Phthalimido-thiazoles as building blocks and their effects on the growth and morphology of Trypanosoma cruzi

Article abstract of DOI:10.1016/j.ejmech.2016.01.010

Chagas disease is a parasitic infection caused by protozoan Trypanosoma cruzi that affects approximately 6-7 million people worldwide. Benznidazole is the only drug approved for treatment during the acute and asymptomatic chronic phases; however, its efficacy during the symptomatic chronic phase is controversial. The present work reports the synthesis and anti-T. cruzi activities of a novel series of phthalimido-thiazoles. Some of these compounds showed potent inhibition of the trypomastigote form of the parasite at low cytotoxicity concentrations in spleen cells, and the resulting structure-activity relationships are discussed. We also showed that phthalimido-thiazoles induced ultrastructural alterations on morphology, flagellum shortening, chromatin condensation, mitochondria swelling, reservosomes alterations and endoplasmic reticulum dilation. Together, these data revealed, for the first time, a novel series of phthalimido-thiazoles-structure-based compounds with potential effects against T. cruzi and lead-like characteristics against Chagas disease.

Full text of DOI:10.1016/j.ejmech.2016.01.010

Accepted Manuscript
Phthalimido-thiazoles as building blocks and their effects on the growth and
morphology of Trypanosoma cruzi
Paulo André Teixeira de Moraes Gomes, Arsênio Rodrigues Oliveira, Marcos
Veríssimo de Oliveira Cardoso, Edna de Farias Santiago, Miria de Oliveira Barbosa,
Lucianna Rabelo Pessoa de Siqueira, Diogo Rodrigo Magalhães Moreira, Tanira
Matutino Bastos, Fábio André Brayner, Milena Botelho Pereira Soares, Andresa
Pereira de Oliveira Mendes, Maria Carolina Accioly Brelaz de Castro, Valéria Rego
Alves Pereira, Ana Cristina Lima Leite
PII:
S0223-5234(16)30010-1
10.1016/j.ejmech.2016.01.010
EJMECH 8300
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 September 2015
Revised Date: 21 December 2015
Accepted Date: 9 January 2016
Please cite this article as: P.A.T. de Moraes Gomes, A.R. Oliveira, M.V. de Oliveira Cardoso, E.
de Farias Santiago, M. de Oliveira Barbosa, L.R.P. de Siqueira, D.R.M. Moreira, T.M. Bastos,
F.A. Brayner, M.B.P. Soares, A.P. de Oliveira Mendes, M.C.A.B. de Castro, V.R. Alves Pereira,
A.C.L. Leite, Phthalimido-thiazoles as building blocks and their effects on the growth and
morphology of Trypanosoma cruzi, European Journal of Medicinal Chemistry (2016), doi: 10.1016/
j.ejmech.2016.01.010.
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ACCEPTED MANUSCRIPT
Phthalimido-thiazoles as building blocks and their effects on the
growth and morphology of Trypanosoma cruzi
a
a
Paulo André Teixeira de Moraes Gomes , Arsênio Rodrigues Oliveira , Marcos
a
a
Veríssimo de Oliveira Cardoso , Edna de Farias Santiago , Miria de Oliveira Barbosa
a, Lucianna Rabelo Pessoa de Siqueira , Diogo Rodrigo Magalhães Moreira b, Tanira
a
Matutino Bastos b, Fábio André Brayner , Milena Botelho Pereira Soares b,d, Andresa
c
Pereira de Oliveira Mendes e, Maria Carolina Accioly Brelaz de Castro e, Valéria Rego
Alves Pereira e and Ana Cristina Lima Leite a, *
a Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Federal
de Pernambuco, 50740-520 Recife, PE, Brazil
b Fundação Oswaldo Cruz, Centro de Pesquisas Gonçalo Moniz, CEP 40296-710 Salvador, BA,
Brazil
c Laboratório de Imunologia Keizo Asami-LIKA/UFPE, CEP, 50670-901 Recife, PE, Brazil
d
Centro de Biotecnologia e Terapia Celular, Hospital São Rafael, Avenida São Rafael, 2152,
São Marcos, CEP 41253-190, Salvador (BA), Brazil
e Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz, CEP, 50670-420 Recife, PE,
Brazil
*Corresponding author. E-mail address: acllb2003@yahoo.com.br

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ABSTRACT
Chagas disease is a parasitic infection caused by protozoan Trypanosoma cruzi that
affects approximately 6 to 7 million people worldwide. Benznidazole is the only drug
approved for treatment during the acute and asymptomatic chronic phases; however, its
efficacy during the symptomatic chronic phase is controversial. The present work
reports the synthesis and anti-T. cruzi activities of a novel series of phthalimido-
thiazoles. Some of these compounds showed potent inhibition of the trypomastigote
form of the parasite at low cytotoxicity concentrations in spleen cells, and the resulting
structure-activity relationships are discussed. We also showed that phthalimido-
thiazoles induced ultrastructural alterations on morphology, flagellum shortening,
chromatin condensation, mitochondria swelling, reservosomes alterations and
endoplasmic reticulum dilation. Together, these data revealed, for the first time, a novel
series of phthalimido-thiazoles-structure-based compounds with potential effects against
T. cruzi and lead-like characteristics against Chagas disease.
Keywords: Chagas disease; Trypanosoma cruzi; Thiazole; Hydrazone; Phthalimide.

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GRAPHICAL ABSTRACT
HIGHLIGHTS
The anti-T. cruzi activity of 26 phthalimido-thiazoles were evaluated;
Five compounds presented better trypanocidal activity than Benznidazole;
Compound 6j was 26-fold more selective than Benznidazole;
The structural upgrade planned present positive effects on trypanocidal activity.

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1. Introduction
Chagas disease, also known as American trypanosomiasis, is a potentially life-
threatening illness caused by the protozoan parasite Trypanosoma cruzi (T. cruzi).
Approximately 6 to 7 million people are estimated to be infected worldwide, mostly in
Latin America, where Chagas disease is endemic[1].
Despite the efforts of many investigators to research new anti-Chagas drugs, only one
drug is currently used in therapy, benznidazole (Bdz)[2,3]. Current chemotherapy for
Chagas disease is unsatisfactory due to the limited efficacy of Bdz, particularly during
the chronic phase, with frequent side effects that can lead to discontinuation of
treatment[4].
One pragmatic way to improve the quality of both the candidate drugs and screening
collections is by improving the quality of the building blocks (reagents) that are used to
synthesize them. Our strategic program focused on substructures and properties that are
known to have imparted biological activity and good ‘drug-like’ properties previously.
Among the chemical groups explored for anti-Chagas activity, thiazolyl hydrazones are
noteworthy because of their wide biological, especially anti-parasitic, activities[5–8].
Caputo et al. have demonstrated trypanocidal activity for a series of 4-
arylthiazolylhydrazones[9], which have broad and potent activities for all forms of the
parasite.
Our efforts toward new antichagasic drugs since 2006 have led us to develop a variety
of thiosemicarbazones and 1,3-thiazolyl hydrazones as trypanocidal agents[4,7,10–14].
In continuation of our search for bioactive molecules, we envisaged that the
derivatization of the thiosemicarbazone group into a thiazole moiety would generate
novel templates that are likely to exhibit anti-T. cruzi activity[4].
However, much effort has been invested to identify the key differences between drugs
and other organic compounds. High-quality libraries are expected to exhibit drug-
likeness to produce compounds with desirable pharmacokinetic and safety profiles. The
phthalimide functional group has been used as an important tool in organic synthesis
because it protects against unwanted reactions. Many research teams have used this
nucleus as a building block to improve compound quality. In fact, phthalimide
derivatives have shown a broad spectrum of pharmacological properties, such as
analgesic[15],
anticonvulsant[16],
antitubercular[17,18],
hypolipidaemic[18],
anxiolytic[15], anti-inflammatory[15], antimicrobial[17,19,20] and antipsychotic[21].

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For this reason, our research group has explored the pharmacological properties of
phthalimide derivatives. As a result, bioactive prototypes were identified with potent
anti-inflammatory[22],
anti-proliferative[23],
immunomodulatory[22,24,25],
antitumor[23], antiangiogenic[26] and schistosomicidal properties[27],
Indeed, Santiago et al. identified phthalimido-thiazole derivatives with potent
schistosomicidal activities. The phthalimide LpQM-45 caused significant ultrastructural
changes, including destruction of the integument in both male and female worms[27],
however, their antichagasic properties have not been explored.
Considering the promising results achieved by compounds bearing a thiazole ring and
phthalimides nuclei, they were chosen as common pharmacophores that exist in diverse
drug classes. In this way, we synthesized a set of molecules with phthalimide and
thiazole nucleus. In this synthetic design of a substructure-based compound library,
substituents around the phenyl ring attached at C4 in the thiazole ring (compounds 2b-
n) were explored. In addition, a spacer group between phthalimide and the thiazole ring
was inserted and a phenyl group at N3 of the thiazole ring was also introduced (6b-l).
To investigate the influence of the phthalimido moiety at the anti-T. cruzi activity, 26
new compounds were tested in vitro against the T. cruzi parasite epimastigote and
trypomastigote forms. Ultrastructural studies and flow cytometry analysis were also
investigated (Figure 1).
PLEASE, INSERT FIGURE 1 HERE
2. Results and discussion
2.1 Chemistry
Initially, 14 phthalimido-thiazoles (2a-n) were synthesized in a two-step reaction,
following the procedures reported by Pessoa et al[25]. Firstly, a reaction of phthalic
anhydride with thiosemicarbazide, in DMF under reflux for 4 h, with a catalytic amount
of DMAP, led us to compound 1. The synthesis of the series 2a-n was performed via
Hantsch cyclization between compound 1 and the appropriate α-halogenated ketone
(1,3-dichloroacetone for compound 2a), under ultrasound irradiation, at room
temperature (rt) for 1 h. This reaction condition led to average yields from 36-65%. To
synthesize the series 6a-l, was followed the reaction protocol reported by Cardoso et

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al.[23]. The desired compounds 6a-l were obtained by the reaction of Intermediate 5b
(or 5a, for compound 6a) with the appropriate α-halogenated ketone (1,3-
dichloroacetone for compound 6a), via Hantsch cyclization, leading good yields (46-
82%) (Scheme 1). All the synthesized compounds were well characterized by infrared
(IR), nuclear magnetic resonance (1H, 13C NMR), mass spectroscopy (ESI-TOF) and, in
case of compounds 2e, 2f and 2g, by single crystal X-ray diffraction analysis (Figure
2).
PLEASE, INSERT SCHEME 1 HERE
PLEASE, INSERT FIGURE 2 HERE
1
The H NMR spectra of some compounds showed that phthalimido-thiazoles 6a-l are
composed by diastereomers. Next, we aimed to define the configuration of the major
isomer by crystallographic analysis. However, we did not succeed in crystallizing
phthalimido-thiazoles 6a-l suitable for X-ray analysis. Based on previous crystallized
compounds by our group, we suggest that the major isomer formed present the E-Z
configuration (Figure 3). Indeed, hydrazine double-bond C2=N2 is commonly assigned
as E configuration[4,23,28]. Concerning the exocyclic double-bond N3=C3, we suggest
that the predominant configuration is in Z-configuration[7,29]. Besides, a representative
1H-NMR spectrum of compound 6i is presented in Supplementary Material.
PLEASE, INSERT FIGURE 3 HERE
2.2 Anti-T. cruzi evaluation
Initially, compounds 2a-n were planned to improve the trypanocidal activity and
cytotoxic tolerance with the cyclization of phthalimido-thiosemicarbazone to the
phthalimido-thiazole ring. From the results, it was observed in most of the cases that
new phthalimido-thiazoles showed high cytotoxic activity in spleen cells. In opposition,
only compounds 2i and 2j showed low cytotoxicity
.
Concerning trypanocidal activity for epimastigotes, it is observed that 16 compounds
(of 28) present better potency than Benznidazole (Table 1). Among series 2a-n,

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compound 2i was the most active, among the series and the entire work. The most
active compound in series 6 is 6k, a 3-NO2 derivative, presenting an IC50 of 6.0 µM.
Observing compounds with withdrawer substituents (2e-h, 2k-n), compound 2,4-
dichloro substituted (2m) was the most active. It analogue disubstituted 3,4-dichloro
(2l) present lower trypanocidal activity and high toxicity for BALB/c mice spleen cells,
denoting that the orientation of the substituents is important for the activity. Observing
bulk substituted compounds (phenyl, 2-naphthyl and 4-biphenylyl), in series 2a-n, a
relationship of LogP and trypanocidal activity (Figure 4) is observed, being compound
2i the most active of this sub-series.
PLEASE, INSERT FIGURE 4 HERE
The trend of bulky substituents (LogP) observed for series 2a-n is not observed for
series 6a-l, being compound 6k (3-NO2) the most active of the series 6a-l.
When comparing the trypanocidal activity against the trypomastigote form of the series
2a-n of phthalimido-thiazole derivatives, compound 2j was found to be the most potent
of this sub-series, presenting lower cytotoxic levels (269.2 µM) and equipotent
trypanocidal activity compared with Bdz (4.7 vs 6.3 µM) (Table 1).
PLEASE, INSERT TABLE 1 HERE
No clear correlation was observed between the substituents at the thiazole C4 and the
biological activity. For example, 2-naphthyl is a bulky substituent present in 2j;
however, compound 2i, which was also substituted with a bulky biphenylyl substituent,
did not present good activity. The influence of the phenyl linked at C4 in the thiazole
ring was also investigated, but no noticeable improvement in activity was observed.
Comparing compound 2a with the phenyl-unsubstituted compound 2b, a decrease in
trypanocidal activity and higher cytotoxicity were observed, while the phenyl-
substituted compounds maintained or increased both activities.
A structural optimization involving addition of a spacer group (-CH2-CH=) between the
phthalimide and the thiazole core was performed in order to increase the flexibility of
the molecules and to investigate its influence on biological activities (Figure 5). A
substitution of H with phenyl at N3 was also performed to explore the influence of

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bulky substituents based on the results of compounds 2i and 2j, both substituted with 4-
biphenylyl and 2-naphthyl at C4, respectively, which displayed lower cytotoxicity.
PLEASE, INSERT FIGURE 5 HERE
Compounds that contained a spacer group (6a-l) and phenyl at N3 (6b-l) in general
showed low cytotoxicity profiles and improved trypanocidal activity for trypomastigote
form, highlighting compounds 6a (2.2 µM), 6h (3.2 µM), 6j (0.5 µM) and 6k (0.9 µM).
In fact, compound 6j presented a selective index (SI, for trypomastigote form) of 409.8,
which was approximately 27-fold more selective than Bdz (SI: 15.25), the standard drug
in clinical use. As observed in the 2a-n series, the compound substituted at C4 with
chloromethyl (6a, 2.2 µM) was more active than compounds substituted with phenyl
(6b, 8.8 µM); indeed, phenyl-substituted compounds 6j (0.5 µM) and 6k (0.9 µM) were
more active than 6a. Comparing compound 6a with 2a, which were both substituted
with chloromethyl at C4 with the unique difference of the presence of the spacer group
in 6a, a 24-fold improvement in trypanocidal activity (2.2 vs 54.4 µM) and a 4-fold
increase in cytotoxicity (73.7 vs 17.0 µM) were observed for 6a. Compounds 6k and
6h, both nitro-substituted compounds at the meta and para positions, respectively,
presented high trypanocidal activity with selective indexes of 114.9 and 64.6,
respectively (Table 1). Compound 6e (with the electron withdrawing fluorine
substituent) and 6i (with 4-biphenylyl substituent) did not present good trypanocidal
activity. Compound 6j, which was substituted with the bulky substituent 2-naphthyl,
presented the highest trypanocidal activity.
The SAR studies revealed that a series of phthalimido-thiazoles were interesting anti-T.
cruzi compounds, which five new compounds showed low cytotoxicity in spleen cells
of BALB/c mice and anti-trypanocidal activity against the trypomastigote form of the
parasite.
Congreve et al. proposed a rule-of-three (RO3)[30] representing a set of guidelines for
constructing a fragment library (molecular weight, <300; cLogP, ≤3; number of
hydrogen bond donors, ≤3; and the number of hydrogen bond acceptors, ≤3). Recently,
RO3 was accredited by most medicinal chemists and could be useful for efficient
fragment selection[31]. As seen in Table 2, the phthalimide and thiazole fragments are
in agreement with RO3.

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PLEASE, INSERT TABLE 2 HERE
The trypanocidal profile of these phthalimido-thiazoles revealed a group of privileged
structure-based compounds that can be used as building blocks to obtain new lead-like
compounds. They possess some structural features, such as lower molecular weights,
decreased complexity and decreased hydrophobicity, which are consistent with lead-like
characteristics. These compounds can be used to produce structurally simple leads with
modest activity, allowing for further derivatization at a later stage to improve affinity
and selectivity while retaining drug-like characteristics.
2.3 Ultra structural studies
In view to investigate the effects of phthalimido-thiazoles on parasite morphology,
compound 6k, one of the most active compound of this work, was selected. The
ultrastructural effects of 6k on trypomastigotes after 24 h were analysed by TEM and
SEM at the IC50 concentration and twice the IC50 value (Table 1), and the ultrastructural
analysis showed several morphological alterations (Figure 6).
SEM analysis revealed that treatment with 0.9 ꢀM (1×IC50) and 1.8 ꢀM (2×IC50) of 6k
caused blebs in the flagellum and shortening of the flagellum with a drastic decrease in
the number of parasites, respectively (Figure 6B-C), while the control group retained
its typical morphology (Figure 6A).
TEM analysis revealed that untreated parasites showed normal ultrastructural
morphologies of organelles, such as kinetoplast, nucleus, nucleolus, flagellum,
ribosomes and microtubule membranes (Figure 6D), whereas parasites treated with 0.9
ꢀM of 6k showed alterations in the reservosomes, and a large number of cells showed
intense cytoplasmic vacuolization. In addition, alterations of the parasite morphology,
large nuclear chromatin clumps that resembled the nuclei of apoptotic cells,
mitochondria swelling and loss of cytoplasmic material were observed (Figure 6E-F).
The parasites treated with 1.8 ꢀM of 6k showed alterations of parasite morphology,
abnormal chromatin condensation, dilated endoplasmic reticula, intense vacuolization in
the cytoplasm, swelling of the kinetoplast and alterations in the reservosomes (Figure
6G).
Ultrastructural analysis was applied to explore the damages induced by the drugs in
trypomastigotes T. cruzi parasites and clearly showed severe morphological changes, of

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which intense cytoplasmic vacuolization, chromatin condensation, alterations in the
reservosomes and swelling of the mitochondria were most frequent. These
ultrastructural alterations are similar to those previously reported[32–36]. The
ultrastructural evaluation indicated a dose-dependent action because a drastic decrease
in the number of parasites was observed with increasing drug doses.
PLEASE, INSERT FIGURE 6 HERE
2.4 Flow cytometry analysis
After confirming that 6k was an antiparasitic compound that affected ultrastructural
cellular organization, we sought to determine whether 6k caused parasite cell death. To
this end, Y strain trypomastigotes were treated with different concentrations of 6k.
After 24 h incubation, parasite cells were stained with propidium iodide (PI) and
annexin-V and analysed by flow cytometry. The results are shown in Figure 7.
PLEASE, INSERT FIGURE 7 HERE
Compared with untreated cells, benznidazole resulted in PI-staining in a concentration-
dependent manner, while no significant annexin-V staining was observed (data not
shown). At 25 µM, benznidazole induced 56±6% PI-staining in parasite cells. At the
same concentration, treatment with compound 6k induced PI-staining in 11±3% of
parasite cells. No PI or annexin-V staining were observed under treatment with 6k (data
not shown). Therefore, these phthalimido-thiazoles do not destroy parasite cells by
classical cell death processes. Based on electronic microscopy observations, it is
possible that phthalimido-thiazoles decrease parasite viability by altering cytosol
organization, mainly by causing intense cytoplasmic vacuolization.
3 Conclusion
Twenty-six phthalimido-thiazoles were obtained in reasonable yields using a simple
methodology. Compounds with important trypanocidal activity, especially compounds
2j, 6a, 6h, 6j and 6k, were identified. Flow cytometry and ultrastructural studies
showed that compound 6k did not kill the parasite via necrosis or apoptosis; however, it

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promoted several morphological changes in the parasite. Compound 6j, the most potent
trypanocidal agent identified in this work, presented a selective index of 409, which was
approximately 26-fold more selective than Bdz, the standard drug in clinical use. Our
results indicated that phthalimido-thiazoles can be used as building blocks to design
promising candidates to treat Chagas disease.
Aknowledgements
This work was funded by Conselho Nacional de Desenvolvimento Científico e
Tecnológico (CNPq), Fundação de Amparo à Ciência e Tecnologia de Pernambuco
(FACEPE, APQ-0549-2.11/14, APQ-0289-4.03/13). P.A.T.M.G. holds a FACEPE
doctoral scholarship. Authors are thankful to the Departamento de Química
Fundamental (DQF-UFPE) for recording the 1H NMR, 13C NMR, LCMS and IR spectra
of compounds. We are also thankful to Julia Campos of Centro de Tecnologias
Estratégicas do Nordeste (CETENE) for recording the MS spectra of compounds 6a-l.
All authors declare no competing financial interest.
4 Experimental section
4.1 Chemistry
4.1.1 Equipment and reagents
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 aluminium foil). The
purities of the target compounds were confirmed by combustion analysis (for C, H, N,
and S) performed using a Carlo-Erba instrument (model EA 1110). Melting points were
determined on a Fisatom 430D electrothermal capillary melting point apparatus and
were uncorrected. NMR spectra were measured on either a Varian UnityPlus 400 MHz
(400 MHz for 1H 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

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constants (J) in hertz. Mass spectrometry experiments were performed on a LC-IT-TOF
(Shimadzu). Unless otherwise specified, ESI was conducted in positive ion mode.
Typical conditions were as follows: capillary voltage of 3 kV, cone voltage of 30 V, and
peak scan between 50 and 1000 m/z. IR spectra were recorded with a Bruker model
IFS66 FT-IR spectrophotometer using KBr pellets.
4.1.2. General procedure for the syntheses of 2a-n
The compound 1 was prepared by reacting commercially available thiosemicarbazide
with the phthalic anhydride (1:1 mol ratio) using DMF under reflux in the presence of a
catalytic amount of DMAP, for 4 h. This reaction condition led to satisfactory yield
(58%). The phthalimido-thiazoles (2a-n) were prepared via cyclization between 1 and
respective α-halogenated ketone (1,3-dichloroacetone for compound 2a), via ultrasound
irradiation for 1 h, as previously related[4]. These reactions proceeded well under
ultrasound conditions at room temperature using 2-propanol as solvent, resulting in
satisfactory yields (36–65%) and shorter reaction times (60 min in most cases).
4.1.2.1. 2-[4-(chloromethyl)thiazol-2-ylamino]isoindoline-1,3-dione (2a)
White crystals; Yield: 50%; m.p. (°C) 207-208; Rf: 0.53 (hexane / ethyl acetate 1:1). IR
1
(KBr, cm-1): 3119.15 (NH), 1743.24 (C=O). H NMR (300 MHz, DMSO-d6), δ ppm
:
4.54 (s, 2H, CH2), 7.03 (s, 1H, thiazole), 7.93-7.99 (m, 4H, Ar), 10.54 (s, 1H, NH). 13C
NMR (75.5 MHz, DMSO-d6), δ ppm: 41.1 (CH2), 109.8 (CH, thiazole), 123.9 (CH, Ar),
129.4 (C, Ar), 135.4 (CH, Ar), 147.1 (C, thiazole), 165.6 (C=O), 168.3 (S-C=N,
thiazole). Anal. Calcd for C12H8ClN3O2S: C, 49.07; H, 2.75; N, 14.31; S, 10.92. found:
C, 48.50; H, 2.81; N, 14.34; S 10.43. HRMS: 294.0097 [M+H]+.
4.1.2.2. 2-(4-phenylthiazol-2-ylamino)isoindoline-1,3-dione (2b)
Light yellow crystals; Yield: 65%; m.p. (°C) 194-196; Rf: 0.60 (hexane / ethyl acetate
1
1:1). IR (KBr, cm-1): 3123.66 (NH), 1742.23 (C=O). H NMR (400 MHz, DMSO-
d6), δ ppm: 7.25 (t, J = 7.4 Hz, 1H, Ar), 7.33 (t, J = 7.4 Hz, 2H, Ar), 7.38 (s, 1H,
13
thiazole), 7.70 (d, J = 7.6 Hz, 2H, Ar), 7.95-8.02 (m, 4H, Ar), 10.65 (s, 1H, NH). C

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NMR (100 MHz, DMSO-d6), δ ppm: 104.8 (CH, thiazole), 123.8 (CH, Ar), 125.5 (CH,
Ar), 127.7 (CH, Ar), 128.6 (CH, Ar) 129.3 (C, Ar), 133.9 (C, Ar), 135.4 (CH, Ar),
149.9 (C, thiazole), 165.5 (C=O), 167.6 (S-C=N, thiazole). Anal. Calcd for
C17H11N3O2S: C, 62.08; H, 3.41; N, 12.98; S, 9.71. found: C, 63.54; H, 3.45; N, 12.53;
S, 9.98. HRMS: 322.0619 [M+H]+.
4.1.2.3. 2-(4-p-tolylthiazol-2-ylamino)isoindoline-1,3-dione (2c)
Light yellow crystals; Yield: 37%; m.p. (°C) 214-216; Rf: 0.65 (hexane / ethyl acetate
1
1:1). IR (KBr, cm-1): 3117.64 (NH), 1738.51 (C=O). H NMR (400 MHz, DMSO-d6),
δ ppm: 2.26 (s, 3H, CH3), 7.14 (d, J = 6.8 Hz, 2H, Ar), 7.30 (s, 1H, thiazole), 7.59 (d, J =
6.8 Hz, 2H, Ar), 7.99 (m, 4H, Ar), 10.41 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6),
δ ppm: 20.8 (CH3), 103.9 (CH, thiazole), 123.9 (CH, Ar), 125.5 (CH, Ar), 129.17 (CH,
Ar), 129.3 (C, Ar), 131.4 (C, Ar), 135.4 (C, Ar), 137.1 (C, Ar), 150.2 (C, thiazole),
165.6 (C=O), 167.5 (S-C=N, thiazole). Anal. Calcd for C18H13N3O2S: C, 63.17; H,
3.81; N, 18.12; S, 9.17. found: C, 64.46; H, 3.91; N, 18.53; S, 9.56. HRMS: 336.0812
[M+H]+.
4.1.2.4. 2-[4-(4-methoxyphenyl) thiazol-2-ylamino]isoindoline-1,3-dione (2d)
Light yellow crystals; Yield: 72%; m.p. (°C) 215-218; Rf: 0.53 (hexane / ethyl acetate
1
3:2). IR (KBr, cm-1): 3232.68 (NH), 1748.64 (C=O). H NMR (300 MHz, DMSO-d6),
δ ppm: 1.025 (s, 3H, CH3), 6.90 (d, J = 11.6 Hz, 2H, Ar), 7.20 (s, 1H, thiazole), 7.62 (d, J
= 11.6 Hz, 2H, Ar), 7.98 (m, 4H, Ar), 10.72 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-
d6), δ ppm: 25.5 (CH3), 102.7 (CH, thiazole), 114.0 (CH, Ar), 123.9 (CH, Ar), 126.7 (C,
Ar), 127.0 (CH, Ar), 129.4 (C, Ar), 135.4 (CH, Ar), 149.5 (C, thiazole), 158.6 (CO, Ar),
165.6 (C=O), 167.6 (S-C=N, thiazole). Anal. Calcd for C18H13N3O3S: C, 58.88; H, 3.87;
N, 11.38; S, 8.77. found: C, 61.53; H, 3.73; N, 11.96; S, 9.13. HRMS: 352.0825
[M+H]+.
4.1.2.5. 2-[4-(4-fluorophenyl)thiazol-2-ylamino]isoindoline-1,3-dione (2e)

ACCEPTED MANUSCRIPT
Yellow crystals; Yield: 51%; m.p. (°C) 208-210; Rf: 0.53 (hexane / ethyl acetate 3:2).
1
IR (KBr, cm-1): 3130.14 (NH), 1743.63 (C=O). H NMR (400 MHz, DMSO-d6), δ ppm
:
7.17 (d, J = 17.6 Hz, 2H, Ar), 7.37 (s, 1H, thiazole), 7.73 (d, J = 14.0 Hz, 2H, Ar), 7.99
(m, 4H, Ar), 10.42 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6), δ ppm: 104.6 (CH,
thiazole), 115.4 (CH, Ar), 115.6 (CH, Ar), 123.9 (CH, Ar), 127.5 (CH, Ar), 129.3 (C,
Ar), 135.4 (CH, Ar), 160.4 (C, thiazole), 162.9 (CF, Ar), 165.5 (C=O), 167.6 (S-C=N,
thiazole). Anal. Calcd for C17H10FN3O2S: C, 60.32; H, 3.02; N, 12.32; S, 9.58. found:
C, 60.17; H, 2.97; N, 12.38; S, 9.45. HRMS: 340.0566 [M+H]+.
4.1.2.6. 2-[4-(4-chlorophenyl)thiazol-2-ylamino]isoindoline-1,3-dione (2f)
Light yellow crystals; Yield: 62%; m.p. (°C) 217-218; Rf: 0.53 (hexane / ethyl acetate
1
1:1). IR (KBr, cm-1): 3119.79 (NH), 1739.00 (C=O). H NMR (400 MHz, DMSO-
d6), δ ppm: 7.39 (d, J = 8.0 Hz, 2H, Ar), 7.46 (s, 1H, thiazole), 7.72 (d, J = 8 Hz, 2H, Ar).
7.99 (m, 4H, Ar), 10.46 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6), δ ppm: 105.7
(CH, thiazole), 123.9 (CH, Ar), 127.3 (CH, Ar), 128.7 (CH, Ar), 129.3 (C, Ar) 132.2
(C, Ar), 132.9 (C, Ar), 135.4 (CCl, Ar), 148.9 (C, thiazole), 165.5 (C=O), 167.7 (S-
C=N, thiazole). Anal. Calcd for C17H10ClN3O2S: C, 57.55; H, 2.96; N, 11.62; S, 9.03.
found: C, 57.39; H, 2.83; N, 11.81; S, 9.01. HRMS: 356.0260 [M+H]+.
4.1.2.7. 2-[4-(4-bromophenyl)thiazol-2-ylamino]isoindoline-1,3-dione (2g)
Light yellow crystals; Yield: 55%; m.p. (°C) 220-221; Rf: 0.68 (hexane / ethyl acetate
1
1:1). IR (KBr, cm-1): 3117.68 (NH), 1739.19 (C=O). H NMR (400 MHz, DMSO-
d6), δ ppm: 7.47 (s, 1H, thiazole), 7.53 (d, J = 8.4 Hz, 2H, Ar), 7.65 (d, J = 8.0 Hz, 2H,
Ar), 7.96 (m, 4H, Ar), 10.46 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6), δ ppm: 105.8
(CH, thiazole), 120.8 (CH, Ar), 123.9 (CH, Ar), 127.6 (CH, Ar), 129.3 (C, Ar) 131.6
(C, Ar), 135.4 (CBr, Ar), 148.9 (C, thiazole), 165.5 (C=O), 167.7 (S-C=N, thiazole).
Anal. Calcd for C17H10BrN3O2S: C, 51.15; H, 2.58; N, 11.62; S, 9.03. found: C, 51.01;
H, 2.52; N, 11.50; S, 8.91. HRMS: 401.9764 [M+H]+.
4.1.2.8. 2-[4-(4-nitrophenyl)thiazol-2-ylamino]isoindoline-1,3-dione (2h)

ACCEPTED MANUSCRIPT
Yellow crystals; Yield: 41%; m.p. (°C) 239-240; Rf: 0.45 (hexane / ethyl acetate 3:2).
1
IR (KBr, cm-1): 3309.85 (NH), 1724.82 (C=O). H NMR (400 MHz, DMSO-d6), δ ppm
:
7.77 (s, 1H, thiazole), 7.97 (m, 4H, Ar), 8.02 (d, J = 8.8 Hz, 2H, Ar), 8.21 (d, J = 8.8
Hz, 2H, Ar), 10.54 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6), δ ppm: 109.6 (CH,
thiazole), 123.9 (CH, Ar), 124.1 (CH, Ar), 126.4 (CH, Ar), 129.3 (CN Ar), 135.4 (CH,
Ar), 139.9 (C, Ar), 146.3 (C, Ar), 147.9 (C, thiazole), 165.382 (C=O), 168,0 (S-C=N,
thiazole). Anal. Calcd for C17H10N4O4S: C, 54.40; H, 2.87; N, 15.23; S, 9.11. found: C,
55.73; H, 2.75; N, 15.29; S, 8.75. HRMS: 367.0517 [M+H]+.
4.1.2.9. 2-[4-(biphenyl-4-yl)thiazol-2-ylamino]isoindoline-1,3-dione (2i)
Light yellow crystals; Yield: 46%; m.p. (°C) 240-241; Rf: 0.65 (hexane / ethyl acetate
1
3:2). IR (KBr, cm-1): 3324.67 (NH), 1660.10 (C=O). H NMR (300 MHz, DMSO-
d6), δ ppm: 7.35 (s, 1H, thiazole), 7.48 (t, J = 10.0 Hz, 1H, Ar), 7.63 (m, 4H, Ar), 7.72 (d,
J = 11.6 Hz, 2H, Ar), 7.82 (m, 4H, Ar) 7.96 (d, J = 11.6 Hz, 2H, Ar), 10.65 (s, 1H, NH).
13C NMR (75.5 MHz, DMSO-d6), δ ppm: 103.4 (CH, thiazole), 126.5 (CH, Ar), 126.9
(CH, Ar), 127.5 (CH, Ar), 128.0 (CH, Ar) 129.0 (CH, Ar), 130.1 (CH, Ar), 131.5 (CH,
Ar), 133.9 (C, Ar), 136.1 (CH, Ar), 139.0 (C, Ar), 139.7 (C Ar), 150.2 (C, thiazole),
167.6 (C=O), 168.3 (S-C=N, thiazole). Anal. Calcd for C23H15N3O2S: C, 66.81; H, 3.98;
N, 10.48; S, 7.95. found: C, 66.50; H, 3.80; N, 10.57; S, 8.07. HRMS: 398.0958
[M+H]+.
4.1.2.10. 2-[4-(naphthalen-2-yl)thiazol-2-ylamino]isoindoline-1,3-dione (2j)
Light yellow crystals; Yield: 47%; m.p. (°C) 225-227; Rf: 0.50 (hexane / ethyl acetate
1
3:2). IR (KBr, cm-1): 3169.87 (NH), 1743.89 (C=O). H NMR (400 MHz, DMSO-
d6), δ ppm: 7.47 (t, 2H, Ar), 7.53 (s, 1H, thiazole), 7.87 (d, J = 11.6 Hz, 4H, Ar), 8.03 (m,
4H, Ar), 8.23 (s, 1H, Ar), 10.48 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ ppm
:
105.5 (CH, thiazole), 123.9 (CH, Ar), 124.2 (CH, Ar), 126.1 (CH, Ar), 126.4 (CH, Ar)
127.1 (CH, Ar), 127.6 (CH, Ar), 128.1 (CH, Ar), 128.2 (CH, Ar), 129.3 (C, Ar), 131.5
(C, Ar), 132.4 (C, Ar), 133.0 (C, Ar), 135.4 (CH, Ar), 150.1 (C, thiazole), 165.6 (C=O),
168.0 (S-C=N, thiazole). Anal. Calcd for C21H13N3O2S: C, 64.89; H, 3.48; N, 11.45; S,
8.26. found: C, 67.91; H, 3.53; N, 11.11; S, 8.63. HRMS: 367.0517 [M+H]+.

ACCEPTED MANUSCRIPT
4.1.2.11. 2-[4-(3-nitrophenyl)thiazol-2-ylamino]isoindoline-1,3-dione (2k)
Light yellow crystals; Yield: 44%; m.p. (°C) 206-207; Rf: 0.55 (hexane / ethyl acetate
1
3:2). IR (KBr, cm-1): 3287.35 (NH), 1732.47 (C=O). H NMR (300 MHz, DMSO-
d6) δ ppm: 7.65 (t, J = 7,2 Hz, 1H, Ar), 7.73 (s, 1H, thiazole), 8.00 (m, 4H, Ar), 8.13 (m,
13
2H, Ar), 8.48 (s, 1H, Ar) 10.36 (s, 1H, NH). C NMR (75.5 MHz, DMSO-d6), δ ppm
:
107.7 (CH, thiazole), 119.9 (CH, Ar), 122.3 (CH, Ar), 123.9 (CH, Ar), 129.3 (CN, Ar)
130.3 (CH, Ar), 131.7 (CH, Ar), 135.5 (CH, Ar), 147.7 (C, Ar), 148.2 (C, thiazole),
165.5 (C=O), 168.2 (S-C=N, thiazole). Anal. Calcd for C17H10N4O4S: C, 53.04; H, 2.88;
N, 15.49; S, 8.90. found: C, 54.73; H, 2.75; N, 15.29; S, 8.75. HRMS: 367.0517
[M+H]+.
4.1.2.12. 2-[4-(3,4-dichlorophenyl)thiazol-2-ylamino]isoindoline-1,3-dione (2l)
Light yellow crystals; Yield: 57%; m.p. (°C) 217-218; Rf: 0.53 (hexane / ethyl acetate
1
3:2). IR (KBr, cm-1): 3337.03 (NH), 1742.53 (C=O). H NMR (300 MHz, DMSO-
d6), δ ppm: 7.52 (s, 1H, thiazole), 7.62 (s, 1H, Ar), 7.68 (d, J = 2.1 Hz, 1H, Ar), 7.94 (d, J
= 1,8 Hz, 1H, Ar), 8.00 (m, 4H, Ar), 10.51 (s, 1H, NH). 13C NMR (75.5 MHz, DMSO-
d6), δ ppm: 107.1 (CH, thiazole), 123.9 (CH, Ar), 127.1 (CH, Ar), 129.3 (CCl, Ar), 129.5
(CH, Ar), 130.9 (CCl, Ar), 131.4 (C, Ar), 134.5 (CH, Ar), 135.4 (C, Ar), 136.0 (CH,
Ar), 147.9 (C, thiazole), 165.4 (C=O), 167.9 (S-C=N, thiazole). Anal. Calcd for
C17H9Cl2N3O2S: C, 51.19; H, 2.43; N, 11.04; S, 8.58. found: C, 52.32; H, 2.32; N,
10.77; S, 8.22. HRMS: 356.0260 [M+H]+.
4.1.2.13. 2-[4-(2,4-dichlorophenyl)thiazol-2-ylamino]isoindoline-1,3-dione (2m)
Colorless crystals; Yield: 46%; m.p. (°C) 217-218; Rf: 0.55 (hexane / ethyl acetate 3:2).
1
IR (KBr, cm-1): 3130.90 (NH), 1741.75 (C=O). H NMR (400 MHz, DMSO-d6), δ ppm
:
7.41 (d, J = 7.2 Hz, 1H, Ar), 7.45 (s, 1H, thiazole), 7.63 (s, 1H, Ar), 7.65 (d, J = 8.4 Hz,
13
1H, Ar). 7.97 (m, 4H, Ar), 10.46 (s, 1H, NH). C NMR (100 MHz, DMSO-d6), δ ppm
:
110.45 (CH, thiazole), 123.8 (CH, Ar), 127.5 (CH, Ar), 129.3 (CCl, Ar), 129.7 (CH,
Ar), 131.5 (CCl, Ar), 131.5 (C, Ar), 132.1 (CH, Ar), 132.7 (C, Ar), 135.4 (CH, Ar),

ACCEPTED MANUSCRIPT
145.4 (C, thiazole), 165.4 (C=O), 166.7 (S-C=N, thiazole). Anal. Calcd for
C17H9Cl2N3O2S: C, 52.25; H, 2.26; N, 10.50; S, 7.91. found: C, 52.32; H, 2.32; N,
10.77; S, 8.22. HRMS: 356.0260 [M+H]+.
4.1.2.14. 2-[4-(4-bromophenyl)-5-methylthiazol-2-ylamino]isoindoline-1,3-dione (2n)
Yellow crystals; Yield: 36%; m.p. (°C) 235-236; Rf: 0.60 (hexane / ethyl acetate 3:2).
1
IR (KBr, cm-1): 3188.90 (NH), 1745.97 (C=O). H NMR (400 MHz, DMSO-d6), δ ppm
:
2.36 (s, 3H, CH3), 7.41 (d, J = 8.4 Hz, 2H, Ar), 7.55 (d, J = 8.4 Hz, 2H, Ar), 7.96 (m,
13
4H, Ar), 10.23 (s, 1H, NH); C NMR (100 MHz, DMSO-d6), δ ppm: 12.1 (CH3), 119.3
(C, thiazole), 120.4 (C, Ar), 123.8 (CH, Ar), 129.3 (C, Ar), 129.8 (CH, Ar), 131.2 (CH,
Ar), 133.7 (CBr, Ar), 135.3 (CH, Ar), 144.0 (C, thiazole), 163.5 (C=O), 165.5 (S-C=N,
thiazole). Anal. Calcd for C17H12BrN3O2S: C, 51.75; H, 3.00; N, 9.93; S, 7.58. found: C,
52.19; H, 2.92; N, 10.10; S, 7.74. HRMS: 401.9764 [M+H]+.
4.1.3. Procedure for the syntheses of intermediate compounds 5a-b
Compound 3 was prepared by the condensation of commercially available
aminoacetaldehyde diethyl acetal with the phthalic anhydride (1:1 mol ratio), using
toluene under reflux, in the presence of a catalytic amount of DMAP (yield 52%) for 2
h. In the next step, 2-(2,2-diethoxyethyl) isoindoline-1,3-dione (3) underwent acid
hydrolysis (sulphuric acid at 70%) in reflux for 2 h. After the reaction was completed, it
was allowed to reach at room temperature and was then cooled to induce precipitation.
The formed precipitate was filtered on a sintered funnel with distilled water, yielding
55% of the pure product. For the synthesis of 5a and 5b, 2-(1,3-dioxoisoindol-2-yl)
acetaldehyde (4) reacted with thiosemicarbazide (in the ratio 1:1) (for 5a) or 4-phenyl-
3-thiosemicarbazide (for 5b), in ethanol, under reflux with catalytic amount of HCl (4
drops) for 4 h. The reactions were followed by thin layer chromatographic plate
analysis. The formed precipitate was filtered on a sintered funnel with ethanol to yield
the pure product (yield 76% for 5a and 70% for 5b).
4.1.3.1. (E)-2-[2-(1,3-dioxoisoindolin-2-yl)ethylidene]hydrazinecarbothioamide (5a)

ACCEPTED MANUSCRIPT
White crystals; Yield: 76%; m.p. (°C) 223-224; Rf 0.45 (hexane / ethyl acetate 3:2); IR
1
(KBr, cm-1): 3423 and 3308 (N-H), 1769 and 1713 (C=O), 1602 (C=N); H NMR (300
MHz, DMSO-d6), δ ppm: 4.36 (d, J = 3.6 Hz, 2H, CH2), 7.36 (s, 1H, NH), 7.42 (t, J = 3.6
13
Hz, 1H, CH=N), 7.83-7.90 (m, 4H, Ar), 8.03 and 11.27 (s, 1H, NH2); C NMR (75.5
MHz, DMSO-d6), δ ppm: 40.1 (CH2), 123.1 (Ar), 131.7 (Ar), 134.4 (Ar), 140.4 (C=N),
167.5 (C=O), 177.9 (C=S). Anal. Calcd for C11H10N4O2S: C, 50.37; H, 3.84; N, 21.36;
S, 12.22. found: C, 50.03; H, 3.45; N, 20.98; S, 12.30. HRMS: 262.3388 [M+H] +
.
4.1.3.2. (E)-2-[2-(1,3-dioxoisoindolin-2-yl)ethylidene]-N-
phenylhydrazinecarbothioamide (5b)
White crystals; Yield: 70%; m.p. (°C) 174-176; Rf 0.52 (hexane / ethyl acetate 3:2); 1H
NMR (300 MHz, DMSO-d6), δ ppm: 4.46 (d, J = 4.2 Hz, 2H, CH3), 7.14 (t, J = 7.8 Hz,
1H, Ar), 7.29 (t, J = 7.8 Hz, 2H, Ar), 7.42 (d, J = 7.8 Hz, 2H), 7.52 (t, J = 4.2 Hz, 1H,
Ar), 7.85-7.99 (m, 4H, Ar), 9.66 (s, 1H, NH), 11.72 (s, 1H, NH); 13C NMR (75.5 MHz,
DMSO-d6), δ ppm: 40 (CH2), 123.2 (Ar), 124.7 (Ar), 125.1 (Ar), 128.1 (Ar), 131.7 (Ar),
134.5 (Ar), 138.7 (Ar), 140.9 (C=N), 167.6 (C=O), 175.8 (C=S). Anal. Calcd for
C17H14N4O2S: C, 60.34; H, 4.17; N, 16.56; S, 9.48. found: C, 60.03; H, 4.45; N, 16.35;
S, 11.30. HRMS: 339.0845 [M+H] +
.
4.1.4. Procedure for the synthesis of 6a
In a round bottom flask was added 2- (2- (1,3-dioxoisoindolin-2-yl) ethylidene)
hydrazinecarbothioamide (5a), 1,3-dichloroacetone and DMF. The reaction mixture was
stirred at room temperature for about 1 h. The reaction was followed by thin layer
chromatographic plate. After addition of distilled water, the pure product precipitates.
4.1.4.1. (E)-2-(2-{2-[4-(chloromethyl)thiazol-2-yl]hydrazono}ethyl)isoindoline-1,3-
dione (6a)
White crystals; Yield: 82%; m.p. (°C) 191-193; Rf 0.23 (hexane / ethyl acetate 3:2); IR
(KBr, cm-1): 3159.36 and 3113.44 (N-H), 1771.01 and 1717.67 (C=O), 1565.77 (C=N).
1H NMR (400 MHz, DMSO-d6), δ ppm: 4.41 (d, J = 3.2 Hz, 2H, CH2), 4.53 (s, 2H, CH2),

ACCEPTED MANUSCRIPT
6.76 (s, 1H, CH, thiazole), 7.32 (s, 1H, CH), 7.86-7.89 (m, 4H, CH Ar), 11.75 (s, 1H,
NH); 13C NMR (75.5 MHz, DMSO-d6), δ ppm: 38 (CH2), 41.7 (CH2), 107.9 (CH,
thiazole), 123.1 (Ar), 131.7 (Ar), 134.5 (Ar), 138.4 (C, thiazole), 147.8 (C=N), 167.5
(C=O), 168.6 (S-C=N, thiazole). Anal. Calcd for C14H11ClN4O2S: C, 50.23; H, 3.31; N,
16.74; S, 9.58. found: C, 49.91; H, 3.30; N, 16.82; S, 10.03. HRMS: 335.0463 [M+H]+.
4.1.5. General procedure for the synthesis of 6b-l
In round bottom flask was added 2-(2-(1,3-dioxoisoindolin-2-yl)ethylidene)-N-phenyl-
hydrazine-carbothioamide (5b), the respective α-halogenated ketone and 2-propanol.
The reaction mixture was kept under magnetic stirring, at room temperature, for 1h. The
reactions were followed by thin layer chromatographic plate. The formed precipitate
was filtered on sintered funnel with distilled water, yielding the pure product.
4.1.5.1. 2-((E)-2-{(Z)-[3,4-diphenylthiazol-2(3H)-ylidene]hydrazono}ethyl)isoindoline-
1,3-dione (6b)
Orange crystals; Yield: 56%; m.p. (°C) 196-198; Rf 0.46 (hexane / ethyl acetate 3:2); IR
1
(KBr, cm-1): 1712.40 (C=O). H NMR (300 MHz, DMSO-d6), δ ppm: 4.42 (d, J = 3 Hz
2H, CH2), 6.45 (s, 1H, CH, thiazole), 7.09-7.32 (m, 10H, CH Ar), 7.44 (t, J = 3 Hz, 1H,
HC=N), 7.91-7.88 (m, 4H, CH Ar, phthalimide); 13C NMR (75.5 MHz, DMSO-d6),
δ ppm: 40.3 (CH2), 104.2 (CH, thiazole), 123.0 (Ar), 125.2 (Ar), 128.0 (Ar), 128.1 (Ar),
128.3 (Ar), 128.6 (Ar), 128.8 (Ar), 130.6 (Ar), 131.8 (Ar), 134.4 (Ar), 137.3 (Ar), 139.3
(C, thiazole), 148.2 (C=N), 167.5 (C=O), 169.9 (S-C-N, thiazole). Anal. Calcd for
C25H18N4O2S: C, 68.48; H, 4.14; N, 12.78; S, 7.31. found: C, 66.88; H, 4.30; N, 12.48;
S, 7.12. HRMS: 439.1028 [M+H] +
.
4.1.5.2. 2-((E)-2-{(Z)-[3-phenyl-4-p-tolylthiazol-2(3H)-
ylidene]hydrazono}ethyl)isoindoline-1,3-dione (6c)
Yellow crystals; Yield: 52%; m.p. (°C) 156-158; Rf: 0.7 (hexane / ethyl acetate 3:2). IR
1
(KBr, cm-1): 1712.39 (C=O). H NMR (300 MHz, DMSO-d6), δ ppm: 2.19 (s, 3H,CH3),
4.42 (d, J = 3 Hz, 2H, CH2), 6.41 (s, 1H, CH thiazole), 6.95-6.99 (m, 4H, CH Ar), 7.17-

ACCEPTED MANUSCRIPT
7.45 (m, 6H, CH Ar), 7.88-7.95 (m, 4H, CH Ar phthalimide); 13C NMR (75.5 MHz,
DMSO-d6), δ ppm: 20.7 (CH3), 38.9 (CH2), 100.7 (CH, thiazole), 123.1 (Ar), 127.7 (Ar),
128.1 (Ar), 128.7 (Ar), 128.8 (Ar), 128.9 (Ar), 129.8 (Ar), 131.8 (Ar), 134.5 (Ar), 137.4
(Ar), 137.9 (Ar), 139.456 (C, thiazole), 148.2 (C=N), 167.6 (C=O), 170.1 (S-C-N,
thiazole). Anal. Calcd for C26H20N4O2S: C, 69.01; H, 4.45; N, 12.38; S, 7.09. found: C,
68.66; H, 4.36; N, 12.12; S, 6.96. HRMS: 453.1138 [M+H] +.
4.1.5.3. 2-((E)-2-{(Z)-[4-(4-methoxyphenyl)-3-phenylthiazol-2(3H)-
ylidene]hydrazono}ethyl)isoindoline-1,3-dione (6d)
Yellow crystals; Yield: 54%; m.p. (°C) 173-175; Rf 0.49 (hexane / ethyl acetate 3:2); IR
(KBr, cm-1): 1713.83 (C=O). 1H NMR (300 MHz, DMSO-d6), δ ppm: 4.18 (s, 3H, CH3),
4.44 (d, J = 3 Hz, 2H, CH2), 6.83-8.10 (m, 14H, Ar); 13C NMR (75.5 MHz, DMSO-d6),
δ ppm: 40.1 (CH2), 56.4 (CH3), 104.3 (CH, thiazole), 123.1 (Ar), 123.3 (Ar), 127.9 (Ar),
128.4 (Ar), 128.8 (Ar), 129.2 (Ar), 131.7 (Ar), 134.5 (Ar), 136.6 (Ar), 136.7 (Ar), 137.6
(Ar), 139.0 (C, thiazole), 146.8 (C=N), 167.5 (C=O), 169.6 (S-C-N, thiazole). Anal.
Calcd for C26H20N4O3S: C, 66.65; H, 4.30; N, 11.96; S, 6.84. found: C, 65.60; H, 4.08;
N, 11.85; S, 6.68. HRMS: 469.1276 [M+H] +
.
4.1.5.4. 2-((E)-2-{(Z)-[4-(4-fluorophenyl)-3-phenylthiazol-2(3H)-
ylidene]hydrazono}ethyl)isoindoline-1,3-dione (6e)
Yellow crystals; Yield: 50%; m.p. (ºC)176-179; Rf: 0.63 (hexane / ethyl acetate 3:2); IR
1
(KBr, cm-1): 1714.47 (C=O); H NMR (300 MHz, DMSO-d6), δ ppm: 4.43 (d, J = 3 Hz,
2H, CH2), 6.47 (s, 1H, CH thiazole), 7.02-7.36 (m, 9H, CH Ar), 7.45 (t, J = 3 Hz, 1H,
13
CH), 7.86-7.95 (m, 4H, CH Ar phthalimide); C NMR (75.5 MHz, DMSO-d6), δ ppm
:
38.9 (CH2), 101.3 (CH, thiazole), 115.1 (Ar), 115.3 (Ar), 123.1 (Ar), 127.1 (Ar), 128.0
(Ar), 128.7 (Ar), 128.9 (Ar), 130.6 (Ar), 131.8 (Ar), 134.5 (Ar), 137.2 (Ar),138.3 (C,
thiazole), 148.3 (C=N), 160.2 and 163.4 (C-F), 167.6 (C=O), 169.9 (S-C-N, thiazole).
Anal. Calcd for C25H17FN4O2S: C, 65.78; H, 3.75; N, 12.27; S, 7.02. found: C, 63.91;
H, 3.79; N, 12.01; S, 6.94. HRMS: 457.1083 [M+H] +.

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4.1.5.5. 2-(2-{(Z)-[4-(4-chlorophenyl)-3-phenylthiazol-2(3H)-
ylidene]hydrazono}ethyl)isoindoline-1,3-dione (6f)
Yellow crystals; Yield: 58%; m.p. (°C) 176-179; Rf: 0.84 (hexane / ethyl acetate 3:2);
1
IR (KBr, cm-1): 1717.30 (C=O). H NMR (300 MHz, DMSO-d6), δ ppm: 4.41 (d, J = 3
Hz, 2H, CH2), 6.50 (s, 1H, thiazole), 7.07-7.37 (m, 9H, Ar), 7.44 (t, J = 3 Hz, 1H) 7.85-
7.94 (m, 5H, Ar); 13C NMR (75.5 MHz, DMSO-d6), δ ppm: 56.0 (CH2), 101.8 (CH,
thiazole), 123.1 (Ar), 127.9 (Ar), 128.1 (Ar), 128.6 (Ar), 128.9 (Ar), 129.5 (Ar), 129.9
(Ar), 131.8 (Ar), 133.1 (Ar), 134.5 (Ar), 137.2 (Ar), 138.7 (C, thiazole), 148.3 (C=N),
167.6 (C=O), 169.9 (S-C-N, thiazole). Anal. Calcd for C25H17ClN4O2S: C, 63.49; H,
3.62; N, 11.85; S, 6.78. found: C, 61.17; H, 3.43; N, 11.47; S, 6.79. HRMS: 473.0793
[M+H] +
.
4.1.5.6. 2-((E)-2-{(Z)-[4-(4-bromophenyl)-3-phenylthiazol-2(3H)-
ylidene]hydrazono}ethyl)isoindoline-1,3-dione (6g)
White crystals; Yield: 55%; m.p. (°C) 189-193; Rf: 0.73 (hexane / ethyl acetate 3:2); IR
(KBr, cm-1): 1717.05 (C=O). 1H NMR (300 MHz, DMSO-d6), δ ppm: 4.42 (d, J = 3 Hz,
2H, CH2), 6.51 (s, 1H, thiazole), 7.01-7.94 (m, 14H, Ar); 13C NMR (75.5 MHz, DMSO-
d6), δ ppm: 39.3 (CH2), 101.87 (CH, thiazole), 121.7 (Ar), 123.1 (Ar), 127.9 (Ar), 128.6
(Ar), 128.9 (Ar), 129.8 (Ar), 130.1 (Ar), 131.2 (Ar), 131.8 (Ar), 134.5 (Ar), 137.2 (Ar),
138.1 (C, thiazole), 148.5 (C=N), 167.6 (C=O), 169.9 (S-C-N, thiazole). Anal. Calcd for
C25H17BrN4O2S: C, 58.03; H, 3.31; N, 10.83; S, 6.20. found: C, 57.27; H, 3.28; N,
10.68; S, 6.06. HRMS: 517.0005 [M+H] +.
4.1.5.7. 2-((E)-2-{(Z)-[4-(4-nitrophenyl)-3-phenylthiazol-2(3H)-
ylidene]hydrazono}ethyl)isoindoline-1,3-dione (6h)
Orange crystals; Yield: 46%; m.p. (°C) 186-189; Rf: 0.75 (hexane / ethyl acetate 3:2).
1
IR (KBr, cm-1): 1713.38 (C=O). H NMR (300 MHz, DMSO-d6), δ ppm: 4.45 (d, J = 3
Hz, 2H, CH2), 6.78-8.10 (m, 15H, Ar); 13C NMR (75.5 MHz, DMSO-d6), δ ppm: 40.1
(CH2), 102.0 (CH, thiazole), 123.2 (Ar), 123.3 (Ar), 124.5 (Ar), 128.0 (Ar), 128.4 (Ar),
129.0 (Ar), 131.7 (Ar), 131.7 (Ar), 134.5 (Ar), 136.7 (Ar), 137.4 (Ar), 138.7 (C,

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thiazole), 146.7 (C=N), 167.5 (C=O), 169.6 (S-C-N, thiazole). Anal. Calcd for
C25H17N5O4S: C, 62.10; H, 3.54; N, 14.48; S, 6.63. found: C, 59.19; H, 3.65; N, 13.76;
S, 6.93. HRMS: 484.0975 [M+H] +
.
4.1.5.8. 2-((E)-2-{(Z)-[4-(biphenyl-4-yl)-3-phenylthiazol-2(3H)-
ylidene]hydrazono}ethyl)isoindoline-1,3-dione (6i)
Yellow crystals; Yield: 73%; m.p. (°C) 157-159; Rf 0.43 (hexane / ethyl acetate 3:2); IR
(KBr, cm-1): 1716.38 (C=O). 1H NMR (400 MHz, DMSO-d6), δ ppm: 4.48 (d, J = 3.6 Hz,
2H, CH2), 6.05 (s, 1H, thiazole), 7.00-7.82 (m, 19H, Ar); 13C NMR (75.5 MHz, DMSO-
d6), δ ppm: 39.2 (CH2), 101.2 (CH, thiazole), 123.3 (Ar), 126.8 (Ar), 126.9 (Ar), 127.7
(Ar), 128.3 (Ar), 128.4 (Ar), 128.5 (Ar), 128.6 (Ar), 128.7 (Ar), 128.8 (Ar), 129.2 (Ar),
129.6 (Ar), 132.3 (Ar), 133.9 (Ar), 133.9 (Ar), 139.9 (C, thiazole), 141.2 (C=N), 167.8
(C=O), 169.0 (S-C-N, thiazole). Anal. Calcd for C31H22N4O2S: C, 72.35; H, 4.31; N,
10.89; S, 6.23. found: C, 70.19; H, 4.32; N, 10.35; S, 6.53. HRMS: 515.1416 [M+H] +.
4.1.5.9. 2-((E)-2-{(Z)-[4-(naphthalen-2-yl)-3-phenylthiazol-2(3H)-
ylidene]hydrazono}ethyl)isoindoline-1,3-dione (6j)
Yellow crystals; Yield: 68%; m.p. (°C) 159-161; Rf 0.43 (hexane / ethyl acetate 3:2); IR
(KBr, cm-1): 1713.93 (C=O). 1H NMR (400 MHz, DMSO-d6), δ ppm: 4.56 (d, J = 4 Hz,
2H, CH2), 6.18 (s, 1H, thiazole), 6.99-7.91 (m, 17H, Ar); 13C NMR (75.5 MHz, DMSO-
d6), δ ppm: 39.2 (CH2), 101.6 (CH, thiazole), 123.3 (Ar), 125.4 (Ar), 126.5 (Ar), 126.7
(Ar), 127.6 (Ar), 127.7 (Ar) 127.8 (Ar), 128.0 (Ar), 128.1 (Ar), 128.3 (Ar), 128.4 (Ar),
128.7 (Ar), 129.2 (Ar), 132.3 (Ar), 132.8 (Ar), 133.9 (Ar), 137.5 (Ar), 140.3 (C,
thiazole), 149.5 (C=N), 167.8 (C=O), 171.0 (S-C-N, thiazole). Anal. Calcd for
C29H20N4O2S: C, 71.29; H, 4.13; N, 11.47; S, 6.56. found: C, 70.09; H, 4.21; N, 11.41;
S, 6.70. HRMS: 489.1296 [M+H] +.
4.1.5.10. 2-((E)-2-{(Z)-[4-(3-nitrophenyl)-3-phenylthiazol-2(3H)-
ylidene]hydrazono}ethyl)isoindoline-1,3-dione (6k)

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Yellow crystals; Yield: 63%; m.p. (°C) 154-154; Rf 0.34 (hexane / ethyl acetate 3:2); IR
(KBr, cm-1): 1712.80 (C=O). 1H NMR (300 MHz, DMSO-d6), δ ppm: 4.43 (d, J = 2.7 Hz,
2H, CH2), 6.76 (s, 1H, CH thiazole), 7.25-7.38 (m, 5H, CH Ar), 7.48-7.51 (m, 3H, CH
13
Ar), 7.86-7.95 (m, 6H, CH Ar), 8.07 (t, J = 2.4 Hz, 1H, HC=N); C NMR (75.5 MHz,
DMSO-d6), δ ppm: 39.3 (CH2), 104.3 (CH, thiazole), 123.2 (Ar), 123.4 (Ar), 123.6 (Ar),
128.8 (Ar), 128.9 (Ar), 129.1 (Ar), 129.3 (Ar), 129.5 (Ar), 129.6 (Ar), 130.0 (Ar), 130.3
(Ar), 135.0 (Ar), 135.6 (Ar), 138.8 (C, thiazole), 149.3 (C=N), 167.5 (C=O), 169.6 (S-
C-N, thiazole). Anal. Calcd for C25H17N5O4S: C, 62.10; H, 3.54; N, 14.48; S, 6.63.
found: C, 60.45; H, 3.70; N, 14.10; S, 6.48. HRMS: 484.0976 [M+H] +.
4.1.5.11. 2-((E)-2-{(Z)-[4-(3,4-dichlorophenyl)-3-phenylthiazol-2(3H)-
ylidene]hydrazono}ethyl)isoindoline-1,3-dione (6l)
Yellow crystals; Yield: 48%; m.p. (ºC) 155-157; Rf: 0.89 (hexane / ethyl acetate 3:2);
1
IR (KBr, cm-1): 1714.45 (C=O). H NMR (300 MHz, DMSO-d6), δ ppm: 4.45 (d, J = 3
Hz, 2H, CH2), 6.81-7.92 (m, 14H, Ar); 13C NMR (75.5 MHz, DMSO-d6), δ ppm: 40.1
(CH2), 104.6 (CH, thiazole), 123.1 (Ar), 128.3 (Ar), 128.6 (Ar), 128.9 (Ar), 129.3 (Ar),
130.3 (Ar), 130.5 (Ar), 130.9 (Ar), 131.4 (Ar), 131.7 (Ar), 134.3 (Ar), 134.6 (Ar), 136.1
(Ar), 137.3 (C, thiazole), 149.2 (C=N), 167.5 (C=O), 169.6 (S-C-N, thiazole); Anal.
Calcd for C25H16Cl2N4O2S: C, 59.18; H, 3.18; N, 11.04; S, 6.32. found: C, 56.85; H,
3.30; N, 10.58; S, 6.02. HRMS: 507.0432 [M+H] +.
4.2. X-ray analysis
X-ray diffraction data collections were performed on an Enraf-Nonius Kappa-CCD
diffractometer (95 mm CCD camera on κ-goniostat) using graphite monochromated
MoKα radiation (0.71073 Å) at room temperature. Data were collected using the
COLLECT software[37] up to 50° (2θ). The final unit cell parameters were based on
15666, 9327, and 12333 reflections to 2e, 2f and 2g, respectively. Integration and
scaling of the reflections as well as the corrections for Lorentz and polarization effects
were performed with the HKL DENZO-SCALEPACK system of programs[38]. The
compound structures were solved by direct methods with SHELXS-97[39]. The model
was refined by full-matrix least squares on F2 using SHELXL-97[39]. The program
ORTEP-3[40] was used for graphic representations, and the program WinGX[41] was

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used to prepare the material for publication. All H atoms were located by geometric
considerations placed (C-H = 0.93 Å; N-H = 0.86 Å) and refined using a riding model
with Uiso(H) = 1.5Ueq(C-methyl) or 1.2Ueq(other). An Ortep-3 diagram of the
molecules is shown in Figure 2, and Table S1 shows the main crystallographic data.
Crystallographic data (excluding structure factors) for the structures reported in this
paper have been deposited with the Cambridge Crystallographic Data Centre as
supplementary material (No. CCDC 1411254, 1411262 and 1411263).
4.3 Biological assays of toxicity to splenocytes
Splenocytes from BALB/c mice were divided into a 96 well plate at a density of 5×106
cells per well in RPMI-1640 medium containing 10% inactivated Foetal Bovine Serum
(FBS). Each chemical inhibitor was dissolved in DMSO at a concentration of 10
mg/mL, and then the sample was serially diluted in RPMI-1640 medium supplemented
with 10% FBS at concentrations of 1.0, 5.0, 10, 25, 50 and 100 ꢀg/mL, in triplicate. As
a positive control, saponin was used at a concentration of 0.1 ꢀg/mL, while RPMI-1640
medium supplemented with 10% FBS and DMSO was used as a negative control. A
total of 1.0 ꢀCi of
3
H-thymidine was added to each well, and the plate was incubated for
24 h at 37 ºC and 5% CO
2
.
The plate was then read in the counter beta irradiation reader (Multilabel Reader,
Finland), and the percent incorporation of tritiated thymidine was determined. Cells that
were not treated with drugs (negative controls) were calculated as 100% of tritiated
thymidine incorporation (100% viable cells). For cells treated with saponin, cell
viability was 5%. When the percentage of incorporation was higher than 90%, the
concentration of the drug was regarded as nontoxic to splenocytes.
4.4 Antiproliferative activity for the epimastigote form
Epimastigotes of Dm28c strain were distributed into a 96 well plate to a final density of
106 cells per well. Each chemical inhibitor was dissolved in the respective wells, in
triplicate. Benznidazole was used as positive controls in this assay. The plate was then
cultivated for 4 days at 27 °C. After this time, aliquots from each well were collected,
and the number of parasites was calculated in a Neubauer chamber. Epimastigotes not
treated with the chemical inhibitors (negative control) were assumed as 100% the

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number of parasites. The dose-response curves were determined, and the IC50 values
were calculated using at least five concentrations (data points) and a nonlinear equation
(Prism, version 4.0).
4.5 Toxicity to trypomastigotes
Strain Y trypomastigotes were collected from Vero cell supernatants and distributed in a
96 well plate to a final density of 4×105 cells per well. Each chemical inhibitor was
added to the wells, in triplicate. Benznidazole was used as positive controls in this
assay. The plate was then cultivated for 24 h at 37 °C and 5% CO . After this time,
2
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).
4.6 Ultrastructural studies
The parasites were cultured for 24 h in RPMI 1640 medium (Sigma-Aldrich, St. Louis,
MO, USA) buffered to pH 7.5 and supplemented with HEPES (20 mM), 10% foetal
bovine serum, penicillin (100 U/mL), and streptomycin (100 ꢀg/mL) containing the
compound 6k at the IC50 concentration and twice the value of IC50. The parasites were
collected, washed in PBS and fixed with 2.5% glutaraldehyde, 4% formaldehyde, and
0.1 M cacodylate buffer at pH 6.8. They were then postfixed in 2% osmium tetroxide
(OsO4) in a 0.1 M cacodylate buffer at pH 6.8 and processed for routine transmission
electron microscopy (TEM) and scanning electron microscopy (SEM).
For SEM analysis, the parasites were dehydrated in graded ethanol and dried by the
critical point method with CO2. The samples were mounted on aluminium stubs, coated
with gold and examined under a JEOL-5600LV microscope.
For TEM analysis, the parasites were dehydrated in a graded series of acetone and
finally embedded in epon. Sections were stained with uranyl acetate and lead citrate and
observed with Tecnai spirit G2 Biotwin microscope.
4.7 Propidium iodide and annexin V staining

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Trypomastigotes (1 × 107) were incubated for 24 h at 37 °C in the absence or presence
of compound 6k. After incubation, the parasites were labelled with propidium iodide
(PI) and annexin V using the annexin V-FITC apoptosis detection kit (Sigma-Aldrich),
according to the manufacturer’s instructions. Acquisition and analyses were performed
using a FACS Calibur flow cytometer (Becton Dickinson, CA, USA) with FlowJo
software (Tree Star, CA, USA). A total of 30,000 events were acquired in the region
previously established as trypomastigote forms of T. cruzi. Two independent
experiments were performed.

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