In vitro and in vivo cytotoxic activity and human serum albumin interaction for a methoxy-styryl-thiosemicarbazone

Article abstract of DOI:10.1007/s10637-018-00722-y

Thiosemicarbazone is a class of compounds with potential applications in medicine, presenting high capacity to inhibit the growth of cancer cells as well as low toxicity. Because of high interest in anticancer studies involving thiosemicarbazones as new c

Full text of DOI:10.1007/s10637-018-00722-y

Investigational New Drugs
https://doi.org/10.1007/s10637-018-00722-y
PRECLINICAL STUDIES
In vitro and in vivo cytotoxic activity and human serum albumin
interaction for a methoxy-styryl-thiosemicarbazone
Otávio Augusto Chaves1,2 & Isabela S. de Castro3 & Carla Marins Goulart2 & Myrtes S. S. Bellieny2
José Carlos Netto-Ferreira2,4 & Juliana Echevarria-Lima3 & Aurea Echevarria2
&
Received: 24 November 2018 /Accepted: 26 December 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2019
Summary
Thiosemicarbazone is a class of compounds with potential applications in medicine, presenting high capacity to inhibit the growth of
cancer cells as well as low toxicity. Because of high interest in anticancer studies involving thiosemicarbazones as new chemothera-
peutic agents, a synthetic thiosemicarbazone derivative, 4-N-(2′-methoxy-styryl)-thiosemicarbazone (MTSC) was evaluated in vivo
against Ehrlich carcinoma in an animal model. In vivo results demonstrated that MTSC treatment induced the survival of mice and
altered significantly the body weight of the surviving mice 12 days after tumor inoculation. Treatment with 30 mg/kg of MTSC
exhibited effective cytotoxic activity with T/C values of 150.49% (1 dose) and 278% (2 doses). Its interaction with human serum
albumin (HSA), which plays a crucial role in the biodistribution of a wide variety of ligands, was investigated by multiple spectroscopic
techniques at 296 K, 303 K, and 310 K, as well as by theoretical calculations. The interaction between HSA and MTSC occurs via
ground-state association in the subdomain IIA (Sudlow’s site I). The binding is moderate (Ka ≈ 104 M–1), spontaneous, entropically, and
enthalpically driven. Molecular docking results suggested hydrogen bonding and hydrophobic interactions as the main binding forces.
Overall, the interaction HSA:MTSC could provide therapeutic benefits, improving its cytotoxic efficacy and tolerability.
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Keywords Thiosemicarbazones Ehrlich carcinoma Human serum albumin Spectroscopy Molecular docking
Introduction
inhibit the growth of various pathogens and their low toxicity
[1]. Among the biological activities, the thiosemicarbazone de-
rivatives show potential application as antiviral [2], antimicrobial
[3, 4], antiparasitic [5–7], and antitumor [8–10] agents. The an-
ticancer effect of thiosemicarbazones is related to the inhibition
of ribonucleotide reductase, an enzyme involved in the rate-
limiting step of DNA synthesis [11]. Significant alterations in
iron metabolism and iron-dependent enzymes of cancer cells
contribute to the antitumor effect of thiosemicarbazones due to
their iron-chelating properties [9, 12]. The antitumor activity of
thiosemicarbazones has been evaluated in vivo, using lympho-
cytic leukemia—L1210, adenocarcinoma 755, Lewis lung carci-
noma, and neuroblastoma, among others [13–15]. Ehrlich’s as-
cites tumor is a very aggressive carcinoma characterized by a
high level of cell proliferation and represents an important
in vivo animal model [16].
Thiosemicarbazones belong to a class of compounds with poten-
tial applications in medicine because of their high ability to
* José Carlos Netto-Ferreira
jcnetto.ufrrj@gmail.com
* Juliana Echevarria-Lima
juechevarria@micro.ufrj.br
1
Instituto SENAI de Inovação em Química Verde (ISI-QV), Rua
Morais e Silva N° 53, Maracanã, Rio de Janeiro, RJ 20271-030,
Brazil
2
Instituto de Química, Departamento de Química Orgânica,
Universidade Federal Rural do Rio de Janeiro (UFRRJ), Rodovia
BR-465, Km 7, Seropédica, RJ 23890-000, Brazil
Human serum albumin (HSA) is the most abundant carrier
protein in the human circulatory system. It plays a crucial role
in the transport, distribution, and metabolism of a wide variety
of endogenous and exogenous ligands [17–19]. The crystallo-
graphic analysis of HSA revealed a monomeric globular pro-
tein structure composed of a single polypeptide chain
3
Laboratório de Imunologia Básica e Aplicada, Depto. Imunologia,
Instituto de Microbiologia Paulo de Góes, Universidade Federal do
Rio de Janeiro (UFRJ), Av. Carlos Chagas Filho, 373, Rio de
Janeiro, RJ 21941-590, Brazil
4
Qualidade e Tecnologia (INMETRO), Divisão de Metrologia
Química, Instituto Nacional de Metrologia, Duque de
Caxias, RJ 25250-020, Brazil

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containing 585 amino acid residues (66 kDa), with three struc-
turally similar domains (I, II, and III), each containing two
subdomains (A and B) [20, 21]. As the therapeutic effect of
a drug is directly related to its free concentration in plasma, it
is of central pharmacological interest to study the drug–
albumin binding ability [22, 23]. Several studies have dealt
with the interaction between serum albumin and
thiosemicarbazone derivatives [24–27]; however, no reports
were found regarding 4-N-(2′-methoxy-styryl)-
thiosemicarbazone (MTSC – Figure 1).
Because of our interest in anticancer/cytotoxic studies, as
well as in thiosemicarbazones as novel chemotherapeutic
agents, the synthetic derivative MTSC and its Cu(II) complex
were assayed as in vitro cell viability as well as in vivo against
Ehrlich carcinoma in an animal model using BALB/c mice.
Because the efficacy of potential drugs is directly related to
HSA binding ability, the present work also reports the study of
interactions between HSA:MTSC using spectroscopic tech-
niques (circular dichroism, steady-state, time-resolved, and
synchronous fluorescence) combined with theoretical calcula-
tions (molecular docking). These data may contribute to a
deeper understanding of the interaction mechanism, distribu-
tion, and transport involved in the association protein–
thiosemicarbazone derivative, which is an important field of
research in drug development.
grade), methanol, ethanol, and sulfuric acid were obtained
from Vetec Quimica Fina Ltda, RJ, Brazil.
Synthesis
MTSC was synthesized and characterized as previously re-
ported in the literature [5]. Briefly, an equimolar mixture of
2-methoxy-benzaldehyde and thiosemicarbazide was dis-
solved in ethanol in the presence of two drops of concentrated
sulfuric acid and stirred by 1 h at 303 K. After removal of the
solvent, the solid was recrystallized from methanol.
4-N-(2’-methoxy-styryl)-thiosemicarbazone (MTSC):
Yield 82%; m. p. = 186-188°C; IR (ATR, cm-1) ν 3420;
3383; 3240 (N-H); 3080 (C-HAr); 2970 (C-Halkyl); 1594
(C=C); 1574; 1483; 1465 (C=C + C=N); 981 (C=Ctrans);
1
823 (C=Sfree). H NMR (DMSO-d6) δ (ppm) 11.34; 8.14;
7.87; 7.57; 7.31; 7.04; 6.98; 6.96; 3.85. 13C NMR (DMSO-
d6) δ (ppm) 177.1; 156.4; 145.3; 133.5; 129.8; 127.0; 125.3;
120.3; 111.1; 55.0.
MTSC-complex was synthesized by the reaction between
an equimolar mixture of MTSC and CuCl2 dissolved in
methanol:water (1:1, v/v) under reflux, for 1 h.
4-N-(2’-methoxy-styryl)-thiosemicarbazone-Cu(II)-com-
plex (MTSC-complex): Yield 57%; m. p. = 207°C; IR (ATR,
cm-1) ν 3396 (N-H); 3070 (C-HAr); 2946 (C-Halkyl); 1596
(C=C); 1548; 1485 (C=C + C=N); 980 (C=Ctrans); 743
1
(C=Scomplex). H NMR (DMSO-d6) δ (ppm) 11.35; 7,89;
7.62; 7.56; 7.32; 7.12; 7.05; 6.96; 6.91; 3.85. 13C NMR
(DMSO-d6) δ (ppm) 178.1; 157.4; 146.0; 134.4; 130.7;
127.9; 126.3; 124.6; 121.3; 112.1; 56.0.
Materials and Methods
Chemicals
Biological assays
2-methoxy-benzaldehyde and thiosemicarbazone, copper (II)
chloride (CuCl2), dimethyl sulfoxide (DMSO), 3-[4,5-dimeth-
ylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), β-
mercaptoethanol, N-(2-hydroxyethyl)piperazine-N′-(2-
ethanesulfonic acid) (HEPES), penicillin, streptomycin,
RPMI-1640 medium, human serum albumin (96-99% of pu-
rity), phosphate buffer solution (PBS), racemic ibuprofen and
racemic warfarin were purchased from Sigma-Aldrich
Chemical Company, St. Louis, USA, and used as received.
One tablet of PBS yields 1.00 × 10-2 M phosphate buffer,
2.70 × 10-3 M potassium chloride and 1.37 × 10-1 M sodium
chloride, pH 7.4, at 298 K. Fetal calf serum was obtained from
Gibco, Life Technologies. Acetonitrile (spectrophotometric
Animals
Inbred female BALB/c mice (2–4 months old) were used in all
in vivo experiments. Animals were housed in a temperature-
controlled room and received free access to water and stan-
dard rat chow (ad libitum). The experimental procedures were
performed in accordance with the Brazilian Guidelines
(Brazilian Directive for Care and Use of Animals for
Teaching and Research-DBCA) published by the Brazilian
Council for Control of Animal Experimentation (Conselho
Nacional de Controle de Experimentação Animal –
CONCEA) and Brazilian Federal Law 11.794 (October 8,
2008). Protocols used in this study were approved by the local
research ethics committee of the Universidade Federal Rural
do Rio de Janeiro, Brazil (Number 6985210617).
H
N
NH2
N
S
OCH3
Cell culture
MTSC
The human erythroleukemia K562, Lucena I (Vincristine-
resistant derivative K562) and the Ehrlich ascites carcinoma
Fig. 1 Chemical structure of 4-N-(2’-methoxy-styryl)-thiosemicarbazone
(MTSC)

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cell line were generously donated by Dr. Vivian M. Rumjanek
from the Instituto de Bioquímica Médica of
Universidade Federal do Rio de Janeiro (UFRJ). Burkett lym-
phoma Daudi cell lines were maintained in culture medium
RPMI-1640, supplemented with β-mercaptoethanol (50
μmol/L), HEPES (25 mmol/L), penicillin (60 mg/L), strepto-
mycin (100 mg/L), and 10% (v/v) fetal calf serum. Cells were
incubated at 310 K in a humidified atmosphere of air and 5%
CO2. The cell lines were passaged twice a week at a density of
1.00 × 104 cells/mL.
HSA binding studies
Steady-state fluorescence experiments
The steady-state fluorescence measurements were performed
on a Jasco model J-815 spectrofluorimeter equipped with a
1.0 cm quartz cell and a Jasco PFD-425S15F thermostat sys-
tem with 0.1°C accuracy. All spectra were recorded with ap-
propriate background corrections. The excitation wavelength
of 280 nm was selected for the experiments and fluorescence
spectra were obtained in the 290–450 nm range, at 296 K, 303
K, and 310 K. For the titration experiment, to a 3.0 mL solu-
tion containing HSA (1.00 × 10–5 M, in phosphate buffered
saline (PBS) solution, pH 7.4), successive aliquots from an
acetonitrile stock solution of MTSC (1.00 × 10–3 M) were
added. The addition was performed manually with a 10 μL
syringe, with varying concentrations of 0.17, 0.33, 0.50, 0.66,
0.83, 0.99, 1.15, and 1.32 × 10–5 M.
Viability assay
Cells (1.00 × 104 cells/mL) were seeded on a 96-well cell culture
plate and incubated in the presence or absence of either the
MTSC or the MTSC–complex at a concentration of 3.12, 6.25,
12.50, 25.00, and 50.00 μM. The control groups comprised cells
cultivated in the presence of either the vehicle (DMSO) or the
medium. Cell viability was assessed by the addition of 20 μL/
well of MTT (5 mg/mL) as described earlier [28]. This colori-
metric assay is based on the conversion of the MTT (yellow) to
the formazan (purple) derivative by mitochondrial enzymes in
viable cells. The absorbance was measured on a microplate read-
er (Sunrise-Basic TECAN) at a wavelength of 490 nm.
To compensate for the inner filter effect, the fluorescence
intensity values of the samples containing HSAwere corrected
for the absorption of MTSC at excitation and emission wave-
lengths using Eq. (1) [29].
½ðAexþAemÞ=2Š
Fcor ¼ Fobs10
ð1Þ
where Fcor and Fobs are the corrected and observed fluores-
cence intensity values, respectively, while Aex and Aem repre-
sent absorbance values at the excitation (λ = 280 nm: ε =
13,998 cm–1M–1, in PBS) and emission wavelengths (λ =
340: ε = 41,762 cm–1M–1, in PBS), respectively.
The quenching of HSA fluorescence in the presence of
increasing MTSC concentrations was analyzed using the
Stern–Volmer equation—Eq. (2A)—and by the kq defini-
tion—Eq. (2B) [30, 31]:
In vivo evaluation of toxicity and cytotoxic activity
The toxicity was analyzed of treated BALB/c adult female mice
with MTSC and MTSC–complex. Mice were treated with ve-
hicle (DMSO; control group), 60 mg/kg of MTSC, or 60 mg/kg
MTSC–complex once a week intraperitoneally (i.p.). After 30
days, the animals’ weight, glucose level, and weight of heart,
kidneys, liver, spleen, and thymus were analyzed. 2.50 × 105 of
Ehrlich ascites carcinoma cells were injected i.p. in saline so-
lution. After 24h mice were treated with vehicle (DMSO; con-
trol group 1), 30 mg/kg (group 2), or 60 mg/kg of MTSC
(group 3) i.p. Seven days after tumor injection, groups 4 and
5 received a second injection (i.p.) of MTSC 30 mg/kg or 60
mg/kg, respectively. Thrice a week the weight of the mice was
analyzed to correlate the tumor volume. After 70 days, the
surviving mice were euthanized, and the weight of the body,
heart, kidney, liver, spleen, and thymus was evaluated.
F0
F
¼ 1 þ kqτ0½QŠ ¼ 1 þ KSV ½QŠ
ð2AÞ
ð2BÞ
KSV
τ0
kq ¼
where F0 and F represent the fluorescence intensity values of
the HSA in the absence and presence of the MTSC, respective-
ly, [Q] is the ligand concentration. KSV and kq are the Stern–
Volmer quenching constant and the bimolecular quenching rate
constant, respectively. τ0 is the lifetime of the fluorophore in the
absence of ligand—the measured average value for the fluores-
cence lifetime of HSA was (5.74 0.18) × 10–9 s.
Data from the fluorescence quenching experiments were
used to calculate the modified Stern–Volmer binding constant
(Ka) of HSAwith MTSC according to the method of the mod-
ified Stern–Volmer equation–Eq. (3) [32, 33]:
Statistical analysis
The values are given as mean SD. The IC50 value was
calculated using the sigmoidaldose–response curve-fitting
model using Prism 5 software. Evidence of drug effect is
described by %T/C = [Δ tumor volume of a treated
group]/[Δ tumor volume of control group] × 100%.
Statistical analysis was performed by the Mann–Whitney
U test. Values of p ≤ 0.05 were considered statistically
significant, using Prism 5 software.
F0
F0−F
1
1
¼
þ
ð3Þ
f ½QŠKa
f

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The intensity of the CD signal was expressed as mean
residue ellipticity (MRE), defined according to Eq. (5) [35]:
where F0 and F are the steady-state fluorescence intensities in
the absence and presence of MTSC, respectively; ƒ is the
fraction of the initial fluorescence intensity corresponding to
a fluorophore that is accessible to the quencher (f ≈ 1.00) and
[Q] the MTSC concentration.
θ
MRE ¼
ð5Þ
ð10:n:l:CPÞ
The thermodynamic parameters (ΔH°, enthalpy change,
and ΔS°, entropy change) for the HSA:MTSC interaction
were obtained from the van’t Hoff plot using Eq. (4A) and
the Gibbs free energy change values (ΔG°) were obtained
from Eq. (4B) [34, 35]:
where θ is the observed CD (in millidegree), n is the number
of amino acid residues (585 for HSA) [36], l is the path length
of the cell (in cm), and Cp is the molar concentration of HSA
(1.00 × 10–6 M). The α-helical contents of free HSA and
bound to MTSC were calculated from the MRE values at
208—Eq. (6A)—and 222 nm—Eq. (6B) [31, 37]:
ΔH0 ΔS0
ln Ka ¼ −
þ
ð4AÞ
ð4BÞ
ꢀ
ꢁ
RT
R
ð−MRE208−4000Þ
ð33000−4000Þ
α−helix% ¼
α−helix% ¼
 100
ð6AÞ
ð6BÞ
ΔG0 ¼ ΔH0−TΔS0
ð−MRE222−2340Þ
where T is the temperature (296 K, 303 K, and 310 K) and R is
 100
the gas constant (8.3145 Jmol–1K–1).
30300
Time-resolved fluorescence experiments
Competitive binding studies
Time-resolved fluorescence measurements were per-
formed on spectrofluorimeter model FL920 CD from
Edinburgh Instruments equipped with an EPL laser
(λexc = 280 10 nm; a pulse of 850 ps with an energy
of 1.8 μW/pulse, monitoring emission at 340 nm).
Fluorescence decay for the free HSA solution (1.00 ×
10–5 M in PBS, pH = 7.4) and for an HSA solution
containing the maximum concentration of MTSC used
in the steady-state fluorescence studies (1.32 × 10–5 M)
was obtained at room temperature (ca 298 K).
Competitive binding studies were carried out using the site
probes for Sites I and II, e.g., warfarin and ibuprofen, respec-
tively. HSA and site probes were used at a fixed concentration
(1.00 × 10–5 M), and the fluorescence quenching titration with
MTSC was performed as described previously for the steady-
state fluorescence quenching procedure, at 310 K. For each
data point, the inner filter correction was made as shown in
Eq. (1) and the Ka value in the presence of each site marker
was obtained from Eq. (3).
Molecular docking
Synchronous fluorescence experiments
The crystallographic structure of HSA was obtained from the
Protein Data Bank (PDB) with access code 1N5U [20]. The
MTSC structure was built and energy-minimized by Density
Functional Theory (DFT) calculations (B3LYP potential) with
basis set 6-31G*, available in the Spartan'14 program
(Wavefunction, Inc.).
The molecular docking studies were performed with the
GOLD 5.2 program (CCDC, Cambridge Crystallographic
Data Centre). Hydrogen atoms were added to the protein ac-
cording to the data inferred by the program on the ionization
and tautomeric states. The docking interaction cavity in the
protein was established with a 10 Å radius from the Trp-214
residue (this amino acid residue was chosen for docking ex-
ploration based on the steady-state fluorescence and compet-
itive binding studies described in the BResults and
Discussion^ section) [21]. The number of genetic operations
(crossover, migration, mutation) in each docking run used in
the search procedure was set to 100,000. The program opti-
mizes hydrogen-bond geometries by rotating the hydroxyl and
the amino groups present on the amino acid. The scoring
Synchronous fluorescence (SF) experiments were performed
in an Edinburgh Instruments fluorimeter model Xe900. SF
spectra for HSA (1.00 × 10–5 M) were recorded with increas-
ing concentration of MTSC (0.17, 0.33, 0.50, 0.66, 0.83, 0.99,
1.15, and 1.32 × 10–5 M) by setting Δλ = 60 nm and Δλ =
15 nm for tryptophan and tyrosine residues, respectively, in
the 240–320 nm range, at room temperature (ca 298 K).
Circular dichroism spectra measurements
CD spectra were obtained using a Jasco J-815 spectropo-
larimeter equipped with a Jasco PFD-425S15F
thermostated system with 0.1°C accuracy. HSA concen-
tration was kept constant at 1.00 × 10–6 M and the li-
gand concentration was set as the maximum concentra-
tion used in the steady-state fluorescence studies (1.32 ×
10–5 M). CD spectra were measured in the 200–260 nm
range, at 310 K. All spectra were recorded with appro-
priate background corrections.

Invest New Drugs
function used was BChemPLP,^ which is the default function
of the GOLD 5.2 program. The score of each pose identified
was calculated as the negative of the sum of a series of energy
terms involved in the protein–ligand interaction process, so
that the more positive the score, the better is the interaction.
The graphical representations of the best scores were generat-
ed with the PyMOL program (DeLano Scientific LLC).
Our results demonstrated that treatment of mice with MTSC
or with MTSC–complex did not alter the weight of heart, kid-
neys, liver, or the body weight. In addition, analysis of the
subpopulations of CD4+ and CD8+ T lymphocytes from lymph
nodes and spleen did not result in any difference (data not
shown). However, treatment with MTSC–complex induced a
dangerous increase in glucose levels (345.0 141.8 mg/dL)
when compared with the vehicle (DMSO) or MTSC (117
19.97 and 98.67 3.18 mg/dL, respectively). Therefore, only
the in vivo cytotoxic activity of MTSC was investigated.
Results and Discussion
Evaluation of cytotoxic activity
Effect on cell viability in vitro
In vivo cytotoxic activity of MTSC
To evaluate the cytotoxic activity of MTSC in vivo, two strat-
egies were used: 1) mice were treated with an injection (i.p.) of
MTSC 24 h after tumor inoculation; 2) mice were treated with
two injections with half the dose of MTSC (i.p.) 24 h and 6
days after tumor inoculation.
To investigate the ability of MTSC and its Cu(II) complex to
induce cell death in vitro, the human K562 erythroleukemia
cell lines and Lucena I (Vincristine-resistant derivative K562)
were used. Cells were incubated with different concentrations
of either MTSC or MTSC–complex for 72h. Using the MTT
assay, it was observed that MTSC induced cell death in K562
cells (IC50= 20.5 1.8 μM) and Lucena I (IC50= 12.8 1.0
μM). Similar results were observed with MTSC–complex
treating Lucena I cells (IC50= 9.1 0.2 μM). In other studies,
thiosemicarbazone derivatives have been shown to have anti-
proliferative activity and to induce cell cycle arrest in the G2/
M phase in tumor cells [38, 39]. In addition, the cytotoxic
activity of thiosemicarbazone derivatives can also be related
to the inhibition of topoisomerase IIα [40, 41]. Although we
have not studied the mechanism of action of MTSC, these
already described routes can be responsible for the results
observed by us.
Our results demonstrated that treatment with 30 mg/mL of
MTSC using both strategies significantly altered the body weight
of surviving mice 12 days after tumor inoculation (Figures 2a
and b). The mean weight of the Ehrlich tumor-inoculated and
vehicle-treated mice was 29.08 g. On the other hand, after treat-
ment with a dose of 30 mg/mL of MTSC the body weight of the
mice was 24.92 g, whereas after treatment with two doses of 30
mg/mL each of MTSC was 25.70 g. In Figures 2c and d, it can be
seen that the treatment with MTSC induced the survival of the
mice. Evidence of compound effect is described by the equation
%T/C = [increase in the survival time of treated mice (T)]/[in-
crease in the survival time of vehicle group (C)] × 100. In
Table 2, it can be observed that 30 mg/kg of MTSC exhibited
effective cytotoxic activity with T/C values of 150.49% (1 dose)
and 278% (2 doses). These values for %T/C indicate a significant
cytotoxic activity. Furthermore, after 70 days, the surviving mice
did not exhibit ascites. The mice were then euthanized and the
analysis of both body weight and heart, kidney, liver, spleen, and
thymus was similar to that observed in healthy mice (without
tumor). The in vivo effect of other thiosemicarbazone derivatives
on the development of Ehrlich solid tumor in mice has been
extensively investigated. As an example, 30 mg/kg of
thiophene-thiosemicarbazone derivatives inhibited tumor growth
by approximately 71% [39]. Interestingly, some phase I clinical
trials using thiosemicarbazones have now been performed,
confirming the promising cytotoxic activity of this class of
compound.
Analysis of toxicity of MTSC and MTSC–complex in vivo
Adult BALB/c female mice were treated with 20 mg/kg
of MTSC or MTSC–complex once a week. After 30
days, the weight of the animal, glucose levels, and the
weight of the heart, kidneys, liver, spleen, and thymus
were analyzed (Table 1).
Table 1 In vivo parameters for the vehicle, MTSC, and MTSC–
complex—the compounds did not induce toxicity in vivo
Parameter
Vehiclea
MTSCa
MTSC–complexa
Body weight
Heart weight
22.000 0.700 22.000 0.700 21.500 0.700
Binding characterization between HSA:MTSC
0.111 0.003
0.116 0.005
0.172 0.012
1.161 0.070
0.110 0.003
0.560 0.001
0.111 0.006
0.157 0.003
0.972 0.050
0.109 0.005
0.560 0.003
Kidney weight 0.164 0.007
Some drugs are able to bind to albumin and this interaction
determines tissue drug delivery [42]. Matsumura and Maeda
[43] demonstrated that albumin accumulated within the inter-
stitium of solid tumors, promoting a retention effect. Steady-
state fluorescence quenching is a sensitive technique for
Liver weight
1.108 0.050
0.101 0.005
Spleen weight
Thymus weight 0.500 0.005
a. The values are expressed as Mean Standard Error

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Fig. 2 Cytotoxic activity of MTSC. 2.50 × 105 of Ehrlich ascites
carcinoma cells were injected (i.p.). Mice were treated with one dose
(i.p.) of MTSC (30 or 60 mg/mL) or vehicle (a and b) 24h after tumor
inoculation; Mice were treated with two doses (c and d). 24h after tumor
inoculation mice were treated with vehicle or 15 or 30 mg/mL of MTSC,
and then 6 days animals received the same treatment (15 or 30 mg/mL of
MTSC or vehicle). The efficiency of the ascitic tumor treatment was
determined by the increase in the survival time of the treated mice as
compared with that of the vehicle group. #,*p< 0.05
monitoring drug binding to serum albumin due to its accuracy,
rapidity, and ease of use at low concentrations under physio-
logical conditions [44]. Figure 3a shows HSA fluorescence
quenching by successive additions of the potential drug
MTSC, at 310 K. This quenching process indicates that
MTSC is located next to the intrinsic albumin fluorophore,
i.e., the Trp-214 residue. Furthermore, the emission maxima
and shape of the peaks changed with increasing concentration
of MTSC (blue shift, from 340 (HSA) to 331 nm
(HSA:MTSC), indicating that MTSC may exert some influ-
ence on the polarity of the microenvironment inside the cavity
where the Trp-214 residue can be found [45].
the Stern–Volmer equation and the kq definition—Eqs.
(2A) and (2B) [46]. As listed in Table 3, the Stern–
Volmer quenching constant values (KSV) decrease with
increasing temperature and the bimolecular quenching
rate constant values (kq) are higher than the limiting dif-
fusion rate constant in water (kdiff ≈ 7.40 × 109 M–1s–1,
according to Smoluchowski–Stokes–Einstein theory at
298 K) [47]. This is a clear indication that the main
fluorescence quenching takes place via a static process.
This static mechanism probably originates from a
ground-state association between Trp-214 and the poten-
tial drug MTSC [48].
To obtain a deeper understanding of the fluorescence
quenching mechanism—if static and/or dynamic—the
steady-state fluorescence data were analyzed using both
To confirm further the fluorescence quenching mechanism
involved in the association HSA:MTSC, time-resolved fluo-
rescence spectroscopy measurements were carried out and the
decay profile obtained is depicted in Figure 3b. Fluorescence
lifetimes for free HSA and for HSA:MTSC, (MTSC at the
maximum concentration used in the steady-state fluorescence
studies) did not vary significantly, ranging from τ1 = 1.46
0.16 (19.0%), τ2 = 5.74 0.18 (81.0%) (χ2 = 1.158, for HSA)
to τ1 = 1.43 0.15 (18.5%), τ2 = 5.70 0.17 (81.5%) (χ2 =
1.171, for HSA:MTSC). From these results, it can be seen that
the fluorescence lifetime of free HSA is unaffected by the
presence of the potential drug, which is an indication that
the fluorescence quenching mechanism is static, and implies
a ground-state association between the tryptophan residue in
HSA and MTSC [27, 49].
Table 2 In vivo effects of MTSC against Ehrlich tumor
Treatment
Days after tumor inoculation Dose (mg/kg) %T/C
Vehicle
1
–
100
Vehicle
1and 6
–
100
MTSC 1 dose
MTSC 1 dose
1
1
30
150.49
73.89
278
60
MTSC 2 doses 1and 6
MTSC 2 doses 1and 6
15 and 15
30 and 30
253.5
a. %T/C ≥ 125 for significant cytotoxic activity

Invest New Drugs
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
296 K
303 K
310 K
b
a
1.4
IRF
1.2
1.0
0.8
0.6
0.4
HSA
HSA:MTSC
1010
3.0x10-6
6.0x10-6
9.0x10-6
1.2x10-5
[MTSC] (M)
109
331 nm
340 nm
0.2
0.0
300
320
340
360
380
400
420
440
0
20
40
Wavelength (nm)
Time (ns)
Fig. 3 a Steady-state fluorescence emission spectra for free HSA and its
quenching by MTSC in PBS (pH = 7.4) at 310 K. Inset: Stern–Volmer
plots for the interaction HSA:MTSC at 296 K, 303 K, and 310 K. b Time-
resolved fluorescence decays and its residuals for HSA solution and
HSA:MTSC at room temperature. [HSA] = 1.00 × 10-5 M, [MTSC] =
0.17, 0.33, 0.50, 0.66, 0.83, 0.99, 1.15, and 1.32 × 10-5
M
The evaluation of the modified Stern–Volmer binding con-
stant (Ka) between serum albumin and a ligand is important to
understand its distribution in plasma and organs. A too-weak
binding can lead to a poor distribution of the molecule in the
body, while strong binding decreases the concentration of free
molecules in plasma. Therefore, moderate binding constant
values can be set as the best parameter to achieve the desired
biological activity for many potential drugs [50]. Table 3 lists
the obtained Kavalues for HSA:MTSC, with the correspond-
ing modified Stern–Volmer plots being presented in Figure 4.
The Ka values are in the order of 104 M–1, indicating a mod-
erate interaction between human serum albumin and MTSC
[25, 26, 45]. Clearly, the decreasing trend of Ka values with
temperature increase is consistent with the KSV dependence on
temperature, being in total agreement with the static fluores-
cence quenching mechanism already discussed [48].
Thermodynamic parameters, including enthalpy change
(ΔH°), entropy change (ΔS°), and Gibbs free energy change
(ΔG°) confirm the driving forces of the binding [48]. To elu-
cidate the interaction of HSAwith MTSC, the thermodynamic
parameters ΔH° and ΔS° were calculated from the van’t Hoff
plot (inset in Figure 4). The negative enthalpy change (ΔH°)
and positive entropy change (ΔS°) values (Table 3) are a con-
sequence of the dominant influence of hydrogen bonding and
hydrophobic forces in the interaction process [52].
Consequently, the negative Gibbs free energy change values
(ΔG°, Table 3) characterize the binding as a spontaneous pro-
cess. From the thermodynamic analysis, binding between
HSA:MTSC is entropically and enthalpically driven because
both enthalpy and entropy change values contribute to the
spontaneity of the binding process [18]. The obtained results
are in agreement with those reported in the literature for benz-
aldehyde thiosemicarbazone and some of its fluorinated de-
rivatives [25, 27].
The main noncovalent forces promoting interactions be-
tween ligands and proteins can be attributed to hydrogen
bonds, van der Waals forces, hydrophobic, and electrostatic
interactions [51].
According to Miller’s theory, when the interval between
the excitation and emission wavelengths (Δλ) is set at
Table 3 Stern–Volmer quenching constant (KSV), bimolecular quenching rate constant (kq), modified Stern-Volmer binding constant (Ka), and
thermodynamic parameters (ΔH°, ΔS° and ΔG°) for the interaction HSA:MTSC at 296 K, 303 K, and 310 K
T (K)
K
SV (× 105) (M–1) kq (× 1013) (M–1s–1) r2
Ka (× 104) (M–1) r2
ΔH° (kJmol–1) ΔS° (kJmol–1K–1) ΔG° (kJmol–1) r2
296
303
1.32 0.05
1.22 0.04
2.30
2.13
0.9967 7.11 0.26
0.9967 6.40 0.26
0.9962 5.75 0.26
0.9999 11.6 1.1
0.9999
0.0537 0.0021
–27.5
–27.9
0.9997
310
1.13 0.04
1.97
0.9998
–28.2

Invest New Drugs
results clearly confirm the data obtained from the steady-state
fluorescence quenching, i.e., that the binding could affect the
polarity around the tryptophan residue.
8
7
6
5
4
3
2
11.15
11.10
11.05
11.00
10.95
Circular dichroism (CD) is an excellent tool for rapid de-
termination of the secondary structure of proteins. As depicted
in Figure 6, the CD spectrum of HSA shows two negative
signals: one at 208 nm and other at 222 nm, characteristic of
π → π* and n → π* transitions of carbonyl groups in a
polypeptide chain, respectively [54]. After the addition of
MTSC, the CD spectrum of HSA shows a gradual loss in
intensity at 222 and 208 nm (becomes less negative), which
indicates the loss of α-helix content [55]. The quantitative
estimation of α-helix content suggests a weak perturbation
on the secondary structures of HSA after the addition of
MTSC, from 63.3% to 61.8%, at 208 nm and from 58.3% to
56.9%, at 222 nm. It has been reported that some fluorinated
derivatives of thiosemicarbazone benzaldehyde also do not
significantly alter the secondary structure of HSA [27].
As previously described by Sudlow et al., there are two
main binding sites in human plasma albumin for different
classes of drugs: one is located in subdomain IIA, namely
Sudlow’s site I (warfarin binding site) and the other is located
in subdomain IIIA, namely Sudlow’s site II (ibuprofen bind-
ing site) [56]. To identify the main binding sites for MTSC on
HSA, site marker competitive experiments were carried out.
The modified Stern–Volmer binding constant (Ka) was calcu-
lated in the presence of each site probe (warfarin and ibupro-
fen), at 310 K and the plots are shown in Figure 7. In the
presence of warfarin, Ka values were significantly changed
(60.9%, from 5.75 × 104 to 2.25 × 104 M–1) while the influ-
ence of ibuprofen in the binding parameter was not significant
(7.83%, from 5.75 × 104 to 5.30 × 104 M–1). These results
indicate a competition between warfarin and the potential drug
for the same binding site in HSA [54]. Therefore, MTSC binds
in Sudlow’s site I (subdomain IIA) of HSA, where the Trp-214
-3
-3
-3
-3
-3
3.2x10 3.3x10 3.3x10 3.3x10 3.4x10 3.4x10
-3
-1
1/T (K )
296 K
303 K
310 K
5
5
5
5
5
5
1x10
2x10
3x10
4x10
5x10
6x10
-1
1/[MTSC] (M )
Fig. 4 Modified Stern-Volmer plots for the interaction between
HSA:MTSC (pH = 7.4) at 296 K, 303 K, and 310 K. Inset: van´t Hoff
plot for HSA:MTSC
15 nm or 60 nm, the synchronous fluorescence spectra (SFS)
of serum albumin can offer characteristic information about
the Tyr or Trp residues, respectively [53]. In other words, SFS
can provide information about the molecular environment in
the vicinity of internal chromophores of a protein, by scanning
the excitation and emission monochromators simultaneously,
while maintaining a constant wavelength interval (Δλ) be-
tween them [54]. As can be seen in Figure 5a, the maximum
fluorescence emission wavelength for the Tyr residue shows
no significant shift after successive additions of MTSC, indi-
cating that the microenvironment around the Tyr residue was
not altered by the presence of MTSC [31]. For the Trp residue,
a clear blue shift in the synchronous fluorescence spectrum
can be observed after successive additions of MTSC
(Figure 5b). This behavior probably expresses an increase in
the hydrophobicity surrounding the Trp residue [51]. These
7
a
= 15 nm
b
= 60 nm
1.0x10
6
6
6
6
6
HSA
HSA
5x10
4x10
3x10
2x10
1x10
6
8.0x10
6
6.0x10
HSA:MTSC
HSA:MTSC
6
4.0x10
6
2.0x10
271 nm
278 nm
0
0.0
240
240
260
280
300
320
260
280
300
320
Wavelength (nm)
Wavelength (nm)
Fig. 5 Synchronous fluorescence measurement for HSAwithout and with the presence of MTSC with Δλ = 15 nm for Tyr (a) and Δλ = 60 nm for Trp
(b). [HSA] = 1.00 × 10-5 M, [MTSC] = 0.17, 0.33, 0.50, 0.66, 0.83, 0.99, 1.15, and 1.32 × 10-5 M and pH = 7.4 at room temperature

Invest New Drugs
20
0
HSA
HSA:MTSC
-20
-40
-60
-80
-100
-120
-140
200
210
220
230
240
250
Wavelength (nm)
Fig. 6 Circular dichroism spectra for free HSA and HSA:MTSC, pH =
7.4, at 310 K. [HSA] = 1.00 × 10-6 M and [MTSC] = 1.32 × 10-5
Fig. 8 Best docking pose for HSA:MTSC in Sudlow’s site I, obtained by
GOLD 5.2 program (ChemPLP function). Ligand structure is represented
in beige, the green color is the HSA structure (PDB: 1N5U), the select
amino acids residues are in cyan. Hydrogen: white; oxygen: red; nitrogen:
dark blue and sulfur: yellow
M
residue can be found. This binding site was previously as-
cribed to the main binding site for some thiosemicarbazone
derivatives [27, 57].
Knowing from fluorescence quenching and competitive
binding studies that MTSC binds in Sudlow’s site I, where
the Trp-214 residue can be found, now it is important to know
which amino acid residues are capable of interacting with this
potential drug inside the protein pocket. For this, molecular
docking calculations were carried out, with Figure 8 depicting
the best docking score pose for HSA:MTSC.
Molecular docking results suggest that MTSC can interact
inside Sudlow’s site I via hydrogen bonding and hydrophobic
forces. The thiosemicarbazone group of the ligand interacts by
hydrogen bonding with the amino acid residues Asp-450 and
Ser-453, within a distance of 2.10 Å for both residues. The
oxygen of the methoxyl group in the aromatic ring of the
ligand is a potential acceptor for hydrogen bonding with
Ser-201 residue, within a distance of 3.80 Å. Furthermore,
the hydrophobic moiety of the ligand is able to interact via
hydrophobic forces with the amino acid residues Ala-209,
Phe-210, Trp-214, Leu-346, Leu-480, and Val-481. In sum-
mary, molecular docking indicates that the intermolecular
forces acting on the HSA:MTSC interaction are in agreement
with those pointed out from the thermodynamic parameters
obtained above (ΔH° and ΔS°).
Conclusions
8
Warfarin
Cancer is the second leading cause of death in the world and
until 2030 this group of diseases will be responsible for 17
million deaths (WHO). Furthermore, aging is directly asso-
ciated with cancer incidence. The treatment can represent an
obstacle in cancer therapy. The discovery of novel com-
pounds that cure or prolong the patient’s life considerably
with the quality of life is necessary to change the future. Our
data demonstrated that the novel thiosemicarbazone com-
pound MTSC has cytotoxic activity in vivo. The
chemotherapy-related toxicity is an important problem in
patients’ cancer treatment. Several reports of adverse anti-
cancer drug reactions have been described, of which 50%
were potentially fatal [58]. The treatment with MTSC did
not induce any toxicity in vivo, suggesting that this com-
pound did not cause adverse reactions and it is safe for mice.
Failure of new anticancer drugs is related to poor absorption
and bioavailability. Tumor cells take up albumin as a source
of nitrogen and energy. To improve its cytotoxic effects,
Ibuprofen
7
6
5
4
3
2
5
5
5
5
5
5
1x10
2x10
3x10
4x10
5x10
6x10
-1
1/[MTSC] (M )
Fig. 7 Modified Stern-Volmer plots for interactions HSA:MTSC in the
presence of site markers warfarin and ibuprofen, in PBS, at 310 K. [HSA]
= 1.00 × 10-5 M, [MTSC] = 0.17, 0.33, 0.50, 0.66, 0.83, 0.99, 1.15, and
1.32 × 10-5 M

Invest New Drugs
Publisher’s Note Springer Nature remains neutral with regard to jurisdic-
tional claims in published maps and institutional affiliations.
some drugs, such as Paclitaxel and 5-Fluorouracil [59, 60],
have been employed coupled with HSA. In our results, we
observed that MTSC binds to HSA, suggesting that the ef-
fects observed in vivo depend on this interaction. These
findings corroborated the higher capacity of this class of
compounds to induce tumor cells’ death. Fluorescence
quenching studies showed that the interaction HSA:MTSC
occurs via a ground-state association. Thermodynamic pa-
rameters indicate that this binding is moderate, spontaneous
(ΔG° < 0), entropically, and enthalpically driven. Circular
dichroism and synchronous fluorescence results suggest that
the binding HSA:MTSC does not perturb significantly the
secondary structure of the protein; however, it can change
the microenvironment around the Trp-214 residue.
Competitive binding studies in the presence of warfarin
and ibuprofen show that the main binding site in HSA is
Sudlow’s site I. Finally, molecular docking results suggest
that the potential drug can interact with the amino acid res-
idues Ser-201, Ala-209, Phe-210, Trp-214, Leu-346, Asp-
450, Asp-453, Leu-480, and Val-481 via hydrogen bonding
and hydrophobic forces. The interaction of MTSC and HSA
could provide therapeutic benefits, improving its cytotoxic
efficacy and tolerability.
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Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and
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