Synthesis, anticancer activity and mechanism of action of new thiazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2017.12.040

Thiazole derivatives are recognized to possess various biological activities as antiparasitic, antifungal, antimicrobial and antiproliferative. The present work reports the synthesis of 22 new substances belonging to two classes of compounds: thiosemicarbazones and thiazoles, with the purpose of developing new drugs that present high specificity for tumor cells and low toxicity to the organism. A cytotoxic screening was performed to evaluate the performance of the new derivatives in five tumor cell lines. Eight compounds were shown to be promising in at least three tumor cell lines. These compounds had their IC₅₀ determined within 72 h and the activity structure ratio was assessed. The effect of the best compounds on PBMC and hemolytic activity assay was then evaluated. The compound 1d was considered the most promising among the samples tested and its influence on cell cycle, DNA fragmentation and mitochondrial depolarization was evaluated.

Full text of DOI:10.1016/j.ejmech.2017.12.040

Accepted Manuscript
Synthesis, anticancer activity and mechanism of action of new thiazole derivatives
Temístocles Italo da Santana, Miria de Oliveira Barbosa, Paulo André Teixeira de
Moraes Gomes, Anne Cecília Nascimento da Cruz, Teresinha Gonçalves da Silva,
Ana Cristina Lima Leite
PII:
S0223-5234(17)31066-8
10.1016/j.ejmech.2017.12.040
EJMECH 10018
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 July 2017
Revised Date: 11 December 2017
Accepted Date: 12 December 2017
Please cite this article as: Temí.Italo. da Santana, M.d.O. Barbosa, Paulo.André.Teixeira.de.Moraes.
Gomes, Anne.Cecí.Nascimento. da Cruz, Teresinha.Gonç. da Silva, A.C.L. Leite, Synthesis, anticancer
activity and mechanism of action of new thiazole derivatives, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2017.12.040.
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GRAPHICAL ABSTRACT

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Synthesis, Anticancer Activity and Mechanism of Action of New
Thiazole Derivatives
Temístocles Italo da Santanaa,1, Miria de Oliveira Barbosab,1, Paulo André Teixeira de
Moraes Gomesb, Anne Cecília Nascimento da Cruza,
Teresinha Gonçalves da Silvaa and Ana Cristina Lima Leiteb*
a
Departamento de Antibióticos, Centro de Ciências da Saúde, Universidade Federal de
Pernambuco, 50740-520, Recife, PE, Brazil.
b
Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade
Federal de Pernambuco, 50740-520, Recife, PE, Brazil.
1 These authors contributed equally to this work.
*To whom correspondence should be addressed: phone +55 81-21268511, fax +55 81-21268510; e-
mail: acllb2003@yahoo.com.br

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Abstract
Thiazole derivatives are recognized to possess various biological activities as
antiparasitic, antifungal, antimicrobial and antiproliferative. The present work reports
the synthesis of 22 new substances belonging to two classes of compounds:
thiosemicarbazones and thiazoles, with the purpose of developing new drugs that
present high specificity for tumor cells and low toxicity to the organism. A cytotoxic
screening was performed to evaluate the performance of the new derivatives in five
tumor cell lines. Eight compounds were shown to be promising in at least three tumor
cell lines. These compounds had their IC50 determined within 72 hours and the
activity structure ratio was assessed. The effect of the best compounds on PBMC and
hemolytic activity assay was then evaluated. The compound 1d was considered the
most promising among the samples tested and its influence on cell cycle, DNA
fragmentation and mitochondrial depolarization was evaluated.
Keywords
Cancer; Thiazoles; Cell cycle; DNA fragmentation; Mitochondrial depolarization

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GRAPHICAL ABSTRACT
HIGHLIGHTS
The cytotoxic activity of 3 thiosemicarbazones and 19 new thiazoles was evaluated;
Eight compounds were shown to be promising in at least three tumor cell lines;
Compound 1d induced mitochondrial depolarization;
Compound 1d induced DNA fragmentation and arrest cell cycle in G1 phase;
Compound 1d presented as a promising antitumor candidate.

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1 Introduction
Cancer is one of the major public health problems in many countries around the world
[1]. A complex disease of genetic trait has long been considered a process of
autonomous cell death, resulting from the acquisition of successive mutations in
oncogenes and tumor suppressor genes that result in a progressive proliferation and
resistance to cell death [2, 3].
The standard treatment for cancer, although not specific, is based on surgery,
radiotherapy, and chemotherapy. Often, surgical resection and radiotherapy are
successful techniques in eradicating the primary tumor, although a problem
commonly found is relapse of the disease due to residual tumor cells and/or
metastasis. Therefore, these therapeutic strategies are usually followed by
chemotherapy [4].
Although many antitumor agents are present in the literature and commercially
available, such compounds exhibit high cytotoxicity and develop resistance, which
may lead to decreased drug efficacy. Thus, cancerology can be considered an area of
great need to be explored [5].
In recent years, compounds with anticancer activity have been extensively designed
and tested as good perspectives by medicinal chemists, such as the possibility of
developing promising new prototypes for the treatment of cancer. Previous studies
have shown that the pharmacophoric group thiosemicarbazones has several
pharmacological properties such, antiparasitic [6, 7, 8, 9], larvicide [10], antimicrobial
[11, 12] and antiproliferative [13, 14].
The antitumor activity of thiosemicarbazones seems to be due to an inhibition of
DNA synthesis produced by the modification in the reductive conversion of
ribonucleotides to deoxyribonucleotides [15]. This biological role is often related with
their capability to inhibit the enzyme, ribonucleotide reductase, similar to what is
observed with potent anticancer drugs such as triapine [16].
Thiazole nucleus, heterocycle derived from thiosemicarbazone, also have a broad
scope in preclinical studies that demonstrate its efficacy in antiparasitic [17, 18, 19],
antifungal and antimicrobial [20, 21] and antiproliferative activities [22, 23, 24, 25] in
addition to a low cytotoxicity. In previous works, our group identified that 1,3-
thiazoles substituted at 2 and 4 positions have potent antitumor and
immunomodulatory activity [24, 25]. Regarding the antiproliferative activity,
according to Morigi et al., thiazole derivatives can act though the inhibition of matrix

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metalloproteinases (MMPs), inhibition of kinases and inhibition of anti-apoptotic
BCL2 family proteins [26].
On the other hand, it is well known that the presence of C–F bonds in organic
molecules has an important effect on properties such as lipophilicity, membrane
permeability, pKa, conformation, metabolic stability and pathways, and
pharmacokinetic properties [27].
Besides, introducing fluorine atoms and/or fluorine-containing groups into bioactive
compounds can improve selectivity, intrinsic potency, increasing efficacy and easiest
to administer [28]. Then, more than 20% of the current approved drugs contain at
least one fluorine atom [29]. Indeed, compounds containing trifluoromethyl in their
structure have shown potent anticancer activity and an increasing number of
fluorinated antimitotic/antitumour agents have now becoming available for cancer
treatment. [30]. For exemple, trifluoromethylated drugs such as flutamide (I),
hydroxyflutamide (II), nilutamide (III) and 5-trifluoromethyl uracil (IV) are widely
used for the treatment of metastatic prostate cancer [31].
In this context, and following the work developed by our research group in the
development of new antitumor agents [24, 25, 32, 33, 34], the present study aimed to
synthesize 1,3-thiazoles derivatives containing 4-(trifluoromethyl)-benzylidene
moiety. The prospect inspiration of the new series is presented in Figure 1.
PLEASE, INSERT FIGURE 1 HERE
Variations were made in the group linked to position 4 of the thiazole ring, through
insertion of aromatic rings with different substitutions in ortho, meta and para
positions and the antiproliferative activity against human tumor cell lines of all
compounds were evaluated. The structural planning of all series is presented in
Figure 2.
PLEASE, INSERT FIGURE 2 HERE
Twenty-two derivatives were tested for cytotoxic activity by the MTT assay in five
tumor lines. Among the molecules tested, 8 showed significant toxicity in at least
three tumor lines tested. The compound 1d presented better performance and was
chosen for studies of the possible mechanism of action.

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2 Results and Discussion
2.1 Chemistry
The intermediate thiosemicarbazones 1-3 (R1 = H, Me, Ph, respectively) were
prepared in reaction between the 4'-trifluoromethyl-benzaldehyde (commercially
obtained) with the corresponding thiosemicarbazide under reflux and catalytic amount
of HCl. These intermediate compounds them react with different α-halogenated
ketones, obtaining the 1a-3d series with yields of 22% to 94% (Scheme 1). All
compounds were identified by infrared (IR) and nuclear magnetic resonance (1H-
NMR and 13C-NMR) spectroscopy, mass spectra (ESI-TOF) and their purity was
established by elemental analysis (EA).
PLEASE, INSERT SCHEME 1 HERE
NMR data are compatible with the proposed compounds. In theory, two geometrical
isomers (E and Z) about the imine (C=N) double bond are possible for the
1
thiosemicarbazones and the respective thiazoles. However, analysis of the H NMR
spectra of the compounds indicated one predominant isomer; the E isomer by
comparison with known analogues [18, 19]. Intramolecular H-bonding involving the
proton attached to N4 (in DMSO) with the imine N-atom leads to a distinctive singlet
around 12 ppm and this is also seen here [18, 19].
Once thiosemicarbazones were characterized, the respective 1,3-thiazoles were
1
characterized by usual spectroscopy. As exemplified with the H NMR analysis of
(E)-4-(2,4-dichlorophenyl)-2-(2-(4-(trifluoromethyl) benzylidene) hydrazinyl)thiazole
(1d), singlet and doublets peaks, corresponding to aromatic protons, were observed at
δ 7.5-7.9. For the thiazole ring, a singlet at δ 7.4 was found. NH proton appeared as
singlet at δ 12.5. In 13C NMR spectrum of 1d, peaks of the aromatic carbons were
found at δ 126.8-138.3 ppm. The presence of peaks at δ 109.7 and 145.8 ppm,
1
confirm the thiazole cyclisation. Besides, representative H-NMR spectrums of some
compounds are presented in Supplementary Material.

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2.2 Cytotoxic screening of thiosemicarbazones and thiazoles derivatives
A cytotoxic screening against five tumor cells lines in the assay single dose of 25
µg/mL is showed in Table 1. Were considered active substances which showed
inhibition above 75% in at least two strains of the type adhered (NCI-H292, HEp-2,
and HT-29) and a suspension type strain (HL-60 and K562).
Among the 22 substances were tested, 8 showed the above criteria (1, 2, 1b, 1c, 1d,
1e, 1h e 2b). Then had IC50 values determined after 72 h.
PLEASE, INSERT TABLE 1 HERE
2.3 IC50 determination of thiosemicarbazones and thiazoles derivatives
Three thiosemicarbazones derivatives were assessed in tumor cell lines NCI-H292,
HEp-2, HT-29, HL-60, and K562. However, only two substances (1 and 2) showed
cytotoxic activity greater than 75% inhibition in at least two cell lines with adhesive
characteristics and a strain in suspension.
Between the three thiosemicarbazones, 1, 2 and 3 it was observed that the simple
addition of phenyl group in the structure reduced the cytotoxicity of the compound 3.
However, 1 (R1 = H) and 2 (R2 = Me) were promising for their antiproliferative
action in all tumor lines tested.
1 and 2 showed cytotoxic activity in all tested tumor cell lines. However, 1 showed
better activity against NCI-H292, HEp-2, K562 and HL-60, with IC50 ranging from
6.27 to 12.74 µM (Table 3).
Of the 19 new thiazoles derivatives tested on tumor cell lines, six had significant
cytotoxic activity and have determined their IC50 (Table 3) and only 1c and 1d
showed significant cytotoxicity to all tested tumor cell lines in the assay single dose
of 25 µg/mL (Table 2).
Compounds which have a strongly electron withdrawing group (NO2) in the para
position (Table 3), as observed in the 1b and 2b, had a large variation of the IC50
values in all tested tumor lines. 1b showed IC50 value ranging from 7.41 to 27.33 µM
while the 2b showed IC50 from 11.83 to 47.77 µM. However, although similar, this
higher range for 2b IC50 value can be related to the presence of a methyl linked to
nitrogen (N-3).

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The presence of electron withdrawing group (NO2) into 1b showed better results for a
NCI-H292 with IC50 = 7.41 µM. In contrast, the addition of electrons withdrawing
group in the meta position (compound 1c) may be associated with the inhibition of all
the cell lines tested in the screening cytotoxic, yet the presence of this group in the
meta position did not show good results when subjected to tests to determine their
IC50 values in the tested tumor cell lines, except in the K562 line cell.
The di-substituted compounds 2,4-dichloro (1d) and 3,4-dichloro (1h) showed
excellent cytotoxic activity with IC50 less than 10 µM in four tumor cells tested. The
variation in the meta and para position do not appear to interfere with high cytotoxic
activity observed.
The 1e compound showed significant cytotoxicity in three tumor cell lines. The
activity of this compound is related to the presence of the methoxy group in the para
position of the aromatic ring.
PLEASE, INSERT TABLE 2 HERE
From the results obtained, an analysis cytotoxic behavior, over time, of the
intermediate compound 1 and the thiazole derivative 1d were determined from serial
dilutions after 24 or 48 h of treatment with K562 cells. The values of IC50 are
presented in Table 3.
PLEASE, INSERT TABLE 3 HERE
The compounds showed low cytotoxicity in K562 cells after 24 hours of treatment. 1
and 1d were active cytotoxic time-dependent with a maximum effect after 72 h of
treatment. From the data obtained, we choose the 1d to verify the mechanism of
action.
2.4 Cytotoxicity in normal cells
The cytotoxicity of the thiosemicarbazones and thiazoles were also evaluated against
peripheral blood mononuclear cell (PBMC). The results presented in Table 4 show
that the cytotoxic effects of these compounds were less pronounced in PBMC when
compared to tumor cells, with a selectivity index (SI) for myeloblastoid leukaemia

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(K562) of 12.49 and 7.85 for 1 and 1d, respectively. The SI was calculated with the
following formula: SI = IC50 (PBMC) / IC50 (K562).
None of the compounds described here were able to cause hemolysis in human
erythrocytes during the study period, even at the highest concentration (250 µg/mL)
(Table 4). The absence of lytic effects suggests that the cytotoxicity of these
compounds is not related to membrane disruption and is likely related to more
specific cellular pathways.
PLEASE, INSERT TABLE 4 HERE
2.5 Measurement of mitochondrial membrane potential (∆Ψm)
Several drugs act on mitochondria. Thus, we evaluated by flow cytometry if there is
involvement of mitochondria in the mechanism of cell death. The assay was based on
incorporation of rhodamine 123 into cells treated with 1d compound after 48 hours of
treatment. The objective was to evaluate the ability of the thiazole derivative to
produce changes in mitochondrial transmembrane potential (∆Ψm).
The integrity of mitochondria and cellular bioenergetic functions are controlled by
maintaining the mitochondrial membrane potential (∆Ψm). During induction of cell
death by drug-induced apoptosis, it is possible to observe mitochondrial alteration,
and the event responsible for this is the increase in mitochondrial depolarization
(decrease of mitochondrial membrane potential - ∆Ψm). This event is related to the
initial events that occurred during the apoptosis process [35]. Mitochondrial
membrane depolarization is one of the main characteristics of apoptosis-induced cell
death [36].
In this study, we evaluated the ability of thiazole derivative 1d (17 µM and 34 µM) to
produce changes in mitochondrial transmembrane potential (∆Ψm) after 48 hours of
treatment. Compound 1d was able to induce mitochondrial depolarization in 25.05%
and 37.12% of cells treated with compound 1d at concentrations of 17 and 34 µM,
respectively. The positive control doxorubicin presented depolarization of 23.6%
while the negative control presented 12.24% of mitochondrial depolarization (Figure
3).
Hsiung and Kadir, [37] observed that mitochondrial depolarization is capable of
inducing caspase-3 activation and consequent externalization of phosphatidylserine

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(OS) on the plasma membrane of cells treated with substances isolated from plants
and thus demonstrated the role of depolarization in apoptosis.
PLEASE, INSERT FIGURE 3 HERE
Recent studies have demonstrated that thiazol-derived compounds are capable of
inducing mitochondrial membrane depolarization and consequent apoptosis in human
acute myeloid leukemia U937 cells [38]. Similarly, benzothiazole YLT322 was able
to depolarize the mitochondrial potential and trigger the release of cytochrome c from
the mitochondria to the cytosol showing the involvement of this derivative in the
intrinsic pathway of apoptosis [39].
Kamal et al. [40] also observed that a benzothiazole linked to phenyl
pyridopyrimidinones was able to induce apoptosis by depolarizing the mitochondrial
membrane of cervical cancer cells ME-180 and activate caspase-3 in cervical cancer
cell line ME-180.
Our results suggest that compound 1d is capable of inducing cell death with
mitochondrial involvement in the two tested doses. However, complementary studies
evaluating the involvement of caspases and cytochrome c must be performed to
elucidate the involvement of mitochondria on the induction of apoptosis in K562 cells
from compound 1d.
2.6 Cell cycle assay
The cell cycle is related to a series of events responsible for cell division and
duplication. The cell cycle presents four distinct phases: G1, S (synthesis), G2
(interphase) and M (mitosis) phases. The harnessing of energy and material for DNA
synthesis occurs in the G1 phase. Then the cell replicates its DNA in the S phase. The
G2 phase prepares the cell for the mitosis itself where division of the nucleus and
cytoplasm of the cell takes place [41]. Due to the importance of the cell cycle in the
process of tumor progression we evaluated if cell growth inhibition in K562 occurred
due to cell cycle arrest by flow cytometry 48 hours after treatment.
Many antitumor treatments have better results when cells are in the process of cell
division. The explanation is because tumor cells show loss of control of
differentiation and alteration in the cell cycle [42]. Due to the importance of this event

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in the process of tumor progression we evaluated the action of compound 1d on the
cell cycle by flow cytometry.
In this study K562 cells were treated with these compounds at concentrations of 17
and 34 µM for 48 h. The data obtained clearly indicated that these compounds show
G1 cell cycle arrest in comparison with the untreated cells. The compound 1d (17 and
34 µM) showed 85.25 and 82.74 % of cells in G1 phase (Figure 4). The positive
control doxorubicin had cell cycle arrest in G2/M.
Several thiazole derivatives have been described in the literature as stop inducers in
different phases of the G0 / G1, S and G2 / M cell cycle and in different tumor cell
lines [41, 42, 43, 44, 45].
Senviki et al. [42] observed that treatment with (5- [5- (2-Hydroxyphenyl) -3-phenyl-
4,5-dihydropyrazol-1-ylmethylene] -3- (3-acetoxyphenyl) -2-thioxothiazolidin-4-one)
after 48 hours of treatment in leukemic cells HL-60 was also able to induce cell cycle
arrest in the G0 / G1 phase.
PLEASE, INSERT FIGURE 4 HERE
2.7 DNA fragmentation assay
An important feature in the process of cell death by apoptosis is the presence of
fragmented DNA. However, it is also possible to observe other changes in nuclear
morphology such as nuclear condensation, chromosomal DNA cleavage, formation of
membrane blebs and presence of apoptotic bodies [46].
In order to evaluate DNA fragmentation, we used the same principle of cell cycle
analysis from the incorporation of propidium iodide in the cells. Cells with
fragmented DNA emit less fluorescence by incorporating less propidium iodide than a
cell with whole DNA. For the tumor line tested, compound 1d induced DNA
fragmentation at the two concentrations analyzed and obtained maximum effect at the
concentration of 34 µM which represented 22.43% of the cells presented fragmented
DNA. Compound 1d at the concentration of 17 µM presented 19.74% of the cells
with fragmented DNA (Figure 5).
PLEASE, INSERT FIGURE 5 HERE

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Compound 1d disrupted the K562-type leukemic cell cycle in G1 phase and was able
to depolarize the mitochondrial membrane. Altogether, these data and the presence of
cells with fragmented DNA may infer that the probable mechanism of cell death
occurs by apoptosis. However, subsequent studies related to this cell death pathway
should be performed.
2.8 Physicochemical and ADME parameters
We evaluated if the synthesized compounds physicochemical properties are within the
Lipinski’s Rule of Five, which are important for pharmacokinetics and drug
development. For this purpose, physicochemical and ADME properties were
calculated using the SwissADME (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 are considered to adhere to Lipinski Rule
[47]. All synthetized compounds are compatible with Lipinski Rule. Another
interesting property is the number of rotatable bonds and the polar surface area (PSA).
A large number of rotatable bonds (≥10) has been associated with poor oral
bioavailability [48]. Compounds with a low PSA (≤ 140 Å2) tend to have higher oral
bioavailability [48, 49]. All synthetized compounds have appropriate PSA and
number of rotatable bonds. These data are presented in the supplementary material.
As demonstrated in Table 5, the most active compounds shown variable permeability
based on gastrointestinal absorption (GI), according to the BOILED-Egg predictive
model (Brain Or IntestinaL EstimateD permeation method). Three compounds
showed high gastrointestinal absorption (1, 2 and 1e). With respect to oral
bioavailability, it's expected 0.55 of probability of oral bioavailability score > 10% in
the rat for all compounds, greater than doxorubicin (0.17). All these data, suggests the
in silico good druglikeness profile and great chemical stabilities for this thiazole
compounds.
PLEASE, INSERT TABLE 5 HERE
3 Conclusion
A series of three thiosemicarbazones and nineteen thiazoles derivatives that insert a 4-
(trifluoromethyl)-benzylidene moiety were synthesized. Between them, 8 compounds

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were identified with significant cytotoxic activity in at least three tumor cell lines.
Their IC50 were determined and their action on red blood cells and PBMC were also
tested. Compound 1d presented as a promising antitumor candidate and had its
mechanism of action evaluated by flow cytometry. Our results showed cell cycle
arrest in G1 phase, DNA fragmentation and alteration in mitochondrial membrane
potential by compound 1d for the two concentrations tested. These data suggest a
process of cell death, at least in part, by apoptosis, with involvement of mitochondrial
pathway in the mechanism of action. Complementary studies, such as involvement of
caspases and cytochrome c should be performed to better elucidate the induction of
cell death produced by 1d. The physicochemical and ADME properties of synthesized
compounds were calculated in silico and the data suggests thiazole compounds fits
druglikeness profile. Thus, our results show that thiazole derivatives can be used in
the development of new drugs with anticancer activity.
4 Experimental Section
4.1 Chemistry
The reagents were purchased from Acros Organics, Fluka, Sigma-Aldrich and Vetec
or Dynamics solvents. Deuterated solvents (DMSO-d 6, CDCl3, D2O) were of the
trademark CIL (Tedia Brazil). The reactions were monitored by thin layer
chromatography using silica gel 60, containing fluorescent indicator F254.
Chromatographic plates were visualized under UV light (with double wavelength 365
or 254 nm). Melting points were measured using a Thomas Hoover capillary and
1
values (°C) were reported thereafter. For all of the novel compounds, H and 13C
NMR analyzes and, where necessary, two-dimensional analysis (DEPT) as well as the
addition of deuterated water to localize NH signals were performed. All compounds
1
13
were solubilized in DMSO-d6. H and C NMR spectra were obtained using Unity
1
13
Plus model Variant instruments (400 MHz to H, 100 MHz to C) or Bruker AMX
1
(300 MHz to H, 75.5 MHz to 13C) using tetramethylsilane (TMS) as internal
1
standard. The number of signals in H NMR spectra was designated as follows:
s/singlet; d/doublet, t/triplet, dd/double doublet, q/quartet, m/multiplet, and the
coupling constants, in hertz, as J. Infrared spectroscopy was performed on a Bruker
instrument (IFS 66 model) using KBr.

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4.1.1 General Procedure for synthesis of compounds 1, 2 and 3
The compounds 1, 2 and 3 was prepared by reacting commercially available
respective thiosemicarbazide with 4-(Trifluoromethyl)benzaldehyde (1:1 mol ratio)
using ethanol under reflux in the presence of catalytic amount of hydrochloric acid,
for 3 hours. Finally, water was added at the end of the reaction for precipitation of the
compounds. This reaction condition led to yield between 85% to 94%. The reaction
was monitored by thin layer chromatography (TLC). The resulting solid was filtered
through a sintered funnel and recrystallized from toluene to give the pure product.
4.1.1.1 (E)-2-[4-(trifluoromethyl)benzylidene]hydrazinecarbothioamide (1)
White crystals; Yield 85%; m.p.(ºC) 166-170; Rf:0,41 (hexane / ethyl acetate 7:3). IR
1
(KBr, cm-1): 3272.63 (NH), 1698.36 (C=N). H NMR (400 MHz, DMSO-d6), δ ppm:
3.350 (s, 2H, NH2), 7.673 (d, J = 7.8 Hz, 2H, Ar), 7.804 (d, J = 8.1 Hz, 2H, Ar), 8.301
(s, 1H, HC=N), 11.598 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6), δ ppm:
124.000(CF3) 125.278 (C, Ar), 125.324 (C, Ar), 127.651 (C, Ar), 129.971 (C, Ar),
134.488 (C, Ar), 138.147 (C, Ar), 140.219 (C=N), 178.297 (C=S). Anal. Calcd for
C9H8F3N3S: C, 43.72; H, 3.26; N, 17.00; S, 12.97. found: C, 43.94; H, 3.04; N, 15.02;
S, 11.89. HRMS: 343.9749 [M+H] +
4.1.1.2
(E)-N-methyl-2-[4-(trifluoromethyl)benzylidene]
hydrazinecarbothioamide (2)
White crystals; Yield 94%; m.p.(ºC) 234-236; Rf:0,75 (hexane / ethyl acetate 7:3). IR
1
(KBr, cm-1): 3158.76 (NH), 1539.93 (C=N). H NMR (400 MHz, DMSO-d6), δ ppm:
3.02 (d, J = 4.2 Hz, 3H, CH3), 7.756 (d, J = 7.8 Hz, 2H, Ar), 8.025 (d, J = 7.5 Hz, 2H,
13
Ar), 8.687 (s, 1H, HC=N), 11.690 (s, 1H, NH). C NMR (100 MHz, DMSO-d6), δ
ppm: 30.923 (CH3), 125.134(CF3), 125.522 (C, Ar), 127.727 (C, Ar), 138.372 (C, Ar),
138.784(C=N), 177.915 (C=S). Anal. Calcd for C10H10F3N3S: C, 45.97; H, 3.86; N,
16.08; S, 12.27. found: C, 46.22; H, 2.77; N, 15.81; S, 11.13. HRMS: 262.0527
[M+H] +
4.1.1.3
(E)-N-phenyl-2-[4-(trifluoromethyl)benzylidene]
hydrazinecarbothioamide (3)
White crystals ; Yield 93%; m.p.(ºC) 190-193; Rf:0,65 (hexane / ethyl acetate 7:3). IR
(KBr, cm-1): 3336.09(NH), 1541.11 (HC=N). 1H NMR (400 MHz, DMSO-d6), δ ppm:

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7.227 (t, J = 7.4 Hz, 1H, Ar), 7.385 (t, J = 7.8 Hz, 2H, Ar), 7.544 (d, J = 8.4 Hz, 2H,
Ar), 7.767 (d J = 8 Hz, 2H, Ar), 8.138 (d, J = 8.4 Hz, 2H, Ar), 8.213 (s, 1H, HC=N),
10.252 (s, 1H, NH), 11.986 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6), δ ppm:
123.2 (C, Ar), 125.9 (CF3), 125.917(C, Ar), 126.0 (C, Ar), 126.6 (C, Ar), 128.5 (C,
Ar), 128.6 (C, Ar), 129.8 (C, Ar), 130.1 (C, Ar), 138,5 (C, Ar), 139.4 (C, Ar), 141.4
(C=N), 176.9 (C=S). Anal. Calcd for C15H12F3N3S: C, 55.72; H, 3.74; N, 13.00; S,
9.92. found: C, 56.12; H,2.91; N, 12.82; S, 8.06. HRMS: 324.0704 [M+H] +
4.1.2 General procedure for synthesis of the series (1a-3d)
In a round bottom flask, arylthiosemicarbazone (1, 2 or 3) and their acetophenone in
2-propanol (20 ml). The reaction mixture was kept at room temperature for 1 h.
Reactions were monitored by thin layer chromatography (TLC). The resulting solid
was filtered through sintered funnel with distilled water, yielding the pure product.
4.1.2.1
(1a)
(E)-4-phenyl-2-{2-[4-(trifluoromethyl)benzylidene]hydrazinyl}thiazole
Light yellow crystals ; Yield 47%; m.p.(ºC) 212-215; Rf: 0,72 (hexane / ethyl acetate
7:3). IR (KBr, cm-1): 3413.82(NH), 1634.54 (C=N); 1H NMR (400 MHz, DMSO-d6),
δ ppm: 12.5(s, 1H, NH), 8.112 (s, 1H, HC=N), 7.857 (d, J = 9,6 Hz 4H, CH Ar),
7.778 (d, J = 10.8 Hz 2H, CH Ar), 7.410 ( m, 3H, CH Ar), 7.304 (t, J = 6.5 Hz 1H,
CH Ar); 13C NMR (100 MHz, DMSO-d6), δ ppm: 167.9 (S-C), 150.3 (C
thiazole),139.6 (C=N), 138.4 (C, Ar), 134.4 (C, Ar), 129.1(C, Ar), 128.7(C, Ar),
127.7 (C, Ar), 126.8 (C, Ar), 125.8 (C, Ar), 125.8 (C, Ar), 125.6 (CF3) e 104.2 (C
thiazole). Anal. Calcd for C17H12F3N3S: C, 58.78; H, 3.48; N, 12.1 ; S, 9.23. found:
C,54.25; H, 2.23; N, 10.15; S, 7.13. HRMS: 348,0710 [M+H] +
4.1.2.2
(E)-4-(4-nitrophenyl)-2-{2-[4-(trifluoromethyl)benzylidene]hydrazinyl}
thiazole (1b)
Light yellow crystals ; Yield 34%; m.p.(ºC) 226-230; Rf: 0,66 (hexane / ethyl acetate
7:3). IR (KBr, cm-1): 3298.22(NH), 1594.12 (C=N); 1H NMR (400 MHz, DMSO-d6),
δ ppm: 8.273 (d, J = 11,6 Hz 2H, CH Ar), 8.109 (m, 3H, CH Ar), 7.862 (d, J = 10.8
Hz 2H, CH Ar), 7.781 ( m, 3H, CH Ar), 4.264 (s, 1H, NH); 13C NMR (100 MHz,
DMSO-d6), δ ppm: 168.4 (S-C), 148.6 (C thiazole), 146.2 (C, Ar), 140.5 (C=N),
139.9 (C, Ar), 138.2 (C, Ar), 130.2 (C, Ar), 129.2 (C, Ar), 126.8 (C, Ar), 126.4 (C,

ACCEPTED MANUSCRIPT
Ar), 125.8 (CF3), 124.2 (C, Ar), 123.9 (C, Ar), 109.1 (C thiazole). Anal. Calcd for
C17H11F3N4O2S: C, 52.04; H, 2.83; N, 14.28; S, 8.17. found: C, 53.51; H, 1.81; N,
12.76; S, 5.34. HRMS: 393,0565 [M+H] +
4.1.2.3
(E)-4-(3-nitrophenyl)-2-{2-[4-(trifluoromethyl)benzylidene]hydrazinyl}
thiazole (1c)
Light yellow crystals ; Yield 63%; m.p.(ºC) 229-231; Rf: 0,61 (hexane / ethyl acetate
7:3). IR (KBr, cm-1): 3049.09(NH), 1618.99 (C=N);1H NMR (400 MHz, DMSO-d6),
δ ppm: 8.658 (s, 1H, CH Ar),8.293 (m, 2H, CH Ar), 8.097 (s, 1H, C=N), 7.783 (m,
6H, CH Ar), 4.295 ( s, 1H, NH);13C NMR (100 MHz, DMSO-d6), δ ppm: 168.7 (S-
C), 167.3 (C-NO2), 148.7 (C=N), 140.2 (C4 thiazole), 138.7 (C, Ar), 136.5 (C, Ar),
132.0 (C, Ar), 130.7 (C, Ar), 127.3 C, Ar), 126.2(C, Ar), 122.6 (C, Ar), 120.4 (CF3),
116.5 (C, Ar), 117.2 (C,Ar), 107.4 (C thiazole). Anal. Calcd for C17H11F3N4O2S: C,
52.04; H, 2.83; N, 14.28; S, 8.17. found: C, 51.36; H, 1.51; N, 11.01; S, 8.86. HRMS:
393,0565 [M+H] +
4.1.2.4
(E)-4-(2,4-dichlorophenyl)-2-{2-[4-(trifluoromethyl)benzylidene]
hydrazinyl}thiazole (1d)
White crystals; Yield 50%; m.p.(ºC) 165-168; Rf: 0,80 (hexane / ethyl acetate 7:3). IR
1
(KBr, cm-1): 1623.63 (C=N); H NMR (400 MHz, DMSO-d6), δ ppm: 12.462 (s, 1H,
NH), 8.128 (s,1H,HC=N), 7.9 (s,1H, CH Ar), 7.862 (d, J = 10 Hz 2H, CH Ar), 7.776
(d, J = 8 Hz 2H, CH Ar), 7.691 ( s, 1H, CH Ar), 7.505 (d, J = 8,4 Hz 2H, CH Ar),
7.443 (s, 1H, CH thiazole);13C NMR (100 MHz, DMSO-d6), δ ppm: 167.2 (S-C),
145.8 (C thiazole),139.7 (C=N), 138.3 (C, Ar), 132.6 (C-Cl), 132.2 (C-Cl), 131.9(C,
Ar), 131.6(C, Ar), 129.8 (C, Ar), 129.0 (C, Ar), 128.7 (C, Ar), 127.5 (C, Ar), 126.8
(C, Ar), 125.7 (CF3), 109.7 (C thiazole). Anal. Calcd for C17H10Cl2F3N3S: C, 49.06;
H, 2.42; N, 10.1; S, 7.7. found: C, 45.71; H, 2.10; N, 7.10; S, 6.30. HRMS: 415,9950
[M+H] +
4.1.2.5
(E)-4-(4-methoxyphenyl)-2-{2-[4-(trifluoromethyl)benzylidene]
hydrazinyl}thiazole (1e)
Yellow crystals; Yield 88%; m.p.(ºC) 210-213; Rf: 0,64 (hexane / ethyl acetate 7:3).
1
IR (KBr, cm-1): 3421.75 (NH); 1611.53 (C=N); H NMR (400 MHz, DMSO-d6), δ
ppm: 12.500 (s, 1H, NH), 8.099 (s,1H,HC=N), 7.858 (d, J = 8 Hz 2H, CH Ar),7.783

ACCEPTED MANUSCRIPT
(d, J = 7,2 Hz 4H, CH Ar), 7.203 (s, 1H,CH thiazole), 6.971 (d, J = 8.4 Hz 2H, CH
Ar) e 3.782 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6), δ ppm: 167.2 (S-C),
145.8 (C thiazole),139.7 (C=N), 138.3 (C, Ar), 132.6 (C-Cl), 132.2 (C-Cl), 131.9(C,
Ar), 131.6(C, Ar), 129.8 (C, Ar), 129.0 (C, Ar), 128.7 (C, Ar), 127.5 (C, Ar), 126.8
(C, Ar), 125.7 (CF3), 109.7 (C thiazole). Anal. Calcd for C18H14F3N3OS: C, 57.29; H,
3.74; N, 11.13; S, 8.5. found: C, 54.93; H, 2.90; N, 9.39; S, 6.77. HRMS: 378,0814
[M+H] +
4.1.2.6 (E)-4-(4-chlorophenyl)-2-{2-[4-(trifluoromethyl)benzylidene]hydrazinyl}
thiazole (1f)
Light Yellow crystals; Yield 90%; m.p.(ºC) 240-243; Rf: 0,74 (hexane / ethyl acetate
7:3). IR (KBr, cm-1): 3451.89 (NH); 1623.25 (C=N);1H NMR (400 MHz, DMSO-d6),
δ ppm: 12.441 (s, 1H, NH), 8.111 (s, 1H,HC=N), 7.861(m ,4H, CH Ar), 7.775 (d, J =
7,2Hz 2H, CH Ar), 7.598 (d, ,J = 10.4 Hz 3H, CH Ar);13C NMR (100 MHz, DMSO-
d6), δ ppm: 168.1 (S-C), 149.2 (C thiazole), 139.7 (C=N), 138.3 (C, Ar), 133.3 (C,
Ar), 132.0 (C-Cl), 129.5 (C, Ar), 128.6 (C, Ar), 127.3 (C, Ar), 126.8 (C, Ar), 125.7
(C,F3), 124.7 (C, Ar) 105 (C thiazole). Anal. Calcd for C17H11ClF3N3S: C, 53.48; H,
2.9; N, 11.01; S, 8.4. found: C, 52.34; H, 2.22; N, 8.83; S, 7.84. HRMS: 382,0342
[M+H] +
4.1.2.7 (E)-4-(4-bromophenyl)-2-{2-[4-(trifluoromethyl)benzylidene]hydrazinyl}
thiazole (1g)
Yellow crystals; Yield 68%; m.p.(ºC) 235-238; Rf: 0,71 (hexane / ethyl acetate 7:3).
1
IR (KBr, cm-1): 3411.28 (NH), 1618.80(C=N); H NMR (400 MHz, DMSO-d6), δ
ppm: 12.501 (s, 1H, NH), 7,820 (m, 6H, CH Ar), 7.602 (d, J = 8,4 Hz 2H, CH Ar),
7.462 (s, 1H, CH thiazole); 13C NMR (100 MHz, DMSO-d6), δ ppm: 168.1 (S-C),
149.3 (C thiazole), 139.6 (C=N), 138.3 (C, Ar), 133.7 (C, Ar), 131.6 (C, Ar), 127.5
(C, Ar), 126.7 (C, Ar), 125.9 (C,Ar) 125.7 (CF3), 120.6(C-Br) e 105.1 (C thiazole).
Anal. Calcd for C17H11BrF3N3S: C, 47.9; H, 2.60; N, 9.86; S, 7.52. found: C, 43.17;
H, 1.98; N, 7.49; S, 6.15. HRMS: 427,9948 [M+H] +
4.1.2.8
(E)-4-(3,4-dichlorophenyl)-2-{2-[4-(trifluoromethyl)benzylidene]
hydrazinyl}thiazole (1h)

ACCEPTED MANUSCRIPT
Beige crystals; Yield 47%; m.p.(ºC) 236-238; Rf: 0,76 (hexane/ethyl acetate 7:3). IR
(KBr, cm-1): 3411.24 (NH), 1610.73 (C=N); 1H NMR (400 MHz, DMSO-d6), δ ppm:
12.452 (s, 1H, NH), 8.1 (s, 1H, HC=N), 7.800 (m ,6H, CH Ar), 7.651 (m, 1H, CH
13
Ar), 7.587 (s, 1H, CH thiazole); C NMR (100 MHz, DMSO-d6), δ ppm: 168.1 (S-
C), 147.9 (C thiazole),139.8 (C=N), 138.2 (C, Ar), 135.0 (C-Cl), 131.4 (C-Cl), 130.9
(C, Ar), 129.8 (C, Ar), 127.7 (C, Ar), 127.160 (C, Ar), 126.7 (C, Ar), 125.6 (C, Ar),
124.5 (CF3), 122.8 (C, Ar) e 106.4 (C, thiazole). Anal. Calcd for C17H10Cl2F3N3S: C,
49.06; H, 2.42; N, 10.1; S, 7.7. found: C, 41.27; H, 1.00; N, 8.23; S, 5.62. HRMS:
414,9924 [M+H] +
4.1.2.9 (E)-5-methyl-4-phenyl-2-{2-[4-(trifluoromethyl)benzylidene]hydrazinyl}
thiazole (1i)
Yellow crystals; Yield 84%; m.p.(ºC) 226-229; Rf: 0,60 (hexane/ethyl acetate 7:3). IR
(KBr, cm-1): 3366.11 (NH), 1616.63 (C=N); 1H NMR (400 MHz, DMSO-d6), δ ppm:
12.441 (s, 1H, NH), 8.142 (s, 1H, HC=N) 7.877(d, J= 7,6 Hz 2H, CH Ar), 7.784 (d, J
= 7,6 Hz 2H, CH Ar), 7.607 (d, J = 7,6 Hz 2H, CH Ar), 7.462 ( t, J = 7,4 Hz 2H, CH
13
Ar), 7.378 (m, 1H, CH Ar), 2.408 (s, 1H, CH3); C NMR (100 MHz, DMSO-d6), δ
ppm: 164.4 (S-C), 146.1 (C thiazole), 142.5 (C=N), 138.2 (C, Ar), 133.5 (C, Ar),
132.3 (C, Ar), 128.4 (C, Ar), 128.1 (C, Ar), 127.7 (C, Ar), 126.9 (C, Ar), 125.7 (C,
Ar), 124.7 (CF3), 117.4 (C thiazole), 12.1 (CH3). Anal. Calcd for C18H14F3N3S: C,
59.82; H, 3.90; N, 15.77; S, 8.87. found: C, 57.87 H, 2.83; N, 9.32; S, 6.63. HRMS:
362,0856 [M+H] +
4.1.2.10
(E)-4-(naphthalen-1-yl)-2-{2-[4-(trifluoromethyl)benzylidene]
hydrazinyl}thiazole (1j)
Yellow crystals ; Yield 76%; m.p.(ºC) 217-220; Rf: 0,71 (hexane/ethyl acetate 7:3).
1
IR (KBr, cm-13421.17 (NH), 1613.46 (C=N); H NMR (400 MHz, DMSO-d6), δ
ppm: 12.456 (s, 1H, NH), 8.402 (s, 1H, CH Ar), 8.156 (s ,1H, C=N), 8.026 (d, J = 8,4
Hz 1H, CH Ar), 7.924 (m, 6H, CH Ar), 7.800(d, J = 7,6 Hz 2H, CH Ar), 7.541 (d,
2H, CH Ar); 13C NMR (100 MHz, DMSO-d6), δ ppm: 168.0 (S-C), 150.3 (C
thiazole), 139.6 (C=N), 138.3 (C, Ar), 133.1 (C, Ar), 132.5 (C, Ar), 131.9 (C, Ar),
128.7 (CF3), 128.1 (C, Ar), 127.8 (C, Ar), 126.7 (C, Ar), 126.4 (C, Ar), 126.1 (C, Ar),
125.7 (C, Ar), 125.5 (C, Ar), 124.1 (C, Ar), 123.9 (C, Ar), 105.0 (C thiazole). Anal.

ACCEPTED MANUSCRIPT
Calcd for C21H14F3N3S: C, 63.47; H, 3.55; N, 10.57; S, 8.07. found: C, 60.08 H, 2.85;
N, 9.60; S, 7.17. HRMS: 398,0866 [M+H] +
4.1.2.11 (E)-4-(p-tolyl)-2-{2-[4-(trifluoromethyl)benzylidene]hydrazinyl}thiazole
(1k)
Yellow crystals ; Yield 76%; m.p.(ºC) 217-220; Rf: 0,71 (hexane/ethyl acetate 7:3).
1
IR (KBr, cm-1): 3421.17 (NH), 1613.46 (C=N); H NMR (400 MHz, DMSO-d6), δ
ppm: 12.418 (s, 1H, NH), 8.109 (s, 1H, C=N), 7.856 (d, J = 7,6 Hz 2H, C Ar), 7.756
(m, 4H, C Ar), 7.291 (s, 1H, CH thiazole), 7.212 (d, J = 7,6 Hz 2H, CH Ar), 2.496 (s,
3H, CH3); 13C NMR (100 MHz, DMSO-d6), δ ppm: 167.8 (S-C), 150.3 (C thiazole),
143.3 (C=N), 139.6 (C, Ar), 138.3 (C, Ar), 136.9 (C, Ar), 131.7 (C, Ar), 129.2 (C,
Ar), 128.9 (C, Ar), 127.1 (C,Ar), 126.7 (C, Ar), 125.6 (CF3), 103.3 (C thiazole), 20.8
(CH3). Anal. Calcd for C18H14F3N3S: C, 59.82; H, 3.90; N, 11.63; S, 8.87. found: C,
56.56 H, 2.73; N, 9.82; S, 6.15. HRMS: 362,0864 [M+H] +
4.1.2.12 (E)-4-(4-fluorophenyl)-2-{2-[4-(trifluoromethyl)benzylidene]hydrazinyl}
thiazole (1l)
Yellow crystals ; Yield 22%; m.p.(ºC) 212-215; Rf: 0,71 (hexane/ethyl acetate 7:3).
1
IR (KBr, cm-1): 3326.72 (NH), 1611.91 (C=N); H NMR (400 MHz, DMSO-d6), δ
ppm: 12.391 (s, 1H, NH), 8.114 s, 1H, C=N), 8.035(d, J = 7,2 Hz 2H, CH Ar), 7.871
(m, 5H, CH Ar), 7.753 (m, 4H, C Ar), 7.361 ( s, 1H, CH thiazole), 7.251 (d, J = 7,6
13
Hz 2H, CH Ar); C NMR (100 MHz, DMSO-d6), δ ppm: 178.3 (S-C, 168.0 (C-F),
150.3 (C thiazole), 140.3 (C=N), 139.5 (C, Ar), 138.3 (C, Ar), 127.8 (C, Ar), 127.6
(C, Ar), 127.5 (C, Ar), 126.5 (C, Ar), 126.7 (C, Ar), 125.6 (CF3), 115.5 (C, Ar) e
108.9 (C thiazole). Anal. Calcd for C17H11F4N3S: C, 55.89; H, 3.03; N, 11.5; S, 8.78.
found: C, 49.67, H, 2.82; N, 10.68; S, 5.66. HRMS: 366.0610 [M+H] +
4.1.2.13
(E)-4-(4-methoxyphenyl)-3-methyl-2-{(E)-[4-(trifluoromethyl)
benzylidene]hydrazono}-2,3-dihydrothiazole (2a)
Yellow crystals ; Yield 72%; m.p.(ºC) 166-168; Rf: 0,71 (hexane/ethyl acetate 7:3).
1
IR (KBr, cm-1): 1590.69 (C=N); H NMR (400 MHz, DMSO-d6), δ ppm: 8.559 (s,
1H, HC=N), 7.968 (d, J = 10 Hz 2H, 2H, CH Ar), 7.834 (d, J = 10,8 Hz 2H, 2H, CH
Ar), 7.481 (d, J = 10 Hz 2H, 2H, CH Ar),7.090 (d, J = 10 Hz 2H, 2H, CH Ar), 6.865
(s, 1H, CH thiazole), 3.824 (s, 3H, O-CH3), 3.471 (s, 3H, CH3); 13C NMR (100 MHz,

ACCEPTED MANUSCRIPT
DMSO-d6), δ ppm: 169.5 (C=N), 160.3 (C, Ar), 150.4 (C-S), 148.7 (C thiazole),
141.7 (C=N), 137.8 (C, Ar), 130.8 (C, Ar), 127.7 (C, Ar), 125.9 (C, Ar), 124.5
(CF3)121.2 (C, Ar), 114.4 (C, Ar), 103.8 (C, thiazole), 55.4 (O-CH3) e 34.9 (CH3). .
Calcd for C19H16F3N3OS: 58.30; H, 4.12; N, 10.74; S, 8.19. found: C, 56.91, H, 2.70;
N, 8.60; S, 6.30. HRMS: 392,1009 [M+H] +
4.1.2.14 (E)-3-methyl-4-(4-nitrophenyl)-2-{[(E)-4-(trifluoromethyl)benzylidene]
hydrazono}-2,3-dihydrothiazole (2b)
White crystals ; Yield 83%; m.p.(ºC) 235-237; Rf: 0,66 (hexane/ethyl acetate 7:3). IR
1
(KBr, cm-1): 1608.64 (C=N); H NMR (400 MHz, DMSO-d6), δ ppm: 8.513 (s, 1H,
HC=N), 8.352 (d, J = 11,2 Hz 2H, CH Ar), 7.953 (d, J = 10,4 Hz 2H, CH Ar), 7.836
(t, J = 13,2 Hz 3H, CH Ar), 7.023 (s, 1H, CH thiazole), 6.865 (s, 1H, CH thiazole),
3.462 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6), δ ppm: 170.7 (C=N), 149.6
(HC=N), 148.2 (C-NO2), 139.9 (C, thiazole), 138.7 (C, Ar), 136.2 (C, Ar), 130.7 (C,
Ar), 128.1 (C, Ar), 127.2 (C, Ar), 126.3 (C, Ar), 125.4 (C, Ar), 124.4 (CF3), 123.3 (C,
Ar), 106.3 (C, thiazole), 35.2 (CH3). Anal. Calcd for C18H13F3N4O2S: C, 53.20; H,
3.22; N, 13.79; S, 7.89. found: C, 54.04, H, 2.31; N, 11.19; S,6.12. HRMS: 407,0711
[M+H] +
4.1.2.15
(E)-4-(2,4-dichlorophenyl)-3-methyl-2-{(E)-[4-(trifluoromethyl)
benzylidene]hydrazono}-2,3-dihydrothiazole (2c)
Yellow crystals ; Yield 70%; m.p.(ºC) 201-204; Rf: 0,65 (hexane/ethyl acetate 7:3).
1
IR (KBr, cm-1): 1540.96 (C=N); H NMR (400 MHz, DMSO-d6), δ ppm: 8.400 (s,
1H, HC=N), 7.786 (d, J = 10 Hz 2H, CH Ar), 7.687 (d, J = 8 Hz 2H, CH Ar), 7.480
(s, 1H, CH Ar), 7.290 (m, 2H, CH Ar), 6.777 (s, 1H, CH thiazole), 3.335(s, 3H,
13
CH3); C NMR (100 MHz, DMSO-d6), δ ppm: 167.2 (S-C), 149.4 (C=N), 144.98
(C, Ar), ), 139.0 (C thiazole), 138.7 (C, Ar132.7 (C,Ar), 132.4 (C,Ar), 129.7 (C, Ar),
129.5 (C, Ar), 128.9 (C, Ar), 126.8 (C, Ar), 125.3 (C, Ar), 125.2 (C, Ar), 124.1 (CF3),
108.7 (C thiazole), 30.85 (CH3). Anal. Calcd for C18H12Cl2F3N3S: C, 50.25; H, 2.81;
N, 9.77; S, 7.45. found: C, 47.68, H, 2.43; N, 8.09; S, 4.12. HRMS: 430.9950
[M+H] +

ACCEPTED MANUSCRIPT
4.1.2.16
(E)-4-(2,4-dichlorophenyl)-3-phenyl-2-{(E)-[4-(trifluoromethyl)
benzylidene]hydrazono}-2,3-dihydrothiazole (3a)
White crystals ; Yield 84%; m.p.(ºC) 2055-208; Rf: 0,79 (hexane/ethyl acetate 7:3).
1
IR (KBr, cm-1): 1540.96 (C=N); H NMR (400 MHz, DMSO-d6), δ ppm: 8.217 (s,
1H, HC=N), 8.140 (d, J = 8 Hz 2H, CH Ar), 7.776 (d, J = 8 Hz 2H, CH Ar), 7.552 (d,
J = 7.2 Hz 3H, CH Ar), 7.384 (m, 4H, CH Ar), 7.227 (t, J = 4.8 Hz 1H, CH Ar), 6.630
13
(s, 1H, CH thiazole); C NMR (100 MHz, DMSO-d6), δ ppm: 158.3 (S-C), 149.4
(C=N), 147.3 (C, thiazole) 141.2 (C, Ar), 139.0 (C, Ar), 133.3 (C, Ar), 133,4 (C, Ar),
132.5 (C-Cl), 129.7 (C, Ar), 129. 6 (C, Ar), 129.5 (C, Ar), 129.4 (C, Ar), 129.3 (C,
Ar), 129.2 (C, Ar), 128.9 (C, Ar), 126.8 (C, Ar), 125.2 (C-Cl), 125.1 (C, Ar), 124.1
(CF3), 122.8 (C, Ar), 122.6 (C, Ar), 122.4 (C, Ar), 106.3 (C, thiazole). Anal. Calcd
for C23H14Cl2F3N3S: C, 56.11; H, 2.87; N, 8.53; S, 6.51. found: C, 55.56, H, 3.79; N,
12.89; S, 5.97. HRMS: 492.0237 [M+H] +
4.1.2.17
(E)-4-(4-methoxyphenyl)-3-phenyl-2-{(E)-[4-(trifluoromethyl)
benzylidene]hydrazono}-2,3-dihydrothiazole (3b)
White crystals ; Yield 75%; m.p.(ºC) 197-200; Rf: 0,71 (hexane/ethyl acetate 7:3). IR
1
(KBr, cm-1): 1589.86 (C=N); H NMR (400 MHz, DMSO-d6), δ ppm: 8.274 (s, 1H,
HC=N), 7.882 (d, J = 8.4 Hz 2H, CH Ar), 7.773 (d, J = 8.4 Hz 2H, CH Ar), 7.362 (m,
5H, CH Ar), 7.198 (d, J = 8.8 Hz 2H, CH Ar), 7.099 (d, J = 8.8 Hz 2H, CH Ar), 6.818
(m, 1H, CH Ar), 6.719 (s, 1H, CH thiazole), 3.697 (s, 3H, CH3); 13C NMR (100
MHz, DMSO-d6), δ ppm: 171.1 (C-O), 159.3 (S-C), 149.6 (C=N), 147.3 (C, Ar)
140.0 (C, Ar), 138.6 (C, Ar), 137.3 (C, Ar), 136.8 (C, Ar), 129.9 (C, Ar), 129.1 (C,
Ar), 128.8 (C, Ar), 128.7 (C, Ar), 128.5 (C, Ar), 127.5 (C, Ar), 125.6 (C, Ar), 125.5
(C, Ar), 122.3 (C, Ar), 113.6 (C, Ar), 55.1 (C thiazole). Anal. Calcd for
C24H18F3N3OS: C, 63.57; H, 4; N, 9.27; S, 7.07. found: C, 62.78, H, 3.07; N, 7.53; S,
7.07. HRMS: 453.1122 [M+H] +
4.1.2.18 (E)-4-(4-chlorophenyl)-3-phenyl-2-{(E)-[4-(trifluoromethyl)benzylidene]
hydrazono}-2,3-dihydrothiazole (3c)
White crystals ; Yield 68%; m.p.(ºC) 198-200; Rf: 0,73 (hexane/ethyl acetate 7:3). IR
1
(KBr, cm-1): 1583.57 (C=N); H NMR (400 MHz, DMSO-d6), δ ppm: 8.249 (s, 1H,
HC=N), 7.877 (d, J = 7.6 Hz 2H, CH Ar), 7.765 (d, J = 7.6 Hz 2H, CH Ar), 7.353 (m,
13
7H, CH Ar), 7.197 (d, J = 8.0 Hz 2H, CH Ar), 6.771 (s, 1H, CH thiazole); C NMR

ACCEPTED MANUSCRIPT
(100 MHz, DMSO-d6), δ ppm: 168.1 (S-C), 150.4 (C=N-S), 148.8 (C=N), 145.20 (C,
Ar) 137.2 (C thiazole), 133.7 (C,Ar), 133.4 (C,Ar), 132.8 (C,Ar), 129.7 (C, Ar), 129.5
(C, Ar), 128.5 (C, Ar), 127.3 (C, Ar), 126.3 (C, Ar), 125.1 (C, Ar), 125.3 (C, Ar),
124.5 (CF3), 108.9 (C thiazole). Anal. Calcd for C23H15ClF3N3S: C, 60.33; H, 3.30; N,
9.18; S, 7. found: C, 58.80, H, 2.46; N, 8,99; S, 5.67. HRMS: 458.9000 [M+H] +
4.1.2.19 (E)-4-(4-fluorophenyl)-3-phenyl-2-{(E)-[4-(trifluoromethyl)benzylidene]
hydrazono}-2,3-dihydrothiazole (3d)
Light Yellow crystals ; Yield 70%; m.p.(ºC) 205-208; Rf: 0,69 (hexane/ethyl acetate
1
7:3). IR (KBr, cm-1): 1585.87 (C=N); H NMR (400 MHz, DMSO-d6), δ ppm: 8.280
(s, 1H, HC=N), 7.880 (d, J = 8.1 Hz 2H, CH Ar), 7.764 (d, J = 8.7 Hz 2H, CH Ar),
7.358 (m, 4H, CH Ar), 7.225 (m, 4H, CH Ar), 7.086 (m, 1H,CH Ar), 6.787 (s, 1H,
CH thiazole); 13C NMR (100 MHz, DMSO-d6), δ ppm: 170.2 (C-F), 168.3 (S-C),
148.3 (C=N), 145.1 (C, Ar) 136.9 (C thiazole), 133.5 (C,Ar), 133.3 (C,Ar), 132.6
(C,Ar), 129.5 (C, Ar), 129.4 (C, Ar), 128.3 (C, Ar), 126.1 (C, Ar), 124.9 (C, Ar),
125.1 (C, Ar), 124.3 (CF3), 108.7 (C thiazole). Anal. Calcd for C23H15F4N3S: C,
62.58; H, 3.43; N, 17.21; S,7.26. found: C, 60.68, H, 2.79; N, 13.95; S, 6.12. HRMS:
441.4435 [M+H] +
4.2 Biological Activity
4.2.1 Ethical aspects for the alamar blue assay and hemolytic activity assay
The project was submitted to the Human Research Ethics Committee of the Federal
University of Pernambuco, which is in accordance with Resolution 466/12 of the
National Health Council and approved with CAAE opinion 62919816.2.0000.5208.
4.2.2 Cell culture
NCI-H292 (human lung carcinoma), HEp-2 (human larynx carcinoma) and HT-29
(human colon adenocarcinoma) were cultured in DMEM medium, while K-562
(human myeloblastic leukaemia) and HL-60 (human promyelocytic leukemia) were
cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 2 mM
glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin, at 37 °C with 5% CO2.
The cell lines used in this work were obtained from Cell Bank of Rio de Janeiro

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(BCRJ), Brazil. Culture medium was changed 2–3 days and subcultured when cell
population density reached to 70–80% confluence.
4.2.3 Cytotoxicity assessment by MTT assay
The cytotoxicity of all compounds was tested using the 3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma Aldrich Co., St. Louis,
MO/USA) against NCI-H292, HEp-2, HT-29, K562 e HL-60 lines cells [50]. Briefly,
cells we added in 96-well plates (3 x 105 cells/mL for K562 and HL-60; and 1 x 105
cells/mL for NCI-H292, HT-29, and HEp-2) with appropriated medium and incubated
for 24 h (37° C and 5% CO2). Compounds were then added in different
concentrations (0.098 to 25.0 µg/mL) and incubated for 72 h. Doxorubicin was used
as a reference drug. Three hours before the end of the incubation time, 25 µL MTT (5
mg/mL in PBS) was added to each well. Culture medium was removed and 100 µL of
DMSO were added. The amount of formazan was determined by measuring the
absorbance at 560 nm. Concentration leading to 50% inhibition of viability (CI50) was
calculated by regression analysis using GraphPad Prism. Each sample was tested in
triplicate in two independent experiments.
4.2.4 Alamar blue assay
To investigate the selectivity of synthetized compounds for tumor cells compared to
normal proliferating cells, the Alamar blue assay was performed using PBMC
(peripheral blood mononuclear cells) from healthy donors, after 72 h of drug
exposure. All studies were performed in accordance with Research Guidelines (Law
196/96, National Council of Health). Heparinised blood (from healthy, non-smoker
donors who had not taken any drug for at least 15 days prior to sampling) was
collected, and the PBMC were isolated by a standard method of density-gradient
centrifugation using Ficoll-Hypaque. PBMC were cultivated in RPMI-1640 medium
supplemented with 20% foetal bovine serum, 2 mM glutamine, 100 U/mL penicillin,
and 100 mg/mL streptomycin at 37 °C with 5% CO2. Phytohaemagglutinin (2%) was
added at the beginning of the culture period. Briefly, the PBMC were plated in 96-
well plates (106 cells/mL). After 24 h, the compounds (0.048–25 µg/ mL), dissolved
in DMSO, were added to each well and incubated for 72 h. Doxorubicin was used as a
positive control. At 24 h before the end of the incubation, 10 µL of a stock solution
(0.312 mg/mL) of Alamar blue (resazurin, Sigma-Aldrich Co., St. Louis, MO, USA)

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was added to each well. The absorbance was measured using a plate reader, and the
effect of the drug was quantified as the percentage of the control absorbance at 570
and 595 nm [51].
4.2.5 Haemolysis assay
The test was performed in 96-well plates using a 2% human erythrocyte suspension in
0.85% NaCl containing 10 mM CaCl2 following the method described by Costa-
Lotufo et al. [52] with modification. The diluted compounds were tested at
concentrations ranging from 0.78 to 250 µg/mL. Triton X-100 (1%) was used as a
positive control. After incubation at room temperature for 1 h followed by
centrifugation, the supernatant was removed, and the liberated haemoglobin was
measured spectrophotometrically at 540 nm.
4.2.6 Flow cytometry analysis
Cells of the K562 strain (3 x 105 cells/ml) were resuspended in RPMI-1640 medium
and treated with 1d compound for 48 h at 37°C with 5% CO2. The cell viability assay
was performed using the count detection kit (Guava technologies) and the cell cycle
assay and DNA fragmentation using the guava cell cycle detection kit (Guava
technologies) according to the manufacturer's instructions. The mitochondrial
depolarization assay was performed using rhodamine. The experiment was performed
on a Guava EasyCyteHT (Merck-Millipore) flow cytometer, acquiring 5,000 events.
The data were analyzed in Guava-SoftTM software version 2.7. Two independent
experiments were performed in triplicate.
Acknowledgments
The authors gratefully acknowledge CNPq (470901/2014-4 and 309889/2015-
4). CNPq hold a CNPq fellowship to A.C.L.L.( 302883/2016-9), P.A.T.M.G.
received a FACEPE fellowship (2016-2018). M. O. B. receives a FACEPE Masters
scholarship. We also thank the Department of Fundamental Chemistry - Federal
University of Pernambuco (DQF-UFPE) and Center for Strategic Technologies of the
Northeast (CETENE) for recording the Elemental Analysis, 1H NMR, 13C NMR,
LCMS and IR spectra of all compounds.

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