Dual Parasiticidal Activities of Phthalimides: Synthesis and Biological Profile against Trypanosoma cruzi and Plasmodium falciparum

Article abstract of DOI:10.1002/cmdc.202000331

Chagas disease and malaria are two neglected tropical diseases (NTDs) that prevail in tropical and subtropical regions in 149 countries. Chagas is also present in Europe, the US and Australia due to immigration of asymptomatic infected individuals. In the absence of an effective vaccine, the control of both diseases relies on chemotherapy. However, the emergence of parasite drug resistance is rendering currently available drugs obsolete. Hence, it is crucial to develop new molecules. Phthalimides, thiosemicarbazones, and 1,3-thiazoles have been used as scaffolds to obtain antiplasmodial and anti-Trypanosoma cruzi agents. Herein we present the synthesis of 24 phthalimido-thiosemicarbazones (3 a–x) and 14 phthalimido-thiazoles (4 a–n) and the corresponding biological activity against T. cruzi, Plasmodium falciparum, and cytotoxicity against mammalian cell lines. Some of these compounds showed potent inhibition of T. cruzi at low cytotoxic concentrations in RAW 264.7 cells. The most active compounds, 3 t (IC₅₀=3.60 μM), 3 h (IC₅₀=3.75 μM), and 4 j (IC₅₀=4.48 μM), were more active than the control drug benznidazole (IC₅₀=14.6 μM). Overall, the phthalimido-thiosemicarbazone derivatives were more potent than phthalimido-thiazole derivatives against T. cruzi. Flow cytometry assay data showed that compound 4 j was able to induce necrosis and apoptosis in trypomastigotes. Analysis by scanning electron microscopy showed that T. cruzi trypomastigote cells treated with compounds 3 h, 3 t, and 4 j at IC₅₀ concentrations promoted changes in the shape, flagella, and surface of the parasite body similar to those observed in benznidazole-treated cells. The compounds with the highest antimalarial activity were the phthalimido-thiazoles 4 l (IC₅₀=1.2 μM), 4 m (IC₅₀=1.7 μM), and 4 n (IC₅₀=2.4 μM). Together, these data revealed that phthalimido derivatives possess a dual antiparasitic profile with potential effects against T. cruzi and lead-like characteristics.

Full text of DOI:10.1002/cmdc.202000331

Accepted Article
Title: Dual Parasiticidal Activities of New Phthalimides: Synthesis and
Biological Profile against Trypanosoma cruzi and Plasmodium
falciparum
Authors: Paulo André T. M. Gomes, Marcos V. O. Cardoso, Ignes
Regina Santos, Fabiano Sousa, Juliana Conceição, Vanessa
Silva, Denise Duarte, Raquel Pereira, Rafael Oliveira, Fátima
Nogueira, Luiz Carlos Alves, Fábio Brayner, Aline Santos,
Valéria Pereira, and Ana Cristina Lima Leite
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000331
Link to VoR: https://doi.org/10.1002/cmdc.202000331
01/2020

10.1002/cmdc.202000331
ChemMedChem
FULL PAPER
Dual Parasiticidal Activities of New Phthalimides: Synthesis and
Biological Profile against Trypanosoma cruzi and Plasmodium falciparum
Paulo André Teixeira de Moraes Gomes[a], Marcos Veríssimo de Oliveira Cardoso[b], Ignes Regina dos
Santos[a], Fabiano Amaro de Sousa[a], Juliana Maria da Conceição[a], Vanessa Gouveia de Melo
Silva[a], Denise Duarte[c], Raquel Pereira[c], Rafael Oliveira[c], Fátima Nogueira[c], Luiz Carlos Alves[d],
Fabio André Brayner[d], Aline Caroline da Silva Santos[e], Valéria Rêgo Alves Pereira[e], Ana Cristina
Lima Leite*[a]
[a]
[b]
Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Federal de Pernambuco, 50740-535, Recife, PE, Brazil.
Laboratório de Prospecção de Moléculas Bioativas, Programa de Pós-Graduação em Ciência e Tecnologia Ambiental para o Semiárido, Universidade de
Pernambuco, 56328-903, Petrolina, PE, Brazil.
[c]
Unidade de Ensino e Investigação de Parasitologia Médica, Global Health and Tropical Medicine, GHTM, Instituto de Higiene e Medicina Tropical, IHMT,
Universidade Nova de Lisboa, UNL, Rua da Junqueira no 100, 1349-008, Lisboa, Portugal.
[d]
[e]
Keizo Asami Immunopathology (LIKA), Campus UFPE, 50670-420, Recife, PE Brazil.
Instituto Aggeu Magalhães, Fundação Oswaldo Cruz, 50670-420, Recife, PE, Brazil.
Abstract: Chagas disease and malaria are two Neglected tropical
diseases (NTDs) that prevail in tropical and subtropical conditions in
149 countries. Chagas is also present in Europe, the United States
and, Australia due to the immigration of asymptomatic infected
individuals. In the absence of an effective vaccine, the control of both
diseases relies on chemotherapy. However, the emergence of
parasite drug resistance is rendering currently available drugs
obsolete. Hence, it is crucial to develop new molecules. Phthalimides,
thiosemicarbazones, and 1,3-thiazoles nucleus have been used as
platform types to obtain antiplasmodial and anti-Trypanosoma cruzi
agents. Here we present the synthesis of 24 phthalimido-
thiosemicarbazones (3a-x) and 14 phthalimido-thiazoles (4a-n) and
the corresponding biological activity against T. cruzi, Plasmodium
falciparum, and cytotoxicity against mammalian cell lines. Some of
these compounds showed potent inhibition of T. cruzi at low
cytotoxicity concentrations in RAW 264.7 cells. The most active
compounds, 3t (IC50= 3.60 M), 3h (IC50= 3.75M), and 4j (IC50= 4.48
M), were more active than the control drug Benznidazole (IC50=
14.6 M). Overall, the phthalimido-thiosemicarbazone derivatives
were more potent than phthalimido-thiazoles derivatives against T.
cruzi. Flow cytometry assay showed that compound 4j was able to
induce necrosis and apoptosis in trypomastigotes. Analysis by
scanning electron microscopy showed that T. cruzi trypomastigote
cells treated with the compounds 3h, 3t, and 4j at IC50 concentrations,
promoted changes in the shape, flagella, and surface of the parasite
body similar to those observed in benznidazole-treated cells. The
compounds with the highest antimalarials activity were the
phthalimido-thiazoles 4l (IC50= 1.2M), 4m (IC50= 1.7M), and 4n
(IC50= 2.4M). Together, these data revealed, phthalimido derivatives
possess a dual antiparasitic profile with potential effects against T.
cruzi and lead-like characteristics.
In the last years, the epidemiology of this infection is changing
due to the unprecedented immigration of asymptomatic infected
individuals from Latin America to Europe, the United States, and
Australia acquired by vertical or through organs or blood products
obtained from infected donors.[5] Chagas disease can be treated
with benznidazole (BDZ) or nifurtimox.[6] Although highly
efficacious, if timely administered, adverse reactions can occur in
up to 40% of treated patients.[4,6] BDZ and nifurtimox
contraindications include pregnancy and kidney or liver
dysfunction.
Malaria is one of the most devastating infectious diseases of our
time, in 2017, there were an estimated 219 million cases of
malaria (more than 99% due to P. falciparum) and 435.000 deaths
in 87 countries.[4] There are relatively few effective treatments for
P. falciparum, apart from artemisinin (ART) combination therapies
(ACTs). Nowadays, extensive P. falciparum malaria treatment
relies on ACT, although resistance is now evident in several
Southeast Asian countries.[7–10] Another relevant issue related to
the widespread application of ART is the difficulty in maintaining
the drug supply; ART is extracted from Artemisia annua in low
yield, and ART-derivatives are obtained by a long and expensive
semi-synthesis process.[11]
There is a recognized need to identify new chemical entities
(NCEs) as drug candidates in order to both improve patient care
and meet targets for elimination, particularly of neglected parasitic
diseases.[12] Phthalimides, thiosemicarbazones and 1,3-thiazoles
nucleus are found in several natural products and have also been
utilized to develop synthetic drugs and drug-like molecules with a
variety of pharmacological effects, binding to a plethora of
different targets across target families.[13,14]
Phthalimides, thiosemicarbazones, and 1,3-thiazoles nucleus
have been used as platform types to obtains antiplasmodial and
[15–22]
anti-T. cruzi agents.
Our research group has explored the
Introduction
pharmacological properties of phthalimide derivatives, and as a
result, phthalimido-thiazole derivatives were identified as building
blocks to promising anti-T. cruzi candidates.[23]
Neglected tropical diseases (NTDs) are a diverse group of
communicable diseases that prevail in tropical and subtropical
conditions in 149 countries. NTDs affect more than one billion
people and impose a heavy economic burden on developing
economies. Drugs available to treat NTDs present some issues:
limited number, low efficacy, toxic side effects, and appearance
of resistant strains.[1,2] Malaria (caused by Plasmodium spp.) and
Chagas disease (caused by Trypanosoma cruzi) are two vector-
borne diseases, with a heavy impact throughout the tropical and
subtropical regions where they remain a life-threatening public
health problem. Chagas disease, also known as American
trypanosomiasis, is a potentially life-threatening illness caused by
the protozoan parasite, Trypanosoma cruzi (T. cruzi) and new
chemotherapies are highly needed.[3] About 6 to 7 million people
worldwide, mostly in Latin America, are estimated to be infected
with T. cruzi.[4]
Here we performed the synthesis of 24 phthalimido-
thiosemicarbazones, (3a-x), and 14 phthalimido-thiazoles (4a-n)
(Figure 1). In this synthetic strategy, a substructure-based
compound library was used to choose different substituents
around the phenyl ring attached in the thiosemicarbazone moiety,
obtaining 3a-x compounds. For 4a-n compounds, substituents
around the phenyl ring attached at C4 in the thiazole ring were
explored. In both series, structural modifications were performed
by insertion of substituents on the N3 position of
thiosemicarbazone (3a-x) or thiazole ring (4a-n). With a view to
investigates the parasitical profile of new phthalimido-
thiosemicarbazones and new phthalimido-thiazoles derivatives,
the compounds were tested in vitro against the T. cruzi parasite
trypomastigote form and P. falciparum.
1
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10.1002/cmdc.202000331
ChemMedChem
FULL PAPER
1
Besides, a representative H-NMR spectrum of compound 4n is
presented in Supplementary Material (Figure S1).
Figure 1. Structural planning of the proposed compounds.
Results and Discussion
Chemistry
Scheme 1. Global synthesis of compounds 3a-x and 4a-n. Reagents and
conditions: (A) Urea, 150°C, 3h; (B) acetone, phthalimide (2), K2CO3, KI,
respective acetophenone, r.t. or reflux, 4h; (C) ethyl alcohol, sulfuric acid,
ultrasound or reflux, 30 min, respective thiosemicarbazide, ultrasound (4-12h)
or reflux (24-48h). (D) compound 3a (for 4a-I; or 3b for 4j-n), isobutanol,
respective acetophenone, ultrasound, 3h.
The details of the synthesis are depicted in Scheme 1. The
composition of newly prepared molecules was confirmed by NMR
(1H and 13C), FT-IR, and HR-MS techniques. In NMR spectra,
protons and carbons were assigned (see supporting information).
All the data were in agreement with our proposed structure.
The 24 phthalimido-thiosemicarbazones (3a-x), as shown in
Scheme 1, were synthesized in a two-step process from
phthalimide (2) and respective acetophenone. The reaction was
carried out in acetone under room temperature (r.t) or reflux for
4h, which led us to intermediate 3. These intermediates were
them reacted with thiosemicarbazides (thiosemicarbazide, 4-
phenylthiosemicarbazide, and 4-methyl-3-thiosemicarbazide)
and catalytic H2SO4 in an ultrasound bath (4-12h) or reflux (24-
48h).[24] After 4-12h, thiosemicarbazones 3a-x precipitated in the
reaction mixture and were collected by filtration. The synthesis of
the series 4a-n was performed via Hantzsch cyclization between
thiosemicarbazones (3a and 3b) and substituted 2-
bromoacetophenones, using isobutanol under ultrasound
irradiation, at room temperature for 3h.[25] This reaction condition
led to average yields from 27 to 94%. All the synthesized
compounds were characterized by infrared (IR), nuclear magnetic
resonance (1H, 13C NMR), and mass spectroscopy (ESI-TOF).
NMR data are compatible with the proposed compounds. In
theory, two geometrical isomers (E and Z) about the imine (CN)
double bond are possible for the thiosemicarbazones and the
respective thiazoles. However, analysis of the 1H NMR spectra of
the compounds indicated one predominant isomer; the E isomer
by comparison with known analogues.[20] Intramolecular H-
bonding involving the proton attached to N4 (in DMSO) with the
imine N-atom leads to a distinctive singlet around 10.2 ppm,[20]
and this is also seen here. Both thiosemicarbazones and
respective 1,3-thiazoles were characterized by usual
spectroscopy.
Cytotoxicity activity against mammalian cells
Initially, we verified the influence of the cyclization of phthalimido-
thiosemicarbazone to the phthalimido-thiazole ring on the
cytotoxic effect against RAW 264.7 (macrophage-like) cells.
Phthalimido-thiosemicarbazones (3a-x), showed a range of IC50
=
42 to 338 M, with 8 compounds presenting IC50 below 100 M.
Compound 3b (IC50= 42.03 M) was the most cytotoxic (Table 1).
In general, compounds 3a-x presented IC50 values higher than
100 M (Table 1). On the other hand, of the 14 phthalimido-
thiazole derivatives (4a-n), only compound 4d showed an IC50
value lower than 100M being the most cytotoxic (IC50= 59.7M)
(Table 2).
Antiparasitic activity against extracellular forms of T. cruzi
Observing the trypanocidal activity for trypomastigote form, we
observed that 13 of 38 compounds present higher anti-T. cruzi
activity than the control drug, BDZ (Table 1) witch exhibit an IC50
value of 14.6M. Compounds that exhibited IC50 lower than BDZ
were considered active anti-T. cruzi agents. Among series 3a-x
and 4a-n, compounds 3t (IC50= 3.60M), 3h (IC50= 3.75M)
(Table 1) and 4j (IC50= 4.48M) (Table 2) were the most active.
Based on the results of anti-T. cruzi activity of the tested
compounds, we first analyzed the effect of substitutions on the
phthalimido-thiosemicarbazones, in which the structural
variations were made in the phenyl ring from 2-
haloacetophenone. No clear correlation was observed between
the substituents, once compound 3a (IC50= 42.88M) which has
no substitution in the phenyl ring, compounds 3g and 3i (fluorine),
as well compounds 3j-l (OCH3), 3m (NO2), 3u (Cl) and 3v (Ph),
presented low activity profile. However, compound 3h presents
The 1H NMR spectra of some compounds showed that
phthalimido-thiazoles 4a-n are composed by diastereomers (supp.
Material, Figures S1 and S2). Based on previous crystallized
compounds by our group, we suggest that the major isomer
formed present the E-Z configuration. Indeed, hydrazine double-
bond C2=N2 is commonly assigned as E configuration.[20,23,25–28]
Concerning the exocyclic double-bond N3=C3, we suggest that
the predominant configuration is in Z configuration.[20,23,28–30]
fluorine in para position, and
a phenyl ring in N4 of
thiosemicarbazone was the most potent of all series, being twice
as potent than reference drug BZD.
2
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10.1002/cmdc.202000331
ChemMedChem
FULL PAPER
Table 1. Cytotoxicity and antiparasitic activity of phthalimido-thiosemicarbazone series (3a-x).
SI
(CC50
RAW264.7/
IC50 Tryp.)
P. falciparum
(D7-GFP)
% of inhibition
at 10µM
IC50
SI
Cytotoxicity
IC50 Trypo.
µM (strain Y)
P. falciparum
(3D7-GFP)
µM ±SD
(CC50 RAW264.7/
IC50
P. falciparum)
Compd.
R1
R2
R3
R4
(RAW264.7)
CC50 µMa
3ª
3b
3c
3d
3e
3f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
H
H
H
H
H
H
-
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
H
H
H
-
H
Ph
Me
H
338.97
42.03
42.88
7.55
7.90
5.57
3.61
26.31
10.34
4.47
4.01
37.46
2.61
4.10
0.76
7.49
4.04
21.88
6.52
9.46
6.53
13.05
10.87
68.21
1.36
1.51
-
19.00
8.97
-
-
-
-
H
331.72
227.55
50.69
91.80
8.65
21.82
45.66
29.96
24.32
42.66
60.36
88.25
48.76
38.43
29.81
0.00
-
-
Br
-
-
Br
Ph
Me
H
4.90
-
-
Br
84.13
18.82
73.66
3.75
-
-
3g
3h
3i
F
296.04
140.47
246.84
311.61
48.80
-
-
F
Ph
Me
H
-
-
F
94.41
76.06
64.52
27.30
52.74
11.51
17.19
16.62
14.23
20.39
22.44
3.60
2.90
85.12
3j
MeO
MeO
MeO
NO2
NO2
NO2
Cl
-
-
3k
3l
Ph
Me
H
-
-
204.55
213.13
251.81
112.08
157.19
92.87
-
-
3m
3n
3o
3p
3q
3r
-
-
Ph
Me
H
22.99
30.56
59.14
0.00
-
-
-
-
-
-
Cl
Ph
Me
H
-
-
Cl
266.08
243.82
245.57
73.75
16.10
36.44
59.82
68.89
8.53
-
-
3s
3t
Cl
-
-
Cl
Ph
Me
H
-
-
3u
3v
3w
3x
BDZ
CQ
Cl
54.40
32.06
ND
3.82
19.31
Ph
Ph
Ph
-
48.54
-
-
Ph
Me
-
49.19
49.05
0.00
-
-
184.73
123.54
77.00
12.09
14.60
-
15.28
8.46
-
-
-
-
-
-
-
-
-
-
99.23*
0.01
7,700.00
BDZ = benznidazole. IC50 = inhibitory concentration for 50%. CC50 = cytotoxic concentration for 50%. CC50 and IC50 values were calculated using
concentrations in triplicate, and experiments were repeated, only values with a standard deviation < 10% mean were considered. SI= Selectivity index.
*challenged with 1M due to the correspondent IC50 values situated at nM range. CQ: chloroquine
which was attached a phenyl ring at 4 position. In this sub-series,
In the same way, compounds with withdrawer substituents, 3p,
3q, 3r (2,3-diCl), 3s, and 3u (2,4diCl) did not present good
trypanocidal activity. In contrast, compound 3t (2,4-diCl) and a
phenyl ring in N4 of thiosemicarbazone moiety, displayed a better
activity profile than benznidazole-treated parasites. Comparing
N4 substitution of thiosemicarbazone moiety, it was observed that
phenyl ring in N4 of thiosemicarbazone was generally beneficial
to the activity against trypomastigote form as can be seen in
compounds 3h and 3t (Table 1).
the majority of compounds exhibited a low antiparasitic activity
profile. Compounds 4b (OCH3) and 4c (Cl) present in para
position displayed trypanocidal effects compared with BDZ. Only
compound 4j, which possess fluorine in para position of phenyl
ring and another phenyl ring in N4, exhibited the most potent
activity of this series (Table 2). Phthalimido-thiosemicarbazones
derivatives, namely 3t (IC50= 3.60M), and 3h (IC50= 3.75M)
were more potent than phthalimido-thiazoles derivatives, of which
only compound 4j (IC50= 4.48M) showed potent trypanocidal
profile (Figure 2).
Concerning compounds 4a-n, they present no substitution in the
phenyl ring from 2-haloacetophenone and a thiazole nucleus,
3
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10.1002/cmdc.202000331
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FULL PAPER
Figure 2. The most active compounds against T. cruzi and P. Falciparum.
In vitro activity of compounds 3a-v and 4a-n against P.
falciparum (3D7-GFP)
production of GFP-protein increases, hence increasing green
fluorescence (GFP fluoresces in the FITC channel). The number
of fluorescent events (parasitized RBCs) detected by the flow
cytometer, represents the number (or %) of the surviving
parasites.[31] Cytotoxicity (CC50) of selected compounds for RAW
264.7 cells and selectivity index (SI) for P. falciparum strain 3D7-
GFP are reported in Tables 1 and 2.
The synthesized derivatives were first evaluated at 10 µM fixed
concentration for their growth inhibitory activity against the
erythrocytic stage of the GFP-expressing P. falciparum (3D7-
GFP). As the parasite develops inside the red blood cell, the
Table 2. Cytotoxicity and antiparasitic activity of phthalimido-thiazoles series (4a-n).
SI
(CC50
RAW264.7/
IC50 Tryp.)
P. falciparum
(3D7-GFP)
% of inhibition
at 10µM
IC50
SI
Cytotoxicity
(RAW264.7)
CC50 µMa
IC50 Trypo.
µM (strain Y)
P. falciparum
(3D7-GFP)
µM ±SD
(CC50 RAW264.7/
IC50
P. falciparum)
Cpd.
R4
R5
Ar
4a
4b
4c
4d
4e
4f
-
-
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
-
3-NO2-Ph
4-MeO-Ph
3,4-diCl-Ph
4-F-Ph
>413
385
51.62
8.94
>8,00
43.06
32.03
>29.49
1.19
14.8
61.5
44.1
50.7
10.5
14.5
17.8
23.8
28.5
70.2
81.9
89.0
82.5
86.8
-
-
-
-
-
-
194.44
>438
59.7
6.07
-
-
-
14.85
50.09
28.12
54.02
12.26
14.06
4.48
-
-
-
4-Br-Ph
2,4-diCl-Ph
2-Naph
4-NO2-Ph
4-Cl-Ph
4-MeO-Ph
3-NO2-Ph
4-F-Ph
-
-
-
-
>394
123
>14.01
2.28
-
4g
4h
4i
-
-
-
-
-
>413
111.9
178
>33.69
3.93
-
-
-
-
4j
Ph
Ph
Ph
Ph
Ph
-
39.73
>35.24
2.00
3.54
3.69
1.24
1.78
2.41
-
50.28
>96.75
>302.42
>198.99
79.79
-
4k
4l
>357
>375
>354.2
192.3
123.54
77.00
10.13
187.76
177.10
39.60
14.60
-
4m
4n
BDZ
CQ
2-Naph
4-NO2-Ph
-
2.00
4.86
8.46
-
-
-
-
99.23*
0.01
7,700.00
BDZ = benznidazole. IC50 = inhibitory concentration for 50%. CC50 = cytotoxic concentration for 50%. CC50 and IC50 values were calculated using
concentrations in triplicate and experiment were repeated, only values with a standard deviation < 10% mean were considered. SI= Selectivity index.
*challenged with 1µM due to the correspondent IC50 values situated at nM range. CQ: chloroquine
4
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Growth inhibition assays (as the estimation of IC50) using flow
cytometry-based methods have been shown to be highly
reproducible, scalable for efficient use in screening antimalarial
activity of new compounds and comparable in terms of sensitivity
to alternative methods such as hypoxanthine uptake.[32] The use
of Plasmodium lines expressing green fluorescent proteins has
enhanced the sensitivity and reproducibility of flow cytometry
methods used to screen the antimalarial activity of new
compounds.[33] Scatter plots data from one representative assay
are shown in Figure 3. The percentage of survival parasites
determined as in Figure 3 was used to determine the % of
inhibition at 10µM, presented in Tables 1 and 2 and to generate
a dose-response curve, from which the correspondent IC50 was
determined (Tables 1 and 2).
lipophilicity, consistent with
a
higher antimalarial activity.
Regarding the selective index against the parasite, compound 4l
presented an SI value higher than 300, a desirable characteristic
(SI>10) for an antimalarial candidate molecule (Table 2).
Compound 4j was active for both parasites tested (IC50= 4.48M
for T. cruzi and 3.54M for P. falciparum) (Table 2).
Flow cytometry analysis for trypomastigote form of T. cruzi
It was possible to evaluate the effects of the most active
compounds using flow cytometry as a tool (3h, 3t and 4j), as well
as BDZ, on the induction of cell death by apoptosis or necrosis. It
was observed that compound 4j (IC50 and 2x IC50) was able to
induce significantly labeling compatible with necrosis and
apoptosis in trypomastigotes. Similar results were found, in higher
proportions, in parasites treated with BDZ, the reference drug and
positive control used in this assay. (supp. material, Figure S2).
Compounds 3h and 3t, despite having demonstrated trypanocidal
potential in the initial evaluation tests, they do not demonstrate
the induction of cell death due to apoptosis or necrosis, with the
methodology used (data not show).
The reference antimalarial chloroquine (CQ) was included as
control and resulted in IC50 value in agreement with previous
results.[34,35]
All compounds were first screened for antimalarial activity against
P. falciparum at a fixed dose of 10µM, and those exhibiting over
around 70% growth inhibition were selected as active and
confirmed by IC50 estimation. For the phthalimido-
thiosemicarbazones (3a-x), only compounds 3i (88%) and 3u
(68.82%) were considered for refinement of activity.
Ultrastructural studies for T. cruzi
In the phthalimido-thiazoles series (4a-n), compounds 4j (70.2%),
4k (81.9%), 4l (89.0%), 4m (82.5%), and 4n (86.8%)
demonstrated considerable antimalarial activity, so were also
further evaluated. Dose-response curves to determine half-
maximal inhibitory concentrations (IC50) against P. falciparum
strain 3D7-GFP were determined based on cytometry evaluation
of parasite growth.
In view to investigate the effects of phthalimido-
thiosemicarbazones and phthalimido-thiazoles on parasite
morphology, the most active compounds of this work (3h, 3t, and
4j) were selected. Analysis by scanning electron microscopy
(SEM) showed that T. cruzi trypomastigote cells treated with the
compounds 3h, 3t and 4j at IC50 and 2x IC50 concentrations (3.75,
3.60, and 4.48µM respectively), promoted changes in the shape,
flagella and surface of the parasite body, compared to the
untreated control group.
Compounds 3h and 3t showing body writhing, appearance
swollen and rounded, flagellum shortening, besides the presence
of filamentous structures on the cell surface, while 4j caused
changes similar to those observed in BDZ-treated cells including
extravasation of the cytoplasmic content. It was also observed
that at IC50 and 2x IC50 values, the compounds promoted the
same morphological changes in the parasites. (supp. material,
Figure S3).
In the present study, the activity of these compounds (3h, 3t and
4j) can be seen through ultrastructural alterations that can lead to
cell unavailability, reinforcing the findings by Menna-Barreto et
al.[36] who analyzing different cell death pathways induced by
drugs in T. cruzi, suggest that
a significant number of
morphological alterations is always related to loss of cell viability
and parasite death. While the changes caused by 3h and 3t are
described in the literature as typical events of death due to
apoptosis even when, despite the changes in the plasma
membrane, it can remain intact,[37] changes by treatment 4j as
leakage of cytoplasmic content suggest death by necrosis or
apoptosis. Thus, all SEM and FACS data are in agreement.
Physicochemical and ADME parameters
Figure 3. Evaluation of Plasmodium falciparum survival using flow cytometry.
Scatter plots showing the gating signals of: uninfected red blood cells as
negative control (top left panel); parasite culture after 72h of incubation without
drug, as positive control (top right panel); parasite culture after 72h of incubation
We evaluated if the synthesized compounds physicochemical
properties are within the Lipinski's Rule of Five, which are
essential for pharmacokinetics and drug development. For this
purpose, physicochemical and ADME properties were calculated
using the Swiss ADME (a free web tool to evaluate
pharmacokinetics, drug-likeness, and medicinal chemistry
friendliness of small molecules). Compound obeying at least three
of the four criteria is considered to adhere to Lipinski Rule.[38] All
3a-x compounds are compatible with Lipinski Rule. For 4a-n,
compounds 4e and 4j-n are not compatible with Lipinski Rule.
Another attractive property is the topological polar surface area
(TPSA). Compounds with a low TPSA (≤140 Å2) tend to have
higher oral bioavailability.[39,40] Must synthesized compounds
have appropriate TPSA. Just 4a, 4k, and 4n have TPSA > 140 Å2.
with 10µM of tested compound 4l (bottom left panel); parasite culture after 72h
of incubation with 1µM of CQ as a control drug (bottom right panel). The
ordinate shows green fluorescence emission from GFP protein (FL1-H::FITC-
H), and the abscissa displays the side scatter (SSC-H::SSC-H) signal intensity.
Inside each panel is represented the percentage (GFP) of the total events
(parasitized red blood cells) recorded in that culture. RBC, red blood cells; GFP,
green fluorescent protein; CQ, chloroquine; 4l tested compound.
For phthalimido-thiosemicarbazone series, compounds 3i and 3u
presented IC50 values of 2.9M and 3.82M, respectively. The
best performing compounds (with lower IC50) belong to the
phthalimido-thiazoles series, being compounds 4l (IC50= 1.2M),
4m (IC50= 1.7M), and 4n (IC50= 2.4M) (Table 2). Phthalimido-
thiazoles derivatives, compounds 4j-m, present a phenyl ring at
N of R5 of thiazole nucleus; hence, they present higher
Compounds
shown
variable
permeability
based
on
gastrointestinal absorption (GI), according to the BOILED-Egg
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000331
ChemMedChem
FULL PAPER
predictive model (Brain or Intestine L Estimate D permeation
method). Most compounds have shown high gastrointestinal
absorption. With respect to oral bioavailability, it is expected 0.55
of the probability of oral bioavailability score > 10% in rat for most
compounds. All these data suggest the in silico good drug-
likeness profile and excellent chemical stabilities for this most
compounds (supp. material, Tables S1 and S2).
for 30 minutes at room temperature. 3.4 mmol of acetophenone (2-
Bromoacetophenone, 2-Bromo-4'-Fluoroacetophenone, 2-Bromo-4'-
Phenylacetophenone,
2,4'-Dibromoacetophenone,
2-Bromo-4'-
Methoxyacetophenone, 2-Bromo-3'-Nitroacetophenone, 2-Bromo-3 ', 4'-
Dichloroacetophenone and 2,2', 4'-Trichloroacetophenone) and then the
system remained under constant stirring for 2-24 h. In all cases, it was
necessary to add excess acetophenone in order to reach the end of the
reaction. To verify the formation of the products, TLCs were performed
with the reagents and the reaction mixture, using as an eluting solvent the
mixture of Ethyl Acetate and Hexane in a ratio of 2: 8.
The
mixture
Conclusion
was vacuum filtered, washed with hot acetone and the supernatant dried
on a rotary evaporator. The dry solid product was subjected to isopropyl
alcohol washing in a vacuum system. The sintered funnel containing the
product was packed in a desiccator for 24 hours, and then the
acetophenone was weighed to obtain the reaction yield data.
Twenty-four phthalimido-thiosemicarbazones (3a-x) and fourteen
phthalimido-thiazoles (4a-n) were obtained in reasonable yields
using
a simple methodology. Compounds with important
trypanocidal activity, especially compounds 3t (IC50= 3.60μM),
and 3h (IC50= 3.75μM), 4j (IC50= 4.48μM) were identified. Flow
cytometry and ultrastructural studies showed that compound 4j
was able to induce both necrosis and apoptosis in
trypomastigotes. Compound 3t, a phthalimido-thiosemicarbazone
derivative, was the most potent trypanocidal agent identified in
this work, presented a selective index of 68.60, which was
approximately 10 times more selective than BDZ, the standard
drug in clinical use. SEM analysis showed that 3h, 3t, and 4j
promoted changes in the shape, flagella, and surface of the
parasite body. Additionally, compound 4l, a phthalimide-thiazole,
exhibited antimalarial activity against sensitive and resistant P.
falciparum in the low micromolar range (IC50 < 10μM), no
cytotoxicity against RAW264.7 cells, a considerable selectivity
index (SI > 300). Our results indicated phthalimide-based
derivatives are promising molecules as potential anti-
Trypanosoma cruzi and/or antiplasmodial agents.
Synthesis of compounds 3a-x: In a round-bottomed flask containing 15
ml of ethyl alcohol, 300 mg of intermediate 3 was added along with 14
drops of P.A. Sulfuric Acid. The mixture was kept under ultrasound or
reflux for about 30 min. After that time, the equivalent molar amount of the
respective
thiosemicarbazide
(thiosemicarbazide,4-
phenylthiosemicarbazide, 4-methyl-3-thiosemicarbazide) was added to
the system. The system was left on ultrasound (4-12h) or reflux (24-48h).
In all cases, it was necessary to add excess thiosemicarbazide in order to
reach the end of the reaction. Thin Layer Chromatography (TLC) with the
reactants and the reaction mixture were prepared using the 3: 7 ethyl
acetate and hexane mixture as eluent solvent. The solid obtained was
subjected to vacuum filtration and washed with Ethyl Alcohol. The sintered
funnel containing the filtered solid was packed in a desiccator for 24 h and
then weighed the final product to obtain the reaction yield data.
(E)-2-(2-(1,3-dioxoisoindolin-2-yl)-1-
phenylethylidene)Hydrazinecarbothioamide (3a): Yield 87.99%; m.p.(°C)
178-180; Rf: 0.48 (hexane/ethyl acetate 7:3). ¹H NMR (DMSO-d6,
300MHz), δ ppm: 4.9 (s, 2H, CH 2), 7.34 (m, 3H, CH- Ar), 7.79 (m, 6H),
8.45 (s, 1H, NH2), 10.67 (s, 1H, NH); 13C NMR (DMSO-d6, 75MHz) δ ppm:
34.42 (CH2), 123.76 (C-Ar), 127.39 (C-Ar), 128.57 (C-Ar), 129.87 (C C-Ar),
131.56 (C-Ar), 135.21 (C-Ar), 135.74 (C-Ar), 145.19 (C = N), 167.90 (C =
O), 179.79 (C = S). HRMS:339.0788 [M - H]+.
Experimental Section
Chemistry: All reagents were used as purchased from commercial
sources (Sigma Aldrich, Acros Organics, Vetec, or Fluka). Reaction
progress was followed by thin-layer chromatography (TLC) analysis
(Merck, silica gel 60 F254 in aluminum foil). Melting points were determined
on a Fisatom 430D electrothermal capillary melting point apparatus and
were uncorrected. NMR spectra were measured on either a Varian Unity
(E)-2-(2-(1,3-dioxoisoindolin-2-yl)-1-phenylethylidene)-N-
phenylhydrazinecarbothioamide (3b): Yield 89.62%; m.p. (°C) 223-224; Rf:
0.76 (hexane/ethyl acetate 7:3). 1H NMR (DMSO-d6, 300MHz), δ ppm:
4.81 (s, 1H, NH), 5.01 (s, 2H, CH2), 7.26 (m, 1H), 7.36 (m, 6H, CH-Ar),
7.58 (m, 1H, CH-Ar), 7.83 (m, 6H, CH-Ar), 10.14 (s, 1H, NH); 13C NMR
(DMSO-d6, 75MHz) δppm: 34.21 (CH2), 123.30 (C-Ar), 123.87 (C-Ar),
125.85 (C-Ar), 127.32 (C C-Ar), 128.09 (C-Ar), 129.30 (C-Ar), 131.10 (C-
Ar), 134.75 (C-Ar), 135.12 (C- 139.00 (C-Ar), 145.57 (C = N) 147.50 (C-
Ar), 167.48 (C = O), 177.42 (C = S). HRMS: 415.0841[M - H]+.
1
Plus 400 MHz (400 MHz for H and 100 MHz for 13C) or a Bruker AMX-
300 MHz (300 MHz for 1H and 75.5 MHz for 13C) instrument. DMSO-d6 and
D2O were purchased from CIL or Sigma Aldrich. Chemical shifts were
reported in ppm, and multiplicities were given as s (singlet), d (doublet), t
(triplet), m (multiplet), dd (double doublet), and coupling constants (J) in
hertz. Mass spectrometry experiments were performed on an LC-IT-TOF
(Shimadzu). Unless otherwise specified, ESI was conducted in positive ion
mode. Typical conditions were as follows: capillary voltage of 3kV, cone
voltage of 30V, and peak scan between 50 and 1000 m/z.
(E)-2-(2-(1,3-dioxoisoindolin-2-yl)-1-phenylethylidene)-N-
methylhydrazinecarbothioamide (3c): Yield 50.75%; m.p. (°C) 148-149; Rf:
0.70 (hexane/ethyl acetate 7:3). 1H NMR (DMSO-d6, 300MHz), δ ppm:
3.06 (d, 3H, CH3), 5.24 (s, 2H, CH2), 7.31 (d, J = 6Hz, 2H, Ar), 7.56 (m, 4H,
CH-Ar), 7.94 (m, 4H, CH-Ar), 8.1 (d, J = 6 Hz, 2H, CH-Ar), 8.54 (d, J = 3
Hz, 1H, NH), 10.70 (s, 1H, NH);13 C NMR (DMSO-d6, 75MHz) δ ppm: 31.17
(CH 3), 44.46 (CH 2), 123.25 (C-Ar), 126.90 (C- Ar), 129.34 (C-Ar), 131.51
(C-Ar), 134.52 (C-Ar), 134.76 (C-Ar), 144.50, (C = O), 178.92 (C = S).
HRMS: 353.0956 [M+H]+.
General procedures for the synthesis of compounds 3a-x:
Synthesis of Intermediate 2: 4.05 g (67.43 mmol) of urea were blended
together with 10.0 g (67.52 mmol) of phthalic anhydride. The powder
mixture was transferred to a round-bottomed flask and left in an oil bath
with heating to reach the temperature of about 156 ° C for approximately
3h with the half-opened flask. During this time, the compounds were
melted, and the new solid formed (a white powder), in addition to gas
evolution. A TLC was carried out with the phthalic anhydride, and the
product obtained, using an eluent system the mixture of Hexane / Ethyl
Acetate in a ratio of 7: 3. For purification, the solid obtained was washed
with water in a vacuum filtration system. The sintered funnel containing the
product was packed in a desiccator for 48h, and then the phthalimide (Int.
1) was weighed to obtain the reaction yield data.
(E)-2-(1-(4-bromophenyl)-2-(1,3-dioxoisoindolin-2-yl)ethylidene)
hydrazinecarbothioamide (3d): Yield 93.89%; m.p. (°C) 206-208; Rf: 0.48
(hexane/ethyl acetate 7:3). 1H NMR (DMSO-d6, 400MHz), δ ppm: 4.90 (s,
2H, CH2), 7.49 (d, J = 8Hz, CH-Ar), 7.77 (m, 4H, CH-Ar), 8.49 (s, 2H, NH2),
10.69 (s, 1H, NH);13 C NMR (DMSO-d6, 100MHz) δ ppm: 33.65 (CH2),
123.32 (C-Ar), 128.99 (C-Ar), 131.02 (C- C-Ar), 134.62 (C-Ar), 134.76 (C-
Ar), 143.21 (C = N), 167.45 (C = O), 179.33 (C = S). HRMS: 418.9856
[M+H]+.
Synthesis of Intermediate 3: Into a round bottom flask, 15 ml of acetone
P.A., 3.4 mmol of phthalimide (0.50 g), 4.6 mmol K2CO3 (0.47 g), and a
solid KI spatula tip were added. The mixture was allowed to stir vigorously
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000331
ChemMedChem
FULL PAPER
(E)-2-(1-(4-bromophenyl)-2-(1,3-dioxoisoindolin-2-yl)ethylidene)-N-
phenylhydrazinecarbothioamide (3e). Yield 94.58%; m.p. (°C) 197-199; Rf:
0.66 (hexane/ethyl acetate 7:3). NMR ¹H (DMSO-d6, 400MHz), δ ppm:
4.99 (s, 2H, CH2), 7.23 (m, 1H, CH- Ar), 7.34 (m, 4H, 7.38 (m, 2H, CH-Ar),
7.54 (m, 2H, CH- Ar), 7.84 (m, 4H, CH- Ar), 10.20 (s, 1H, NH), 11.05 (s,
1H, NH);13C NMR (DMSO-d6, 100MHz) δ ppm: 33.97 (CH2), 122.95 (C-Ar),
123.39 (C-Ar), 125.63 (C-Ar), 126.12 ( C-Ar), 128.14 (C-Ar), 129.27 (C-Ar),
131.07 (C-Ar), 131.13 (C-Ar), 134.47 134.84 (C-Ar), 139.02 (C-Ar), 144.13
(C = N), 167.55 (C = O), 177.47 (C = S). HRMS: 493.0031 [M+H]+.
(C = N), 160.31 (C-Ar), 167.51 (C = O), 177.12 (C = S). HRMS: 445.1262
[M+H]+
.
(E)-2-(2-(1,3-dioxoisoindolin-2-yl)-1-(4-methoxyphenyl)ethylidene)-N-
methylhydrazinecarbothioamide (3l): Yield 77.77%; m.p. (°C) 189-191; Rf:
0.76 (hexane/ethyl acetate 7:3). NMR ¹H (DMSO-d6, 400MHz), δ ppm:
3.05 (d, J = 4.4 Hz, 3H, CH3), 3.31 (s, 3H, CH3), 4.88 (s, 2H (M, 2H, CH-
Ar), 10.54 (m, 6H, CH-Ar), 8.4 (s, 1H, NH); 13C-NMR (DMSO-d6, 100MHz)
δ ppm: 31.13 (CH 3), 30.70 (CH2), 55.11 (CH 3 -O) 113.51 (C-Ar), 123.16
(C-Ar) ), 127.64 (C-Ar), 128.42 (C-Ar), 131.10 (C-Ar), 138.30 (C-Ar),
144.28 (C = (CO), 167.32 (C = O), 178.78 (C = S). HRMS: 383.1166
(E)-2-(1-(4-bromophenyl)-2-(1,3-dioxoisoindolin-2-yl)ethylidene)-N-
methylhydrazinecarbothioamide (3f): Yield 75.96%; m.p. (°C) 191-193; Rf:
0.70 (hexane/ethyl acetate 7:3). NMR ¹H (DMSO-d6, 400MHz), δ ppm:
3.05 (d, J = 4.8 Hz, 3H, CH3), 4.91 (s, 2H, CH2), 7.52 (d, J = 8). (D, J = 8.4
Hz, 2H, CH-Ar), 7.81 (m, 4H, CH-Ar), 8.60 (d, J = 4.4 Hz, 1H, NH), 10.72
(s, 1H, NH);13C NMR (DMSO-d6, 100MHz) δ ppm: 31.18 (CH3), 33.70
(CH2), 122.65 (C-Ar), 123.30 (C- Ar), 131.02 (C-Ar), 131.09 (C-Ar), 134.69
(C-Ar), 134.74 (C-Ar), 143.00 (C = O), 178.86 (C = S). HRMS: 430.9623
[M+H]+
.
2-(2-(4-nitrophenyl)-2-oxoethyl)isoindoline-1,3-dione (3m): Yield 91.29%;
m.p. (°C) 217-218; Rf: 0.38 (ethyl acetate/hexane 3:7). ¹H NMR (DMSO-
d6, 300MHz), δ ppm: 5.00 (s, 2H, CH2), 7.61 (m, 2H, CH-Ar), 7.89 (m, 2H,
CH-Ar), 8,26 (m, 2H, CH-Ar), 8.58 (m, 2H, CH-Ar), 8.76 (s, 2H, NH2), 10.79
(s,1H, NH). 13C NMR (DMSO-d6, 75MHz) δ ppm: 44.71 (CH2), 123.31 (C-
Ar), 128.32 (C-Ar), 130.76 (C-Ar), 131.44(C-Ar), 134.47 (C-Ar), 137.18 (C-
Ar), 141.87 (C=N), 147.88 (C-Ar), 167.53 (C=O), 179.45 (C=S). HRMS:
384.0742 [M+H]+.
[M+H]+
.
(E)-2-(2-(1,3-dioxoisoindolin-2-yl)-1-(4-
fluorophenyl)ethylidene)hydrazinecarbothioamide (3g): Yield 63.33%; m.p.
(°C) 198-200; Rf: 0.28 (hexane/ethyl acetate 7:3). 1H NMR (DMSO-d6,
400MHz), δ ppm: 4.92 (s, 2H, CH2), 7.15 (t, J = 8.8 Hz, 2H, 6H, CH-Ar),
8.47 (s, 1H, NH), 10.67 (s, 2H, NH2); 13C NMR (DMSO-d6, 100MHz) δ ppm:
34.31 (CH2), 115.34 (C-Ar), 123.61 (C-Ar), 129.66 (C-Ar), 132.27 (C C-Ar),
134.98 (C-Ar), 143.97 (C = N), 146.58 (C-Ar), 161.85 (CF), 167.90 (C = O)
(E)-2-(2-(1,3-dioxoisoindolin-2-yl)-1-(4-nitrophenyl)ethylidene)-N-
phenylhydrazinecarbothioamide (3n): Yield 94.81%; %; m.p. (°C) 211-213;
Rf: 0.44 (ethyl acetate/hexane 3:7). ¹H NMR (DMSO-d6, 400MHz), δ
:
ppm
5.08 (s, 2H, CH2), 7.24 (m, 1H, CH-Ar), 7.41 (m, 2H, CH-Ar), 7.54 (d, J=7,2
Hz, 2H, CH-Ar), 7.85 (m, 4H, CH-Ar), 8.17 (d, J=8,4 Hz, 2H, CH-Ar), 8.39
(d, J=8 Hz, 2H, CH-Ar), 10.33 (s, 1H, NH), 11,12 (s, 1H, NH). 13C NMR
(DMSO-d6, 100MHz) δ ppm: 33.96 (CH2), 121.95 (C-Ar), 123.38 (C-Ar),
125.74 (C-Ar), 126.21 (C-Ar), 128.17 (C-Ar), 129.61 (C-Ar), 131.16 (C-Ar),
133.65 (C-Ar), 134.80 (C-Ar), 137.05 (C-Ar), 139.00 (C-Ar), 142.78 (C=N),
179.74 (C = S). HRMS: 357.0707 [M+H]+
.
(E)-2-(2-(1,3-dioxoisoindolin-2-yl)-1-(4-fluorophenyl)ethylidene)-N-
phenylhydrazinecarbothioamide (3h): Yield 67.62%; m.p. (°C) 205-207; Rf:
0.88 (hexane/ethyl acetate 7:3). 1H NMR (DMSO-d6, 400MHz), δ ppm:
5.00 (s, 2H, CH2), 7.21 (m, 2H, CH- Ar), 7.39 (m, 2H, 7.5 (m, 2H, CH-Ar),
7.58 (d, J = 8Hz, 2H, CH- Ar), 7.83 (m, 4H, , CH-Ar), 10.18 (s, 1H, NH),
11.01 (s, 1H, NH); 13C NMR (DMSO-d6, 100MHz) δppm: 34.63 (CH2),
115.39 (C-Ar), 123.80 (C-Ar), 125.99 (C-Ar), 126.45 C-Ar), 128.55 (C-Ar),
129.96 (C-Ar), 132.10 (C-Ar), 132.12 (C-Ar), 135.24 139.48 (C-Ar), 144.82
(C = N), 164.45 (CF), 167.98 (C = O), 178.88 (C = S). HRMS: 433.1117
147.87 (C-N), 167.63 (C=O), 177.65 (C=S). HRMS: 460.0442 [M+H]+
.
(E)-2-(2-(1,3-dioxoisoindolin-2-yl)-1-(4-nitrophenyl)ethylidene)-N-
methylhydrazinecarbothioamide (3o): Yield 86.97%; m.p. (°C) 148-149; Rf:
0.4 (ethyl acetate/hexane 3:7). ¹H NMR (DMSO-d6, 400MHz), δ ppm: 3.07
(d, J=3.6 Hz, 3H, CH3), 5.00 (s, 2H, CH2), 7.81 (m, 4H, CH-Ar), 8.16 (d,
J=8.4Hz, 2H, CH-Ar), 8.32 (d, J=7.6 Hz, 2H, CH-Ar), 8.74 (d, J=3.6 Hz, 1H,
NH2), 10.81 (s, 1H, NH). 13C NMR (DMSO-d6, 100MHz) δ ppm: 31.26 (CH3),
33.76 (CH2), 123.34 (C-Ar), 129.58 (C-Ar), 131.45 (C-Ar), 133.35 (C-Ar),
134.74 (C-Ar), 137.29 (C-Ar), 141.89 (C=N), 147.89 (C-Ar), 167.57 (C=O),
178.88 (C=S). HRMS: 398.0510 [M+H]+.
[M+H]+
.
(E)-2-(2-(1,3-dioxoisoindolin-2-yl)-1-(4-fluorophenyl)ethylidene)-N-
methylhydrazinecarbothioamide (3i): Yield 55.42%; m.p. (°C) 188-190; Rf:
1
55.42 (hexane/ethyl acetate 7:3). H NMR (DMSO-d6, 400MHz), δ ppm:
3.06 (d, J = 4.8 Hz, 3H, CH3), 4.92 (s, 2H, CH2), 7.17 (t, J = 9 Hz, 2H, CH-
Ar), 7.84 (m, 6H, CH-Ar), 8.58 (d, J = 4 Hz, 1H, NH), 10.69 (s, 1H, NH);
13C NMR (DMSO-d6, 100MHz) δ ppm: 31.12 (CH3), 33.86 (CH 2), 114.86
(C-Ar), 123.13 (C- Ar), 131.04 (C-Ar), 131.82 (C-Ar), 134.51 (C-Ar), 143.32
(C = N), 161.34 (CF), 167.43 (C C = O), 178.83 (C = S). HRMS: 371.0997
(E)-2-(1-(3,4-dichlorophenyl)-2-(1,3-dioxoisoindolin-2-
yl)ethylidene)hydrazinecarbothioamide (3p). Yield 94.5%; m.p. (°C) 233-
234; Rf: 0,56 (ethyl acetate/hexane 3:7) ¹H NMR (DMSO-d6 400MHz),, δ
ppm: 4.91 (s, 2H, CH2), 7,57 (d, J=8.4 Hz, 1H, CH-Ar), 7.84 (m, 4H, CH-Ar),
8.18 (s, 1H, CH-Ar), 8.27 (s, 1H, CH-Ar), 8.54 (s, 2H, NH2), 10.71 (s,1H,
NH). 13C NMR (DMSO-d6. 100MHz) δ ppm: 33.44 (CH2), 123.32 (C-Ar),
126.90 (C-Ar), 128.83 (C-Ar), 130.09 (C-Ar), 131.10 (C-Ar), 131.21 (C-Ar),
131.70 (C-Ar), 134.74 (C-Cl), 136.03 (C-Cl), 141.53 (C=N), 167.55 (C=O),
179.33 (C=S). HRMS: 406.9683 [M+H]+.
[M+H]+
.
(E)-2-(2-(1,3-dioxoisoindolin-2-yl)-1-(4-
methoxyphenyl)ethylidene)hydrazinecarbothioamide (3j): Yield 94.10%;
1
m.p. (°C) 179-181; Rf: 0.34 (hexane/ethyl acetate 7:3). H NMR (DMSO-
d6, 400MHz), δ ppm: 3.71 (s, 3H, CH3), 4.88 (s, 2H, CH2), 6.85 (d, J = 8.8
Hz, CH-Ar), 7.80 (m, 6H, CH-Ar), 8.41 (s, 2H, NH2), 10.60 (s, 1H, NH); 13
C NMR (DMSO-d6, 100MHz) δ ppm: 33,69 (CH2), 55,13 (CH3-O), 113,53
(C-Ar), 123,32 (C-Ar), 127,57 (C-Ar), 128,46(C-Ar), 131,11 (C-Ar), 134,76
(C-Ar), 144,48 (C=N), 160,14 (C-O), 167,48 (C=O), 179,05 (C=S). HRMS:
(E)-2-(1-(3,4-dichlorophenyl)-2-(1,3-dioxoisoindolin-2-yl)ethylidene)-N-
phenylhydrazinecarbothioamide (3q). Yield 90.99; m.p. (°C) 214-216; Rf:
0.82. (ethyl acetate/hexane 3:7). ¹H NMR (DMSO-d6 400MHz), δ ppm: 4.99
(s, 2H, CH2), 7.30 (m, 1H, CH-Ar), 7.40 (m, 2H, CH-Ar), 7.56 (m, 3H, CH-
Ar), 7.86 (m, 6H, CH-Ar); 10.29 (s, 1H, NH), 11.05 (s, 1H, NH). 13C NMR
(DMSO-d6 100MHz) δ ppm: 33.76 (CH2), 123.38 (C-Ar), 125.76 (C-Ar),
126.40 (C-Ar), 127.21 (C-Ar), 128.14 (C-Ar), 129.98 (C-Ar), 130.15 (C-Ar),
131.16 (C-Ar), 131.88 (C-Ar), 134.81 (C-Ar), 135.92 (C-Ar), 139.03 (C-Ar),
369.1122 [M+H]+
.
(E)-2-(2-(1,3-dioxoisoindolin-2-yl)-1-(4-methoxyphenyl)ethylidene)-N-
phenylhydrazinecarbothioamide (3k): Yield 96.67%; m.p. (°C) 219-221; Rf:
0.80 (hexane/ethyl acetate 7:3). ¹H NMR (DMSO-d6, 400MHz), δ ppm:
3.73 (s, 3H, CH3), 4.96 (s, 2H, CH2), 6.88 (d, J = 8.8 Hz, 2H, CH-Ar), 7.22
(m, 1H, CH-Ar), 7.39 (m, 2H, CH- Ar), 7.59 (d, J = 7.6 Hz, , 7.85 (m, 6H,
CH-Ar), 10.09 (s, 1H, NH), 10.92 (s, 1H, NH). 13C NMR (DMSO-d6,
100MHz) δ ppm: 33.90 (CH2), 55.24 (CH3 -O), 113.54 (C-Ar), 123.35 (C-
Ar), 125.73 ( C-Ar), 127.39 (C-Ar), 128.05 (C-Ar), 128.22 (C-Ar), 128.71
(C-Ar), 129.04 (C-Ar), 131.12 (C-Ar), 134.78 (C-Ar), 139.03 (C-Ar), 147.38
142.47 (C=N), 167.63 (C=O), 177.60 (C=S). HRMS: 483.0058 [M+H]+
.
(E)-2-(1-(3,4-dichlorophenyl)-2-(1,3-dioxoisoindolin-2-yl)ethylidene)-N-
methylhydrazinecarbothioamide (3r): Yield 59,52; m.p. (°C) 209-210; Rf:
0,78. (ethyl acetate/hexane 3:7). ¹H NMR (DMSO-d6. 400MHz), δ ppm: 3.07
(d, J=4.8Hz, 3H, CH3), 4.91 (s, 2H, CH2), 7.59 (d, J=8,4 Hz, 1H, CH-Ar),
7.82 (m, 4H, CH-Ar), 8.15 (d, J=2 Hz, 2H, CH-Ar), 8.71 (d, J=4.4 Hz, 1H,
NH), 10.74 (s, 1H, NH). 13C NMR (DMSO-d6. 100MHz) δ ppm: 31.27 (CH3),
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000331
ChemMedChem
FULL PAPER
33.52 (CH2), 123.35 (C-Ar), 126.96 (C-Ar), 128.78 (C-Ar), 130.18 (C-Ar),
131.13 (C-Ar), 131.19 (C-Ar), 131.67 (C-Cl), 134.78 (C-Cl), 141.43 (C=N),
Synthesis of compounds 4a-i: 500mg (14.8mmol) of compound 3a was
solubilized in 20mL of isobutanol at reflux, and different acetophenones
were added as shown in table 2, after 6h a plate was made, and the
reaction was confirmed to have come to an end as soon as possible. Then
the product was filtered and washed with ethanol and taken to the
desiccator for subsequent reaction yield.
167.60 (C=O), 178.79 (C=S). HRMS: 421.0617 [M+H]+
.
(E)-2-(1-(2,4-dichlorophenyl)-2-(1,3-dioxoisoindolin-2-
yl)ethylidene)hydrazinecarbothioamide (3s): Yield 64.10%; m.p. (°C) 194-
195; Rf: 0.52 (ethyl acetate/hexane 3:7). ¹H NMR (DMSO-d6 400MHz), δ
ppm: 3.32 (s, 2H, CH2), 7.30 (d, J=8,4 Hz, 1H, CH-Ar), 7.47 (d, J=8 Hz, 1H,
CH-Ar), 7.69 (s, 1H, CH-Ar), 7.84 (m, 4H, CH-Ar), 8.32 (s, 1H, NH), 10.04
(s, 2H, NH2). 13C NMR (DMSO-d6 100MHz) δ ppm: 42.52 (CH2), 123.21 (C-
Ar), 128.02 (C-Ar), 129.42 (C-Ar), 129.84 (C-Ar), 131.35 (C-Ar), 132.70 (C-
Ar), 134.58 (C-Ar), 135.23 (C-Ar), 142.00 (C=N), 167.19 (C=O), 179.10
(E)-2-(2-(2-(4-(3-nitrophenyl)thiazol-2-yl)hydrazono)-2-
phenylethyl)isoindoline-1,3-dione (4a): Yield 81.25%; m.p.(°C) 198-202;
Rf: 0.45 (hexane/ethyl acetate 7:3). ¹H NMR (DMSO-d6. 400 MHz), δppm
4.70 (s, 2H, CH2), 5.02 (s, 1H, N-H); 7.43 (s, 1H, C5-Hthiazole); 7.53 (s, 1H,
CHar); 8.74-7.72 (m, 12H, CH-Ar). 13C NMR (DMSO-d6. 100 MHz), δppm
:
:
(C=S). HRMS: 406.9683 [M+H]+
34.24 (-CH2); 107.16 (C5-thiazole); 143.94 (C=N), 167.56 (C=O), 167.28
(C=O), 169,93 (C2-thiazole), 148,28 (C4-thiazole), 136.10 (CH-Ar), 135.42
(CH-Ar), 134.7 (CH-Ar), 123.25 (CH-Ar), 122.04 (CH-Ar), 121.90 (CH-Ar),
119.94 (CH-Ar), 119.80 (CH-Ar), 131.07-126.0 (CH-Ar). HRMS: 483.5244
.
(E)-2-(1-(2,4-dichlorophenyl)-2-(1,3-dioxoisoindolin-2-yl)ethylidene)-N-
phenylhydrazinecarbothioamide (3t): Yield 81.47%; m.p. (°C) 177-179; Rf:
0.88 (ethyl acetate/hexane 3:7). ¹H NMR (DMSO-d6 400MHz), δ ppm: 4.75
(s, 2H, CH2), 7.16 (1H, CH-Ar), 7.32 (m, 2H, CH-Ar), 7.44 (m, 2H, CH-Ar),
7.56 (m, 2H, CH-Ar), 7.71 (s, 1H, C-Ar), 7.85(m, 4H, C-Ar). 13C NMR
(DMSO-d6 100MHz) δ ppm: 42.54 (CH2), 123.31 (C-Ar), 125.35 (C-Ar),
127.00 (C-Ar), 127.98 (C-Ar), 128.07 (C-Ar), 128.64 (C-Ar), 129.82 (C-Ar),
131.04 (C-Ar), 131.34 (C-Ar), 132.81 (C-Ar), 133.90 (C-Ar), 134.70 (C-Ar),
135.31 (C-Ar), 138.71 (C-Ar), 142.25 (C=N), 167.26 (C=O), 177.61 (C=S).
[M-H]+
.
(E)-2-(2-(2-(4-(4-methoxyphenyl)thiazol-2-yl)hydrazono)-2-
phenylethyl)isoindoline-1,3-dione (4b): Yield 94.80%; m.p. (°C) 174-178;
Rf: 0.40 (hexane/ethyl acetate 7:3). ¹H NMR (DMSO-d6. 400 MHz), δppm
5.01 (s, 2H, CH2); 3.79 (t, 3H, CH3); 7.17ppm (C5-H-thiazole); 5.01ppm (N-
H); 7.80 -7.6 (m, 12H, C-H Ar). 13C NMR (DMSO-d6. 100 MHz), δppm
:
:
HRMS: 483.0327 [M+H]+
169.82 31 (C2-thiazole), 167.63 (C=O), 167.40 (C=O), 159,03 (C4-
thiazole); C=N (144.28); 101.95 (C5-thiazole); 55.62 (CH2); 34.06 (CH3);
.
135.54 (C-OCH3); 134.69 – 12.318 (CH-Ar). HRMS: 466.5547 [M-H]+
.
(E)-2-(1-(2,4-dichlorophenyl)-2-(1,3-dioxoisoindolin-2-yl)ethylidene)-N-
methylhydrazinecarbothioamide (3u): Yield 62.69; m.p. (°C) 204-205; Rf:
0,72 (ethyl acetate/hexane 3:7). ¹H NMR (DMSO-d6 . 400MHz), δ ppm: :
2,95 (d, J=4.4Hz, 3H, CH3), 4.89 (s, 2H, CH2), 7.27 (s, 1H, CH-Ar), 7.43
(d, J= 2Hz, 1H, CH-Ar), 7.65 (d, J=2 Hz, 1H, CH-Ar), 7.83 (m, 4H, CH-Ar),
8,45 (d, J=4.8 Hz, 1H, NH), 9,95 (s, 1H, NH). 13C NMR (DMSO-d6.
100MHz) δ ppm: 31.07 (CH3), 42.79 (CH2), 123.22 (C-Ar), 127.01 (C-Ar),
128,58 (C-Ar), 129,36 (C-Ar), 131,32 (C-Ar), 13,53 (C-Ar), 133,13 (C-Ar),
134. 06 (C-Ar), 134.59 (C-Ar), 141.,37 (C=N), 167.19 (C=O), 179.15 (C=S).
(E)-2-(2-(2-(4-(3,4-dichlorophenyl)thiazol-2-yl)hydrazono)-2-
phenylethyl)isoindoline-1,3-dione (4c): Yield 54.32%; m.p. (°C) 189-193;
Rf: 0.66 (hexane/ethyl acetate 7:3). ¹H NMR (DMSO-d6. 400 MHz), δppm
:
4.69 (s, 2H, CH2); 5.00 (s, 1H, N-H); 7.33 (s, 1H, C5-H-thiazole); 7.51 (s,
1H, CH-Ar); 7.30 – 7.58 (m, 12H, CH-Ar). 13C NMR (DMSO-d6. 100 MHz),
δppm: 167.56; 167.30] (2C=O); 169,03 (C2-thiazole); 106.72 (C5-thiazole);
144.01 (C4-thiazole); 42,35 (CH2); 153.96 (C=N); 135.65 – 123.25 (CH-
HRMS: 421.0082 [M+H]+
Ar). HRMS: 507.6055 [M-H]+
.
.
(E)-2-(1-([1,1'-biphenyl]-4-yl)-2-(1,3-dioxoisoindolin-2-
(E)-2-(2-(2-(4-(4-fluorophenyl)thiazol-2-yl)hydrazono)-2-
yl)ethylidene)hydrazinecarbothioamide (3v). Yield 78.02; m.p. (°C) 232-
234; Rf: 0,46 (ethyl acetate/hexane 3:7). ¹H NMR (DMSO-d6 . 400MHz), δ
ppm: 4.96 (s, 2H, CH2), 7.45 (m, 1H, CH-Ar), 7.63 (m, 4H, CH-Ar), 7.70 (d,
J=7.6 Hz, 4H, CH-Ar), 7.81 (m, 2H, CH-Ar) 7.92 (d, J=78.4 Hz, 2H, CH-
Ar), 9,21 (s, 2H, NH2), 10.69 (s, 1H, NH). 13C NMR (DMSO-d6. 100MHz)
δ ppm: 42.57 (CH2), 123.35 (C-Ar), 126.28 (C-Ar), 127.52 (C-Ar), 128.92(C-
Ar), 129.82 (C-Ar), 131.46 (C-Ar), 134.53 (C-Ar), 140.70 (C-Ar), 141.50
(C=N), 143.93 (C-Ar), 146.81 (C-Ar), 167.54 (C=O), 179.30 (C=S). HRMS:
phenylethyl)isoindoline-1,3-dione (4d): Yield 50.88%; m.p. (°C) 180-184;
Rf: 0.59 (hexane/ethyl acetate 7:3). ¹H NMR (DMSO-d6. 400 MHz), δppm
:
4.70 (s, 2H, CH2); 5.01 (s, 1H, N-H); 7.07 (s, 1H, C5-thiazole); 7.84 - 7.13
(CH-Ar). 13C NMR (DMSO-d6. 100 MHz), δppm: 169,61 (C2-thiazole);
167.57; 157.31 (2C=O); 163.10 (C4-thiazole); 159.88 (C2-thiazole);
144.70 (C=N); 103.35 (C6-thiazole); 42.34 (CH2); 134.62 – 115.19 (CH-
Ar). HRMS: 457.0678 [M-H]+
.
415.1059 [M+H]+
.
(E)-2-(2-(2-(5-methyl-4-phenylthiazol-2-yl)hydrazono)-2-
phenylethyl)isoindoline-1,3-dione (4e): Yield 53.81%; m.p. (°C) 226-230;
(E)-2-(1-([1,1'-biphenyl]-4-yl)-2-(1,3-dioxoisoindolin-2-yl)ethylidene)-N-
phenylhydrazinecarbothioamide (3w): Yield 69.83; m.p. (°C) 203-205; Rf:
0.82 (ethyl acetate/hexane 3:7). ¹H NMR (DMSO-d6 400MHz), δ ppm: 5.04
(s, 2H, CH2), 7.23 (m, 2H, CH-Ar), 7.41 (m, 4H, CH-Ar), 7.66 (m, 6 H, CH-
Ar), 7.86 (m, 4H, CH-Ar), 8,00 (d, J= 8Hz, 2H, CH-Ar), 10.20 (s, 1H, CH-
Ar), 11.03 (s, 1H, CH-Ar). 13C NMR (DMSO-d6 100MHz) δ ppm: 33.96 (CH2),
123.35 (C-Ar), 125.50 (C-Ar), 125.87 (C-Ar), 126.28 (C-Ar), 126.71 (C-Ar),
127.76 (C-Ar), 128.10 (C-Ar), 128.25 (C-Ar), 128.92 (C-Ar), 129,03(C-Ar),
131,51(C-Ar), 134.21 (C-Ar), 134.78 (C-Ar), 139,13 (C-Ar), 140,85 (C-Ar),
Rf: 0,66 (hexane/ethyl acetate 7:3). ¹H NMR (DMSO-d6. 400 MHz), δppm:
3,59 (s, 3H, CH3); 4.97 (s, 2H, CH2); 7.80 (s, 1H, N-H); 7.90 – 7.27 (m,
14H, CH-Ar). 13C NMR (DMSO-d6. 100 MHz), δppm: 175.22 (C2-thiazole);
169.35; 167.83 (2C=O); 144.44 (C=N); 163.,38; (C4-thiazole); 110,27 (C5-
thiazole); 62.75 (CH2); 34.46 (CH3), 136.22 – 123.17 (CH-Ar). HRMS:
452.0856 [M-H]+
.
(E)-2-(2-(2-(4-(2,4-dichlorophenyl)thiazol-2-yl)hydrazono)-2-
phenylethyl)isoindoline-1,3-dione (4f): Yield 27.73%; m.p. (°C) 165-270;
144.78 (C=N), 167.57 (C=O), 177.34 (C=S). HRMS: 491,1315 [M+H]+
.
Rf: 0.79 (hexane/ethyl acetate 7:3). ¹H NMR (DMSO-d6. 400 MHz), δppm
:
5.04 (s, 2H, CH2); 7.28 (s, 1H, C5-thiazole); 8,43 (s, 1H, NH); 8,05 – 7.34
(m, 12H, CH-Ar). 13C NMR (DMSO-d6. 100 MHz), δppm: 169.76 (C2-
thiazole); 167.33 and 167.30 (2C=O); 149.72 (C4-thiazole); 144.05 (C=N);
106.03 (C5-thiazole); 61.90 (CH2); 135.54 – 123.25 (CH-Ar). HRMS:
(E)-2-(1-([1,1'-biphenyl]-4-yl)-2-(1,3-dioxoisoindolin-2-yl)ethylidene)-N-
methylhydrazinecarbothioamide (3x): Yield 85.37; m.p. (°C) 241-243; Rf:
0.72 (ethyl acetate/hexane 3:7). ¹H NMR H (DMSO-d6. 300MHz), δ
:
ppm
3.08 (d, J=4.4Hz, 3H, CH3), 4.95 (s, 2H, CH2), 7.36 (m, 1H, CH-Ar), 7.46
(m, 2H, CH-Ar), 7.53 (m, 2H, CH-Ar), 7.65 (m, 2H, CH-Ar), 7.82 (m, 2H,
CH-Ar), 7.91 (m, 2H, CH-Ar), 7.97 (m, 2H, CH-Ar), 8.60 (d, J=4.4 Hz, 1H,
NH), 10,70 (s, 1H, NH). 13C NMR (DMSO-d6. 100MHz) δ ppm: 31.19(CH3),
33.19 (CH2), 123.30 (C-Ar), 126.26 (C-Ar), 126.53 (C-Ar), 127.48 (C-Ar),
127.69 (C-Ar), 128.65 (C-Ar), 131.13 (C-Ar), 134.44 (C-Ar), 139.14 (C=N),
140.63 (C-Ar), 143.70 (C-Ar), 167.51 (C=O), 178.87 (C=S). HRMS:
506.6013 [M-H]+
.
(E)-2-(2-(2-(4-(naphthalen-2-yl)thiazol-2-yl)hydrazono)-2-
phenylethyl)isoindoline-1,3-dione (4g): Yield 52.33%; m.p. (°C) 226-228;
Rf: 0,72 (hexane/ethyl acetate 7:3). ¹H NMR (DMSO-d6. 400 MHz), δppm
4.70 (s, 2H, CH2); 5.03 (s, 1H, N-H); 7.36 (s, 1H, C5-thiazole); 7.53 (s, 1H,
CH-Ar); 8.10 – 7.23 (m, 15H, CH-Ar). 13C NMR (DMSO-d6. 100 MHz), δppm
169.77 (C2-thiazole); 167.56 e 167.33 (2C=O); 144.01 (C4-thiazole);
:
:
429.1289 [M+H]+
.
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000331
ChemMedChem
FULL PAPER
144.75 (C=N); 106.11 (C5-thiazole); 42.19 (CH2); 133.09 – 123.00 (CH-
(C=O), 167.40 (C=O), 148.92 (C4-thiazole), 146.69 (C=N), 105.32 (C5-
Ar). HRMS: 488.6852 [M-H]+
thiazole), 41.45 (CH2), 137.04 – 122.97 (CH-Ar). HRMS: 560.0671 [M-H]+
.
.
2-((E)-2-((Z)-(4-(4-nitrophenyl)thiazol-2(3H)-ylidene)hydrazono)-2-
phenylethyl)isoindoline-1,3-dione (4h). Yeld 83.29%; m.p. (°C): 218-221;
Rf: 0.72(hexane/ethyl acetate 7:3). ¹H NMR (DMSO-d6. 400 MHz), δppm
4.7 (s, 2H, CH2); 5.01 (s, 1H, N-H); 7.6 (s,1H, C5-thiazole); 7.3 – 7.5 (m,
5H, CH-Ar); 7.8 - 8.3 (m, 9H, CH-Ar). 13C NMR (DMSO-d6. 100 MHz), δppm
Sample preparation: Each compound was solubilized in DMSO (Sigma-
Aldrich) to obtain a final concentration of 5 M. Intermediate dilutions were
then prepared in RPMI-1640 (Invitrogen ™) supplemented with AlbuMAXII
(Invitrogen ™).
:
:
169,94 (C2-thiazole); 167.56 e 167.31 (2C=O); 146.16 (C4-thiazole);
Biological assays of toxicity to macrophage RAW264.7 lineage cells:
RAW264.7 macrophages were seeded in 96-well plates containing
complete DMEM medium in a 5% CO2 atmosphere at 37ºC for 24h.
Subsequently, the compounds were added at different concentrations
(6.25μg/ml; 12.5μg/ml; 25μg/ml; 50μg/ml; 100μg/ml, 200μg/ml) and
incubated again for 48 h. Each compound was tested in duplicate.
144.74 (C=N); 108,96 (C5-thiazole); 25.45 (CH2); 134.70 – 123.21 (CH-
Ar). HRMS: 483.0395 [M-H]+
.
(E)-2-(2-(2-(4-(4-chlorophenyl)thiazol-2-yl)hydrazono)-2-
phenylethyl)isoindoline-1,3-dione (4i): Yield 72.85%; m.p. (°C): 220 - 224;
Rf: 0.72(hexane/ethyl acetate 7:3). ¹H NMR (DMSO-d6. 400 MHz), δppm
:
4.69 (s, 2H, CH2); 5.00 (s, 1H, N-H); 7.16 (s,1H, C5-thiazole); 7.27 – 7.95
(m, 13H, CH-Ar). 13C NMR (DMSO-d6. 100 MHz), δppm: 169,71 (C2-
thiazole), 167.60 (C=O), 167.30 (C=O), 135.51 (C4-thiazole), 134.71
(C=N), 104.83 (C5-thiazole), 34.09 (CH2), 134.64 – 123.22 (CH-Ar).
After this period, MTT (5mg / mL in PBS) was added, followed by further
incubation for 2h. DMSO was added to dissolve the formazan crystals, and
the absorbance was read at 570nm. Negative reaction control was
obtained in wells containing only culture medium and cells (untreated). The
activity of the benznidazole reference drug was also evaluated. From the
culture inhibition values, the CC50 was obtained by simple linear regression
using GraphPad Prism 5.0 software.
HRMS: 473.0412 [M-H]+
.
Synthesis of compounds 4j-n: 500mg (14.8mol) of compound 3b was
solubilized in 20mL of isobutanol at reflux, and different acetophenones
were added as shown in table 2, after 6h a plate was made and it was
confirmed that the reaction had come to an end shortly after the product
was filtered and washed with ethanol and taken to the desiccator for
subsequent reaction yield.
Toxicity to trypomastigotes: Strain Y trypomastigotes were collected
from L929 cell supernatants and distributed in a 96 well plate to a final
density of 4 x 105 cells per well. Each chemical inhibitor was added to the
wells in triplicate. Benznidazole was used as a positive control in this assay.
The plate was then cultivated for 24 h at 37 ºC and 5% CO2. After this time,
aliquots from each well were collected, and the number of viable parasites
(i.e., with apparent motility) was counted in a Neubauer chamber. The
wells that did not receive the chemical inhibitors were assumed as 100%
the number of viable parasites. The dose-response curves were
determined, and the IC50 values were calculated by nonlinear regression
(Prism, version 4.0) using at least seven concentrations (data points).
2-((E)-2-((Z)-(4-(4-methoxyphenyl)-3-phenylthiazol-2(3H)-
ylidene)hydrazono)-2-phenylethyl)isoindoline-1,3-dione
(4j):
Yield
34.55% ; m.p. (°C) 214-216; Rf: 0.70 (hexane/ethyl acetate 7:3). ¹H NMR
(DMSO-d6. 400 MHz), δppm: 3.69 (s, 3H, CH3); 4.82 (s, 2H, CH2); 6.55 (s,
1H, C5-thiazole) 6.55, 7.74 – 6.78 (m, 14H, CH-Ar). 13C NMR (DMSO-d6.
100 MHz), δppm: 167.64 (C2-thiazole), 159.50 (C=O), 159.40 (C=O),
153.69 (C4-thiazole), 139.99 (C=N), 101.33 (C5-thiazole), 55.54 (CH2),
35.93 (CH3), 133.64 – 123.44 (CH-Ar). HRMS: 544.9669 [M-H]+
.
Plasmodium falciparum in vitro culture: Laboratory-adapted P.
falciparum line 3D7-GFP (MRA-1029, MR4. ATCC® Manassas Virginia),
a chloroquine-sensitive strain, was continuously cultured using the method
of Trager and Jensen, with previously described modifications [29].
Parasites were cultivated at 5% hematocrit, 37°C and atmosphere with 5%
of CO2, human serum was replaced with 0.5% AlbuMAXII (Invitrogen™) in
the culture medium. Synchronized cultures were obtained by treatments
with a 5% (m/v) solution of D-sorbitol (Sigma-Aldrich) [30].
2-((E)-2-((Z)-(4-(3-nitrophenyl)-3-phenylthiazol-2(3H)-ylidene)hydrazono)-
2-phenylethyl)isoindoline-1,3-dione (4k): Yield 59.70%; m.p. (°C): 179-
181; Rf: 0.75 (hexane/ethyl acetate 7:3). ¹H NMR (DMSO-d6. 400 MHz),
δppm: 4.84 (s, 2H, CH2), 6.72 (s,1H,C5-thiazole), 6.96 (s, CH-Ar), 8,10 –
7.23 (m, 17H, CH-Ar). 13C NMR DMSO-d6. 100 MHz), δppm: 170.13 (C2-
thiazole), 167.75 (C=O), 167.00 (C=O), 153.84 (C4-thiazole), 148.59
(C=N), 104.67 (C5-thiazole), 41.45 (CH2), 137.32 – 122.54 (CH-Ar).
HRMS: 559.9842 [M-H]+
.
Antimalarial activity primary screening using flow cytometry: All
compounds were screened for their in vitro antimalarial activity against
chloroquine-susceptible 3D7 strain GFP expressing P. falciparum (3D7-
GFP). Unsynchronized culture with 2% hematocrit and 1% parasitaemia,
was incubated in a 96-well flat-bottom plate with 1uM and 10uM of each
compound for 72h (37 °C and 5% CO2). Each plate also included growth
controls wells: no drug added and 5nM and 500 nM of chloroquine.
Parasite growth was assessed by flow cytometry (Beckman Coulter,
Cytoflex) with a 96-well plate reader, using Fl-1 (green fluorescent protein
[GFP]; excitation wavelength, 488 nm). Typically, 20,000 to 40,000 RBCs
were counted for each well. Samples were analyzed using FlowJo
software (Tree Star Inc.).
2-((E)-2-((Z)-(4-(4-fluorophenyl)-3-phenylthiazol-2(3H)-
ylidene)hydrazono)-2-phenylethyl)isoindoline-1,3-dione
(4l):
Yield
59,65%; m.p. (°C): 213-216); Rf: 0.86 (hexane/ethyl acetate 7:3). ¹H NMR
(DMSO-d6. 400 MHz), δppm: 4.73 (s, 2H, CH2); 6. (s,1H, C5-thiazole); 7.74
– 7.04 (m, 19H, CH-Ar). 13C NMR (DMSO-d6. 100 MHz), δppm: 170.42 (C2-
thiazole); 167.40 (C=O), 167.00 (C=O), 160.24 (C2-thiazole), 153.58
(C=N), 138.58 (C4-thiazole), 102.23 (C5-thiazole), 35.51 (CH2), 137.12 –
115.01 (C-Ar). HRMS: 532.8695[M-H]+.
2-((E)-2-((Z)-(4-(naphthalen-2-yl)-3-phenylthiazol-2(3H)-
ylidene)hydrazono)-2-phenylethyl)isoindoline-1,3-dione
(4m).
Yield
81.64%; m.p. (°C): 225 - 228; Rf: 0.84 (hexane/ethyl acetate 7:3). ¹H NMR
(DMSO-d6. 400 MHz), δppm: 4.85 (s, 2H, CH2); 6.80 (s,1H, C5-thiazole),
7.12 – 7.83 (m, 21H, CH-Ar). 13C NMR (DMSO-d6. 100 MHz), δppm: 170.45
(C2-thiazole), 167.05 (C=O), 167.40 (C=O), 153.49 (C4-thiazole), 139.65
(C=N), 102.75 (C5-thiazole), 35.5 (CH2), 137.33 – 122.96 (CH-Ar). HRMS:
Dose-response assay: IC50 estimation: Compounds exhibiting over 70%
growth inhibition in the primary screening assay, were selected as active
and confirmed in the dose-response assay to estimate the correspondent
IC50. Unsynchronized culture with 2% hematocrit and 1% parasitaemia,
was incubated with the tested compounds in 3-fold serial dilutions ranging
from 10 to 0.014 μM. After 72h at 37 °C and 5% CO2, parasite growth was
assessed by flow cytometry as described for the antimalarial activity
565.0973 [M-H]+
.
primary screening assay. Half-maximal inhibitory concentrations (IC50
)
2-((E)-2-((Z)-(4-(4-nitrophenyl)-3-phenylthiazol-2(3H)-ylidene)hydrazono)-
2-phenylethyl)isoindoline-1,3-dione (4n). Yield 96.74%; m.p. (°C): 250 -
252; Rf: 0.81 (hexane/ethyl acetate 7:3). ¹H NMR (DMSO-d6. 400 MHz),
δppm: 4.82 (s, 2H, CH2), 6.75 (s,1H, C5-thiazole), 7.18 – 8,08 (m, 18H, CH-
Ar). 13C NMR (DMSO-d6. 100 MHz), δppm: 167.76 (C2-thiazole), 167.04
were determined with GraphPad Prism 5 (trial version). At least three
experiments, each in duplicate, were performed to obtain the mean IC50
presented.
9
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10.1002/cmdc.202000331
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Acknowledgements
The authors thank the Brazilian National Research Council (CNPq),
the Research Foundation of Pernambuco State (FACEPE), and
CAPES for student fellowships. Ana Cristina Lima Leite is a CNPq
senior fellow. M.V.O.C. is thankful for CNPq (427078/2016-4) and
FACEPE (APQ-0350-4.03/18) fundings. Authors are thankful for the
Centro de Tecnologias Estratégicas do Nordeste (CETENE) for
recording LCMS of all compounds and Department of Chemistry at
the Federal University of Pernambuco (UFPE) for recording the NMR
(1 H and 13C). We also thank MR4 for providing us with malaria
parasites contributed by Andrew Talman & Robert Sinden. This
research was partially supported by Fundação para a Ciência e a
Tecnologia for funds to GHTM—UID/Multi/04413/2013, Portugal. All
authors declare no competing financial interest.
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Keywords: Phthalimide • Plasmodium falciparum • Thiazole •
Thiosemicarbazone • Trypanosoma cruzi
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GRAPHICAL ABSTRACT
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