Facile synthesis of a series of non-symmetric thioethers including a benzothiazole moiety and their use as effcient in vitro anti-trypanosoma cruzi agents

Article abstract of DOI:10.3390/molecules24173077

A series of 2-benzylsulfanyl benzothiazole (BTA) derivatives were synthesized and fully characterized and in vitro tested against two strains of T. cruzi (NINOA and INC-5), exhibiting good activities at low concentrations.

Full text of DOI:10.3390/molecules24173077

molecules
Article
Facile Synthesis of a Series of Non-Symmetric
Thioethers Including a Benzothiazole Moiety and
Their Use as Efficient In Vitro anti-Trypanosoma
cruzi Agents
Alcives Avila-Sorrosa 1,
*
, Jazz D. Tapia-Alvarado 1, Benjamín Nogueda-Torres 2,
Karla Fabiola Chacón-Vargas 2, Francisco Díaz-Cedillo 1, María Elena Vargas-Díaz 1 and
David Morales-Morales 3,
*
1
Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Departamento de Química Orgánica,
Carpio y Plan de Ayala S/N, Colonia Santo Tomás, 11340 Ciudad de México, México
2
3
Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas, Departamento de Parasitología,
Carpio y Plan de Ayala S/N, Colonia Santo Tomás, 11340 Ciudad de México, México
Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior s/n, Ciudad
Universitaria, C.P. 04510, Ciudad de México, México
*
Correspondence: aavilas@ipn.mx (A.A.-S.); damor@unam.mx (D.M.-M.); Tel.: +52-555-729-6000 (A.A.-S.);
+52-55-56224514 (D.M.-M.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 24 July 2019; Accepted: 19 August 2019; Published: 24 August 2019
Abstract: A series of 2-benzylsulfanyl benzothiazole (BTA) derivatives were synthesized and fully
characterized and in vitro tested against two strains of T. cruzi (NINOA and INC-5), exhibiting good
activities at low concentrations.
Keywords: Trypanosoma cruzi; Chagas disease; benzothiazole derivatives; non-symmetric thioethers;
neglected diseases
1. Introduction
The benzothiazole (BTA) is a heterocyclic compound of great interest in different areas of chemistry,
materials, and biological sciences. The BTA core is rarely found in nature (e.g., luciferin, marine natural
compounds, etc.); thus, most of the known derivatives have been prepared by chemical synthesis.
Nevertheless, compounds including these structures have found applications in the synthesis of polymers,
dyes, chemical sensors, herbicides, etc. In addition, they represent a wide variety of important biologically
active xenobiotics, including antidiabetic [1], anticonvulsant agent [2], anticancer [3], anti-inflammatory [4],
and antiparasitic [ ]. Thus, this privileged pharmacological unit is one of the "master keys" for the design
5
and synthesis of bioactive compounds with applications in medicinal chemistry and pharmaceuticals.
Several reports have shown that functionalization of the BTA system on position 2 is key for their
enhanced biological activity mainly as antimicrobial agents. Thus, there is currently a growing interest
in obtaining 2-substituted BTAs to direct them as significant active antiparasitic agents [6–10].
In this line, American trypanosomiasis or Chagas disease is caused by the hemoflagellate protozoan
Trypanosoma cruzi (T. cruzi) that is transmitted by vectors, mainly triatomine insects [11]. This parasitic
infection is considered neglected and affects mainly countries of tropical zones of Americas, where
it is estimated that between 6 to 7 million people are infected with T. cruzi [12], and approximately
100 million people are at risk of infection with this protozoan [13]. Due to the increased number of
cases of infections registered in recent decades in non-endemic areas, such as the United States, Canada,
and European countries [14], Chagas disease is a serious emerging public health problem worldwide.
Molecules 2019, 24, 3077; doi:10.3390/molecules24173077
www.mdpi.com/journal/molecules

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Clinically, Chagas disease occurs in acute and chronic phases. The first can be asymptomatic or
manifest with fever or edema, or both, on the face and extremities. This phase is characterized by the
presence of many parasites in the trypomastigote stadium circulating through the bloodstream, while
in the chronic phase, there is a lower blood parasitaemia, but manifestations of the disease are most
serious, leading to heart problems, megaviscera, neurological changes, and sudden death [15].
Regarding treatment, there are only two nitrated chemotherapeutic agents: nifurtimox (Nfx)
((R,S)-3-methyl-N-[(1E)-(5-nitro-2-furyl)methylene]thiomorpholin-4-amine-1,1-dioxide) and benznidazole
(Bnz) (N-benzyl-2-(2-nitro-1H-imidazol-1-yl)acetamide) (Figure 1), which are usually more effective in the
acute stage. However, therapy with both drugs shows serious side effects, as well as the development of
resistance to parasites [16].
O
N
NO2
N
O
O
H
O
S
N N
NO2
N
Nifurtimox (Nfx)
Benznidazole (Bnz)
Figure 1. Main antichagasic chemotherapeutic agents.
Thus, based on the above, we would like to report the facile synthesis of a series of non-symmetric
thioethers, including a BTA moiety, and their use as efficient in vitro anti-Trypanosoma cruzi agents.
2. Results and Discussion
2.1. Synthesis
The structures of the series of BTA derivatives obtained are shown in Table 1. Their synthesis was
achieved in a facile, single step procedure from the stoichiometric reaction of 2-mercaptoBTA
different benzyl chlorides (2a ), according to Scheme 1. Affording derivatives 3a (yields ranging
were obtained as liquids) with melting
1 with
–g
–g
from 75 to 87%) as microcrystalline solids (derivatives
points lower than 100 ◦C (Table 1).
5 and 6
Scheme 1. Synthesis of 2-benzylsulfanyl-BTA derivatives.
Analysisofcompounds2-(benzylthio)benzo[d]thiazole(3a)[17], 2-(4-chlorobenzylthio)benzo[d]thiazole
(3b) [18], 2-(4-methoxybenzylthio)benzo[d]thiazole (3c) [19], 2-(4-fluorobenzylthio)benzo[d]thiazole (3d) [17],
and 2-(3-fluorobenzylthio)benzo[d]thiazole (3e) [20] are coherent with those reported in the literature.

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Table 1. Structures and yields of synthesized BTA derivatives.
Entry
Benzyl chloride
Compound
Yield a (%)
N
S
S
Cl
2a
1
75
3a
Cl
Cl
N
S
2
3
4
80
80
73
S
S
S
2b
Cl
3b
3c
Cl
OCH3
F
N
S
2c
OCH3
Cl
N
S
2d
F
3d
F
F
Cl
5
6
83
77
N
S
S
2e
3e
F
F
Cl
N
S
S
F
2f
F
3f
F
F
F
F
F
F
Cl
F
N
S
7
87
S
F
2g
F
3g
F
a
Isolated yield based on 2-mercaptoBTA.
Spectroscopic Characterization
The series of compounds were characterized by spectroscopic methods (IR, NMR), mass
spectrometry (EM-ESI), and elemental analysis. In general, the IR vibration analysis shows the
main diagnostic signals for the different BTA derivatives. Hence, C-H stretching bands are observed
in the range of
1610 and 1360 cm−1, a series of sharp and intense bands due to the vibrations of the BTA moiety are
observed. In the case of the fluorinated compounds, a very intense signal around
990 cm−1 due to
ν ν
3080 to 2840 cm−1 due to the aromatic and aliphatic hydrocarbon units. Between
ν
the C-F stretch is observed. Mass spectrometry analysis by electrospray ionization showed, in all cases,
a single peak in the corresponding spectra due to the molecular ion plus one mass unit (M+ + 1).
1
Further, analysis by H, 13C{1H}, and 19F{1H} NMR afforded spectra exhibiting a number of
signals on the proper chemical shifts coherent with the proposed structures of the series of BTAs. Thus,
1H NMR spectra showed signals in the range of 7.84–7.30 ppm due to the protons of the BTA moiety
(4H). In addition, a diagnostic signal due to the benzylic protons (2H) was found at about 5 ppm.
Moreover, the spectra of the BTA derivatives having a para-substituted benzene ring, exhibited a typical
set of signals corresponding to an AA’BB’ system (4H) in the aromatic zone between 7.9 and 6.7 ppm.

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Besides, the 13C{1H} NMR spectra exhibited the seven expected signals for the BTA fragment in the
range of 135–110 ppm. The signal due to the benzylic carbon was observed between 24–36 ppm. In the
case of the fluorine-bonded carbons, these exhibited coupling constants of J = 244 Hz, this value being
characteristic for this type of couplings. Finally, the 19F{1H} NMR spectra obtained were consistent
with the different fluorine substituted derivatives 3d–g.
2.2. Biological Evaluation
As mentioned above, the acute phase of American trypanosomiasis is characterized by a high
parasitaemia and bloodstream presence trypomastigotes, these parasitic forms are highly infectious
for myocardial cells and enterocytes among others, where they can remain for years as intracellular
amastigotes resulting in the chronic phase of the disease [21]. Thus, given the fact that parasites on the
bloodstream trypomastigotes stadium are the infective form, in mammals it becomes important the
performing of trypanocidal evaluation of new compounds considering at least two or more strains
endemic in the region.
In this line, the trypanocidal activity of the series of 2-benzylsulfanyl-BTA derivatives was
evaluated in vitro against bloodstream trypomastigotes of two strains of T. cruzi endemic in Mexico
(Table 2). Hence, different concentrations were evaluated to determine the LC50 of each compound.
Additionally, the cytotoxic effect and the selectivity index (SI) were determined. Table 2 shows the
results obtained for each of the compounds, as well as the reference drugs employed.
Table 2. Biological activity of BTA derivatives on T. cruzi NINOA and INC-5 strain.
Bloodstream Trypomastigotes
SI
Macrophages
J774A.1
LC50 (µM)
(CC50 /LC50)
Compound
T. cruzi
NINOA
T. cruzi
INC-5
CC50 (µM)
T. cruzi
NINOA
T. cruzi
INC-5
3a
3b
123.44 ± 21.86
>350
335.59 ±35.7
262.62 ± 12.33
275.67 ± 22.96
>350
>900
>7.29
Nd
>2.68
>3.42
>3.26
Nd
>900
3c
307.82 ± 28.88
>350
>900
>2.92
Nd
3d
3e
869.91 ± 26.8
784.82 ± 25.45
491.88 ± 10.09
249.56 ± 9.44
347.32 ± 18.26
223.43 ± 11.23
>350
>350
Nd
Nd
3f
109.76 ± 23.18
146.95 ± 21.01
96.26 ± 11.48
173.46 ± 15.89
259.81 ± 25.56
185.35 ± 12.95
127.63 ±14.97
216.57 ± 23.08
4.48
1.69
3.61
1.28
1.91
1.34
2.72
1.05
3g
Nfx
Bnz
Nd: Not determined.
From Table 2, it can be observed that compounds 3a
bloodstream trypomastigotes of T. cruzi NINOA, with lower values of LC50 than Bnz (LC50 = 173.46
and having compound 3f as the best of this group (LC50 = 109.76 M) exhibiting similar activity to
that of Nfx (LC50 = 96.96 M). However, when the evaluation was performed on trypomastigotes of
T. cruzi INC-5, compound 3g (LC50 = 185.35 M) exhibited the best trypanocidal effect when compared
with Bnz (LC50 = 216.57 M). In addition, compounds 3b (LC50 = 262.62 M), 3c (LC50 = 275.67 M)
and 3f (LC50 = 259.81 M) exhibited similar activities, however, still better that those observed with
,
3f, and 3g exhibited trypanocidal activity in
µM),
µ
µ
µ
µ
µ
µ
µ
both reference drugs, being only closer to that produced by Bnz. Finally, compound 3b only showed
activity against T. cruzi INC-5, while compounds 3d and 3e showed null activity in neither of both
strains. None of the synthesized compounds exceeded the activity of Nfx in both strains.
Interestingly, when the cytotoxic effect in macrophages was studied, six of the seven BTA
derivatives evaluated exhibited a reduced cytotoxic effect on mammalian cells, compared with both

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reference drugs. Hence, compounds 3a
,
3b
,
3c,
3d, and 3e were over two times less toxic than Nfx
and about three-fold less toxic than Bnz. Compounds 3d and 3e were not considered in this analysis
because they did not exhibit trypanocidal activity. Moreover, although compound 3g resulted to be the
most toxic compound of the series, it is still slightly less toxic than Bnz.
As a consequence of the combination of trypanocidal effect and low toxicity observed with
compounds 3a, 3b, 3c, and 3f, their SI were better. Thus, all active compounds exhibited better SI
values than Bnz in both strains. In the case of Nfx, its SI value was surpassed by the values exhibited
by compounds 3a and 3f on T. cruzi NINOA and by compounds 3a and 3c on T. cruzi INC-5. However,
although compound 3b produced the best SI values for T. cruzi INC-5, the most interesting compounds
of the series are 3a and 3c, since they were the only species showing trypanocidal activity in both
strains with minimum cytotoxic effect. Thus, these compounds are the best candidates for further
studies since they have similar or better selectivity than that of the reference drugs.
The bloodstream trypomastigotes of T. cruzi NINOA were more sensitive to both series of BTA and
reference drugs, requiring lower concentrations to reach the 50% lysis of the parasites in comparison
with those of the T. cruzi INC-5 string. This is probably due to the different origin of both strains
because the T. cruzi NINOA strain was isolated form a patient with acute Chagas, while the T. cruzi
INC-5 strain was obtained from a chronic Chagas patient. Besides, it is well known that among the
T. cruzi strains, there is a wide genetic diversity, and thus their biological behavior may vary [22].
Interestingly, structure-activity relationship analysis (SAR) showed that the amount of fluorine in
the BTA molecules plays an important role on their trypanocidal activity. Thus, compounds 3d and 3e
did not show lytic activity on either of the strains, having only one fluorine on their structures, and
their trypanocidal activity is low in comparison with that observed, in both strains, for compounds 3f
and 3g, which have two or more fluorines on their structures. In addition, compound 3b has a single
chlorine proved to be active with the T. cruzi INC-5 strain, however, not with T. cruzi NINOA strain.
On the other hand, compounds 3a and 3c, which do not have halogens on their structures, only
exhibited moderated trypanocidal activity. While derivatives 3a, 3b, and 3c were the less cytotoxic
(Table 2). Noteworthy is the fact that cytotoxicity increases for those BTA’s with fluorines on their
structures, with a clear trend of increase in their cytotoxicity as the number of fluorines on their
structures increases, i.e., 3g > 3f > 3e > 3d.
Improving selectivity in drug design is a must. Thus, from the series of BTA’s compounds attained,
it was important to determine whether their biological activity was a consequence of being selective
or due to their general toxicity [23]. Thus, results from the experiments showed that in the case of
compounds 3f and 3g their trypanocidal effect is due to toxicity and not to selectivity. Meanwhile,
those compounds without halogens on their structures, i.e., 3a and 3c, exhibited the best SI indexes
(Table 2) for both strains.
In summary, we synthesized, in a facile manner, a series 2-substituted-BTA’s, that exhibited good
trypanocidal activity at low concentrations, thus being promising candidates for further, more detailed
studies in both in vitro and in vivo or to be used as a base for the further improved design of other
similar molecules in the search for more efficient and more selective compounds (in silico studies).
Some of these approaches, including the anti-inflammatory and antioxidant studies of the series of
BTA compounds, are currently under development in our laboratories.
3. Materials and Methods
3.1. Reagents and Apparatus
All reagents used were commercially obtained from Sigma-Aldrich Chemical Co., Inc. (St Louis,
MO, USA), and were used as received without further purification. Solvents were supplied by J.T.
Baker (Phillipsburg, NJ, USA), which were dried and distilled prior to use, using standard procedures
established under dinitrogen atmosphere. The melting points were determined and are reported
without correction using a MELT-TEMP II Laboratory Devices, and vibrational spectroscopy IR was

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performed in the range of 4000 to 350 cm−1 in a NICOLET MAGNA spectrometer 750 FT-IR in KBr
discs. MS-ESI were carried out using a JEOL JMS-SX102A spectrometer. NMR spectra were recorded
in DMSO-d6 at room temperature on a JEOL spectrometer GX300 ECLIPSE with 300 Hz frequency for
1H, 75 Hz for 13C{1H}, 282 Hz for 19F{1H}. The chemical shifts ( ) for 1H and 13C{1H} are reported in
δ
ppm at low field in relation to TMS or the residual signal of the solvents employed. In the case of the
19F{1H}, F3CCO2H was used as external reference. All reactions were performed in open atmosphere.
3.2. Synthesis of 2-benzylsulfanyl BTAs Derivatives (1–7)
The BTA derivatives were obtained by the following general procedure: A suspension of
2-mercaptoBTA (1.0 eq) and K2CO3 (1.1 eq) in THF, where the corresponding benzyl chloride (1.1
eq) was added dropwise and, thereafter, a catalytic amount of KI was added. The reaction mixture
was stirred at room temperature for 24 h. The course of the reaction was monitored by thin-layer
chromatography. Over time, the reaction mixture was filtered and washed with CH2Cl2 (3
×
20 mL).
The organic filtrate was washed with brine (20 mL), dried with anhydrous Na2SO4, and evaporated in
vacuo to give solids, in most of cases, which were purified by recrystallization from hexane: AcOEt 9:1
to afford amorphous solids.
2-(benzylthio)benzo[d]thiazole (3a). Light yellow amorphous solid (1.5 g, 5.8 mmol, 75%), mp 34–36 ◦C.
IR (KBr),
ν
(cm−1): 3106, 3083, 3056, 3030, 2926, 2838, 1898, 1817, 1688, 1584, 1558, 1492, 1453, 1421,
1307, 1274, 1236, 1190, 1120, 1074, 994, 933, 916, 889, 858, 811, 768, 748, 706, 673, 620. MS-ESI, m/z: 258
1
(100, [M+ + 1]). H NMR (300 MHz, DMSO-d6),
δ
(ppm): 8.14 (d, J = 7.9 Hz, 1H, Ar), 8.04 (d, J = 8.1 Hz,
1H, Ar), 7.50–7.45 (m, 3H, Ar), 7.37–7.32 (m, 3H, Ar), 7.29–7.26 (m, 1H, Ar), 4.80 (s, 2H, CH2). 13C{1H}
NMR (75 MHz, DMSO-d6),
δ (ppm): 166.03, 152.56, 136.47, 134.62, 128.99, 128.50, 127,52, 126.32, 124.44,
121.72, 121.13, 36.57. Elem. Anal. Anal. Calc. for C14H11NS2 (257.37 gmol−1): C, 65.33; H, 4.31; N, 5.44;
S, 24.92 Found: C 64.83, H 4.29, N, 5.40; S 24.46.
2-(4-chlorobenzylthio)benzo[d]thiazole (3b). Yellowish amorphous solid (1.2 g, 4.11 mmol, 80%), mp
78–80 ◦C. IR (KBr),
ν
(cm−1): 3060, 2923, 1589, 1555, 1487, 1453, 1421, 1307, 1273, 1235, 1187, 1120, 1088,
997, 939, 892, 862, 828, 805, 751, 726, 701, 679, 645. MS-ESI m/z: 292 (100, [M+ + 1]), 294 (45, [M+ +
1
2]), 295 (10), 258 (20),125 (12). H NMR (300 MHz, DMSO-d6)
δ (ppm): 7.84 (d, J =8.0 Hz, 1H, Ar),
7.75 (d, J = 8.1 Hz, 1H, Ar), 7.38 (d, J = 6.8 Hz, 1H, Ar), 7.33 (t, J = 7.2 Hz, 1H, Ar), 7.25-7.20 (m, 3H,
Ar), 4.63 (s, 2H, CH2). 13C{1H} NMR (75 MHz, DMSO-d6),
δ
(ppm): 165.77, 152.52, 136.86, 134.69,
132.17, 131.91, 128.46, 126.38, 124.54, 122.79, 121.22, 36.71. Elem. Anal. Anal. Calc. for C14H10Cl1N1S2
(291.82 gmol−1): C, 57.62; H, 3.45; N, 4.80; S, 21.98 Found: C 57.80, H 3.42, N, 4.68; S 22.00.
2-(4-methoxybenzylthio)benzo[d]thiazole (3c). Yellow amorphous solid (1.17 g, 4.07 mmol, 80%), mp
63–65 ◦C. IR (KBr),
ν
(cm−1): 3050, 3009, 2949, 2930, 2903, 2832, 1985, 1878, 1724, 1610, 1581, 1507,
1456, 1424, 1301, 1279, 1235, 1197, 1174, 1124, 1093, 1071, 1025, 995, 935, 898, 823, 752, 698. MS-ESI
1
m/z: 288 (30, [M+ + 1]), 121 (100). H NMR (300 MHz, DMSO-d6)
δ
(ppm): 8.00 (d, J =7.9 Hz, 1H, Ar),
7.89 (d, J = 8.1 Hz, 1H, Ar), 7.50–7.33 (m, 4H, Ar), 6.9 (d, J = 8.6 Hz, 2H, Ar), 4.72 (s, 2H, CH2), 3.86
(s, 3H, OCH3). 13C{1H} NMR (75 MHz, DMSO-d6),
δ
(ppm): 166.22, 158.72, 152.62, 134.62, 130.33,
128.11, 126.34, 124.45, 121.75, 121.14, 113.95, 55.04, 36.36. Elem. Anal. Anal. Calc. for C15H13N1O1S2
(287.4 gmol−1): C, 62.69; H, 4.56; N, 4.87; S, 22.31 Found: C 62.57, H 4.54, N, 4.74; S 22.13.
2-(4-fluorobenzylthio)benzo[d]thiazole (3d). Yellowish amorphous solid (1.47 g, 6.24 mmol, 73%), mp
61–63 ◦C. IR (KBr),
ν
(cm−1): 3429, 3064, 2928, 2850, 1735, 1596, 1503, 1453, 1422, 1370, 1308, 1270, 1243,
1219, 1154, 1122, 1108, 1077, 1054, 1016, 990, 940, 899, 832, 751, 725, 704, 690, 668. MS-EI m/z: 275(38,
[M+ + 1]), 109 (100), 242 (20), 179 (2), 166 (5), 139 (2), 122 (3), 83 (12), 69 (3), 63(3), 39(3). 1H NMR
(300 MHz, DMSO-d6)
7.58 (t, Ar), 7.47 (t, J = 7.5 Hz, 1H, Ar), 7.27 (t, J = 8.7 Hz, 2H, Ar), 4.75 (s, 2H, CH2). 13C{1H} NMR
(75 MHz, DMSO-d6), (ppm):165.94, 161.54 (d, J = 243.9 Hz), 152.58, 134.70, 132.92 (d, J = 3.1 Hz), 131.12
(d, J = 8.3 Hz), 126.40, 124.54, 121.80, 121.20, 115.36 (d, J = 29.5 Hz), 35.74. 19F{1H} NMR (282 MHz,
δ (ppm): 8.10 (d, J =7.9 Hz, 1H, Ar), 8.0 (d, J = 8.1 Hz, 1H, Ar), 7.68-7.63 (m, Ar),
δ

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DMSO-d6) δ
(ppm): -114.58. Elem. Anal. Anal. Calc. for C14H10F1N1S2 (275.36 gmol−1): C, 61.06; H,
3.66; N, 5.09; S, 23.29 Found: C 61.02, H 3.58, N, 5.11; S 23.23.
2-(3-fluorobenzylthio)benzo[d]thiazole (3e). Amber liquid (1.25 g, 4.54 mmol, 83%). IR (KBr),
ν
(cm−1):
3060, 2931, 1738, 1614, 1587, 1486, 1453, 1424, 1307, 1256, 1236, 1135, 1074, 993, 942, 879, 784, 751, 723,
1
707, 678. MS-ESI m/z: 276(100, [M+ + 1]), 277 (18), 278 (10). H NMR (300 MHz, DMSO-d6)
δ
(ppm):
8.00 (d, J = 7.9 Hz, 1H, Ar), 7.90 (d, J = 8.1 Hz, 1H, Ar), 7.47 (t, J = 7.6 Hz, 1H, Ar), 7.36 (t, 4H, Ar), 7.11
(t, J = 7.9 Hz, 1H, Ar), 4.77 (s, 2H, CH2). 13C{1H} NMR (75 MHz, DMSO-d6),
(ppm):165.79, 162.02 (d,
J = 243.9 Hz), 152.54, 140.11 (d, J = 7.7 Hz),134.73, 130.95 (d, J = 8.5 Hz), 126.40, 125.19, 124.55, 121.81,
δ
121.20, 116.30 (d, J = 21.9 Hz), 114.90 (d, J = 20.9 Hz), 33.59. 19F{1H} NMR (282 MHz, DMSO-d6)
(ppm):
S, 23.29 Found: C 60.98, H 3.63, N, 5.01; S 23.15.
δ
−
112.97. Elem. Anal. Anal. Calc. for C14H10F1N1S2 (275.36 gmol−1): C, 61.06; H, 3.66; N, 5.09;
2-(2,6-difluorobenzylthio)benzo[d]thiazole (3f). Amber liquid (1.152 g, 3.93 mmol, 77%). IR (KBr),
ν
(cm−1):
3376, 3062, 3000, 2953, 1738, 1623, 1589, 1561, 1509, 1465, 1424, 1375, 1308, 1272, 1236, 1164, 1131, 1074,
1
993, 936, 853, 826, 786, 753, 725, 702, 661. MS-ESI m/z: 294 (100, [M+ + 1]), 295 (42), 296 (25). H NMR
(300 MHz, DMSO-d6)
3H, Ar), 7.26 (t, J = 8.0, 2H, Ar), 4.81 (s, 2H, CH2). 13C{1H} NMR (75 MHz, DMSO-d6),
160.62 (dd, J = 249.8, 8.8 Hz), 152.54, 134.96,131.05 (t, J = 10.4 Hz), 126.50, 124.80, 121.93, 112.27 (m),
δ
(ppm): 8.14 (d, J =7.8 Hz, 1H, Ar), 8.02 (d, J = 8.0 Hz, 1H, Ar), 7.63–7.48 (m,
δ
(ppm):164.73,
24.49. 19F{1H} NMR (282 MHz, DMSO-d6)
δ
(ppm):
−
113.36. Elem. Anal. Anal. Calc. for C14H9F2N1S2
(293.35 gmol−1): C, 57.32; H, 3.09; N, 4.97; S, 23.08 Found: C 57.13, H 3.02, N, 5.01; S 23.15.
2-(perfluorobenzylthio)benzo[d]thiazole (3g). Yellowish amorphous solid (1.298 g, 3.73 mmol, 86%), mp
54–56 ◦C. IR (KBr),
ν
(cm−1): 3065, 2963, 1737, 1654, 1501, 1454, 1423, 1306, 1274, 1237, 1169, 1123, 1078,
1038, 985, 967, 882, 756, 726, 703, 682, 640, 603. MS-ESI m/z: 348(100, [M+ + 1]), 349 (45), 350 (25). H
1
NMR (300 MHz, DMSO-d6)
δ (ppm): 8.04 (d, J = 7.90 Hz, 1H, Ar), 7.87 (d, J = 8.1 Hz, 1H, Ar), 7.50 (t,
J = 7.60 Hz, 1H, Ar), 7.40 (t, J = 7.6, 1H, Ar), 4.77 (s, 2H, CH2). 13C{1H} NMR (75 MHz, DMSO-d6),
δ
(ppm):164.01, 152.37, 144.84 (dt, J = 244.2, 13.5 Hz), 140.15 (dt, J = 248.8, 13.5 Hz), 136.90 (dt, J = 249.9,
15.8 Hz), 135.01, 126.53, 124.88, 122.00, 121.35, 111.36 (t, J = 19.5 Hz), 24.21. 19F{1H} NMR (282 MHz,
DMSO-d6)
δ
(ppm):
−
140.92 (dd, J = 22.8, 7.2 Hz),
−
154.91, -155.11 (m), 162.54–162.71 (m). Elem.
−
Anal. Anal. Calc. for C14H6F5N1S2 (347.33 gmol−1): C, 48.41; H, 1.74; N, 4.03; S, 18.46 Found: C 48.37
H 1.67, N, 4.05; S 18.50.
3.3. Biological Activity
3.3.1. Trypanocidal Activity Evaluation
Compounds and reference drugs, nifurtimox (Nfx: Lampit™, Bayer) and Bnz (Rochagan™,
Roche), were dissolved at 10 mg/mL in dimethyl sulfoxide (DMSO, Sigma-Aldrich), and corresponding
dilutions were carried out in phosphate-buffered saline (PBS).
Bloodstream trypomastigotes of two strain of T. cruzi were used: NINOA strain
(MHOM/MX/1994/NINOA, isolated from a patient in acute phase in Oaxaca, Mexico) and INC-5 strain
(MHOM/MX/1994/INC5, isolated from a patient in chronic phase in Guanajuato, Mexico). CD1 mice
6–8 weeks old were intraperitoneally infected; at the maximum peak of parasitaemia (4 weeks), infected
blood was obtained by cardiac puncture using heparin as anticoagulant. Infected blood was adjusted
to 1
×
106 parasites/mL and seeded into 96-well microplates. Animal experiments were performed
according to Norma Oficial Mexicana (NOM-062-Z00-1999) published on august 22, 2009 entitled
Technical specifications for the production, care and use of laboratory animals. The compounds were
evaluated at different concentrations obtained by serial dilutions starting from 100 µg/mL. In each well,
90 µL of infected blood and 10 µL of the corresponding compound or reference drug were deposited.
A positive control of lysis reference drugs were used, and DMSO 1% was used as the negative control.
All assays were performed in triplicate. The microplate was incubated 24 h at 4 ◦C.

Molecules 2019, 24, 3077
8 of 9
The quantification of bloodstream trypomastigotes was performed by the Brener-Pizzi method,
L of blood was deposited between a slide and a coverslip (18 18 mm), and all bloodstream
trypomastigotes present in 20 fields were quantified in an optical microscope at 40 . The amount
5
µ
×
×
of trypomastigotes from each sample was compared to the negative control and the percentage of
lysis was determined; finally, the lytic concentration of 50% parasites (LC50) were calculated for each
compound and converted to micromolar data [23].
3.3.2. Cytotoxic Activity Evaluation and SI
For this assay, murine macrophage cell line J774A.1 (TIB-61 ATCC) were used in RPMI 1640 medium
(Gibco, Carlsbad, CA, USA), enriched with 10% inactivated fetal bovine serum (FBS, Gibco Carlsbad,
CA, USA), 1% penicillin-streptomycin (In vitro S.A., Mexico City, Mexico) and 1% MEM-NEA medium
(Gibco, Carlsbad, CA, USA), the cells were kept at 37 ◦C with 5% CO2 and moisture atmosphere. Initially,
on a 96-well microplate, they were placed 5
were tested at different concentrations, starting from 100
negative control untreated cells were used. The microplate was incubated for 20 h under the same
conditions; afterward, 10 L of resazurin (Invitrogen, Grand Island, NY, USA) 0.01% was added and
×
104 per well and incubated for 24 h. The compounds
g/mL and by triplicate, as cytotoxicity
µ
µ
incubated for an additional 4 h period. The microplate was analyzed on a fluorometer at 544 nm
excitation/590 nm emission (Spectramax Plus; Molecular Devices, Sunnyvale, CA, U.S.). As a negative
control of cytotoxicity, macrophages treated with 0.1% DMSO were used. The cytotoxic concentration
of 50% of population (CC50) was determined using the de Probit statistical tool and converted to
micromolar data. Finally, the ratio between CC50 of macrophages and LC50 of the bloodstream
trypomastigotes (CC50/ CL50) was calculated to get SI values [23].
Author Contributions: A.A.-S. and D.M.-M. conceived and designed the experiments; A.A.-S., J.D.T.-A., F.D.-C.,
and M.E.V.-D. performed the chemical and spectroscopic experiments; B.N.-T. and K.F.C.-V. performed the
biological experiments. All authors discussed the results and commented on the manuscript.
Funding: This research was funded by CONACYT 62267, SIP-IPN (grants 20170697, 2018500) and PAPIIT (grants
No. IN207317).
Acknowledgments: A.A.-S. would like to acknowledge CONACYT 62267, SIP-IPN (grants 20170697, 2018500).
We would like to thank Luis Velasco Ibarra, Dr. Francisco Javier P
é
rez Flores, Q. Er
é
ndira Garc
í
a R
í
os, Lucia del
Carmen M
á
rquez Alonso, Lucero R
í
os Ruiz, Alejandra N
ú
ñez Pineda (CCIQS), Q. Mar
í
a de la Paz Orta Pé
rez, and
Q. Roció Patiño-Maya for technical assistance. PAPIIT (grants No. IN207317) is gratefully acknowledged.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).