 7.33 (t, 1H,
H-5′), 7.36–7.42 (m, 3H, H-3, H-4 and H-5), 7.88–7.95 (m,
2H, H-2 and H-6), 8.11 (br s, 1H, NH2), 8.49 (br s, 1H,
NH2), 10.88 (br s, 1H, NH). MS (m/z, %) 319 (M+,<1), 256
(3), 178 (23), 160 (2), 131 (100), 128 (9), 116 (59), 89 (12),
60 (31), 57 (25). Anal. Calcd for C15H14ClN3OS: C, 56.34;
H, 4.41; N, 13.14. Found: C, 56.15; H, 4.53; N, 13.10.
2‑(2‑Phenoxy‑1‑phenylethylidene)‑N‑phenylhydra‑
zine‑1‑carbothioamide (4g)
White solid; yield: 69%; m.p: 133–135 °C; IR (KBr,
cm−1): 3273, 3182, 2889, 1594, 1492, 1228, 1180, 1030,
1
794, 686. H NMR (400 MHz, DMSO-d6): δ 5.40 (s, 2H,
CH2O), 6.97–7.05 (m, 3H, H-2′, H-4′ and H-6′), 7.23 (t,
1H, J=7.2 Hz, H-4″), 7.30–7.39 (m, 4H, H-3′, H-5′, H-3″
and H-5″), 7.39–7.44 (m, 3H, H-3, H-4 and H-5), 7.56 (dd,
2H, J=7.2 and 1.2 Hz, H-2″ and H-6″), 7.97–8.04 (m, 2H,
H-2 and H-6), 10.20 (br s, 1H, NH), 10.92 (br s, 1H, NH).
MS (m/z, %) 361 (M+,<1), 268 (8), 254 (7), 210 (15), 195
(15), 169 (7), 152 (7), 135 (21), 107 (13), 105 (86), 98 (22),
93 (100), 77 (81), 51 (27). Anal. Calcd for C21H19N3OS: C,
69.78; H, 5.30; N, 11.63. Found: C, 69.55; H, 5.17; N, 11.80.
2‑(2‑(2‑Methoxyphenoxy)‑1‑phenylethylidene)hydra‑
zine‑1‑carbothioamide (4d)
White solid; yield: 85%; m.p: 114–117 °C; IR (KBr, cm−1):
1
3288, 2961, 1596, 1498, 1477, 1263, 1130, 996, 731. H
NMR (400 MHz, DMSO-d6): δ 3.83 (s, 3H, OCH3), 5.23
(s, 2H, CH2O), 6.88–6.96 (m, 1H, H-3′), 6.98–7.06 (m, 2H,
H-4′ and H-5′), 7.15 (d, 1H, J=8.4 Hz, H-6′), 7.45–7.44 (m,
3H, H-3, H-4 and H-5), 7.95–8.01 (m, 2H, H-2 and H-6),
8.14 (br s, 1H, NH2), 8.53 (br s, 1H, NH2), 10.75 (br s, 1H,
NH). MS (m/z, %) 315 (M+, 1), 298 (7), 233 (4), 210 (11),
178 (100), 133 (30), 124 (90), 110 (54), 103 (58), 81 (27),
77 (48), 51 (14). Anal. Calcd for C16H17N3O2S: C, 60.93; H,
5.43; N, 13.32. Found: C, 60.89; H, 5.61; N, 13.21.
2‑(2‑(4‑Fluorophenoxy)‑1‑phenylethylidene)hydra‑
zine‑1‑carbothioamide (4h)
Yellow solid; yield 81%; m.p: 103–105 °C; IR (KBr, cm−1):
3358, 2765, 1601, 1597, 1497, 1320, 1240, 1226, 1128,
1
1004, 829, 751, 554. H NMR (400 MHz, DMSO-d6): δ
5.58 (s, 2H, CH2O), 7.69–7.31 (m, 4H, H-2′, H-3′, H-5′ and
H-6′), 7.62–7.75 (m, 3H, H-3, H-4 and H-5), 8.31 (d, 2H,
J=7.6 Hz, H-2 and H-6), 8.59 (br s, 1H, NH), 8.83 (br s, 1H,
NH). MS (m/z, %) 303 (M+,<1), 256 (4), 230 (10), 179 (10),
178 (100), 160 (4), 133 (27), 112 (24), 105 (51), 103 (52),
77 (41), 60 (19), 51 (11). Anal. Calcd for C15H14FN3OS: C,
59.39; H, 4.65; N, 13.85. Found: C, 59.33; H, 4.39; N, 14.01.
2‑(1‑Phenyl‑2‑(3,4,5‑trimethoxyphenoxy)ethylidene)
hydrazine‑1‑carbothioamide (4e)
White solid; yield: 84%; m.p: 178–180 °C; IR (KBr, cm−1):
3413, 3016, 2964, 1610, 1592, 1508, 1493, 1235, 1130,
1018, 808, 762. 1H NMR (400 MHz, DMSO-d6): δ 3.57 (s,
1 3

Molecular Diversity
2‑(2‑(4‑Fluorophenoxy)‑1‑phenylethylidene)‑N‑phenylhy‑
drazine‑1‑carbothioamide (4i)
1H NMR (400 MHz, DMSO-d6): δ 3.80 (s, 3H, OCH3),
5.29 (s, 2H, CH2O), 6.94 (d, 2H, J=8.8 Hz, H-3 and H-5),
7.00 (dd, 2H, J = 9.2 and 4.4 Hz, H-2′, H-6′), 7.17 (t, 2H,
J=8.8 Hz, H-3′ and H-5′), 7.90 (d, 2H, J=8.8 Hz, H-2 and
H-6), 8.07 (br s, 1H, NH2), 8.44 (br s, 1H, NH2), 10.70 (br
s, 1H, NH). MS (m/z, %) 333 (M+, 6), 316 (12), 259 (3),
222 (2), 208 (100), 180 (4), 163 (28), 148 (12), 133 (100),
122 (55), 112 (24), 95 (19), 77 (16), 60 (14). Anal. Calcd
for C16H16FN3O2S: C, 57.64; H, 4.84; N, 12.60. Found: C,
57.31; H, 4.81; N, 12.73.
White solid; yield 73%; m.p: 125–127 °C; IR (KBr, cm−1):
3302, 3273, 2972, 1551, 1508, 1497, 1441, 1287, 1213, 1178,
897, 689, 566. 1H NMR (400 MHz, DMSO-d6): δ 5.38 (s,
2H, CH2O), 7.04 (dd, 2H, J=9.2 and 4.4 Hz, H-2′ and H-6′),
7.18 (t, 2H, J=8.8 Hz, H-3′ and H-5′), 7.30 (t, 1H, J=7.2 Hz,
H-4″), 7.38 (t, 2H, J=7.6 Hz, H-3″ and H-5″), 7.39–7.45
(m, 3H, H-3, H-4 and H-5), 7.56 (d, 2H, J=7.2 Hz, H-2″
and H-6″), 7.96–8.03 (m, 2H, H-2 and H-6), 10.19 (br s,
1H, NH), 10.78 (br s, 1H, NH). MS (m/z, %) 379 (M+,<1),
286 (21), 254 (36), 178 (28), 133 (41), 112 (30), 103 (100),
94 (84), 77 (53), 51 (17). Anal. Calcd for C21H18FN3OS: C,
66.47; H, 4.78; N, 11.07. Found: C, 66.40; H, 4.74; N, 10.89.
2‑(2‑(4‑Fluorophenoxy)‑1‑(4‑methoxyphenyl)
ethylidene)‑N‑phenylhydrazine‑1‑carbothioamide (4m)
White solid; yield 91%; m.p: 156–158 °C; IR (KBr, cm−1):
3210, 2935, 1610, 1596, 1542, 1445, 1470, 1253, 1180,
1
2‑(1‑(4‑Methoxyphenyl)‑2‑phenoxyethylidene)hydra‑
zine‑1‑carbothioamide (4j)
1016, 829, 650, 556. H NMR (400 MHz, DMSO-d6): δ
3.82 (s, 3H, OCH3), 5.37 (s, 2H, CH2O), 6.97 (d, 2H,
J=8.8 Hz, H-3, and H-5), 7.04 (dd, 2H, J=8.8 and 4.0 Hz,
H-2′ and H-6′), 7.20 (t, 2H, J=8.8 Hz, H-3′ and H-5′), 7.24
(t, 1H, J=7.2 Hz, H-4″), 7.40 (t, 2H, J=7.6 Hz, H-3″and
H-5″), 7.58 (d, 2H, J=7.6 Hz, H-2″ and H-6″), 7.99 (d, 2H,
J=8.8 Hz, H-2 and H-6), 10.17 (br s, 1H, NH), 11.04 (br s,
1H, NH). MS (m/z, %) 409 (M+ <1), 316 (52), 284 (28), 258
(7), 205 (10), 163 (4), 147 (18), 133 (100), 122 (95), 118 (9),
93 (68), 77 (25), 51 (8). Anal. Calcd for C22H20FN3O2S: C,
64.53; H, 4.92; N, 10.26. Found: C, 64.50; H, 5.04; N, 10.33.
White solid; yield 74%, m.p: 156–158 °C; IR (KBr, cm−1):
3343, 3433, 2807, 1598, 1587, 1502, 1481, 1296, 1257,
1234, 1171, 1114, 835, 760, 630, 555. 1H NMR (400 MHz,
DMSO-d6): δ 3.81 (s, 3H, OCH3), 5.31 (s, 2H, CH2O), 6.94
(d, 2H, J=8.4 Hz, H-3 and H-5), 6.97–7.06 (m, 3H, H-2′,
H-4′ and H-6′), 7.33 (t, 2H, J=8.0 Hz, H-3′ and H-5′), 7.91
(d, 2H, J=8.8 Hz, H-2 and H-6), 8.07 (br s, 1H, NH2), 8.43
(br s, 1H, NH2), 10.70 (br s, 1H, NH). MS (m/z, %) 315
(M+,<1), 242 (12), 135 (100), 121 (7), 107 (6), 92 (7), 77
(18), 51 (3). Anal. Calcd for C16H17N3O2S: C, 60.93; H,
5.43; N, 13.32. Found: C, 61.20; H, 5.29; N, 13.16.
2‑(1‑(4‑Chlorophenyl)‑2‑phenoxyethylidene)hydra‑
zine‑1‑carbothioamide (4n)
2‑(1‑(4‑Methoxyphenyl)‑2‑phenoxyethylidene)‑N‑phenyl‑
hydrazine‑1‑carbothioamide (4k)
White solid; yield 46%; m.p: 164–166 °C; IR (KBr, cm−1):
3259, 3164, 2911, 1619, 1607, 1508, 1210, 1199, 1130,
1
831, 556. H NMR (400 MHz, DMSO-d6): δ 5.31 (s, 2H,
White solid; yield 84%; m.p: 162–164 °C; IR (KBr, cm−1):
3281, 3220, 2838, 1591, 1544, 1487, 1256, 1183, 1033,
CH2O), 6.93–7.07 (m, 3H, H-2′, H-4′ and H-6′), 7.17 (t, 2H,
J=8.4 Hz, H-3′ and H-5′), 7.45 (d, 2H, J=8.0 Hz, H-3 and
H-5), 7.97 (d, 2H, J=8.4 Hz, H-2 and H-6), 8.19 (br s, 1H,
NH2), 8.54 (br s, 1H, NH2), 10.89 (br s, 1H, NH). MS (m/z,
%) 320 (M+1, 9), 262 (2), 247 (2), 226 (8), 214 (62), 212
(100), 184 (4), 167 (43), 137 (75), 125 (22), 102 (22), 75
(22), 60 (7). Anal. Calcd for C15H14ClN3OS: C, 56.34; H,
4.41; N, 13.14. Found: C, 56.61; H, 4.30, N, 13.16.
1
759, 518. H NMR (400 MHz, DMSO-d6): δ 3.82 (s, 3H,
OCH3), 5.39 (s, 2H, CH2O), 6.97 (d, 2H, J = 8.8 Hz, H-3
and H-5), 7.00–7.08 (m, 3H, H-2′, H-4′ and H-6′), 7.24 (t,
1H, J = 7.2 Hz, H-4″), 7.34 (d, 2H, J = 7.6 Hz, H-3, and
H-5), 7.40 (t, 2H, J=8.0 Hz, H-3″ and H-5″), 7.58 (d, 2H,
J=8.0 Hz, H-2″ and H-6″), 8.00 (d, 2H, J=8.4 Hz, H-2 and
H-6), 10.17 (br s, 1H, NH), 11.04 (br s, 1H, NH). MS (m/z,
%) 391 (M+,<1), 298 (59), 240 (27), 225 (6), 210 (8), 163
(4), 133 (100), 122 (81), 107 (28), 93 (96), 77 (72), 51 (16).
Anal. Calcd for C22H21N3O2S: C, 67.50; H, 5.41; N, 10.73.
Found: C, 67.46; H, 5.77; N, 10.79.
2‑(1‑(4‑Chlorophenyl)‑2‑phenoxyethylidene)‑N‑phenylhy‑
drazine‑1‑carbothioamide (4o)
White solid; yield 63%; m.p: 154–156 °C; IR (KBr, cm−1):
3181, 3058, 1614, 1597, 1549, 1490, 1232, 1178, 1023,
985, 822, 744. 1H NMR (400 MHz, DMSO-d6): δ 5.40 (s,
2H, CH2O), 6.98–7.06 (m, 3H, H-2′, H-4′ and H-6′), 7.24
(t, 1H, J=7.6 Hz, H-4″), 7.35 (t, 2H, J=8.0 Hz, H-3′ and
H-5′), 7.41 (t, 2H, J=7.6 Hz, H-3″ and H-5″), 7.48 (d, 2H,
J = 8.4 Hz, H-3 and H-5), 7.55 (d, 2H, J = 7.6 Hz, H-2″
2‑(2‑(4‑Fluorophenoxy)‑1‑(4‑methoxyphenyl)ethylidene)
hydrazine‑1‑carbothioamide (4l)
White solid; yield 75%; m.p: 140–142 °C; IR (KBr, cm−1):
3420, 2998, 1605, 1589, 1466, 1257, 1212, 1014, 809, 569.
1 3

Molecular Diversity
and H-6″), 8.06 (d, 2H, J = 8.8 Hz, H-2 and H-6), 10.26
(br s, 1H, NH), 11.23 (br s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6): 177.81, 158.01, 144.42, 139.49, 134.89, 134.62,
130.00, 129.65, 128.71, 128.63, 126.68, 126.16, 121.89,
115.30, 61.69. MS (m/z, %) 395 (M+,<1), 378 (3), 302 (21),
290 (36), 288 (100), 244 (26), 229 (22), 212 (21), 180 (12),
167 (19), 137 (100), 125 (20), 111 (12), 102 (29), 93 (57),
77 (73), 66 (28), 51 (20). Anal. Calcd for C21H18ClN3OS: C,
63.71; H, 4.58; N, 10.61. Found: C, 63.65; H, 4.77; N, 10.46.
of 20 to 25 °C) and were given drinking water and a regular
mouse diet. This research was performed according to insti-
tutional animal ethics guidelines of the Animal Research
Center, Mazandaran University of Medical Sciences (ARC-
MUMS). The experimental protocols for animal use in this
study were approved by the Mazandaran University of Medi-
cal Sciences Ethics Committee (MUMSEC).
Vero cells (ATCC No. CCL-81) were cultivated in RPMI-
1640 medium with the addition of 10% inactivated FBS,
100 units/mL of penicillin and 100 μg/mL of streptomycin.
For cytotoxicity assay, 2×104 Vero cells/mL were seeded in
96-well plates. The compounds were initially dissolved in
dimethyl sulfoxide (DMSO) to form a stock solution. Then,
this stock solution was diluted in PBS as required [7].
After 24 h, the cells were exposed to compounds and
pyrimethamine (positive control) at the fnal concentrations
of 1.56, 3.12, 6.25, 12.5, 25, 50 and 100 μg/mL. In the next
day, the viabilities of the Vero cells were evaluated by using
a colorimetric MTT assay [9]. For efects of the compounds
on T. gondii infection, 2×104 Vero cells/mL (suspended in
RPMI-1640 supplemented with 10% FBS) were seeded in
96-well plates. After 24 h of seeding, the cells were infected
with RH strain of T. gondii (2 × 105 tachyzoites/mL) and
placed in a 37 °C incubator maintained at 5% CO2 for 24 h.
After that, the cells were exposed to the compounds and
pyrimethamine at fnal concentrations of 1.56–100 μg/mL.
After 24 h, the viability of T. gondii-infected Vero cells was
evaluated by the MTT assay [20]. The 50% inhibitory con-
centrations (IC50s) were calculated by the GraphPad Prism
6.0.
2‑(1‑(4‑Chlorophenyl)‑2‑(4‑fuorophenoxy)ethylidene)
hydrazine‑1‑carbothioamide (4p)
White solid; yield 45%; m.p: 159–161 °C; IR (KBr, cm−1):
3421, 3060, 1610, 1596, 1486, 1437, 1287, 1241, 1092,
823, 753, 519. 1H NMR (400 MHz, DMSO-d6): δ 5.33 (s,
2H, CH2O), 6.90–7.08 (m, 2H, H-2′ and H-6′), 7.33 (t, 2H,
J=8.0 Hz, H-3′ and H-5′), 7.45 (d, 2H, J=8.4 Hz, H-3 and
H-5), 7.99 (d, 2H, J=8.8 Hz, H-2 and H-6), 8.20 (br s, 1H,
NH2), 8.54 (br s, 1H, NH2), 10.90 (br s, 1H, NH). MS (m/z,
%) 337 (M+,<1), 302 (2), 244 (3), 212 (100), 184 (4), 167
(32), 137 (60), 125 (16), 102 (6), 77 (33), 60 (25). Anal.
Calcd for C15H13ClFN3OS: C, 53.34; H, 3.88; N, 12.44.
Found: C, 53.27; H, 4.01; N, 12.40.
2‑(1‑(4‑Chlorophenyl)‑2‑(4‑fuorophenoxy)
ethylidene)‑N‑phenylhydrazine‑1‑carbothioamide (4q)
White solid; yield 72%; m.p: 168–170 °C; IR (KBr,
cm−1): 3295, 3063, 1606, 1595, 1547, 1490, 1204, 1179,
1
1096, 824, 662, 535. H NMR (400 MHz, DMSO-d6): δ
5.38 (s, 2H, CH2O), 7.04 (dd, 2H, J=8.8 and 4.4 Hz, H-2′
and H-6′), 7.20 (t, 2H, J =8.4 Hz, H-3′ and H-5′), 7.26 (t,
1H, J = 7.6 Hz, H-4″), 7.41 (t, 2H, J = 7.6 Hz, H-3″ and
H-5″), 7.48 (d, 2H, J=8.4 Hz, H-3 and H-5), 7.55 (d, 2H,
J = 8.4 Hz, H-2″ and H-6″), 8.05 (d, 2H, J = 8.4 Hz, H-2
and H-6), 10.26 (br s, 1H, NH), 11.23 (br s, 1H, NH). MS
(m/z, %) 413 (M+,<1), 320 (29), 288 (25), 262 (8), 247 (5),
209 (11), 180 (8), 155 (5), 139 (34), 137 (100), 125 (30),
111 (16), 93 (70), 75 (27), 66 (22), 51 (8). Anal. Calcd for
C21H17ClFN3OS: C, 60.94; H, 4.14; N, 10.15. Found: C,
61.15; H, 4.10; N, 10.23.
In vitro evaluation of compounds against tachyzoites
To evaluate the efect of compounds 4l and 4p on tachy-
zoites, a concentration (5 μg/mL) of compounds were
assessed for 24 h on mortality of T. gondii tachyzoites. Equal
proportions of each compound and 2×105 fresh tachyzoites
were placed in test tube. After slow mixing, the contents of
the tubes were incubated at 37 °C for 24 h. Then, 0.5 mL
of 0.5% trypan blue stain was added to the settled tachy-
zoites. The percentage of dead tachyzoites was determined
by counting 100 tachyzoites with hemocytometer under a
light microscope. PBS and pyrimethamine (50 μg/mL) were
considered as negative and positive control groups, respec-
tively. The experiments were performed in triplicate [21].
Biological evaluations
Anti‑T. gondii assay using MTT
Anti‑parasitic evaluation of compounds against the inva‑
Tachyzoites (RH strain of T. gondii) were collected from the
peritoneal cavity of Balb/c mice after intraperitoneal injec-
tion with 1×105 of the parasite. Six-week-old inbred female
Balb/c mice weighing 20–22 g were used for this experi-
ment. All experimental animals were housed in cages under
standard laboratory conditions (with an average temperature
sion and intracellular proliferation of T. gondii
Vero cells were cultured on 13-mm round glass slides in
24-well plates (1 × 104 cells/200 μL per well). After 72 h
incubation in 37 °C and 5% CO2, the cells were infected
with tachyzoites at a ratio of 5:1 for 3 h incubation. Then,
1 3

Molecular Diversity
activities of anti-Toxoplasma drugs and compounds (2006–2016).
Front Microbiol 8:25. https://doi.org/10.3389/fmicb.2017.00025
7. Melo EJT, Beiral HJ (2003) Efect of hydroxyurea on the intracel-
lular multiplication of Toxoplasma gondii, Leishmania amazonen-
sis and Trypanosoma cruzi. Braz J Med Biol Res 36:65–69. https
://doi.org/10.1590/s0100-879x2003000100009
the cells were washed to remove extracellular parasites, and
the cells were treated with the compounds or pyrimethamine
(5 μg/mL). The slides with adherent cells were washed with
sterile cold PBS, fxed with bufered formalin 10% for 24 h
and stained with 1% toluidine blue for 10 s [22]. After that,
the cells were counted under a light microscope (Nikon,
Japan) in order to determine the infection index (number
of infected cell per 100 examined cells) and T. gondii intra-
cellular proliferation (total number of tachyzoites per 100
examined cells).
8. Melo EJT, Souza W (2000) Efect of hydroxyurea on intracellular
Toxoplasma gondii. FEMS Microbiol Lett 185:79–85. https://doi.
org/10.1016/s0378-1097(00)00074-4
9. Gomes MAGB, Carreira GM, Souza DPV, Nogueira PMR, de
Melo EJT, Maria EJ (2013) Study of the efect of thiosemicarba-
zones against Toxoplasma gondii. CR Biol 336:203–206. https://
doi.org/10.1016/j.crvi.2013.03.002
10. Liu MC, Lin TS, Sartorelli AC (1992) Synthesis and antitumor
activity of amino derivatives of pyridine-2-carboxaldehyde
thiosemicarbazone. J Med Chem 35:3672–3677. https://doi.
org/10.1021/jm00098a012
Computational studies
11. Tenório RP, Carvalho CS, Pessanha CS, de Lima JG, de Faria
AR, Alves AJ, de Melo EJ, Góes AJ (2005) Synthesis of thio-
semicarbazone and 4-thiazolidinone derivatives and their in vitro
anti-Toxoplasma gondii activity. Bioorg Med Chem Lett 15:2575–
2578. https://doi.org/10.1016/j.bmcl.2005.03.048
Gaussian 09 was used to fully optimize the structures at the
B3LYP level of theory [23]. For all the calculations, solvent
efects were considered using the SMD solvation model with
water as the solvent [24]. The 6-31G(d) basis set was used
for all atoms [25]. Frequency calculations were carried out
at the same level of theory as those for the structural opti-
mization. To further refne the energies obtained from the
B3LYP-SMD/6-31g(d) calculations and to consider disper-
sive interactions [26], we carried out single-point energy
calculations using the M06-D3 functional method for all of
the structures with a larger def2-TZVP basis set on all atoms
[27].
12. Silva BV, Silva BNM (2017) Thio- and semicarbazones: hope in
the search for treatment of Leishmaniasis and chagas disease. Med
Chem 13:110–126. https://doi.org/10.2174/157340641266616
0909152614
13. Beraldo H, Gambino D (2004) The wide pharmacological
versatility of semicarbazones, thiosemicarbazones and their
metal complexes. Mini Rev Med Chem 4:31–39. https://doi.
org/10.2174/1389557043487484
14. Walcourt A, Loyevsky M, Lovejoy DB, Gordeuk VR, Richardson
DR (2004) Novel aroylhydrazone and thiosemicarbazone iron che-
lators with anti-malarial activity against chloroquine-resistant and
-sensitive parasites. Int J Biochem Cell Biol 36:401–407. https://
doi.org/10.1016/S1357-2725(03)00248-6
Acknowledgements This project was supported by a Grant (No. 2571)
from the Research Council of Mazandaran University of Medical Sci-
ences, Sari, Iran.
15. Ansari M, Emami S, Khalilzadeh MA, Maghsoodlou MT,
Foroumadi A, Faramarzi MA, Samadi N, Ardestani SK (2012)
Replacement of the methylene of dihydrochalcones with oxy-
gen: synthesis and biological evaluation of 2-phenoxyacetophe-
nons. Chem Biol Drug Des 80:591–597. https://doi.org/10.111
1/j.1747-0285.2012.01432.x
Compliance with ethical standards
Conflict of interest The authors declare no confict of interest.
16. Landge SM, Tkatchouk E, Benítez D, Lanfranchi DA, Elhabiri
M, Goddard WA, Aprahamian I (2011) Isomerization mecha-
nism in hydrazone-based rotary switches: lateral shift, rotation,
or tautomerization? J Am Chem Soc 133:9812–9823. https://doi.
org/10.1021/ja200699v
References
17. Dehestani L, Ahangar N, Hashemi SM, Irannejad H, Honarchian
Masihi P, Shakiba A, Emami S (2018) Design, synthesis, in vivo
and in silico evaluation of phenacyl triazole hydrazones as new
anticonvulsant agents. Bioorg Chem 78:119–129. https://doi.
org/10.1016/j.bioorg.2018.03.001
1. Dubey JP (2008) The history of Toxoplasma gondii the frst 100
years. J Eukaryot Microbiol 55:467–475. https://doi.org/10.111
1/j.1550-7408.2008.00345.x
2. Montoya JG, Liesenfeld O (2004) Toxoplasmosis. Lancet
363:1965–1976. https://doi.org/10.1016/S0140-6736(04)16412-X
3. Gangneux FR, Murat JB, Hidalgo HF, Brenier-Pinchart MP,
Gangneux JP, Pelloux H (2011) The placenta: a main role in con-
genital toxoplasmosis? Trends Parasitol 27:530–536. https://doi.
org/10.1016/j.pt.2011.09.005
18. Pires DEV, Blundell TL, Ascher DB (2015) pkCSM: predicting
small-molecule pharmacokinetic and toxicity properties using
graph-based signatures. J Med Chem 58:4066–4072. https://doi.
org/10.1021/acs.jmedchem.5b00104
19. King LC, Ostrum GK (1964) Selective bromination with
copper(II) bromide. J Org Chem 29:3459–3461. https://doi.
org/10.1021/jo01035a003
4. Neville AJ, Zach SJ, Wang X, Larson JJ, Judge AK, Davis LA,
Vennerstrom JL, Davis PH (2015) Clinically available medicines
demonstrating anti-Toxoplasma activity. Antimicrob Agents
Chemother 59:7161–7169. https://doi.org/10.1128/AAC.02009
-15
20. Galván-Ramírez Mde L, Dueñas Jiménez JM, Rocío Rodríguez
Pérez L, Troyo-Sanroman R, Ramírez-Herrera M, García-Iglesias
T (2013) Efect of nitaxozanide and pyrimethamine on astrocytes
infected by Toxoplasma gondii in vitro. Arch Med Res 44:415–
421. https://doi.org/10.1016/j.arcmed.2013.07.002
5. Alday PH, Doggett JS (2017) Drugs in development for toxoplas-
mosis: advances, challenges, and current status. Drug Des Devel
Ther 11:273–293. https://doi.org/10.2147/DDDT.S60973
6. Montazeri M, Sharif M, Sarvi S, Mehrzadi S, Ahmadpour E,
Daryani A (2017) A systematic review of in vitro and in vivo
21. Daryani A, Ebrahimzadeh MA, Sharif M, Ahmadpour E, Eda-
latian S, Esboei BR et al (2015) Anti-Toxoplasma activities of
methanolic extract of Sambucus nigra (Caprifoliaceae) fruits and
1 3

Molecular Diversity
leaves. Rev Biol Trop 63:7–12. https://doi.org/10.15517/rbt.v63i1
.14545
26. Grimme S, Antony J, Ehrlich S, Krieg H (2010) A consistent and
accurate ab initio parametrization of density functional disper-
sion correction (DFT-D) for the 94 elements H-Pu. J Chem Phys
132:154104. https://doi.org/10.1063/1.3382344
22. Barbosa BF, Gomes AO, Ferro EAV, Napolitano DR, Mineo JR,
Silva NM (2012) Enrofoxacin is able to control Toxoplasma gon-
dii infection in both in vitro and in vivo experimental models. Vet
Parasitol 187:44–52. https://doi.org/10.1016/j.vetpar.2011.12.039
23. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA,
Cheeseman JR et al (2009) Gaussian 09, Revision D.01. Gaussian
Inc, Wallingford
27. Marenich AV, Cramer CJ, Truhlar DG (2009) Universal solva-
tion model based on solute electron density and on a continuum
model of the solvent defned by the bulk dielectric constant and
atomic surface tensions. J Phys Chem B 113:6378–6396. https://
doi.org/10.1021/jp810292n
24. Cossi M, Rega N, Scalmani G, Barone V (2003) Energies, struc-
tures, and electronic properties of molecules in solution with the
C-PCM solvation model. J Comput Chem 24:669–681. https://doi.
org/10.1002/jcc.10189
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
25. Hariharan PC, Pople JA (1973) The infuence of polarization func-
tions on molecular orbital hydrogenation energies. Theor Chim
Acta 28:213–222. https://doi.org/10.1007/BF00533485
Afliations
Mahsa Ansari1 · Mahbobeh Montazeri2,3,4 · Ahmad Daryani2,4 · Kaveh Farshadfar5 · Saeed Emami6
1
4
5
6
Pharmaceutical Sciences Research Center, Student Research
Committee, Faculty of Pharmacy, Mazandaran University
of Medical Sciences, Sari, Iran
Department of Parasitology, Faculty of Medicine,
Mazandaran University of Medical Sciences, Sari, Iran
Department of Chemistry, Islamic Azad University, Central
Tehran Branch, Poonak, Tehran, Iran
2
3
Toxoplasmosis Research Center, Mazandaran University
of Medical Sciences, Sari, Iran
Department of Medicinal Chemistry and Pharmaceutical
Sciences Research Center, Faculty of Pharmacy, Mazandaran
University of Medical Sciences, Sari, Iran
Student Research Committee, Mazandaran University
of Medical Sciences, Sari, Iran
1 3