Antileishmanial activity evaluation of thiazolidine-2,4-dione against Leishmania infantum and Leishmania braziliensis

Article abstract of DOI:10.1007/s00436-020-06706-3

Leishmaniasis is responsible for approximately 65,000 annual deaths. Despite the mortality data, drugs available for the treatment of patients are insufficient and have moderate therapeutic efficacy in addition to serious adverse effects, which makes the development of new drugs urgent. To achieve this goal, the integration of kinetic and DSF assays against parasitic validated targets, along with phenotypic assays, can help the identification and optimization of bioactive compounds. Pteridine reductase 1 (PTR1), a validated target in Leishmania sp., is responsible for the reduction of folate and biopterin to tetrahydrofolate and tetrahydrobiopterin, respectively, both of which are essential for cell growth. In addition to the in vitro evaluation of 16 thiazolidine-2,4-dione derivatives against Leishmania major PTR1 (LmPTR1), using the differential scanning fluorimetry (ThermoFluor), phenotypic assays were employed to evaluate the compound effect over Leishmania braziliensis (MHOM/BR/75/M2903) and Leishmania infantum (MHOM/BR/74/PP75) promastigotes viability. The ThermoFluor results show that thiazolidine-2,4-dione derivatives have micromolar affinity to the target and equivalent activity on Leishmania cells. 2b is the most potent compound against L. infantum (EC₅₀ = 23.45 ± 4.54 μM), whereas 2a is the most potent against L. braziliensis (EC₅₀ = 44.16 ± 5.77 μM). This result suggests that lipophilic substituents on either—meta and/or—para positions of the benzylidene ring increase the potency against L. infantum. On the other hand, compound 2c (CE₅₀ = 49.22 ± 7.71 μM) presented the highest selectivity index.

Full text of DOI:10.1007/s00436-020-06706-3

Parasitology Research
https://doi.org/10.1007/s00436-020-06706-3
PROTOZOOLOGY - ORIGINAL PAPER
Antileishmanial activity evaluation of thiazolidine-2,4-dione
against Leishmania infantum and Leishmania braziliensis
Flávio Simas Moreira Neri1 & David Bacelar Costa Júnior1 & Thamires Quadros Froes2
&
Priscila Brandão Gomes da Silva3 & Micalyne Soares do Egito3 & Paulo Otávio Lourenço Moreira4
&
Fernando de Pilla Varotti4 & Marcelo Santos Castilho2 & Rafael Gonçalves Teixeira-Neto5
Jullianna Ferreira Cavalcanti de Albuquerque3 & Franco Henrique Andrade Leite1
&
Received: 20 December 2019 /Accepted: 5 May 2020
#
Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract
Leishmaniasis is responsible for approximately 65,000 annual deaths. Despite the mortality data, drugs available for the treatment of
patients are insufficient and have moderate therapeutic efficacy in addition to serious adverse effects, which makes the development
of new drugs urgent. To achieve this goal, the integration of kinetic and DSF assays against parasitic validated targets, along with
phenotypic assays, can help the identification and optimization of bioactive compounds. Pteridine reductase 1 (PTR1), a validated
target in Leishmania sp., is responsible for the reduction of folate and biopterin to tetrahydrofolate and tetrahydrobiopterin,
respectively, both of which are essential for cell growth. In addition to the in vitro evaluation of 16 thiazolidine-2,4-dione derivatives
against Leishmania major PTR1 (LmPTR1), using the differential scanning fluorimetry (ThermoFluor®), phenotypic assays were
employed to evaluate the compound effect over Leishmania braziliensis (MHOM/BR/75/M2903) and Leishmania infantum
(MHOM/BR/74/PP75) promastigotes viability. The ThermoFluor® results show that thiazolidine-2,4-dione derivatives have mi-
cromolar affinity to the target and equivalent activity on Leishmania cells. 2b is the most potent compound against L. infantum
(EC50 = 23.45 4.54 μM), whereas 2a is the most potent against L. braziliensis (EC50 = 44.16 5.77 μM). This result suggests that
lipophilic substituents on either—meta and/or—para positions of the benzylidene ring increase the potency against L. infantum. On
the other hand, compound 2c (CE50 = 49.22 7.71 μM) presented the highest selectivity index.
.
.
.
.
.
Keywords Dissociation constant Phenotypic assay Leishmania braziliensis Leishmania infantum Leishmania major
.
.
Pteridine reductase 1 ThermoFluor® Thiazolidine-2,4-dione
Introduction
Leishmaniasis is an infectious disease caused by protozoa of the
genus Leishmania sp., which is endemic in more than 98 coun-
tries, distributed throughout Africa, Asia, and Latin America
(WHO 2017; 2019). According to World Health Organization
(WHO 2019), the incidence of new cases of leishmaniasis per
year is estimated at 700,000 to 1 million, and up to 65 thousand
deaths are caused by the different clinical forms (WHO 2019).
Despite the public health impact, the therapeutic arsenal to
fight leishmaniasis is limited, what underscores the need for
new alternatives to treat the patients infected with different spe-
cies of Leishmania (Gontijo and Carvalho 2003; Swinney and
Anthony 2011; Alvar and Arana 2018). One way to achieve this
goal is through the inhibition of targets that are unique for the
parasite (Eggert 2013). Leishmania genus is auxotrophic for free
or conjugated pteridines, such as folic acid and biopterin, which
Section Editor: Ramaswamy Kalyanasundaram
* Franco Henrique Andrade Leite
fhenrique@uefs.br
1
Departamento de Saúde, Universidade Estadual de Feira de Santana,
Feira de Santana, Brazil
2
Faculdade de Farmácia, Universidade Federal da Bahia,
Salvador, Brazil
3
Departamento de Antibióticos, Universidade Federal de
Pernambuco, Recife, Brazil
4
Laboratório de Bioquímica Medicinal, Universidade Federal de São
João del Rei, Campus Centro-Oeste, Divinópolis, Brazil
5
Laboratório de Doenças Parasitárias e Infecciosas, Universidade
Federal de São João del Rei, Campus Centro-Oeste,
Divinópolis, Brazil

Parasitol Res
are cofactors in the process of DNA synthesis only in their fully
reduced forms (tetrahydrofolate and tetrahydrobiopterin, respec-
tively) (Nare et al. 2009; Leprohon et al. 2015). As a conse-
quence, these parasites developed a complex and versatile bio-
chemical pathway capable of using conjugated and unconjugated
pteridines from the host (Nare et al. 1997; Gourley et al. 1999).
In Leishmania sp., the preferred route to obtain tetrahydrofo-
late (H4F) is through the action of the enzyme dihydrofolate
reductase-thymidylate synthase (DHFR-TS; E. C 1.5.1.3/
2.1.1.45), which is considered a validated target for the discovery
of new drugs. However, the use of classic antifolates such as
methotrexate and trimethoprim is ineffective for the treatment
of patients with leishmaniasis. The therapeutic failure is associ-
ated with the presence of pteridine reductase 1 (PTR1; EC
1.5.1.33) (Nare et al. 1997). Gene-silencing assays showed that
PTR1 is essential for the growth and development of the parasite
(Cavazzuti et al. 2008). The selective inhibition of PTR1 is suf-
ficient to kill the parasite, what suggests this is a promising target
for the development of new antileishmanial drugs (Vickers and
Beverley 2011). Leite et al. (2016) showed that thiazolidine-2,4-
dione derivatives are micromolar inhibitors of Leishmania major
PTR1 that display competitive behavior with the enzyme cofac-
tor (NADPH) (Fig. 1). In order to improve the compound’s
biological profile, 16 novel thiazolidine-2,4-dione derivatives
were designed, synthesized, and evaluated against L. major
PTR1. This most active compound (4 Kd = 18.56 μM) has a
1.3-fold-higher affinity for LmPTR1 than our previous hit (2d
Kd = 25.35 μM). In addition, cell-based assays suggest that high/
moderate selectivity (Leishmania braziliensis (MHOM/BR/75/
M2903) and Leishmania infantum (MHOM/BR/74/PP75) ver-
sus WI-26VA4 fibroblast cells) can be achieved, depending on
the substituents decorating the benzylidene ring.
coupling constants (J) were reported in Hertz. Mass spectra
were recorded on a Varian MAT 711 spectrometer 70-eV
electron impact. Thin-layer chromatography (TLC) was per-
formed on pre-coated silica plates (Merck Kiesegel 60 F254),
and the spots were visualized under ultravioleta light (254 nm/
long (365 nm) UV wavelength). The chemical reagents were
supplied by Sigma-Aldrich (USA) and were used without fur-
ther purification. Purity of the compounds was confirmed by
using TLC (Kiesel gel 60 G F 254) using the appropriated
system for each compound.
Procedure for preparation synthesis of thiazolidine-2,4-dione
The thiazolidine-2,4-dione was obtained by using the method
described by Libermann in 1948 and modified by
Albuquerque in 1995 (Albuquerque et al. 1995; Libermann
et al. 1948; Gouveia et al. 2009). This reaction occurs by
condensation of monochloroacetic acid and thiourea in an
aqueous medium under reflux for 24 h (Bozdag-Dündar
et al. 2007; Bruno et al. 2002) (Fig. 2). Molecular formula
C3H3O2NS; yield 78%; mp 118–120 °C; Rf 0.48 (0.9:0.1
CHCl3/MeOH). Recrystallization: water.
General method for the synthesis
of 5-arylidene-thiazolidine-2,4-diones (1–16)
The compounds of 1–16, in turn, have the description of the
synthesis below.
General method for the synthesis of thiazolidine-2,4-dione
The compounds (1–16) were synthesized in two steps. In step
1, the thiazolidine 2,4-dione compound was synthesized using
monochloric acetic acid and thiourea. The solvent used was
water. The reagents were heated at 100 °C for 16 h. The
compounds 2a-s was prepared by using a simple method,
which involve the Knoevenagel condensation with different
aromatic aldehydes. The structures of the desired compounds
Materials and methods
Chemistry
1
Experiment
were determined by H NMR, 13C NMR, IR, and mass
spectrometry.
All melting points were measured in a capillary tube on a
Buchi apparatus. Infrared spectra (1% KBr, cm−1) pellets were
recorded on a Bruker IFS66 spectrophotometer and are uncor-
rected. The 1H NMR and 13C NMR spectra were recorded on
General method for the synthesis
of 5-arylidene-thiazolidine-2,4-dione (1–16)
1
a VARIAN VNMRS 400-MR, using 400 MHz for H and
The compounds 1–16 and compounds 2a–2e were synthe-
sized from a mixture of thiazolidine-2,4-dione (1—Fig. 2)
(2.5 g, 21.36 mmol), aldehyde (21.36 mmol), piperidine
(14.11 mmol), and ethanol (150 mL). The reaction mixture
was heated under reflux and continuously stirred for a period
of 16 h. The course of the reaction was monitored by using
TLC. The reaction mixture was poured into water and acidi-
fied with acetic acid. The resulting precipitate was filtered off
and recrystallized from acetic acid to give (2—Fig. 2).
75.4 MHz in DMSO-dδ, using tetramethylsilane (TMS) as
the internal standard. Chemical shift values are reported in
parts per million units. The 13C NMR in CDCl3 and DMSO
was maintained at 25 °C using Me4Si (TMS) as an internal
standard. The following abbreviations were used to indicate
the peak multiplicity: s (singlet), d (doublet), dd (double dou-
blet), ddd (double doublet, doublet), t (triplet), and m (multi-
plet). The chemical shifts were reported in δ units, and the

Parasitol Res
Fig. 1 Inhibition profile of 2d
against LmPTR1. Kinetic
experiments were conducted in
the presence of increasing
concentrations of the inhibitor:
● = 0 μM; Δ = 25 μM; ☐ =
45 μM. a [Biopterin] = 40 μM. b
[NADPH] = 40 μM
(Z)-5-(3-methoxy-4-hydroxy-benzilydene)-thiazolidine-2,4-
dione (1) Yield 70%; mp 260 °C; Rf 0.55(CH2Cl2/MeOH 9:1)
Recrystallization: ethanol; IR (KBr 1%, νmax cm−1) 1566
(imide)); 8. 57 (d 1H C-H), 7.81 (dd 2H, H4, J = 7.3), 7.77
(dd 1H, H5) J = 7.6; J = 1.5, 8.13 d,1H J = 7.6 (H6). HRMS+,
calcd. 250.0048; found 250.0049.
1
(C=C); 1730–1670 (C=O). H NMR (CDCl3, 300 MHz, δ
ppm): 8.48 (s 1H, NH); 7.71 (s 1H, CH=); 3.77 (s 3H,
OCH3); 6.02 (s 1H, OH). 6.92 (s 1H(2)); 6.86 (d 1H(5) J =
8.33); 6.91 (d 1H(6) J = 8.34); 13C NMR (Acetone-d6,
75.4 MHz, δ ppm): 169.81 (C=O2); 166.89 (C=O4); 119.55
(C5, heterocycle); 143.28 (CH=); 56.50 (OCH3); 129.20 (C1);
112.58 (C2), 148.06 (C3); 144.76 (C4); 115.56 (C5); 122.30
(C6). Anal. Calcd. for C11H9NO4S: C, (52.58%); H, (3.61%);
N, (5.57%); Found: C, 53.01%; H, 3.83%; N, 5.25%.
HRMS+: calcd 251.0252; found 251.0253.
(Z) 5-(2,3,5-Trichloro-benzylidene)-thiazolidine-2,4-dione (4)
Yield 73%; MP 170.5–171.0 °C; MF C10H4Cl3NO2S; MW
308.5683. Rf 0.50 (CHCl3/MeOH 9:1) Recrystallization: eth-
anol, IR (KBr 1%, νmax cm−1) 1569 (C=C); 1738–1667
1
(C=O). HNMR (CDCl3, 300 MHz, δ ppm): 11.97 (s 1H,
NH (imide)); 8. 35 (s 1H C=H), 7.55 (s 1H, (H4). 7.23 (s
1H, H6) HRMS+, calcd. 306.9028; found 306.9028.
(Z)-5-(3-methoxybenzylidene) thiazolidine-2,4-dione (5) Yield
83%; MP 170–171 °C; MF C11H9NO3S; MW 235.2591; Rf
0.50 ((Hex: Ethyl acetate 5:5) Recrystallization: ethanol, IR
(KBr 1%, νmax cm−1) 1564 (C=C); 1733–1665 (C=O).
1HNMR (CDCl3, 300 MHz, δ ppm): 11.87 (s 1H, NH (im-
ide)); 7.87 (s 1H C=H), 7.19 (d 1H, H2–4, J = 7.5), 3.83 (s 3H
OCH3) 7.56 (t 1H, H5) J = 7.6; J = 1.5), 6.90.(d,1H J = 7.6
(H6). HRMS+, calcd. 235.0303; found 235.0303.
(Z) 5-(2,4-dimethoxy-arylidene)-thiazolidine-2,4-dione (2)
Yield 67%; MP 250 °C; MF C12H11NO4S; MW 265.2850;
Rf 0.50 (CHCl3/MeOH 9:1 Recrystallization: ethanol; IR
1
(KBr 1%, νmax cm−1) 1571 (C=C); 1733–1691 (C=O). H
NMR (CDCl3, 300 MHz, δ ppm): 11.30 (s 1H, NH); 8.25 (s
1H, CH=); 3.86 (s 3H, OCH3 (orto); 6.57 (s 1H, H3); 3.87 (s
3H, OCH3 para); 6.0 (d 1H(6) J = 8.34); 11.40 (s 1H, NH, (im-
ide). HRMS+, calcd 250.0048; found 265.0409.
(Z) 5-(3-hydroxy-benzylidene)-thiazolidine-2,4-dione (6)
Yield 69%; MP 265–266 °C; MF C10H7NO3S; MW
221.2025; Rf 0.50 ((Hex: Ethyl acetate 5:5)
Recrystallization: ethanol, IR (KBr 1%, νmax cm−1) 1568
(Z) 5-(2-nitro-benzylidene)-thiazolidine-2,4-dione (3) Yield
83%; MP 205–206 °C; MF C10H6N2O4S; MW 250.2306;
Rf 0.50 (Hex: Ethyl acetate 9:1) Recrystallization: ethanol,
IR (KBr 1%, νmax cm−1) 1569 (C=C); 1736–1660 (C=O).
1HNMR (CDCl3, 300 MHz, δ ppm): 11.97 (s 1H, NH
1
(C=C); 1738–1669 (C=O). HNMR (CDCl3, 300 MHz, δ
ppm): 12.31 (s 1H, NH (imide)); 7.97 (s 1H C=H), 6.72 (d
1H, H2, J = 7.48), 5.37 (s 1H OH) 6.86 (d 1H, H4) 7.56.(s 1 H)
Fig. 2 Synthetic route of
thiazolidine-2,4-dione
compounds

Parasitol Res
7.56; J = 1.5), 7.19 (d,1H, J = 7.16; (H6). (d,1H) HRMS+,
calcd. 235.0303; found 235.0303.
(Z)5-((6-methylene-4-oxo-4H-chromen-3-yl) methylene))
thiazolidine-2,4-dione (12) Yield 72%; MP 287.5- C; MF
0
C14H9NO4S; MW 287.2906. Rf 0.5 ((Hex: Ethyl acetate
6:4) Recrystallization: ethanol, IR (KBr 1%, νmax cm−1)
1559 (C=C); 1730–1666 (C=O). 1HNMR (CDCl3,
300 MHz, δ ppm): 12.29 (s 1H, NH (imide)); 7.43 (s 1H
C=H); 6.71 (s 1H (H1)) 7.63 (d 1H (H2)) 7.67 (d 1H (H3),
Ar) J = 7.47.2.44 (s 3H, (CH3) 7.41 (s 1H), HRMS+, calcd.
289.0409; found 289. 0409.
(Z) 5-(3,4-dimethoxy-benzylidene)-thiazolidine-2,4-dione (7)
Yield 73%; MP 213–214 °C; MF C12H11NO4S; MW 265.
0409; Rf 0.46 ((Hex: Ethyl acetate 7:3) Recrystallization: etha-
nol, IR (KBr 1%, νmax cm−1) 1568 (C=C); 1735–1668 (C=O).
1HNMR (CDCl3, 300 MHz, δ ppm): 12.31 (s 1H, NH (imide));
7.95 (s 1H C=H), 7.24 (s 1H, H2), 3.84 (s 3H OCH3) 3.86 (s 3H,
OCH3) 6.96 (d,1H, J = 7.7; (H5). 7.21 (d,1H, J = 7.6; (H6).
HRMS+, calcd. 265.0409; found 265.0409.
(Z)-5-((4-nitro-1H-indol-3-yl)methylene)thiazolidine-2,4-dione
(13) Yield 57%; MP 129.5–131.0 0C; MF C12H7N3O4S;
MW 289.2667; Rf 0.54 (Dichloro metane: Methanol
9.9:0.1) Recrystallization: ethanol, IR (KBr 1%, νmax cm-1)
1560 (C=C); 1729–1657 (C=O). 1HNMR (CDCl3, 300 MHz,
δ ppm): 12.29 (s 1H, NH (imide)); 7.21 (s 1H C-H); 7.95 (t 2H
(H2) J = 7.63, J = 1.4 J = 1.6). 7.23 (d 1H (H3) J = 7.47); 9.9 (d
1H J = 7.80 (NH), 8.81 (d 1H, J = 7.79). HRMS+, calcd.
289.0157; found 289.0156.
(Z) 5-(4-methylthio)-benzylidene)-thiazolidine-2,4-dione (8)
Yield 63%; MP 209–210 °C; MF C11H9NO2S2; MW
251.3247–251.3251. Rf 0.65 (Hex: Ethyl acetate 6:4)
Recrystallization: ethanol, IR (KBr 1%, νmax cm−1) 1571
1
(C=C); 1735–1668 (C=O). HNMR (CDCl3, 300 MHz, δ
ppm): 12.31 (s 1H, NH (imide)); 7.95 (s 1H C=H), 7.24 (s
1H, H2), 3.84 (s 3H OCH3) 3.86 (s 3H, OCH3) 6.96 (d,1H, J =
7.7; (H5). 7.21 (d,1H, J = 7.6; (H6). HRMS+, calcd. 265.0409;
found 265.0409.
(Z)-5-(quinolin-4-ylmethylene)-thiazolidine-2,4-dione (14)
Yield 83%; MP 273–274 °C; MF C14H10N2O2S; MW
270.3036. Rf 0.50 ((Hexane Ethyl acetate 9:1)
Recrystallization: ethanol, IR (KBr 1%, νmax cm−1) 1559
(C=C); 1733–1661 (C=O). 1HNMR (CDCl3, 300 MHz, δ
ppm): 12.26 (s 1H, NH (imide)); 7.82 (s 1H C-H); 6.73 (t
2H (H2) J = 7.60, J = 1.5 J = 1.5). 7.76 (t 1H (H3) J =
7.45;1.5,1.5) 8.12 (d 1H (H4), 8.75 (d 1H, J = 7.47, J = 1.5)
7.12 (d 1H, J = 7.3, J = 1.5 HRMS+, calcd. 256.0306; found
256.0306.
(Z) 5-(2-Chloro benzylidene)-thiazolidine-2,4-dione (9) Yield
70%; MP 165.9–166.5 °C; MF C10H6ClNO2S; MW
239.6781. Rf 0.50 (Hex: Ethyl acetate 6:4) Recrystallization:
ethanol, IR (KBr 1%, νmax cm−1) 1568 (C=C); 1733–1667
(C=O). 1HNMR (CDCl3, 300 MHz, δ ppm): 12.29 (s 1H, NH
(imide)); 8.23 (s 1H C=H); 7.54 (d 1H, H3 J = 7.5; 1.4), 7.05 (t
3H (H4) J = 7.4; 1.5) H5 7.25 (t 2H, (H5) J = 7.4; 1.5) 7.28
(d,1H, J = 7.7) (H5). 7.28 (d,1H, J = 7.6; (H6). HRMS+, calcd.
238.9808.; found 238.9808.
(Z)-5-((5-Bromothiophen-2-ylmethylene)-thiazolidine-2,4-
dione (15) Yield 83%; MP 180.3–180.7 °C; MF
C8H4BrNO2S2; MW 290.1490. Rf 0.50 (CHCl3/MeOH 9:1)
Recrystallization: ethanol, IR (KBr 1%, νmax cm−1) 1558
(C=C); 1735–1662 (C=O). 1HNMR (CDCl3, 300 MHz, δ
ppm): 12.25 (s 1H, NH (imide)); 7.55 (s 1H C-H); 7.55 (d
1H (H1, C=H J = 7.50); 7.24 (d 2H (H2,-H2´ J = 7.46; J = 1.45;
J = 1.45) 7.45 (d d H3-H5 CH) J = 7.24. HRMS+, calcd.
288.01878; found 288.01878.
(Z) 5-([1,1′-biphenyl]-4-ylmethylene) thiazolidine-2,4-dione
(10) Yield 78%; MP 238.5–239.8 °C; MF C16H11NO2S;
MW 281.0510. Rf 0.53 (CH2Cl2/Acetone (0.53)
Recrystallization: ethanol, IR (KBr 1%, νmax cm−1) 1567
1
(C=C); 1731–1665 (C=O). HNMR (CDCl3, 300 MHz, δ
ppm): 12.29 (s 1H, NH (imide); 7.99 (s 1H C=H); 7.56 (d
1H, H2-H2’;J = 7.5; 1.4), 7.74. (d 2H (H3-H3’) J = 7.4; 1.5)
7.40 (t 2H (H6-H6’) J = 7.5–1.50; 1.50. (t 2H, (H7−H7’) J =
7.6; 1.5; 1.5). 7.40 (t 2H, (H6−H6’) J = 7.6; 1.5; 1.5).
HRMS+, calcd. 281.0510.; found 281.0510.
(Z)-5-((E)-2-methyl-phenylallylidene (thiazolidine-2,4-dione)
(16) Yield 79%; MP 204.6–205.2 °C; MF C13H11NO2S;
MW 245–246. Rf 0.49 ((Hexane: Ethyl acetate 9.9:0.1)
Recrystallization: ethanol, IR (KBr 1%, νmax cm−1); 1555
(Z) 5-((1H-indol-3-yl) methylene) thiazolidine-2,4-dione (11)
Yield 78%; MP 165.9–166.5 °C; MF C12H8N2O2S; MW
239.6781. Rf 0.50 (Hex: Ethyl acetate 6:4) Recrystallization:
ethanol, IR (KBr 1%, νmax cm−1) 1567 (C=C); 1733–1667
(C=O). 1HNMR (CDCl3, 300 MHz, δ ppm): 12.29 (s 1H, NH
(imide)); 8.11 (s 1H C=H); 7.76 (d 1H, H2 indol J = 7.5; 1.4);
12.0 (d 1H (NH)) 7.05 (d 1H (H1, Ar) J = 7.4; 1.5); 7.11 (t 2H,
(H2) J = 7.4; 1.5; 1.5) 7.18 (t, 2H, (H3). J = 7.7; 1.5, 1.5)) 7.57
(d, 1H, J = 7.6, H6). HRMS+, calcd. 244.0306; found
244.0306.
1
(C=C); 1733–1665 (C=O). HNMR (CDCl3, 300 MHz, δ
ppm): 12.27 (s 1H, NH (imide)); 7.41 (C=C; 6.37 (s
1H C-H); 2.21 (s 3H CH3); 6.34 (s 1H CH) 7.61 (d
2H (H2,-H2`) J = 7.46; J = 1.45; J = 1.45) 7.45 (d 2H
(H3-H5´) CH) J = 1.45; J = 1.45) 7.33 (t 2H. HRMS+,
calcd. 245.0510 found 245.0510.
The 5-arylidene thiazolidine-2,4-diones compounds (2a–e)
were synthesized as described in Leite et al. 2016.

Parasitol Res
In vitro assays
Determination of the dissociation constant by
ThermoFluor®
ThermoFluor® assays
Compounds that show a concentration-response behavior, in
the previous step, had their dissociation constants (Kd) deter-
mined according to the equation established by Vivoli et al.
(2014), using data from ThermoFluor assays carried out in the
presence of the following concentrations of each compound:
6.25, 12.5, 25, 50, 100.0, 200.0, and 400.0 μM. The five
compounds that presented the best ΔTm values in the study
by Leite et al. (2016) also had their dissociation constants
determined, following the same concentrations and previous
parameters of ThermoFluor® assays. The assays were per-
formed in triplicate.
Thermal shift assays were carried out using a CFX Touch RT-
PCR Detection System (Bio-Rad CFX Manager®, Bio-Rad
Laboratories, Inc.) fitted with custom filter sets. The data was
recorded in the CFX Manager™ v 3.1 Software. Initially,
conditions of the assay (choice of buffer, protein concentra-
tion, and DMSO concentration) were optimized for LmPTR1.
Each parameter was tested in triplicate on a 96-well PCR plate
(PCR plates 96-well Bio-Rad®), manually sealed with trans-
parent capping strips (Flatcap strips Bio-Rad®). The final
protein concentration, for optimization trials, was set to
5 μM, except for the determination of optimum PTR1 con-
centration, which varied from 1 to 5 μM. Fluorescence chang-
es were monitored with SYPRO Orange® dye, using 492- and
610-nm wavelengths for excitation and emission respectively.
Thermal shift assays were carried out from 25 to 85 °C in
increments of 1 °C per minute. Fluorescence data (raw data)
was recorded using the CFX Manager™ v 3.1 Software, and
then exported to Excel 2007 worksheet (ftp://ftp.sgc.ox.ac.uk/
pub/biophysics) for processing and analysis. Tm values
employed for compound comparison were those calculated
from the non-linear fitted melting curves (Boltzmann sigmoi-
dal function) implemented in GraphPad Prism version 5.0 for
Windows (GraphPad® Software, San Diego, CA, USA,
www.graphpad.com).
Cellular assays
Cultivation of cell lines
The human cell line WI-26VA4 (pulmonary fibroblast ATCC
CCL-75) was used for toxicity analysis. This lineage is part of
the animal cell bank of the Cell Biology Service (SBC) of the
Ezequiel Dias Foundation (FUNED) in Belo Horizonte -
Minas Gerais.
Cells were cultured from cryopreserved ampoule; they
were thawed at 37 °C; their contents were transferred to a
15-mL tube containing 10-mL RPMI 1640 medium. Cells
were centrifuged at 1200×g for 5 min; the supernatant was
discarded, and the pellet was resuspended in RPMI 1640 me-
dium supplemented with 10% heat-inactivated fetal bovine
serum (SBF) (complete medium). The cells were transferred
to plastic bottles of treated cell culture to promote cell adhe-
sion, T75 (75 cm2) 10 mL (Corning Costar Inc., USA), and
maintained as monolayers at 37 °C in a greenhouse (Thermo
electron co.) humid atmosphere of 5% CO2. The culture me-
dium was replaced every 48 h of incubation. Cellular mor-
phology and monolayer formation were observed with the
aid of an inverted microscope at × 100 magnification
(Olympus model, CKX 41). After confluence of 80% of the
culture in the T75 bottle, the cells were replicated or used in
the cytotoxicity assays (Patra et al. 2011; Pereira et al. 2012).
Single-concentration assays
The PTR1 expression and purification steps were carried out
as described previously (Leite et al. 2016). All compounds
were assayed at the final concentration of 50 μM. In each well,
a solution containing 4 μL of LmPTR1 (to obtain the final
concentration, 5 μM), 14 μL of 20-mM sodium acetate buffer
pH 4.7, 1-μL SYPRO Orange (Invitrogen™) (5X), and 1-μL
of each compound was added. The values of Tm obtained in
the presence of each compound were compared with the value
of the reference solution, which contains 5% DMSO P.A.
(v/v), instead of the inhibitor. The assays were performed in
triplicate.
Cytotoxicity assays
Cells with 80% confluency were trypsinized with 1.5 mL of
trypsin (1:250 Sigma), incubated at 37 °C for 5 min, resus-
pended in 10 mL of complete RPMI medium, and centrifuged
at 1200×g for 5 min. The supernatant was discarded, and the
pellet was resuspended with complete RPMI medium. Cells
were distributed into 96-well microplates at a density of 4 ×
105 cells/100 μL per well and incubated in a CO2 oven at
37 °C for 24 h for adhesion to the plate. After this period of
adhesion, the medium was removed, a new RPMI medium
Concentration-response assays
Compounds with ΔTm > 1.0 °C, in single-concentration as-
says, also had their effect evaluated at the following concen-
trations: 6.25, 12.5, 25, 50, and 100 μM. The obtained Tm
values were compared with the value of the reference solution,
which contains 5% DMSO P.A. (v/v), instead of the inhibitor.
All assays were carried out in triplicate.

Parasitol Res
supplemented with 1% FBS was added, and then the diluted
compounds were added in RPMI medium containing different
concentrations (0.01, 0.10, 1.0, 10, and 100 μg/mL). The
plates were incubated for an additional 48 h.
50, and 100 μg/mL in triplicate for the compounds and 50 μg/
mL for amphotericin B deoxycholate) and 180 μL of parasite
at the concentration of 1.11 × 106 parasites/mL for each well
in 96-well microplates. From this, the incubation was incubat-
ed at 26 °C for 48 h.
At the end of the treatment period, 100 μL/well of a solu-
tion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole
(MTT) (Sigma-Aldrich) bromide, tetrazole salt, concentration
of 0.5 mg/mL in RPMI 1640 without phenol red (Denizot and
Lang 1986) were add in the plate. After 3 h of incubation in
the CO2 incubator at 37 °C in the dark with MTT, the super-
natant was aspirated and the formazan crystals were dissolved
in 50 μL/well of DMSO P.A. and held at 37 °C for 10 min.
The absorbance per well was measured at a wavelength of
550 nm using the Gen5 software (Data-Analysis Software-
Bio-Tek). The assays were performed in triplicate.
At the end of the incubation period, the MTT was diluted in
incomplete Schneider’s medium (without fetal bovine serum),
so that it was at a concentration of 5 mg/mL. From this mo-
ment, the experiments were performed with the laminar flow
hood light off, since the MTT is photosensitive. A volume of
20-μL MTT was added to the wells. The plates were then
wrapped in aluminum foil and incubated for 4 h at 37 °C.
The plates are maintained at this temperature, as the com-
pounds have already acted, the temperature no longer affects
the viability of Leishmania protozoan, and some proliferative
capacity is diminished. It is also worth noting that in low
temperatures, the production of formazan salts is lower.
After 4 h of incubation with MTT, the plates were centri-
fuged at 3500×g for 10 min at 4 °C in the plate centrifuge. At
the end of centrifugation, the supernatant was carefully re-
moved to preserve the formazan crystals and parasites,
50 μL/well of DMSO P.A. was added to dissolve the
formazan, and the plates were homogenized and allowed to
stand for 15 min for reading in ELISA reader at 570 nm using
the Gen5 software (Data-Analysis Software-Bio-Tek). The
assays were performed in triplicate.
Cultivation of Leishmania sp.
The parasite cultures were carried out with reference strains of
Leishmania (V.) braziliensis (MHOM/BR/75/M2903) and
Leishmania (L.) infantum (MHOM/BR/74/PP75) belonging
to the Laboratory of Parasitic Infectious Diseases of the
UFSJ/CCO (LAB-DIP/CCO).
The parasites were cultured in biphasic medium, the liquid
phase being composed of Schneider’s Insect Medium with L-
glutamine and sodium bicarbonate (Sigma®), plus 10% Fetal
Bovine Serum (FBS) and 1% antibiotic Pen Strep-GIBCO
(10,000 units/mL penicillin and 10,000-μg/mL streptomycin),
and maintained in solid phase with NNN culture medium
(Novy, McNel, and Nicolle) plus 1% antibiotic Pen Strep for
storage. The parasites were kept in bottles for cell culture in
polystyrene, growth volume of 25 cm3, sterile, and in BOD
greenhouse at 25 °C. For the maintenance of the parasites,
peaks were performed every 4 days. All procedures involving
the maintenance and cultivation of the strains were carried out
in a laminar flow hood, avoiding any type of contamination by
microorganisms. Follow-up of the cultures was carried out by
observation in a ZEISS optical microscope, AXIOSTAR
PLUS model, by the preparation of crop slides, in order to
observe flagellar mobility and absence of contamination.
Calculation of the selectivity index
After determination of the effective concentration to kill 50%
of the parasites (CE50) and determination of the lethal dose
capable of killing 50% of human cells (LD50), the selectivity
index (SI) was calculated according to the equation:
SI ¼ LD50=CE50:
Results and discussion
Synthesis and characterization of thiazolidine-2,4-
dione
Antileishmania activity assays
Antileishmania activity assays were performed by the same
cytotoxicity test, i.e., the MTT chemosensitivity test. After
48 h of boiling, to allow the parasites to grow, that is, in the
log phase (ideal phase for testing the compounds), the viability
of the parasites was evaluated by microscopy. Aliquots of the
viable parasites were removed from the growth bottles, and
the ideal volume was calculated for a final concentration of
1.0 × 106 parasites/well. The reaction volume per well was
200 μL, with 20 μL corresponding to the compounds to be
tested (at the concentrations of 1.5625, 3.123, 6.25, 12.5, 25,
Leite et al. (2016) showed that thiazolidine-2,4-dione deriva-
tives inhibit LmPTR1 at micromolar range concentration.
Compound 2d, the most promising compound identified in
that study, was used as a prototype to design novel com-
pounds with optimized effect against Leishmania cells
in vitro. A total of 16 compounds have been synthetized based
on features of the prototype and their structural variations:
compounds 1 to 10 (except for 2, 3, and 6) were synthetized
based on chemical viability, while 11–15 were designed to
explore additional LmPTR1 pockets nearby the active site.

Parasitol Res
Compounds 2, 3, and 9 were designed to the effect of bulk
substituents (steric clashes) on binding affinity, whereas com-
pound 16 aimed at investigating the effect of a spacer moiety,
between the two rings on the binding profile.
show that compound 12 shows negative thermal displace-
ment. Some studies have shown that compounds with this
profile can stabilize the target with whom they interact in a
conformation other the native one. However, a direct relation-
ship between negative values of thermal shift and biological
profile has not been described (Waldron and Murphy 2003;
Dupeux et al. 2011; Garbett and Chaires 2012; Sancho 2013;
Contessoto et al. 2018).
Thus, only molecules 4 and 9 were evaluated at different
concentrations (6.25, 12.5, 25, 50, and 100 μM) to probe if the
thermal stabilization follows a dose-dependent behavior. Both
compounds were found to increase LmPTR1 thermal stability
at higher concentrations, with ΔTm values for compound 4
higher than those seen for compound 6 (Graph 2). For com-
pound 4, a significant increase (p < 0.01) in ΔTm is observed
between 6.25-μM and 12.5-μM and between 12.5-μM and
25-μM concentrations. Higher concentrations afford negligi-
ble increases in LmPTR1 Tm. Noteworthy is an eight-fold
increase in thermal shift between the lowest and highest ligand
concentration. In contrast, compound 9 showed a significant
increase (p < 0.01) in ΔTm only at low concentrations (be-
tween 6.25 μM and 12.5 μM), and despite the overall tenden-
cy to increase LmPTR1 TM at higher concentrations, only a 6-
fold increase in thermal shift is observed if one compares the
lowest and the highest concentration of compound 9.
ThermoFluor® assays
Single-concentration assays for the identification of LmPTR1
inhibitors
Sixteen structural analogs of compound 2d were evaluated for
their ability to interact with LmPTR1. ThermoFluor® is a
high/medium throughput screening that has enabled the prior-
itization of potential molecules due to the ability to evaluate
the (de)stabilizing effect of hundreds of compounds over the
tertiary structure of the protein and, therefore, their ability to
bind to the target and generate a biological response.
Molecules with ΔTm > 1.0 °C are described as promising
and tend to confirm the biological potential in secondary as-
says (Pantoliano et al. 2001; Ericsson et al. 2006;
Cimmperman and Matulis 2011; Kopec and Schneider
2011). This technique has already been employed to identify
thiazolidine-2,4-dione that inhibit LmPTR1 (Leite et al. 2016)
or that inhibit both DHFR-TS and PTR1 (Teixeira et al. 2019).
Compounds with higher degree of affinity to the target tend to
have higher ΔTm when all other variables are kept equal (Zaia
et al. 1998; Pantoliano et al. 2001; Ericsson et al. 2006; Niesen
et al. 2007; Zhang and Monsma 2010). Among the 16 novel
thiazolidine-2,4-dione derivatives evaluated by a single-
concentration ThermoFluor® assay against LmPTR1
(Graph 1), only two compounds, 4 (ΔTm = 12.12 0.34 °C)
and 9 (ΔTm = 4.69 0.21 °C), may be considered as potential
inhibitors against pteridine reductase 1 of L. major.
Determination of apparent dissociation constant
by ThermoFluor®
The inhibition constant (Kd) can also be determined by
ThermoFluor® assays (Lo et al. 2004). This assay does not
require a high degree of sample purity and does not have to
include solution enthalpy in the Kd calculation, which some-
times makes it difficult to characterize (Cimmperman and
Matulis 2011; Craig and Newton 1991; Vivoli et al. 2014).
Alternatively, Vivoli et al. (2014) developed a method to cal-
culate the apparent dissociation constant (Kdapp) from
However, both compounds have lower ΔTm values than
compound 2d, (compound 4 ΔTm = 12.12 0.34 °C and
compound 9 ΔTm = 4.69 0.21 °C versus compound 2d
ΔTm = 44.67 1.74 μM). Single-concentration assays also
*
16
**
14
**
12
10
8
6
4
*
1
0
-2
1
2
3
4
5
6
7
8
9
0
d
1
11
12
13
14
15
16
2
Molecules (50 M)
Graph 1 Thermal stability of LmPTR1 in the presence of the
thiazolidine-2,4-dione derivatives in the single-concentration assay. The
values represent the median and interquartile range of the variation of Tm
(ΔTm). Statistical differences were calculated between each compound
(n = 3) and compound 2d, ***p < 0.001 (Kruskal-Wallis test with Dunn’s
post-test), previously reported by Leite et al. (2016)

Parasitol Res
Graph 2 Effect of different concentrations of compound 4 (a) or 9 (b) over the LmPTR1 thermal stability. The values represent the median and
interquartile range of the variation of Tm (ΔTm) by multiple comparisons (n = 3), **p < 0.01. (Kruskal-Wallis test with Dunn post-test)
information, provided by differential scanning fluorimetry as-
says, which in most cases is very close to the real Kd value
(Petrauskas et al. 2019).
In order to make a fair comparison of the current com-
pounds to those already published (Leite et al. (2016)), not
only compounds 4 and 9 had their Kdapp calculated but also
compounds 2a–2e (Table 1).
According to this method, compound 4 (18.56 μM) has the
lowest Kdapp value, followed by compound 2b (23.12 μM),
2d (25.35 μM), 2e (89.98 μM), and 9 (98.11 μM). Hence,
compound 4 seems to have higher affinity to LmPTR1 than
our previous lead compound (2d). Moreover, compounds with
Kdapp ≤ 100 μM are expected to have moderate to high affin-
ity for the target (Vivoli et al. 2014). As compound 2d is
among the most promising compounds, the assumptions made
by Cimmperman and Matulis (2011) and Vivoli et al. (2014)
are in good agreement with our previous results (Leite et al.
2016), since the IC50 is related to the ability of a compound to
inhibit the catalytic activity of the target at a fixed substrate
concentration and Kd accounts for the ability of a ligand to
bind with the target.
since the advent of molecular cloning (Pikkemaat et al.
2002; Gasteiger et al. 2005; Sievers et al. 2011; Priest and
Erdemli 2014). However, this approach is considered a sim-
plistic view of the effect of the compound against the pathol-
ogy (Grasberger et al. 2005; Lesyk et al. 2006; Vicini et al.
2006; Silva et al. 2014; Wagner and Schreiber 2016). An
example to be highlighted is the research carried out by
Sienkiewicz et al. (2010), which, after genetic validation of
Trypanosoma brucei PTR1 as a potential target, developed a
potent and selective inhibitor of TbPTR1 (IC50 for TbPTR1 =
7.0 nM; TbDHFR ≥ 30.0 μM) that is unable to inhibit parasite
growth. Thus, enzymatic assays only account for the com-
pound’s ability to interact with the target, whereas its perme-
ability through the cell membranes, efflux process of the com-
pounds, the retention capacity in lipophilic compartments, the
high rate non-specific binding to proteins, among other factors
remain largely ignored by such assays (Gilbert 2013).
Although the in vitro assays were performed on pteridine re-
ductase 1 from Leishmania major, in Brazil, the endemic spe-
cies responsible for the clinical manifestations of CL, CML,
and VL are, mainly, L. braziliensis and L. infantum, respec-
tively. Nevertheless, the NADPH-dependent oxidoreductase
enzymes present conservation of their supersecondary struc-
ture (Vidal et al. 2018) and highly similar active sites. In fact, a
sequential identity analysis of PTR1 (Fig. 3) among the three
species reveals 77.8% sequence identity between LmPTR1
and LbPTR1 and 88.9% sequence identity between LmPTR1
and LiPTR1 or LiPTR1 and LbPTR1. Despite overall similar-
ity, the cofactor-binding site has two substitutions, His-241 for
a Tyr-241 and a Tyr-194 for a Phe-194, that might impact the
binding of ligands. In fact, Tyr-194 is considered an essential
amino acid in the mechanism proposed by Gourley et al.
(2001). However, previous results from our group (Leite
et al. 2016) suggest that this residue does not interact with
2d, which only interact with residues that are conserved in
LbPTR1 and LiPTR1. Thus, based on the sequential align-
ment data as well as the interaction profile described for the
2d, it is reasonable to assume that the L. major PTR1 inhibi-
tors described in this work also bind to PTR1 from other
Leishmania species. In order to support this hypothesis, com-
pounds 2a, 2b, 2c, 2d, 2e, 4, and 9 were evaluated against the
Cellular assays
It is known that the use of enzymatic assays for the character-
ization of specific target inhibitors has been commonly used
Table 1 Apparent dissociation constant values (Kd) of the seven
compounds screened with ΔTm > 1 °C. The assays were performed in
triplicate, and these were analyzed by non-linear regression with p < 0.05
by using the program GraphPad Prima version 5.0
Compounds
Kd (μM)
R2
2a
2b
2c
2d
2e
4
43.42
23.12
196.40
25.35
89.98
18.56
98.11
0.97
0.94
0.97
0.99
0.99
0.92
0.99
9

Parasitol Res
Fig. 3 Multiple sequential alignment of PTR1 of L. major (LmPTR1),
L. infantum (LiPTR1), and L. braziliensis (LbPTR1). Multiple alignment
was generated on the online Clustal Omega server. The rectangles delimit
the residues present at cofactor binding site. (“*” = Identical, “:” = similar,
“.” = not conserved, “-” = absent)
strains of L. braziliensis and L. infantum. In addition, their
cytotoxicity profile in human cells was also evaluated.
The evaluation of promising compounds against
Leishmania sp. consists in determining the efficacy in
inhibiting parasite growth by 50% (CE50). However, cytotox-
icity assays in human cell lines are essential for evaluating the
possible damages caused by the compounds on the human
organism and to understand these compound selectivity
profile.
Initially the seven compounds were analyzed for cytotox-
icity against the WI-26VA4 cell line (ATCC CCL-75 lung
fibroblast) in order to determine the dose capable of killing
50% of cells (LD50) (Table 2). The seven compounds evalu-
ated had an LD50 value higher than the drug used as reference
(amphotericin B deoxycholate). Compounds 2b and 2c are the
least cytotoxic in the series. The high standard deviation of
error presented by compound 9 prevents a correct
classification of its toxicity. Perhaps the structure of the com-
pound enables it to emit fluorescence at the same wavelength
as MTT, causing interference. The action of the compounds
on human cells is unknown, since humans do not possess the
enzyme PTR1. Although there are NADPH-dependent oxido-
reductase enzymes in human cells, this does not guarantee the
action of the compounds on them. Teles et al. (2018) have
shown that analogous compounds, which bind to DHFR of
Schistosoma mansoni (SmDHFR), have a competitive mech-
anism of inhibition towards the substrate. However, com-
pounds 2a, 2b, and 2c were inactive against the SmDHFR.
Thus, it is not possible infer these compounds’ mechanism
of action from those studies, since interference with normal
physiology can be triggered by the impairment of basic cellu-
lar functions, such as energy metabolism, cytoskeleton orga-
nization, membrane integrity, glycogen storage, electrical
conductivity, glutathione depletion, radical formation, or
Table 2 Evaluation of the biological activities and selectivity index for the compounds evaluated against Leishmania braziliensis, Leishmania
infantum, and the WI-26VA4 cell line
Compounds
Cells line
WI-26VA4 LD50 (μM)
L. braziliensis
EC50 (μM)
L. infantum
EC50 (μM)
SI WI-26VA4/
L. braziliensis
SI WI-26VA4/
L.
infantum
2a
2b
2c
2d
2e
4
33.41 8.90
43.29 10.05
> 100
44.16 5.77
65.69 13.85
49.22 7.71
48.23 13.05
47.60 12.51
46.44 9.58
70.98 11.25
4.23 0.44
40.89 5.65
23.45 4.54
66.09 14.17
35.90 6.93
51.24 3.39
30.36 5.11
68.77 12.62
3.12 0.74
0.76
0.66
0.82
1.85
> 2.03
0.55
> 1.51
0.74
26.67 5.27
31.24 7.13
29.22 6.37
61.29 26.85
3.89 1.64
0.66
0.61
0.63
0.96
9
0.86
0.89
Amphotericin
0.92
1.24
B deoxycholate

Parasitol Res
Fig. 4 Structure-activity relationship of the 7 compounds tested against L. infantum (EC50). Shaded atoms in blue promote improved potency, and those
shaded in red promote potency loss
alteration of uptake or excretion (Schoonen et al. 2005). It
should be noted that high standard deviations are characteris-
tic for MTT phenotypic assays (Stockert et al. 2012).
Next, the effective concentration to inhibit 50% growth
(EC50) of Leishmania (V.) braziliensis (MHOM/BR/75/
M2903) and Leishmania (L.) infantum (MHOM/BR/74/
PP75) was determined (Table 2).
For the L. braziliensis species, all compounds show similar
EC50 values; however, compound 2c shows higher selectivity
for L. braziliensis than the other compounds. In contrast, the
biological activity of the compounds against the species of
L. infantum exhibited a wider distribution of EC50 values,
compound 2b was the most potent (EC50 = 23.5 4.5) and
the most selective (SI 1.85). On the other hand, compound
2c is among the least potent ((EC50 = 66.1 14.2), despite
its selectivity profile remains high. Thus, compound 2b is
the best lead compound. No compound showed superior bio-
logical potency than amphotericin B deoxycholate, the control
drug for both species.
compounds have lower biological activity than amphotericin
B deoxycholate, they are less cytotoxic. Lipophilic substituents
at the orto position affect the affinity of the compounds for the
L. infantum species and, consequently, the biological activity.
The relationship between the biological affinity of compounds
2a, 2b, 2d, 2e, and 4 against LmPTR1 and the biological poten-
cy of these compounds compared with the L. infantum species
may be associated with greater conservation of residues at the
binding site of LiPTR1 and LmPTR1, since the same behavior
is assumed in the mechanism of action of these compounds
with the prototype compound 2d. Hence, the conservation of
the Tyr-194 residue, essential for the catalytic mechanism of
PTR1, in the species LiPTR1 and LmPTR1, may be responsible
for the improved activity profile of the compounds against
L. infantum (Fig. 4). This line of thought does not hold for the
L. braziliensis species, since there is no relationship between
structure and activity. This difference, therefore, is expected,
since L. major and L. infantum are closer, from the phylogenetic
point of view, than L. braziliensis, a neotropical species.
Considering the structure-activity relationship data, it can be
seen that both Kd and EC50 values follow the same structural
relationship profile.
Given that compound 2b presented the best biological activ-
ity (EC50 = 23.45 4.54 μM) and one of the best Kd values
(23.12 μM), it seems that the presence of small lipophilic sub-
stituents in the meta and/or para position is essential for effec-
tive compounds against Leishmania species. Although these
Following the determination of the LD50 of the compounds
against the human cell line and the EC50 against the species of

Parasitol Res
Leishmania, the selectivity index was calculated (Table 2) in
order to assess to which organism the compounds are most
harmful.
for the Leishmania species circulating in Brazil is something
to be explored, given the diversity of species in this country
and their respective epidemiological impacts.
Only compounds 2b for L. infantum and 2c for both
L. braziliensis and L. infantum had a selectivity index greater
than 1 and were more effective against the parasite than cyto-
toxic to human cells. Although the literature adopts a value
greater than 10 for the selectivity index, in order to consider
that a compound is promising in relation to phenotypic assays
(Di Giorgio et al. 2004; De Mesquita et al. 2009), this does not
mean that compounds with the values below are insignificant.
Thus, compound 2b, due to both enzymatic and cellular char-
acteristics, is considered the most promising lead compound.
Funding information I would like to thank FAPESB and CNPq (479160/
2013-9) for the financial support to carry out this work.
References
Albuquerque JFC et al (1995) Synthèse et étude structurale des 5-
arilidène thiazolidine-2,4-diones et 4-thio-imidazolidine-2-ones-3-
substituées. Ann Pharm Fr 53:209–214
Alvar J, Arana B (2018) Leishmaniasis, impact and therapeutic needs. In:
Rivas L and Gil C (ed) Drug Discov Leishmaniasis, Royal Society
of Chemisty, pp 1–23
Bozdag-Dündar O et al (2007) Synthesis and antimicrobial activity of
some new thiazolyl thiazolidine-2,4-dione derivatives. Bioorg Med
Chem 15:6012–6017
Conclusions
Bruno G, Costantino L, Curinga C, Maccari R, Monforte F, Nicolò F,
Ottanà R, Vigorita MG (2002) Synthesis and aldose reductase in-
hibitory activity of 5-Arylidene-2,4-thiazolidinedionesy. Bioorg
Med Chem 10:1077–1084
Cavazzuti A, Paglietti G, Hunter WN, Gamarro F, Piras S, Loriga M,
Allecca S, Corona P, McLuskey K, Tulloch L, Gibellini F, Ferrari S,
Costi MP (2008) Discovery of potent pteridine reductase inhibitors
to guide antiparasite drug development. Proc Natl Acad Sci U S A
105(5):1448–1453
Cimmperman P, Matulis D (2011) Chapter 8. Protein Thermal
Denaturation Measurements via a Fluorescent Dye. In: Protein ther-
mal denaturation measurements via a fluorescent dye. Royal Society
of Chemistry
Contessoto VG et al (2018) Introdução ao problema de enovelamento de
proteínas: uma abordagem utilizando modelos computacionais
simplificados. Rev Brasil Ensino Física 40(4):1–13
Craig DQM, Newton JM (1991) Characterisation of polyethylene glycol
solid dispersions using differential scanning calorimetry and solu-
tion calorimetry. Int J Pharm 76:17–24
De Mesquita ML et al (2009) Cytotoxic activity of Brazilian Cerrado
plants used in tradicional medicine against câncer cell lines. J
Ethnopharmacol 123(3):439–445
Denizot F, Lang R (1986) Rapid colorimetric assay for cell growth and
survival. Modifications to the tetrazolium dye procedure giving im-
proved sensitivity and reliability. J Immunol Methods 89(2):271–
277
Di Giorgio C et al (2004) In vitro activity of the b-carboline alkaloids
harmane, harmine, and harmaline toward parasites of the species
Leishmania infantum. Exp Parasitol 106:67–74
Dupeux F, Röwer M, Seroul G, Blot D, Márquez JA (2011) A thermal
stability assay can help to estimate the crystallization likelihood of
biological samples. Biol Crystallogr 67:915–919
Eggert US (2013) The why and how of phenotypic small-molecule
screens. Nat Chem Biol 9(4):206–209
Ericsson UB et al (2006) Thermofluor-based high-throughput stability
optimization of proteins for structural studies. Anal Biochem 357:
289–298
Garbett NC, Chaires JB (2012) Thermodynamic studies for drug design
and screening. Expert Opin Drug Discovery 7(4):299–314
Gasteiger E et al (2005) Protein identification and analysis tools on the
ExPASy Server. In: Walker JM. The Proteomics Protocols
Handbook, Humana Press 571–607
The knowledge about compounds’ biophysical characteristics
is critical to set a suitable screening process. The information
provided by LmPTR1 thermal shift assays suggest that com-
pound 2c has low affinity to the target, whereas compounds
2a, 2b, 2d, and 4 have micromolar affinity to LmPTR1. As
might be expected, these compounds are also active against
other Leishmania species, as they share greater phylogenetic
proximity. The most potent compound for the L. infantum is
compound 2b (CE50 = 23.45 4.54 μM), while the most po-
tent compound for the L. braziliensis species is compound 2a
(CE50 = 44.16 5.77 μM). These results highlight that kinetic
characterization is the first step to identify a novel inhibitor;
however, subsequent phenotypic assays make it possible to
probe the compound behavior in complex media that allow
pharmacokinetic parameters to be extrapolated for in vivo
assays.
Results from phenotypic assays have been routinely
employed, by research groups and the pharmaceutical indus-
try alike, to decide which compound to move forward, or not,
based on their pharmacokinetic and toxicological profile.
Accordingly, it is found that only compounds 2b and 2c for
L. infantum and 2c for L. braziliensis showed an
antileishmania activity superior to their cytotoxic effect.
Although, cell permeability assays are required to make a final
decision on these compounds, some knowledge on their
structure-activity relationships is crucial to unravel structural
modifications that maintain the activity, but reduce toxicity to
human cells, or increase their lipophilicity. Considering the
molecular diversity of the compounds presented in this work
(modification of the substituents of the -meta and -para, ad-
dition of other lateral lipophilic residues of different sizes),
compound 2b shows the best selectivity index and the highest
potency against L. infantum. This results are in good agree-
ment with the Kd value for LmPTR1, which might be related
to their high sequential identity within the active site. It should
also be considered that a future analysis of the PTR1 structure
Gilbert IH (2013) Drug discovery for neglected diseases: molecular
target-based and phenotypic approaches. J Med Chem 56(20):
7719–7726

Parasitol Res
Gontijo B, Carvalho MLR (2003) Leishmaniose tegumentar americana.
Rev Soc Bras Med Trop 36(1):71–80
Gourley DG, Luba J, Hardy LW, Beverley SM, Hunter WN (1999)
Crystallization of recombinant Leishmania major pteridine reduc-
tase 1 (PTR1). Acta Crystallogr Sec D 55:1608–1610
Pikkemaat MG, Linssen ABM, Berendsen HJC, Janssen DB (2002)
Molecular dynamics simulations as a tool for improving protein
stability. Protein Eng 15:185–192
Priest B, Erdemli G (2014) Phenotypic screening in the 21st century.
Front Pharmacol 5(264):1–2
Sancho J (2013) The stability of 2-state, 3-state and more-state proteins
from simple spectroscopic techniques... plus the structure of the
equilibrium intermediates at the same time. Arch Biochem
Biophys 531:4–13
Gourley DG, Schüttelkopf AW, Leonard GA, Luba J, Hardy LW,
Beverley SM, Hunter WN (2001) Pteridine reductase mechanism
correlates pterin metabolism with drug resistance in trypanosomatid
parasites. Nat Struct Biol 8(6):521–525
Schoonen WGEJ, Westerink WM, de Roos JA, Débiton E (2005)
Cytotoxic effects of 100 reference compounds on Hep G2 and
HeLa cells and of 60 compounds on ECC-1 and CHO cells. I
Mechanistic assays on ROS, glutathione depletion and calcein up-
take. Toxicol in Vitro 19:505–516
Gouveia FL, de Oliveira RMB, de Oliveira TB, da Silva IM, do
Nascimento SC, de Sena KXFR, de Albuquerque JFC (2009)
Synthesis, antimicrobial and cytotoxic activities of some 5-
arylidene-4-thioxo-thiazolidine-2-ones. Eur J Med Chem 44:2038–
2043
Sienkiewicz N, Ong HB, Fairlamb AH (2010) Trypanosoma brucei pter-
idine reductase 1 is essential for survival in vitro and for virulence in
mice. Mol Microbiol 77(3):658–671
Sievers F et al (2011) Fast, scalable generation of high-quality protein
multiple sequence alignments using Clustal Omega. Mol Syst Biol
7(539):1–6
Silva IM et al (2014) Synthesis and antimicrobial activities of 5-
arylidene-thiazolidine-2,4-dione derivatives. Biomed Res Int 2014:
1–8
Stockert JC, Blázquez-Castro A, Cañete M, Horobin RW, Villanueva Á
(2012) MTT assay for cell viability: intracellular localization of the
formazan product is in lipid droplets. Acta Histochem 114(8):785–
796
Swinney DC, Anthony J (2011) How were new medicines discovered?
Nat Rev Drug Discov 10(7):507–519
Teixeira BVF et al (2019) Dual and selective inhibitors of pteridine re-
ductase 1 (PTR1) and dihydrofolate reductase-thymidylate synthase
(DHFR-TS) from Leishmania chagasi. J Enzyme Inhib Med Chem
27(6):1439–1450
Teles ALB et al (2018) Identification, characterization and molecular
modelling studies of Schistosoma mansoni dihydrofolate reductase
inhibitors: from assay development to hit identification. Curr Top
Med Chem 18:1–10
Grasberger BL et al (2005) Discovery and cocrystal structure of
benzodiazepinedione HDM2 antagonists that activate p53 in cells.
J Med Chem 4(48):909–912
Kopec J, Schneider G (2011) Comparison of fluorescence and light scat-
tering based methods to assess formation and stability of protein-
protein complexes. J Struct Biol 175(2):216–223
Leite FHA, Santiago PBGS, Froes TQ, da Silva Filho J, da Silva SG,
Ximenes RM, de Faria AR, Brondani DJ, de Albuquerque JFC,
Castilho MS (2016) Structure-guided discovery of thiazolidine-2,
4-dione derivatives as a novel class of Leishmania major pteridine
reductase 1 inhibitors. Eur J Med Chem 123:639–648
Leprohon P, Fernandez-Prada C, Gazanion É, Monte-Neto R, Ouellette
M (2015) Drug resistance analysis by next generation sequencing in
Leishmania. Int J Parasitol Drugs Drug Resist 5(1):26–35
Lesyk R, Zimenkovsky B, Atamanyuk D, Jensen F, Kiec-Kononowicz K,
Gzella A (2006) Anticancer thiopyrano[2,3-d][1,3]thiazol-2-ones
with norbornane moiety. Synthesis, cytotoxicity, physico-chemical
properties, and computational studies. Bioorg Med Chem 14:5230–
5240
Libermann D, Himbert J, Hengl L (1948) La thiazolidione, point de
depart d’une synthèse des acides thiopyruviques et thioglyoxiliques
substituèes. Bull Soc Chim Fr 11–12:1120–1124
Lo MC, Aulabaugh A, Jin G, Cowling R, Bard J, Malamas M, Ellestad G
(2004) Evaluation of fluorescence-based thermal shift assays for hit
identification in drug discovery. Anal Biochem 332:153–159
Nare B, Hard LW, Beverley SM (1997) The roles of pteridine reductase 1
and synthase in pteridine metabolism in the protozoan parasite
Leishmania major. J Biol Chem 272(21):13883–13891
Nare B, Garraway LA, Vickers TJ, Beverley SM (2009) PTR1-dependent
synthesis of tetrahydrobiopterin contributes to oxidant susceptibility
in the trypanosomatid protozoan parasite Leishmania major. Curr
Genet 55:287–299
Vicini P, Geronikaki A, Anastasia K (2006) Synthesis and antimicrobial
activity of novel 2-thiazolylimino-5-arylidene-4-thiazolidinones.
Bioorg Med Chem 14:3859–3864
Vickers TJ, Beverley SM (2011) Folate metabolic pathways in
Leishmania. Essays Biochem 51:63–80
Vidal LS et al (2018) Review of NAD(P)H-dependent oxidoreductases:
properties, engineering and application. BBA Proteins Proteom
1866(2):327–347
Vivoli M et al (2014) Determination of protein-ligand interactions using
differential scanning fluorimetry. J Vis Exp 91:1–13
Wagner BK, Schreiber SL (2016) The power of sophisticated phenotypic
screening and modern mechanism-of-action methods. Cell Chem
Biol 23(1):3–9
Niesen FH, Berglund H, VEdadi M (2007) The use of differential scan-
ning fluorimetry to detect ligand interactions that promote protein
stability. Nat Protoc 2(9):2212–2221
Waldron TT, Murphy KP (2003) Stabilization of proteins by ligand bind-
ing: application to drug screening and determination of unfolding
energetics. Biochemistry 42:5058–5064
World Health Organization (2017) http://www.who.int/gho/neglected_
diseases/leishmaniasis/en/. Accessed 04 Set 2017
World Health Organization. Leishmaniasis (2019) https://www.who.int/
news-room/fact-sheets/detail/leishmaniasis. Accessed 12 Abr 2019
Zaia DAM, Zaia CTBV, Lichtig J (1998) Determinação de proteínas
totais via espectrofotometria: vantagens e desvantagens dos
métodos existentes. Química Nova 21(6):787–793
Pantoliano MW, Petrella EC, Kwasnoski JD, Lobanov VS, Myslik J,
Graf E, Carver T, Asel E, Springer BA, Lane P, Salemme FR
(2001) High-density miniaturized thermal shift assays as a general
strategy for drug discovery. J Biomol Screen 6(6):429–440
Patra N, de U, Kang JA, Kim JM, Ahn MY, Lee J, Jung JH, Chung HY,
Moon HR, Kim HS (2011) A novel epoxypropoxy flavonoid deriv-
ative and topoisomerase II inhibitor, MHY336, induces apoptosis in
prostate cancer cells. Eur J Pharmacol 658:98–107
Pereira JR et al (2012) Cytotoxicity evaluation of marine alkaloid ana-
logues of viscosaline and theonelladin C. Biomed Prev Nutrit 2:
145–148
Petrauskas V et al (2019) Inhibitor binding to carbonic anhydrases by
fluorescent thermal shift assay. In: Matulis D (ed) Carbonic
anhydrase as drug target: thermodynamics and structure of inhibitor
binding, Springer, pp 63–78
Zhang R, Monsma F (2010) Fluorescence-based thermal shift assays.
Curr Opin Drug Discov Dev 13(4):389–402
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