Synthetic (E)-3-phenyl-5-(phenylamino)-2-styryl-1,3,4-thiadiazol-3-ium chloride derivatives as promising chemotherapy agents on cell lines infected with HTLV-1

Article abstract of DOI:10.3390/molecules25112537

Synthesis of four compounds belonging to mesoionic class, (E)-3-phenyl-5-(phenylamino)-2-styryl-1,3,4-thiadiazol-3-ium chloride derivatives (5a-d) and their biological evaluation against MT2 and C92 cell lines infected with human T-cell lymphotropic virus type-1 (HTLV-1), which causes adult T-cell leukemia/lymphoma (ATLL), and non-infected cell lines (Jurkat) are reported. The compounds were obtained by convergent synthesis under microwave irradiation and the cytotoxicity was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Results showed IC50 values of all compounds in the range of 1.51-7.70 μM in HTLV-1-infected and non-infected cells. Furthermore, it was observed that 5b could induce necrosis after 24 h for Jurkat and MT2 cell lines. The experimental (fluorimetric method) and theoretical (molecular docking) results suggested that the mechanism of action for 5b could be related to its capacity to intercalate into DNA. Moreover, the preliminary pharmacokinetic profile of the studied compounds (5a-d) was obtained through human serum albumin (HSA) binding affinity using multiple spectroscopic techniques (circular dichroism, steady-state and time-resolved fluorescence), zeta potential and molecular docking calculations. The interaction HSA:5a-d is spontaneous and moderate (Ka ～ 104 M-1) via a ground-state association, without significantly perturbing both the secondary and surface structures of the albumin in the subdomain IIA (site I), indicating feasible biodistribution in the human bloodstream.

Full text of DOI:10.3390/molecules25112537

molecules
Article
Synthetic (E)-3-Phenyl-5-(phenylamino)
-2-styryl-1,3,4-thiadiazol-3-ium Chloride Derivatives
as Promising Chemotherapy Agents on Cell Lines
Infected with HTLV-1
Danilo Sousa-Pereira 1, Thais Silva de Oliveira 2, Rojane O. Paiva 2, Otávio Augusto Chaves 1,3
,
José C. Netto-Ferreira 1,4,*, Juliana Echevarria-Lima 2,* and Aurea Echevarria 1,
*
1
Instituto de Química, Universidade Federal Rural do Rio de Janeiro (UFRRJ), Seropédica, Rio de Janeiro
23.890-000, Brazil; sousadanilo90@gmail.com (D.S.-P.); otavioaugustochaves@gmail.com (O.A.C.)
Laboratório de Imunologia Básica e Aplicada, Departamento de Imunologia, Instituto de Microbiologia
Paulo de Góes, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro 21.941-590, Brazil;
thais.silvadeoliveira@yahoo.com.br (T.S.d.O.); rojanedeoliveirapaiva@gmail.com (R.O.P.)
Instituto SENAI de Inovação em Química Verde (ISI QV), Maracanã, Rio de Janeiro 20.271-030, Brazil
Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMETRO), Divisão de Metrologia Química,
Duque de Caxias, Rio de Janeiro 25.250-020, Brazil
2
3
4
*
Correspondence: jcnetto.ufrrj@gmail.com (J.C.N.-F.); juechevarria@micro.ufrj.br (J.E.-L.);
echevarr@ufrrj.br (A.E.); Tel.: +55-21-2682-1078 (A.E.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Academic Editor: Beatriz De Pascual-Teresa
Received: 4 May 2020; Accepted: 26 May 2020; Published: 29 May 2020
Abstract:
Synthesis
of
four
compounds
belonging
to
mesoionic
class,
(E)-3-phenyl-5-(phenylamino)-2-styryl-1,3,4-thiadiazol-3-ium chloride derivatives (5a
–
d
) and
their biological evaluation against MT2 and C92 cell lines infected with human T-cell
lymphotropic virus type-1 (HTLV-1), which causes adult T-cell leukemia/lymphoma (ATLL),
and non-infected cell lines (Jurkat) are reported.
The compounds were obtained by
convergent synthesis under microwave irradiation and the cytotoxicity was evaluated using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Results showed
IC50 values of all compounds in the range of 1.51–7.70 µM in HTLV-1-infected and non-infected
cells. Furthermore, it was observed that 5b could induce necrosis after 24 h for Jurkat and MT2
cell lines. The experimental (fluorimetric method) and theoretical (molecular docking) results
suggested that the mechanism of action for 5b could be related to its capacity to intercalate into DNA.
Moreover, the preliminary pharmacokinetic profile of the studied compounds (5a–d) was obtained
through human serum albumin (HSA) binding affinity using multiple spectroscopic techniques
(circular dichroism, steady-state and time-resolved fluorescence), zeta potential and molecular
is spontaneous and moderate (Ka ~ 104 M−1
docking calculations. The interaction HSA:5a–d )
via a ground-state association, without significantly perturbing both the secondary and surface
structures of the albumin in the subdomain IIA (site I), indicating feasible biodistribution in the
human bloodstream.
Keywords: mesoionic compounds; HTLV-1; HSA interaction; multiple spectroscopic; molecular
docking
1. Introduction
In 2018, Global Cancer Statistics estimated that 18.1 million new cases of cancer and 9.6 million
deaths (excluding nonmelanoma skin cancer) would occur around the world. Among these new
Molecules 2020, 25, 2537; doi:10.3390/molecules25112537
www.mdpi.com/journal/molecules

Molecules 2020, 25, 2537
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cases, it was estimated that the incidence of leukemia and lymphoma (non-Hodgkin) would be
approximately 1 million cases worldwide [ ]. Leukemia was characterized by substantial impairment
of bone marrow and the presence of circulating tumor cells in peripheral blood. Leukemia can be
classified based on disease progression and related cell lines [ ]. Human T-cell lymphotropic virus
type 1 (HTLV-1) is an etiological agent of the adult T-cell leukemia/lymphoma (ATLL) malignancy [
and the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), an inflammatory
disease [ ]. The viral particles and reverse transcriptase enzyme were identified from a peripheral blood
sample from a T-cell leukemia patient, leading HTLV-1 to be described as the first neoplastic-associated
retrovirus in humans [ ]. It is estimated that 10 to 20 million individuals worldwide are infected with
HTLV-1. The HTLV-1 infection is higher prevalent in Japan, Africa, and Central and South America [ ].
1
2
3
]
4
5
6
On the other hand, Japan has the highest prevalence of ATLL in the world and 1000 new cases are
diagnosed annually [7].
The predominant target of HTLV-1 infection is the CD4+ T lymphocyte, which induces
activation and leads to spontaneous proliferation of these cells [
8]. Cell proliferation induces
genomic instability, leading to chromosomal abnormalities, culminating in cell transformation [
9].
Unfortunately, treatment with ATLL is restricted and patients with aggressive disease have poor
prognosis due to the state of immunosuppression and resistance of the leukemic cells to multiple
drugs. ATLL is classified into four subtypes, based on clinical differences and prognostic factors:
acute, lymphoma, chronic, and smoldering [10–12]. Acute or lymphoma subtypes represent 60% of
ATLL types, with a one-year prognosis of median survival, approximately [13]. The clinical features
are heterogeneous lymphadenopathy, splenomegaly, hepatomegaly, hypercalcemia, skin and lung
lesions, and lymphocyte infiltration [12].
The literature reports several treatment protocols for ATLL with variable results. The treatment
strategy depends on ATLL subtype. Some protocols employ chemotherapy, with the use of ziduvidine
(AZT)/interferon-
or not depending on the aggressiveness of the disease [14]. The AZT/IFN-
results, considering the acute, chronic, and indolent forms of ATLL, since the immunological effect of
IFN- is associated with the cytostatic effect of AZT on lymphoid cells [15]. The most-used treatment
α
(IFN-α
) and hematopoietic stem cell transplantation, which may occur in association
α
association allowed good
α
in the face of the lymphomatous and acute clinical forms of ATLL (more aggressive) is the use of
the CHOP cocktail: (C) cyclophosphamide; (H) hydroxyldaunorubicin (doxorubicin); (O) oncovin
(vincristine); and (P) prednisone. Cyclophosphamide is an alkylating agent that causes the formation
of cross-links with deoxyribonucleic acid (DNA), hydroxyldaunorubicin damages DNA by inserting
itself between the bases (intercalating agent), oncovin prevents cell duplication through binding to
tubulin protein, and prednisone acts on cytokines (inflammatory process) [13]. Figure 1 shows the
structures for CHOP and AZT chemotherapeutic drugs.
The AZT/IFN-
α and CHOP combination showed more prolonged survival in patients
when compared to chemotherapy alone. However, resistance to conventional chemotherapy,
the increase in proteins responsible for resistance to multiple drugs (MDR—multiple drugs
resistance), hepatic or renal dysfunction, and the state of immunodeficiency during chemotherapy
are still factors associated with failure in the treatments used. In addition, the long latency of
HTLV-1 infection may contribute to the failure of ATLL therapy [13,16,17]. Moreover, ATLL trial
treatments using mogamulizumab (anti-CCR4 monoclonal antibody) or/and lenalidomide
((RS)-3-(4-amino-1-oxo-1,3-dihydro-2H-isoindol-2-yl)piperidine-2,6-dione), a thalidomide derivate,
were employed in Japan [18]. Mogamulizumab was also used in combination with multi-agent
chemotherapy, improving the therapy response of 33% to 52% [19].

Molecules 2020, 25, 2537
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Cl
O
O
OH
OH
O
OH
OH
O
Cl
N
O
P
H
H
O
N
CH3
O
O
CH3
OH
CH3
O
H
N
N
O
Doxorubicin
N
Ciclophosphamide
O
NH2
HO
OH
Azidotimidine - AZT
O
OH
N+
–N
CH3
O
N
OH
CH3
H
N
R
H
H
R=CO2CH3
OCOCH3
CO2CH3
H3CO
Vincristine
N
O
CHO OH
Prednisone
Figure 1. Chemical structures for the chemical compounds that make up the CHOP cocktail
(cyclophosphamide, doxorubicin, prednisone, and vincristine) and AZT (ziduvidine) used in adult
T-cell leukemia/lymphoma (ATLL) chemotherapy.
Heterocyclic compounds of the mesoionic class, especially those with 1,3,4-thiadiazole moiety,
have been synthesized and their biological effects investigated, confirming their excellent potential
biological activity. Different biological activities have been described for these compounds, such as
anticancer [20–23], antiparasitic [24–26], anti-inflammatory, analgesic [27,28], and antimicrobial [29,30].
Mesoionic compounds have a betaine-like characteristic, with a positive charge on the heterocyclic
ring and a negative charge on the exocyclic atom and, therefore, can be represented only by dipolar
structures [31].
In our previous studies with 1,3,4-thiadiazolium salts (mesoionic compounds) containing a
substituted styryl moiety, important results for in vitro cytotoxic activity in various types of tumor
cells, including leukemias [23
,
32–
34], and in vivo antitumor effects have been observed [35
,36].
Besides, results on the antiviral activity of 1,3,4-thiadiazole derivatives against HIV-1 [37
–
40], which also
belong to the same Retroviridae family, have also been reported in the literature. These results motivated
us to synthesize new 1,3,4-thiadiazolium salts containing an unsubstituted styryl group, as well as a
substituted phenyl ring linked to the exocyclic nitrogen (Scheme 1).
Human serum albumin (HSA), one of major proteins in the human blood circulation system,
takes part in many physiological functions. Its proportion in plasma proteins is about 55% [41].
HSA has a remarkable capacity for reversible binding with drugs, which helps in improving their
solubility, protect their oxidation in plasma, and decrease toxicity. Because of these exceptional
abilities, HSA is one of the key targets to predict the pharmacokinetic profile for development of
a potential therapeutic agent [42]. From the structural point of view, HSA is a non-glycosylated
heart-shaped single chain polypeptide with molecular weight of 66.5 kDa (80
×
80
×
30 Å in size),
being divided into three analogous domains (I, II, and III) having amino acid residues of 1–195, 196–383,
and 384–585, respectively [43]. Each domain comprises two subdomains (A and B) and all domains are
balanced through a bridge of 17-disulfide bonding. HSA has two main hydrophobic cavities for ligand
binding: subdomain IIA (site I) and subdomain IIIA (site II) [44]. The main fluorophore of HSA is
the tryptophan (Trp-214) residue, which is located in a large hydrophobic cavity in subdomain IIA
(site I) [45]. Therefore, the intrinsic fluorescence intensity of Trp-214 residue is a common phenomenon
used to investigate the interaction of ligands with HSA [46–48].
Based on the importance of 1,3,4-thiadiazolium salts in the medicinal chemistry,
the present study reports the synthesis and characterization of four heterocyclic
compounds, namely (E)-3-phenyl-5-(phenylamino)-2-styryl-1,3,4-thiadiazol-3-ium chloride derivatives,

Molecules 2020, 25, 2537
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where R = CH3 (5a), OCH3 (5b), Cl (5c), and Br (5d). Each compound was assayed against C91 and
MT2 cell lines infected with HTLV-1 and a Jurkat cell line (lymphocytic leukemia). In addition,
it was also investigated how the mesoionic compounds 5a–d induce apoptotic cell death, as well as
their preliminary pharmacokinetic profile via affinity for HSA by multiple spectroscopic techniques
combined with molecular docking. It is important to highlight that the synthesis of the mesoionic
compounds 5a, 5b, and 5d have already been described [29] (5c is a new synthetic mesoionic),
however, this work reports for the first time the evaluation of 5a–d as promising chemotherapy agents.
2. Results and Discussion
2.1. Synthesis
The compounds (E)-3-phenyl-5-(phenylamino)-2-styryl-1,3,4-thiadiazol-3-ium chloride derivatives
5a–d) were prepared following Scheme 1 as previously reported [23] using microwave irradiation as
(
the power supply. Initially, the aryl-thioureas were prepared from substituted anilines and ammonium
isothiocyanate in the presence of concentrated HCl and water as solvent. The aryl-thioureas obtained
(
(
2a–d) were submitted under 8 h of reflux in chlorobenzene as solvent, and the aryl-isothiocyanates
3a–d) were used immediately in the subsequent step as crude products [49] to obtain the
N-aryl-2-phenyl-hydrazinecarbothioamides (4a–d). The intermediates 4a–d were prepared in solvent
free conditions using a grinding method with aryl-isothiocyanates and phenyl-hydrazine at room
temperature for 2 min. These compounds were obtained in quantitative yields and high purity, and their
structures were confirmed by FT-IR spectroscopy and NMR 1H and 13C spectrometry, in accordance
with previous publications on mesoionic compounds [29,50].
H
NCS
N
NH2
ii
NH2
i
NCS
NH4
+
S
R
R
3
R
2
1
iii
H
H
N
Cl
N N
R
iv
N
N
H
S
N
H
R
S
4
5a-d
R = CH3 (5a), OCH3 (5b), Cl (5c), Br (5d)
i: HCl, H2O, 6 h reflux; ii:chlorobenzene, 8 h, reflux; iii: phenyl-hydrazine,
grinding, 2 min; iv: cinnamaldehyde, SOCl2. 1,4-dioxane drops, microwave
irradiation 5 min
Scheme 1. Synthetic route for obtaining the target compounds 5a–d.
The (E)-3-phenyl-5-(phenylamino)-2-styryl-1,3,4-thiadiazol-3-ium chloride derivatives (5a–d
were prepared by reaction of cinnamaldehyde and the N-aryl-2-phenyl-hydrazinecarbothioamides
4a–d) in equimolar amounts, thionyl chloride (excess of three times), and enough drops of 1,4-dioxane
)
(
to homogenize the reagents. The mixture was submitted to microwave irradiation for 5 min and
then poured into 1,4-dioxane. After standing for 24 h, the products were isolated and washed
thoroughly with ice water and obtained in good yields (96% to 98%). The compounds 5a–d were
characterized by FT-IR, 1H, and 13C-NMR spectroscopies (Figures S1–S4 in the Supplementary Material),
and their purity was confirmed by elemental analysis. The infrared spectra showed the characteristic

Molecules 2020, 25, 2537
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absorptions in the range of 3431–3433 cm−1 assigned to C=N, 2717 cm−1 and 2746 cm−1 to 5b and 5a
,
respectively, and 2586 cm−1 and 2663 cm−1 to 5c and 5d, respectively, attributed to C=NH+ (stretching),
according to other compounds of this class [23]. In the 1H-NMR spectra the chemical shift attributed to
the exocyclic amino hydrogen was in the range of 12.58 to 12.86 ppm with a singlet feature. The signals
of H-6 were assigned at 7.04–7.24 ppm, and of H-7 at 8.02–8.10 ppm, as doublets (J = 14 Hz). In the
13C-NMR spectra the characteristic signals of heterocyclic carbons were attributed to C-2 at 128.5 ppm
(5a), 156.0 ppm (5b), 158.8 ppm (5c), and 158.9 ppm (5d), and to C-5 159.8 ppm (5a), 161.3 ppm (5b),
163.2 ppm (5c), and 163.4 ppm (5d). The carbon atoms C-6 and C-7 showed signals coherent with
the transmission of the electronic effect of substituents in the N-X-phenylamine group linked to the
heterocyclic ring, that is, the C-6 had more shielding in 111.5 ppm to 115.5 ppm and C-7 had more
deshielding in 142.8 ppm to 148.2 ppm. The other carbon atoms appeared with the chemical shifts as
expected according to the literature [15].
2.2. Biological Activity
The anti-leukemia activity of mesoionic compounds has been investigated since the
1970s [51], with their anti-leukemia effects having been described both in vivo and in vitro [51–53].
However, the cytotoxicity effect of this class of compounds on cells infected with HTLV-1 has not yet
been evaluated. In the present work, it was demonstrated for the first time that the cell lines (MT2 and
C91/PL) infected with HTLV-1 and treated with mesoionic compounds 5a–d significantly reduced cell
viability (Figures 2 and 3). In addition, 5a–d also exhibited a cytotoxicity effect against Jurkat cells,
an uninfected cell line in adult T leukemia (Figure 2). The results obtained demonstrated that treatment
with these mesoionic compounds reduced cell viability in a dose-dependent manner (Figures 2–4).
From Figure 5 it can be seen that 5b was able to induce necrosis in Jurkat cells (75.37 ± 3.63%),
MT2 (35.34 ± 7.80%), and C91/PL (33.75 ± 1.65%) after 24 h of cell treatment.
IC50= 1.51 μM
IC50= 6.15 μM
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
*
*
*
*
*
5a (μM)
5c (μM)
IC50= 7.70 μM
IC50= 5.09 μM
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
*
*
*
*
*
5b (μM)
5d (μM)
Figure 2. Effect of mesoionic compounds 5a–d on MT2 cells. The HTLV-1-infected cell line, i.e., MT2,
was incubated in the presence or absence of 5a–d (0.78–25
M) at 37 ◦C in a humid atmosphere
µ
containing 5% CO2. After 72 h, the cells were incubated with MTT for 3 h under the same conditions.
Then, cells were centrifuged, and DMSO was added to dissolve the crystals formed by reducing the
MTT. The bars represent mean
* p < 0.05 were considered statistically significant compared to control and DMSO.
±
SEM values of % of viable cells from 4 to 5 independent experiments.

Molecules 2020, 25, 2537
6 of 22
IC50= 2.82 μM
IC50= 4.50 μM
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
*
*
*
*
5a (μM)
5c (μM)
IC50= 4.50 μM
IC50= 5.25 μM
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
*
*
*
*
*
*
5b (μM)
5d (μM)
Figure 3. Effect of mesoionic compounds 5a–d on HTLV-1-infected cells. C91/PL cells were incubated
in the presence or absence of 5a–d (0.78–25
M) at 37 ◦C in a humid atmosphere containing 5% CO2.
µ
After 72 h, the cells were incubated with MTT for 3 h under the same conditions. Then, cells were
centrifuged, and DMSO was added to dissolve the crystals formed by reducing the MTT. The bars
represent mean
±
SEM values of % of viable cells from 4 to 7 independent experiments. * p < 0.05 were
considered statistically significant compared to control and DMSO.
IC50= 2.15 μM
IC50= 5.6 μM
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
*
*
*
*
*
*
5c (μM)
5a (μM)
IC50= 2.27 μM
IC50= 1.74 μM
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
*
*
*
*
*
*
*
*
5d (μM)
5b (μM)
Figure 4. Effect of mesoionic compounds 5a–d on Jurkat cells. Cells were incubated in the presence or
absence of 5a–d (0.78–25
M) at 37 ◦C in a humid atmosphere containing 5% CO2. After 72 h, the cells
were incubated with MTT for 3 h under the same conditions. Then, cells were centrifuged and DMSO
was added to dissolve the crystals formed by the MTT reduction. Bars represent mean SEM values of
µ
±
% of viable cells from 4 to 5 independent experiments. * p < 0.05 were considered statistically significant
compared to control and DMSO.

Molecules 2020, 25, 2537
7 of 22
100
80
60
40
20
0
CTR
DMSO
5b
*
*
*
MT2
C91/PL
Jurkat
Figure 5. Effect of compound 5b on induction of necrosis in HTLV-1 infected MT2, C91/PL, and Jurkat
cells. HTLV-1-infected cells and Jurkat cells were incubated with or without the compound 5b (25 M)
µ
at 37 ◦C in a humid atmosphere containing 5% CO2. After 24 h, the cells were collected, and the cell
viability was measured using PI (propidium iodide) labeling. Then, the fluorescence was examined
using flow cytometry. A total of 10,000 events was acquired for the experimental data. Bars represent
mean
±
SEM values of % of necrotic cells from three independent experiments. * p < 0.05 were
considered statistically significant compared to control and DMSO.
Each cell line showed different sensitivity to the 5a–d mesoionic compounds, as evidenced by
the IC50 values shown in Table 1. Interestingly, our results indicated that the values obtained for
IC50 were less than 10
cytotoxicity value against HTLV-1 infected cell lines, i.e., MT2 or C91/PL, with IC50 values of 1.51
and 2.82 M, respectively. On the other hand, compound 5b showed significant inhibitory activity in
Jurkat cells with an IC50 value of 1.74 M. It is important to note that the mesoionic compounds with
µM. Furthermore, these results clearly show that compound 5a has the highest
µ
µ
greater activity, 5a (R = CH3) and 5b (R = OCH3), have a substituent group displaying an electron
donor property, which suggests that this characteristic may be related to the results of cytotoxicity.
In addition, values of log Po/w of 3.74 and 3.35 for 5a (R = CH3) and 5b (R = OCH3), respectively,
indicate that these compounds are less hydrophobic than compounds 5c (R = Cl) and 5d (R = Br),
for which log Po/w are 3.97 and 4.10, respectively. In the literature, several authors have suggested that
reduced hydrophobicity can be associated with the efficiency of cytotoxicity, as this property is directly
related to the binding to cellular targets [54,55]. Besides, retrovirus infection alters lipid synthesis,
leading to an alteration in the cell membrane [56]. The HTLV-1 genome has a pX region, which encodes
viral accessory genes, such as tax. The transcription of the tax gene is a Tax protein responsible for
modifying or decreasing the modulation of many cellular genes, interfering with cell proliferation,
apoptosis, secretion, and others [57]. Thus, the difference observed between the effect of cytotoxicity in
cells infected with HTLV-1 and Jurkat cells may be directly related to viral modulation in the synthesis
of lipids and proteins.
Table 1. In vitro anti-cancer activity results for the mesoionic compounds 5a–d.
Cell Lines
IC50 Values for the Mesoionic Compounds 5a–d (µM)
5a
5b
5c
5d
MT2
C91/PL
Jurkat
1.51
2.82
2.15
5.09
4.50
1.74
6.15
4.50
5.60
7.70
5.25
2.27
IC50 = drug concentration required to inhibit the cell viability by 50% after 72 h of incubation.
It is known that mesoionic compounds can interact with the DNA molecule and their biological
activity can be related with this interaction [32,58]. Based on this, the interaction of compounds 5a–d
with DNA was investigated using spectroscopic analysis. The fluorescence emission spectra of pure

Molecules 2020, 25, 2537
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DNA and the corresponding DNA complex with the mesoionic compounds under study are shown
in Figure 6 for 5b, whereas Figure S5 in the Supplementary Material shows the DNA assays for 5a
5c, and 5d. The experimental results suggested that only compound 5b can interact with the DNA
strands; however, the mechanism of this interaction remains unknown, and more tests are needed to
indicate its nature.
,
100000
5b
DNA 2
10000
1000
100
10
DNA 2+(5b)
1
300
400
500
600
nm
100000
10000
1000
100
5b
DNA 10
DNA 10+(5b)
10
1
300
400
500
600
nm
100000
10000
1000
100
5b
DNA 100
DNA 100+(5b)
10
1
300
400
500
600
nm
Figure 6. Fluorescence emission spectra of 5b (25
(2, 10, and 100 ng/mL). A.U. = arbitrary unit.
µM) in the presence of increasing amounts of DNA
Some anticancer drugs approved by the FDA (US Agency for Food and Drug Administration) can
induce cytotoxicity effects through reactive oxygen species (ROS) modulation, including antileukemia
drugs, such as L-asparaginase, N-benzyloxycarbonyl-IIe-Glu-(O-t-butyl)-Ala-leucinal, anthracyclines
(doxorubicin and daunorubicin), arsenic trioxide, and 2-methoxyestradiol. These drugs diminished the
reduced glutathione intracellular levels and induced mitochondrial dysfunction or ROS generation by
other pathways [59]. Thus, the capacity of mesoionic compounds 5a–d to produce ROS was evaluated.
Using the fluorescent dye dihydrorhodamine 123 (DHR), it was observed that a short treatment (30 min)
of the cells (MT2 and Jurkat) with these compounds induced an increase in ROS levels, indicating that
the oxidative stress can be a mechanism of action of these substances (Figure 7). These mesoionic
compounds can also induce nitric oxide production, which inhibits cellular proliferation and promotes
cytostatic action [53
,60]. Overall, our results suggest that the mesoionic compounds 5a–d constitute a
promising class of new anticancer drugs.

Molecules 2020, 25, 2537
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B
A
1.5
1.5
1.0
0.5
0.0
1.0
0.5
0.0
DMSO 5a
5b
5c
5d
DMSO 5a
5b
5c
5d
12.5 μM
12.5 μM
Figure 7. Effect of mesoionic compounds 5a–d on reactive oxygen species (ROS) production. Jurkat (
A)
and MT2 ( ) cells were incubated with or without 5a–d (12.5
B
µ
M) at 37 ◦C in a humid atmosphere
containing 5% CO2. After 30 min, the cells were washed and incubated with the ROS probe DHR
(Dihydrorhodamine 123). Then, the fluorescence was examined using flow cytometry. A total of
10,000 events was acquired for the experimental data. Bars represent mean
production (DMSO was considered as a control) from two independent experiments.
±
fold change of ROS
2.3. Interaction with HSA
2.3.1. Experimental Binding Ability
The steady-state fluorescence spectra for HSA (1.00
successive additions of mesoionic compounds 5a–d (concentration up to 1.32
Figure S6 in the Supplementary Material. In the presence of these compounds, it is possible to observe
the quenching of the fluorescence emission of the protein ( em = 340 nm), which indicates the existence
of a possible interaction between the Trp-214 residue, responsible for the emission of albumin, and
5a–d 50 61]. The addition of any of these mesoionic compounds to the HSA solution did not result in a
×
10−5 M) in the absence and presence of
10−5 M) are shown in
×
λ
[
,
shift in the maximum fluorescence emission, indicating that the HSA:mesoionic compound interaction
does not disturb the microenvironment surrounding the Trp-214 residue [46].
The steady-state fluorescence quenching of a protein can be classified as static and/or dynamic [62].
A Stern–Volmer analysis of the HSA quenching process for 5a–d (Figure S7 in the Supplementary
Material) shows values for the bimolecular quenching rate constant of the order of kq ~ 1013
M
−1s−1
(Table 2). These values are about four orders of magnitude higher than for the water limiting diffusion
rate constant (kdiff ~ 7.40
109 M−1s−1, at 298K [63]). This is an indication that the fluorescence
quenching mechanism must be static, involving an association in the ground-state between albumin
and mesoionic compounds 5a–d 50]. In addition, the values for the Stern–Volmer constant (KSV
×
[
)
at different temperatures are similar (Table 2), suggesting that 5a–d show similar binding capacity
towards HSA.
To further confirm which mechanism is operating in the fluorescence quenching of HSA
by mesoionic compounds 5a–d, time-resolved fluorescence measurements were performed.
The fluorescence decay for HSA is bi-exponential, with lifetimes of τ1 = 1.73
τ2 = 5.54 ± 0.12 ns (Figure S8, Supplementary Material), which is in accordance with literature
data [64 65]. Both fluorescence lifetimes are independent of the addition of any of the mesoionic
compounds 5a–d (1.35
10−5 M concentration) (Table 3), which is a clear indication of a static process
±
0.16 ns and
,
×
as the main mechanism fluorescence quenching in the interaction between HSA and the mesoionic
compounds under study [47]. Similar results have been reported previously for the interaction between
HSA and piperonal mesoionic derivatives [66].

Molecules 2020, 25, 2537
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Table 2. Binding constant values for the interaction between human serum albumin (HSA) and the
mesoionic compounds 5a–d.
kq (× 1013
)
KSV (× 105)
Ka (× 104)
∆H◦
∆S◦ (× 10−2
)
∆G◦
Sample
HSA:5a
T (K)
(M−1s−1
)
(M−1
)
(M−1
)
(kJ.mol−1
)
(kJ.mol−1K−1
)
(kJ.mol−1
)
296
303
310
1.19 ± 0.03
1.20 ± 0.03
1.21 ± 0.03
1.10 ± 0.02
1.14 ± 0.03
1.15 ± 0.03
1.30 ± 0.03
1.36 ± 0.04
1.40 ± 0.03
1.45 ± 0.02
1.54 ± 0.03
1.60 ± 0.03
2.15
2.16
2.18
7.68 ± 0.26
7.51 ± 0.26
7.32 ± 0.26
8.57 ± 0.26
8.44 ± 0.26
8.27 ± 0.26
8.53 ± 0.26
9.06 ± 0.26
9.57 ± 0.26
8.71 ± 0.26
9.02 ± 0.26
9.28 ± 0.26
−27.7
−28.3
−28.9
−28.0
−28.6
−29.2
−28.1
−28.9
−29.7
−27.9
−28.7
−29.4
−2.64 ± 0.15
−1.88 ± 0.17
6.27 ± 0.10
3.43 ± 0.16
8.46 ± 0.05
296
303
310
1.99
2.06
2.07
HSA:5b
HSA:5c
HSA:5d
8.81 ± 0.06
11.6 ± 0.1
10.6 ± 0.1
296
303
310
2.35
2.45
2.53
296
303
310
2.61
2.79
2.89
r2 for KSV and kq: 0.9986–0.9999; r2 for Ka: 0.9961–0.9998; r2 for ∆H◦, ∆S◦ and ∆G◦: 0.9920–0.9979.
Table 3. Time-resolved fluorescence parameters for the interaction between HSA and the mesoionic
compounds 5a–d.
Sample
τ1 (ns)
Relative %
τ2 (ns)
Relative %
χ2
HSA
1.73 ± 0.16
1.61 ± 0.13
1.66 ± 0.12
1.68 ± 0.11
1.65 ± 0.10
20.7
26.4
23.9
33.0
29.8
5.54 ± 0.12
5.38 ± 0.13
5.25 ± 0.11
5.66 ± 0.16
5.37 ± 0.13
79.3
73.6
76.1
67.0
70.2
1.095
1.140
1.106
1.155
1.038
HSA:5a
HSA:5b
HSA:5c
HSA:5d
The modified Stern–Volmer binding constant (Ka) is related to the protein–ligand interaction, and its
magnitude is important for understanding the distribution of a drug in the human bloodstream [47 61].
Moderate values for Ka, in the order of 104 M−1, at 296, 303, and 310 K, were obtained for the interaction
HSA:5a (Figure S9 in the Supplementary Material and Table 2), indicating the viability of the
absorption and distribution of these mesoionic compounds to various tissues [46,48,61].
The thermodynamic parameters of enthalpy (
H◦) and entropy ( S◦) variation for the interaction
between HSA and 5a were calculated according to the van’t Hoff approximation (Figure S10,
Supplementary Material, and Table 3). The positive values of
S◦ for all investigated compounds
,
–
d
∆
∆
–
d
∆
are related to hydrophobic forces, as a consequence of an interaction between the non-polar amino
acid residues and the non-polar portions of the ligand structure. Furthermore, the same hydrophobic
effect may also be related to the fact that both the water molecules responsible for the solvation of the
ligand and those that occupied the protein cavity are expelled out of the cavity during the HSA:ligand
interaction process. In this case, these water molecules present higher increased degrees of freedom
and, therefore, an increase in the system disorder (∆S◦ > 0) [67,68].
The
∆
H◦ < 0 values for HSA:5a and HSA:5b indicate a significant contribution of hydrogen
bonding, whereas for HSA:5c and HSA:5d
,
∆
H◦ > 0 and, in this case, the presence of electrostatic
interaction forces can be suggested [67,69].
The negative sign for Gibb’s free energy values (
∆
G◦ < 0, Table 2) for HSA:mesoionic compounds
is a clear indication of the spontaneity of the binding process. A general analysis of the thermodynamic
parameters leads us to conclude that the interaction between HSA:5a and HSA:5b is enthalpically and
entropically driven, while for HSA:5c and HSA:5d it is only entropically driven [69].
2.3.2. Perturbation on the Surface and Secondary Structure of Albumin
Circular dichroism spectroscopy (CD) is a sensitive technique used to investigate the secondary
structure of proteins after binding to a ligand [69]. The CD spectrum for HSA exhibits two negative
peaks, at 208 nm and 220 nm, characteristic of the typical α-helix structure of proteins. These two

Molecules 2020, 25, 2537
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peaks correspond to the
mesoionic compounds 5a–d (1.32
π
–
π
* and n–
π
* transitions, respectively [70]. After adding a solution of the
×
10−5 M) to the HSA solution (1.00
×
10−5 M), a small decrease in
intensity at 208 nm and 222 nm was clearly observed (Figure S11, Supplementary Material), indicating a
slight change in the secondary structure of the HSA [50]. The variation in the secondary structure
of albumin is about 5.0% in all cases, which is evidence that the 5a–d interaction can occur with a
very slight disturbance in the secondary structure of albumin (Table 4) [71]. The same trend has been
reported previously for the interaction between HSA and piperonal mesoionic derivatives [66].
The zeta potential (ZP, ζ) is a useful and feasible parameter for characterizing the surface charge
properties of proteins. Changes in the ZP for a protein may imply conformational changes in its
structure, modifications on the surface, and/or unfolding or denaturation processes after its binding
to the ligand [72]. Table 4 shows the ZP value for HSA without and in the presence of 5a–d. The ZP
values for HSA did not change significantly with the addition of these mesoionic compounds, with
this change being within the standard error of the measurement. These results indicated that there is
no significant structural change on the surface of the protein after its interaction with 5a–d [73] and are
fully in accordance with the results for circular dichroism described above.
Table 4. α-helix % values at 208 nm and 222 nm (Circular dichroism spectroscopy (CD) measurements)
and , conductance and current values (zeta potential (ZP) measurements) for HSA without and in the
ζ
presence of mesoionic compounds 5a–d at 310 K.
CD Results
ZP Results
Sample
208 nm
222 nm
ζ (mV)
Conductance (µs)
Current (mA)
HSA
86.7%
84.0%
82.4%
83.8%
82.0%
80.1%
78.5%
77.3%
78.8%
77.7%
−9.15
−8.62
−8.55
−8.77
−8.69
±
±
±
±
±
1.79
1.40
1.46
1.52
1.50
29,645
29,615
29,642
29,622
29,638
150
150
150
150
150
HSA:5a
HSA:5b
HSA:5c
HSA:5d
2.4. Molecular Docking Evaluation
Molecular docking calculations have become an increasingly important tool for drug discovery,
since they can offer a molecular level explanation on the binding ability of potential compounds
towards biomacromolecules, as well as evaluate physical-chemical properties related to their biological
activity [74]. In this sense, molecular docking calculations were carried out for both DNA and HSA
binding ability to evaluate the main binding region and intermolecular forces related between the
mesoionic compounds 5a–d and nucleobases/amino acid residues.
As previously described in Section 2.2, it is known that some mesoionic compounds are able
to interact with DNA. However, the experimental data indicated that only 5b could interact with
DNA strands. Molecular docking runs for the interaction DNA:5a–d offered higher molecular docking
score values for the minor groove than for the major groove of DNA strands, i.e., 84.8 and 10.6,
respectively for DNA:5b, suggesting minor groove as the main possible region of DNA to interact with
this mesoionic compound. Differently from the preliminary experimental data, molecular docking
results suggested that the mesoionic compounds 5a–d could interact with DNA, corroborating with
the statement that more tests are needed to evaluate the biological mechanism. Figure 8 shows the
best docking pose for DNA:5a–d via top view representation of the minor groove, reinforcing that the
mesoionic compounds can interact with DNA strands mainly via van der Waals interaction (interaction
forces were not discussed in the present work).

Molecules 2020, 25, 2537
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.
Figure 8. Top view representation for the best docking pose of the interaction (
A
) DNA:5a, (B) DNA:5b,
(
C) DNA5c, and (D) DNA:5d in the minor groove. Mesoionic compounds 5a, 5b, 5c, and 5d are in
stick representation in beige, pink, cyan, and brown, respectively. Elements’ colors: hydrogen: white;
oxygen: red; nitrogen: dark blue; sulfur: yellow; bromine: wine; and chloro: green.
The HSA structure is composed of three domains (I, II, and III), and each domain is divided into
two subdomains (A and B). Pioneering studies by Sudlow using the fluorescent probes displacement
method showed the presence of two main specific drug binding sites: Sudlow’s site I, also named the
warfarin binding site (subdomain IIA—where Trp-214 residue can be found), and Sudlow’s site II,
also named the ibuprofen binding site (subdomain IIIA) [75,76]. Compounds that strongly bind in
the Sudlow’s site I are generally bulky heterocyclic molecules with a positive charge localized in its
center. On the other hand, Sudlow’s site II is a largely non-polar cavity with a single dominant polar
patch near the pocket entrance. This arrangement of polar and non-polar features is consistent with
the typical structures of site II drugs, such as aromatic carboxylic acids with a negatively charged acid
group at one end of the molecule, separated by a hydrophobic center [77]. Overall, Sudlow’s site II
was proposed to be a smaller or narrower site than Sudlow’s site I, because large molecules rarely bind
to site II [78]. Recently, subdomain IB, known as site III, was also characterized as a potential binding
pocket for different ligands to the albumin structure [47].
Table 5 shows the docking score values for each of the mesoionic compounds 5a–d to the three main
binding sites of HSA. Since the highest docking score value was obtained for site I, molecular docking
calculations suggested subdomain IIA, where Trp-214 can be found, as the main binding pocket for
the compounds under study. From previous HSA fluorescence quenching studies and the chemical
characteristics of each of the mesoionic compounds 5a–d, it was expected that they should be located
next to the amino acid Trp-214 residue in the subdomain IIA. Figure 9 shows the best molecular docking
pose for the interaction HSA:5a–d. From this Figure, it can be observed that the mesoionic compounds
5a/5b and 5c/5d present a similar docking pose, in accordance with the very close experimental
binding parameters obtained from the fluorescence quenching experiments (see Table 2 in Section 2.3.1).
In addition, molecular docking results also suggested van der Waals and hydrogen bonding as the
main intermolecular forces involved in the binding process, fully in accordance with the experimental
data described using the van’t Hoff approximation.

Molecules 2020, 25, 2537
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Table 5. Molecular docking score values (dimensionless) for the interaction between DNA:5a–d and
HSA:5a–d.
DNA
Minor Groove
HSA
Compound
Major Groove
Site I
Site II
Site III
5a
5b
5c
83.9
84.8
83.4
87.4
11.2
10.6
13.8
13.6
78.3
80.1
86.2
85.0
40.2
40.0
45.6
42.9
39.5
38.1
41.1
40.8
5d
Figure 9. Superposition of the best docking pose for (
A
) HSA:5a
/
5b and (B) HSA:5c/5d in the subdomain
IIA (site I). Representation of the main amino acid residues from the HSA structure, which can interact
with ( 5a, ( 5b, ( 5d, and ( 5c. Selected amino acid residues and the mesoionic compounds
5a 5b 5c, and 5d are in stick representation in orange, beige, pink, cyan, and brown, respectively.
C)
D
)
E
)
F)
,
,
Elements’ colors: hydrogen: white; oxygen: red; nitrogen: dark blue; sulfur: yellow; bromine: wine;
and chlorine: green.
3. Materials and Methods
3.1. General Information
Nuclear magnetic resonance (1H- and DEPTQ 13C-NMR) spectra were recorded on a Bruker Avance
III Ultrashield Plus spectrometer (1H, 500 MHz; DEPTQ, 125 MHz, Bruker, Rheinstetten, Germany) using
DMSO-d6 as the solvent and tetramethylsilane (TMS) as the internal standard. Fourier-transformed
infrared spectra (FT-IR) (ATR) were recorded on a Bruker Vertex 70 spectrophotometer. The elemental
analyses were carried out with a Carlo Erba EA-1110 CHNS-O Elemental Analyzer (Elemental
Microanalysis, New Jersey, NJ, USA). The melting points were determined on a Gehaka (PF1500 Farma,
São Paulo, SP, Brazil) capillary melting point apparatus and uncorrected. The microwave-assisted
organic reactions were performed in a CEM Discovery System reactor (CEM Corporation,
North Carolina, NC, USA).
Human acute lymphocytic leukemia (Jurkat cells) were generously provided by Professor Ana
Giannini from Universidade Federal do Rio de Janeiro (UFRJ, RJ, Brazil) and HTLV-1 transformed
cell lines (MT-2 and C91/Pl) were a gift from the Fundação Oswaldo Cruz, RJ, Brazil. All cells were
used and cultured in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 10%
fetal bovine solution (FBS) (Gibco/Thermo Fisher), 60 mg/mL penicillin and 100 mg/mL streptomycin

Molecules 2020, 25, 2537
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(LGC, Brazil). They were incubated at 37 ◦C in a humidified atmosphere of air and 5% CO2. The cell
line was passaged twice a week.
3.2. General Procedure for the Synthesis of Aryl-thioureas (2a–2d)
The different aryl anilines (23.5 mmol) and ammonium isothiocyanate (23.5 mmol) were mixed
in the presence of concentrated hydrochloric acid (23.5 mmol) and 3.17 mL of water as solvent.
The reaction mixture was kept under reflux for 6 h. The solid product was obtained by filtration and
washed with ice-cold water. All thioureas were identified by the comparison of analytical data (FT-IR,
NMR data, and melting points) with literature reports [49].
3.3. General Procedure for the Synthesis of Aryl-isothiocyanates (3a–d)
The aryl-thioureas (7 mmol) was submitted under 8 h of reflux in chlorobenzene as
solvent. The solution was then distilled under reduced pressure for chlorobenzene removal.
The aryl-isothiocyanates (3a–d) were used immediately in the subsequent step as crude products [49].
3.4. General Procedure for the Preparation of Hydrazinecarbothiamides (4a–d)
The phenylhydrazine (0.74 mmol) and aryl-isothiocyanate (0.74 mmol) were mixed at room
temperature for 2 min in a porcelain mortar and pestle in the absence of any organic solvent.
The formed solid was monitored by TLC and was obtained in high purity without recrystallization.
All hydrazinecarbothiamides were identified by the comparison of analytical data (FT-IR, NMR data,
and melting points) with literature reports [50].
3.5. General Procedure for the Preparation of Mesoionic Chlorides (5a–d)
The mixture containing the N-(4-R-phenyl)-2-phenylhydrazinecarbothioamide (0.38 mmol) and
cinnamaldehyde (0.38 mmol) in the presence of SOCl2 and a few drops of 1,4-dioxane was submitted
to microwave irradiation for 5 min at 100 W. After this continuous time, the mixture was added to
1,4-dioxane and left to stand overnight at room temperature. The solid obtained was filtered and
washed with ice-cold 1,4-dioxane and exhaustively with water. The solid formed was obtained in high
purity without recrystallization. All mesoionic chlorides (Figure 10) were identified by melting point,
FT-IR, NMR data, and elemental analyses [29].
3''
4''
2''
5''
1'
5'''
Cl
1''
4'''
R
6'''
6''
N
N
2'
7
3'''
3'
4'
2 S
N
H
5
1'''
2'''
6
6'
5'
R = CH3 (5a), OCH3 (5b), Cl (5c), Br (5b)
Figure 10. Mesoionic chlorides 5a–d numbered to the NMR attributions.
(E)-3-Phenyl-5-(40-methylphenylamino)-2-styryl-1,3,4-thiadiazol-3-ium chloride (5a).
◦
Orange solid; yield 98%; mp 287–286 C; FT-IR (ATR,
(C-H), 2746 (C=NH+), 1614 (C=C), 1564, 1452 (C=C), 1523 (C=N), 1340 (C-S), 1114 (C-H), 823, 763, 688
(C-H); 1H-NMR (500 MHz, DMSO-d6) 12.58 (s, 1H, N-H), 8.25 (d, J = 7.9 Hz, 2H, H-30’, H-50’), 8.10
d, J = 15.9 Hz, 1H, H-7), 8.01 (d, J = 9.5 Hz, 2H, H-20’, H-60’), 7.83-7.75 (m, 6H, H-20, H-30, H-40, H-50,
H-60, H-40’), 7.47 (d, J = 7.9 Hz, 2H, H-30”, H-50”), 7.25 (d, J = 15.9 Hz, 1H, H-6), 7.22 (d, J = 6.3 Hz, 2H,
H-20”, H-60”), 2.27 (s, 3H, CH3); 13C-NMR (125 MHz, DMSO-d6)
159.8 (C-2), 148.5 (C-5), 144.5 (C-7),
139.9 (C-10”), 136.9 (C-10), 133.6 (C-10’), 131.8 (C-40’), 130.2 (C-20, C-60), 130.1 (C-30, C-50), 129.9 (C-30’,
υ
cm−1): 3431 (N-H), 3039 (C-H), 2922
δ
(
δ

Molecules 2020, 25, 2537
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C-50’), 126.1 (C-40”), 125.2 (C-20’, C-60’), 124.1 (C-30”, C-50”), 118.8 (C-20”, C-60”), 115.5 (C-6), 20.5 (CH3).
Elemental analysis found: C, 67.98%; H, 4.92%; N, 10.41%. Calculated for C23H20ClN3S (MW 405.94):
C, 68.05%; H, 4.97%; N, 10.35%.
(E)-3-Phenyl-5-(40-methoxyphenylamino)-2-styryl-1,3,4-thiadiazol-3-ium chloride (5b).
Red solid; yield 96%; mp 292–291 ◦C; FT-IR (ATR,
υ
cm−1): 3431 (N-H), 3043 (C-H), 2929 (C-H),
2717 (C=NH+), 1622 (C=C), 1595, 1575, 1454 (C=C), 1512 (C=N), 1342 (C-S), 1244 (C-O), 1109 (OCH3),
954 (C-H), 829, 773, 694 (C-H); 1H-NMR (500 MHz, DMSO-d6)
δ 12.73 (s, 1H, N-H), 8.24 (d, J = 9.7 Hz,
2H, H-30’, H-50’), 8.09 (d, J = 16.0 Hz, 1H, H-7), 8.00 (d, J = 9.7 Hz, 2H, H-20’, H-60’), 7.83–7.75 (m, 6H,
H-20, H-30, H-40, H-50, H-60, H-40’), 7.52 (d, J = 9.7 Hz, 2H, H-20”, H-60”), 7.24 (d, J = 16.0 Hz, 1H, H-6),
6.99 (d, J = 9.7 Hz, 2H, H-30”, H-50”), 3.73 (s, 3H, OCH3); 13C-NMR (125 MHz, DMSO-d6)
δ 161.3 (C-2),
156.0 (C-5), 148.5 (C-40”), 144.2 (C-7), 140.0 (C-10), 137.0 (C-10’), 131.8 (C-40’), 131.7 (C-10”), 130.2 (C-20,
C-60), 130.0 (C-30’, C-50’), 126.1 (C-20’, C-60’), 124.1 (C-20”, C-60”), 120.5 (C-40), 115.5 (C-6), 114.6 (C-30”,
C-50”), 55.3 (OCH3). Elemental analysis found: C, 65.05%; H, 4.58%; N, 10.04%. Calculated for
C23H20ClN3OS (MW 421.94): C, 65.47%; H, 4.78%; N, 9.96%.
(E)-3-Phenyl-5-(40-chlorophenylamino)-2-styryl-1,3,4-thiadiazol-3-ium chloride (5c).
Orange solid; yield 97%; mp 284–282 ◦C; FT-IR (ATR,
υ
cm−1): 3433 (N-H); 2922 (C-H);
2586 (C=NH+); 1620 (C=C); 1562, 1492 (C=C); 1537 (C=N); 1249 (C-S); 1097 (C-H); 1033 (C-Cl);
817 (C-H); 761e 686 (C-H). 1H-NMR (500 MHz, DMSO-d6)
12.82 (s, 1H, N-H), 8.02 (d, J = 19.7 Hz
1H, H-7), 7.84 (dd, J = 3.9 Hz, 2H, H-30’, H-50’), 7.75 ( m, 5H, H-20’, H-60’, H-40’, H-30, H-50),
7.61 (d, J = 10.5 Hz, 2H, H-20, H-60), 7.47 (m, 5H, H-20”, H-60”, H-30”, H-50”, H-40), 7.04 (d, J = 19.7 Hz
1H, H-6); 13C-NMR (125 MHz, DMSO-d6) 163.2 (C-2), 158.8 (C-5), 148.2 (C-7), 137.4 (C-10”), 136.9 (C-10),
δ
,
,
δ
133.8 (C-10’), 132.0 (C-40’), 131.7 (C-40), 130.2 (C-30, C-50), 129.4 (C-30”, C-50”), 129.1 (C-20”, C-60”),
129.2 (C-20, C-60), 127.7 (C-40”), 126.2 (C-20’, C-60’), 120.1 (C-30’, C-50’), 111.4 (C-6). Elemental analysis
found: C, 61.84%; H, 3.97%; N, 9.98%. Calculated for C22H17Cl2N3S (MW 426.36): C, 61.98%; H, 4.02%;
N, 9.86%.
(E)-3-Phenyl-5-(40-bromophenylamino)-2-styryl-1,3,4-thiadiazol-3-ium chloride (5d).
Orange solid; yield 96%; mp 298–296 ◦C; FT-IR (ATR,
υ
cm−1): 3432 (N-H); 3037 (C-H);
2663 (C=NH+); 1604 (C=C); 1560, 1484 e 1442 (C=C); 1540 (C=N); 1309 (C-S); 1114 (C-H); 948, 669 (C-Br);
827 (C-H); 755, 690 (C-H). 1H-NMR (500 MHz, DMSO-d6)
δ 12.62 (s, 1H, N-H), 8.03 (d, J = 19.7 Hz,
1H, H-7), 7.83 (dd, J = 4.5 Hz, 2H, H-30’, H-50’), 7.75 (m, 5H, H-20’, H-60’, H-40’, H-30, H-50),
7.60 (d, J = 12.1 Hz, 2H, H-20, H-60), 7.54 (d, J = 12.1 Hz, 2H, H-30”, H-50”), 7.47 (m, 5H, H-20”, H-60”,
H-40), 7.04 (d, J = 19.7 Hz, 1H, H-6); 13C-NMR (125 MHz, DMSO-d6)
δ 163.4 (C-2), 158.9 (C-5), 148.2 (C-7),
137.9 (C-10”), 136.9 (C-10), 133.8 (C-10’), 132.3 (C-30”, C-50”), 132.0 (C-40’), 131.8 (C-40), 130.2 (C-30, C-50),
129.2 (C-20”, C-60”), 129.2 (C-20, C-60), 126.2 (C-20’, C-60’), 120.5 (C-30’, C-50’), 115.8 (C-40”), 111.5 (C-6).
Elemental analysis found: C, 56.08%; H, 3.57%; N, 9.02%. Calculated for C22H17BrClN3S (MW 470.81):
C, 56.12%; H, 3.64%; N, 8.92%.
3.6. Biological Assays
3.6.1. Cell Viability Assays
The 1
DMSO (Sigma-Aldrich/Merck, St.
with a 5% CO2. After 72 h, the cellular viability was assessed using 5 mg/mL of
×
105 cells/mL were incubated with or without compounds 5a
Louis, MO, USA) at 37 ◦C in humidified atmosphere
, 5b, 5c, 5d or
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich/Merck). Cells were
incubated for 3 h, centrifuged, and formazan crystal was dissolved with DMSO. Plates were read on
a Paradigma® spectrofluorometer (Molecular Devices, San Jose, CA, USA) at a 490 nm wavelength.
Cell death was also evaluated using Propidium Iodide (Biolegend) exclusion after a 24 h culture under
the same conditions described above. The fluorescence was examined by FACSCalibus flow cytometry
(BD) and 10,000 events were acquired for the experimental data. Analysis was accomplished using
Summit V4.0 or FlowJo software (Becton, Dickingson and Company, Santa Barbara, CA, USA).

Molecules 2020, 25, 2537
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3.6.2. Reactive Oxygen Species (ROS) Detection
The 1
×
105 cells/mL were incubated with or without compounds 5a–d (12.5
µ
M) or DMSO
for 30 min at 37 ◦C in a humidified atmosphere with a 5% CO2. Cells were washed with
phosphate buffer (Sigma-Aldrich/Merck) and incubated with 50 µM of Dihydrorhodamine 123 (DHR;
Sigma-Aldrich/Merck). After 15 min at 37 ◦C, cells were washed and the fluorescence was examined
using FACSCalibus flow cytometry. A total of 10,000 events was acquired for the experimental data,
and a gate based on forward and side scatter parameters was made to analyze only viable cells.
Analysis was accomplished using Summit V4.0 or FlowJo software.
3.6.3. DNA Interaction Assay
Fluorescence emission spectroscopic titration experiments of DNA were conducted in the absence
and presence of a constant concentration of the mesoionic compounds 5a–d. The assay was performed
using 2 and 10 ng/mL of salmon DNA (Sigma-Aldrich/Merck) at 37 ◦C. After that, the fluorescence
emission was recorded after 1 h of DNA incubation with or without compounds 5a–d (25 µM) or
DMSO, over a wavelength range of 300 to 600 nm in a Tris-acetate-EDTA buffer (Promega) at room
temperature. The slit widths for excitation and emission were set to 5.0 nm. The fluorescence was
examined using a F4500 fluorometer (Hitachi, New Jersey, NJ, USA).
3.6.4. Statistical Analysis
Statistical analysis was performed by one-way analysis of variance (ANOVA) and means were
compared using a Tukey post-test. The IC50 values were obtained using nonlinear regression. Data were
analyzed using GraphPad Prism-5 software (GraphPad software, San Diego, CA, USA), and p ≤ 0.05
was considered statistically significant.
3.7. Spectroscopic HSA Binding Studies
The steady-state fluorescence spectra were measured in the 290–450 nm range, at 296, 303,
and 310 K, with the excitation wavelength at
λ
= 295 nm. To a 3.0 mL solution containing an appropriate
concentration of HSA −(13.00
×
10−5 M), successive aliquots from a stock solution of each mesoionic
under study (1.00
×
10 M in DMSO) were added, with final concentrations of 0.17, 0.33, 0.50, 0.66,
0.83, 0.99, 1.15, and 1.32
×
10−5 M. The addition was done manually by using a micro syringe. The inner
filter correction for the observed steady-state fluorescence of HSA:mesoionic compounds was done in
order to compensate for the absorption value of 5a–d at the excitation and emission wavelengths (295
and 340 nm, respectively) when applying Equation (1) [50]:
A
+A
exc
em
]
Fcor = Fobs10[
(1)
2
where Fcor and Fobs are the corrected and observed steady-state fluorescence intensity values,
respectively, while Aex and Aem represent absorbance values for each ligand at the excitation and
emission wavelengths (λ = 295 and 340 nm, respectively).
The binding properties of each ligand toward HSA—Stern–Volmer quenching constant (KSV),
bimolecular quenching rate constant (kq), modified Stern–Volmer binding constant (Ka), enthalpy
change (∆ ∆ ∆
H◦), entropy change ( S◦), and Gibbs’ free energy change ( G◦)—were calculated from the
steady-state fluorescence quenching data according to Equations (2)–(5) [47,48]:
F0
F
= 1 + kqτ0[Q] = 1 + KSV[Q]
(2)
(3)
F0
1
1
=
+
F0−F
f
f[Q]Ka

Molecules 2020, 25, 2537
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∆H0
RT
∆S0
R
ln Ka = −
+
(4)
(5)
∆G0 = ∆H0 − T∆S0
where F0 and F are the steady-state fluorescence intensities of HSA in the absence and presence
of mesoionics, respectively. [Q] and τ0 are the ligand concentration and fluorescence lifetime,
respectively, of the fluorophore in the absence of quencher (5.54
×
10−9 s—experimental data,
see Section 2.3.1). T, , and R are the temperature (296, 303, and 310 K), fraction of the initial
ƒ
fluorescence intensity that corresponds to fluorophore that is accessible to the quencher (f
and gas constant (8.3145 J/mol.K), respectively.
≈
1.0),
The time-resolved fluorescence decay of a 3.0 mL solution of HSA (1.00
buffer solution-PBS) was measured in the absence and presence of the maximum concentration of each
mesoionic used in the steady-state fluorescence studies (1.32
10−5 M) at room temperature (ca 298 K).
×
10−5 M, in phosphate
×
The CD spectra were recorded in the 200–250 nm range at 310 K for free serum albumin
(
1.00 × 10−6 M) and for HSA with the higher concentration of each mesoionic used in the steady-state
fluorescence studies (1.32
The obtained data were further converted into mean residue ellipticity (MRE) according to
×
10−5 M). Each spectrum was taken on the average of three scans.
Equation (6) [50]:
θ
MRE = ꢀ
ꢁ
(6)
10.n.l.Cp
where
θ
is the observed ellipticity (mdeg); n is the number of amino acid residues (585 to HSA) [45],
l is the length of the optical cuvette (1.0 cm), and Cp is the molar concentration of HSA (1.00 × 10−6 M).
The 5a–d induced helical content perturbation at 208 and 222 nm were calculated using Equation (7) [46]:
208−4000)
(A) α − helix%(−MRE
× 100
× 100
(33000−4000)
(7)
222−2340)
(B) α − helix%(−MRE
(30300)
where MRE208 and MRE222 are the significant molar residual ellipticities (deg.cm2/dmol) at 208 nm
and 222 nm, respectively.
3.8. Zeta Potential Measurements for HSA Surface Perturbation
All zeta potential (ZP) measurements were performed with 10 runs at 298 K and results were
reported in terms of ZP
±
SD. The ZP was measured for the HSA solution (1.0
×
10−5 M in PBS solution)
without and in the presence of the mesoionic compounds at the maximum concentration used in the
steady-state fluorescence experiments (1.32 × 10−5 M) at room temperature (ca. 298 K).
3.9. Molecular Docking Procedure for DNA and HSA
The chemical structure for the mesoionic compounds (5a–d) was built and energy-minimized
with Density Functional Theory (DFT), with the Becke-3-Lee Yang Parr (B3LYP) method with the
standard 6-31G* basis set, available in Spartan’18 software (Wavefunction, Inc., Irvine, CA, USA) [79].
The crystallographic structures for the DNA and HSA were obtained from the Protein Data Bank (PDB)
with access codes 1BNA [80] and 1N5U [45], respectively. Molecular docking was performed with
GOLD 5.7 software (CCDC) [81]. Hydrogen atoms were added to the macromolecules according to
the data inferred by the GOLD 5.7 software (Cambridge Crystallographic Data Centre, Cambridge,
CB2 1EZ, UK) on the ionization and tautomeric states. For DNA, a 10 Å radius spherical cavity around
minor and major groove was defined as the binding site for the molecular docking calculations and for
has, and a 10 Å radius spherical cavity around subdomains IIA, IIIA, and IB was defined. The scoring
function used was “ChemPLP”, which is the standard function of GOLD 5.7 software. The figures of the

Molecules 2020, 25, 2537
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docking poses for the largest docking score value were generated with the PyMOL Delano Scientific
LLC program [82]. For more details, see previous publications [46–48].
4. Conclusions
The results obtained in this work show new promising chemotherapy agents against MT2 and
C92 cell lines infected with HTLV-1, which causes adult T-cell leukemia/lymphoma (ATLL), and in
uninfected Jurkat cells with IC50 values lower the 10 µM. The compounds containing electron donor
substituents (R=CH3 or OCH3) were more active and with lower hydrophobicity. Furthermore, the
compounds showed cell death by necrosis and DNA interaction, especially those containing electron
donor substituents.
The interaction between HSA and the mesoionic compounds is spontaneous and occurs via
a ground-state association (static fluorescence quenching mechanism). The binding is moderate
(
Ka ~ 104 M−1), being enthalpically and entropically driven for HSA:5a and HSA:5b, while for the other
mesoionic compounds, i.e., 5c and 5d, is only entropically driven. The binding does not significantly
perturb both the secondary and surface structures of the albumin, and the main binding pocket is
site I (subdomain IIA), where Trp-214 residue can be found. Overall, all the mesoionic compounds
presented good binding parameters toward serum albumin, indicating feasible biodistribution in the
human bloodstream.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/11/2537/s1,
Figures S1–S4: FTIR, 1H-NMR and 13C-NMR spectra for 5a
emission spectra for 5a 5c, and 5d (25
quenching for HSA without and in the presence of 5a 5b, 5c, or 5d at 310 K. Figure S7: Stern–Volmer plots for
, 5b, 5c, and 5d, respectively. Figure S5: Fluorescence
,
µM) in the presence of 100 ng/mL of DNA. Figure S6: Steady-state fluorescence
,
the interaction between HSA and the mesoionic compounds at 296, 303, and 310 K. Figure S8: Time-resolved
fluorescence decay for the interaction between HSA and the mesoionic compounds 5a–d in a PBS solution.
[HSA] = 1.00 × 10−5
M
and [mesoionic] = 1.32
×
10−5 M. IRF is the instrument response factor. Figure S9: Modified
Stern–Volmer plots for the interaction between HSA and the mesoionic compounds at 296, 303, and 310 K. Figure
S10: Van’t Hoff plot for the interaction between HSA and the mesoionic compounds. Figure S11: Circular
dichroism spectra for HSA without and in the presence of 5a, 5b, 5c, or 5d at 310 K.
Author Contributions: Conceptualization, A.E., J.E.-L., J.C.N-F. and O.A.C.; methodology, D.S.-P., T.S.d.O., R.O.P.
and O.A.C.; software, O.A.C.; validation, A.E., J.E.-L. and J.C.N.-F.; formal analysis, A.E., J.E.-L., O.A.C., D.S-P.
and J.C.N.-F.; investigation, D.S.-P., T.S.d.O., R.O.P. and O.A.C.; resources, A.E.; data curation, A.E., D.S.-P., J.E.-L.,
O.A.C. and J.C.N-F.; writing, A.E., D.S.-P., J.E.-L., O.A.C. and J.C.N-F.; funding acquisition, A.E. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by the following Brazilian agencies: Coordenaç
de Pessoal de N vel Superior (CAPES), Conselho Nacional de Desenvolvimento Cient
(CNPq–Process: 310345/2015-4) and Fundaç o de Amparo Pesquisa do Estado do Rio de Janeiro (FAPERJ).
ã
o de Aperfeiçoamento
í
í
fico e Tecnol gico
ó
ã
à
J.C.N.-F. acknowledges Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMETRO) for a Visiting
Professor Fellowship.
Acknowledgments: C.M.R. Sant’Anna from Laboratório de Modelagem Molecular (Institute of Chemistry-UFRRJ)
for the molecular docking facilities and N.C.L. Garden (Institute of Chemistry—UFRJ) for the time-resolved and
synchronous fluorescence facilities.
Conflicts of Interest: The authors declare no conflict of interest.
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