Synthesis and evaluation of the trypanocidal activity of a series of 1,3,4-thiadiazoles derivatives of R-(+)-limonene benzaldehyde-thiosemicarbazones

Article abstract of DOI:10.1007/s00044-016-1561-7

In this study, we synthesized a series of 1,3,4-thiadiazole derivatives of R-(+)-limonene benzaldehyde-thiosemicarbazones (2a–k). We also determined the cytotoxicity in LLCMK₂ cells and the activity against epimastigote and trypomastigote forms

Full text of DOI:10.1007/s00044-016-1561-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1561-7
ORIGINAL RESEARCH
Synthesis and evaluation of the trypanocidal activity of a series
of 1,3,4-thiadiazoles derivatives of R-(+)-limonene benzaldehyde-
thiosemicarbazones
2
2
•
Solange C. Martins1 Vania C. Desoti Danielle Lazarin-Bidoia
•
•
ˆ
´
3,4
•
•
•
´
Fabio Vandresen
Cleuza C. da Silva3 Tania Ueda-Nakamura2
Sueli de O. Silva2 Celso V. Nakamura1,2
•
Received: 15 December 2015 / Accepted: 19 March 2016
Ó Springer Science+Business Media New York 2016
Abstract In this study, we synthesized a series of 1,3,4-
thiadiazole derivatives of R-(?)-limonene benzaldehyde-
thiosemicarbazones (2a–k). We also determined the cyto-
toxicity in LLCMK2 cells and the activity against epi-
mastigote and trypomastigote forms of Trypanosoma cruzi,
of these synthetic compounds and also of a series of 1,3,4-
thiadiazole without the monoterpene R-(?)-limonene (4a–
k). 1,3,4-Thiadiazole compounds showed significant try-
panocidal activity and a high selectivity indexes. The vast
majority of the monoterpene derivatives, substituted by R-
(?)-limonene, presented better anti-T. cruzi activity than
the non-substituted compounds. Regarding the cytotoxic
profile, the compounds without the monoterpene R-(?)-
limonene were, in general, less toxic. The present findings
indicate that the 1,3,4-thiadiazoles derivatives of R-limo-
nene have potential trypanocidal activity that justify further
studies to better understand the mechanism of action of
these substances on T. cruzi.
Keywords Chagas’ disease Á Trypanosoma cruzi Á
Trypanocidal activity Á Limonene derivatives Á Cytotoxicity
Introduction
Chagas’ disease, also known as American trypanosomiasis,
was discovered and described by Carlos Chagas in 1909
(Chagas, 1909). This disease is caused by the protozoan
parasite Trypanosoma cruzi, which currently affects
approximately 6–7 million people worldwide, mainly in
endemic areas in 21 countries in Latin America (WHO,
2015). In Brazil, benznidazole nitroderivative is the only
drug that is available for the treatment of chagasic patients,
but it has serious side effects, such as skin disorders (rash),
fever, generalized edema, lymphadenopathy, muscle and
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-016-1561-7) contains supplementary
material, which is available to authorized users.
Sueli de O. Silva
lautenschlager@uem.br
& Celso V. Nakamura
cvnakamura@uem.br
1
Solange C. Martins
scardosomartins@yahoo.com.br
´
Programa de Pos-graduac¸a˜o em Ciencias Biologicas,
Universidade Estadual de Maringa, Av. Colombo 5790,
ˆ
´
´
´ ´
Maringa, Parana CEP 87020-900, Brazil
ˆ
Vania C. Desoti
vaniadesoti@gmail.com
2
3
4
´
ˆ
ˆ
Programa de Pos-graduac¸a˜o em Ciencias Farmaceuticas,
Universidade Estadual de Maringa, Av. Colombo 5790,
´
´
Danielle Lazarin-Bidoia
dlbidoia@gmail.com
´ ´
Maringa, Parana CEP 87020-900, Brazil
´
Departamento de Quımica, Universidade Estadual de
Maringa, Av. Colombo 5790, Maringa, Parana
´
Fabio Vandresen
fabiovandresen@utfpr.edu.br
´
´
´
CEP 87020-900, Brazil
Cleuza C. da Silva
ccsilva@uem.br
´
Departamento de Quımica, Universidade Tecnologica
Federal do Parana, Av. dos Pioneiros 3131, Londrina, Parana
´
´
´
Tania Ueda-Nakamura
tunakamura@gmail.com
CEP 86036-370, Brazil
123

Med Chem Res
joint pain, bone marrow depression (agranulocytosis and
thrombocytopenic purpura), and peripheral neuropathy
(Castro et al., 2006; Maya et al., 2007). These side effects
are often decisive for treatment dropout (Longo, 2015).
Considering these facts, the search for new substances with
satisfactory trypanocidal action and minimal adverse
effects is essential for the treatment of patients with Cha-
gas’ disease.
resonance spectra were recorded in a Varian model Mer-
cury plus 300 spectrometer at 300 and 75.45 MHz,
respectively, with CDCl3 as the solvent and tetramethyl-
silane (TMS) as the internal standard. The chemical shifts
(d) are given in parts per million relative to TMS. Ele-
mental analyses were carried out on an Elemental Analyzer
Perkin-Elmer 2400 Series II. The column chromatography
was performed on silica gel Merck 230–400 mesh ASTM.
All of the reagents were purchased from commercial
suppliers.
Thiosemicarbazones are compounds with broad profiles
of biological and pharmacological activities that have
gained considerable scientific interest (Geng et al., 2013;
´
Tenorio et al., 2005). Several studies have demonstrated
Synthesis of the compounds
the actions of thiosemicarbazones, including antitumor
(Vandresen et al., 2014), antioxidant (Al-Amiery et al.,
2012), antibacterial (Mohamed et al., 2014), antifungal,
antiviral (Glisoni et al., 2012), antimalarial (Pingaew et al.,
2010), and antitrypanosomal effects (Fatondji et al., 2013;
Soares et al., 2011). These molecular substances are con-
sidered very versatile ligands and show high stability under
various reaction conditions. They have many applications
as intermediates for the synthesis of important heterocycles
General procedure for the preparation of 1,3,4-
thiadiazoles (4a–k)
1,3,4-Thiadiazoles (4a–k) were prepared based on a pre-
viously reported procedure (Gireesh et al., 2011), and the
structures were established by comparison with data in the
literature (Rajak et al., 2011).
´
and can be used to obtain 1,3,4-thiadiazole (Tenorio et al.,
2005).
General procedure for the preparation of 1,3,4-
thiadiazoles derivatives of R-(?)-limonene benzaldehyde-
thiosemicarbazones (2a–k)
1,3,4-Thiadiazole is one of the most well-known com-
pounds studied to date. It is widely used in the pharma-
ceutical, agricultural, and chemical industries because of
the large number of biological activities attributed to the
heterocyclic nucleus (Hu et al., 2014; Jain et al., 2013).
Among these activities are antioxidant (Chhajed et al.,
2014), anticancer (Hosseinzadeh et al., 2013), antituber-
culosis (Oruc¸ et al., 2004), antitrypanosomal (Carvalho
et al., 2008; Saloma˜o et al., 2010), antimicrobial (Farghaly
et al., 2012; Kadi et al., 2010), and antiinflammatory
effects (Kadi et al., 2010).
Benzaldehyde-thiosemicarbazones (1a–k) were initially
prepared according to a previously described method
(Vandresen et al., 2014), using equimolar amounts of
benzaldehyde or derivatives and (?)-N(4)-{R-[2-(4-
methylcyclohex-3-en-1-yl)-propan-2-yl]}-thiosemicar-
bazide. 1,3,4-Thiadiazoles (2a–k) were prepared using a
mixture of thiosemicarbazones (1a–k) (0.22 mmol), FeCl3
(1.10 mmol), and ethanol/water (3:1; 10 mL), with heating
at reflux for 2–20 h. Upon completion of the reaction, as
indicated by thin layer chromatography (TLC), the reaction
mixture was cooled in an ice bath and then filtered, washed
with water, and recrystallized from acetone or ethanol.
The aim of the present study was to synthesize a series
of 1,3,4-thiadiazole derivatives of R-(?)-limonene ben-
zaldehyde-thiosemicarbazones (2a–k, Scheme 1) and to
determine their cytotoxicity in LLCMK2 cells and their
activity against epimastigote and trypomastigote forms of
T. cruzi in order to check the influence of this lipophilic
component on its trypanocidal activity.
N-{1-Methyl-1-[(1R)-4-methylcyclo-hex-3-em-yl]ethyl}-5-
phenyl-1,3,4-thiadiazol-2-amine 2a White solid (MeOH);
yield 46 %; mp. 169–171 °C; IR (KBr) mmax: 3226, 1551,
1
1095 cm-1; H NMR (CDCl3, 300 MHz): d = 7.80 (2H,
m, H-200, H-600), 7.39 (3H, m, H-300,H-400, H-500), 5.75 (1H,
bs, NH), 5.36 (1H, m, H-30), 2.16 (1H, m, H-10), 2.01 (2H,
m, H-50), 1.99 (2H, m, H-20), 1.86 (2H, m, H-60), 1.64 (3H,
s, H-100), 1.41 (3H, s, H-90), 1.38 (3H, s, H-80); 13C NMR
(CDCl3, 75.5 MHz,): d = 166.9 (C, C-2), 158.1 (C, C-5),
134.3 (CH, C-40), 131.2 (C, C-1¢¢¢), 129.9 (CH, C-400), 129.1
(CH, C-300, C-500), 127.0 (CH, C-200, C-600), 120.5 (CH,
C-30), 58.3 (C, C-70), 41.4 (CH, C-10), 31.2 (CH2, C-50),
26.9 (CH2, C-20), 24.5 (CH3, C-90), 24.3 (CH2, C-60), 23.5
(CH3, C-100), 24.2 (CH3, C-80); EI-MS m/z 313 (M?•);
Materials and methods
General experimental procedure
´
Melting points were determined in a Micro-Quımica
MQAPF-301 apparatus. Infrared (IR) spectra were recor-
ded on a BOMEM model MB-100 spectrometer. Low-
resolution mass spectra were recorded using a SHI-
MADZU-GC/MS model QP 2000A spectrometer at 70 eV
with a probe for solids. 1H- and 13C-nuclear magnetic
123

Med Chem Res
Scheme 1 Synthetic routes for the 1,3,4-thiadiazole derivatives of R-(?)-limonene benzaldehyde-thiosemicarbazones (2a–k) and 1,3,4-
thiadiazoles (4a–k)
Anal.Calc. for C18H23N3S: C, 68.97 %; H, 7.40 %; N,
13.41 %. Found: C, 69.16 %; H, 7.49 %; N, 13.86 %.
1.41 (3H, s, H-90), 1.37 (3H, s, H-80); 13C NMR (CDCl3,
75.5 MHz,): d = 166.3 (C, C-2), 161.0 (C, C-400), 158.2 (C,
C-5), 134.4 (C, C-40), 128.4 (2 CH, C-200, C-600), 124.0 (C,
C-100), 120.5 (CH, C-30), 114.5 (2 CH, C-300, C-500), 58.3
(OCH3), 55.6 (C, C-70), 41.4 (C, C-10), 31.2 (CH2, C-50), 26.9
(CH2, C-20), 24.5 (CH3, C-90), 24.3 (CH2, C-60), 23.9 (CH3,
C-80), 23.5 (CH3, C-100); EI-MS m/z 343 (M?•); Anal.Calc.
for C19H25N3OS: C, 66.44 %; H, 7.34 %; N, 12.23 %.
Found: C, 66.65 %; H, 7.28 %; N, 12.44 %.
N-{1-Methyl-1-[(1R)-4-methylcyclohex-3-en-1-yl]ethyl}-5-
(4-methylphenyl)-1,3,4-thiadiazol-2-amine
solid (MeOH); yield 60 %; mp. 173–175 °C; IR (KBr)
mmax
3211, 1510, 1102 cm-1 1H NMR (CDCl3,
2b White
:
;
300 MHz): d = 7.68 (2H, d, J 7.9 Hz, H-200, H-600), 7.23
(2H, d, J 7.9 Hz, H-300, H-500), 6.22 (1H, bs, NH), 5.37 (1H,
m, H-30), 2.39 (3H, s, 4-CH3), 2.16 (1H, m, H-10), 2.12 (2H,
m, H-50), 2.00 (2H, m, H-20), 1.86 (2H, m, H-60), 1.64 (3H,
s, H-100), 1.41 (3H, s, H-90), 1.38 (3H, s, H-80); 13C NMR
(CDCl3, 75.5 MHz,): d = 166.4 (C, C-2), 157.4 (C, C-5),
140.9 (C, C-400), 134.4 (C, C-40), 129.1 (2 CH, C-300,C-500),
127.6 (C, C-100), 127.0 (2 CH, C-200,C-600), 120.3 (CH,
C-30), 59.1 (C, C-70), 41.6 (CH, C-10), 31.2 (CH2, C-50),
26.9 (CH2, C-20), 24.5 (CH3, C-90), 24.3 (CH2, C-60), 23.9
(CH3, C-80), 23.5 (CH3, C-100), 21.7 (CH3); EI-MS m/z 327
(M?•); Anal.Calc. for C19H25N3S: C, 69.68 %; H, 7.69 %;
N, 12.83 %. Found: C, 69.36 %; H, 7.39 %; N, 13.04 %.
5-[4-(Dimethylamino)phenyl]-N-{1-methyl-1-[(1R)-4-meth-
ylcyclohex-3-en-1-yl]ethyl}-1,3,4-thiadiazol-2-amine
Orange solid (MeOH); yield 35 %; mp. 154 °C; IR (KBr)
mmax
3199, 1579, 1101 cm-1 1H NMR (CDCl3,
2d
:
;
300 MHz): d = 7.66 (2H, d, J 8.6 Hz, H-200, H-600), 6.70
(2H, d, J 8.6 Hz, H-300, H-500), 1.36 (3H, s, H-80), 5.36 (1H,
m, H-30), 5.21 (1H, bs, NH), 3.01 (6H, s, N(CH3)2, 2.14
(1H, m, H-10), 2.02 (2H, m, H-50), 2.00 (2H, m, H-20), 1.85
(2H, m, H-60), 1.64 (3H, s, H-100), 1.39 (3H, s, H-90); 13C
NMR (CDCl3, 75.5 MHz,): d = 165.5 (C, C-2), 156.0 (C,
C-5), 151.6 (C, C-400), 134.4 (C, C-40), 128.4 (2 CH, C-200,
C-600), 120.5 (CH, C-30), 119.0 (C, C-100), 112.1 (2 CH,
C-300, C-500), 58.2 (C, C-70), 41.3 (CH, C-10), 40.4
(N(CH3)2, 31.2 (CH2, C-50), 26.9 (CH2, C-20), 24.5 (CH3,
C-90), 24.3 (CH2, C-60), 23.9 (CH3, C-80), 23.5 (CH3,
C-100); EI-MS m/z 356 (M?•); Anal.Calc. for C20H28N4S:
C, 67.38 %; H, 7.92 %; N, 15.71 %. Found: C, 67.25 %;
H, 7.75 %; N, 15.46 %.
5-(4-Methoxyphenyl)-N-{1-methyl-1-[(1R)-4-methylcyclo-
hex-3-en-1-yl]ethyl}-1,3,4-thiadiazol-2-amine 2c White
solid (MeOH); yield 65 %; mp. 173–175 °C; IR (KBr)
m
max: 3183, 1611, 1096 cm-1; 1H NMR (CDCl3, 300 MHz):
d = 7.73 (2H, d, J 8.2 Hz, H-200, H-600); 6.93 (2H, d,
J 8.2 Hz, H-300, H-500), 5.45 (1H, bs, NH), 5.37 (1H, m, H-30),
3.84 (3H, s, OCH3), 2.16 (1H, m, H-10), 2.01 (2H, m, H-50),
1.99 (2H, m, H-20), 1.86 (2H, m, H-60), 1.64 (3H, s, H-100),
123

Med Chem Res
N-{1-Methyl-1-[(1R)-4-methylcyclohex-3-en-1-yl]ethyl}-5-
(2-nitrophenyl)-1,3,4-thiadiazol-2-amine 2e Yellow solid
(MeOH); yield 75 %; mp. [ 250 °C; IR (KBr) mmax: 3184,
C18H22N4O2S: C, 60.31 %; H, 6.19 %; N, 15.63 %. Found:
C, 60.45 %; H, 6.06 %; N, 15.89 %.
5-(2-Chlorophenyl)-N-{1-methyl-1-[(1R)-4-methylcyclohex-
1557, 1100, 1530, 1359 cm-1
;
1H NMR (CDCl3,
3-en-1-yl]ethyl}-1,3,4-thiadiazol-2-amine 2h White solid
300 MHz): d = 7,88 (1H, dd, J 7,6 and 1,5 Hz, H-400); 7.76
(1H, dd, J 7,6 and 1,5 Hz, H-300), 7.65 (1H, m, H-500), 7.57
(1H, m, H-600), 6.55 (1H, bs, NH), 5.36 (1H, m, H-30), 2.11
(1H, m, H-10), 2.07 (2H, m, H-50), 2.00 (2H, m, H-20), 1.87
(2H, m, H-60), 1.64 (3H, s, H-100), 1.42 (3H, s, H-90), 1.39
(3H, s, H-80); 13C NMR (CDCl3, 75.5 MHz,): d = 168.3
(C, C-2), 153.5 (C, C-5), 148.4 (C, C-200), 134.1 (C, C-40),
132.8 (CH, C-500), 131.9 (CH, C-400), 130.6 (CH, C-600),
124.8 (C, C-100), 124.6 (CH, C-300), 120.5 (C, C-30), 58.6
(C, C-70), 41.4 (CH, C-10), 31.2 (CH2, C-50), 26.9 (CH2,
C-20), 24.5 (CH3, C-90), 24.3 (CH2, C-60), 23.9 (CH3, C-80),
23.5 (CH3, C-100); and EI-MS m/z 358 (M?•); Anal.Calc.
for C18H22N4O2S: C, 60.31 %; H, 6.19 %; N, 15.63 %.
Found: C, 60.45 %; H, 6.06 %; N, 15.89 %.
(acetone); yield 75 %; mp. 166–169 °C; IR (KBr) mmax
:
3174, 1549, 1104 cm-1 1H NMR (CDCl3, 300 MHz):
;
d = 8.16 (1H, m, H-300), 7.44 (1H, m, H-600), 7.33 (2H, m,
H-400, H-500), 6.36 (1H, bs, NH), 5.36 (1H, m, H-30), 2.19
(1H, m, H-10), 2.02 (2H, m, H-50), 1.98 (2H, m, H-20), 1.87
(2H, m, H-60), 1.65 (3H, s, H-100), 1.44 (3H, s, H-90), 1.41
(3H, s, H-80); 13C NMR (CDCl3, 75.5 MHz,): d = 168.8
(C, C-2), 153.0 (C, C-5), 134.1 (C, C-40), 131.5 (C, C-200),
130.5 (CH, C-400), 130.4 (2 CH, C-500, C-600), 129.1 (C,
C-100), 127.2 (CH, C-300), 120.4 (CH, C-30), 58.2 (C, C-70),
41.4 (CH, C-10), 31.1 (CH2, C-50), 26.9 (CH2, C-20), 24.5
(CH3, C-90), 24.3 (CH2, C-60), 23.9 (CH3, C-80), 23.3 (CH3,
C-100); EI-MS m/z 347 (M?•); Anal.Calc. for C18H22
ClN3S: C, 62.14 %; H, 6.37 %; N, 12.08 %. Found: C,
62.23 %; H, 6.66 %; N, 11.94 %.
N-{1-Methyl-1-[(1R)-4-methylcyclohex-3-en-1-yl]ethyl}-5-
(3-nitrophenyl)-1,3,4-thiadiazol-2-amine 2f Yellow solid
5-(3-Chlorophenyl)-N-{1-methyl-1-[(1R)-4-methylcyclohex-
(MeOH); yield 68 %; mp. 198–200 °C; IR (KBr) mmax
:
3-en-1-yl]ethyl}-1,3,4-thiadiazol-2-amine 2i White solid
1
3171, 1557, 1102, 1530 cm-1, 1348; H NMR (CDCl3,
300 MHz): d = 8.58 (1H, d, J 1,5 Hz, H-200), 7.22 (2H, dd,
J 7.9 and 1.5 Hz, H-400, H-600), 7.61 (1H, t, J 7,9 Hz, H-500),
6.49 (1H, bs, NH), 5.36 (1H, m, H-30), 2.20 (1H, m, H-10),
2.00 (2H, m, H-50), 2.00 (2H, m, H-20), 1.88 (2H, m, H-60),
1.64 (3H, s, H-100), 1.46 (3H, s, H-90), 1.42 (3H, s, H-80);
13C NMR (CDCl3, 75.5 MHz,): d = 167.7 (C, C-2), 154.8
(C, C-5), 134.3 (C, C-40), 148.7 (C, C-300), 133.0 (C, C-100),
132.2 (CH, C-600), 130.2 (CH, C-500), 124.1 (CH, C-400),
121.6 (CH, C-200), 120.4 (CH, C-30), 58.6 (C, C-70), 41.2
(CH, C-10), 31.2 (CH2, C-50), 26.9 (CH2, C-20), 24.5 (CH3,
C-90), 24.3 (CH2, C-60), 24.1 (CH3, C-80), 23.4 (CH3,
C-100), and EI-MS m/z 358 (M?•); Anal.Calc. for C18
H22N4O2S: C, 60.31 %; H, 6.19 %; N, 15.63 %. Found: C,
60.45 %; H, 6.06 %; N, 15.89 %.
(MeOH); yield 70 %; mp. 139–140 °C; IR (KBr) mmax
:
3253, 1502, 1078 cm-1 1H NMR (CDCl3, 300 MHz):
;
d = 7.80 (1H, s, H-200), 7.69 (1H, m, H-600), 7.35 (2H, m,
H-400, H-500), 5.64 (1H, bs, NH), 5.37 (1H, m, H-30), 2.16
(1H, m, H-10), 2.02 (2H, m, H-50), 2.00 (2H, m, H-20), 1.85
(2H, m, H-60), 1.64 (3H, s, H-100), 1.42 (3H, s, H-90), 1.38
(3H, s, H-80); 13C NMR (CDCl3, 75.5 MHz,): d = 167.1
(C, C-2), 156.6 (C, C-5), 135.1 (C, C-300), 134.4 (C, C-40),
132.9 (C, C-100), 130.3 (CH, C-400), 129.8 (CH, C-500), 126.9
(CH,C-200), 125.0 (CH, C-600), 120.4 (CH, C-30), 58.5 (C,
C-70), 41.4 (CH, C-10), 31.2 (CH2, C-50), 26.9 (CH2, C-20),
24.5 (CH3, C-90), 24.3 (CH2, C-60), 24.2 (CH3, C-80), 23.5
(CH3, C-100); EI-MS m/z 347 (M?•); Anal.Calc. for C18
H22ClN3S: C, 62.14 %; H, 6.37 %; N, 12.08 %. Found: C,
62.23 %; H, 6.66 %; N, 11.94 %.
N-{1-Methyl-1-[(1R)-4-methylcyclohex-3-en-1-yl]ethyl}-5-
5-(4-Chlorophenyl)-N-{1-methyl-1-[(1R)-4-methylcyclohex-
(4-nitrophenyl)-1,3,4-thiadiazol-2-amine 2g Yellow solid
3-en-1-yl]ethyl}-1,3,4-thiadiazol-2-amine 2j White solid
(MeOH); yield 85 %; mp. 234–238 °C; IR (KBr) mmax
:
(MeOH); yield 65 %; mp. 172–174 °C; IR (KBr) mmax
:
3176, 1557, 1102, 1517,1341 cm-1 1H NMR (CDCl3,
;
3179, 1552, 1088 cm-1 1H NMR (CDCl3, 300 MHz):
;
300 MHz): d = 8.28 (2H, d, J 8.4 Hz, H-300, H-500); 7.97
(2H, d, J 8.4 Hz, H-200, H-600), 5.67 (1H, bs, NH), 5.37 (1H,
m, H-30), 2.15 (1H, m, H-10), 2.06 (2H, m, H-50), 2.01 (2H,
m, H-20), 1.87 (2H, m, H-60), 1.65 (3H, s, H-100), 1.44 (3H,
s, H-90), 1.36 (3H, s, H-80); 13C NMR (CDCl3, 75.5 MHz,):
d = 167.8 (C, C-2), 155.4 (C, C-5), 148.2 (C, C-400), 137.1
(C, C-100), 134.4 (C, C-40), 127.4 (2 CH, C-200 C-600), 124.4
(2 CH, C-300, C-500), 120.5 (CH, C-30), 58.9 (C, C-70), 41.4
(CH, C-10), 31.2 (CH2, C-50), 26.9 (CH2, C-20), 24.5 (CH3,
C-90), 24.3 (CH2, C-60), 23.9 (CH3, C-80), 23.5 (CH3,
C-100); EI-MS m/z 358 (M?•); Anal.Calc. for
d = 7.72 (2H, d, J 8.1 Hz, H-200, H-600), 7.38 (2H, d,
J 8.4 Hz, H-300, H-500), 6.18 (1H, bs, NH), 5.37 (1H, m,
H-30), 2.16 (1H, m, H-10), 2.12 (2H, m, H-50), 1.99 (2H, m,
H-20), 1.87 (2H, m, H-60), 1.63 (3H, s, H-100), 1.44 (3H, s,
H-90), 1.38 (3H, s, H-80); 13C NMR (CDCl3, 75.5 MHz,):
d = 167.1 (C, C-2). 156.6 (C, C-5), 135.6 (C, C-400), 134.4
(C, C-40), 129.7 (C, C-100), 129.3 (2 CH, C-300, C-500), 128.3
(2 CH, C-200, C-600), 120.5 (CH, C-30), 58.4 (C, C-70), 41.3
(CH, C-10), 31.2 (CH2, C-50), 26.9 (CH2, C-20), 24.5 (CH3,
C-90), 24.3 (CH2, C-60), 23.9 (CH3, C-80), 23.5 (CH3,
C-100); EI-MS m/z 347 (M?•); Anal.Calc. for
123

Med Chem Res
C18H22ClN3S: C, 62.14 %; H, 6.37 %; N, 12.08 %. Found:
C, 62.23 %; H, 6.66 %; N, 11.94 %.
in triplicate for 96 h under the same conditions as above.
Control wells without treatment were included. After treat-
ment, the medium was removed and washed with phosphate-
buffered saline (PBS), and 50 lL of MTT, a water-soluble
5-(4-Fluorophenyl)-N-{1-methyl-1-[(1R)-4-methylcyclohex-
3-en-1-yl]ethyl}-1,3,4-thiadiazol-2-amine 2k White solid
tetrazolium
salt
(3-[4,5-dimethylthiazol-2-yl]-2,5-
(acetone); yield 59 %; mp. 180–183 °C; IR (KBr) mmax
:
diphenyltetrazolium bromide) (2 mg/mL), was added to
each well for 4 h in a 5 % CO2–air mixture at 37 °C. DMSO
(150 lL) was then added to solubilize the formazan crystals,
and the plates were homogenized. Absorbance was read in a
96-well plate reader (BIO-TEK Power Wave XS spec-
trophotometer) at 570 nm. The percentage of viable cells
was calculated relative to controls, consisting of cells that
were cultured in medium alone, according to CC50 values
(i.e., 50 % cytotoxicity concentration). The CC50 was
determined by logarithm regression analysis.
3206, 1566, 1090 cm-1 1H NMR (300 MHz, CDCl3)
;
d = 7.80 (2H, m, H-200, H-600), 7.15 (2H, t, J 8.5 Hz, H-300,
H-500), 5.55 (1H, bs, NH), 5.37 (1H, m, H-30), 2.15 (1H, m,
H-10), 2.04 (2H, m, H-50), 2.01 (2H, m, H-20), 1.86 (2H, m,
H-60), 1.64 (3H, s, H-100), 1.42 (3H, s, H-90), 140 (3H, s,
H-80). 13C NMR (CDCl3, 75.5 MHz) d = 167.9 (C, C-2),
164.4 (C, C-400), 156.0 (C, C-5), 134.4 (C, C-40), 129.9 (2
CH, C-200, C-600), 127.9 (C, C-100), 116.9 (2CH, C-300, C-500),
120.5 (CH, C-30), 58.0 (C, C-70), 41.3 (CH, C-10), 31.2
(CH2, C-50), 26.9 (CH2, C-20), 24.5 (CH3, C-90), 24.3 (CH2,
C-60), 23.9 (CH3, C-80), 23.5 (CH3, C-100). EI-MS m/z 331
(M?Á); Anal.Calc. for C18H22FN3S: C, 65.23 %; H, 6.69 %;
N, 12.68 %. Found: C, 65.03 %; H, 6.78 %; N, 12.89 %.
Antiproliferative activity against epimastigote forms
Epimastigote forms of T. cruzi in the logarithmic phase
were used for this assay. The compounds were dissolved in
DMSO and LIT medium and used at increasing concen-
trations. A cell density of 1.0 9 106 cells/mL in LIT
medium supplemented with 10 % FBS was incubated in
duplicate at 28 °C for 96 h in 24-well microplates in the
absence or presence of different concentrations of the tes-
ted compounds. Afterward, cell growth was estimated by
counting the parasites using a Neubauer hemocytometer
(Improved Double Neubauer), and IC50 values (concen-
trations that inhibit cell growth in 50 %) were determined.
The results are expressed as a percentage of growth inhi-
bition compared with control cultures.
Compound preparation
The compounds were dissolved in dimethylsulfoxide
(DMSO) and diluted in culture medium for use in the assay.
The final concentration of DMSO did not exceed 1 %.
Parasites and cell cultures
LLCMK2 cells [epithelial kidney cells (Macaca mulatta);
CCL-7; American Type Culture Collection, Rockville, MD,
USA] were maintained in Dulbecco’s Modified Eagle’s
Medium (DMEM; Gibco Invitrogen) supplemented with
2 mM L-glutamine, 10 % heat-inactivated fetal bovine
serum (FBS), 5000 U/mL penicillin and 5 mg/mL strepto-
mycinsolutionat37 °Cina humidified5 % CO2 atmosphere.
Epimastigote forms of the Y strain of T. cruzi were
axenically maintained at 28 °C with weekly transfers in
liver infusion tryptose (LIT) medium supplemented with
10 % FBS at pH 7.4 (Camargo, 1964). Trypomastigotes
were collected from the supernatant of LLCMK2 cells that
were preinfected by bloodstream trypomastigotes that were
aseptically obtained by heart puncture of infected Swiss
mice at the peak of parasitemia. The animal protocol was
approved by the Ethics Committee of the Universidade
Effect on viability of trypomastigote forms
Tissue culture-derived trypomastigote forms (1.0 9 107
cells/mL) were resuspended in DMEM and added in
duplicate to 96-well microplates in the absence or presence
of different concentrations of the compounds. Parasites
were incubated for 24 h at 37 °C in a 5 % CO2 atmosphere.
The results were obtained by observing motility, allowing
the determination of viability of the parasites, using the
Pizzi–Brener method (Brener, 1962). The EC50 value (i.e.,
the concentration that inhibited 50 % of viability of the
parasites) was then calculated.
´
Estadual de Maringa/PR/Brazil (no 074/2011).
Cytotoxicity assay
Results and discussion
Chemistry
A suspension of 2.5 9 105 LLCMK2 cells/mL was seeded in
a 96-well microplate and maintained at 37 °C in a 5 % CO2
atmosphere for 24 h until a confluent cell monolayer was
obtained. Thereafter, the medium was replaced with DMEM
that contained different concentrations of the test compounds
The synthesis of 1,3,4-thiadiazoles derivatives of R-(?)-
limonene benzaldehyde-thiosemicarbazones (2a–k) was
based on the monocyclic monoterpene R-(?)-limonene,
123

Med Chem Res
4-isopropenyl-1-methylcyclohexene, as a synthetic sub-
strate. R-(?)-limonene is part of the chemical composition
of over 300 plant species and a major component of the
essential oils of citrus fruits in general. Furthermore, it can
be used as a building block in organic synthesis, primarily
because of exocyclic double-bond chemoselectivity (Mar-
Compound 4a had an IC50 of 14.8 5.1 lM for epi-
mastigotes and EC50 of 395.7 11.5 lM for trypomastig-
otes. The CC50 in LLCMK2 cells was 892.3 63.9 lM,
resulting in SIs of 60.3 and 2.3, respectively. Compound 2a
had a CC50 of 424.3 41.3 lM, IC50 of 10.3 1.7 lM,
and SI of 41.2.
´
´
ostica-Junior and Pastore, 2007; Sun, 2007). Derivatives of
R-(?)-limonene have several biological activities, includ-
ing anti-Leishmania, anti-T. cruzi, and anticancer (Graebin
et al., 2010; Vandresen et al., 2014).
The introduction of substituents in the para position of
the benzene ring of the 1,3,4-thiadiazoles derivatives of R-
(?)-limonene benzaldehyde-thiosemicarbazones (2a–
k) differentially influenced the behavior of the resulting
compounds compared with derivative 2a. The 2b com-
Synthetic routes for the 1,3,4-thiadiazoles derivatives of
R-(?)-limonene benzaldehyde-thiosemicarbazones (2a–
k) and 1,3,4-thiadiazoles (4a–k) are shown in Scheme 1.
The series of 1,3,4-thiadiazole derivatives of R-(?)-limo-
nene benzaldehyde-thiosemicarbazones (2a–k) were syn-
thesized with chloro (–Cl) and nitro (–NO2) substituents in
the ortho, meta, and para positions, hydrogen (–H) and
fluorine (–F) substituents as well as methyl (–CH3),
methoxy (–OCH3) and dimethylamino (–N[CH3]2) groups
in the para position of the benzene ring. The chemical
structures of the compounds are given in Table 1.
pound with –CH3 group was the least toxic (CC50
=
795.0 77.8 lM) and more selective for epimastigote
forms (SI = 53.0). The 2c compound with –OCH3 group
exhibited an increase in antiproliferative activity against
epimastigotes and excellent activity against trypomastig-
otes (EC50 = 4.7 0.0 lM), with SIs of 21.5 and 29.7,
respectively. The 2d compound with –N(CH3)2 group also
exhibited activity against epimastigotes (IC50 = 8.4
2.2 lM) and trypomastigotes (EC50 = 9.5 0.2 lM),
with SIs of 27.0 and 23.8, respectively. The presence of –F
substituent in the 2k compound led to a significant increase
in activity against epimastigotes (IC50 = 6.9 1.6 lM)
and low cytotoxicity in LLCMK2 cells, promoting a
14-fold increase in the SI (565.1).
Eleven heterocycle 1,3,4-thiadiazoles derivatives of R-
(?)-limonene benzaldehyde-thiosemicarbazones (2a–
k) had yields that ranged from 35 to 85 %. The melting
point (mp) ranged from 139 to[250 °C. The IR spectra of
the synthesized compounds showed characteristic bands of
the expected functional groups, with bands of (NH) ranging
from 3171 to 3253 cm-1, bands of (C=C) ranging from
1502 to 1611 cm-1, and bands of (N=C–S–C=N) ranging
Comparisons of the structures of compounds 2e, 2f, and
2g, which possess the –NO2 group, revealed better activity
against epimastigotes (IC50 = 22.4 1.3 lM) in the ortho
position. The substituent in the meta position resulted in
better activity against trypomastigotes (EC50 = 25.7
2.7 lM). The –NO2 in the para position considerably
decreased the cytotoxicity profile (CC50 = 4371.5
262.3 lM).
For compounds 2h, 2i, and 2j, the presence of the –Cl
substituent in the meta position (2i) resulted in excellent
trypanocidal activity, with IC50 = 1.6 0.0 lM, EC50 =-
7.2 0.8 lM, and SI = 104.2 and 23.2 for epimastigotes
and trypomastigotes, respectively. With the substituent in
the ortho position (2h), a significant decrease in cytotoxi-
city was observed.
from 1078 to 1104 cm-1. H NMR and 13C NMR showed
1
signals for the terpene moiety at dH/dC 5.36–5.37 (1H, s,
H-30)/120.3–120.5 (C-30), 1.36–1.42 (3H, s, H-80)/
23.9–24.2 (C-80), 1.39–1.46 (3H, s, H-90)/24.5 (C-90), and
1.63–1.65 (3H, s, H-100)/23.3–23.9 (C-100). The 5-amino-
1,3,4-thiadiazole moiety was characterized by signals at dH
5.21–6.55 (NH group) and dC 153.0–158.2 (C-2) and dC
165.5–168.8 (C-5). Based on the spectroscopic data, it was
possible to characterize the 1,3,4-thiadiazole derivatives of
R-(?)-limonene benzaldehyde-thiosemicarbazones (2a–k).
The present data are consistent with the structures sug-
gested for the products.
We found that the vast majority of the non-substituted
1,3,4-thiadiazole (4a–k) derivatives presented trypanocidal
activity that was lower than the derivatives substituted by a
terpene unit, except for compounds 2b, 2g, and 2j, which
exhibit, respectively, substituents –CH3, –NO2, and chlo-
rine in para position of the benzene ring and also for 2h
compound which has chlorine in ortho position of the
benzene ring. One explanation for the best activity of the
R-(?)-limonene 1,3,4-thiadiazole derivatives (4a–k) may
be the presence of the terpenic group, which improved the
lipophilic character of molecules and allowed higher
plasmatic membrane permeability, resulting in better
bioavailability and an improved pharmacological profile
Biology
1,3,4-Thiadiazoles derivatives of R-(?)-limonene ben-
zaldehyde-thiosemicarbazones (2a–k) and 1,3,4-thiadia-
zoles (4a–k) were initially tested in in vitro assays against
epimastigote and trypomastigote forms of T. cruzi, and the
cytotoxicity was demonstrated in LLCMK2 cells. All these
evaluations are important in the search for active com-
pounds with trypanocidal activity. The selectivity index
(SI) was also determined, and the results are reported in
Table 1.
123

Med Chem Res
Table 1 Effect of 1,3,4-thiadiazole derivatives of R-(?)-limonene benzaldehyde-thiosemicarbazones 2a–k and 1,3,4-thiadiazoles 4a–k against
epimastigote and trypomastigote forms of T. cruzi, cytotoxicity in LLCMK2 cells, and selectivity index
Compound
Structure
ICa50 (lM)
CCb50 (lM)
SIc
ECd50 (lM)
SIe
2a
10.3 1.7
424.3 41.3
41.2
NR
NR
2b
15.0 3.0
795.0 77.8
53.0
170.0 22.5
4.7
2c
6.5 2.1
139.7 22.2
21.5
4.7 0.0
29.7
2d
2e
2f
8.4 2.2
226.3 20.7
27.0
9.5 0.2
23.8
22.4 1.3
106.0 9.9
4.7
69.3 2.2
25.7 2.7
[279.0 0.0
12.2 3.2
1.5
28.7 1.9
28.4 6.8
1.0
1.1
2g
2h
[ 279.0 0.0
4371.5 262.3
\15.7
\ 15.7
20.6 7.2
950.0 25.4
46.1
77.9
123

Med Chem Res
Table 1 continued
Compound
Structure
ICa50 (lM)
1.6 0.0
CCb50 (lM)
SIc
ECd50 (lM)
SIe
2i
166.7 17.9
104.2
7.2 0.8
23.2
2j
43.3 3.7
6.9 1.6
14.8 5.1
6.5 0.3
793.3 60.4
3899.4 253.9
92.3 63.9
18.3
565.1
60.3
84.4 6.6
9.4
2k
4a
4b
199.4 10.2
395.7 11.5
64.9 9.6
19.6
2.3
973.4 107.3
149.8
15.0
4c
10.7 1.5
43.5 0.0
4.1
59.0 4.8
0.7
4d
11.3 0.5
3785.2 140.0
335.0
[460.5 0.0
\8.2
123

Med Chem Res
Table 1 continued
Compound
Structure
ICa50 (lM)
CCb50 (lM)
SIc
3.4
ECd50 (lM)
SIe
4e
121.0 8.8
409.8 57.3
[450.3 0.0
\0.9
4f
55.2 8.3
454.8 194.2
8.2
269.7 5.5
1.7
4g
17.2 3.8
1152.8 130.6
67.0
285.3 2.2
4.0
4h
7.3 1.0
770.7 83.6
105.6
65.6 3.6
11.7
4i
4j
6.6 1.5
293.1 13.4
44.4
344.2 23.8
0.9
5.7 0.0
364.0 69.5
63.9
67.3 5.7
5.4
4k
55.2 10.0
630.6 43.5
11.4
[512.7 0.0
\1.2
a
Concentration that inhibited 50 % of growth of epimastigotes
50 % cytotoxicity concentration on LLCMK2 cells
b
c
d
e
Selectivity index (SI; CC50 LLCMK2 cells/IC50
Concentration that inhibited 50 % of viability of the trypomastigotes
Selectivity index (SI; CC50 LLCMK2 cells/EC50
)
)
123

Med Chem Res
´
(Alvarez et al., 2010; Barreiro and Fraga, 2008). When
New limonene-hybrid derivatives with anti-T. cruzi activity. Lett
Drug Des Discov 7:452–460
evaluating the cytotoxicity profile, with the exception of
the substituted compounds 2g, 2h, 2j, and 2k, the non-
substituted compounds presented less cytotoxic action.
Vandresen et al. (2014) reported promising results with
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drugs used to treat Chagas’ disease (American trypanosomiasis).
Hum Exp Toxicol 25:471–479
´
results corroborate with Alvarez et al. (2010) that identified
new limonene-hybrid derivatives with anti-T. cruzi activity
against Tulahuen 2 strain, whose presence of limonene
substructure may be responsible for modifying the
lipophilicity of these heterocycles, promoting adequate
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antimicrobial activity of some new 1,3,4-thiadiazole derivatives.
Molecules 17:14625–14636
Fatondji HR, Kpoviessi S, Gbaguidi F, Bero J, Hannaert V, Quetin-
Leclercq J, Poupaert J, Moudachirou M, Accrombessi GC (2013)
Structure-activity relationship study of thiosemicarbazones on an
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In conclusion, these compounds presented relevant try-
panocidal activity with promising selectivity indexes.
The majority of the monoterpene derivatives containing
R-(?)-limonene presented better trypanocidal activity
than the non-substituted compounds. The present findings
indicate that the 1,3,4-thiadiazoles derivatives of R-
limonene have potential for further studies to better
understand the mechanism of action of these substances
on T. cruzi.
Acknowledgments This work was supported by grants from the
Glisoni RJ, Cuestas ML, Mathet VL, Oubin˜a JR, Moglioni AG,
Sosnik A (2012) Antiviral activity against the hepatitis C virus
(HCV) of 1-indanone thiosemicarbazones and their inclusion
complexes with hydroxypropyl-b-cyclodextrin. Eur J Pharm Sci
47:596–603
´
Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico
(CNPq), Capacitac¸a˜o de Aperfeic¸oamento de Pessoal de Nıvel
´
´
´
ˆ
Superior (CAPES), Programa de Nucleos de Excelencia (PRONEX/
Fundac¸a˜o Araucaria), Programa de Pos-Graduac¸a˜o em Ciencias
´
´
ˆ
´
Biologicas, and Pos-Graduac¸a˜o em Ciencias Farmaceuticas da
Universidade Estadual de Maringa.
´
ˆ
ˆ
Graebin CS, Madeira MF, Yokoyama-Yasunaka JK, Miguel DC,
´
´
Uliana SR, Benitez D, Cerecetto H, Gonzales M, Rosa RG,
Eifler-Lima VL (2010) Synthesis and in vitro activity of
limonene derivatives against Leishmania and Trypanosoma.
Eur J Med Chem 45:1524–1528
Compliance with ethical standards
Conflict of interest The authors declare no conflicts of interest.
Hosseinzadeh L, Khorand A, Aliabadi A (2013) Discovery of
2-phenyl-N-(5-(trifluoromethyl)-1,3,4-thiadiazol-2-yl)acetamide
derivatives as apoptosis inducers via the caspase pathway with
potential anticancer activity. Arch Pharm (Weinheim)
346:812–818
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