Synthesis and trypanocidal activity of novel pyridinyl-1,3,4-thiadiazole derivatives

Article abstract of DOI:10.1016/j.biopha.2020.110162

Herein, we present the design, synthesis and trypanocidal evaluation of sixteen new 1,3,4-thiadiazole derivatives from N-aminobenzyl or N-arylhydrazone series. All derivatives were assayed against the trypomastigote form of Trypanosoma cruzi, showing IC₅₀ values ranging from 3 to 226 μM, and a better trypanocidal profile was demonstrated for the 1,3,4-thiadiazole-N-arylhydrazones (3a-g). In this series, the 2-pyridinyl fragment bound to the imine subunit of the hydrazine moiety presented pharmacophoric behavior for trypanocidal activity. Compounds 2a, 11a and 3e presented remarkable activity and excellent selectivity indexes. Compound 2a was also active against the intracellular amastigote form of T. cruzi. Moreover, its corresponding hydrochloride, compound 11a, showed the most promising profile, producing phenotypic changes similar to those caused by posaconazole, a well-known inhibitor of sterol biosynthesis. Thus, 1,3,4-thiadiazole derivative 11a could be considered a good prototype for the development of new drug candidates for Chagas disease therapy.

Full text of DOI:10.1016/j.biopha.2020.110162

Biomedicine&Pharmacotherapy127(2020)110162
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy
journal homepage: www.elsevier.com/locate/biopha
Synthesis and trypanocidal activity of novel pyridinyl-1,3,4-thiadiazole
derivatives
Rosana H.C.N. Freitasa,1, Juliana M.C. Barbosab,1, Patrícia Bernardinob, Vitor Sueth-Santiagoc,
Solange M.S.V. Wardelld, James L. Wardelle, Débora Decoté-Ricardof, Tatiana G. Melog,
Edson F. da Silvah,i, Kelly Salomãob,2, Carlos A.M. Fragaa,*,2
a Laboratório de Avaliação e Síntese de Substâncias Bioativas (LASSBio), Instituto de Ciências Biomédicas, Universidade Federal do Rio de Janeiro, 21941902, Rio de
Janeiro, RJ, Brazil
b Laboratório de Biologia Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, 21040-360, Rio de Janeiro, RJ, Brazil
c Instituto Federal de Educação, Ciência e Tecnologia do Rio de Janeiro, Campus São Gonçalo, 24425-004, São Gonçalo, RJ, Brazil
d CHEMSOL, 1 Harcourt Road, Aberdeen, AB15 5NY, Scotland, UK
e Department of Chemistry, University of Aberdeen, Old Aberdeen, AB 24 3 UE, Scotland, UK
f Departamento de Microbiologia e Imunologia Veterinária, Instituto de Veterinária, Universidade Federal Rural do Rio de Janeiro, 23890-000, Seropédica, RJ, Brazil
g Laboratório de Ultraestrutura Celular, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, 21040-360, Rio de Janeiro, RJ, Brazil
h Instituto de Tecnologia em Fármacos e Farmanguinhos, Fundação Oswaldo Cruz, 21041-250, Rio de Janeiro, RJ, Brazil
i Escola de Ciência e Tecnologia, Universidade do Grande Rio, 25071-202, Duque de Caxias, RJ, Brazil
A R T I C L E I N F O
A B S T R A C T
Keywords:
Herein, we present the design, synthesis and trypanocidal evaluation of sixteen new 1,3,4-thiadiazole derivatives
from N-aminobenzyl or N-arylhydrazone series. All derivatives were assayed against the trypomastigote form of
Trypanosoma cruzi, showing IC50 values ranging from 3 to 226 μM, and a better trypanocidal profile was de-
monstrated for the 1,3,4-thiadiazole-N-arylhydrazones (3a-g). In this series, the 2-pyridinyl fragment bound to
the imine subunit of the hydrazine moiety presented pharmacophoric behavior for trypanocidal activity.
Compounds 2a, 11a and 3e presented remarkable activity and excellent selectivity indexes. Compound 2a was
also active against the intracellular amastigote form of T. cruzi. Moreover, its corresponding hydrochloride,
compound 11a, showed the most promising profile, producing phenotypic changes similar to those caused by
posaconazole, a well-known inhibitor of sterol biosynthesis. Thus, 1,3,4-thiadiazole derivative 11a could be
considered a good prototype for the development of new drug candidates for Chagas disease therapy.
Chagas disease
Trypanocidal activity
Inhibitor of sterol biosynthesis
2-amino-1,3,4-thiadiazole
1,3,4-thiadiazole-N-arylhydrazone
1. Introduction
the treatment of Chagas disease [5]. Thus, intensive research programs
have focused on the search for alternative natural or synthetic lead
Chagas disease, also known as American trypanosomiasis, is caused
by the flagellate protozoan Trypanosoma cruzi. The disease was dis-
covered in 1909 and is currently considered by the World Health
Organization (WHO) as one of the twenty neglected tropical diseases,
affecting more than 5 million people worldwide [1,2]. Even 110 after
years its discovery, the etiological treatment for Chagas disease is re-
stricted to two nitroheterocyclic drugs: benznidazole (Bz) and ni-
furtimox (Nif) (Fig. 1A) [3]. Their effectiveness varies with the phase of
the infection, dose, period of treatment, and age and geographical
origin of the patient [4]. Severe adverse reactions and limited efficacy
in the chronic phase justify the need for new drugs/combinations for
trypanocidal compounds for drug development.
Currently, two approaches are commonly used in the development
of drugs for neglected diseases: combination and repositioning [6]. In
the context of repositioning, inhibitors of the biosynthetic pathway of
ergosterol, originally developed against fungi, are being intensively
investigated for pathogenic trypanosomatids. Like fungi, T. cruzi has a
strict requirement for specific endogenous ergosterol, and for this
reason, enzymes of sterol metabolism have been intensively studied as
drug targets [7]. C14-α-sterol demethylase of T. cruzi (TcCYP51) is a
key enzyme for the biosynthesis of ergosterol, catalyzing the removal of
the C14 methyl group of lanosterol [8,9]. The majority of CYP51
⁎ Corresponding author at: Centro de Ciências da Saúde, Av. Carlos Chagas Filho, 373, PO Box 68023, Ilha do Fundão, 21941-971, Rio de Janeiro, RJ, Brazil.
E-mail address: cmfraga@ccsdecania.ufrj.br (C.A.M. Fraga).
1 R.H.C.N. Freitas and J.M.C. Barbosa contributed equally to this article.
2 Both authors contributed equally as senior authors.
https://doi.org/10.1016/j.biopha.2020.110162
Received 25 February 2020; Received in revised form 8 April 2020; Accepted 13 April 2020
0753-3322/©2020TheAuthor(s).PublishedbyElsevierMassonSAS.ThisisanopenaccessarticleundertheCCBYlicense
(http://creativecommons.org/licenses/BY/4.0/).

R.H.C.N. Freitas, et al.
Biomedicine&Pharmacotherapy127(2020)110162
Fig. 1. (A) Drugs employed for the etiological treatment of Chagas disease. (B) Examples of azole TcCYP51 inhibitors.
inhibitor studies involve the repositioning of antifungal azoles. Several
azoles at nanomolar concentrations have displayed a strong binding
affinity for TcCYP51 and trypanocidal activity, interfering with sterol
biosynthesis in intracellular amastigotes, and in vivo assays have led to
high levels of parasitological cure [10–14]. In phase II clinical trials
with chronic patients, monotherapy with both azole compounds posa-
conazole [15] and ravuconazole (E-1224) [16] (Fig. 1B) failed to
maintain a sustained response. Recent results of a phase II clinical trial
showed that both monotherapy with Bz and the combination of Bz/
E1224 were efficacious [17]. Thus, TcCYP51 inhibition remains a
promising target for the development of novel trypanocidal compounds
[18].
Among the TcCYP51 inhibitors described in the literature, prototype
1 (Fig. 2) was active at the nanomolar level in vitro against intracellular
amastigotes with an excellent selectivity index (SI) and high efficacy in
mouse models [19]. Since the 3-pyridine moiety of 1 (A, Fig. 2) is
considered pharmacophoric for the inhibition of TcCYP51 due to its
ability to coordinate with heme iron, we planned a new series of re-
gioisomeric pyridinyl-1,3,4-thiadiazole derivatives 2a-f (Fig. 2). These
compounds were designed by replacing the amide subunit of 1 (B,
Fig. 2) with the isosteric 1,3,4-thiadizole ring [20], which has been
described as a stable peptidomimetic group [21,22], aiming to prevent
the characteristic hydrolytic lability of peptide bonds. Moreover, an
azamethylene linker was attached to the heterocyclic subunit in order
to mimic the basic behavior of the N-arylpiperazine unit of 1 (C, Fig. 2).
During the structural planning of 2a-f, a series of molecular sim-
plifications [23] of the auxophoric groups of 1 (E, Fig. 2) were per-
formed. In addition, compounds containing a carbon-fluorine subunit
instead of the pharmacophoric aromatic nitrogen of the 4-pyridine ring
were also planned, exploiting the well-known isosteric relationship
between these groups [20] as represented by retroisosteres 2a and 2d
(Fig. 2). Finally, a more rigid series of aza-homolog derivatives (3a-f)
presenting a hydrazone group was also developed (Fig. 3), aiming to
better understand the influence of this spacer unit on the trypanocidal
activity of these pyridinyl-1,3,4-thiadiazoles. This spacer group was
selected in function of the conformational restriction promoted by the
presence of conjugated imine double bond and good chemical stability
[24], which makes it very attractive for the construction of bioactive
compounds of different therapeutic classes [25].
Therefore, in this paper, we describe the synthesis, structural
characterization and trypanocidal profile of pyridinyl-1,3,4-thiadiazole
Fig. 2. Design concept of pyridinyl-1,3,4-thiadiazole derivatives 2a-f.
2

R.H.C.N. Freitas, et al.
Biomedicine&Pharmacotherapy127(2020)110162
1112 series CHN-Analyzer, using a Mettler MX5 electronic balance.
2.1.1. General procedure for the synthesis of methyl esters (5b-e)
To
a solution of the respective aryl-carboxylic acid (4c-e,
4.09 mmol) in 10 mL methanol was added 15 mL toluene and 3 drops of
concentrated sulphuric acid. The obtained mixture was refluxed at
110 °C for 48 h. After that, the solvent was concentrated under reduced
pressure and then 10 mL of 10 % aqueous sodium carbonate solution
was added to the residue, forming an emulsion. Next, this emulsion was
partitioned between dichloromethane (3 × 30 mL) and brine
(3 × 30 mL). The organic phases were combined, dried with anhydrous
sodium sulphate and evaporated under reduced pressure to furnish the
corresponding methyl esters (5c-e) [26], as described next. Methyl ni-
cotinate (5b) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.1.1.1. Methyl picolinate (5c). Compound (5c) was obtained in 44 %
yield as a colorless oil. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 3.87
(3H, s, CH3), 7.61–7.65 (1H, m), 7.95–8.06 (2H, m), 8.70 (1H, d, J
=6 Hz); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 52.4 (CH3), 124.8 (C3),
127.4 (C5), 137.5 (C4), 147.5 (C2), 149.7 (C6), 165.2 (C=O); IR (KBr)
cm−1: 1249 (C-O), 1585 and 1651 (C = C), 1731 (C = O).
2.1.1.2. Methyl 4-fluorobenzoate (5d). Compound (5d) was obtained in
70 % yield as a colorless oil. 1H NMR (300 MHz, DMSO-d6) δ (ppm):
3.84 (3H, s, CH3), 7.34 (2H, t, J = 9 Hz), 7.99–8.04 (2H, m);13C NMR
(75 MHz, DMSO-d6) δ (ppm): 52.3 (CH3), 115.9 (C3 and C5), 126.3
(C1), 132.1 (C2 and C6), 163.5 and 166.8 (C4, C-F coupling), 165.4
(C = O); IR (KBr) cm−1: 1278 (C-O), 1582 and 1601 (C = C), 1724
(C=O).
2.1.1.3. Methyl benzoate (5e). Compound (5e) was obtained in 82 %
yield as a colorless oil. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 3.85
(3H, s, CH3),7.52 (2H, t, J = 9 Hz), 7.65 (1H, d, J = 9 Hz), 7.96 (2H, d,
J =3 Hz); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 52.2 (CH3), 128.8 (C3
and C5), 129.2 (C2 and C6), 129.7 (C1), 133.3 (C4), 166.3 (C=O); IR
(KBr) cm−1: 1238 and 1281 (C-O), 1509 and 1602 (C = C), 1728
(C = O).
Fig. 3. Design of 1,3,4-thiadiazole-arylhydrazone derivatives 3a-f.
derivatives 2a-f and 3a-f against the clinically relevant forms of T. cruzi.
2. Material and methods
2.1. Chemistry
2.1.2. General procedure for the synthesis of hydrazides (6b-e)
To a solution of the methyl ester (5b-e, 3.68 mmol) in 20 mL of
ethanol was added 2.68 mL of 80 % aqueous hydrazine hydrate
(55.2 mmol) and the mixture was refluxed for 6 h. After that, the excess
of solvent was removed under reduced pressure and the residue was
General information: Purification of the products was performed by
column chromatography using silica gel (230–400) flash (Merck,
Darmstadt) as the stationary phase and mixtures of N-hexane and ethyl
acetate or dichloromethane and methanol in various proportions as the
mobile phase. 1H NMR and 13C NMR were obtained on a Bruker DPX-
200 (4.7 T), Bruker DRX-300 (7.05 T), Bruker AVHD400 (9.4 T), or
Bruker AVIII500 (11.7 T) spectrometer operating at 200, 300, 400 or
500 MHz, respectively. Chemical shifts (δ) are given in parts per million
(ppm) with tetramethylsilane (TMS) as the internal standard and cou-
pling constant (J) values are given in Hertz (Hz). The deuterated solvent
used to obtain the spectra was dimethyl sulfoxide-d6 (DMSO-d6) using
NMR-specific glass tubes (5 mm diameter).
partitioned
between
chloroform
(3 × 30 mL)
and
brine
(3 × 30 mL).The organic phases were combined, dried with anhydrous
sodium sulphate and evaporated under reduced pressure to furnish the
corresponding hydrazides (6b and 6c). Alternatively, in the case of the
hydrazides 6d and 6e a mixture of ice/water was added to promote the
formation of precipitates, which were collected by filtration under re-
duced pressure. The structural characterization and yields of the hy-
drazides (6b-6e) are described next [27].
Infrared (IR) spectra were obtained in a Nicolet 6700 Fourier
transform IR (FT-IR) spectrophotometer apparatus a using potassium
bromide pellets.
2.1.2.1. Nicotinylhydrazide (6b). Compound (6b)was obtained in 55 %
yield as a white solid, mp. 159−160 °C. 1H NMR (500 MHz, DMSO-d6)
δ (ppm): 7.48 (1H, t, J =5 Hz), 8.14 (1H, d, J = 10 Hz), 8.67 (1H, d, J
=5 Hz), 8.94 (1H, s), 9.99 (1H, br., NH);13C NMR (125 MHz, DMSO-d6)
δ (ppm): 123.7 (C5), 129.0 (C3), 134.9 (C4), 148.2 (C2), 151.9 (C6),
164.6 (C = O); IR (KBr) cm−1: 1596 (C = C), 1673 (C = O), 3206 and
3323 (N-H).
The chromatographic purity of the final products was determined by
HPLC on a Shimadzu LC-20CE apparatus with a Kromasil 100-5 C18
column (4.6 mm × 250 mm) and an SPD-M20A diode array detector.
Quantification of the final compounds was performed at a wavelength
of 300 nm. The mobile phase used was acetonitrile and water
(60–80%), varying the running time between 10 and 20 min.
Melting points were determined on a Quimis Q340.23 apparatus
and were not corrected.
2.1.2.2. Picolinylhydrazide (6c). Compound (6c) was obtained in 50 %
yield as a colorless oil. 1H NMR (300 MHz, DMSO-d6) δ (ppm):4.61 (2H,
br., NH2), 7.52–7.58 (1H, m), 7.94–7.98 (2H, m), 8.6 (1H, d, J =4 Hz),
9.88 (1H, br., NH); 13C NMR (75 MHz, DMSO-d6)δ (ppm): 121.7 (C3),
126.2 (C5), 137.6 (C4), 148.5 (C6), 149.8 (C2), 162.6 (C = O); IR (KBr)
cm−1: 1585 and 1651 (C = C), 1731 (C = O), 3433 (N-H).
Determination of the aqueous solubility was performed using a
scanning UV–vis Femto spectrophotometer (model 800XI).
Microanalyses were carried out using a Thermo Scientific Flash EA
3

R.H.C.N. Freitas, et al.
Biomedicine&Pharmacotherapy127(2020)110162
2.1.3.1. 4-Fluorobenzoylhydrazide (6d). Compound (6d) was obtained
in 53 % yield as a white solid, mp. 162−164 °C. 1H NMR (300 MHz,
DMSO-d6) δ (ppm): 4.49 (2H, br., NH2), 7.28 (2H, t, J = 9 Hz),
7.86–7.91 (2H, m), 9.79 (1H, br., NH); 13C NMR (75 MHz, DMSO-d6)
δ (ppm):115.8 (C3 and C5), 130.1 (C2 and C6), 162.6 and 165.9 (C4, C-
F coupling), 165.4 (C = O); IR (KBr) cm−1: 1619 (C = C), 1663
(C = O), 3222 and 3303 (N-H).
156.4 (C5), 168.5 (C2); IR (KBr) cm−1: 1635 and 1685 (C = C), 3257
(N-H).
2.1.5. General procedure for the synthesis of the 1,3,4-thiadiazole
derivatives (2a-f)
In a flask coupled to a reflux condenser were added equimolar
amounts of 1,3,4-thiadiazole-amine (7a-e, 2.25 mmol), the corre-
sponding aromatic or heteroaromatic aldehyde (8a-c, 2.25 mmol), 5 mL
of a 25 % solution of zinc chloride in N,N-dimethylformamide (DMF)
and trimethylsilyl-acetate (TMSOAc, 6.75 mmol, 2.5 equivalents). The
mixture was refluxed at 100 °C for 12 h, sufficient time to formation of
the imine intermediate (observed by TLC). Then, sodium triacetox-
yborohydride (4.4 mmol, 1.5 equivalent) was added to the flask and the
mixture was stirred at room temperature for 72 h. After this time, 2 mL
water and 4 mL saturated sodium carbonate solution were added to the
mixture. For derivatives 2a, 2d and 2e precipitates were formed and
the final products were obtained by vacuum filtration using a Büchner
funnel. However, to obtain 2b and 2c it was necessary a partition be-
tween chloroform (50 mL) and brine (50 mL). Then, organic phase was
dried with anhydrous sodium sulphate and evaporated under reduced
pressure. The obtained residue was purified by column chromatography
using a gradient mixture of N-hexane and ethyl acetate [29].
2.1.3.2. Benzoylhydrazide (6e). Compound (6e) was obtained in 63 %
yield as a white solid, mp. 114−115 °C. 1H NMR (300 MHz, DMSO-d6)
δ (ppm): 4.53 (2H, br., NH2), 7,42-7,53 (3H, m), 7.82–7.84 (2H, d, J
=6 Hz), 9. (1H, br., NH); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 127.2
(C2 and C6), 128.6 (C3 and C5), 131,3 (C4), 133.6 (C1), 166.2 (C = O);
IR (KBr) cm−1: 1579 e 1616 (C = C), 1662 (C = O), 3197 and 3300 (N-
H).
2.1.4. Synthesis of 2-amino-1,3,4-thiadiazoles (7b-e)
To a solution of the corresponding hydrazide (6b-e, 4 mmol) in
20 mL of ethanol equimolar amount of trimethylsilylisothiocyanate
(TMSNCS) was added. The resulting mixture was refluxed at 80 °C for
4 h. Subsequently, the solvent was reduced at reduced pressure, re-
sulting in the formation of a solid mass that was collected by filtration
on a Büchner funnel. After drying, these solids were transferred to an
Erlenmeyer flask and 3 mL of sulfuric acid was added. This suspension
was agitated at room temperature for 12 h. The flask was cooled at 0 °C
with an ice bath and a mixture of ice and water was added, increasing
the temperature and promoting gas release. After cooling, the pre-
cipitate obtained was filtered at reduced pressure on Büchner for the
isolation of the 2-amino-1,3,4-thiadiazoles 7d and 7e. For the 2-amino-
1,3,4-thiadiazoles 7b and 7c, it was necessary to increase the pH to 8
using ammonium hydroxide and maintain the mixture at 0 °C. The solid
formed was filtered on a Büchner funnel and the compounds 7b-c were
obtained as described next [28]. 5-(Pyridin-4-yl)-1,3,4-thiadiazol-2-
amine (7a) was purchased from Sigma-Aldrich.
2.1.5.1. N-(4-Fluorobenzyl)-5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine
(2a). Compound 2a was obtained in 95 % yield as a pale yellow solid,
m.p. 154−155 °C. 1H NMR(200 MHz, DMSO-d6) δ (ppm): 4.55 (2H, d, J
=6 Hz, CH2), 7.18 (2H, t, J =8 Hz), 7.40–7.47 (2H, m), 7.69 (2H, d, J
=6 Hz), 8.64 (2H, d, J =6 Hz), 8.71 (1H, t, J =6 Hz); 13C NMR
(50 MHz, DMSO-d6) δ (ppm): 47.3 (CH2), 115.1 (C3’’ and C5’’ of Ar),
120.2 (C3’ and C5’ of 4-Py), 129.6 (C2’’ and C6’’ of Ar), 134.4 (C1’’ of
Ar), 137.6 (C4’ of 4-Py), 150.5 (C2’ and C6’ of 4-Py), 154.0 (C5), 159.0
and 163.8 (C4’’ of Ar, C-F coupling), 169.4 (C2);IR (KBr) cm-1: 1603 and
1542 (C = C), 3217 (N-H). HPLC purity (λ 300 nm): 98.69 %. Anal.
Calcd for C14H11FN4S: C, 58.73; H, 3.87; N, 19.57. Found: C, 58.85; H,
3.86; N, 19.51.
2.1.4.1. 5-(Pyridin-3-yl)-1,3,4-thiadiazol-2-amine (7b). Compound 7b
was obtained in 52
%
yield as
a
light yellow solid, m.p.
2.1.5.2. N-(4-Fluorobenzyl)-5-(pyridin-3-yl)-1,3,4-thiadiazol-2-amine
(2b). Compound 2b was obtained as a yellow solid in 33 % yield, m.p.
162−164 °C. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 4.54 (2H, d, J
=3 Hz, CH2), 7.18 (2H, t, J = 9 Hz), 7.42 (2H, t, J = 9 Hz), 7.48–7.52
(1H, m), 8.12 (1H, d, J = 9 Hz), 8.61–8.64 (2H, m, 3-Py-H + NH), 8.93
(1H, s). 13C NMR (75 MHz, DMSO-d6) δ (ppm): 47.3 (CH2), 115.2 (C3’’
and C5’’ of Ar), 124.3 (C5’ of 3-Py), 127.0 (C3’ of 3-Py), 129.7 (C2’’ and
C6’’), 133.7 (C4’ of 3-Py), 134.7 (C1’’ of Ar), 147.0 (C2’ of 3-Py), 150.4
(C6’ of 3-Py), 153.4 (C5), 159.8 and 163.1 (C4’’ of Ar), 168.9 (C2); IR
(KBr) cm−1: 1555 and 1509 (C = C), 3177 (N-H). HPLC purity (λ
300 nm): 97.31 %. Anal. Calcd for C14H11FN4S: C, 58.73; H, 3.87; N,
19.57. Found: C, 58.70; H, 3.88; N, 19.62.
237−238 °C.1H NMR (500 MHz, DMSO-d6) δ (ppm): 7.47–7.51 (1H,
m), 7.55 (2H, br, NH2), 8.13 (1H, dt, J = 10 Hz, J =5 Hz), 8.61 (1H, dd,
J =5 Hz and J =5 Hz), 8.94 (1H, s); 13C NMR (125 MHz, DMSO-d6) δ
(ppm): 124.1 (C5’ of 3-Py), 127.1 (C3’ of 3-Py), 133.5 (C4’ of 3-Py),
146.9 (C2’ of 3-Py), 150.2 (C6’ of 3-Py), 153.3 (C5), 169.1 (C2); IR
(KBr) cm-1: 1644 and 1514 (C = C), 3277 (N-H).
2.1.4.2. 5-(Pyridin-2-yl)-1,3,4-thiadiazol-2-amine (7c). Compound 7c
was obtained in 47
% yield as a light yellow solid, m.p.
263−265 °C.1H NMR (300 MHz, DMSO-d6) δ (ppm): 7.40 (1H, t, J
=4 Hz), 7.51 (2H, br, NH2), 7.90 (1H, t, J =8 Hz) 8.04 (1H, d, J
=8 Hz); 8.57 (1H, d, J =4 Hz); 13C NMR (75 MHz, DMSO-d6) δ
(ppm):119.0 (C3’ of 2-Py), 124.2 (C5’ of 2-Py), 137.3 (C4’ of 2-Py),
149.5 (C6’ of 2-Py), 158.5 (C5), 170.0 (C2); IR (KBr) cm−1: 1585 and
1620 (C = C), 3262 (N-H).
2.1.5.3. N-(4-Fluorobenzyl)-5-(pyridin-2-yl)-1,3,4-thiadiazol-2-amine
(2c). Compound 2c was obtained as a yellow solid in 11 % yield, m.p.
131−133 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 4.52 (2H, d, J
=4 Hz, CH2), 7.18 (2H, t, J =8 Hz), 7.40–7.44 (3H, m), 7.90 (1H, t, J
=8 Hz), 8.04 (1H, d, J =8 Hz), 8.57 (2H, s, 2-Py-H + NH). 13C NMR
(100 MHz, DMSO-d6) δ (ppm): 47.3 (CH2), 115.1 (C3’’ and C5’’ of Ar),
119.1 (C3’ of 2-Py), 124.4 (C5’ of 2-Py), 129.6 (C2’’ and C6’’ of Ar),
134.6 (C1’’ of Ar), 137.4 (C4’ of 2-Py), 149.3 (C2’ of 2-Py), 149.6 (C6’ of
2-Py), 158.5 (C5), 160.2 and 162.6 (C4’’ of Ar, C-F coupling), 169.8
(C2); IR (KBr) cm−1: 1603 and 1677 (C = C), 3216 (N-H). HPLC purity
(λ 300 nm): 98.79 %. Anal. Calcd for C14H11FN4S: C, 58.73; H, 3.87; N,
19.57. Found: C, 58.77; H, 3.88; N, 19.63.
2.1.4.3. 5-(4-Fluorophenyl)-1,3,4-thiadiazol-2-amine (7d). Compound
7d was obtained in 34 % yield as a grey solid, m.p. 238−240 °C.1H
NMR (200 MHz, DMSO-d6) δ (ppm): 7.30 (2H, t, J =8 Hz), 7.42 (2H, br,
NH2), 7.77–7.83 (2H, m); 13C NMR (75 MHz, DMSO-d6) δ (ppm):116.7
(C3’ and C5’ of Ar), 128.1 (C1’ of Ar), 129.0 (C2’and C6’ of Ar), 155.8
(C5), 160.8 and 165.6 (C4’ of Ar, C-F coupling), 169.1 (C2); IR (KBr)
cm−1: 1615 and 1516 (C = C), 3338 and 3248 (N-H).
2.1.4.4. Phenyl-1,3,4-thiadiazol-2-amine (7e). Compound 7e was
obtained in 67 % yield as a white solid, m.p. 223−225 °C.1H NMR
(300 MHz, DMSO-d6) δ (ppm): 7.41–7.49 (5H, m, Ph + NH2), 7.74 (2H,
d, J =6 Hz); 13C NMR (75 MHz, DMSO-d6) δ (ppm):126.3 (C2’ and C6’
of Ar), 129.1 (C3’ and C5’ of Ar), 129.5 (C4’ of Ar), 130.9 (C1’ of Ar),
2.1.5.4. 5-(4-Fluorophenyl)-N-(pyridin-4-ylmethyl)-1,3,4-thiadiazol-2-
amine (2d). Compound 2d was obtained as a pale yellow solid in 53 %
yield, m.p. 210−212 °C. 1H NMR (200 MHz, DMSO-d6) δ (ppm): 4.59
(2H, d, J =2 Hz, CH2), 7.31 (2H, t, J =4 Hz), 7.36 (2H, d, J =2 Hz),
4

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Biomedicine&Pharmacotherapy127(2020)110162
7.78–7.82 (2H, m), 8.53 (2H, d, J =2 Hz), 8.58 (1H, t, J =2 Hz,
NH).13C NMR (50 MHz, DMSO-d6) δ (ppm): 46.2 (CH2), 115.8 (C3’ and
C5’ of Ar), 121.8 (C3’’ and C5’’ of 4-Py), 126.9 (C1’ of Ar), 128.1 (C2’
and C6’ of Ar), 147.3 (C4’’ of 4-Py), 149.1 (C2’’ and C6’’ of 4-Py), 155.1
(C5), 159.9 and 164.8 (C4’ of Ar, C-F coupling), 167.8 (C2); IR (KBr)
cm−1: 1603 and 1502 (C = C), 3191 (N-H). HPLC purity (λ 300 nm):
95.41 %. Anal. Calcd for C14H11FN4S: C, 58.73; H, 3.87; N, 19.57.
Found: C, 58.83; H, 3.86; N, 19.52.
(300 MHz, DMSO-d6) δ (ppm): 7.60−7.4 (1H, m), 8.34 (1H, d,
J = 9 Hz), 8.78 (1H, s), 9.13 (1H, s,); 13C NMR (75 MHz, DMSO-d6) δ
(ppm): 124.1 (C5’ of 3-Py), 125.0 (C3’ of 3-Py), 134.8 (C4’ of 3-Py),
147.8 (C2’ of 3-Py), 152.1 (C6’ of 3-Py), 153.8 (C5), 167.6 (C2); IR
(KBr) cm−1: 823 (C-Cl), 1567 and 1584 (C = C).
2.1.6.3. 2-(5-Chloro-1,3,4-thiadiazol-2-yl)pyridine (9c). Compound (9c)
was obtained as a crystalline light yellow solid in 70 % yield. 1H NMR
(300 MHz, DMSO-d6) δ (ppm): 7.62 (1H, t, J =6 Hz), 8.05 (1H, t,
J = 9 Hz), 8.21 (1H, d, J = 9 Hz), 8.7 (1H, d, J =6 Hz); 13C NMR
(75 MHz, DMSO-d6) δ (ppm): 120.2 (C5’ of 2-Py), 126.6 (C4’ of 2-Py),
138.2 (C3’ of 2-Py), 147.3 (C6’ of 2-Py), 150.3 (C2’ of 2-Py), 155.3 (C5),
172,6 (C2); IR (KBr) cm−1: 787 (C-Cl), 1567 and 1581 (C = C).
2.1.5.5. 5-(4-Fluorophenyl)-N-(pyridin-3-ylmethyl)-1,3,4-thiadiazol-2-
amine (2e). Compound 2e was obtained as a dark yellow solid in 75 %
yield, m.p. 117−119 °C. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 4.58
(2H, d, J =6 Hz, CH2), 7.31 (2H, t, J = 9 Hz), 7.4–7.44 (1H, m),
7.78–7.84 (3H, m), 8.48–8.54 (2H, m, 3-Py-H + NH), 8.61 (1H, s). 13C
NMR (50 MHz, DMSO-d6) δ (ppm): 45.0 (CH2), 116.2 (C3’ and C5’ of
Ar), 123.9 (C5’’ of 3-Py), 127.3 (C1’ of Ar), 128.6 (C2’ and C6’ of Ar),
134.6 (C3’’ of 3-Py), 136.3 (C4’’ of 3-Py), 148.1 (C2’’ of 3-Py), 148.6
(C6’’ of 3-Py), 155.5 (C5), 160.3 and 165.2 (C4’ of Ar, C-F coupling),
168.1 (C2); IR (KBr) cm−1: 1669 and 1557 (C = C), 3269 (N-H). HPLC
purity (λ 300 nm): 96.82 %. Anal. Calcd for C14H11FN4S: C, 58.73; H,
3.87; N, 19.57. Found: C, 58.90; H, 3.86; N, 19.51.
2.1.6.4. 2-Chloro-5-(4-fluorophenyl)-1,3,4-thiadiazole (9d). Compound
(9d) was obtained as an amorphous gray solid in 88 % of yield. 1H
NMR (300 MHz, DMSO-d6) (300 MHz, DMSO-d6) δ (ppm): 7.42 (2H, t, J
=6 Hz), 7.99–8.04 (2H, m); 13C NMR (75 MHz, DMSO-d6) δ (ppm):
116.7 (C3’ and C5’), 125.5 (C1’ of Ar), 130.1 (C2’ and C6’), 153.1 (C5),
162.4 and 165.7 (C4’ of Ar, C-F coupling), 169.4 (C2); IR (KBr) cm−1
813 (C-Cl), 1515 and 1600 (C = C).
:
2.1.5.6. N-(4-Fluorobenzyl)-5-phenyl-1,3,4-thiadiazol-2-amine
(2f). Compound 2f was obtained as a beige solid in 39 % yield, m.p.
150−152 °C. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 4.52 (2H, d, J
=6 Hz, CH2), 7.18 (2H, t, J = 9 Hz), 7.41–7.47 (5H, m), 7.74 (2H, d,
J = 9 Hz), 8.47 (1H, t, J =6 Hz, NH); 13C NMR (50 MHz, DMSO-d6) δ
(ppm): 47.2 (CH2), 115.1 (C3’’ and C5’’ of Ar), 126.3 (C2’ and C6’ of
Ph), 129.1 (C3’ and C5’ of Ph), 129.5 (C4’ of Ph), 129.6 (C2’’ and C6’’ of
Ar), 130.7 (C1’ of Ph), 134.7 (C1’’ of Ar), 156.3 (C5), 158.9 and 163.7
(C4’’ of Ar, C-F coupling), 168.2 (C2); IR (KBr) cm−1: 1562 and 1509
(C = C), 3176 (N-H). HPLC purity (λ 300 nm): 95.52 %. Anal. Calcd for
C15H12FN3S: C, 63.14; H, 4.24; N, 14.73. Found: C, 63.22; H, 4.25; N,
14.67.
2.1.6.5. 2-Chloro-5-phenyl-1,3,4-thiadiazole (9e). Compound (9e) was
obtained as an amorphous yellow solid in 71 % yield.1H NMR
(300 MHz, DMSO-d6) δ (ppm): 7.57–7.62 (3H, m), 7.94–7.96 (2H, m);
13C NMR (75 MHz, DMSO-d6) δ (ppm): 127.5 (C2’ and C6’), 128.8 (C4’
of Ar), 129.5 (C3’ and C5’ of Ar), 131.9 (C1’ of Ar), 153.1 (C5), 170.5
(C2); IR (KBr) cm−1: 769 (C-Cl), 1427 and 1456 (C = C).
2.1.7. General procedure for the synthesis of heteroarylhydrazines (10a-e)
To an ethanolic suspension of the heteroaryl chlorides (9a-e,
2.03 mmol, 1 equiv.) was added 80 % aqueous hydrazine hydrate
(30 mmol, 20 equiv.) at room temperature. Then, the mixture was re-
fluxed at 80 °C for 2 h. After this time, it was possible to observe the
total consumption of the starting material and the formation of the
desired hydrazine. Then, the solvent was reduced at reduced pressure
and an ice/water mixture was added to the resultant residue, promoting
immediate precipitation. The solid was collected by filtration on a
Büchner funnel to yield the desired heteroarylhydrazine (10a-e), as
described next [31].
2.1.6. General procedure for the synthesis of 1,3,4-thiazolyl-chlorides (9a-
e)
A solution of corresponding 2-amino-1,3,4-thiadiazole derivative
(7a-e, 2.81 mmol, 1 equiv.) in a mixture of 7 mL concentrated hydro-
chloric acid (HCl) and 3 mL water was maintained at 0 °C. Then, sodium
nitrite (NaNO2, 14 mmol, 5 equiv.) was added to the flask, resulting in
intense gas release and the visual modification of the reaction medium
2.1.7.1. 2-Hydrazinyl-5-(pyridin-4-yl)-1,3,4-thiadiazole
%
to a strong yellow colour. Then, hydrated copper chloride (CuCl2 2H2O,
(10a). Compound 10a was obtained in 81 % yield, as an amorphous
beige solid, m.p. 229−230 °C. 1H NMR (300 MHz, DMSO-d6) δ (ppm):
5.25 (2H, br., NH2), 7.7 (2H, d, J = 9 Hz), 8.63 (2H, d, J =6 Hz), 9.18
(1H, br, NH); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 119.9 (C3’ and C5’
of 4-Py), 138.0 (C4’ of 4-Py), 150.4 (C2’ and C6’ of 4-Py), 154.0 (C5),
178.1 (C2);).IR (KBr) cm−1: 1147 (C-N), 1601 and 1636 (C = C), 3291
and 3310 (N-H).
14 mmol, 5 equiv.) was added and the solution darkened. The mixture
was kept under magnetic stirring at 0 °C for 2 h. Then, still in an ice
bath, 4 mL water was added to the reaction medium, followed by
neutralization to pH 8 with the addition of ammonium hydroxide. Next,
the suspension was extracted with ethyl acetate and saturated NaCl
solution. The organic phase was dried with anhydrous sodium sulphate
and evaporated under reduced pressure to yield the desired heteroaryl
chlorides (9a-e). For the derivatives 9d and 9e, neutralization and ex-
traction were not necessary. The isolation occurred by adding water
and ice to the flask followed by vacuum filtration to collect the pre-
cipitates formed [30]. The yields and the careful structural character-
ization of the 1,3,4-thiadiazolyl-chlorides (9a-e) are described next.
2.1.7.2. 2-Hydrazinyl-5-(pyridin-3-yl)-1,3,4-thiadiazole
(10b). Compound 10b was obtained in 58 % yield, as an amorphous
beige solid, m.p. 160−161 °C. 1H NMR (300 MHz, DMSO-d6) δ (ppm):
5.32 (2H, br, NH2), 7.47–7.51 (1H, m), 8.14 (1H, d, J = 9 Hz), 8.60
(1H, d, J =3 Hz), 8.95 (1H, s), 9.14 (1H, br, NH); 13C NMR (75 MHz,
DMSO-d6) δ (ppm): 124.1 (C5’ of 3-Py), 127.3(C3’ of 3-Py), 133.2 (C4’
of 3-Py), 146.6 (C2’ of 3-Py), 150.1 (C6’ of 3-Py), 153.3 (C5), 177.7
(C2); IR (KBr) cm−1: 1153 (C-N), 1586 and 1633 (C = C), 3343 (N-H).
2.1.6.1. 4-(5-Chloro-1,3,4-thiadiazol-2-yl)pyridine
(9a). Compound
(9a) was obtained as a crystalline solid in 55 % yield. 1H NMR
(300 MHz, DMSO-d6) δ (ppm): 7.92 (2H, d, J =6 Hz), 8.79 (2H, d, J
=6 Hz); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 121.2 (C3’ and C5’ of 4-
Py), 135.7 (C6), 150.9 (C2’ and C6’ of 4-Py), 155.0 (C5), 168.6 (C2); IR
(KBr) cm−1: 827 (C-Cl), 1554 and 1593 (C = C).
2.1.7.3. 2-Hydrazinyl-5-(pyridin-2-yl)-1,3,4-thiadiazole
(10c). Compound 10c was obtained in 67 % yield, as an amorphous
yellow solid, m.p. 214−216 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm):
5.23 (2H, br, NH2), 7.40(1H, t, J =6 Hz), 7.89 (1H, t, J =9 Hz), 8.04
(1H, d, J = 9 Hz), 8.58 (1H, d, J =6 Hz), 9.11 (1H, br., NH); 13C NMR
(75 MHz, DMSO-d6) δ (ppm):118.7 (C3’ of 3-Py), 124.0 (C5’ of 3-Py),
2.1.6.2. 3-(5-Chloro-1,3,4-thiadiazol-2-yl)pyridine
(9b). Compound
(9b) was obtained as a crystalline beige solid in 68 % yield. 1H NMR
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137.2 (C4’ of 3-Py), 149.5 (C6’ of 3-Py), 149.8 (C2’ of 3-Py), 158.3 (C5),
178.3 (C2); IR (KBr) cm−1:1139 (C-N), 1587 and 1633 (C = C), 3343
(N-H).
amorphous yellow solid, m.p. 249−250 °C. 1H NMR (400 MHz, DMSO-
d6) δ (ppm): 7.38–7.41 (1H, m), 7.48–7.51 (1H, m), 7.87 (1H, t, J
=8 Hz), 7.92–8.00 (2H, m), 8.11–8.13 (2H, m), 8.60 (1H, d, J =4 Hz),
8.65 (1H, d, J =4 Hz), 12.8 (1H, br, NH); 13C NMR (75 MHz, DMSO-d6)
δ (ppm): 119.1 (C3’ of 2-Py), 119.5 (C3’’ of 2-Py), 124.0 (C5’ of 2-Py),
124.8 (C5’ of 2-Py), 136.8 (C4’ of 2-Py), 137.5 (C4’’ of 2-Py), 144.6
(imine CH), 148.9 (C2’ of 2-Py), 149.4 (C6’ of 2-Py), 149.7 (C6’’ of 2-
Py), 152.8 (C5), 170.9 (C2); IR (KBr) cm−1: 1159 (C-N), 1564 and 1575
(C = C), 3440 (N-H). HPLC purity (λ 300 nm): 95.48 %. Anal. Calcd for
2.1.7.4. 2-(4-Fluorophenyl)-5-hydrazinyl-1,3,4-thiadiazole
(10d). Compound 10d was obtained in 63 % yield, as an amorphous
beige solid, m.p. 230−231 °C. 1H NMR (200 MHz, DMSO-d6) δ (ppm):
5.22 (2H, br., NH2), 7.3 (2H, t, J =6 Hz), 7.79–7.83 (2H, m), 8.97 (1H,
br, NH); 13C NMR (75 MHz, DMSO-d6) δ (ppm): 116.1 (C3’ and C5’ of
Ar), 127.8 (C1’ of Ar), 128.2 (C2’ and C6’ of Ar), 155.3 (C5), 161.3 and
163.8 (C4’ of Ar, C-F coupling), 177.3 (C2); IR (KBr) cm−1: 1615 and
1516 (C = C), 3338 and 3248 (N-H).
C
13H10N6S: C, 55.30; H, 3.57; N, 29.77. Found: C, 55.36; H, 3.57; N,
29.75.
2.1.8.4. (E)-2-(4-Fluorophenyl)-5-(2-(pyridin-2-ylmethylene)hydrazinyl)-
1,3,4-thiadiazole (3d). Compound 3d was obtained in 85 % yield, as an
amorphous yellow solid, m.p. 243−245 °C. 1H NMR (300 MHz, DMSO-
d6) δ (ppm): 7.34–7.37 (3H, m), 7.87 (4H, m), 8.11 (1H, s, imine CH),
8.59 (1H, m), 12.8 (1H, br., NH); 13C NMR (75 MHz, DMSO-d6) δ
(ppm):116.3 (C3’ and C5’ of Ar), 119.6 (C3’’ of 2-Py), 124.0 (C5’’ of 2-
Py), 127.0 (C1’ of Ar), 128.8 (C3’ and C5’ of Ar), 136.8 (C4’’ of 2-Py),
144.5 (C7’) 149.5 (C6’’ of 2-Py), 152.8 (C5), 161.4 and 164.7 (C4’ of Ar,
C-F coupling), 169.7 (C2). IR (KBr) cm−1: 1161 (C-N), 1577 and 1600
(C = C), 3201 (N-H). HPLC purity (λ 300 nm): 96.28 %. Anal. Calcd for
2.1.7.5. 2-Hydrazinyl-5-phenyl-1,3,4-thiadiazole (10e). Compound 10e
was obtained in 67% yield, as an amorphous yellow solid, m.p.
201−203 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 5.21 (2H, br,
NH2), 7.41–7.49 (3H, m), 7.75–7.78 (2H, m), 8.97 (1H, br, NH); 13C
NMR (100 MHz, DMSO-d6) δ (ppm): 126.0 (C3’ and C5’ of Ar), 129.1
(C2’ and C6’ of Ar), 129.4 (C4’ of Ar), 131.2 (C1’ of Ar), 156.4 (C5),
177.1 (C2).IR (KBr) cm−1: 1140 (C-N), 1572 and 1645 (C = C), 3265
(N-H).
2.1.8. General procedure for the synthesis of 1,3,4-thiadiazolyl-N-
arylhydrazone derivatives (3a-f)
C14H10FN5S: C, 56.18; H, 3.37; N, 23.40. Found: C, 56.09; H, 3.38; N,
23.46.
Equimolar amounts of heteroaryl-hydrazines (11a-e, 1.55 mmol)
and 4-fluorbenzaldehyde (8a, 1.55 mmol, 1 equiv.) or pyridine-2-car-
boxaldehyde (8d, 1.55 mmol, 1 equiv.) were added in ethanol in a
50 mL flask. Then, two drops of 10 % HCl were added and after a few
minutes it was possible to observe the formation of precipitates in the
flask. The mixture was stirred at room temperature for 4 h. After con-
firming the end of the reaction through the TLC, the solvent volume was
reduced in a rotary evaporator and then ice and ice water were added to
the flask. Final compounds (3a-f) were obtained after vacuum filtration
on Büchner funnel [31].
2.1.8.5. (E)-2-Phenyl-5-(2-(pyridin-2-ylmethylene)hydrazinyl)-1,3,4-
thiadiazole (3e). Compound 3e was obtained in 65 % yield, as an
amorphous yellow solid, m.p. 220−222 °C. 1H NMR (500 MHz, DMSO-
d6) δ (ppm): 7.38 (1H, m), 7.49–7.51 (3H, m), 7.85–7.88 (4H, m), 8.13
(1H, s, imine CH), 8.59 (1H, m), 12.8 (1H, br., NH); 13C NMR
(125 MHz, DMSO-d6) δ (ppm): 119.6 (C3’’ of 2-Py), 124.0 (C5’’ of 2-
Py), 126.5 (C2’ and C6’ of Ar), 129.3 (C3’ and C5’ of Ar), 130.3 (C4’ of
Ar), 130.4 (C6), 136.9 (C4’’ of 2-Py), 144.4 (C2’’ of 2-Py), 148.2 (imine
CH), 149.5 (C6’’ of 2-Py), 152.8 (C5), 169.6 (C2). IR (KBr) cm−1: 1156
(C-N), 1574 and 1601 (C = C), 3481 (N-H). HPLC purity (λ 300 nm):
96.55 %. Anal. Calcd for C14H11N5S: C, 59.77, H, 3.94, N, 24.89. Found:
C, 59.91; H, 3.93; N, 24.81.
2.1.8.1. (E)-2-(2-(Pyridin-2-ylmethylene)hydrazinyl)-5-(pyridin-4-yl)-
1,3,4-thiadiazole (3a). Compound 3a was obtained in 75 % yield, as an
amorphous dark yellow solid, m.p. 239−240 °C. 1H NMR (400 MHz,
DMSO-d6) δ (ppm): 7.40 (1H, q, J =4 Hz), 7.84 (2H, d, J =8 Hz), 7.89
(2H, d, J =4 Hz), 8.16 (1H, s, imine CH), 8.61 (1H, d, J =8 Hz), 8.71
(2H, d, J =4 Hz), 13.01 (1H, br., NH); 13C NMR (100 MHz, DMSO-d6) δ
(ppm): 118.2 (C3’’ of 2-Py), 119.0 (C3’ and C5’ of 4-Py), 120.9 (C5’’ of
2-Py), 135.7 (C4’’ of 2-Py), 139.7 (C4’ of 4-Py), 140.1 (imine CH), 148.0
(C2’’ of 2-Py), 148.9 (C6’’ of 2-Py), 149.9 (C2’ and C6’ of 4-Py), 156.4
(C5), 183.4 (C2);IR (KBr) cm−1: 1147 (C-N), 1600 and 1636 (C = C),
3310 (N-H). HPLC purity (λ 300 nm): 99.01 %. Anal. Calcd for
C13H10N6S: C, 55.31; H, 3.57; N, 29.77. Found: C, 55.17; H, 3.58; N,
29.87.
2.1.8.6. (E)-2-(2-(4-Fluorobenzylidene)hydrazinyl)-5-phenyl-1,3,4-
thiadiazole (3f). Compound 3f was obtained in 90 % yield, as an
amorphous yellow solid, m.p. 259−261 °C. 1H NMR (400 MHz, DMSO-
d6) δ (ppm): 7.28 (2H, t, J =8 Hz), 7.46–7.53 (3H, m), 7.72–7.75 (2H,
m), 7.85 (2H, d, J =4 Hz) 8.12 (1H, s, imine CH), 12.5 (1H, br., NH);
13C NMR (100 MHz, DMSO-d6) δ (ppm): 115.9 (C3’’ and C5’’ of Ar),
126.3 (C2’ and C6’ of Ar), 128.6 (C2’’ and C6’’ of Ar), 129.2 (C3’ and C5’
of Ar), 130.0 (C4’ of Ar), 130.5 (C1’ of Ar), 130.7 (C1’’ of Ar), 143.4
(imine CH), 156.9 (C5), 161.6 and 164.0 (C4’’ of Ar, C-F coupling),
169.6 (C2);IR (KBr) cm−1: 1148 (C-N), 1604 and 1621 (C = C), 3445
(N-H). HPLC purity (λ 300 nm): 99.68 %. Anal. Calcd for C15H11FN4S:
C, 60.39, H, 3.72; N, 18.78. Found: C, 60.52; H, 3.73; N, 18.71.
2.1.8.2. (E)-2-(2-(Pyridin-2-ylmethylene)hydrazinyl)-5-(pyridin-3-yl)-
1,3,4-thiadiazole (3b). Compound 3b was obtained in 79 % yield, as an
amorphous yellow solid, m.p. 226−227 °C. 1H NMR (400 MHz, DMSO-
d6) δ (ppm): 7.37–7.40 (1H, m, H10), 7.52–7.56 (1H, m), 7.88 (2H, d, J
=4 Hz), 8.12 (1H, s, imine CH), 8.24 (1H, d, J =8 Hz), 8.60 (1H, d, J
=4 Hz), 8.66 (1H, s), 9.05 (1H, s), 12.9 (1H, br, NH); 13C NMR
(100 MHz, DMSO-d6) δ (ppm): 119.6 (C3’’ of 2-Py), 124.0 (C4’ of 3-Py),
124.2 (C5’’ of 2-Py), 133.7 (C5’ of 3-Py), 136.8 (C4’’ of 2-Py), 144.5
(imine CH), 147.0 (C2’ of 3-Py), 149.5 (C6’’ of 2-Py), 150.8 (C6’ of 3-
Py), 152.7 (C5), 170.0 (C2); IR (KBr) cm−1: 1157 (C-N), 1575 and 1603
(C = C), 3462 (N-H). HPLC purity (λ 300 nm): 97.23 %. Anal. Calcd for
2.1.9. Synthesis of hydrochlorides (11a-d)
Hydrochlorides 11a-d were obtained in good yields after bubbling
hydrochloric acid gas to a solution of corresponding 1,3,4-thiadiazole
derivatives 2a, 2d, 2e and 3e in CHCl3, as previously described [32].
Since hydrochlorides are not new chemical entities, we chose to only
characterize these molecules by using 1H NMR and elemental analysis.
2.1.9.1. N-(4-Fluorobenzyl)-5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine
hydrochloride (11a). Compound 11a was obtained in 72 % yield, as an
amorphous yellow solid, m.p. 118−119 °C. 1H NMR (400 MHz, DMSO-
d6) δ (ppm):4.62 (2H, s), 7.19 (2H, t, J =8 Hz), 7.42–7.46 (2H, m), 8.27
(2H, d, J =4 Hz), 8.88 (2H, d, J =4 Hz), 9.43 (1H, br., NH). Anal. Calcd
for C14H12ClFN4S: C, 52.09; H, 3.75; N, 17.36. Found: C, 51.98; H, 3.76;
N, 17.41.
C
13H10N6S: C, 55.30; H, 3.57; N, 29.77. Found: C, 55.39; H, 3.58; N,
29.65.
2.1.8.3. (E)-2-(Pyridin-2-yl)-5-(2-(pyridin-2-ylmethylene)hydrazinyl)-
1,3,4-thiadiazole (3c). Compound 3c was obtained in 81 % yield, as an
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2.1.9.2. 5-(4-Fluorophenyl)-N-(pyridin-4-ylmethyl)-1,3,4-thiadiazol-2-
amine hydrochloride (11b). Compound 11b was obtained in 62 % yield,
as an amorphous pale-yellow solid, m.p. 219−220 °C. 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 4.92 (2H, s), 7.32 (2H, t, J =8 Hz),
7.79–7.82 (2H, q), 8.04 (2H, d, J =4 Hz), 8.89 (2H, d, J =4 Hz), 9.48
(1H, br, NH). Anal. Calcd for C14H12ClFN4S: C, 52.09; H, 3.75; N, 17.36.
Found: C, 52.21; H, 3.74; N, 17.31.
ketoconazole, posaconazole and Bz were used as control. The activity of
the 1,3,4-thiadiazole derivatives against intracellular amastigote forms
of the Y strain was evaluated using primary cultures of mouse embryo
heart muscle cells (HMC) as host cells. Briefly, hearts of 18-day-old
mouse embryos were fragmented and dissociated with trypsin and
collagenase in phosphate-buffered saline (PBS), pH 7.2 and the cells
were resuspended in Dulbecco’s modified Eagle’s medium (Sigma-Al-
drich, St. Louis, MO) supplemented with horse and foetal calf serum,
chicken embryo extract, CaCl2, and L-glutamine (DMEM) and plated
onto gelatine-coated glass coverslips [36]. HMC was infected with
bloodstream trypomastigotes (MOI 10:1), and after 24 h of infection,
the cells were treated with the compounds. After 24 and 48 h of
treatment, the cultures were rinsed with saline, fixed and stained with
Diff-Quick Staining (Laborclin), examined by light microscopy and
counted and the infection index (II) was determined, this parameter
corresponds to the number of parasites/100 cells, obtained by the
multiplication of the percentage of infection by the number of para-
sites/infected cell) [34]. The IC50 values for the different days of
treatment, corresponding to the concentration that led to 50 % in-
hibition of this parameter was calculated. Ketoconazole, posaconazole
and Bz were used as control.
2.1.9.3. 5-(4-Fluorophenyl)-N-(pyridin-3-ylmethyl)-1,3,4-thiadiazol-2-
amine hydrochloride (11c). Compound 11c was obtained in 98 % yield,
as an amorphous dark yellow solid, m.p. 105−107 °C. 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 4.76 (2H, s), 7.32 (2H, t, J =8 Hz),
7.79–7.83 (2H, m), 8.04–8.08 (1H, m), 8.58 (1H, d, J =8 Hz), 8.85 (2H,
d, J =4 Hz), 8.95 (1H, br., NH). Anal. Calcd for C14H12ClFN4S: C,
52.09; H, 3.75; N, 17.36. Found: C, 51.99; H, 3.74; N, 17.40.
2.1.9.4. (E)-2-Phenyl-5-(2-(pyridin-2-ylmethylene)hydrazinyl)-1,3,4-
thiadiazole hydrochloride (11d). Compound 11d was obtained in 73 %
yield, as an amorphous yellow solid, m.p. 205−206 °C. 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 7.50–7.53 (3H, m), 7.59 (1H, t, J
=8 Hz), 7.83–7.86 (2H, m), 8.03 (1H, d, J =8 Hz), 8.13 (1H, d, J
=8 Hz), 8.22 (1H, s), 8.66 (1H, d,
J =4 Hz). Anal. Calcd for
C
14H12ClN5S: C, 52.91; H, 3.81; N, 22.04. Found: C, 53.03; H, 3.80;
2.4.2. In vitro toxicity of pyridinyl-1,3,4-thiadiazoles to mammalian cells
Mouse peritoneal macrophages were obtained from Swiss mice, and
for the cytotoxicity assays, 2.5 × 104 cells in 200 μL RPMI-1640
medium (pH 7.2, plus 10 % foetal bovine serum (FBS) and 2 mM L-
glutamine) were added to each well of a 96-well microliter plate and
incubated with the 1,3,4-thiadiazole derivatives for 24 h at 37 °C. For
the assays with HMC, the cells were plated at the concentration of
5 × 104 in 200 μL in DMEM and treated with the compounds for 24 and
48 h at 37 °C [37]. For both mammalian cells, after treatment, Pre-
stoBlue (Invitrogen) was added in the proportion 1:10, and the micro-
plates were incubated for 2 h and the fluorescence measured at 560 and
590 nm, as recommended by the manufacturer. The results were ex-
pressed as the difference in the percentage of reduction between treated
and untreated cells being LC50 the concentration that leads to damage
of 50 % of the mammalian cells [38]. The selectivity index (SI) was
calculated by the ratio between LC50 and IC50 at 24 h for peritoneal
macrophages, in experiments with bloodstream trypomastigotes and at
both 24 and 48 h for HMC, in experiments with intracellular amasti-
gotes.
N, 21.99.
2.2. Single crystal X-ray diffraction
After the synthesis and purification procedures, a well-shaped clear
single crystal of compound 3d, was selected for the X-ray diffraction
experiment. Atomic coordinates, bond lengths, angles and thermal
parameters have been deposited at the Cambridge Crystallographic
Data Centre, deposition number CCDC 1968552. Molecules are linked
into spiral chains by N1-Hn1—N5 strong hydrogen bonds: also present
are weak C18-H188-F1 hydrogen bonds.
2.3. Aqueous solubility determination
Aqueous solubility of some target 1,3,4-thiadiazole derivatives was
determined by UV/Vis spectroscopy as described by Schneider et al.
[33].
2.4. Biological activity
2.4.3. Transmission and scanning electron microscopy analysis
All experiments dealing with animals were performed in accordance
with the Brazilian Law 11.794/2008 and regulations of the National
Council of Animal Experimentation Control under the license L038/
2018 from the Ethics Committee for Animal Use of the Oswaldo Cruz
Institute (CEUA/IOC). The mice were housed at a maximum of 6 in-
dividuals per cage, kept in a specific-pathogen-free (SPF) room at
20–22 °C under a 12/12 h light/dark cycle, 50–60% humidity and
provided sterilized water and chow ad libitum.
Trypomastigotes (Y strain, 5 × 106 cells/mL) were treated for 24 h
with the selected compound sat the concentrations corresponding to the
IC50/24 h and 2x IC50/24 h values. Afterward, they were fixed with 2.5
% glutaraldehyde in 0.1 M Na-cacodylate buffer (pH 7.2) for 40 min at
25 °C and post-fixed with 1% OsO4, 0.8 % potassium ferricyanide and
2.5 mM CaCl2 in the same buffer for 20 min at 25 °C. The cells were
dehydrated in an ascending acetone series and embedded in PolyBed
812 resin. Ultrathin sections were stained with uranyl acetate and lead
citrate and examined in a Jeol JEM1011 transmission electron micro-
scope (Tokyo, Japan) (Technological Platform of Electronic Microscopy
at Instituto Oswaldo Cruz). Alternatively, dehydrated samples were
dried by the critical point method with CO2, mounted on aluminium
stubs, coated with a 20 nm thick gold layer and examined on a Jeol
JSM6390LV scanning electron microscope (Tokyo, Japan)
(Technological Platform of Electronic Microscopy at Oswaldo Cruz
Institute).
2.4.1. In vitro activity of pyridinyl-1,3,4-thiadiazoles against
trypomastigotes and intracellular amastigotes of T. cruzi
For all the experiments stock solutions of the 1,3,4-thiadiazole de-
rivatives, ketoconazole, posaconazole and Bz were prepared in di-
methyl sulfoxide, with the final concentration of the solvent in the
experiments never exceeding 0.6 %, concentration known to exert no
toxicity to the parasite or host cells [34]. Bloodstream trypomastigotes
of Y strain [35] were obtained from infected Swiss Webster mice at the
peak of parasitemia by differential centrifugation. The parasites
(5 × 106 cells/mL) were incubated for 24 h at 37 °C and 5% CO2 at-
mosphere in absence or presence of the compounds. Cell counts were
performed in a Neubauer chamber, by light microscopy and the activity
of the compounds was expressed as the IC50/24 h, corresponding to the
concentration that led to 50 % lysis of the parasites. The standard drugs
2.4.4. Determination of mitochondrial membrane potential and reactive
oxygen species production
Trypomastigotes (Y strain, 5 × 106/mL) were treated for 24 h with
the selected compounds at concentrations corresponding to half the
value of IC50/24 h, IC50/24 h and 2x IC50/24 hand the mitochondrial
membrane potential (ΔΨm) and reactive oxygen species (ROS)
7

R.H.C.N. Freitas, et al.
Biomedicine&Pharmacotherapy127(2020)110162
Scheme 1. Synthesis of pyridinyl-1,3,4-thia-
diazole derivatives 2a-f. Reagents and condi-
tions: (a) MeOH, MePh, H2SO4 cat., 110 °C,
48 h (44-82 %); (b) NH2NH2 80 %, EtOH,
80 °C, 6 h (50-63 %); (c) 1. TMSNCS, EtOH,
80 °C, 4 h; 2. H2SO4, 12 h, r.t. (47-69 %); (d) 1.
Aromatic aldehyde (8a-c), ZnCl2, TMSOAc,
DMF, 12 h, 100 °C; 2. NaBH(OAc)3, r.t., 72 h
(11-95 %).
Scheme 2. Synthesis of 1,3,4-thiadiazolyl-N-arylhydrazone derivatives 3a-f. Reagents and conditions: (a) 1. NaNO2, HCl, H2O; 2. CuCl2.2H2O, 0 °C, 2 h (55–88 %);
(b) NH2NH2 80 %, EtOH, 80 °C, 2 h (52-81 %); (c) 8a or 8d, 10 % HCl (cat.), EtOH, r.t., 4 h (65-90 %).
production were determined. For ΔΨm analysis, the parasites were
incubated with 30 μg/mL propidium iodide (PI) plus 50 nM tetra-
methylrhodamine (TMRE) (Molecular Probes, Carlsbad, USA) for
30 min at 37 °C, using 10 μM carbonyl cyanide 4-(trifluoromethoxy)
phenylhydrazone (FCCP) (Sigma-Aldrich) as a control for ΔΨm dis-
sipation. Alterations in TMRE fluorescence were quantified using an
index of variation (IV), which was calculated using the equation (MT -
MC)/MC, where MT is the median of fluorescence for treated parasites
and MC is the median of fluorescence of the control parasites. Negative
IV values correspond to depolarization of the mitochondrial membrane.
To evaluate ROS generation, labelling with 10 μM dihydroethidium
(DHE) (Molecular Probes) for 30 min at 37 °C was performed, using
22 μM antimycin A (AA) (Sigma-Aldrich) as the positive control. The
samples were analyzed in a FACSCalibur flow cytometer (Becton
Dickinson, CA, USA) (Flow Cytometry and Electron Microscopy
Facilities from Instituto Oswaldo Cruz). The Summit program was used
for data analysis. A total of 10,000 events were acquired in the region
previously established as that of the parasites.
3. Results and discussion
Compounds 2a-f were obtained by using a 4-stage linear route ex-
ploiting classical reactions of functional group interconversions
(Scheme 1). Carboxylic acids (4c-e) were converted to their respective
methyl esters (5c-e) in good yield by the Fischer esterification reaction
in methanol-toluene with sulfuric acid catalysis [26]. Thereafter, the
methyl esters (5c-e) were subjected to a hydrazinolysis reaction after
treatment with 80 % aqueous hydrazine hydrate in ethanol under reflux
to afford the corresponding hydrazides (6b-e) in moderate yield
[27,39]. Then, equimolar amounts of the hydrazides (6b-e) and com-
mercially available trimethylsilyl isothiocyanate (TMSNCS) were
maintained under reflux in ethanol to form the N-acyl-thiosemicarba-
zide intermediates, which were next treated with sulfuric acid at room
temperature to promote cyclization and subsequent dehydration to
furnish the 2-amino-1,3,4-thiadiazoles (7b-e) in yields ranging from 47
to 69% [28]. 5-(Pyridin-4-yl)-1,3,4-thiadiazol-2-amine (7a) was pur-
chased from Sigma-Aldrich (St. Louis, MO, USA). Finally, the desired
1,3,4-thiadiazole derivatives (2a-f) were synthesized in acceptable
8

R.H.C.N. Freitas, et al.
Biomedicine&Pharmacotherapy127(2020)110162
yield through reductive amination of the amines (7a-e) with the se-
lected aromatic aldehydes (8a-d) by using sodium triacetoxyborohy-
dride as the reducing agent after previous formation of the imine in-
termediate promoted by ZnCl2/TMSOAc in DMF [29].
The synthesis of 1,3,4-thiadiazolyl-N-arylhydrazones 3a-g started
from the Sandmeyer reaction of the 1,3,4-thiadiazolylamines (7a-e)
after treatment with sodium nitrite in hydrochloric acid and water,
followed by nucleophilic displacement of the diazonium intermediate
with copper chloride to furnish to the corresponding heteroaryl chlor-
ides (9a-e) (Scheme 2) [30]. Then, the 1,3,4-thiadiazolyl chlorides (9a-
e) were subjected to an aromatic nucleophilic substitution reaction with
excess hydrazine hydrate in ethanol to give the corresponding hetero-
arylhydrazines (10a-e) in good yield [31]. Finally, the last step for the
synthesis of the desired compounds 3a-f involved the acid-catalyzed
condensation between heteroarylhydrazines 11a-e and the selected
aromatic aldehydes (8a or 8d) in ethanol at room temperature [31]. All
1,3,4-thiadiazolyl-N-arylhydrazones (3a-f) were obtained in high yield.
Formation of the hydrazone derivatives as single diastereomers was
confirmed due to the presence of a single signal from the hydrogen
attached to the imine subunit at approximately 8.1 ppm in the 1H NMR
spectra.
Fig. 4. (A) ORTEP representation of the structure of 1,3,4-thiadiazolyl-N-(2-
pyridinyl)hydrazine 3d in the crystalline state. Probability ellipsoids are drawn
at the 50 % level. (B) Side-on view of compound 3d.
is considered adequate for the next step of biological evaluation.
Since in vitro assays are strongly influenced by the aqueous solubi-
lity of the compound [43], studies were conducted to determine the
aqueous solubility of the 1,3,4-thiadiazole derivatives 2a-f [33]. The
results are presented in Table 1. As predicted, compounds having the
pyridinyl subunit (2a-e) in their structure showed greater aqueous so-
lubility than 2f, which does not have the azaheterocyclic subunit in its
structure. As expected, regioisomers 2a-c and 2d-e demonstrated si-
milar aqueous solubility profiles. The aqueous solubilities of the 1,3,4-
thiadiazolyl-N-arylhydrazone derivatives were similar, and the pyr-
idinyl derivatives 3a-c showed a greater solubility profile. With respect
to compounds 3d and 3e, it is relevant to note that insertion of the
fluorine atom into 3d slightly improved the solubility profile of this
compound compared to its unsubstituted analog 3e. Compound 3f, the
only compound in this series in which the pyridinyl subunit is absent,
also showed a suitable solubility profile for pharmacological assays.
In general, all 1,3,4-thiadiazole derivatives presented a satisfactory
aqueous solubility profile for pharmacological tests [44]. However, as
anticipated, we were able to demonstrate an improvement in the aqu-
eous solubility profile of the hydrochlorides 11a-d of approximately 2-
to 4-fold when compared with the solubility of the corresponding free
bases (2a, 2d, 2e and 3e, respectively) (Table 1). Furthermore, the
water solubility profile of the target compounds was similar to that of
other previously published 1,3,4-thiadiazole derivatives [45].
It has been reported in the literature that the reaction between
hydrazines and pyridine-2-carboxaldehyde (8d) leads to the generation
of the corresponding hydrazone with the relative Z configuration due to
the possibility of forming an intramolecular hydrogen bond between
the amine hydrogen of the hydrazone group and the pyridine nitrogen,
leading to the formation of a stable six-membered ring. Nevertheless,
the (E)-diastereoisomer would be formed in the minority or would not
be formed, since it is not capable of forming this type of intramolecular
interaction [40,41] (Scheme 3).
However, although the literature reports the formation of the (Z)-
diastereomer in the case of 2-pyridinyl-hydrazone, the single diaster-
eoisomer observed among our 1,3,4-thiadiazolyl-N-arylhydrazones was
characterized as the (E)-configuration, since it is known to be the
thermodynamic diastereoisomer, with less electronic repulsion that is
therefore more stable [32,42].
In order to confirm which diastereomer was obtained, X-ray crys-
tallography studies were conducted. Fortunately, it was possible to
obtain adequate resolution of the crystalline structure of compound 3d
(Fig. 4). After observing the ORTEP representation of 1,3,4-thiadiazolyl
2-pyridinylhydrazone 3d, it was unequivocally confirmed that the
diastereoisomer formed presents in the relative (E)-configuration.
Some of the 1,3,4-thiadiazole derivatives were subjected to trans-
formation into their corresponding hydrochlorides in order to improve
their aqueous solubility and evaluate how this physicochemical beha-
vior could affect their trypanocidal profile. Therefore, compounds 2a,
2d, 2e and 3e were treated with hydrochloric acid after solubilization
in chloroform to furnish the corresponding hydrochlorides 11a, 11b,
11c and 11d in yields ranging from 62 to 98% (Scheme 4) [32].
The obtained pyridinyl-1,3,4-thiadiazole compounds 2a-f, 3a-f and
11a-d were fully spectroscopically characterized, and their degree of
purity was determined by HPLC analysis to be greater than 95 %, which
It has been reported in the literature that azaheterocyclic fragments
are capable of inhibiting the TcCYP51 enzyme; for example, 3-pyridinyl
and 4-pyridinyl-containing fragments [19,46]. For this reason, we also
chose to test our 1,3,4-thiadiazolylamine intermediates (7a-e) in the
pharmacological assays.
Initially, we evaluated the effects of the 21 compounds on the in-
fective bloodstream trypomastigotes of the Y strain of T. cruzi (Table 2).
Our findings demonstrated that the 2-amino-1,3,4-thiadiazoles 7a-e
did not present an important trypanocidal effect, with IC50 values
Scheme 3. Representation of the possible (E)- and (Z)-diastereoisomers of the 1,3,4-thiadiazolyl-N-arylhydrazones 3a-f.
9

R.H.C.N. Freitas, et al.
Biomedicine&Pharmacotherapy127(2020)110162
Scheme 4. Synthesis of hydrochlorides 11a-d. Reagents and conditions: (a) HCl(g), CHCl3, 0 °C, 30 min (62-98 %).
Table 1
Table 2
Aqueous solubility of the 1,3,4-thiadiazole derivatives 2a-f and 3a-e and the
hydrochlorides 11a-d.
In vitro activity of the 1,3,4-thiadiazole derivatives on the trypomastigotes of T.
cruzi (Y strain), cytotoxicity to peritoneal macrophages and selectivity index.
Compound
Aqueous solubilitya
Compound
Bloodstream
Peritoneal
SIc
trypomastigotesa IC50/24 h
(μM)
macrophagesb LC50
24 h (μM)
/
mg/mL
μM
1.41 × 10−2
1.11 × 10−2
1.23 × 10−2
1.82 × 10−2
2.04 × 10−2
3.17 × 10−3
1.40 × 10−2
1.51 × 10−2
5.06 × 10−3
2.64 × 10−3
1.48 × 10−3
1.74 × 10−3
6.70 × 10−2
5.70 × 10−2
9.02 × 10−2
1.05 × 10−2
4.06
3.10
3.52
5.21
5.84
0.90
3.97
4.27
1.43
0.79
0.42
0.52
21.60
18.25
29.10
3.36
7a
215.0
832.9
338.4
340.5
> 1000
6.9
-
–
d
2a
2b
2c
7b
48.1
33.0
45.0
–
–
–
–
–
7c
–
2d
2e
7d
–
7e
–
2f
2a
16.6
168.6
182.9
226.5
81.2
190.4
23.3
21.6
9.5
0.9
401.3
412.8
391.7
> 1000
119.7
192.6
41.4
41.2
23.7
24.2
2.4
2.1
> 4.4
1.5
1.0
3.5
4.8
7.7
6.0
66.1
2.3
66.6
0.4
0.9
26.9
2.3
< 0.2
> 113.6
3a
2b
25.8
11.7
17.6
3b
3c
2c
2d
3d
3e
2e
5.9
8.8
5.8
2f
19.7
5.2
3.7
3f
3a
81.4
5.3
11a
11b
11c
11d
3b
102.7
72.8
10.6
3c
2.3
2.5
3d
9.3
3.0
0.8
56.1
4.8
3e
6.7
443.3
118.3
682.0
206.0
147.5
96.7
13.7
3f
52.2
10.2
541.3
170.8
3.6
7.1
13.9
57.9
17.5
22.6
a
11a
2.7
38.2
13.7
0.5
16.1
Determined by the spectrophotometric method described by Schneider
et al. [33] on a scanning UV–vis Femto spectrophotometer (model 800XI).
11b
11c
11d
8.3
higher than 200 μM (Table 2). Compound 7a (4-pyridinyl) showed only
modest activity (IC50 = 215.0 μM), followed by its ortho-regioisomer
7c (2-pyridinyl) (IC50 = 338.4 μM), while 7b, with a 3-pyridinyl sub-
unit, was approximately 4-fold less active.
Ketoconazole
Posaconazole
Benznidazole
118.3
> 500
271.7
104.6
> 1000
37.8
19.6
8.8
1.1
a
b
c
Treatment for 24 h at 37 °C of the trypomastigotes of T. cruzi (Y strain).
Treatment for 24 h at 37 °C of non-infected peritoneal macrophages.
Selectivity index (SI) = LC50 of macrophages/IC50 of trypomastigotes.
not determined.
The pyridinyl-1,3,4-thiadiazole derivatives 2a-f showed
a sig-
nificant increase in trypanocidal activity, with lower IC50 values (from
16.6–226.5 μM) relative to their respective amino intermediates 7a-e,
demonstrating that the lipophilic 4-fluorobenzyl unit introduced into
the structure contributed considerably to the biological effects. From
this series, compound 2a was the most active, with low toxicity to
mammalian macrophages, leading to an excellent selectivity index (SI)
of 24.2. Compared to the standard drugs, 2a was more active than
ketoconazole or posaconazole but 2 times less active than benznidazole.
It is interesting to note that similar to the results obtained with inter-
mediates 7a-e, regioisomer 2a with a 4-pyridinyl subunit showed the
highest activity. Thus, despite the classical isosteric relationships be-
tween 3-pyridinyl and 2-pyridinyl subunits with the 4-pyridinyl group
[20], in the present case, concerning the trypanocidal profile, com-
pounds 2a, 2b and 2c could not be characterized as bioisosteres. In-
terestingly, although 2a and 2d are regioisomers and retroisomers, they
showed different activities on trypomastigotes, with 2d being ap-
proximately 14-fold less active. In addition, derivative 2e, containing a
3-pyridinyl subunit, was more active than 2d (IC50 24 h of 81.2 μM
versus 226.5 μM). Thus, despite their high structural similarity, these
compounds present different mechanisms of action. It is interesting to
note that the pyridinyl group is considered a pharmacophoric group for
d
the inhibition of TcCYP51; therefore, 2f, in which this subunit is absent,
and 2d, one of compounds with a 4-pyridinyl group attached in place of
the lipophilic anchor D (Fig. 2), both showed only moderate activity
against the parasite. This fact suggests that the trypanocidal activity of
2f and 2d might not involve TcCYP51 inhibition, and further studies are
needed.
To circumvent the solubility issues of 2a-f, facilitating permeability
through biological membranes, hydrochloride analogs were synthe-
sized, resulting in 11a-d. Interestingly, their activity against T. cruzi
was inferior to that of the corresponding free bases, with the exception
of 11a, which was more active than 2a and displayed activity similar to
that of Bz and with an SI equal to 66.6 (Table 2).
The series of 1,3,4-thiadiazole-N-arylhydrazone derivatives (3a-f)
showed promising results with excellent IC50 values in the range of
6.7–52.2 μM (Table 2). The importance of an adequate water solubility
profile for the biological activity is highlighted by the performance of
hydrochloride 11d compared to its free base, 3e (IC50 3.6 μM versus 6.7
μM). Compounds 3a and 3b with 4-pyridinyl and 3-pyridinyl subunits,
10

R.H.C.N. Freitas, et al.
Biomedicine&Pharmacotherapy127(2020)110162
respectively, showed similar activity and were less active than 3c, their
regioisomer with a 2-pyridinyl subunit. The bioisosteric relationship of
3c-e with the subunits of 2-pyridinyl, p-fluorophenyl and phenyl, re-
spectively, and their similar effects on trypomastigotes suggest that a
similar mechanism of action could be involved. In series 3 (3a-3f), 3f
presented the lowest trypanocidal activity and is the compound that
does not present a 2-pyridinyl subunit compared to 3e (IC50 52.2 μM
versus 6.7 μM). Thus, it is clear that for this series, the presence of a 2-
pyridinyl subunit linked to the imine functionality of the hydrazone
moiety is important for activity against the parasite. In addition, 2-
pyridinyl contains a site for metal coordination, so the trypanocidal
activity could be related to the coordination of iron ions, for example,
which are essential for the survival of T. cruzi. The remarkable activity
of 3e can be explained by the insertion of a second nitrogen atom in the
side chain, linking the 1,3,4-thiadiazole ring with a 2-pyridinyl group.
In this context, it is important to correlate the activity of 3e with that of
1,3,4-thiadiazole-N-arylhydrazones previously reported by our group
[31,47], corroborating the pharmacophoric character of the 2-pyridinyl
subunit. Compounds 3e and 11a demonstrated a low toxicity profile in
assays with macrophages, presenting, after 24 h of treatment, LC50
values of 443.3 and 682.0 μM, respectively, leading to high selectivity
indexes (SI values > 66).
Light microscopy analysis illustrated the results obtained for the
inhibition of amastigote proliferation by pyridinyl-1,3,4-thiadiazole
derivative 2a and its hydrochloride 11a (Fig. 6). Both compounds, even
at the highest concentration used, did not interfere with the contract-
ibility and cellular organization of the cardiac cells.
The ultrastructural alterations induced by compound 11a in the T.
cruzi trypomastigote forms were investigated by electron microscopy
using the concentration corresponding to its IC50/24 h value (10 μM).
TEM analysis revealed that 11a led to blebbing of the plasma mem-
brane and the appearance of atypical vacuoles (Fig. 7B–E). The most
prominent phenotypic changes observed in the treated parasites was
the alteration in the shape of the kinetoplast, presenting with a rod-like
appearance, similar to amastigote kinetoplasts (Fig. 7B–E). Untreated
parasites exhibited a typical morphology with characteristic rounded
kinetoplasts (Fig. 7A (K)).
Corroborating the TEM data, which revealed parasites with altera-
tions in the kinetoplast appearance and probably alteration in the form
of the parasite, trypomastigotes treated with 11a and observed by SEM
also displayed a rounded body. SEM analysis revealed that 11a induced
severe morphological changes in trypomastigotes (Fig. 8), with
rounding of the parasite’s body and flagellum shortening (Fig. 8B–D)
compared to untreated cells (Fig. 8A). These alterations, such as surface
shrinkage, induced by this derivative may indicate destabilization of
cytoskeleton components or microtubule-associated proteins [49].
Veiga-Santos and coworkers [50] observed that treatment of epi-
mastigote forms of T. cruzi with posaconazole led to intense alteration
of the cell shape, similar to that observed by our group in the organi-
zation of the Golgi complex, leading to the formation of autophagic
vacuoles and the loss of kDNA compaction. Treating L. amazonensis
promastigotes with posaconazole, De Macedo-Silva et al. [51] observed
retraction of the parasite's body and several alterations in the mi-
tochondrion-kinetoplast complex, including images suggesting decom-
paction of the kinetoplast, the presence of cytoplasmic vacuoles con-
taining vesicles and profound changes in the plasma membrane. It was
further described by TEM that treatment for 48 h with ketoconazole in
epimastigotes of T. cruzi also induced alterations in the membrane,
mitochondrion and reservosomes [52]. Both studies, with different
trypanosomatids treated with posaconazole, showed alterations in the
shape of the kinetoplast and plasma membrane, as well as the presence
of cytoplasmatic vacuoles, as observed by our group after treatment of
In addition, other compounds structurally similar to those synthe-
sized here, including the thiazolyl-N-(2-pyridinyl)hydrazone deriva-
tives described by Da Silva et al. [48], demonstrated excellent trypa-
nocidal action (IC50 < 10 μM), with their mode of action interfering
with the cell cycle and inducing apoptosis in the parasite, while in-
hibition of TcCYP51 was not investigated.
The most selective compounds were tested for their cytotoxicity to
heart muscle cells, and 2a (456.5
26.9 μM) and 11a (423.8
52.7
μM) showed low toxicity to these cells with values above 400 μM after
24 h of treatment, while the corresponding values for 3e and 11d were
29.9
4.3 and 120.9
2.9 μM.
Whereas compound 3e exhibited low selectivity in HMC and 11d
had the lowest aqueous solubility, only compound 2a and its hydro-
chloride 11a were selected for evaluation of their activity against in-
tracellular amastigotes. A dose- (20–80 μM) and time- (24 and 48 h)
dependent effect on the proliferation of intracellular amastigotes was
observed for both compounds, with SI values between 3.4 and 11.0
(Fig. 5).
Fig. 5. Infection indexes for T. cruzi-infected
HMCs after treatment for 24 and 48 h with (A)
20-80 μM 2a and (B) its hydrochloride 11a.
HMCs were infected with bloodstream trypo-
mastigotes (MOI 10:1), and after 24 h of in-
fection, the cells were treated with 2a or 11a
for 24 or 48 h. The infection index corresponds
to the number of parasites/100 cells, obtained
by the percent of infection multiplied by the
number of parasites/infected cell, and the IC50
values (Table) with 50 % inhibition of this
parameter. The selectivity index (SI) corre-
sponds to the ratio between the LC50 for HMCs
and the IC50 for the infection index. Asterisks
indicate significant differences in relation to
the untreated control group (p ≤ 0.05) by
ANOVA followed by Bonferroni’s posttest.
11

R.H.C.N. Freitas, et al.
Biomedicine&Pharmacotherapy127(2020)110162
Fig. 6. Effect of 2a and 11a on HMCs infected with T. cruzi (Y strain) after 24 and 48 h. (A) Control/24 h; (B) 80 μM 2a/24 h; (C) 80 μM 11a/24 h; (D) control/48 h;
(E) 80 μM 2a/48 h; (F) 80 μM 11a/48 h. Bars = 20 μm. Black arrows indicate intracellular parasites.
Fig. 7. Transmission electron microscopy analysis of T. cruzi trypomastigotes. (A) Untreated trypomastigotes showing normal ultrastructural aspects and presenting
typical morphologies of the mitochondria (M), kinetoplast (K), flagellum (F), nucleus (N) and Golgi apparatus (G). (B-E) Trypomastigotes treated with IC50
/
24 h = 10 μM 11a showing blebs in the plasma membrane (thin black arrows), alterations in the shape of the kinetoplast, presenting with a rod-like appearance,
similar to the amastigote kinetoplast (white arrowheads) and an increase in the number of cytoplasmic vacuoles (black asterisk). Bars =2 μm.
12

R.H.C.N. Freitas, et al.
Biomedicine&Pharmacotherapy127(2020)110162
Fig. 8. Scanning electron microscopy analysis
of trypomastigotes: (A) Untreated trypomasti-
gotes with typical morphology; an elongated
shape and flagellum located laterally in the
posterior region of the parasite; (B-D) trypo-
mastigotes treated with 10 μM 11a for 24 h
showing the retraction and rounding of the
parasite body and loss of the flagellar structure
(thin arrow).
against the trypomastigote form of T. cruzi. The IC50 values of the
amino-substituted 1,3,4-thiadiazole derivatives (2a-f) ranged from
16.6–226.5 μM, and the values for the 1,3,4-thiadiazole-N-arylhy-
drazone series (3a-f) ranged from 6.7–52.2 μM. Regarding the deriva-
tives 3a-f, it was evident that the 2-pyridinyl fragment bound to the
imine subunit of the hydrazine moiety presents pharmacophoric be-
havior for trypanocidal activity.
Table 3
Flow cytometry analysis of the mitochondrial membrane, generation of ROS
and plasma membrane integrity in T. cruzi trypomastigotes treated with 11a for
24 h.
μM
IV TMRE
% TMRE +
% DHE +
% PI +
Control
11a
–
1.00a
1.15
1.13
1.06
90.4
89.7
88.6
80.4
5.8b
6.2
15.3
6.7
8.2
4.2
4.2
3.3
6.0
2.0
5
5.2
3.8
4.1
2.9
1.6
3.6
Four hydrochlorides (11a-d) were also synthesized and, as planned,
presented a better aqueous solubility profile than their corresponding
free bases. Considering the potencies, two of these hydrochlorides, i.e.,
11b and 11c, showed a significant decrease in trypanocidal action,
while the other two, 11a and 11d, potentiated the trypanocidal action
of their respective free bases (2a and 3e, respectively).
10
15
5.8
8.1
10.1
9.9
a
IV = (MT–MC)/MC, where MT corresponds to the median fluorescence for
treated parasites and MC corresponds to that of control parasites.
b
Mean
standard deviation of three independent experiments.
The 1,3,4-thiadiazole prototypes 2a and 11a presented remarkable
trypanocidal action on trypomastigotes and excellent selectivity in-
dexes. Additionally, 2a and 11a demonstrated trypanocidal effects on
the amastigote form of T. cruzi. Moreover, the corresponding hydro-
chloride 11a presented the most promising profile, producing pheno-
typic changes similar to those promoted by treatment with the standard
drug posaconazole. For this reason, 1,3,4-thiadiazole derivative 11a
could be considered a good prototype for the development of drug
candidates for Chagas disease therapy.
trypomastigotes with 11a.
Following the ultrastructural analysis of 11a, the mitochondrial
membrane potential, generation of reactive oxygen species and plasma
membrane integrity were evaluated by flow cytometry. Our results
suggest that treatment with 11a does not alter these parameters even at
concentrations corresponding to 2× IC50/24 h (Table 3). Previous
studies indicate that sterol biosynthesis inhibitors are able to induce
alterations in mitochondrial structure and function, as this organelle
has a special requirement for sterols [52,53]. De Macedo-Silva and
coworkers [51] reported that posaconazole treatment in L. amazonensis
promastigotes compromised plasma membrane integrity after 48 h of
treatment. In another study with posaconazole-treated L. amazonensis,
the De Macedo-Silva group showed that this drug was able to induce an
increase in ROS production and collapse the mitochondrial transmem-
brane electric potential [54]. Additionally, treatment with ketoconazole
in T. cruzi epimastigotes also induced time-dependent mitochondrial
depolarization and a loss of plasma membrane integrity [55]. This
finding suggests that these phenotypic alterations caused by inhibitors
of sterol biosynthesis could be generated only with longer treatment
times, unlike the trypomastigote treatment time in these studies.
Funding source
None.
Declaration of Competing Interest
None.
Acknowledgments
The authors thank Dr. Solange Lisboa de Castro for critical reading
of the manuscript. We also thank the Technological Platform of
Electronic Microscopy and Flow Cytometry and Electron Microscopy
Facilities at Instituto Oswaldo Cruz. The authors are also grateful to
Fundação de Amparo à Pesquisa do Rio de Janeiro (FAPERJ, BR),
4. Conclusion
Fortunately, all 1,3,4-thiadiazole derivatives presented activity
13

R.H.C.N. Freitas, et al.
Biomedicine&Pharmacotherapy127(2020)110162
Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES,
BR), Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq, BR) and Fundação Oswaldo Cruz (FIOCRUZ, BR) for financial
support and fellowships.
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