4-Phenyl-1,3-thiazole-2-amines as scaffolds for new antileishmanial agents

Article abstract of DOI:10.1186/s40409-018-0163-x

Background: There is still a need for new alternatives in pharmacological therapy for neglected diseases, as the drugs available show high toxicity and parenteral administration. That is the case for the treatment of leishmaniasis, particularly to the cutaneous clinical form of the disease. In this study, we present the synthesis and biological screening of eight 4-phenyl-1,3-thiazol-2-amines assayed against Leishmania amazonensis. Herein we propose that these compounds are good starting points for the search of new antileishmanial drugs by demonstrating some of the structural aspects which could interfere with the observed activity, as well as suggesting potential macromolecular targets. Methods: The compounds were easily synthesized by the methodology of Hantzsch and Weber, had their purities determined by Gas Chromatography-Mass spectrometry and assayed against the promastigote forms of Leishmania amazonensis as well as against two white cell lines (L929 and THP-1) and the monkey's kidney Vero cells. PrestoBlue and MTT viability assays were the methodologies applied to measure the antileishmanial and cytotoxic activities, respectively. A molecular modeling target fishing study was performed aiming to propose potential macromolecular targets which could explain the observed biological behavior. Results: Four out of the eight compounds tested exhibited important anti-promastigote activity associated with good selectivity indexes when considering Vero cells. For the most promising compound, compound 6, IC₅₀ against promastigotes was 20.78 while SI was 5.69. Compounds 3 (IC₅₀: 46.63μM; SI: 26.11) and 4 (IC₅₀: 53.12μM; SI: 4.80) also presented important biological behavior. A target fishing study suggested that S-methyl-5-thioadenosine phosphorylase is a potential target to these compounds, which could be explored to enhance activity and decrease the potential toxic side effects. Conclusions: This study shows that 4-phenyl-1,3-thiazol-2-amines could be good scaffolds to the development of new antileishmanial agents. The S-methyl-5-thioadenosine phosphorylase could be one of the macromolecular targets involved in the action.

Full text of DOI:10.1186/s40409-018-0163-x

Rodrigues et al. Journal of Venomous Animals and Toxins including Tropical Diseases
2018) 24:26
(
https://doi.org/10.1186/s40409-018-0163-x
RESEARCH
Open Access
4
-Phenyl-1,3-thiazole-2-amines as scaffolds
for new antileishmanial agents
Carina Agostinho Rodrigues , Paloma Freire dos Santos , Marcela Oliveira Legramanti da Costa ,
Thais Fernanda Amorim Pavani , Patrícia Xander , Mariana Marques Geraldo , Ana Mengarda ,
Josué de Moraes and Daniela Gonçales Galasse Rando
1
1
1
1
2
2
3
3
1*
Abstract
Background: There is still a need for new alternatives in pharmacological therapy for neglected diseases, as the
drugs available show high toxicity and parenteral administration. That is the case for the treatment of leishmaniasis,
particularly to the cutaneous clinical form of the disease. In this study, we present the synthesis and biological
screening of eight 4-phenyl-1,3-thiazol-2-amines assayed against Leishmania amazonensis. Herein we propose that
these compounds are good starting points for the search of new antileishmanial drugs by demonstrating some of
the structural aspects which could interfere with the observed activity, as well as suggesting potential
macromolecular targets.
Methods: The compounds were easily synthesized by the methodology of Hantzsch and Weber, had their purities
determined by Gas Chromatography-Mass spectrometry and assayed against the promastigote forms of Leishmania
amazonensis as well as against two white cell lines (L929 and THP-1) and the monkey’s kidney Vero cells.
PrestoBlue® and MTT viability assays were the methodologies applied to measure the antileishmanial and cytotoxic
activities, respectively. A molecular modeling target fishing study was performed aiming to propose potential
macromolecular targets which could explain the observed biological behavior.
Results: Four out of the eight compounds tested exhibited important anti-promastigote activity associated with
good selectivity indexes when considering Vero cells. For the most promising compound, compound 6, IC50
against promastigotes was 20.78 while SI was 5.69. Compounds 3 (IC : 46.63 μM; SI: 26.11) and 4 (IC : 53.12 μM; SI:
50
50
4
.80) also presented important biological behavior. A target fishing study suggested that S-methyl-5-thioadenosine
phosphorylase is a potential target to these compounds, which could be explored to enhance activity and decrease
the potential toxic side effects.
Conclusions: This study shows that 4-phenyl-1,3-thiazol-2-amines could be good scaffolds to the development of
new antileishmanial agents. The S-methyl-5-thioadenosine phosphorylase could be one of the macromolecular
targets involved in the action.
Keywords: 2-aminothiazoles, Antikinetoplastids, Antileishmanial, Cutaneous, Target fishing
*
Correspondence: dgrando@unifesp.br
1
Chemical and Pharmaceutical Research Group, Department of
Pharmaceutical Sciences, Institute of Environmental, Chemical and
Pharmaceutical Sciences, Federal University of São Paulo (UNIFESP), Rua São
Nicolau, 210, 2o andar, Diadema, SP 09913-030, Brazil
Full list of author information is available at the end of the article
©
The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Rodrigues et al. Journal of Venomous Animals and Toxins including Tropical Diseases (2018) 24:26
Page 2 of 10
Background
Methods
Neglected diseases affect thousands of people, pre- Eight different aromatic ketones were applied in accord-
dominantly in developing countries [1]. The available ance with their 4-phenyl ring substituent, considering
pharmacological therapies, however, are far from sat- their effect on the lipophilicity of the system (Table 1).
isfactory, since the existing drugs exhibit a moderate
efficacy associated with toxicity. This is the case of
Synthesis
leishmaniasis, a tropical endemic disease caused by
The 4-phenyl-2-aminothiazoles were synthesized accord-
protozoa of the genus Leishmania [2]. The most com-
ing to the methodology of Hantzsch and Weber [12],
mon leishmaniasis treatments frequently require
from their respective arylketones and thiourea (1:2), in
parenteral administration and chronic therapeutic
the presence of iodine (4 equivalents), by heating the
schemes, which result in high costs and low accessi-
mixture for 4 h. At the end of the reaction, hot water
bility to the farthest endemic regions [3]. For these
was added to the raw mixture, which was cooled to
reasons, the need for new alternatives to treat
room temperature and washed with three portions of
neglected diseases remains urgent.
ethyl ether (~ 10 mL each) to remove the residual iodine.
The 2-aminothiazole scaffold is present in the
The water phase was neutralized with a saturated solu-
structure of antiviral [4], antifungal [5], antimicrobial
tion of ammonium hydroxide and washed with distilled
[
6], anti-cancerous [7], and anti-inflammatory com-
water to yield a solid isolated by filtration under reduced
pressure. The pure 4-phenyl-1,3-thiazol-2-amines were
obtained by crystallization from Ethanol:water (4:1) solu-
tion [13].
pounds [8]. Recent papers have reported different
-aminothiazole derivatives as promising agents
2
against kinetoplastids such as Trypanosoma cruzi,
Trypanosoma brucei, Leishmania donovani, and
Leishmania infantum [9–11].
Kaiser et al. screened a set of 400 drug-like compounds Antileishmanial assay
from the chemical library known as “Malaria Box”, made All compounds were assayed against promastigotes of
available by the Medicines for Malaria Venture initiative, Leishmania amazonensis (MHOM/BR/1973/M2269).
for potential repurposing of these compounds as antikine- Promastigotes were grown in 199 media (Sigma, USA)
toplastids. Their findings showed that 2-aminothiazole de- supplemented with 10% (v/v) of fetal bovine serum, 1%
rivatives might be potential hits to be explored against penicillin (10,000 UI/mL)/streptomycin (10.0 mg/mL)
trypanosomatids, specifically against Trypanosomes [9].
(Sigma, USA). The parasites were collected at log phase,
More recently, Papadopoulou et al. synthesized a small they were counted at the Neubauer chamber, their
6
series of 5-nitro-2-aminothiazoles derivatives which pre- concentration was adjusted to 1 × 10 cell/mL, and
sented better antitrypanosomal activity than the stand- their cells were seeded at a 96-wells plate. The
ard drug benznidazole. Antileishmanial activity was also 4-phenyl-2-aminothiazoles were added to the culture,
observed in some of the derivatives; however, toxicity considering concentrations ranging from 3.2 to 142 μM.
against L6 cells was also verified [10].
Compounds were diluted in Dimethyl sulfoxide (DMSO)
The 2-aminothiazole ring has gained increasing at- (25 mg/mL) and diluted in 199 media to generate a 1%
tention as an antiprotozoal hit. Nevertheless, the DMSO starting solution of each test compound. Positive
4
-phenyl-1,3-thiazole-2-amine scaffold alone has never control with no compounds and DMSO control were
been explored by its biological potential, mainly re- considered at the assay. All plates were incubated at
garding the antileishmanial activity.
25 °C for 48 h, and PrestoBlue® vital dye was added
In this paper, we report the synthesis and biological to the wells.
screening of eight 4-phenyl-1,3-thiazol-2-amines assayed
Table 1 The 4-Phenyl-1,3-thiazol-4-amines series
against L. amazonensis, a species of leishmania from the
L. mexicana complex, which is responsible for the cuta-
neous form of the disease. The compounds were also ex-
plored as to assess their cytotoxicity, selectivity index,
and potential macromolecular targets.
The main objective of these studies was to understand
whether 4-phenyl-1,3-thiazol-2-amines could be good start-
ing points in the search for new antileishmanial drugs, as
well as to assess the chemical aspects that could interfere
with the activity. Potential macromolecular targets, which
could explain the observed actions, were also investigated
by target fishing approach.
a
b
Compound
R
CLogP
2.61
3.07
3.47
3.64
3.49
4.23
2.32
3.12
MR
Yield %
61
01
H
50.20
55.25
59.85
59.81
56.18
68.87
52.19
55.01
02
03
4-CH
3
72
4-CH CH3
2
59
0
0
0
0
4
5
6
7
3,4-Cl
53
4-CF
3
18
4-C(CH
4-OH
4-Cl
3
)
3
27
47
08
67
(a) Calculated n-Octanol/Water Partition Coefficient; (b) Molar Refractivity

Rodrigues et al. Journal of Venomous Animals and Toxins including Tropical Diseases (2018) 24:26
Page 3 of 10
Viability was accessed with PrestoBlue® (Invitrogen, Selectivity index calculation
ThermoFisher) according to the manufacturer’s instruc- Selectivity indexes (SI) were calculated by dividing CC50
tions. The reagent was added in each well (10 μL) and against each mammalian cell line by the IC50 against L.
plates incubated for 2 h. Then, plates were read on a amazonensis (CC /IC ).
50
50
microplate reader (PowerWave HT, Biotek) with excita-
tion/emission 560/590 nm (nm). The relative fluores Structure-activity relationship studies
cence units were used to generate the quantitative results. Regarding the studies of the structure-activity relation-
All compounds were analyzed as duplicates, and the ship, both the partition coefficient and the molar refrac-
parameters to calculate IC50 were obtained by GraphPad tivity (CLogP and MR, respectively) were calculated with
Prism, version 5.0. IC50 values were determined by the Hill Marvin Sketch 6.2.2 software [18]. Electronic parame-
equation rearranged [14].
ters, such as punctual Mulliken charges and dipole
moment values, were calculated to the minimum energy
conformer obtained after geometry optimization pro-
cessing at Gaussian 09 W software. Electrostatic poten-
tial charges (ChelpG) were calculated by the ab initio
method HF/6-31 g* (Gaussian 09 W) and mapped on
the Connolly surface of each molecule [19].
PaDel-descriptor calculator was used to obtain the topo-
LogIC50 ¼ logEC50−f logðTop−Bottom=Y−Bottom−1Þ=HillSlopeg
Cytotoxicity assay
The cytotoxicity was measured in L929 and THP1 logical indexes [20]. Only qualitative structure-activity
cell lines using PrestoBlue®Cell Viability Reagent relationship studies were performed, as well as only the
(
Thermo Scientific, Invitrogen) as described by Lall et linear correlation coefficients above 0.8 were taken into
al. with modifications [15]. First, cells were main- consideration.
tained in RPMI 1640 (Gibco) supplemented with 10%
fetal bovine serum (FBS) and 1% antibiotics (100 U/mL Target fishing and docking studies
penicillin, 100 μg/mL streptomycin). Cell cultures were Target fishing studies were performed applying the
grown and maintained at 37 °C in a humidified incubator Web-based Software Pharmmapper [21], considering the
with 5% CO . Then, L929 cells were seeded in 96-wells 3D-optimized structure of the 4-phenyl-1,3-thiazole-2-a-
2
5
flat microtiter plate at a concentration of 1 × 10 cells/mL mines and the ChelpG charges as the input files. The
(
100 μL/well). To THP-1 cells, cultures were differentiated best 300 results were analyzed based on their fit and
into macrophages by adding 50 ng/mL 12-myristate z’score functions.
3-acetate phorbol (PMA) (Calbiochem, San Diego, USA) The docking protocol was established after performing
1
in RPMI-free FBS. After 24 h of culturing, the adhered redocking studies with the crystallographic data re-
cells were washed with PBS and RPMI with 10% FBS were trieved from Protein Data Bank, entry code 1CG6
added. Both cell lines were treated with 4-phenyl-2-ami- (resolution: 1.7 Å), using the Gold 5.4.1 software [22].
nothiazoles at concentrations ranging from 3.2 to This protocol was applied to the docking simulation
1
42 μM. The plates were incubated for 48 h and, with the test compounds, mainly to the 6 one, the best
thereafter, 20 μL/well PrestoBlue® was added. The plates compound of the series. The binding site was defined by
were incubated for further 2 h and the fluorescence the centroid between Met196 and Asp220 and consider-
was read at bottom using 540–570 nm excitation and ing a region of 14 Å around this point. Ten runs with 10
5
80–610 nm emission. The average of the fluorescence solutions each were performed to generate a set of 100
values of the control wells (medium plus PrestoBlue®) complexes, which were examined by the incidence of
was subtracted from the fluorescence value of the positive a recurrent pose as well as by their Goldscore and
controls (cells, medium, and PrestoBlue®) and from Chemscore values.
each experimental well (cells, medium, compounds and
PrestoBlue®).
Results
Cytotoxicity was also evaluated using Vero cells, a Synthesis
monkey kidney cell line obtained from the American The compounds were synthesized and isolated as solids
Type Culture Collection (ATCC CCL-81; Manassas, of good purity and yields. Structural characterization of
1
13
VA). The cytotoxicity was determined by the 3-(4,5di- these compounds was performed by H-/ C-Nuclear
methylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide Magnetic Resonance (NMR) and Infrared spectra (IR).
(
MTT) method according to a previously described pro- Gas Chromatography-Mass Spectrometry (GC-MS)
cedure [16, 17].
analysis was performed to verify the purity, as well as, the
In all cases, the CC50 was calculated as described as in structure of the analogues. All compounds presented pu-
the Antileishmanial assay Section. rities varying from 98 to 100% and molecular ion values

Rodrigues et al. Journal of Venomous Animals and Toxins including Tropical Diseases (2018) 24:26
Page 4 of 10
(
m/z) and fragmentation patterns correspondent to what (C and C ); 126.46 (C and C ); 127.45 (C ); 127.98
9 11 8 12 10
was expected for each molecule (Additional file 1).
(C ); 138.95 (C ); 151.47 (C ); 168.96 (C ); IR (KBr):
7 13 4 2
Compound 1 was obtained as a pale yellow powder 3479 (νassNH ); 3298 (ν NH ); 3143 (νC-H aromatic);
2
sim
2
1
(
(
9
7
[
61%): H-NMR (300 MHz, [D6] DMSO, ppm): δ=7.00 1537 (νC=N); 754 (νC-F); GC (retention time, relative
s, 1H, H ); 7.05 (s, 2H, NH ); 7.24 (t, 1H, J = 6 Hz, J = intensity in %): 14,13 (100); LRMS (EI, 70 eV) MS m/z
Hz, H ); 7.35 (t, 2H, J = 6 Hz, J = 9 Hz, H and H ); calculated for C H F N S 244.24, found 244.
Compound 6 was obtained as a pale yellow powder
D6] DMSO, ppm): δ=101.94 (C ); 125.99 (C and C ); (27%): mp: 227-230 °C; H-NMR (300 MHz, [D6]
5
2
1
2
1
3
1
2
9
11
10
7
3
2
1
3
.79 (d, 2H, J = 9 Hz, H and H ); C-NMR (75 MHz,
8
12
1
5 8, 12
1
27.62 (C , C and C ); 128.91 (C ); 135.40 (C ); IR DMSO, ppm): δ=1.29 (s, 9H, H , H and H ); 6.91 (s,
9 10 11 4 2 14 15 16
(
KBr): 3436 (νassNH ); 3254 (ν NH ); 3155 (νC-H aro- 1H, H ); 7.02 (s, 2H, NH ); 7.37 (d, 2H, J = 9 Hz, H and
2 sim 2 5 2 8
matic); 1599 (νC=N), 715 (νC-S-C); GC (retention time, H ); 7.70 (d, 2H, J = 9 Hz, H and H ). Melting point:
12
9
11
relative intensity in %): 14.10 (100); LRMS (EI, 70 eV) Experimental (227-230 °C); Reference (228-230 °C).
13
m/z calculated for C H N S 176.23, found 176.
C-NMR, IR and GC-MS acquisitions not performed.
9
8
2
Compound 2 was obtained as a pale yellow powder
72%): H-NMR (300 MHz, [D6] DMSO, ppm): δ=2.29 (47%): H-NMR (300 MHz, [D6] DMSO, ppm): δ=6.74–
Compound 7 was obtained as a pale yellow powder
1
1
(
(
2
s, 3H, CH ); 6.91 (s, 1H, H ); 7.01 (s, 2H, NH ); 7.16 (d, 6.69 (m, 3H, H , H and H ); 6.92 (s, 2H, NH ); 7.60–
3
5
2
5
8
12
2
3
1
H, J = 6 Hz, H a and H ); 7.67 (d, 2H, J = 9 Hz, H and 7.55 (m, 2H, H and H ); 9.42 (s, 1H, H ); C-NMR
9 11 8 9 11 13
1
3
H ); C-NMR (75 MHz, [D6] DMSO, ppm): δ=21.25 (75 MHz, [D6] DMSO, ppm): δ=98.87 (C ); 115.60 (C9
1
2
5
(
C ); 101.01(C ); 125.93 (C ); 129.48 (C and C12); and C ); 127.35 (C ); 150.57 (C and C ); 157.19 (C );
13 5 7 8 11 7 8 12 10
1
32.76 (C and C ); 136.81 (C ); 150.36 (C ); 168.53 168.41 (C ); 206.96 (C ); IR (KBr): 3487 (ν NH ); 3377
9 11 10 4 4 2 ass 2
(
(
(
C ); IR (KBr): 3454 (ν NH ); 3300 (ν NH ); 3117 (νsimNH ); 3126 (νC-H aromatic); 2984 (νO-H); 1535
2 ass 2 sim 2 2
νC-H aromatic); 2954 (νC-H CH ); 1537 (νC=N), 731 (νC=N); GC (retention time, relative intensity in %):
3
νC-S-C); GC (retention time, relative intensity in %): 17,69 (100); LRMS (EI, 70 eV) MS m/z calculated for
1
5,46 (100); LRMS (EI, 70 eV) m/z calculated for C H N OS 192.24, found 192.
9 8 2
C H N S 190.26, found 190.
Compound 8 was obtained as a white powder (67%):
H-NMR (300 MHz, [D6] DMSO, ppm): δ=7.07 (s, 1H,
10
10
2
1
Compound 3 was obtained as a dark yellow powder
1
(
59%): H-NMR (300 MHz, [D6] DMSO, ppm): δ=1.18 H ); 7.08 (s, 2H, NH ); 7.39–7.44 (m, 2H, H and H );
5 2 8 9
13
(
t, 3H, J = 9 Hz, J = 6 Hz, CH ethyl); 2.59 (q, 2H, J =
7.83–7.79 (m, 2H, H9 and H11); C-NMR (75 MHz,
1
2
3
1
9
Hz, J = 6 Hz, CH ethyl); 6.91 (s, 1H, H ); 7.01 (s, 2H, [D6] DMSO, ppm): δ=102.77 (C ); 127.69 (C ); 128.93
2
2
5
5
7
NH ); 7.19 (d, 2H, J = 9 Hz, H and H ); 7.69 (d, 2H, J
(C and C ); 131.98 (C and C ); 134.23 (C ); 143.05
9 11 8 12 10
2
9
11
1
3
8 12 4 2 ass 2
=
9 Hz, H and H ); C-NMR (75 MHz, [D6] DMSO, (C ); 168.81 (C ); IR (KBr): 3437 (ν NH ); 3282
ppm): δ=15.97 (C ); 28.35 (C ); 101.06 (C ); 126.00 (νsimNH ); 3109 (νC-H aromatic); 1533 (νC=N); 826
1
4
13
5
2
(
C ); 128.27 (C and C ); 133.00 (C and C ); 143.17 (νC-Cl); GC (retention time, relative intensity in %):
7 9 11 8 12
(
C ); 150.36 (C ); 168.54 (C ); IR (KBr): 3425 (νassNH2); 16,50 (99,6); LRMS (EI, 70 eV) MS m/z calculated for
10 4 2
3
265 (νsimNH ); 3116 (νC-H aromatic); 2958 (νC-H CH ); C H ClN S 210.68, found 210.
2 3 9 7 2
2
870 (νC-H CH );1516 (νC=N); GC (retention time, rela-
2
tive intensity in %): 16,54 (100); LRMS (EI, 70 eV) MS m/z Biological assays
calculated for C H N S 204.29, found 204. All compounds were tested against L. amazonensis pro-
1
1 12 2
Compound 4 was obtained as a white powder (53%): mastigotes. The cytotoxicity was also assessed against
H-NMR (300 MHz, [D6] DMSO, ppm): δ=7.14 (s, 2H, L929 and THP-1 by using the PrestoBlue® cell viability
1
NH ); 7.22 (s, 1H, H ); 7.61 (d, 1H, J = 9 Hz, H ); 7.77 reagent, as well as against Vero cell lines by using the
2
5
12
(
d, 1H, J = 9 Hz, H ); 8.01 (d, 1H, J = 3 Hz, H ); MTT viability assay. The results are presented in
C-NMR (75 MHz, [D6] DMSO, ppm): δ=104.27 (C5); Table 2.
11 8
1
3
1
1
3
1
26.00 (C ); 127.60 (C ); 129.72 (C ); 131.17 (C );
1
2
7
8
11
31.74 (C and C ); 135.88 (C ); 147.65 (C ); IR (KBr):
Structure-activity relationships and molecular modeling
9
10
4
2
446 (νassNH ); 3282 (ν NH ); 3124 (νC-H aromatic); studies
2
sim
2
527 (νC=N); 1022 and 1041(νC-Cl); GC (retention time, Structure-activity relationship analyses revealed a good
relative intensity in %): 18,96 (97,8); LRMS (EI, 70 eV) MS parabolic correlation between CLogP and antileishma-
m/z calculated for C H Cl N S 245.13, found 244.
nial activity (Additional file 2A), with a correlation coef-
9
6
2 2
Compound 5 was obtained as a pale yellow powder ficient of 0.968. The same behavior was also observed
1
(
18%): H-NMR (300 MHz, [D6] DMSO, ppm): δ=7.18 when regarding the Molar Refractivity (MR) values of
(
s, 1H, NH ); 7.26 (S, 1H, H ); 7.73 (d, 2H, J = 9 Hz, H
the compounds, a parameter which also describes mo-
2
5
8
1
3
and H ); 8.00 (d, 2H, J = 9 Hz, H and H 11); C-NMR lecular hydrophobicity associated to molecular volume
1
2
9
(
75 MHz, [D6] DMSO, ppm): δ=104.84 (C ); 125.89 (Additional file 2B).
5

Rodrigues et al. Journal of Venomous Animals and Toxins including Tropical Diseases (2018) 24:26
Page 5 of 10
Table 2 IC50 values against L. amazonensis promastigotes and the cytotoxicity assay results, expressed as CC50 values
a)
b)
c)
Compound
IC50 (μM)
957.56
107.68
46.63
pIC50
3.02
3.97
4.33
4.27
4.27
4.68
inactive
3.94
–
CC50 (μM) THP1 cells
SI
CC50 (μM) L929 cells
198.26
159.52
95.45
SI
CC50 (μM) VERO cells
SI
1
143.57
135.40
117.27
84.65
92.21
45.73
inactive
82.15
ND
0.15
1.26
2.51
1.59
1.73
2.20
ND
0.70
–
0.20
1.48
2.05
2.28
2.00
1.30
ND
0.99
–
710.06
657.51
1217.5
255.03
511.76
118.17
651.29
595.94
ND
0.74
6.11
26.11
4.80
9.59
5.69
ND
5.14
–
2
3
4
53.12
121.12
106.08
27.07
5
53.37
6
20.78
7
inactive
115.95
16.23
Inactive
115.06
ND
8
Amphotericin B
Pentamidine
10.76
–
ND
–
ND
–
ND
–
(a) THP1 cells: human monocytic cell line derived from acute monocytic leukemia patients; (b) L929 cells: fibroblasts from subcutaneous connective tissue; c)
Vero cells: kidney epithelial cells extracted from African green monkeys. IC50: Half-maximal inhibitory concentration on promastigotes; pIC50: Half-maximal
inhibitory concentration in log units; CC50: Half cytotoxic concentration; SI: Selectivity Indexes, ND: Not determined. pIC50 calculates as pIC50 = −log10 (IC50
)
2
No significant correlation (r = > 0.8) was found between most probable targets to compounds with defined chem-
activity and electronic parameters, such as electrostatic ical structures [21].
potential charges, punctual charges or dipole moments
The energetically optimized structure of each com-
(
see Additional file 3 to verify the parameters and pound, considering their ChelpG charges, was employed
Additional file 4 for electrostatic potential maps).
as input, and the best 300 targets – human and
However, some relationships could be observed regarding non-human – were retrieved. Table 3 lists the five best
the Electrostatic Potential Maps. Compounds 3 and 6, for targets common to these compounds, along with the
instance, showed a green left-side region (Additional file 4) maximum z’score values that were obtained.
whereas compounds 4 and 5, which presented an inter-
The human S-Methyl-5-thioadenosine phosphorylase
mediary activity, exhibited a yellowish left-side region was elected as the best potential target, based in two
similarly to the one observed with compound 7, an inactive criteria: the best Max z’score values found to each
analogue. These findings could reveal some potential influ- compound under analysis combined to the fact that this
ence of the electronic distribution on the activity, although target was returned to all compound submitted to the
further studies must be performed to confirm these target fishing.
relationships.
A BLAST (Basic Local Alignment Search Tool, Na-
As the cytotoxic effects on Vero cells were substan- tional Center for Biotechnology Information) search
tially different from those obtained against L. amazonensis, was, then, performed to find homologous proteins in the
L-929 and THP-1 cells, a target fishing study was proposed L. amazonensis available proteome [23]. The best nine
to suggest potential targets and, consequently, mechanisms results are listed in Table 4, together with the statistical
which could explain these observations.
analyses which corroborate their significance.
The web-based software Pharmmapper was applied to
Identity values above 35% were observed for the Leish-
search pharmacophore databases, such as the Target mania species enzymes, so a docking study considering
Bank, Drug Bank, Binding DB, and Potential Drug the human enzyme was also performed to elucidate the
Target Database. Such software also takes advantage of a potential binding mode of the compounds into the cata-
normalized score function, the z’score, to present the lytic site. The results are illustrated in Fig. 1.
Table 3 Pharmmapper results
a
Target
PDB Code
1CG6
Ligand Compounds
All derivatives
Max z’score value
Function
S-Methyl-5-thioadenosine phosphorylase
Adenosine deaminase
3.69
4.72
3.59
3.12
3.02
Nucleotide transport and metabolism
Nucleotide transport and metabolism
Replication, recombination, and repair
Coenzyme transport and metabolism
1 V79/1 V89
1C40
All but compound 3
All but compound 4
All but compound 4
UvrABC System Protein B
Dihydrofolate reductase
Queuine t-RNA-ribosyltransferase
1IA2
1Q66
All but compounds 3
and 4
Translation, ribosomal structure and
biogenesis
(
a) among all compounds tested

Rodrigues et al. Journal of Venomous Animals and Toxins including Tropical Diseases (2018) 24:26
Page 6 of 10
Table 4 BLAST results for Leishmania sp protein sequences producing significant alignment with human S-methyl-5-thioadenosine
phosphorylase
Target
Max Score Total Score Query Cover (%) E value Identity (%)
Putative methylthioadenosine phosphorylase [Leishmania infantum JPCM 5]
Putative methylthioadenosine phosphorylase [L. major strain Friedlin]
Putative methylthioadenosine phosphorylase [L. guyanensis]
182
180
182
182
180
182
182
179
179
87
87
95
95
95
95
2e-55
1e-54
3e-55
2e-55
2e-54
2e-54
38
38
37
37
37
37
Putative methylthioadenosine phosphorylase [L. mexicanaMHOM/GT/2001/U1103] 182
Putative methylthioadenosine phosphorylase [L. panamensis] 179
Putative methylthioadenosine phosphorylase [L. brasiliensis MHOM/BR/75/M2904] 179
Discussions
The compounds were obtained as expected with good activity was verified solely considering this scaffold.
yields and purities. The fact of the easy synthetic A suitable cytotoxic profile was obtained to compound
results are still encouraging because the antileishmanial
preparation constitutes one of the major advantages 6 when considering the THP-1 cells, with a selectivity
for the future application of these analogues as bio- index of 2.20, but this index was lower when considering
logical agents, mainly regarding the therapeutics of L929 cells (1.30). The THP-1 cell line is a frequent
neglected diseases.
model for imitating the function and regulation of mac-
Out of the eight tested analogues, four of them rophages, the host cells of L. amazonensis.
showed activity against the promastigotes of L. amazo-
Similarly, Compounds 3, 4 and 5 exhibited selectivity
nensis (Table 2), within the range of 20 to 60 μM. Com- indexes suitable to proceed to the next steps of the de-
pound 6 was the most active one (21 μM), exhibiting an velopment, since the toxic dose was about two times
IC50 value close to the one obtained with the standard higher than the effective dose, both when considering
drug of the assay, the Amphotericin B (16.23 μM). Com- L929 and THP-1 cells. Moreover, no cytotoxic effect was
pound 3 was the second best of the series, followed by observed against Vero cells at the same concentration
Compounds 4 and 5, both presenting very similar antil- range tested, except for compound 1, which had a SI of
eishmanial behavior. However, no compound was better 0.74. This last compound was inactive against L. amazo-
than the pentamidine standard drug (10.76 μM).
nensis, though.
Even though compounds that were more active against
These observations encouraged us to proceed with fur-
leishmaniasis have been reported in literature, our ther studies, aiming to understand which of the
Fig. 1 Docking at the S-methyl-5-thioadenosine phosphorylase. (a) Co-crystallized ligand. (b) Compound 6 docked at the active site. Blue dashed
lines: pi-stacking interactions; black dashed lines: hydrogen bonds (For color version, please access the online version of the article)

Rodrigues et al. Journal of Venomous Animals and Toxins including Tropical Diseases (2018) 24:26
Page 7 of 10
structural aspects could be related to the activity or pdb entry). This enzyme catalyzes the conversion of
cytotoxicity of the compounds. 5′-deoxy-5′-methylthioadenosine in adenine and 5-met
CLogP values ranging from 3.5 to 4.5 seem to be ideal hylthio-D-ribose-1-phosphate in a fundamental step of
to the biological activity of the series observed. Moreover, the polyamine biosynthesis [24]. This pathway is essen-
molar refractivity (MR) also presented a good correlation tial to the production of nucleotides and, therefore, es-
with antileishmanial activity (Additional file 2), indicating sential to cell growth and proliferation.
that not just the lipophilicity contributes to the antileish-
manial activity, but also molar volume and polarizability.
Unfortunately, the phosphorylase isoform pointed out
as a target to these compounds is the human one, and it
The observed relations with hydrophobic, volume, and would not be able to explain the antileishmanial action
polarizability parameters strongly suggest the importance observed. However, this enzyme has its correspondent
of membrane permeation to the activity of these scaffolds.
Such findings are also in accordance with the observa-
isoform in several living organisms.
Investigation for potential Leishmania sp isoforms was
tions of Papadopoulou et al., in which linear correlation performed with the BLAST. The search found nine protein
coefficients of 0.979 and 0.977 were found between the sequences with identity values ranging from 37 to 38%, all
antitrypanosomal activity and the CLogP values of their from different Leishmania species, including one from the
piperazine and non-piperazine derivatives [10]. Their L. mexicana complex. The Expectation values (E-values) of
−
54
results against L. donovani are significant and have also 10
obtained in this study corroborates with the signifi-
demonstrated good correlation with the lipophilicity of cance of the alignment, since the lower the E-value, the
2
their analogues (r = 0.886). These compounds, however, more significant the alignment and its scores (Table 4).
presented cytotoxicity toward L6 cells, which were also
related to the lipophilicity.
Most of the conserved residues are located at the
binding site region, as revealed by the alignment of the
Aiming to explain the differential cytotoxic profile ob- putative L. mexicana phosphorylase and the human
served among the eukaryotic cells, molecular modeling isoform (Fig. 2).
studies were also performed.
The binding mode of 5′-deoxy-5′-methylthioadenosine
Initially, a target fishing study was carried out to iden- on the human isoform is sustained by three main
tify potential targets to the compounds. Most of the hydrogen-bound interactions with the Met196, Asp220 and
returned targets are related to nucleotide recognition, Asp222. The alignment with the enzyme from L. mexicana
processing, transport, and/or metabolism. This could showed that these residues are conserved at the binding
indicate structural similarity between the scaffold and site, corresponding to the Met200, Asp224 and Asp226.
these endogenous substrates. It also could explain, to
some degree, the observed cytotoxicity toward some of icity observed toward the L929 fibroblasts and THP-1
the tested cell lines. cells, but not toward the Vero cells, which is a
This similarity could be the basis for the cytotox-
The single common target to all tested compounds S-methyl-5-thioadenosine phosphorylase negative cell
was the S-methyl-5-thioadenosine phosphorylase (1CG6, line [25].
Fig. 2 (a) Human and (b) L. Mexicana S-methyl-5-thioadenosine phosphorylase binding site alignment

Rodrigues et al. Journal of Venomous Animals and Toxins including Tropical Diseases (2018) 24:26
Page 8 of 10
In the absence of crystallographic data from the L. diseases, just like observed in infections caused by the L.
mexicana enzyme, docking studies with the human iso- braziliensis. This clinical manifestation is characterized
form were performed to verify the potential binding by deforming lesions that often compromise the patient’s
mode of the 4-phenyl-2-aminothiazole to the phosphor- social life.
ylase (Fig. 1).
Moreover, leishmania species from the L. mexicana
The most prevalent pose of compound 6 at the phos- complex are widely known to be resistant to treatment,
phorylase binding site was found at 89% frequency. This mainly those based on in situ free-radical production, as
pose showed values 43.34Goldscore and 18.93 Chem- is the case of nitroderivatives. Such mechanism of action
score, which are compatible with the good fitness of implies low selectivity and, consequently, a broad panel
compound 6 at the binding site.
of side effects. Ironically, several of the promising new
The docked compound 6 also showed good corres- compounds explored against trypanosomatidae parasites
pondence with the binding mode exhibited by the are nitroaromatic compounds [30, 31].
5
′-deoxy-5′-methylthioadenosine, the original ligand of
Finding compounds that are active against such para-
the phosphorylase.
sites with IC50 values lower than 10 μM and with good
The amino group of 4-phenyl-2-aminothiazole com- toxicological profile is still a challenging task. From this
pound 6 (Fig. 1b) established hydrogen bonds, as a point of view, the 4-phenyl-2-aminothiazole scaffold
donor, with the Asp220 and Asp222 residues, just like arises as a non-nitroaromatic alternative to the develop-
observed for the co-crystallized ligand (Fig. 1a). Com- ment of new antileishmanial drugs.
pound 6 also positioned its aromatic systems suitably to
establish pi-stacking interactions with Phe117, which, Conclusion
once more, mimics the 5′-deoxy-5′-methylthioadenosine Considering the results herein reported, the 4-phenyl
binding mode. The tert-butyl group from the 4-phenyl -2-aminothiazoles might be potential scaffolds to be
ring was positioned close to a set of hydrophobic resi- explored in the search for new antileishmanial hits. Four
dues, Thr18, Pro69 and Val231, occupying the same site out of the eight tested compounds exhibited important
of the methylthio moiety from the co-crystallized ligand. anti-promastigote activity associated with good selectiv-
Conversely, no interaction between compound 6 and ity indexes, except in the cases of compounds 1 and 6
Met196 was observed.
(in the latter, only when considering its activity against
Compounds 3, 4 and 5 were also theoretically docked L929 cells).
to the phosphorylase and exhibited the same pattern of
This is the first report of their effectiveness against the
interactions described above when regarding their L. amazonensis, one of the species of leishmania respon-
most frequent poses, 96%, 94% and 100%, respectively sible for the cutaneous form of the disease. Currently,
(
Additional file 5).
studies using the most promising compounds have been
Literature on S-methyl-5-thioadenosine phosphorylase conducted to evaluate their effects on the amastigote
expression in different cell lines reports that almost all form of the parasite, which is found in the mammalian
non-neoplastic mammal cells express this enzyme [26–28]. host. Such tests are important and fundamental for a
Most of the S-methyl-5-thioadenosine phosphorylase better evaluation of the applicability of the compounds
lacking cells are malignant cells, as discussed by for the treatment of leishmaniasis.
Kamatani et al. [29].
The 4-phenyl-2-aminothiazole scaffold arises, as
Even though these studies were performed taking the discussed, as a non-nitroaromatic alternative to the
human enzyme into consideration, the results could be development of new antileishmanial drugs. As scaffolds,
used as a guide to future design new antileishmanial these structures could be rationally modified to present
compounds. For instance, homology modeling studies selectivity along with improved antileishmanial activity,
can be useful in order to improve such studies.
since the differences between the binding sites of the
Derivatives of 2-aminothiazoles have been reported as human and leishmania phosphorylases can be explored.
predominantly active against trypanosomes, such as in
the case of Kaiser et al., but they have not shown expres-
sive activity against leishmanias. Nearly all literature
Additional files
reports are based on studies with L. donovani or L.
infantum, both responsible for the visceral leishmaniasis,
since this is the lethal form of the disease [9].
Additional file 1: GC-MS results, under the following conditions: Inlet
temperature: 250 °C; Oven: initial temperature 80 °C, 10 °C/min up to
250 °C, kept for 13 min; Column RTX-5MS (30 m × 0.25 mm × 0.25 μm).
(
DOCX 342 kb)
Unfortunately, cutaneous leishmaniasis is thought to
be less dangerous, and because of it, it is believed to
potentially lead to a spontaneous cure. Several cases,
however, evolve to the mucocutaneous form of the
Additional file 2: Structure-Activity Relationships. (a) CLogP versus
Biological Activity Graphic. Parabolic Correlation coefficient, r = 0.968. (b)
MR versus Biological Activity Graphic. Parabolic Correlation coefficient, r
2
2
=
0.945. (DOCX 40 kb)

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Additional file 4: Electrostatic potential maps. (DOCX 2496 kb)
Additional file 5: GPQF-03, GPQF-04, and GPQF-05 best docking poses
at S-methyl-5-thioadenosine phosphorylase (1CG6). (DOCX 284 kb)
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The authors are grateful to FAPESP for the financial support (FAPESP 2013/
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1875-0), and to CAPES and CNPq for the grants.
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Gorczynski MJ, Leal RM, Mooberry SL, Bushweller JH, Brown ML. Synthesis
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The authors would like to thank the São Paulo Research Foundation
(
FAPESP) for the financial support (FAPESP 2013/01875–0) and for the
software licenses, when applicable; and the Coordination for the
Improvement of Higher Education Personnel (CAPES) and the National
Council for Scientific and Technological Development (CNPq) for the grants
of MOLC and PFS (PIBIC).
The publication of this work was also partially supported by the
Coordination for the Improvement of Higher Education Personnel (CAPES)
through Programa Editoração CAPES (edital n. 13/2016, auxílio n. 0722/2017,
processo n. 88881.142062/2017–01) and by the National Council for Scientific
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1
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2
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The authors contributed equally to the final version of the manuscript. CAR,
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