Antileishmanial thioureas: Synthesis, biological activity and in Silico evaluations of new promising derivatives

Article abstract of DOI:10.1248/cpb.c17-00293

Leishmaniasis is a neglected tropical disease caused by protozoan parasites belonging to the genus Leishmania. Currently, the drugs available for treatment of this disease present high toxicity, along with development of parasite resistance. In order to overcome these problems, efforts have been made to search for new and more effective leishmanicidal drugs. The aim of this study was to synthesize and investigate the leishmanicidal effect of N,N′-disubstituted thioureas against Leishmania amazonensis, with evaluation of their in silico pharmacokinetics and toxicity profiles. Our results showed that different thioureas could be obtained in high to moderate yields using simple reaction conditions. Nine thiourea derivatives (3e, 3i, 3k, 3l, 3p, 3q, 3v, 3x and 3z) were active against parasite promastigotes (IC₅₀ 21.48–189.10μM), with low cytotoxicity on mice peritoneal macrophages (CC₅₀>200μM), except for thiourea 3e (CC₅₀=49.22μM). After that, the most promising thioureas (3k, 3l, 3p, 3q and 3v) showed IC₅₀ ranging from 70 to 150μM against L. amazonensis amastigotes in infected macrophages. Except for thiourea 3p, the leishmanicidal activity of the derivatives were independent of nitric oxide (NO) production. Thioureas 3q and 3v affected promastigotes cell cycle without disturbing the mitochondrial membrane potential. Furthermore, our derivatives showed satisfactory theoretical absorption, distribution, metabolism, excretion, toxicity (ADMET) properties. These data indicate that thiourea derivatives are good candidates as leading compounds for the development of new leishmanicidal drugs.

Full text of DOI:10.1248/cpb.c17-00293

Vol. 65, No. 10
Chem. Pharm. Bull. 65, 911–919 (2017)
911
Regular Article
Antileishmanial Thioureas: Synthesis, Biological Activity and in Silico
Evaluations of New Promising Derivatives
Gil Mendes Viana,a,# Deivid Costa Soares,b,# Marcos Vinicius Santana,c
Lilian Henriques do Amaral,a Paloma Wetler Meireles,a Raquel Pinto Nunes,a
Luiz Cláudio Rodrigues Pereira da Silva,a Lúcia Cruz de Sequeira Aguiar,d
Carlos Rangel Rodrigues,e Valeria Pereira de Sousa,a Helena Carla Castro,c
,a
Paula Alvarez Abreu,f Plínio Cunha Sathler,a Elvira Maria Saraiva,*,b and Lucio Mendes Cabral*
a Universidade Federal do Rio de Janeiro, LabTIF, Faculdade de Farmácia, Ilha do Fundão; CEP 21941–902, Rio
b
de Janeiro, RJ, Brazil: Universidade Federal do Rio de Janeiro, Laboratório de Imunobiologia das Leishmanioses,
c
Instituto de Microbiologia Paulo de Góes; CEP 21941–902 Rio de Janeiro, RJ, Brazil: Universidade Federal
Fluminense, LABiEMol, Departamento de Biologia Celular e Molecular, Outeiro de São João Baptista; CEP 24020–
d
141, Niterói, RJ, Brazil: Universidade Federal do Rio de Janeiro, Instituto de Química, Ilha do Fundão; CEP 21941–
e
909, Rio de Janeiro, RJ, Brazil: Universidade Federal do Rio de Janeiro, ModMolQSAR, Faculdade de Farmácia,
f
Ilha do Fundão; CEP 21941–902, Rio de Janeiro, RJ, Brazil: and Laboratório de Modelagem Molecular e Pesquisa
em Ciências Farmacêuticas, LaMCiFar. Universidade Federal do Rio de Janeiro-Campus Macaé; Av. São José do
Barreto, Macaé 27965–045, RJ, Brazil.
Received April 7, 2017; accepted July 10, 2017
Leishmaniasis is a neglected tropical disease caused by protozoan parasites belonging to the genus
Leishmania. Currently, the drugs available for treatment of this disease present high toxicity, along with
development of parasite resistance. In order to overcome these problems, efforts have been made to search
for new and more effective leishmanicidal drugs. The aim of this study was to synthesize and investigate
the leishmanicidal effect of N,N′-disubstituted thioureas against Leishmania amazonensis, with evaluation
of their in silico pharmacokinetics and toxicity profiles. Our results showed that different thioureas could
be obtained in high to moderate yields using simple reaction conditions. Nine thiourea derivatives (3e, 3i,
3k, 3l, 3p, 3q, 3v, 3x and 3z) were active against parasite promastigotes (IC50 21.48–189.10 µM), with low
cytotoxicity on mice peritoneal macrophages (CC50>200µM), except for thiourea 3e (CC50=49.22 µM). After
that, the most promising thioureas (3k, 3l, 3p, 3q and 3v) showed IC50 ranging from 70 to 150µM against L.
amazonensis amastigotes in infected macrophages. Except for thiourea 3p, the leishmanicidal activity of the
derivatives were independent of nitric oxide (NO) production. Thioureas 3q and 3v affected promastigotes
cell cycle without disturbing the mitochondrial membrane potential. Furthermore, our derivatives showed
satisfactory theoretical absorption, distribution, metabolism, excretion, toxicity (ADMET) properties. These
data indicate that thiourea derivatives are good candidates as leading compounds for the development of new
leishmanicidal drugs.
Key words thiourea; antileishmanial; in silico evaluation
Leishmaniasis comprises a group of neglected tropical
In order to overcome this challenging problem, there is an
diseases caused by parasites of the genus Leishmania and is urgent need for the development of new leishmanicidal com-
transmitted by the bite of phlebotomine female sand flies. This pounds, which is stimulated by Drugs for Neglected Diseases
disease remains as a major public health issue occurring in Initiative (DNDi), a non-profit drug research and development
98 countries distributed in 5 continents, with approximately organization.4)
1.7 million symptomatic people annually, most of them poor
Thioureas are an important class of substances contain-
people.1)
ing sulfur, widely described in literature.5–13) Due to its great
Treatment for this disease is available; however, like most versatility, these substances have been applied in medicinal
infectious diseases, cost, toxicity, and resistance are constant chemistry. Thiourea compounds have proven recently as
obstacles of mass treatment. Pentavalent antimonials (Sb+5) promising new drugs and its broad activities spectrum has
remain as the first choice treatment, regardless of their toxicity been quite investigated, including: anti-human immunode-
and low patient tolerance, followed by pentamidine or ampho- ficiency virus (HIV),14) anticancer,15) anti-diabetes,13) anti-
tericin B as second line treatments.2) Miltefosine is the first hepatitis C virus (HCV)6) and antimicrobial agents.7) Recently,
oral drug approved for treatment of visceral leishmaniasis, our group reported the antiplatelet activity of alkyl/aryl asym-
however, it has been reported low efficacy against cutaneous metric thioureas.8)
leishmaniasis and side effects such as vomiting, diarrhea, and
Thiourea derivatives have already been identified as poten-
transient liver enzyme level elevation, besides its teratogenic- tial antiprotozoal agents. Several works demonstrate their ac-
ity.3)
tivity against Plasmodium falciparum,11,16) Trichomonas vagi-
nalis,17) Trypanosoma cruzi16,18) and Leishmania infantum,18)
and some examples of these compounds are given in Fig. 1.
# These authors contributed equally to this work.
*To whom correspondence should be addressed. e-mail: esaraiva@micro.ufrj.br; lmcabral@pharma.ufrj.br
© 2017 The Pharmaceutical Society of Japan

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Chem. Pharm. Bull.
Vol. 65, No. 10 (2017)
Fig. 1. Thiourea Derivatives as Antiprotozoal Agents
Table 1. Reaction Conditions and Yields for Thioureas 3a–z
Thioureaa)
Methodb)
Time (h)
Yield (%)c)
R
R1
R2
n
3a
3b
3c
3d
3e
3f
A
A
A
A
A
A
B
B
B
B
B
B
A
A
B
B
B
A
A
A
A
B
B
A
B
B
4.0
2.5
6.0
7.0
7.0
8.0
4.0
4.5
4.0
8.0
10.0
7.0
3.0
6.0
4.0
10.0
10.0
5.0
4.5
4.0
5.5
12.0
8.0
4.0
6.0
8.0
98
98
95
91
78
90
97
80
82
82
92
86
93
95
95
85
70
90
89
87
88
94
87
80
82
84
H
Me
H
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
2
2
2
2
2
2
0
0
0
H
iPr
H
H
Et
Et
H
CH2CH2OH
H
H
CH2CH2OH
CH2CH2OH
H
Cy
H
H
H
H
H
H
H
H
Et
H
H
Ph
H
H
Et
H
H
H
H
H
H
3g
3h
3i
H
Ph
H
2,5-OMe-Ph
H
2,4-OMe-Ph
3j
H
3,4,5-OMe-Ph
3k
3l
H
3,4-Methylenedioxy-Ph
H
α-Naph
3m
3n
3o
3p
3q
3r
3s
H
iPr
H
Et
H
Ph
H
3,4-Methylenedioxy-Ph
H
Ph
H
iPr
H
Bu
3t
H
Et
Cy
3u
3v
3w
3x
3y
3z
H
H
3,4-Methylenedioxy-Ph
α-Naph
H
3,4,5-OMe
3,4,5-OMe
3,4,5-OMe
Cy
3,4,5-OMe-Ph
3,4-Methylenedioxy-Ph
a) Products were characterized by physical and spectroscopic methods. b) Method A: Isothiocyanate (1.0mmol), amine (1.2mmol), dichloromethane, room temperature (r.t.)
Method B: Isothiocyanate (1.0mmol), amine (1.2mmol), tert-butanol, reflux. c) Isolated yield.
In view of these observations, we have designed and syn-
Experimental
thesized thiourea derivatives (3a–z, Table 1) to investigate
Reagents All reagents were purchased and used without
their leishmanicidal activities and performed an in silico eval- further purification. 1H-NMR and 13C-NMR spectra were
uation of structure–activity relationship and pharmacokinetics recorded on a Bruker Avance 200MHz spectrometer using
and toxicity profiles.
CDCl3 or dimethyl sulfoxide (DMSO)-d6 as solvent. Chemical
shifts were given in ppm (δ scale) and coupling constants (J)
were given in hertz (Hz). Infrared (IR) spectra were recorded

Vol. 65, No. 10 (2017)
Chem. Pharm. Bull.
913
on a Shimadzu IRPrestige-21 FTIR spectrometer using KBr
pellets. High-resolution (HR)-MS were obtained on a Bruker
N-(2,4-Dimethoxyphenyl)-N′-phenylthiourea (3i)
Gray solid; mp 125–127°C; IR (KBr): 3376, 3162, 3000,
micrOTOF II mass spectrometer using electrospray ionization 1548, 1517, 1313, 1285, 1247, 1212, 1161, 1126, 1040, 506cm−1;
(ESI). Melting points were measured on a Shimadzu DSC-60 1H-NMR (CDCl3) δ: 8.08 (brs, 1H), 7.85 (brs, 1H), 7.74
thermal analyzer at a heating rate of 10°C/min, room tempera- (d, J=9.0Hz, 1H); 7.43–7.35 (m, 4H), 7.31–7.20 (m, 1H),
ture to 200°C under a nitrogen flow rate of 50mL/min and 6.55–6.43 (m, 2H); 13C-NMR (50MHz, CDCl3) δ: 179.8, 159.3,
using an aluminum standard. Analytical TLC was performed 153.7, 137.5, 129.6, 127.0, 126.8, 125.2, 119.7, 104.3, 99.4,
on precoated silica gel plates (aluminum sheets 60 F254, 56.0, 55.7; HR-MS-ESI: m/z [M+Na]+ Calcd for C15H16N2O2S:
Merck) using ethyl acetate–hexane (1:5 v/v) as the eluent.
Preparation of Thioureas 3a–z
Method A
311.0825. Found: 311.0814.
N-Benzyl-N′-phenylthiourea (3o)21)
Brown solid; mp 154–156°C (Lit.21) 153–154°C); IR (KBr):
To a solution of isothiocyanate 1 (1.0mmol) in dichlo- 3366, 3152, 1545, 1510, 1297, 1247, 973, 744, 695cm−1;
romethane (10mL) was added the corresponding amine 2 1H-NMR (CDCl3) δ: 8.25 (brs, 1H), 7.47–7.18 (m, 10H), 6.33
(1.2mmol), and the mixture was stirred at room temperature (brs, 1H), 4.88 (d, J=5.5Hz, 2H); 13C-NMR (CDCl3) δ: 181.0,
until isothiocyanate was consumed (TLC). After that, the or- 137.4, 136.2, 130.3, 128.9, 127.8, 127.7, 127.4, 125.4, 49.5; HR-
ganic phase was washed with 5% aqueous hydrochloric acid MS-ESI: m/z [M+Na]+ Calcd for C14H14N2S: 265.0770. Found:
(3×10mL), dried with anhydrous sodium sulfate and evaporat- 265.0765.
ed in a rotary evaporator to afford the pure thiourea 3, which
did not require any further purification.
Method B
N-Benzyl-N′-N′-diphenylthiourea (3q)
Brown solid; mp 176–179°C; IR (KBr): 3379, 1587, 1507,
1494, 1340, 746, 706, 692, 626cm−1; 1H-NMR (CDCl3) δ:
To a solution of isothiocyanate 1 (1.0mmol) in tert-butanol 7.60–7.25 (m, 15H), 6.05 (brs, 1H), 4.90 (d, J=5.3Hz, 2H);
(10mL) was added the corresponding amine 2 (1.2mmol), and 13C-NMR (CDCl3) δ: 183.4, 144.1, 137.7, 129.9 (2C), 128.8,
the mixture was refluxed until isothiocyanate was consumed 128.5 (2C), 127.7, 50.0; HR-MS-ESI: m/z [M+Na]+ Calcd for
(TLC). Then, the solvent was evaporated, 10mL of CH2Cl2 C20H18N2S: 341.1083. Found: 341.1076.
was added and the same work up used in method A was fol-
lowed.
N-Cyclohexyl-N′-(3,4,5-trimethoxyphenyl)thiourea (3x)
White solid; mp 139–141°C; IR (KBr): 3348, 2940, 2848,
The following shows the characterization data of thiourea 1603, 1534, 1508, 1232, 1125, 1008, 987cm−1; 1H-NMR
derivatives, except for thioureas 3a, 3c–d, 3f, 3j–n, 3p, 3r–w (CDCl3) δ: 8.16 (brs, 1H), 6.43 (s, 2H), 5.99 (brd, J=8.3Hz,
and 3z, since these data have been previously reported.8)
N-Isopropyl-N′-phenylthiourea (3b)19)
1H), 4.35–4.12 (m, 1H), 3.85–3.79 (m, 9H), 2.07–0.98 (m,
10H); 13C-NMR (CDCl3) δ: 179.0, 154.0, 136.8, 131.7, 102.6,
Pale yellow solid; mp 99–103°C; IR (KBr): 3272, 3246, 60.8, 56.1, 53.8, 32.5, 25.3, 24.6; HR-MS-ESI: m/z [M+Na]+
2970, 1545, 1501, 1396, 1238, 778, 693cm−1; 1H-NMR Calcd for C16H24O3N2S: 347.1400. Found: 347.1390.
(CDCl3) δ: 8.25 (brs, 1H), 7.46–7.36 (m, 2H), 7.32–7.15 (m,
3H), 5.85 (brs, 1H), 4.72–4.42 (m, 1H), 1.19 (d, J=6.5Hz,
N-N-Bis(3,4,5-trimethoxyphenyl)thiourea (3y)
Pale brown solid; mp 197–199°C; IR (KBr): 3283, 3161,
6H); 13C-NMR (CDCl3) δ: 179.5, 136.6, 130.5, 127.3, 125.3, 2959, 2933, 1603, 1544, 1501, 1455, 1340, 1233, 1135,
47.7, 22.7; HR-MS-ESI: m/z [M+Na]+ Calcd for C10H14N2S: 1004 cm−1; 1H-NMR (CDCl3) δ: 7.97 (brs, 2H), 6.65 (s,
217.0770. Found: 217.0770.
4H), 3.84 (s, 18H); 13C-NMR (CDCl3) δ: 179.9, 153.9, 137.2,
132.8, 103.2, 61.1, 56.5; HR-MS-ESI: m/z [M+Na]+ Calcd for
N-N-Bis(2-hydroxyethyl)-N′-phenylthiourea (3e)
Yellow oil; IR (KBr): 3278, 2935, 2880, 1643, 1598, 1554, C19H24O6N2S: 431.1247. Found: 431.1255.
1
1536, 1359, 1254, 1209, 1070, 1041, 758, 694cm−1; H-NMR
Ethics Statement All animal experiments were performed
(CDCl3) δ: 9.58 (brs, 1H), 7.51–6.99 (m, 5H), 3.87 (s, 8H); in strict accordance with the Brazilian animal protection law
13C-NMR (50MHz, CDCl3) δ: 184.71, 140.20, 128.68, 125.10, (Lei Arouca number 11.794/08) of the National Council for
124.23, 61.38, 54.07; HR-MS-ESI: m/z [M+Na]+ Calcd for the Control of Animal Experimentation (CONCEA, Brazil).
C11H16N2O2S: 263.0825. Found: 263.0827.
The protocol was approved by the Committee for Animal Use
Ethics of the Universidade Federal do Rio de Janeiro (Permit
N-N-Diphenylthiourea (3g)20)
White solid; mp 153–156°C (Lit.20) 153–154°C); IR (KBr): Number: 128/15).
3207, 3037, 3005, 1600, 1549, 1495, 1450, 1348, 758, 698,
Anti-promastigote Activity Leishmania amazonensis
631 cm−1; 1H-NMR (CDCl3) δ: 8.13 (brs, 2H), 7.45–7.21 (WHOM/BR/75/Josefa) promastigotes were cultured at 26°C
(m, 10H); 13C-NMR (50MHz, CDCl3) δ: 179.7, 137.1, 129.5, in Schneider insect medium (Sigma-Aldrich, St. Louis, MO,
126.9, 125.2; HR-MS-ESI: m/z [M+Na]+ Calcd for C13H12N2S: U.S.A.), 10% fetal calf serum (FCS, Gibco-BRL, MD, U.S.A.)
251.0613. Found: 251.0621.
N-(2,5-Dimethoxyphenyl)-N′-phenylthiourea (3h)
and 40µg/mL of gentamycin (Schering-Plough, Rio de Ja-
neiro, Brazil).
The leishmanicidal properties of thioureas 3a–z were
Brown solid; mp 134–136°C; IR (KBr): 3347, 3164, 1593,
1
1545, 1506, 1374, 1282, 1048, 1023, 755, 726cm−1; H-NMR evaluated by measuring promastigote viability through the
(CDCl3) δ: 8.29–8.22 (m, 2H), 8.07 (d, J=2.8Hz, 1H), dehydrogenases activity using the XTT method (2,3 bis-[2-
7.48–7.24 (m, 5H), 6.78 (d, J=8.9Hz, 1H), 6.65 (dd, J=8.9Hz methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxani-
and J=2.8Hz, 1H), 3.78 (s, 3H), 3.72 (s, 3H); 13C-NMR lida; Sigma-Aldrich) as described.22) Briefly, stationary-phase
(50MHz, CDCl3) δ: 178.75, 153.80, 145.07, 137.08, 130.05, promastigotes were treated with the thioureas for 48h at 26°C,
128.10, 127.40, 125.47, 112.14, 111.11, 109.71, 56.75, 56.17; and then incubated with XTT activated with phenazine metho-
HR-MS-ESI: m/z [M+Na]+ Calcd for C15H16N2O2S: 311.0825. sulfate (Sigma-Aldrich) for 3h. The reaction product was read
Found: 311.0828.
at 450nm. Meglumine antimoniate (MA) was used as refer-

914
Chem. Pharm. Bull.
Vol. 65, No. 10 (2017)
ence compound.
PBS, fixed in 70% (v/v) ice-cold methanol/PBS for 1h at 4°C,
Anti-amastigote Activity Mice peritoneal macrophages washed again once with PBS and then incubated in PBS sup-
obtained after stimulation with 3% thioglycolate for 3d were plemented with 10µg/mL propidium iodide (PI) and 20µg/mL
harvested in RPMI 1640 medium (LGC Biotec, São Paulo, ribonuclease (RNase) at 37°C for 45min, according to Ambit
Brazil) and cultured inside 24-well plates for 2h adherence et al.26) For each sample, 10000 events were collected on a BD
at 35°C, 5% CO2. Non-adherent cells were removed, and FACScalibur (Becton Dickinson, San Jose, CA, U.S.A.) and
macrophages were incubated overnight in RPMI-10% FCS as analyzed using CellQuest software.22)
above. Adhered macrophages were infected with Leishmania
Measurement of Mitochondrial Membrane Potential
amazonensis promastigotes (stationary growth phase) at a (∆Ψm) Promastigotes were treated or not with 100µM 3k,
10:1 parasite/macrophage ratio during 1h at 35°C, 5% CO2. 3l, 3p, 3q or 3v for 48h and then incubated with JC-1 solu-
Free parasites were washed out with 0.01M phosphate buffered tion (5µg/mL, Sigma-Aldrich) for 20min at 37°C according
saline (PBS), and the cultures maintained for 24h at 35°C, 5% to the manufacturer’s instructions. ∆Ψm was measured in
CO2. Infected macrophages cultures were treated with differ- 96-well opaque plates using 490/530nm excitation/emission
ent concentrations of the thiourea derivatives during 24h at (JC-1 monomers) and 525/590nm excitation/emission (J-
the same culture conditions mentioned above. Cultures were aggregates) in a SpectraMax Paradigm (Molecular Devices),
washed with PBS and incubated with 0.01% sodium dodecyl as described.27)
sulfate for 10min followed by 1mL of Schneider’s medium
supplemented with 10% FCS, and maintained at 26°C for
2d. The viable intracellular L. amazonensis amastigotes, that
Molecular Modeling
Structure–Activity Relationship (SAR) Studies
All molecular computations were performed using
survived after treatment, were measured after promastigote SPARTAN’10 (Wavefunction Inc., Irvine. CA, U.S.A.). Brief-
transformation, using Alamar Blue (Invitrogen), as previously ly, the structures were optimized to a local minimum and the
described.23) After 4h of Alamar Blue incubation, the fluores- equilibrium geometry was obtained in a vacuum using RM1
cence was read at 540/610nm excitation/emission wavelenghts semi empirical methods. Subsequently the structures were
in a SpectraMax Paradigm (Molecular Devices). MA was used submitted to a single-point energy Density Functional Theory
as reference compound.
(DFT) calculation using B3LYP/6-311G* method to calculate
Cytotoxicity for Host Macrophages Thioglycolate-stim- some stereoelectronic properties and to perform SAR studies.
ulated mice peritoneal macrophages adhered to 96-well plates Thus, we calculated the descriptors: highest occupied molecu-
obtained as above were treated with different concentrations lar orbital (HOMO) and lowest unoccupied molecular orbital
of thioureas for 24h, and cell viability was determined using (LUMO) energies, molecular weight (MW), molecular surface
1mg/mL XTT (2,3-Bis[2-Methoxy-4-nitro-5-sulfophenyl]-2H- area, polar surface area (PSA), dipole moment and lipophilic-
tetrazolium-5-carboxinilide inner salt, Sigma-Aldrich), 200µM ity (cLogP).28)
Phenazine Methosulfate (PMS). After 3h incubation, the reac-
In Silico Absorption, Distribution, Metabolism, Excretion,
tion product was read at 450nm. The results are expressed in Toxicity (ADMET) Studies
percentage of viable cells compared to untreated control.24)
MA was used as reference compound.
The structures of the thiourea derivatives were screened for
theoretical oral bioavailability presence of Pan assay interfer-
Nitric Oxide (NO) Production Thioglycolate-stimulated ing compounds (PAINS) and toxicophores with FAF-DRUGS3
mice peritoneal macrophages were obtained as above (106 server using the Lead-like physicochemical filter available on
cells/well in 24-well plate) and activated with 1µg/mL li- the server (http://fafdrugs3.mti.univ-paris-diderot.fr/).29) Be-
popolysaccharide (LPS) and 100ng/mL interferon-γ (IFN-γ) sides, the toxicity risks of the compounds were analyzed using
(Sigma-Aldrich) or left untreated. After incubating the cells Osiris Property Explorer (http://www.organicchemistry.org/
for 24h at 37°C, 5% CO2, they were treated with 100µM of prog/peo/). The toxicity risks are evaluated as low medium or
3k, 3l, 3p, 3q or 3v. The nitrite concentrations in the culture high risk based on the Registry of Toxic Effects of Chemical
supernatants were determined using the Griess method. The Substances (RTECS) database.
reaction was read at 540nm, and the concentration of nitrite
determined using a standard curve of sodium nitrite. The re-
Results
Chemistry A series of 26 N,N′-disubstituted thioureas
sults are expressed as micromolar concentrations of nitrite.25)
Cell Cycle Analysis Promastigotes were incubated in was synthesized according to Chart 1. Thioureas 3a–z were
Schneider’s complete medium, with or without 100µM 3k, prepared through the reaction of the isothiocyanate 1 and an
3l, 3p, 3q or 3v for 48h. The cells were then washed with excess of the appropriate amine 2, in a dichloromethane or
Chart 1. Synthesis of Thiourea Derivatives 3a–z

Vol. 65, No. 10 (2017)
Chem. Pharm. Bull.
915
tert-butanol solution (Table 1). The products were obtained in of thioureas during 48h and parasite viability evaluated. By
good to excellent yields (70–98%) and no further purification this initial screening, thioureas 3e, 3i, 3k, 3l, 3p, 3q, 3v, 3x,
was required after isolation from the crude reaction mixture. 3y and 3z were active (data not shown), and then selected for
The proposed structures of all prepared thioureas were con- IC50 determination. As shown in Table 2, nine thioureas pre-
1
firmed by H-NMR, 13C-NMR, FT-IR and HR-MS.
sented IC50 values against promastigotes under 200µM, rang-
Activity of the Thioureas Initially, the antileishmanial ing between 21.48 and 189.10µM.
activity of the thioureas 3a–z was determined in vitro against
Next, the safety of the ten selected thioureas on host mac-
Leishmania amazonensis promastigotes using the XTT assay. rophages was accessed, evaluating the dehydrogenases activity
Thus, stationary-phase promastigotes were treated with 100µM using the XTT method. Our results showed that macrophage
treatment with all thioureas only affected dehydrogenase
activities at higher concentrations (CC50>200µM), except for
Table 2. In vitro Antileishmanial Activity and Cytotoxicity Results for
thiourea 3e, which CC50 was 49.22µM, and thus it was ex-
Thiourea Derivatives
cluded from further tests (Table 2).
Following we assessed the activity of the most promis-
ing thiourea derivatives (3k, 3l, 3p, 3q and 3v) against L.
amazonensis amastigotes, which is the intracellular stage
of the parasite responsible for the clinical manifestations of
leishmaniasis. Hence, Leishmania-infected macrophages were
treated with thiourea derivatives during 24h resulting in IC50
values ranging from 70 to 150µM, indicating the relevance of
this class of compounds in the search for new leishmanicidal
drugs. Among these compounds the lowest IC50 were obtained
for 3q and 3v, 81.4 and 70.0µM, respectively, presenting
greater effectiveness than MA (IC50=212.3 µM), a drug used in
clinical practice (Table 2).
Macrophages 50% cytotoxic Promastigotes
Amastigotes
IC50 (µM)
Thiourea
concentration (µM)
IC50 (µM)
3e
3i
49.22
>200
>200
>200
>200
>200
>200
>200
>200
26.04 1.61
127.50 12.81
80.09 13.87
21.94 1.19
46.08 8.791
21.48 0.001
54.14 6.769
139.60 10.84
>200
ND
ND
3k
3l
150 20
110 12.14
145 4.70
81.4 10.54
70.0 6.15
ND
3p
3q
3v
3x
3y
3z
MA
ND
>200
189.10 7.9
ND
174 38.9
ND
212.30 3.7
Thiourea derivatives tested against amastigotes were also
investigated by its ability to modulate NO production, and
ND=Not Determined. MA=Meglumine antimoniate.
Fig. 2. Role of Thioureas on NO Production
Macrophages infected with Leishmania amazonensis were stimulated or not with IFN-γ+LPS and incubated in the presence or absence of 100µM of 3k (A), 3l (B), 3p
(C), 3q (D) or 3v (E). NO production was evaluated after 48h of treatment by the Griess method. The results of three independent experiments performed in duplicate
are shown as the mean nitrite concentration standard error of the mean (S.E.M.). ***p<0.0001 in relation of control (CT) and **p<0.0001 in relation of untreated or
IFN-γ+LPS controls.

916
Chem. Pharm. Bull.
Vol. 65, No. 10 (2017)
Table 3. Effect of Thioureas on Leishmania amazonensis Promastigotes Cell Cycle
% Cells in different stages of the cell cycle#
Treatment
Sub-G0/G1
G0/G1
S
G2/M
Control
3k
0.866 0.1281
3.810 0.3483**
3.110 0.900*
1.700 0.3408
1.447 0.1988*
5.280 0.5658**
35.38 2.073
35.84 0.9020
36.06 1.925
38.66 1.811
27.00 0.9526
30.39 1.883
12.72 0.8517
4.23 0.3984***
9.47 1.190**
8.01 1.626
28.77 1.092
25.42 0.4768*
27.14 1.780
23.05 0.6895*
31.82 0.4719
23.86 3.300
3l
3p
3q
11.24 1.250
3v
8.15 0.9355***
All thioureas were tested at 100µM. # Results are the mean S.E.M. of four independent experiments. *p<0.05, **p<0.01, ***p<0.001, in relation to control.
effect on the parasite cell cycle and mitochondrial membrane
potential (ΔΨm). NO is an important mediator of Leishmania
death into the macrophage phagolysosomes, therefore we
investigated the capacity of thiourea derivatives to modulate
NO production in infected macrophages stimulated or not with
IFN-γ+LPS. Results showed that leishmanicidal activity of
derivatives is independent of NO production, since there was a
decrease of 81, 69, 100 and 79% in NO production on unstim-
ulated infected macrophages treated with 100µM of 3k, 3l, 3q
and 3v, respectively. Among all thiourea compounds tested,
only 3p, was able to modulate NO production in IFN-γ+LPS-
stimulated infected macrophages (Fig. 2).
To further investigate the leishmanicidal effect of thiourea
derivatives we analyzed its effect on the parasite cell cycle.
Our results demonstrated that promastigotes treated with
100 µM of 3k, 3l, 3q and 3v during 48h, increased 4.40, 3.59,
1.67 and 6.09-fold respectively, cells in Sub-G0/G1 phase.
Fig. 3. Mitochondrial Membrane Potential of Leishmania amazonensis
Promastigotes Treated with 3k, 3l, 3p, 3q and 3v Thioureas
The mitochondrial membrane potential (ΔΨm) was measured by JC-1 assay after
treating promastigotes with 3k, 3l, 3p, 3q and 3v at 100µM for 24h. Miltefosine at
30µM was used as positive control and untreated promastigotes were the negative
control (CT). Results represent the mean S.E.M. of 3 independent experiments.
Cells in S phase were decreased 3.00, 1.34 and 1.56-fold after
treatment with 3k, 3l and 3v, respectively. Derivatives 3k and
*p<0.05 and **p<0.02, in relation to control.
3p decreased 1.13- and 1.24-fold, respectively, cells in G2/M
phase (Table 3). In a previous work published by our group30)
amphotericin B (0.1µM) did not affect the cell cycle of the position, the activity was abolished. It is important to notice
parasite.
that changing substituents can affect the ability of the mole-
The single mitochondrion of Leishmania parasites is in- cule to interact with the target, besides, it also changes various
volved in death by apoptosis, and the increase in the number parameters such as its partition coefficient, electronic density,
of cells in the sub-G0/G1 hypodiploid DNA (Table 3) might steric environment, membrane permeability and others.32) We
represent promastigotes that undergo this type of death.31) observed that more than 5 hydrogen bond acceptors (HBA)
Thus, we measured whether thiourea derivatives induced pro- reduced activity on promastigotes and, in general, lower PSA
mastigote mitochondrial alteration by the reduction of the mi- and higher cLogP values were better for antileishmanial ac-
tochondrial membrane potential (ΔΨm) using JC-1 assay. Our tivity. Thiourea 3e was an exception, with one of the lowest
data showed that 3k and 3l reduced ΔΨm of promastigotes IC50 in promastigote (26.04µM) and highest PSA and lower
by 25 and 30%, respectively. Similarly, promastigotes treated cLogP values, but as it was toxic for macrophages, we con-
with 30µM miltefosine, which was used as a positive control, sidered it as a non-promising compound for further analysis.
had their ΔΨm reduced by 27% (Fig. 3).
Thiourea 3e was the only one with good activity in promas-
SAR
tigote that present aliphatic chain in R1 and R2. We observed
Herein we calculated some stereoeletronic properties of the that higher activity was achieved by compounds with aromatic
ten most active thiourea derivatives. The descriptors HOMO ring in R1. Derivatives bearing the rings benzo1,3)dioxole (3k,
and LUMO energies, lipophilicity (cLogP), molecular volume, 3v and 3p), naphthalene (3l) and 1,1-diphenyl (3q) showed the
molecular area, number of hydrogen bond donors (HBD), ac- best activities in promastigotes, pointing that more bulky rings
ceptors (HBA) groups and dipole moment were evaluated in this position could improve activity. Furthermore, deriva-
in order to correlate to the leishmanicidal activity (Table 4). tives bearing less bulky ring (i.e., phenyl ring or hexyl) in R1
The parameters related to molecular shape (Area, Volume and as 3f, 3g, 3h, 3j, 3o and 3u were inactive, while 3i, 3x and
PSA) and weight (MW) varied within the series but we did 3y showed some activity in promastigotes, but they were less
not observe a clear correlation between these parameters and expressive than others. Thiourea 3q (R1=R2=phenyl) revealed
activity. Although, in case of 3x, 3y and 3z (R=3,4,5-OMe), it the importance of the second substituent for the activity and
seems that higher MW, volume and area were correlated to the it can be shown by the comparison with the inactive 3g with
reduced activity of these compounds on promastigotes. Also, phenyl only in R1.
in case of 3j and 3y with 3,4,5-OMe-C6H5 substituent in R1
Thiourea 3v showed the best activity in amastigotes

Vol. 65, No. 10 (2017)
Chem. Pharm. Bull.
917
Table 4. Stereoelectronic Properties of N,N′-Disubstituted Thioureas Including: Molecular Weight (MW), Area (Å2), Volume (Å3), Polar Surface Area
(PSA, Å2), Dipole Moment (μ, Debye); HOMO and LUMO Energies (eV), Hydrogen Bond Acceptors (HBA), Hydrogen Bond Donors (HBD) and Octa-
nol–Water Partition Coefficient (cLogP)
#
MW
Volume (Å3) Area (Å2)
PSA (Å2)
DM
EHOMO (eV) ELUMO (eV)
HBA
HBD
cLogP
3e
3i
240.33
288.37
272.33
278.38
286.36
318.44
300.38
408.48
324.45
362.41
243.46
291.61
259.97
285.87
281.62
338.54
299.61
397.09
332.41
341.05
264.59
315.02
272.98
293.93
303.53
348.59
320.68
416.37
358.22
356.02
44.36
32.68
37.83
20.91
35.49
11.06
34.24
58.16
36.89
56.28
3.62
4.91
6.07
5.81
5.95
4.88
5.79
6.77
6.51
6.83
−5.36
−5.30
−5.51
−5.63
−5.57
−5.56
−5.52
−5.56
−5.50
−5.54
−0.70
−0.95
−1.11
−1.67
−0.82
−1.06
−0.83
−1.17
−0.81
−1.17
5
5
5
3
5
3
5
9
6
8
3
2
2
2
2
1
2
2
2
2
1.34
3.41
3.44
4.66
3.51
5.10
3.79
2.90
3.16
3.06
3k
3l
3p
3q
3v
3y
3x
3z
Table 5. Structural Alerts Identified with FAF-DRUGS3 and Analysis of GSK 4/400 (Higher Risks of Toxicity if LogP> 4 and MW>400)
Drug safety profile
#
GSK 4/400
Pfizer 3/75
Lilly rules
Alerts
3e
3i
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Good
Bad
Pass
Low risk thiourea
Low risk thiourea
Pass
3k
3l
Warning
Bad
Pass
Low risk benzodioxolane and thiourea
Low risk thiourea and cLogP
Warning-naphthalene
3p
3q
3v
3x
3y
3z
Warning
Bad
Pass
Pass
Pass
Pass
Pass
Pass
Low risk benzodioxolane and thiourea
Low risk thiourea and cLogP
Bad
Low risk benzodioxolane and thiourea
Low risk thiourea
Warning
Good
Good
Low risk thiourea and H-bond acceptors
Low risk benzodioxolane and thiourea
Pfizer 3/75 (lower toxicity when cLogP< 3 and (tPSA)>75Å2) and Lilly rules for the most active thiourea derivatives.
(IC50=70µM) and some features seemed to be important for promiscuous compounds in biochemical assays (PAINS) using
the activity, as the presence of benzo1,3) dioxole ring as well as FAF-DRUGS3.29)
the length of aliphatic chain, as can be seen by the compari-
son with 3k (IC50=150 µM) and 3p (IC50=145 µM).
FAF-DRUGS3 predicted a good theoretical oral bioavail-
ability for the most active compounds using Lipinski, Veber
Thiourea derivatives did not show toxicity on macrophages, and Egan rules. Our analysis of toxicophores and problematic
with CC50>200µM, with exception of 3e (CC50=49.22 µM), groups showed an alert of low risk associated with the thio-
which exhibited cytotoxic profile, despite its good activity urea group. In addition, no PAINS were identified indicat-
against promastigotes (IC50=26.04µM). Cytotoxic profile of 3e ing that their activity is might not be a false positive. The
is probably related to the primary alcohol as substituent in R1 drug safety of the most active compounds was promising
and/or R2. All other nontoxic thiourea derivatives presented with 3e, 3i, 3k, 3l, 3p, 3q, 3v, 3x, 3y and 3z passing GSK
aromatic substituents in R1 or hexyl group (e.g. 3x). In addi- 4/400 rule. On the other hand, most of these compounds did
tion, it was also possible to note that R1 and R2 substitution not pass Pfizer 3/75 rule since most of them have cLogP>3
by phenyl group resulted in compound 3q that also presented and PSA<75 Å2. Interestingly, derivatives with best activity
good cytotoxic profile.
against promastigotes, 3k, 3p, 3q and 3v, with exception of
In Silico ADMET Studies The most active thiourea 3l, passed Lilly rules pointing that these compounds could be
derivatives (3e, 3i, 3k, 3l, 3p, 3q, 3v, 3x, 3y and 3z) were promising, specially 3q and 3v, the most active compounds
screened for an ADMET study using FAF-DRUGS3 server. (Table 5).
The theoretical oral bioavailability was analyzed using
The ten most active thiourea derivatives were submitted to
Lipinski rule of Five (cLogP≤5, MW≤500Da, HBD≤5, a toxicity analysis using Osiris Property Explorer to identify
HBA≤10)33) Egan Rule (0≥tPSA≤132)34) and Veber Rule structures with mutagenic, tumorigenic, irritant and potential
(rotatable bonds≤10 and tPSA≤140 Å).35) Drug safety profile effects on reproduction. Only 3l showed high mutagenic and
of the derivatives was evaluated using well known rules devel- tumorigenic risks, related to the naphthalene ring. Interest-
oped by the pharmaceutical industry such as GSK 4/400 rule ingly, the naphthalene group is not included on FAF-DRUGS3
(higher risks of toxicity if cLogP>4 and MW>400),36) Pfizer list of toxicophores29) but this derivative was promptly rejected
3/75 rule (lower toxicity when cLogP<3 and (tPSA)>75 Å2)37) using Lilly rules for problematic compounds. The other com-
and a set of 275 rules applied by Lilly to identify compounds pounds presented no risks alert in Osiris. These results sug-
that may interfere with biological assays.38) Furthermore, we gested a satisfactory theoretical toxicity profile of the most ac-
analyzed the derivatives for the presence of toxicophores and tive compounds 3k, 3p, 3q and 3v pointing them as promising

918
Chem. Pharm. Bull.
Vol. 65, No. 10 (2017)
lead structures for future analysis.
We observed that higher activity was found for compounds
with aromatic and bulky ring in R1, lower PSA and higher
cLogP values were better for antileishmanial activity, while
Discussion
Here we report the leishmanicidal activity of thiourea de- higher HBA reduced activity on promastigotes. Also, we
rivatives against L. amazonensis, a New World species that found that cytotoxic profile of 3e is probably related to the
can cause cutaneous, diffuse cutaneous and visceral leish- primary alcohol as substituent. Together these features can
maniasis.39)
guide the synthesis of new derivatives. It is important to high-
It has been shown that different compounds affect the light that the analysis of compounds’ structural features and
leishmanial cell cycle, some with characteristics suggesting properties should be done together as in some cases there is
the induction of incidental death, an apoptosis-like mecha- no significant difference in molecular properties but the com-
nism.22,30,40) Therefore, we analyzed the effect of derivatives pound was not able to bind to the target.
on parasite cell cycle.
Our derivatives showed satisfactory theoretical ADMET
Among the 26 derivatives assayed, 3q and 3v were highly properties, fulfilling Lipinski, Veber and Egan bioavailabil-
active against amastigotes with low toxicity for host macro- ity guidelines, suggesting that they could be administered by
phages. The 3v derivative showed the lowest amastigote IC50 oral route. We also observed that these compounds were not
value and both, 3q and 3v derivatives affected promastigotes labeled as PAINS, indicating that the activity observed might
cell cycle without disturb the mitochondrial membrane poten- not be result of a false positive.45) Thus, our derivatives could
tial. Although their mechanism of action was not fully under- represent a starting point for new analogs series. However,
stood, the induction of promastigote apoptosis was evident in FAF-DRUGS3 flagged the thiourea moiety as problematic, due
by the cell cycle data. Interestingly, 3k and 3l significantly in- to oxidative metabolism and formation of reactive metabo-
creased the number of cells in sub G0/G1 phase and decreased lites.46) Our theoretical analysis of physicochemical properties,
the mitochondrial membrane potential, reflecting a pro apo- such as LogP, PSA and MW, showed that most derivatives
ptotic mechanism in promastigotes. On the other hand, these did not pass Pfizer 3/75 guideline due to high lipophilicity.
compounds exhibited lower activity than 3v and 3q against On the other hand, most active derivatives passed GSK 4/400
amastigotes.
and Lilly set of guidelines, suggesting that although our de-
Other groups have studied thiourea derivatives as antileish- rivatives do not have optimal physicochemical properties,
manial agents. Using a phenotypic fragment-based screening, they might be modified in order to search for better analogs.
Blaazer et al. identified compounds with thiourea fragments In addition, since these guidelines use different threshold
as promising antiprotozoal candidates. However, these com- for LogP, PSA and MW, thus it is expected that compounds
pounds did not show activity against Leishmania infantum present different results for each industrial guideline used
amastigotes. It is interesting to point that the prototype used in drug discovery.36–38) The analysis of ADMET properties
by this group was similar to our derivatives, bearing two late in preclinical or clinical stages results in higher costs in
substituents on nitrogens and varying the length between the drug design, while in silico evaluation of these properties in
thiourea group and substituents.41) Saudi et al. identified nine preliminary stages may help the selection of the most prom-
urea and thiourea derivatives with antileishmanial activity in ising compounds and elimination of problematic molecules
vitro (L. major promastigotes), and four of them also showed earlier. Several rules have been developed to select molecules
in vivo activity.42) Among 31 monosubstituted thiourea de- for optimization, they are very usefull but should not be used
rivatives which showed promastigote IC50 values above 100µM blindly to avoid discarding interesting molecules.29) Herein,
Boechat described three derivatives which were more than more than developing a new prototype, we described the an-
50-fold less active than pentamidine against L. amazonensis tileishmanial activity of thiourea derivatives and the potential
promastigotes and were not tested against amastigotes. In ad- of this class for further modifications to find compounds with
dition, authors proposed the enzyme arginase as a supposed higher activity. Overall, this study opens new possibilities of
target for those compounds.43)
exploring thiourea derivatives to improve and/or design new
NO production by macrophages is a potent mechanism antiparasitics.
involved in Leishmania killing, thus we tested the capacity
of the thioureas derivatives to modulate NO production by in-
Conclusion
fected macrophages. Although both 3q and 3v derivatives de-
In this report, we synthesized different N,N′-disubstituted
creased NO production of unstimulated infected macrophages, thioureas in high to moderate yields, using simple reaction
they did not change NO production in LPS-IFN-γ stimulated conditions. The thiourea derivatives exhibited anti-leishmanial
infected macrophages, suggesting that the leishmanicidal ac- activity against promastigote and amastigote forms of Leish-
tivity of these derivatives could occur by a mechanism differ- mania amazonensis. Thioureas 3q and 3v were the most ac-
ent from NO activation.
tive compounds against amastigote forms (81.4 and 70.0µM,
In this study, we used molecular modeling approaches to respectively), with low cytotoxicity on host macrophages.
understand the relationship of the structures of a series of These two thiourea derivatives did not change NO production,
thiourea derivatives and their antileishmanial activity. The but they affected promastigotes cell cycle, without disturbing
basis of SAR method is that structurally similar compounds the mitochondrial membrane potential. Besides, the thioureas
tend to show similar biological activity and based on this fact, of this study showed satisfactory in silico pharmacokinetics
it is possible to found the structural characteristics and prop- and toxicity profiles. The presented work shows that thiourea
erties correlated with the activity for a series of compounds derivatives may be used to search new compounds for leish-
that act in the same target, even when the potential target is maniasis treatment.
unknown.44)

Vol. 65, No. 10 (2017)
Chem. Pharm. Bull.
919
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ordenação de Aperfeiçoamento de Pessoal de Nível Superior
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