Synthesis and biological activities of ethyl 2-(2-pyridylacetate) derivatives containing thiourea, 1,2,4-triazole, thiadiazole and oxadiazole moieties

Article abstract of DOI:10.3390/molecules22030409

Thirty six novel heterocyclic derivatives of ethyl 2-(2-pyridylacetate) were efficiently synthesized. The new compounds involve the linkage of a 2-pyridyl ring with thiosemicarbazide (compounds 1-7), 1,2,4-triazole (compounds 1a-7a), 1,3,4-thiadiazole (compounds 1b-7b), and 1,3,4-oxadiazole (compounds 1f-7f) moieties. The last group of compounds 1e-7e involves the connection of a 2-pyridyl ring with 1,2,4-triazole and thiourea. ¹H-NMR, ¹³C-NMR and MS methods were used to confirm the structures of the obtained derivatives. The molecular structures of 3, 3b, 7a and 7f were further confirmed by X-ray crystallography. All obtained compounds were tested in vitro against a number of microorganisms, including Gram-positive cocci, Gram-negative rods and Candida albicans. In addition, the obtained compounds were tested for cytotoxicity and antiviral activity against HIV-1.

Full text of DOI:10.3390/molecules22030409

molecules
Article
Synthesis and Biological Activities of Ethyl
2-(2-pyridylacetate) Derivatives Containing Thiourea,
1,2,4-triazole, Thiadiazole and Oxadiazole Moieties
Daniel Szulczyk 1,2,*, Piotr Tomaszewski 3, Michał Józ´wiak 1,2,3, Anna E. Kozioł 4, Tadeusz Lis 5,
David Collu 6, Filippo Iuliano 7 and Marta Struga 1,2
1
Department of Biochemistry and Pharmacogenomics, Faculty of Pharmacy, Medical University of Warsaw,
02-097 Warsaw, Poland; michal.jozwiak@wum.edu.pl (M.J.); marta.struga@wum.edu.pl (M.S.)
Laboratory of Centre for Preclinical Research, Medical University of Warsaw, Banacha 1B,
2
02-097 Warsaw, Poland
3
Department of Biochemistry, Second Faculty of Medicine, Medical University of Warsaw, 02-097 Warsaw,
Poland; piotr.tomaszewski@wum.edu.pl
Faculty of Chemistry, Maria Curie-Sklodowska University, 20-031 Lublin, Poland;
4
anna.koziol@poczta.umcs.lublin.pl
Faculty of Chemistry, University of Wroclaw, 50-383 Wroclaw, Poland; tadeusz.lis@chem.uni.wroc.pl
Department of Life and Environmental Sciences, Section of Microbiology and Virology,
5
6
University of Cagliari, 09042 Cittadella Universitaria Monserrato, Italy; david.collu@unica.it
Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9,
7
845 05 Bratislava, Slovak; filippo.iuliano@unica.it
*
Correspondence: daniel.szulczyk@wum.edu.pl; Tel.: +48-22-572-07-35
Academic Editor: Diego Muñoz-Torrero
Received: 14 January 2017; Accepted: 1 March 2017; Published: 6 March 2017
Abstract: Thirty six novel heterocyclic derivatives of ethyl 2-(2-pyridylacetate) were efficiently
synthesized. The new compounds involve the linkage of a 2-pyridyl ring with thiosemicarbazide
(compounds
1
–
7
), 1,2,4-triazole (compounds 1a
–
7a), 1,3,4-thiadiazole (compounds 1b
–
7b), and
1,3,4-oxadiazole (compounds 1f
–
7f) moieties. The last group of compounds 1e
–
7e involves the
1
connection of a 2-pyridyl ring with 1,2,4-triazole and thiourea. H-NMR, 13C-NMR and MS methods
were used to confirm the structures of the obtained derivatives. The molecular structures of 3, 3b,
7a and 7f were further confirmed by X-ray crystallography. All obtained compounds were tested
in vitro against a number of microorganisms, including Gram-positive cocci, Gram-negative rods
and Candida albicans. In addition, the obtained compounds were tested for cytotoxicity and antiviral
activity against HIV-1.
Keywords: 1,2,4-triazole; 1,3,4-thiadiazole; 1,3,4-oxadiazole; thiourea; X-ray crystal structure analysis;
biological activity
1. Introduction
Heterocyclic compounds are well-known for their pharmaceutical importance, and the research
for simple and efficient methods of synthesis of compounds incorporating heterocyclic rings has
brought a new dimension to drug development [1]. In past decades heterocyclic scaffolds such as
the 1,2,4-triazole, 1,3,4-oxadiazole and 1,3,4-thiadiazole rings and their derivatives were intensively
investigated by researchers because of their biological activity. Among many heterocyclic compounds,
these five-membered structures display unique spatial arrangements, which give possibilities to
develop the synthetic pathways for microbiologically active compounds that can have potential
therapeutic applications as new medicines. Currently, there are a lot of pharmaceutical products
Molecules 2017, 22, 409; doi:10.3390/molecules22030409
www.mdpi.com/journal/molecules

Molecules 2017, 22, 409
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available on the market, which contain the previously mentioned structures such as fluconazole,
efinaconazole, terconazole and fosfluconazole. These are used as antifungal agents and their structures
are based on the 1,2,4-triazole core. The 1,3,4-thiadiazole scaffold can be found in other active
substances like acetazolamide, methazolamide and megazol. Finally, the 1,3,4-oxadiazole arrangement
is present in raltegravir and zibotentan. Exemplary structures of some of the previously listed
medicines containing mentioned active scaffolds are presented in Figure 1 below.
Figure 1. Active substances containing 1,2,4-triazole, 1,3,4-thiadiazole and 1,3,4-oxadiazole scaffold.
Currently researchers continue to design and sythesize new 1,2,4-triazole, 1,3,4-oxadiazole
and 1,3,4-thiadiazole derivatives because of their high antimicrobial activity against a wide range
of Gram-negative and Gram-positive bacteria and yeasts. Compounds that contain one of
mentioned heterocyclic motifs are often inhibitory or lethal against different types of microorganisms.
Derivatives containing the 1,2,4-triazole core are still intensively studied as they possess potent
antifungal properties [
anticonvulsant [ ] and anti-HIV agents [
using the 1,3,4-thiadiazole motif were found to have number of biological activities such as
anticonvulsant [10 11], antihypertensive [12 13], local anesthetic [14], anticancer [15 16], anti-HIV [17],
anti-inflammatory [18 19], and hypoglycemic activities [20]. 1,3,4-Oxadiazoles are intensively
evaluated for their activity against HIV-1 and HIV-2 in MT-4 cells [21–24].
2]. Recently, 1,2,4-triazole-based compounds were reported as potential
3
–7
8,9]. Among the thiadiazole derivatives, those synthesized
,
,
,
,
All three scaffolds are fundamental in designing novel antimicrobial agents. Compounds built on
these heterocyclic cores represent the non-steroidal antimicrobial drugs (NSAMDs). A study of the
literature confirms that 1,3,4-oxadiazoles, 1,3,4-thiadiazoles, 1,2,4-triazoles and their amino derivatives
are recognized as promising candidates for medicines used against bacteria and yeast [25–29]. These
compounds often show higher activity than standard antibiotics commonly used as reference materials.
Available data presume that the introduction of halogen atoms into the pharmacophore structure can
be favourable for preventing the resistant microorganisms from spreading and emerging. These kinds
of structural modifications help to increase the lipid-solubility of the studied scaffolds [30,31]. Ureas,
thioureas, hydrazones, semicarbazides or thiosemicarbazides are found to posses similar properties
and are useful as starting material in the synthesis of the three title heterocyclic arrangements and their
structural modification. Prompted by this fact and encouraged by the results of recent studies [32–34]
we decided to design synthetic paths to produce a battery of 1,3,4-oxadiazole, 1,3,4-thiadiazole, and
1,2,4-triazole derivatives.
2. Results and Discussion
2.1. Chemistry
Our aim was to obtain a small library of ethyl 2-(2-pyridylacetate) derivatives. The planned
synthetic route is depicted in Scheme 1.

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Scheme 1. Synthesis of ethyl 2-(2-pyridylacetate) derivatives. Reagents and conditions: (
80%, EtOH, rt. 2 h ( ) appropriate isothiocyanate, acetonitrile, reflux, 6 h ( ) H2SO4(conc.)
reflux, 1 h (e) KOH, EtOH, CS2 (f) NH2NH2 80%, reflux (g) Et3N, HgCl2, DMF.
a
) NH2NH2
b
c
(d) 2% NaOH,
In first step the ester ethyl 2-(2-pyridylacetate) was transformed into 2-(pyridin-
2-yl)acetohydrazide (A) [35 36]. This was a starting material to obtain the 2-pyridylacetate
,
thiosemicarbazide and oxazole derivatives. Derivatives of N-(phenylsubstituted)-2-(pyridin-2-yl
acetyl) hydrazinecarbothioamide (thiosemicarbazide derivatives) were prepared by reaction of
hydrazide (A) with an appropriate aryl isothiocyanate. Briefly, 2-(pyridin-2-yl)-acetohydrazide (A) and
different isothiocyanates were allowed to react in boiling acetonitrile for 6 h. N-(Phenylsubstituted)-
2-(pyridin-2-yl-acetyl)hydrazinecarbothioamide derivatives 1–7 were the starting materials to obtain
the corresponding 1,2,4-triazole, 1,3,4-thiadiazole and 1,3,4-oxadiazole derivatives 1a
1f–7f, respectively.
–7a
,
1b–7b and
The methods for obtaining the 1,2,4-triazole and 1,3,4-thiadiazole derivatives 1a
–
7a and 1b
–
7b
was similar. For both groups of compounds this required cyclisation, however for 1,2,4-triazole process
was accomplished under basic conditions (2% NaOH) while for 1,3,4-thiadiazoles acidic conditions
(conc. H2SO4) were used, respectively [37,38].

Molecules 2017, 22, 409
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2-(Pyridin-2-yl)acetohydrazide (A) was also used to obtain 5-(pyridin-2-ylmethyl)-1,3,4-
oxadiazole-2(3H)-thione (c). This compound was known [39], but in the present work a different
synthesis method was applied. Hydrazide A was added to the solution of ethanol and KOH and next
carbon disulfide was added to the mixture. The reaction mixture was refluxed on a steam bath for 5 h,
then the solid formed was filtered off and recrystallized from butanol. 1,3,4-Oxadiazole-2-thione (c)
was transformed into 4-amino-5-(pyridin-2-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (d) by
reaction with hydrazine hydrate. The free amino group offered a possibility for further reaction with
isothiocyanates to obtain the thiourea derivatives 1e
similar to previously reported thiourea derivative syntheses [32,34].
Oxadiazoles 1f 7f were prepared according to a new procedure developed by our team. This
–7e. This reaction was performed in acetonitrile
–
synthesis relies on a desulfurization/cyclization reaction of thiosemicarbazides to oxadiazoles using
mercuric chloride. Development of this synthetic procedure was inspired by a method already used for
preparation of a library of amino acid derived 2-arylamino-[1,3,4]-oxadiazoles [40]. The mechanism of
this type of reaction is already established, therefore our goal was to find suitable reaction conditions.
It was found that reflux is not needed, furthermore catalytic amounts of triethylamine (1–3 drops) are
optional. Applications of this synthetic route will be presented in future publications. The molecular
structures of one of the parent derivatives, compound
3, and three representatives of the cyclization
products (3b 7a and 7f) are illustrated in Figure 2. The non-covalent intermolecular contacts in the
,
crystals are shown in the Supplementary Materials. The crystal data and experimental parameters are
given in Table 1, below.
Figure 2. Perspective view of the molecular structures of the derivatives 2-(pyridin-2-yl-acetyl)-N-
[3-(trifluoromethyl)phenyl]hydrazinecarbothioamide (3), 5-(pyridin-2-yl-methyl)-N-[3-(trifluoromethyl)
phenyl]-1,3,4-thiadiazol-2-amine (3b), 4-(5-chloro-2-methylphenyl)-5-(pyridin-2-yl-methyl)-2,4-dihydro-
3H-1,2,4-triazole-3-thione (7a) and N-(2-chloro-5-methylphenyl)-5-(pyridin-2-ylmethyl)-1,3,4-oxadiazol-
2-amine (7f).

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Table 1. Crystal data and structure refinement parameters for 3, 3b, 7a and 7f.
Compound
3
3b
7a
7f
Empirical formula
Formula weight
Temperature
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
C15H13F3N4OS
354.35
80(2) K
0.71073
monoclinic
P21/n
C15H11F3N4S
336.34
100(2) K
0.71073
monoclinic
P21/c
C16H14ClN3S
315.81
120(2) K
1.54184
triclinic
P-1
C15H13ClN4O
300.74
295(2) K
1.54184
monoclinic
I2/a
4.6532(9)
34.942(18)
9.800(3)
90
91.17(5)
90
1593.1(10)
4
728
1.477
16.152(5)
7.668(2)
11.594(4)
90
90.99(3)
90
1435.7(8)
4
688
1.556
7.417(2)
8.601(2)
12.111(3)
106.72(2)
92.49(2)
95.86(2)
733.9(3)
2
15.738(2)
13.945(2)
13.254(2)
90
90.94(1)
90
2908.4(7)
8
b (Å)
c (Å)
◦
α ( )
◦
β ( )
◦
γ ( )
−3
Volume (Å
Z
)
F(000)
328
1.429
3.591
1248
1.374
2.364
Density (calcd) (g/cm3)
Absorpt. coeff. (mm-1
)
0.246
0.263
Max. and min. transmission
Crystal size (mm)
θ range for data coll. ( )
0.985 and 0.8996
0.44 × 0.07 × 0.06
0.9844 and 0.8886
0.46 × 0.13 × 0.06
0.990 and 0.9833
0.22 × 0.20 × 0.18
0.798 and 0.6493
0.20 × 0.20 × 0.10
◦
◦
◦
◦
◦
2.72 to 30.39 .
2.94 to 28.75 .
3.82 to 73.65 .
4.24 to 73.95 .
Index ranges
Reflections collected
−5 ≤ h ≤ 5, −49 ≤ k ≤ 49, −12 ≤ l ≤ 13
−
21
≤
h
≤
21,
−
9
≤
k
≤
10,
−
11
≤
l
≤
15
−9 ≤ h ≤ 9, −10 ≤ k ≤ 10, −11 ≤ l ≤ 15
−13 ≤ h ≤ 19, −16 ≤ k ≤ 11, −16 ≤ l ≤ 12
8587
8611
4813
3609
Independent reflections
4085 [R(int) = 0.0466]
3364 [R(int) = 0.0353]
2849 [R(int) = 0.0251]
2419 [R(int) = 0.0189]
Data/parameters
4085/229
1.109
3364/239
1.061
2849/195
1.086
2419/196
1.049
Goodness-of-fit on F2
R1 = 0.0594
wR2 = 0.1229
R1 = 0.0871
wR2 = 0.1354
--
R1 = 0.0398
wR2 = 0.0956
R1 = 0.0600
wR2 = 0.1001
--
R1 = 0.0432
wR2 = 0.1012
R1 = 0.0490
wR2 = 0.1050
--
R1 = 0.0448
wR2 = 0.1240
R1 = 0.0580
wR2 = 0.1382
0.0017(2)
Final R indices [I > 2σ (I)]
R indices (all data)
Extinction coefficient
−3
0.38/−0.38
0.32/−0.24
0.58/−0.33
0.18/−0.29
∆ρmax/min (e Å
)
CCDC No.
1539080
1539081
1539083
1539082

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2.2. Biological Studies
All obtained compounds were tested in vitro against a number of bacteria, including
Gram-positive cocci, Gram-negative rods and fungi. Microorganisms used in this study have common
applications in the antimicrobial tests for many substances like antibiotics, antiseptic drugs and in the
search for new antimicrobial agents [32–34,41]. The procedure started with a preliminary screening by
the disc diffusion method, in order to select derivatives with antimicrobial properties. All obtained
compounds are inactive in microbiological screening.
With the aim to evaluate more widely the biological properties of synthesized compounds (
1–7,
1a
–
7a and 1b 7b) on the basis of reported anti-HIV activities of the similar derivatives, compounds
–
were tested in cell-based assay against the human immunodeficiency virus type-1 (HIV-1), using
efavirenz as reference inhibitor. The cytotoxicity against the MT-4 cells was evaluated in parallel with
the antiviral activity (Table 2). None of the tested compounds showed selective anti-HIV-1 activity.
Almost all tested compounds are non-cytotoxic for exponentially growing MT-4 cells (CC50 >100 µM).
Table 2. Cytotoxicity and anti-HIV-1 activity of compounds (1–7, 1a–7a and 1b–7b).
MT-4
HIV-1IIIB
a
b
Compounds
CC50
EC50
(µM)
1
2
3
4
5
6
7
1a
2a
3a
4a
5a
6a
7a
1b
2b
3b
4b
5b
6b
7b
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
47
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>47
70.6
74.0
>100
>100
>100
>70.6
>74.0
>100
>100
>100
EFV
45.0
0.003
a
Compound concentration (
µ
M) required to reduce the viability of mock-infected MT-4 cells by 50%, as determined
by the MTT method; b Compound concentration (
µM) required to achieve 50% protection of MT-4 cells from the
HIV-1 induced cytopathogenicity, as determined by the MTT method.
3. Materials and Methods
3.1. Apparatus, Materials and Analysis
The NMR spectra were recorded on an AVANCE DMX 400 spectrometer (Bruker, Billerica MA,
USA) operating at 300 MHz (1H-NMR) and 75 MHz (13C-NMR). The chemical shift values are expressed
in ppm relative to TMS used as an internal standard. Mass spectral ESI measurements were carried out
on ZQ Micromass instrument (Waters, Milford, MA, USA) equipped with a quadrupole mass analyzer.
The spectra were run in the positive ion mode at a declustering potential of 40–60 V. The samples
were previously separated on a UPLC column (C18) using an UPLC ACQUITY
™ system by Waters

Molecules 2017, 22, 409
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connected with a DPA detector. Flash chromatography was performed on silica gel 60 (200–400 mesh,
Merck, Kenilworth, NJ, USA) using chloroform/methanol (19:1 vol) mixture as eluent. Analytical TLC
was carried out on silica gel F254 (Merck) plates (0.25 mm thickness). The diffraction data for
3 and 3b
were collected on an Xcalibur instrument (Thermo Fisher Scientific, Waltham, MA, USA), while that for
7a and 7f was collected on a SuperNova diffractometer (Agilent Technologies, Santa Clara, CA, USA).
The CRYSALIS program system [42] was used for data collection, cell refinement and data reduction.
The data were corrected for Lorentz and polarization effects. A multi-scan absorption correction was
applied. The structure was solved using direct methods using the SHELXS-97, and refined by the
full-matrix least-squares on F2 with the SHELXL-97 program [43]. All non-H atoms were refined with
anisotropic displacement parameters. The H-atoms attached to carbon were positioned geometrically
and refined using the riding model with Uiso(H) = 1.2Ueq(C). The nitrogen bonded H-atoms were
found in the difference-Fourier map and refined with isotropic displacement parameters. The crystal
data and experimental parameters used in the X-ray structure analysis are given in Table 1.
3.2. General Procedure of the Synthesis of N-(Phenylsubstituted)-2-(pyridin-2-ylacetyl)hydrazinecarbo-
thioamide Derivatives
2-(Pyridin-2-yl)acetohydrazide (A 0.01 mol, 1.51 g) and 0.01 mol of an appropriate isothiocyanate
were refluxed in acetonitrile for 6 h. The product was filtered and washed carefully with diethyl
ether in order to remove unreacted isothiocyanate, and then with water in order to remove hydrazide
residues. The product was dried and then crystallized from an ethanol solution.
N-(3,4-Dichlorophenyl)-2-(pyridin-2-ylacetyl)hydrazinecarbothioamide (
1H-NMR (DMSO-d6)
(ppm): 3.72 (s, 2H, CH2); 7.24 (t, 1H, CHarom., J = 6.20 Hz); 7.37 (d, 1H, CHarom.
1
). Yield 85%. m.p. 173–174 ◦C.
δ
,
J = 7.60 Hz); 7.44–7.46 (dd, 1H, CHarom., J1 = 3.46 Hz, J2 = 1.50 Hz); 7.59 (d, 1H, CHarom., J = 8.80 Hz);
7.74 (t, 1H, CHarom., J = 7,40 Hz), 7.80 (s, 1H, CHarom.); 8.33 (s, 1H, CHarom.); 9.95 (s, 1H, NH); 9.98 (s,
1H, NH); 10.32 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ
(ppm): 42.73, 122.17, 124.18, 125.46, 127.0, 130.08
(2C), 130.26, 136.9, 139.31, 148.8, 155.65, 168.82, 180.72. ESI MS: m/z = 377.00 [M + Na] (100%).
◦
N-(3-Chloro-4-methylphenyl)-2-(pyridin-2-ylacetyl)hydrazinecarbothioamide ( ). Yield 83%. m.p. 183–184 C.
1H-NMR (DMSO-d6)
(ppm): 2.36 (s, 3H, CH3), 3.90 (s, 2H, CH2); 7.21 (t, 1H, CHarom., J = 6.20 Hz);
7.30 (d, 1H, CHarom., J = 7.60 Hz); 7.20 (m, 2H, CHarom.); 7.52 (s, 1H, CHarom.); 7.72 (t, 1H, CHarom.
J = 7,40 Hz); 8.40 (s, 1H, CHarom.); 9.19 (s, 1H, NH); 9.21 (s, 1H, NH); 10.30 (s, 1H, NH). 13C-NMR
(DMSO-d6) (ppm): 19.07, 42.69, 122.16, 124.19, 125.71, 130.66, 132.27, 136.94, 138.22 (2C), 148.74 (2C),
2
δ
,
δ
155.76, 168.73, 180.72. ESI MS: m/z = 357.00 [M + Na] (100%).
2-(Pyridin-2-ylacetyl)-N-[3-(trifluoromethyl)phenyl]hydrazinecarbothioamide (
3
).
Yield 72%.
m.p.
◦
1
177–178 C. H-NMR (DMSO-d6)
7.50 (m, 2H, CHarom.); 7.70 (s, 2H, CHarom.); 8.39 (d, 1H, CHarom., J = 4.00 Hz); 9.49 (s, 1H, NH); 9.52
(s, 1H, NH); 10.30 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 42.75, 118.63 (q) 121.67 (q, J = 3.70
δ (ppm): 3.94 (s, 2H, CH2); 7.32 (d, 1H, CHarom., J = 8.00 Hz);
δ
Hz), 122.14, 124.2, 125.85 (q, J = 272.80 Hz), 128.63 (q, J = 33.40 Hz), 129.42, 168.86, 180.84. ESI MS:
m/z = 377.00 [M + Na] (100%).
◦
N-(3-Bromophenyl)-2-(pyridin-2-ylacetyl)hydrazinecarbothioamide (
1H-NMR (DMSO-d6)
8.35 (d, 1H, CHarom., J = 4.00 Hz); 9.49 (s, 1H, NH); 9.96 (s, 1H, NH); 10.33 (s, 1H, NH). 13C-NMR
(DMSO-d6) (ppm): 42.72, 120.59, 121.82, 122.16, 124.19, 127.87, 130.16 (2C), 136.92, 140.76, 148.75,
155.72, 148.76, 168.79, 180.68. ESI MS: m/z = 366.30 [M + H] (100%).
4). Yield 80%. m.p. 176–177 C.
δ
(ppm): 3.76 (s, 2H, CH2); 7.35–7.49 (m, 5H, CHarom.); 7.75–7.80 (m, 2H, CHarom.);
δ
◦
N-(3-Chlorophenyl)-2-(pyridin-2-ylacetyl)hydrazinecarbothioamide ( ). Yield 78%. m.p. 171–172 C.
1H-NMR (DMSO-d6)
(ppm): 3.76 (s, 2H, CH2); 7.25–7.44 (m, 5H, CHarom.); 7.64 (br. s, 1H, CHarom.);
7.78 (tt, 1H, CHarom., J = 4.56 Hz); 8.35 (d, 1H, CHarom., J = 4.00 Hz); 9.90 (s, 1H, NH); 9.97 (s, 1H, NH);
10.34 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 42.71, 122.15, 124.18, 124.98, 129.87, 132.25, 136.9,
140.63 (2C), 148.76 (2C), 155.72, 168.77, 180.73. ESI MS: m/z = 319.30 [M − H] (100%).
5
δ
δ

Molecules 2017, 22, 409
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◦
N-(3-Chloro-4-fluorophenyl)-2-(pyridin-2-ylacetyl)hydrazinecarbothioamide (
6
). Yield 72%. m.p. 188–189 C.
1H-NMR (DMSO-d6)
δ
(ppm): 3.76 (s, 2H, CH2); 7.25–7.29 (dd, 1H, CHarom.
7.41 (m, 3H, CHarom.); 7.71 (d, 1H, CHarom., J = 6.80 Hz); 7.78 (tt, 1H, CHarom., J = 4.56 Hz); 8.34 (d, 1H,
CHarom., J = 4.00 Hz); 9.91 (s, 1H, NH); 9.93 (s, 1H, NH); 10.33 (s, 1H, NH). 13C-NMR (DMSO-d6)
, J1 = 5.10 Hz, J2 = 1.50 Hz);
δ
(ppm): 42.7, 116.51, 118.66, 122.15, 124.18, 126.17, 127.58, 136.36, 148.77 (2C), 153.05, 155.69, 168.78,
180.99. ESI MS: m/z = 337.30 [M − H] (100%).
◦
N-(5-Chloro-2-methylphenyl)-2-(pyridin-2-ylacetyl)hydrazinecarbothioamide (
7
). Yield 78%. m.p. 192–193 C.
1H-NMR (DMSO-d6)
δ
(ppm): 2.24 (s, 3H, CH3), 3.90 (s, 2H, CH2); 7.18–7.22 (dd, 1H, CHarom.,
J1 = 4.53 Hz, J2 = 1.80 Hz); 7.30 (d, 1H, CHarom., J = 10.80 Hz); 7.2 (t, 1H, CHarom., J = 8.40 Hz); 7.62 (d,
1H, CHarom., J = 10.00 Hz); 7.52 (tt, 1H, CHarom., J = 4.48 Hz); 8.2 (d, 1H, CHarom., J = 2.80 Hz); 8.40 (d,
1H, CHarom., J = 4.00 Hz); 9.18 (s, 1H, NH); 9.21 (s, 1H, NH); 10.30 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ
(ppm): 17.05, 43.07, 122.2, 124.19, 126.67, 128.57, 129.5, 131.68, 134.82, 136.98, 139.3, 148.83, 156.16,
168.7, 181.47. ESI MS: m/z = 357.00 [M + Na] (100%).
3.3. General Procedure for the Synthesis of 5-(Pyridin-2-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-
3-thione Derivatives
N-(Substituted-phenyl)-2-(pyridin-2-ylacetyl)hydrazinecarbothioamide (0.01 mol) was refluxed
with 2% NaOH solution (40–50 mL) for 4 h. After cooling the solution was neutralized with dilute
hydrochloric acid. The precipitated compound was filtered and then crystallized from ethanol.
4-(3,4-Dichlorophenyl)-5-(pyridin-2-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (1a). Yield 79%. m.p.
192–193 ◦C. 1H-NMR (DMSO-d6)
δ
(ppm): 4.13 (s, 2H, CH2); 7.08 (d, 1H, CHarom., J = 10.40 Hz); 7.19
(dd, 1H, CHarom., J1 = 5.20 Hz, J2 = 2.40 Hz); 7.28 (d, 1H, CHarom., J = 4.93 Hz); 7.57 (d, 1H, CHarom.
J = 3.20 Hz); 7.61 (tt, 1H, CHarom., J = 4.08 Hz), 7.68 (d, 1H, CHarom., J = 11.20 Hz); 8.37 - 8.39 (dd, 1H,
CHarom., J1 = 4.80 Hz, J2 = 0.60 Hz); 13.88 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 33.99, 122.15,
,
δ
123.41, 128.88, 130.51, 130.93, 131.34, 132.28, 133.46, 136.64, 148.99, 150.25, 154.59, 167.82. ESI MS:
m/z = 361.00 [M + Na] (100%).
4-(3-Chloro-4-methylphenyl)-5-(pyridin-2-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2a). Yield 75%.
◦
1
m.p. 188–189 C. H-NMR (DMSO-d6)
CHarom., J = 10.40 Hz); 7.11–7.12 (dd, 1H, CHarom., J1 = 8.10 Hz, J2 = 1.80 Hz); 7.17–7.21 (dd, 1H, CHarom.
J1 = 7.20 Hz, J2 = 2.10 Hz); 7.28 (d, 1H, CHarom., J = 2.40 Hz); 7.38 (d, 1H, CHarom. J = 10.80 Hz); 7.61
(tt, 1H, CHarom., J = 4.56 Hz); 8.39 (d, 1H, CHarom., J = 4.80 Hz); 13.81 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 19.31, 34.01, 122.12, 123.33, 127.03, 128.58, 131.41, 132.43, 133.15, 136.6, 136.96, 148.97, 150.36,
δ (ppm): 2.32 (s, 3H, CH3); 4.07 (s, 2H, CH2); 7.07 (d, 1H,
,
,
δ
154.72, 167.90. ESI MS: m/z = 339.00 [M + Na] (100%).
5-(Pyridin-2-ylmethyl)-4-[3-(trifluoromethyl)phenyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (3a). Yield 70%.
m.p. 178–179 ◦C. 1H-NMR (DMSO-d6)
δ
(ppm): 4.13 (s, 2H, CH2); 6.99 (d, 1H, CHarom., J = 10.40 Hz);
7.13–7.17 (dd, 1H, CHarom., J1 = 6.60 Hz, J2 = 1.50 Hz); 7.52–7.64 (m, 4H, CHarom.); 7.77 (d, 1H, CHarom.
J = 9.60 Hz); 8.35 (d, 1H, CHarom., J = 4.80 Hz); 13.90 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 34.08,
,
δ
118.01 (q), 121.62 (q, J = 4.20 Hz), 122.07, 123.29, 125.6 (q, J = 272.60 Hz), 126. 03 (q, 30.70 Hz) 129.51 (q,
30.80 Hz), 134.41, 136.6, 154.56, 167.91. ESI MS: m/z = 359.00 [M + Na] (100%).
4-(3-Bromophenyl)-5-(pyridin-2-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (4a). Yield 79%. m.p.
◦
1
222–223 C. H-NMR (DMSO-d6)
δ (ppm): 4.09 (s, 2H, CH2); 7.02 (d, 1H, CHarom., J = 10.40 Hz);
7.17–7.21 (dd, 1H, CHarom., J1 = 6.60 Hz, J2 = 1.80 Hz); 7.27 (m, 1H, CHarom.); 7.36 (t, 1H, CHarom.
J = 10.80 Hz); 7.42 (t, 1H, CHarom., J = 2.40 Hz); 7.57–7.62 (m, 2H, CHarom.); 8.38 (d, 1H, CHarom.
,
,
J = 4.80 Hz); 13.83 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ
(ppm): 34.04, 121.24, 122.11, 123.34, 127.55,
130.87, 131.15, 132.24, 134.95, 136.62, 148.96, 150.27, 154.59, 167.82. ESI MS: m/z = 347.20 [M] (100%).
4-(3-Chlo◦rophenyl)-5-(pyridin-2-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (5a). Yield 73%. m.p.
1
223–224 C. H-NMR (DMSO-d6)
δ (ppm): 4.10 (s, 2H, CH2); 7.03 (d, 1H, CHarom., J = 10.40 Hz);
7.16–7.23 (m, 2H, CHarom.); 7.31 (t, 1H, CHarom., J = 2.40 Hz); 7.39–7.50 (m, 2H, CHarom.); 7.60 (tt, 1H,

Molecules 2017, 22, 409
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CHarom., J = 4.56 Hz); 8.38 (d, 1H, CHarom., J = 5.20 Hz); 13.84 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ
(ppm): 34.04, 119.93, 122.14, 123.41, 127.19, 130.59, 133.1, 134.89, 136.61, 149.01, 150.3, 154.6, 167.98. ESI
MS: m/z = 334.90 [M + Na] (100%).
4-(3-Chloro-4-fluorophenyl)-5-(pyridin-2-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (6a). Yield 74%.
m.p. 224–225 ◦C. 1H-NMR (DMSO-d6)
δ (ppm): 4.11 (s, 2H, CH2); 7.05 (d, 1H, CHarom., J = 10.40 Hz);
7.17–7.21 (dd, 1H, CHarom., J1 = 6.60 Hz, J2 = 1.50 Hz); 7.26–7.31 (m, 1H, CHarom.); 7.46 (t, 1H, CHarom.
J = 11.00 Hz); 7.51 (t, 1H, CHarom., J = 4.40 Hz); 7.61 (tt, 1H, CHarom., J = 4.56 Hz); 8.39 (d, 1H, CHarom.
,
,
J = 5.10 Hz); 13.85 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ
(ppm): 34.03, 117.40, 119.90, 122.13, 123.40,
129.63, 130.63, 136.64, 149.01, 150.40, 154.59, 155.71, 159.01, 167.97. ESI MS: m/z = 319.30 [M] (100%).
4-(5-Chloro-2-methylphenyl)-5-(pyridin-2-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (7a). Yield 73%.
m.p. 225 ◦C. 1H-NMR (DMSO-d6)
(ppm): 1.81 (s, 3H, CH3); 4.00 (s, 2H, CH2); 6.95 (d, 1H, CHarom.
δ
,
J = 10.40 Hz); 7.18–7.22 (dd, 1H, CHarom., J1 = 6.60 Hz, J2 = 1.80 Hz); 7.25–7.28 (m, 2H, CHarom.);
7.40–7.43 (dd, 1H, CHarom., J1 = 8.10 Hz, J2 = 2.10 Hz); 7.59 (tt, 1H, CHarom., J = 4.56 Hz); 8.39 (d, 1H,
CHarom., J = 5.10 Hz); 13.86 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ (ppm): 16.60, 34.11, 122.24, 123.32,
128.43, 129.79, 130.47, 132.21, 133.52, 135.76, 136.63, 149.03, 150.20, 154.37, 167.24. ESI MS: m/z = 339.00
[M + Na] (100%).
3.4. General Procedure for the Synthesis of 5-(Pyridin-2-ylmethyl)-1,3,4-thiadiazol-2-amine Derivatives
N-(Substituted-phenyl)-2-(pyridin-2-ylacetyl)hydrazinecarbothioamide (0.01 mol) was mixed for
4 h with concentrated sulfuric acid (0.5 mL). Then to the solution crushed ice was added. After cooling
the solution was neutralized with dilute NaOH. The precipitated compound was filtered and then
crystallized from ethanol.
N-(3,4-Dichlorophenyl)-5-(pyridin-2-ylmethyl)-1,3,4-thiadiazol-2-amine (1b). Yield 71%. m.p. 201–202 ◦C.
1H-NMR (DMSO-d6)
δ
(ppm): 4.45 (s, 2H, CH2); 7.28–7.32 (m, 1H, CHarom.); 7.42–7.46 (m, 2H, CHarom.);
7.55 (d, 2H, Carom., J = 8.70 Hz); 7.78 (tt, 1H, CHarom., J = 4.56 Hz); 8.05 (d, 1H, CHarom., J = 2.40 Hz);
8.54 (d, 1H, CHarom., J = 6.40 Hz); 10.55 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 38.01, 117.38,
δ
118.30, 122.31, 122.70, 123.33, 130.78, 131.22, 137.18, 140.55, 149.26, 156.58, 158.08, 164.19. ESI MS:
m/z = 361.00 [M + Na] (100%).
N-(3-Chloro-4-methylphenyl)-5-(pyridin-2-ylmethyl)-1,3,4-thiadiazol-2-amine (2b). Yield 78%. m.p.
◦
1
181–182 C. H-NMR (DMSO-d6)
CHarom.); 7.43 (d, 1H, CHarom., J = 8.00 Hz); 7.79 (tt, 1H, CHarom., J = 4.56 Hz); 7.59 (d, 1H, CHarom.
J = 2.80 Hz); 8.55 (d, 1H, CHarom., J = 6.40 Hz); 10.31 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 18.77,
δ (ppm): 2.26 (s, 3H, CH3); 4.43 (s, 2H, CH2); 7.25–7.33 (m, 3H,
,
δ
38.08, 115.98, 117.18, 122.28, 123.32, 128.0, 131.37, 133.31, 137.16, 139.78, 149.27, 156.7, 157.4, 164.52. ESI
MS: m/z = 339.00 [M + Na] (100%).
5-(Pyridin-2-ylmethyl)-N-[3-(trifluoromethyl)phenyl]-1,3,4-thiadiazol-2-amine (3b). Yield 69%. m.p.
◦
1
179–180 C. H-NMR (DMSO-d6)
δ (ppm): 4.46 (s, 2H, CH2); 7.28–7.33 (m, 2H, CHarom.); 7.44 (d,
1H, CHarom., J = 10.40 Hz); 7.55 (t, 1H, CHarom., J = 10.60 Hz); 7.71–7.75 (dd, 1H, CHarom., J1 = 8.40 Hz,
J2 = 1.50 Hz); 7.81 (tt, 1H, CHarom., J = 4.56 Hz); 8.15 (s, 1H, CHarom.,); 8.55 (d, 1H, CHarom., J = 6.60 Hz);
10.59 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ (ppm): 38.05, 113.18 (q, 4.20 Hz), 117.75 (q, 4.20 Hz), 120.77,
122.37, 123.35, 125.97 (q, 271.20 Hz), 129.79 (q, 33.20 Hz), 130.14. 137.19, 141.29, 149.27, 156.63, 157.96,
164.43. ESI MS: m/z = 359.0 [M + Na] (100%).
◦
N-(3-Bromophenyl)-5-(pyridin-2-ylmethyl)-1,3,4-thiadiazol-2-amine (4b). Yield 76%. m.p. 156–157 C.
1H-NMR (DMSO-d6)
δ
(ppm): 4.45 (s, 2H, CH2); 6.99–7.03 (m, 1H, CHarom.); 7.28–7.45 (m, 4H, CHarom.);
7.78 (tt, 1H, CHarom., J = 4.56 Hz); 7.86 (t, 1H, CHarom., J = 2.60 Hz); 8.54 (d, 1H, CHarom., J = 6.40 Hz);
10.44 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 37.31, 116.10, 119.46, 121.92, 122.86, 124.06, 130.88,
138.63, 142.09, 148.15, 148.75, 155.85, 157.38, 164.47. ESI MS: m/z = 347.20 [M] (100%).
δ

Molecules 2017, 22, 409
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◦
N-(3-Chlorophenyl)-5-(pyridin-2-ylmethyl)-1,3,4-thiadiazol-2-amine (5b). Yield 78%. m.p. 149–150 C.
1H-NMR (DMSO-d6)
7.79 (tt, 1H, CHarom., J = 4.56 Hz); 7.85 (t, 1H, CHarom., J = 2.60 Hz); 8.54 (d, 1H, CHarom., J = 6.40 Hz);
10.48 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 37.94, 115.69, 116.62, 121.12, 122.41, 123.47, 130.58,
δ (ppm): 4.44 (s, 2H, CH2); 6.96–7.08 (m, 1H, CHarom.); 7.27–7.40 (m, 4H, CHarom.);
δ
133.43, 137.43, 142.00, 149.08, 156.53, 157.74, 164.43. ESI MS: m/z = 311.90 [M] (100%).
N-(3-Chloro-4-fluorophenyl)-5-(pyridin-2-ylmethyl)-1,3,4-thiadiazol-2-amine (6b). Yield 75%. m.p.
◦
1
196–197 C. H-NMR (DMSO-d6)
1H, CHarom. J = 4.64 Hz); 7.97–7.99 (dd, 1H, CHarom., J1 = 6.00 Hz, J2 = 2.70 Hz); 8.54 (d, 1H, CHarom.
J = 2.76 Hz); 10.43 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 38.06, 116.97 (d, 22.20 Hz), 117.45
δ (ppm): 4.44 (s, 2H, CH2); 6.28–7.45 (m, 4H, CHarom.); 7.79 (tt,
,
,
δ
(d, 7.80 Hz) 118.38, 119.48 (d, 18.60 Hz), 122.29, 123.32, 137.16, 137.91 (d, 3.30 Hz), 149.26, 153.62
(242.60 Hz), 156.63, 157.68, 164.49. ESI MS: m/z = 319.30 [M + H] (100%).
N-(5-Chloro-2-methylphenyl)-5-(pyridin-2-ylmethyl)-1,3,4-thiadiazol-2-amine (7b). Yield 71%. m.p.
145–146 ◦C. 1H-NMR (DMSO-d6)
δ (ppm): 2.24 (s, 3H, CH3); 4.53 (s, 2H, CH2); 6.99 (dd, 1H, CHarom.,
J1 = 8.10 Hz, J2 = 2.10 Hz); 7.22 (d, 1H, CHarom., J = 10.80 Hz); 7.51 (t, 1H, CHarom., J = 8.40 Hz); 7.63 (d,
1H, CHarom., J = 10.00 Hz); 8.02 (tt, 1H, CHarom., J = 4.48 Hz); 8.20 (d, 1H, CHarom., J = 2.80 Hz); 8.65 (d,
1H, CHarom., J = 6.40 Hz); 10.31 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ (ppm): 17.62, 36.64, 119.01, 122.21,
123.32, 124.70, 125.99, 130.69, 131.83, 139.48, 140.01, 147.19, 155.23, 157.24, 165.65. ESI MS: m/z = 334.80
[M] (100%).
3.5. 5-(Pyridin-2-ylmethyl)-1,3,4-oxadiazole-2(3H)-thione (c)
Compound was synthesized and described previously [39,44].
3.6. 4-Amino-5-(pyridin-2-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
5-(Pyridin-2-ylmethyl)-1,3,4-oxadiazole-2(3H)-thione (c, 0.01 mole, 1.93 g) was refluxed with 80%
hydrazine hydrate (0.5 mL) and ethanol (20 mL) for 4 h. The precipitated compound was filtered and
then crystallized from ethanol. Yield 80%. m.p. 210–211 ◦C. 1H-NMR (DMSO-d6)
δ
(ppm): 4.20 (s, 2H,
CH2); 5.5 (br. s, 2H, NH2); 7.27 (d, 1H, CHarom. J = 4.10 Hz); 7.35 (d, 1H, CHarom. J = 7.80 Hz); 7.75 (tt,
1H, CHarom. J = 3.43 Hz); 8.45 (d, 1H, CHarom. J = 3.90 Hz); 13.53 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ
(ppm): 33.47, 122.32, 123.65, 136.88, 139.40, 149.10, 167, 31, 181.70. ESI MS: m/z = 206.50 [M
−
H] (100%).
3.7. General Procedure for the Synthesis of 1-(Subtituted
phenyl)-3-[3-(pyridin-2-ylmethyl)-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]thiourea Derivatives
4-Amino-5-(pyridin-2-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (0.01 mol, 2.0 g) and an
appropriate isothiocyanate (0.01 mol) were refluxed in acetonitrile for 6 h. The product was filtered and
washed carefully with diethyl ether in order to remove unreacted isothiocyanate. The product was then
dried and then the residue was purified by column chromatography (eluent: chloroform–methanol
9.5:0.5).
1-(3,4-Dichlorophenyl)-3-[3-(pyridin-2-ylmethyl)-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]-thiourea
◦
1e). Yield 68%. m.p. 156–157 C. 1H-NMR (DMSO-d6)
δ (ppm): 4.14 (s, 2H, CH2); 7.28 (d, 1H, CHarom.,
(
J = 7.80 Hz); 7.37 (d, 1H, CHarom., J = 8.50 Hz); 7.48 (m, 1H, CHarom.); 7.59 (d, 1H, CHarom., J = 9.00 Hz);
7.56 (tt, 1H, CHarom., J = 4.08 Hz), 7.68 (m, 1H, CHarom.); 8.49 (d, 1H, CHarom., J = 2.80 Hz); 10.78 (br.
s, 2H, NH); 13.72 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ (ppm): 33.47, 122.35, 122.78, 123.63, 127.25,
130.30, 131.20, 136.71, 136.90, 138.62, 149.11, 151.15, 154.48, 167.25, 181.72. ESI MS: m/z = 409.90
[M − H] (100%).
1-(3-chloro-4-methylphenyl)-3-[3-(pyridin-2-ylmethyl)-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]thiourea (2e).
◦
Yield 65%. m.p. 177–178 C. 1H-NMR (DMSO-d6)
δ (ppm): 2.30 (s, 3H, CH3); 4.14 (s, 2H, CH2); 7.29 (d,
1H, CHarom., J = 10.40 Hz); 7.31 (s, 1H, CHarom.); 7.34 (d, 1H, CHarom., J = 5.40 Hz); 7.37 (d, 1H, CHarom.
J = 2.40 Hz); 7.40 (d, 1H, CHarom., J = 10.80 Hz); 7.76 (tt, 1H, CHarom., J = 4.56 Hz); 8.49 (d, 1H, CHarom.
,
,

Molecules 2017, 22, 409
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J = 2.40 Hz); 10.00 (br. s, 1H, NH); 10.59 (br. s, 1H, NH); 13.70 (s, 1H, NH). 13C-NMR (DMSO-d6)
δ
(ppm): 19.07, 33.42, 116.73, 122.34, 123.62, 127.42, 131.33, 131.41, 133.31, 136.92, 137.52, 149.06, 154.50,
167.26, 181.20. ESI MS: m/z = 389.00 [M − H] (100%).
1-[3-(pyridin-2-ylmethyl)-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]-3-[3-(trifluoromethyl)-phenyl]thiourea
◦
(
3e). Yield 80%. m.p. 172–173 C. 1H-NMR (DMSO-d6)
δ
(ppm): 4.15 (s, 2H, CH2); 2.26 (d, 1H, CHarom.
,
,
J = 8.50 Hz); 7.38 (d, 1H, CHarom., J = 7.80 Hz); 7.49–7.64 (m, 4H, CHarom.); 7.75 (tt, 1H, CHarom.
J = 9.60 Hz); 8.46 (d, 1H, CHarom., J = 2.40 Hz); 10.70 (br. s, 2H, NH); 13.83 (s, 1H, NH). 13C-NMR
(DMSO-d6)
δ
(ppm): 33.44, 121.81 (q), 122.32, 123.65 (q), 125.77 (q, 270.20 Hz), 129.66 (q, J = 33.2 Hz),
136.88, 139.40, 149.09, 151.07, 154.47, 167.31, 180.31. ESI MS: m/z = 409.0 [M − H] (100%).
1-(3-Bromophenyl)-3-[◦3-(pyridin-2-ylmethyl)-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]-thiourea (4e). Yield
1
75%. m.p. 178–179 C. H-NMR (DMSO-d6)
δ
(ppm): 4.14 (d, 2H, CH2, J = 8.40 Hz); 7.29 (m, 6H,
CHarom.); 7.76 (tt, 1H, CHarom., J = 2.40 Hz); 8.49 (d, 1H, CHarom. J = 8.40 Hz.); 10.67 (br. s, 2H, NH);
13.71(s, 1H, NH). 13C-NMR (DMSO-d6)
δ
(ppm): 34.47, 121.24, 122.37, 123.64, 128.05, 130.43, 136.29,
140.11, 149.12, 151.22, 154.52, 155.2, 167.28, 181.41. ESI MS: m/z = 429.90 [M − H] (100%)
1-(3-Chlorophenyl)-3-[3-(pyridin-2-ylmethyl)-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]-thiourea (5e). Yield
◦
66%. m.p. 170 C. 1H-NMR (DMSO-d6)
δ
(ppm): 4.15 (d, 2H, CH2, J = 9.00 Hz); 7.14–7.39 (m, 6H,
CHarom.); (tt, 1H, CHarom., J = 4.56 Hz); 8.47 (d, 1H, CHarom., J = 5.20 Hz); 10.67 (br. s, 2H, NH); 13.71(s,
1H, NH). 13C-NMR (DMSO-d6)
(ppm): 34.45, 122.37, 123.81, 125.15, 127.19, 130.15, 132.60, 133.39,
δ
136.87, 137.88, 139.96, 148.84, 151.13, 154.52, 167.98, 181.36. ESI MS: m/z = 375.00 [M − H] (100%).
1-(3-Chloro-4-fluorophenyl)-3-[3-(pyridin-2-ylmethyl)-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]thiourea (6e).
◦
Yield 65%. m.p. 190–192 C. 1H-NMR (DMSO-d6)
δ
(ppm): 4.15 (d, 2H, CH2, J = 9.00 Hz); 7.29 (t,
1H, CHarom., J = 6.00 Hz); 7.39–7.42 (m, 4H, CHarom.); 7.75 (tt, 1H, CHarom., J = 9.00 Hz); 8.49 (d, 1H,
CHarom., J = 6.00 Hz); 10.71 (br. s, 2H, NH); 13.72 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 33.43,
δ
116.44 (d, 22.40 Hz), 118.73 (d, 19.40 Hz), 121.22 (d), 121.42, 123.63, 125.08 (d), 135.59 (d), 136.9, 149.09,
152.16, 154.47 (d, 24.30 Hz), 156.37, 167.97, 181.79. ESI MS: m/z = 393.00 [M − H] (100%).
1-(2-Chloro-5-methylphenyl)-3-[3-(pyridin-2-ylmethyl)-5-thioxo-1,5-dihydro-4H-1,2,4-triazol-4-yl]thiourea
(
7e). Yield 63%. m.p. 180–181 ◦C. 1H-NMR (DMSO-d6)
δ
(ppm): 2.07 (s, 3H, CH3); 4.37 (s, 2H, CH2);
7.27 (s, 1H, CHarom.); 7.65–7.72 (m, 3H, CHarom.); 8.17 (t, 1H, CHarom., J = 4.56 Hz); 8.70 (d, 1H, CHarom.
J = 2.40 Hz); 10.37 (br. s, 1H, NH); 10.69 (br. s, 1H, NH); 13.82 (s, 1H, NH). 13C-NMR (DMSO-d6)
,
δ
(ppm): 17.41, 29.65, 118.09, 125.25, 126.95, 127.23, 130.47, 132.85, 142.94, 144.76, 148.62, 150.38, 150.87,
167.44, 181.67. ESI MS: m/z = 389.00 [M − H] (100%).
3.8. General Procedure for the Synthesis of
N-(Substituted-phenyl)-5-(pyridin-2-ylmethyl)-1,3,4-oxadiazol-2-amine Derivatives
Triethylamine (2–3 drops) was added to a suspension of thiosemicarbazide (1 mmol) and mercuric
chloride (1.25 mmol) in dry DMF (5 mL). The resulting mixture was stirred for a maximum of 8 h
at room temperature or until TLC showed the end of the reaction. The suspension was filtered
through paper filter, then washed with CHCl3. The filtrate was diluted with water, extracted three
times with CHCl3 (15 mL), the combined organic fractions were dried over MgSO4, filtered and
concentrated under reduced pressure. The resulting residue was purified by silica gel chromatography
(chloroform–methanol; 9.5:0.5).
N-(3,4-Dichlorophenyl)-5-(pyridin-2-ylmethyl)-1,3,4-oxadiazol-2-amine (1f). Yield 72%. m.p. 198–199 ◦C.
1H-NMR (DMSO-d6)
δ
(ppm): 4.36 (s, 2H, CH2); 7.28–7.33 (m, 1H, CHarom.); 7.41–7.45 (dd, 2H, CHarom.
,
,
J1 = 11.40 Hz, J2 = 2.70 Hz); 7.55–7.58 (d, 1H, CHarom., J = 8.70 Hz); 7.71–7.72 (td, 1H, CHarom.
J = 5.70 Hz); 7.85–7.86 (d, 1H, CHarom., J = 2.40 Hz); 8.49–8.52 (m, 1H, CHarom.); 10.81 (s, 1H, NH).
13C-NMR (DMSO-d6)
(ppm): 33.48, 117.11, 117.93, 122.48, 123.05, 123.39, 130.90, 131.31, 137.12, 138.82,
149.37, 154.75, 158.30, 159.44. ESI MS: m/z = 319.00 [M − H] (100%).
δ

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N-(3-Chloro-4-methylphenyl)-5-(pyridin-2-ylmethyl)-1,3,4-oxadiazol-2-amine (2f). Yield 75%. m.p.
◦
1
176–177 C. H-NMR (DMSO-d6)
CHarom.); 7.38–7.44 (m, 1H, CHarom.); 7.67–7.68 (d, 1H, CHarom., J = 0.90 Hz); 7.71-7.72 (td, 1H, CHarom.
J = 2.90 Hz); 7.95 (s, 1H, CHarom.); 8.50–8.51 (m, 1H, CHarom.); 10.52 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 18.74, 33.50, 115.65, 116.67, 122.47, 123.37, 128.09, 131.49, 133.35, 137.12, 137.88, 149.36, 154.83,
δ (ppm): 1.22 (s, 3H, CH3); 4.34 (s, 2H, CH2); 7.26–7.35 (m, 2H,
,
δ
157.98, 159.92 ESI MS: m/z = 301.20 [M] (100%).
5-(Pyridin-2-ylmethyl)-N-[3-(trifluoromethyl)phenyl]-1,3,4-oxadiazol-2-amine (3f). Yield 67%. m.p.
145–146 ◦C. 1H-NMR (DMSO-d6)
δ (ppm): 4.37 (s, 2H, CH2); 7.29–7.33 (m, 2H, CHarom.); 7.42–7.45
(m, 1H, CHarom.); 7.53–7.58 (t, 1H, CHarom., J = 4.20 Hz); 7.71–7.75 (m, 1H, CHarom.); 7.77–7.83 (td, 1H,
CHarom., J = 3.90 Hz); 7.97–7.98 (m, 1H, CHarom.); 8.49–8.52 (dq, 1H, CHarom., J = 2.40 Hz); 10.83 (s, 1H,
NH). 13C-NMR (DMSO-d6)
δ (ppm): 33.51, 112.69, 117.90 (q, 4.00 Hz), 120.54 (q, 4.10 Hz), 123.40 (q,
27.40 Hz), 125.91, 130.03 (q, 33.20 Hz), 130.26 (q, 33.20 Hz), 137.13, 139.52, 149.37, 154.80, 158.25, 159.66.
ESI MS: m/z = 319.10 [M − H] (100%).
◦
N-(3-Bromophenyl)-5-(pyridin-2-ylmethyl)-1,3,4-oxadiazol-2-amine (4f). Yield 70%. m.p. 164–165 C.
1H-NMR (DMSO-d6)
δ
(ppm): 4.36 (s, 2H, CH2); 7.13–7.17 (m, 1H, CHarom.); 7.25–7.33 (m, 2H, CHarom.);
7.42–7.47 (m, 2H, CHarom.); 7.77–7.84 (m, 2H, CHarom., J = 3.90 Hz); 8.49–8.52 (m, 1H, CHarom.); 10.65 (s,
1H, NH). 13C-NMR (DMSO-d6)
(ppm): 33.51, 115.78, 119.01, 121.94, 122.47, 123.37, 124.19, 130.97,
137.11, 140.30, 149.36, 154.80, 158.14, 159.59. ESI MS: m/z = 343.10[M − H] (100%).
δ
◦
N-(3-Chlorophenyl)-5-(pyridin-2-ylmethyl)-1,3,4-oxadiazol-2-amine (5f). Yield 72%. m.p. 147–148 C.
1H-NMR (DMSO-d6)
(ppm): 4.36 (s, 2H, CH2); 7.00–7.03 (m, 1H, CHarom.); 7.28–7.45 (m, 4H, CHarom.);
7.69–7.70 (t, 1H, CHarom., J = 2.10 Hz); 7.76–7.82 (m, 1H, CHarom.); 8.50–8.51 (d, 1H, CHarom. J = 4.80 Hz);
10.67 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm): 33.51, 115.42, 116.20, 121.29, 122.47, 123.38, 130.68,
133.48, 137.11, 140.18, 149.37, 154.81, 158.14, 159.64. ESI MS: m/z = 329.10 [M − H] (100%).
δ
δ
N-(3-Chloro-4-fluorophenyl)-5-(pyridin-2-ylmethyl)-1,3,4-oxadiazol-2-amine (6f). Yield 72%.
178–179 ◦C. 1H-NMR (DMSO-d6)
(ppm): 4.35 (s, 2H, CH2); 7.28–7.45 (m, 4H, CHarom.); 7.76-7.22 (m,
2H, CHarom.); 8.49–8.51 (d, 1H, CHarom., J = 2.40 Hz); 10.66 (s, 1H, NH). 13C-NMR (DMSO-d6)
(ppm):
m.p.
δ
δ
33.52, 117.10, 117.90, 122.47, 123.38, 136.03, 136.06, 137.11, 149.37, 150.60, 153.79, 154.80, 158.12, 159.66.
ESI MS: m/z = 317.10 [M − H] (100%).
N-(2-Chloro-5-methylphenyl)-5-(pyridin-2-ylmethyl)-1,3,4-oxadiazol-2-amine (7f). Yield 69%. m.p.
◦
215–216 C. 1H-NMR (DMSO-d6)
δ
(ppm): 3.36 (bs, 3H, CH3); 4.36 (s, 2H, CH2); 7.28–7.33 (m, 1H,
CHarom.); 7.41–7.45 (m, 2H, CHarom.); 7.55–7.57 (d, 1H, CHarom., J = 4.30 Hz); 7.77–7.82 (td, 1H, CHarom.
J = 3.90 Hz); 7.85–7.86 (d, 1H, CHarom., J = 1.20 Hz); 8.49–8.52 (m, 1H, CHarom.). 13C-NMR (DMSO-d6)
(ppm): 33.49, 33.51, 117.14, 117.95, 122.49, 123.03, 123.40, 130.91, 131.31, 137.14, 138.87, 149.37, 154.37,
,
δ
158.30, 159.48. ESI MS: m/z = 301.20 [M] (100%).
3.9. Biological Assays
3.9.1. In Vitro Evaluation of Antimicrobial Activity
Microorganisms used in this study were as follows: Gram-positive bacteria: S. aureus NCTC
4163, S. aureus ATCC 25923, S. aureus ATCC 6538, S. aureus ATCC 29213, S. epidermidis ATCC 12228,
S. epidermidis ATCC 35984, Enterococcus hirae ATCC 10541, Enterococcus faecalis ATCC 29212, Bacillus
subtilis ATCC 6633, Bacillus cereus ATCC 11778, Micrococcus luteus ATCC 9341, M. luteus ATCC 10240;
Gram-negative rods: E. coli ATCC 10538, E. coli ATCC 25922, E. coli NCTC 8196, P. vulgaris NCTC 4635,
P. aeruginosa ATCC 15442, P. aeruginosa NCTC 6749, P. aeruginosa ATCC 27863, B. bronchiseptica ATCC
4617 and yeasts: C. albicans ATCC 10231, C. albicans ATCC 90028, C. parapsilosis ATCC 22019. All of
listed microorganisms used were obtained from the collection of the Department of Pharmaceutical
Microbiology, Medical University of Warsaw, Poland.

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3.9.2. Media, Growth Conditions and Antimicrobial Activity Assays
Antibacterial activity was examined by the disc-diffusion method under standard conditions
using Mueller-Hinton II agar medium (Becton Dickinson, Franklin Lakes, NJ, USA) according to CLSI
(previously NCCLS) guidelines [41]. Antifungal activities were assessed using Mueller-Hinton agar
2% glucose and 0.5 mg/ml Methylene Blue Dye Medium [41]. Sterile filter paper discs (9 mm diameter,
Whatman No 3 chromatography paper) were dripped with tested compound solutions (in DMSO) to
load 400 mg of a given compound per disc. Dry discs were placed on the surface of appropriate agar
medium. The results (diameter of the growth inhibition zone) were read after 18 h of incubation at
35 ◦C.
3.9.3. Cytotoxicity and Antiviral Assays
CD4+ human T-cells containing an integrated HTLV-1 genome (MT-4) were purchased from
American Type Culture Collection (ATCC). Laboratory strain IIIB of Human Immunodeficiency Virus
type-1 (HIV-1) was obtained from the supernatant of the persistently infected H9/IIIB cells (NIH 1983).
Compounds’ activity against HIV-1 was based on inhibition of virus-induced cytopathogenicity in MT-4
cell acutely infected with a multiplicity of infection (MOI) of 0.01, as described in Drzewiecka et al. [45]
.
After a 4-day incubation at 37 ◦C, cell viability was determined by the MTT method [46]. Efavirenz
was used as reference inhibitor.
4. Conclusions
We successfully synthesized five groups of compounds: thiosemicarbazides , 1,2,4-triazoles
1–7
1a
–
7a, 1,3,4-thiadiazoles 1b 7b, 1,3,4-oxadiazoles 1f 7f and thiourea derivatives 1e–7e to check their
–
–
microbiological activity, cytotoxicity and anti HIV activity. Introduction of the 1,2,4-triazole, thiadiazole
and oxazole ring to a 2-pyridyl moiety by a methylene linker decreased the antimicrobial activity and
cytotoxicity. A second substituent like a phenyl ring connected with halogen atoms (chlorine, bromine
or fluorine) had no influence on the biological activity. It could be summarized that all obtained
derivatives possess moderate activity and their structure-activity relationships need to be evaluated.
Supplementary Materials: Supplementary Materials are available online.
Acknowledgments: This work was supported by the Medical University of Warsaw and carried out with the use
of CePT infrastructure financed by the European Union—the European Regional Development Fund within the
Operational Programme Innovative Economy for 2007–2013.
Author Contributions: Daniel Szulczyk performed the syntheses of triazoles, thiadiazoles and oxadiazoles,
Piotr Tomaszewski collected biological data and wrote part of the paper, Michał Józ´wiak did the spectral data
analyses, Anna E. Kozioł and Tadeusz Lis collected and analyzed X-ray diffraction data, David Collu and
Filippo Iuliano performed cytotoxicity and antiviral activity assay, Marta Struga was the principal investigator of
the project and provided the research funding. All authors approved the final version of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available.
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