Synthesis and biological evaluation of new Mannich and Schiff bases containing 1,2,4-triazole and 1,3,4-oxadiazole nucleus

Article abstract of DOI:10.1007/s00044-016-1640-9

5-(Pyridine-3-yl)-1,3,4-oxadiazole-2-thiole 2, obtaining starting from nicotinic acid hydrazide were converted to the corresponding Mannich bases (3a–c) by the reaction with several heterocyclic amines in the presence of formaldehyde. 1,2,4-Triazole-3-thi

Full text of DOI:10.1007/s00044-016-1640-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1640-9
ORIGINAL RESEARCH
Synthesis and biological evaluation of new Mannich and Schiff bases
containing 1,2,4-triazole and 1,3,4-oxadiazole nucleus
Sule Ceylan1
Received: 25 May 2015 / Accepted: 28 June 2016
© Springer Science+Business Media New York 2016
Abstract 5-(Pyridine-3-yl)-1,3,4-oxadiazole-2-thiole 2,
obtaining starting from nicotinic acid hydrazide were con-
verted to the corresponding Mannich bases (3a–c) by the
reaction with several heterocyclic amines in the presence of
formaldehyde. 1,2,4-Triazole-3-thiole, (4) prepared from
1,3,4-oxadiazole-2-thiole (2) was converted to the corre-
sponding Mannich bases (5a–e) by several steps. The
synthesis of Schiff bases (6a–d) was performed from the
reaction of the corresponding triazol-3-thioles with various
aromatic aldehydes. The treatment of Schiff bases con-
taining 1,2,4-triazoles 6c and 6d with morpholine or thio-
morpholine generated the corresponding Mannich bases 7a,
b and 8a, b. The synthesized compounds were screened for
their antimicrobial, antilipase, and antiurease activities.
Some of them were found to possess good-moderate anti-
microbial, antiurease, and/or antilipase activity.
design of new compounds to deal with resistant bacteria has
become one of the most important work of medicinal che-
mists today. In addition, primary and opportunistic fungal
infections continue to increase rapidly because of the
increased number of immunosuppressed patients. Bio-
chemical similarity of the human cell and fungi as well
as the easily gained resistance constitute a handicap in
developing safe and efficient antifungals (Demirci et al.,
2014; Bayrak et al., 2009a; Bayrak et al., 2009b; Basoglu
et al., 2013; Mentese et al., 2013; Guzeldemirci and
Kucukbasmacı, 2010; Bektas et al., 2010; Ceylan et al.,
2013).
Another problematic infection, Mycobacterium tubercu-
losis remains a serious disease causing death in the world
today. The incidence of tuberculosis is increasing world-
wide, partly due to poverty and inequity and partly to the
HIV/AIDS pandemic, which greatly increase the risk of
infection proceeding to overt disease (Guzeldemirci and
Kucukbasmacı, 2010). As per the survey reported by Global
Alliances, there are 8–10 million new active cases of TB
with approximately three million deaths each year (Chandra
et al., 2006; Yu and Huiyuan, 2002; Bonde and Gaikward,
2004; Dixit et al., 2006; Hubschwerlen et al., 2003; Yolal
et al., 2012).
●
●
Keywords 1, 2, 4-Triazole 1, 3, 4-Oxadizole Nicotonic
●
●
●
acid hydrazide Mannich base Schiff base Biological
activity
Introduction
In particular, the appearance of multi-drug-resistant
strains of Mycobacterium tuberculosis, which exhibit
in vitro resistance to at least two major antituberculosis
drugs (usually Isoniazide and Rifampicin) and cause
intractable tuberculosis, has greatly contributed to the
increased incidence of tuberculosis (Guzeldemirci and
Kucukbasmacı, 2010). Moreover, the absence of an effec-
tive vaccine makes the treatment a main tool for controlling
the dissemination of TB, however, the length of treatment
(usually 6 months) makes it difficult for patients to comply
with treatment (Carneiro et al., 2011).
The resistance of pathogenic bacteria toward available
antibiotics has become a rapid increasing worldwide con-
cern leading to mortality and morbidity. Due to this reason,
* Sule Ceylan
sulecanim@hotmail.com
1
Department of Occupational Health and Safety, Artvin Çoruh
University, Artvin 08000, Turkey

Med Chem Res
The problem with clinically used drugs is not only the
increasing microbial resistance; also, administration of such
drugs is accompanied by toxic side effects that are often
dose limiting.
as Mannich bases. Mannich bases are regarded as deriva-
tives of the substrates obtained through substitution by an
aminoalkyl moiety. Although primary amines and even
ammonia may be used as amine component in a Mannich
reaction, secondary aliphatic amines are the most com-
monly encountered as amine reagents in the Mannich
reaction (Roman, 2015; Oloyede et al., 2014). The group
linked to parent amine by Mannich reaction is believed to
increase the lipophilicity of molecule at physiological pH
values by decreasing their protonation, and this restriction
of protonation results in enhancement of absorption through
bio-membranes (Bundgaard and Johnson, 1980; Johnson
et al., 1982). N-Mannich bases have been used successfully
to obtain prodrugs of amine as well as amide-containing
drugs. Some Mannich base derived from 1,2,4-triazole
nucleus have been reported to possess protozocidal and
antibacterial activity (Bektas et al., 2010; Demirbas et al.,
2009; Basoglu et al., 2014).
As a part of our continuing study on the synthesis of
biologically active compounds and on the basis of the fact
that more efficacious antibacterial compounds can be
designed by joining two or more biologically active het-
erocyclic systems together in a single molecular framework
(Bektas et al., 2010; Demirbas et al., 2009; Basoglu et al.,
2014), this paper presents the synthesis of new pyridine
derivatives incorporating different heterocycles as hybrid
molecules possessing antimicrobial activity.
1,2,4-Triazoles and their heterocyclic derivatives con-
stitute an important class of compounds exhibiting a wide
spectrum of biological activities including antibacterial
(Bayrak et al., 2009a; Bayrak et al., 2009b; Guzelde-
mirci and Kucukbasmacı, 2010), anti-inflammatory
(Tozkoparan et al., 2007), CNS depressant (Stefanska
et al., 2012), antitubercular (Kumar et al., 2010) anti-HIV
(Kucukguzel et al., 2008), and antiproliferative (Rashid
et al., 2013). 1,2,4-Triazole system is a structural element of
many drugs that have antifungal activity such as flucona-
zole, itraconazole, and voriconazole (Johnsona et al., 2008).
Also, there are other known drugs containing 1,2,4-triazole
nucleus, for example, triazolam, a benzodiazepine class
psychotropic drug, rizatriptan (antimigraine), nefazodone
(antidepressant), ribavirin (antiviral), alprazolam (analge-
sic), and etizolam (hypnotic and sedative). Vorozole,
letrozole, and anastrozole are aromatase inhibitors and used
especially for the treatment of breast cancer, which inter-
rupts synthesis of estrogen in the body (Cai et al., 2007; Rao
et al., 2006; Hancu et al., 2007; Bajetti et al., 2000;
Demirbas et al., 2007).
Pyridine known as the most ubiquitous heterocyclic
compounds in nature (e.g., in the coenzyme vitamin B6
family and in numerous alkaloids) plays a major role as
versatile building block in the synthesis of natural products
as well as biologically active compounds. Pyridine bases
have been widely used in pharmaceuticals as nicotinamides
and nicotinic acid derivatives, especially; pyridine-3-
carboxylic acid derivatives are very useful as anti-
microbial, fungicidal, antitubercular agricultural, and
industrial chemicals (Patel et al., 2013).
Another class of heterocyclic bioactive compounds is
1,3,4-oxadiazoles, the widespread use of which is a
scaffold in medicinal chemistry makes this moiety as a
member of the privileged structures (Rane et al., 2013).
Differently substituted 1,3,4-oxadiazoles have been found
to exhibit anti-inflammatory (Palaska et al., 2002),
hypoglycemic (Ramalingam and Sattur, 1990), antianxiety
(Harfenist et al., 1996), antidepressant (Ergun et al.,
2010), antiproliferative (Jin et al., 2006), antifungal
(Liu et al., 2008; Chen et al., 2007), antibacterial (Xu
et al., 2012), and antitubercular activities (Ahsan et al.,
2011).
The classical Mannich reaction, a three-component
condensation between structurally diverse substrates con-
taining at least one active hydrogen atom (ketones,
nitroalkanes, b-ketoesters, and b-cyanoacids), an aldehyde
component (generally formaldehyde) and an amine reagent
leads to the formation of aminoalkylated compounds named
Results and discussion
Chemistry
The main aim of the present study is to synthesize and
investigate the antimicrobial and antiurease activities of
some new nicotinic acid hydrazide derivatives also con-
taining 1,2,4-triazole, 1,3,4-oxadiazole, penicillanic acid,
morpholine thiomorpholine, piperazine, or pyrimidine
nucleus in one molecular framework. Synthesis of the
intermediate and target compounds was performed accord-
ing to the reactions outlined in Schemes 1, 2, and 3. The
starting compound of nicotinic acid hydrazide (1) was
provided commercially.
In the present study, compound 2 was obtained from the
cyclocondensation of nicotinic hydrazide (1) with carbon-
disulfide in the presence of potassium hydroxide with the
aim to introduce a 1,3,4-oxadiazole nucleus to the cepha-
losporanic acid skeleton. Then the treatment of 2 with
hydrazine hydrate generated the corresponding 1,2,4-tria-
zole compound (4). Both the compounds including 1,3,4-
oxadiazole or 1,2,4-triazole nucleus synthesized in the
present study (2 and 4) are useful intermediates for joining
different bioactive molecular frameworks with each other

Med Chem Res
O
Scheme 1 Synthetic pathway
for the preparation of
compounds 2 and 3a–c
N
NHNH2
1
KOH / CS2
12 h, reflux
N NH
N
S
O
2
O
NH
S
NH
F
N
NH
HCHO, THF, 4 h, rt
HCHO, THF, 4 h, rt
HCHO, THF, 4 h, rt
N
O
N
S
N
N
N
N
F
N
N
N
N
N
S
O
N
S
O
N
S
O
3a
3b
3c
by Mannich reaction, beside their importance as pharma-
cophores. Compounds 2 and 4 displayed spectral data
consistent with the assigned structures.
signals derived from amine moiety were observed at related
chemical shift values. Moreover these compounds (3a–c,
5a–e, 7a, b, and 8a, b) exhibited mass spectral data con-
sistent with the assigned structures.
The synthesis of Schiff bases (6a–d) was performed from
the reaction of compound 4 with several aldehydes namely
2-hydroxybenzaldehyde, 3-hydroxy-4-methoxybenzaldehyde,
4-methoxybenzaldehyde, and indol-3-carbaldehyde, respec-
tively. This idea originated from the fact that several com-
pounds with imine bond were reported as antimicrobial and/
or antitumoral agents (Bayrak et al., 2009a; Bayrak et al.,
2009b; Demirbas et al., 2002; Demirbas et al., 2004;
Demirbas et al., 2005). Moreover, some Mannich bases
possessing antimicrobial activity were synthesized by using
1,2,4-triazole-Schiff bases and methyl piperazine or
morpholine (Bayrak et al., 2009a; Bayrak et al., 2009b;
Ashok et al., 2007). In addition, the presence of a phenyl
ring in the structure of 1,2,4-triazoles is important to
increase the lipophilicity of the molecule, because it is well
known that the lipophilic character in a bioactive molecule
facilitates the penetration of it into the cell. Another factor
to facilitate penetration is the presence of a heteroatom that
can do H-bonding with the targets in the bacterial cell
(Basoglu et al., 2013; Kucukguzel et al., 2002; Eftekhari-Sis
et al., 2006).
Biological activity
Antimicrobial activity
All the newly synthesized compounds were screened for
their antimicrobial activities and the obtained results were
presented in Table 1. According to the results obtained, all
compounds except 6a–d and 8a, b displayed activity
against the test microorganisms with the mic values varying
15.6–500 µg/mL.
Compound 2, that is, a 1,3,4-oxadiazole derivative
exhibited moderate activity selectively toward to Candida
albicans (Ca), Saccharomyces cerevisiae (Sc), which are
yeast like fungi and was found to have slight activity toward
Escherichia coli (Ec), enteric bacteria, Yersinia pseudotu-
berculosis (Yp), which are Gram positive cocci, Bacillus
cereus (Bc) that is Gram positive spore bacillus. Mannich
bases, 3a–c, which were obtained from the reaction com-
pound 2 with morpholine, thiomorpholine, 1-(4-fluor-
ophenyl) piperazine displayed excellent antibacterial
activities against all of the test microorganisms with the
MIC values of 15.6 or 31.3 mg/mL. Nevertheless, when
compound 2 were converted to the corresponding 1,2,4-
triazole derivative 4, the antibacterial activities significantly
decreased, even activities disappeared on most of the test
microorganisms.
The amino alkylation of 1,3,4-oxadiazole (3) and 1,2,4-
triazoles (4, 6a–d) with several amines namely morpholine,
thiomorpholine, 1-(4-fluorophenyl)piperazine, 6-aminopenicil-
lanic acid, and 5,6-dimethylpyrazin-2-amine at room tem-
perature in the presence of formaldehyde afforded the
corresponding Mannich bases, 3a–c, 5a–e, 7a, b, and 8a, b
1
in good yields. In the H and 13C nuclear magnetic reso-
nance spectra of the obtained Mannich bases, additional

Med Chem Res
O
OH
O
N
CH3
CH3
S
NH
N
N
N
S
N
NH2
5e
NH
2
S
H3C
H3C
O
HCHO, THF, 4 h, rt
N
OH
CH3
O
H3C
N
N
N
NH
2
N NH
N NH
NH
N
CH3
H3C
N
N
6 h, reflux
+ NH2NH2.H2O
N
S
N
S
O
N
HCHO, THF, 4 h, rt
N
S
N
NH2
2
NH2
4
5d
O
NH
S
NH
F
N
NH
HCHO, THF, 4 h, rt
N
HCHO, THF, 4 h, rt
HCHO, THF, 4 h, rt
N
O
N
S
N
N
N
F
N
N
N
N
N
S
N
N
S
N
N
S
N
NH2
NH2
NH2
5a
5b
5c
Scheme 2 Synthetic pathway for the preparation of compounds 4 and 5a–e
Compound 5a, that is, a 1,2,4-triazole derivative con-
taining a morpholine moiety exhibited moderate anti-
bacterial activity only against Ec and Yp with the MIC
probably due to the presence of morpholine or thiomor-
pholine ring.
values of 62.5 mg/mL. However, Mannich compounds 5b–e
which contain a thiomorpholine, 1-(4-fluorophenyl) piper-
azine, 2-amino-4,6-dimethyl pyrimidine, 6-aminopenicillanic
acid moiety in the 1,2,4-triazole skeleton, demonstrated
excellent activities on the test microorganisms except
Ca and Sc. Moreover, as seen in Table 1, compounds
5b–e have better activity than the standard drug
Ampicillin.
4-Amino-5-pyridine-3-yl-2,4-dihydro-3H-1,2,4-triazole-
3-thione (4) were converted to the corresponding Schiff
bases, 6a–d, which were found to be inactive toward all
the test microorganisms. On the other hand, compounds 7a
and 7b, which were obtained from the condensation of 6c
with several amines in the presence of formaldehyde,
exhibited good activity on most of the test microorganisms
Anti-urease activity
Moreover, the synthesized compounds were assayed for
their in vitro inhibitory activity against Jack bean urease.
Seven of those compounds showed excellent urease inhi-
bition. Thiourea with IC50 value 39.5 7.0 µg/mL was used
as standard inhibitor. Potent compounds have their IC50
values in the range of 36.7 µg/mL to 101.2 µg/mL (Table 2).
Among investigated compounds 3c which is 1,3,4-oxadia-
zole derivatives including also 1-(4-fluorophenyl)piperazine
nucleus, was found to have the best inhibitory effect against
urease with an IC50 value of 36.7 12.5 µg/mL. Dose
dependent inhibitory effects of compounds were depicted in
Fig. 1. These compounds might be considered as potential
antibiotics to treat infections.

Med Chem Res
Scheme 3 Synthetic pathway
for the preparation of
compounds 6a–d, 7a, b, and
8a, b
N NH
N NH
N
S
N
N
N
S
N
N
O
OH
OCH
3
H
HO
OH
O
OH
OCH3
6 h, reflux
H
N NH
N
6b
6a
6 h, reflux
O
N
S
O
H
NH2
OCH3
H
4
N
H
6 h, reflux
6 h, reflux
N NH
N NH
N
S
N
N
N
S
N
N
N
H
OCH3
6d
6c
HCHO, THF, 4 h, rt
X
NH
HCHO, THF, 4 h, rt
X
NH
N
X
N
X
N
N
N
N
N
S
N
S
N
N
N
N
N
H
OCH3
8a-b
7a-b
8a: X= O
8b: X= S
7a: X= O
7b: X= S
Anti-pancreatic lipase activity
98.6 % at a concentration of 6.25 µg/mL, respectively
(Table 3). Orlistat, a known pancreatic lipase inhibitor
used as antiobesity drug, showed an inhibitory effect by
99.1 % at the same concentration. IC50 values for com-
pounds 5b and 5c were calculated as 0.42 0.03 and 0.27
0.04 µg/mL, respectively (Fig. 2). Orlistat is the only
approved antiobesity medication (Jandacek and Woods,
2004) but it has some side effects, such as fecal incon-
tinence, flatulence, and steatorrhea (Birari and Bhutani,
2007; Weigle, 2003). The synthesized compounds 5b and
5c have a significant potential to become alternatives of
Orlistat.
All compounds were evaluated with regard to pancreatic
lipase activity and 5b and 5c, which contain a phenylpi-
perazine or thiomorpholine nucleus as different from the
other compounds 5, showed anti-lipase activities at various
concentrations (Table 3). No significant inhibitory effect
was detected for the other compounds. Among the tested
compounds, 5b and 5c, which are 1,2,4-triazole derivatives
including also thiomorpholine or 1-(4-fluorophenyl) piper-
azine, nucleus, showed the best anti-lipase activity. These
compounds inhibited pancreatic lipase activity by 98.2 and

Med Chem Res
Table 1 Antimicrobial activity
of the compounds (μg/mL)
Compound no.
Minimal inhibition concentration values (µg/mL)
Ec
Yp
Pa
Sa
Ef
Bc
Ms
Ca
Sc
3a
31.3
31.3
31.3
62.5
31.3
31.3
31.3
15.6
62.5
62.5
2
31.3
31.3
31.3
62.5
31.3
31.3
31.3
15.6
62.5
62.5
32
31.3
31.3
31.3
—
31.3
31.3
31.3
—
15.6
15.6
15.6
—
31.3
31.3
31.3
500
62.5
62.5
31.3
31.3
250
250
< 1
31.3
31.3
62.5
—
125
62.5
62.5
500
—
62,5
31.3
31.3
250
—
3b
3c
5a
5b
62.5
62.5
31.3
62.5
62.5
62.5
>128
—
62.5
62.5
15.6
31.3
500
500
2
15.6
15.6
15.6
31.3
—
62.5
62.5
62.5
62.5
62.5
62.5
—
5c
—
—
5d
—
—
5e
—
—
7a
—
500
500
—
7b
—
—
Amp.
Strep.
Flu.
2
—
—
—
—
—
—
4
—
—
—
—
—
—
—
—
—
< 8
< 8
Ec E. coli ATCC 35218, Yp Y. pseudotuberculosis ATCC 911, Pa P. aeruginosa ATCC 10145, Sa S. aureus
ATCC 25923, Ef E. faecalis ATCC 29212, Bc B. cereus 709 Roma, Ms M. smegmatis ATCC607, Ca C.
albicans ATCC 60193, S. cerevisiae RSKK 251, Amp. Ampicillin, Strep. Streptomycin, Flu. Fluconazole,
(—): no activity of test concentrations
Table 2 Inhibitory activities of the synthesized compounds against
urease. All compounds and Thiourea were assayed at final
concentration of 83 µg/mL
3b
3c
100
80
60
40
20
0
5b
Thiourea
Compound
% Inhibition
IC50 (µg/mL)
3a
73.9 0.0
83.0 1.7
92.1 7.9
85.1 8.7
40.5 4.0
38.5 7.4
69.5 1.5
100.0 0.0
59.3 5.2
41.7 2.3
36.7 12.5
40.2 6.8
96.5 12.0
101.2 18.1
50.6 3.6
39.5 7.0
3b
3c
5b
5c
7a
7b
1
10
100
Thiourea
Concentration (µg/mL)
Fig. 1 Dose-dependent inhibitory effect of some synthesized com-
pounds. Thiourea was used as standard inhibitor. Inhibitory effect of
all compounds and Thiourea were measured at the range of 250–0.114
µg/mL concentrations. Residual activities of compounds are expressed
as the mean S.D. in triplicate
Conclusion
This study reports synthesis of some new hybrid molecules
containing morpholine or penicillanic acid moieties with
some other pharmacophore heterocycles in a single struc-
ture. Hence herein we combined all these potential
chemotherapeutic units, namely 1,2,4-triazole, 1,3,4-
oxadiazole, thiomorpholine, penicillanic acid, moieties.
Their structures were confirmed by Infrared, 1H NMR,
13C NMR, Mass spectroscopic, and elemental analysis
techniques. The antimicrobial, antiurease, and antilipase
screening studies were also performed in the study.
or penicillanic acid moiety, displayed good-moderate
activity on some of the test microorganisms. The excellent
antimicrobial activity was observed for compounds (3a–c),
which are Mannich bases containing 1,2,4-triazole nucleus.
Moreover, seven of those compounds (3a–c, 5b, 5c, 7a,
7b) showed excellent urease inhibition. Among investigated
compounds 3-{[4-(4-fluorophenyl) piperazin-1-yl]methyl}-
5-pyridin-3-yl-1,3,4-oxadiazole-2(3H)-thione 3c, which is
1,3,4-oxadiazole derivatives including also 1-(4-fluor-
ophenyl) piperazine nucleus, was found to be the best
Among the synthesized compounds, the compounds
containing 1,2,4-triazole, morpholine, thiomorpholine, 1-(4-
fluorophenyl) piperazine, 2-amino-4,6-dimethyl pyrimidine,

Med Chem Res
Table 3 Residual lipase activity of synthesized compounds. All
compounds were screened at concentration of 6.25 µg/mL
120
100
80
60
40
20
0
5b
5c
Orlistat
Compound
Inhibition %
IC50 (µg/mL)
3a
34.9 3.8
32.5 0.1
29.4 5.2
35.2 0.5
98.2 1.2
98.6 0.8
30.5 2.0
62.2 1.4
35.7 10.9
99.1 0.2
—
3c
—
4
—
5a
—
5b
0.42 0.03
0.27 0.04
—
5c
6b
0.1
1
10
6d
—
Concentration (µg/mL)
7b
—
Orlistat
0.42 0.01
Fig. 2 Dose-dependent inhibitory effect of synthesized compounds.
Orlistat was used positive control. All compounds and Orlistat were
measured at final concentrations of 0.037 to 21 µg/mL. Residual
activities of compounds are expressed as the mean S.D. in triplicate
inhibitory effect against urease with an IC50 value of 36.7
12.5 µg/mL.
Furthermore, compounds 5b and 5c which are 1,2,4-
triazole derivatives including also thiomorpholine or 1-(4-
fluorophenyl) piperazine, nucleus, showed the best anti-
lipase activities at final concentration of 6.25 µg/mL.
According to the results obtained it can be concluded that
the conversion of 1,3,4-oxadiazole or 1,2,4-triazole to the
derivatives which contain a 1-(4-fluorophenyl) piperazine
(3c and 5c) or thiomorpholine (5b) resulted in complete
activation of these compounds against the test micro-
organisms. In other words, compounds 3c, 5b, and 5c exi-
hibited the best activity in terms of antimicrobial, antilipase,
and anti-urease activities.
The chemical shifts are given in part per million relative to
Me4Si as an internal reference, J values are given in Hz. The
elemental analysis was performed on a Costech Elemental
Combustion System CHNS-O elemental analyzer. All the
compounds gave C, H, and N analysis within 0.4 % of the
theoretical values. The Mass spectra were obtained on a
Quattro LC-MS (70 eV) ınstrument.
5-(Pyridine-3-yl)-1,3,4-oxadiazole-2(3H)thione (2) (Xu
et al., 2011) Nicotinic acid hydrazide (1) (1.37 g, 0.01
mol) and CS2 (1.21 mL, 0.02 mol) were added to a solution
of KOH (0.56 g, 0.01 mol) in 50 mL H2O and 50 mL
butanol. The reaction mixture was refluxed for 12 h. Then,
the resulting solution was cooled to room temperature and
acidified to pH 5 with 37 % HCl. The precipitate was fil-
tered off, washed with water and recrystallized to afford the
desired compound 2. Recrystallization from EtOH to give
Experimental
Chemistry
white solid; yield 82 %; m.p. 230–231 °C; IR (KBr) νmax
:
3055, 1531, 1208, 1155 cm−1; H NMR (DMSO-d6): δ =
7.58–7.64 (m, 1H, arH), 8.24 (d, 1H, arH, J = 8.2 Hz), 8.78
(d, 1H, arH, J = 8.2 Hz), 9.03 (s, 1H, arH), 10.03 (s, 1H,
NH); 13C NMR (DMSO-d6): δ = 119.98, 125.02, 134.50,
147.45, 153.25 (aromatic carbons), 159.54 (oxadiazole
C-5), 169.46 (oxadiazole C-2); EI MS m/z (%): 229.29 (81),
217.21(50), 214.14 (38), 203.13 ([M+1+Na]+, 100), 189.11
(44), 177.03 (44), 134.98 (31); Anal. calcd. for C7H5N3OS:
C, 46.92; H, 2.81; N, 23.45 %. Found: C, 46.62; H, 2.50; N,
23.31 %.
1
General information for chemicals
All the chemicals were purchased from Fluka Chemie AG
Buchs (Switzerland) and used without further purification.
Melting points of the synthesized compounds were deter-
mined in open capillaries on a Büchi B-540 melting point
apparatus and are uncorrected. Reactions were monitored
by thin-layer chromatography (TLC) on silica gel 60 F254
aluminium sheets. The mobile phase was ethyl acetate:
diethyl ether (1:1) and detection was made using ultraviolet
light. Fourier transform infrared (FTIR) spectra were
recorded as potassium bromide pellets using a Perkin-Elmer
General synthesis method for compounds (3a–c) To a
solution of corresponding compound 2 (1.79 g, 0.01 mol) in
tetrahydrofuran, morpholine (for 3a) (0.87 mL, 0.01 mol),
thiomorpholine (for 3b) (0.94 mL, 0.01 mol), or 1-(4-
fluorophenyl) piperazine (1.80 g, 0.01 mol) (for 3c) was
added in the presence of formaldehyde (37 %, 3.72 mL,
1
1600 series FTIR spectrometer. H NMR and 13C NMR
spectra were registered in dimethyl sulfoxide (DMSO)-d6
on a BRUKER AVENE II 400 MHz NMR Spectrometer
(400.13 MHz for 1H and 100.62 MHz for 13C).

Med Chem Res
0.05 mol) and the mixture was stirred at room temperature
for 4 h. After evaporating the solvent under reduced pres-
sure, a solid appeared.
The resulting solid separated was collected by filtration and
recrystallized to afford the desired product 4. Recrystalli-
zation from EtOH to give cream solid; yield: 87 %; m.p.
145–146 °C; IR (KBr) νmax: 3437, 3233, 1565, 1208 cm−1;
1H NMR (DMSO-d6): δ = 2.63 (brs, 4H, N-2CH2), 3.04
(brs, 4H, S-2CH2), 5.04 (s, 2H, CH2), 7.64 (d, 1H, arH, J =
5.0 Hz), 8.26 (d, 1H, arH, J = 7.8 Hz), 8.80 (s, 1H, arH),
9.04 (s, 1H, arH). 13C NMR (DMSO-d6): δ = 27, 82 (N-
2CH2), 52, 56 (S-2CH2), 72, 16 (CH2), 119.90, 125.04,
134.59, 147.54, 153.35 (aromatic carbons), 157.75 (oxa-
diazole C-5), 178.18 (oxadiazole C-2); EI MS m/z (%):
314.21 ([M+2+H2O]+, 14), 312.08 ([M+H2O]+, 12), 296.23
([M+2]+, 279.18 (100); anal. calcd. for C12H14N4OS2: C,
48.96; H, 4.79; N, 19.03 %. Found: C, 48.81; H, 4.68; N,
19.31 %.
3-(Morpholin-4-ylmethyl)-5-pyridin-3-yl-1,3,4-oxadiazole-
2(3H)-thione (3a) Recrystallization from EtOH to give
white solid; yield 83 %; m.p. 180–182 °C. IR (KBr) νmax
:
1454, 1212, 1101 cm−1; 1H NMR (DMSO-d6): δ = 4.55
(8H, d, 4CH2+H2O), 5.41 (2H, s, CH2), 7.72–7.77 (1H, m,
arH), 8.39 (1H, d, J = 7.4 Hz, arH), 8.84 (1H, s, arH), 9.11
(1H, s, arH); 13C NMR (DMSO-d6): δ = 43.43 (N-2CH2),
63.93 (O-2CH2), 72.33 (CH2), 120.16, 125.74, 136.15,
146.41, 152.18 (aromatic carbons), 157.29 (oxadiazole
C-5), 176.69 (oxadiazole C-2); EI MS m/z (%): 305.35
(100), 205.28 (78); anal. calcd. for C12H14N4O2S: C, 51.78;
H, 5.07; N, 20.13 %. Found: C, 51.40; H, 5.35; N, 20.31 %.
General method for the synthesis of compounds 5a–e To a
solution of corresponding compound 4 (1.92 g, 0.01 mol) in
tetrahydrofuran, morpholine (for 5a) (0.87 mL, 0.01 mol),
thiomorpholine (for 5b) (0.94 mL, 0.01 mol), 1-(4-fluor-
ophenyl) piperazine (for 5c) (1.80 g, 0.01 mol), 2-amino-
4,6-dimethyl pyrimidine (for 5d) (1.23 g, 0.01 mol), or 6-
aminopenicillanic acid (for 5e) (2.16 g, 0.01 mol) was added
in the presence of formaldehyde (37 %, 3.72 mL, 0.05 mol)
and the mixture was stirred at room temperature for 4 h.
After evaporating the solvent under reduced pressure, a
solid appeared.
5-Pyridin-3-yl-3-(thiomorpholin-ylmethyl)-1,3,4-oxadia-
zole-2(3H)-thione (3b) Recrystallization from EtOH to
give white solid; yield 85 %; m.p. 146–147 °C; IR (KBr)
1
νmax: 1451, 1209, 1138 cm−1; H NMR (DMSO-d6): δ =
2.63 (4H, brs, N-2CH2), 3.04 (4H, brs, S-2CH2), 5.04 (2H,
s, CH2), 7.64 (1H, d, J = 5.0 Hz arH), 8.26 (1H, d, J = 7.8
Hz, arH), 8.80 (1H, s, arH), 9.04 (1H, s, arH). 13C NMR
(DMSO-d6): δ = 27.82 (N-2CH2), 52.56 (S-2CH2), 72.16
(CH2), 119.90, 125.04, 134.59, 147.54, 153.35 (aromatic
carbons), 157.75 (oxadiazole C-5), 178.18 (oxadiazole
C-2); EI MS m/z (%): 314.21 ([M+2+H2O]+, 14), 312.08
([M+H2O]+, 12), 296.23 ([M+2]+, 279.18 (100); anal. calcd.
for C12H14N4OS2: C, 48.96; H, 4.79; N, 19.03 %. Found: C,
48.60; H, 5.05; N, 19.31 %.
4-Amino-2-(morpholin-ylmethyl)-5-pyridin-3-yl-2,4-dihy-
dro-3H-1,2,4-triazole-3-thione (5a) Recrystallization from
dimethyl sulfoxide–water (1:3) to give gray solid; yield 76 %,
m.p. 218–220 °C; IR (KBr) νmax: 3400, 1506, 1214, 1114
3-{[4-(4-Fluorophenyl)piperazin-1-yl]methyl}-5-pyridin-3-yl-
1,3,4-oxadiazole-2(3H)-thione (3c) Recrystallization from
EtOH to give white solid; yield 87 %, m.p. 125–126 °C;
1
cm−1; H NMR (DMSO-d6): δ = 2.07 (4H, s, 2CH2), 3.09
(4H, brs, 2CH2), 5.11 (2H, brs, CH2), 5.57 (2H, brs, NH2),
7.03–7.73 (1H, m, arH), 8.01 (1H, brs, arH), 8.29 (1H, brs,
arH), 8.74 (1H, s, arH); 13C NMR (DMSO-d6): δ = 31.12
(N-2CH2), 58.71 (O-2CH2), 71.84 (CH2), 124.20, 127.85,
133.58, 148.15, 151.24 (aromatic carbons), 154.67 (triazole
C-3), 170.38 (triazole C-5); EI MS m/z (%): 293.33 ([M
+1]+, 19), 279.25 (34), 206.23 (41), 194.03 (53), 114.28
(62), 100.44 (88), 100.19 (100); anal. calcd. for
C12H16N6OS: C, 49.30; H, 5.52; N, 28.75 %. Found: C,
49.18; H, 5.55; N, 28.59 %.
1
IR (KBr) νmax: 1511, 1205, 1168 cm−1; H NMR (DMSO-
d6): δ = 2.92 (4H, brs, N-2CH2), 3.06 (4H, brs, N-2CH2),
5.11 (2H, s, CH2), 6.92–7.05 (4H, m, arH), 7.61 (1H, d, J =
7.0 Hz, arH), 8.26 (1H, d, J = 7.8 Hz, arH), 8.79 (1H, d, J =
3.0 Hz, arH), 9.04 (1H, s, arH); 13C NMR (DMSO-d6): δ =
49.77 (N-2CH2), 50.16 (N-2CH2), 70.52 (CH2), 115.71,
116.14, 118.02, 118.17, 119.79, 125.00, 143.60, 147.57,
148.55, 153.40, 154.43 (aromatic carbons), 157.63 (oxa-
diazole C-5), 178.24 (oxadiazole C-2); EI MS m/z (%):
420.48 (81), 413.41 ([M+1+Na+H2O]+, 19), 398.33 (69),
373.30 ([M+2]+, 69), 372.30 ([M+1]+ , 68), 305.47 (100);
anal. calcd. for C18H18FN5OS: C, 58.21; H, 4.88; N, 18.86
%. Found: C, 58.40; H, 5.05; N, 18.59 %.
4-Amino-5-pyridine-3-yl-2-(thiomorpholin-4-ylmethyl)-
2,4-dihydro-3H-1,2,4-triazole-3-thione (5b) Recrystalliza-
tion from dimethyl sulfoxide–water (1:3) to give gray solid;
yield 77 %; m.p. 169–171 °C; IR (KBr) νmax: 3435, 1505,
1
4-Amino-5-pyridine-3-yl-2,4-dihydro-3H-1,2,4-triazole-3-
thione (4) (Omprakash et al., 2011) A solution of com-
pound 2 (1.79 g, 0.01 mol) in n-butanol was refluxed with
hydrazine hydrate (1.2 mL, 0.025 mol) for 6 h (controlled
with TLC). After cooling the reaction mixture to room
temperature, the mixture was kept overnight in cold.
1208 cm−1; H NMR (DMSO-d6): δ = 3.38–3.57 (8H, m,
4CH2), 5.08 (2H, brs, CH2), 5.51 (2H, brs, NH2), 7.25 (1H,
brs, arH), 7.57 (1H, brs, arH), 8.33 (1H, d, J = 17.2 Hz,
arH), 8.73 (1H, s, arH); 13C NMR (DMSO-d6): δ = 27.15
(N-2CH2), 55.32 (S-2CH2), 70.51 (CH2), 125.33, 128.61,
149.12, 152.10, 155.02 (aromatic carbons), 158.60 (triazole

Med Chem Res
C-3), 174.12 (triazole C-5); EI MS m/z (%): 310.38
([M+2]+, 28), 284.34 (28), 190.25 (30), 169.25 (100), 169.06
(88), 104.15 (88); anal. calcd. for C12H16N6S2: C, 46.73; H,
5.23; N, 27.25 %. Found: C, 46.40; H, 5.05; N, 27.49 %.
(triazole C-3), 163.90 (triazole C-5), 168.71 (C=O), 172.52
(COOH); EI MS m/z (%): 464.21 ([M+2+Na+H2O]+, 100),
454.51 (50), 444.38 ([M+Na]+, 50), 421.23 ([M]+, 38),
384.62 (47), 366.29 (63); anal. calcd. for C16H19N7O3S2: C,
45.59; H, 4.54; N, 23.26 %. Found: C, 45.40; H, 4.35; N,
23.59 %.
4-Amino-2-{[4-(4-fluorophenyl)piperazin-1-yl]methyl}-5-
pyridin-3-yl-2,4-dihydro-3H-1,2,4-triazole-3-thione (5c)
Recrystallization from dimethyl sulfoxide–water (1:3) to
give white solid; yield 81 %; m.p. 202–203 °C; IR (KBr)
General method for the synthesis of compounds 6a–d A
solution of compound 5 (1.92 g, 0.01 mol) in ethanol was
refluxed with salicyl aldehyde (for 6a) (1.22 g, 0.01 mol), 3-
hydroxy-4-methoxy benzaldehyde (for 6b) (1.37 g, 0.01
mol), 4-methoxy benzaldehyde (for 6c) (1.22 mL, 0.01 mol)
or indole-3-carbaldehyde (for 6d) (1.45 g, 0.01 mol) and
one drop H2SO4 for 6 h. Then, the mixture was cooled to
room temperature and left overnight in cold. The formed
solid was filtered and it was recrystallized to afford the
desired products.
1
νmax: 3410, 1508, 1209 cm−1; H NMR (DMSO-d6): δ =
2.07 (4H, s, N-2CH2), 2.53 (4H, brs, N-2CH2), 5.06 (2H,
brs, CH2), 5.45 (2H, brs, NH2), 7.01 (1H, d, J = 9.4 Hz,
arH), 7.26 (1H, brs, arH), 7.56 (1H, brs, arH), 7.72 (1H, brs,
arH), 8.03 (1H, brs, arH), 8.30–8.46 (2H, m, arH), 8.74 (1H,
brs, arH); 13C NMR (DMSO-d6): δ = 49.85 (N-2CH2),
50.62 (N-2CH2), 71.02 (CH2), 113.70, 114.14, 116.01,
118.11, 120.82, 125.50 , 138.48, 146.57, 151.27, 153.40 ,
154.43 (aromatic carbons), 161.63 (triazole C-3), 174.31
(triazole C-5); EI MS m/z (%): 444.63 ([M+Na+K]+, 19),
409.71 ([M+1+Na]+, 19), 387.31 ([M+2]+, 25), 365.29 (31),
135.04 (100), 134.85 (94), 134.04 (72); anal. calcd. for
C18H20FN7S: C, 56.09; H, 5.23; N, 25.44 %. Found: C,
56.40; H, 5.07; N, 25.59 %.
4-{[(2-Hydroxyphenyl)methylene]amino}-5-pyridin-3-yl-
2,4-dihydro-3H-1,2,4-triazole-3-thione (6a) Recrystalliza-
tion from dimethyl sulfoxide–water (1:3) to give yellow
solid; yield 92 %; m.p. 227–228 °C; IR (KBr) νmax: 3025,
2636, 1512, 1208 cm−1; 1H NMR (DMSO-d6): δ =
6.92–7.01 (2H, m, arH), 7.49 (2H, d, J = 6.8 Hz, arH), 7.83
(1H, d, J = 6.8 Hz, arH), 8.25 (1H, d, J = 7.0 Hz, arH), 8.70
(1H, brs, arH), 9.04 (1H, brs, arH), 10.04 (1H, brs, N=CH),
10.51 (1H, brs, NH), OH was not observed; 13C NMR
(DMSO-d6): δ = 117.40 (CH, N=CH), 118.90, 120.40,
122.78, 124.36, 127.73, 135.23, 136.63, 147.49, 149.44,
151.86, 163.39 (aromatic carbons), 159.30 (triazole C-3),
163.07 (triazole C-5); EI MS m/z (%): 298.30 ([M+1]+,
100), 179.18 (22), 104.28 (25); anal. calcd. for
C14H11N5OS: C, 56.55; H, 3.73; N, 23.55 %. Found: C,
56.40; H, 3.43; N, 23.59 %.
4-Amino-2-{[(4,6-dimethylpyrimidin-2-yl)amino]methyl}-
5-pyridin-3-yl-2,4-dihydro-3H-1,2,4-triazole-3-thione (5d)
Recrystallization from dimethyl sulfoxide–water (1:3) to
give white solid; yield 78 %; m.p. 177–178 °C. IR (KBr)
1
νmax: 3437, 3290, 1586, 1213 cm−1; H NMR (DMSO-d6):
δ = 2.06 (6H, s, 2CH3), 5.48 (2H, d, J = 7.4 Hz, CH2), 5.81
(1H, s, NH), 5.90 (1H, s, NH2), 7.07 (1H, t, J = 7.4 Hz,
arH), 7.59 (1H, t, J = 4.8 Hz, arH), 8.37 (1H, d, J = 7.8 Hz,
arH), 8.73 (1H, d, J = 3.2 Hz, arH), 9.15 (1H, s, arH); 13C
NMR (DMSO-d6): δ = 31.41 (2CH3), 71.80 (CH2), 122.42,
123.79, 124.34, 136.55, 149.21, 149.34, 152.00, 167.42
(aromatic carbons), 168.10 (triazole C-3), 168.95 (triazole
C-5); EI MS m/z (%): 326.12 ([M-2]+, 12), 271.19 (16).
Anal. calcd. for C14H16N8S: C, 51.20; H, 4.91; N, 34.12 %.
Found: C, 51.40; H, 4.65; N, 34.33 %.
4-{[(3-Hydroxy-4-methoxyphenyl)methylene]amino}-5-
pyridin-3-yl-2,4-dihydro-3H-1,2,4-triazole-3-thione (6b)
Recrystallization from dimethyl sulfoxide–water (1:3) to
give white solid; yield 88 %; m.p. 231–232 °C; IR (KBr)
1
νmax: 3100, 2938, 1501, 1201 cm−1; H NMR (DMSO-d6):
δ = 3.84 (3H, s, OCH3), 7.06 (1H, d, J = 8.2 Hz, arH),
7.19–7.35 (2H, m, arH), 7.53–7.59 (1H, m, arH), 8.23 (1H,
d, J = 7.0 Hz, arH), 8.72 (1H, s, arH), 9.07 (1H, s, arH),
9.48 (1H, s, N=CH), 9.61 (1H, s, NH), OH was not
observed; 13C NMR (DMSO-d6): δ = 112.58 (N=CH),
113.65, 122.76, 124.04, 124.42, 125.04, 136.47, 147.23,
147.77, 149.28, 151.88, 167.63 (aromatic carbons), 152.92
(triazole C-3), 163.13 (triazole C-5); EI MS m/z (%): 358.36
(100), 339.31 (21), 329.31 ([M+Na]+, 19), 328.30 ([M+1]+,
81); anal. calcd. for C15H13N5O2S: C, 55.03; H, 4.00; N,
21.39 %. Found: C, 55.40; H, 4.05; N, 21.11 %.
7-{[(4-Amino-3-pyridin-3-yl-5-thioxo-4,5-dihydro-1H-
1,2,4-triazol-1-yl)methyl]amino}-3,3-dimethyl-6-oxo-2-
thiabicyclo[3.2.0]heptane-4-carboxylic acid (5e) Recrys-
tallization from dimethyl sulfoxide–water (1:3) to give
yellow solid; yield 71 %; m.p. 183–185 °C; IR (KBr) νmax
:
3429, 3274, 1771, 1418, 1208 cm−1; 1H NMR (DMSO-d6):
δ = 1.73 (3H, s, CH3), 2.39 (3H, s, CH3), 4.52 (1H, s, CH),
4.75 (1H, s, CH), 5.25 (1H, s, CH), 5.77 (2H, d, J = 4.0 Hz,
CH2), 5.97 (2H, s, NH2), 7.01 (1H, s, J = 3.2 Hz, arH), 7.72
(1H, brs, arH), 8.73 (1H, d, J = 7.8 Hz, arH), 9.15 (1H, s,
arH), 10.05 (1H, s, NH), 11.89 (1H, brs, OH); 13C NMR
(DMSO-d6): δ = 28.53 (CH3), 33.11 (CH3), 60.02 (C),
64.91 (CH2), 66.01 (CH), 68.71 (CH), 72.20 (CH), 121.17,
124.82, 137.11, 149.58, 153.05 (aromatic carbons), 158.54
4-{[(4-Methoxyphenyl)methylene]amino}-5-pyridin-3-yl-
2,4-dihydro-3H-1,2,4-triazole-3-thione (6c) Recrystalliza-
tion from dimethyl sulfoxide–water (1:3) to give cream

Med Chem Res
solid; yield 86 %; m.p. 224–225 °C; IR (KBr) νmax: 3120,
1567, 1210 cm−1; 1H NMR (DMSO-d6): δ = 3.84 (3H, s, O-
CH3), 7.11 (2H, d, J = 8.6 Hz, arH), 7.56 (1H, brs, arH),
7.85 (2H, d, J = 8.2 Hz, arH), 8.25 (1H, d, J = 8.2 Hz, arH),
8.70 (1H, s, arH), 9.02 (1H, s, arH), 9.58 (1H, s, N=CH),
10.21 (1H, s, NH); 13C NMR (DMSO-d6): δ = 56.28 (CH3),
115.48 (N=CH), 122.73, 124.43, 124.91, 131.52, 136.48,
147.26, 149.30, 151.92, 167.35 (aromatic carbons), 163.15
(triazole C-3), 163.80 (triazole C-5). EI MS m/z (%): 313.14
([M+2]+, 22), 312.20 ([M+1]+, 100), 280.38 (34), 179.31
(50). Anal. calcd. for C15H13N5OS: C, 57.86; H, 4.21; N,
22.49 %. Found: C, 57.71; H; 4.05; N, 22.71 %.
58.22; H, 5.40; N, 20.47 %. Found: C, 58.40; H, 5.05; N,
20.59 %.
4-{[(4-Methoxyphenyl)methylene]amino}-5-pyridin-3-yl-
2-(thiomorpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-tria-
zole-3-thione (7b) Recrystallization from ethonol–water
(1:3) to give white solid; yield 82 %; m.p. 124–125 °C; IR
1
(KBr) νmax: 1512, 1201 cm−1; H NMR (DMSO-d6): δ =
2.62 (brs, 4H, N-2CH2), 3.06 (brs, 4H, S-2CH2), 3.84
(s, 3H, CH3), 5.19 (s, 2H, CH2), 7.11 (d, 2H, arH, J = 7.0
Hz), 7.60 (d, 1H, arH, J = 5.8 Hz), 7.86 (d, 2H, arH, J = 7.8
Hz), 8.26 (d, 1H, arH, J = 6.6 Hz), 8.73 (s, 1H, arH), 9.04
(s, 1H, arH), 9.52 (s, 1H, N=CH); 13C NMR (DMSO-d6): δ
= 27.86 (N-2CH2), 52.98 (S-2CH2), 56.30 (CH3), 71.26
(CH2), 115.50 (CH, N=CH), 122.37, 124.49, 124.80,
131.64, 136.69, 146.04, 149.45, 152.12, 168.13, 168.38
(aromatic carbons), 162.92 (triazol C-3), 163.91 (triazol
C-5); EI MS m/z (%): 427.17 ([M+1]+, 12), 313.30 (19),
312.23 (100), 179.09 (51), 115.95 (16). Anal. calcd. for
C20H22N6OS2: C, 56.31; H, 5.20; N, 19.70 %. Found: C,
56.51; H, 4.89; N, 19.89 %.
4-{[(1H-indol-3-ylmethylene]amino}-5-pyridin-3-yl-2,4-
dihydro-3H-1,2,4-triazole-3-thione (6d) Recrystallization
from dimethyl sulfoxide–water (1:3) to give yellow solid;
yield 83 %; m.p. 285–286 °C; IR (KBr) νmax: 3190, 1578,
1
1215 cm−1; H NMR (DMSO-d6): δ = 7.21 (4H, d, J = 6.8
Hz, arH), 7.49 (1H, d, J = 6.8 Hz, arH), 7.93 (1H, s, arH),
8.37 (1H, d, J = 6.2 Hz, arH), 8.93 (1H, s, N=CH), 11.71
(1H, s, indole-NH), NH was not observed; 13C NMR
(DMSO-d6): δ = 112.65 (N=CH), 112.81, 122.26, 122.84,
123.35, 125.43, 132.56, 137.91, 155.84 (aromatic carbons),
150.08 (triazole C-3), 163.27 (triazole C-5); EI MS m/z (%):
360.57 ([M+1+K]+, 30), 359.50 ([M+K]+, 31), 343.58 ([M
+Na]+, 16), 341.38 (31), 339.37 ([M+1+H2O]+, 31), 327.30
(63), 323.33 (100); anal. calcd. for C16H12N6S: C, 59.98; H,
3.78; N, 26.23 %. Found: C, 59.76; H, 3.45; N, 26.01 %.
General method for the synthesis of compounds 8a and
8b To a solution of corresponding compound 6d (3.20 g,
0.01 mol) in tetrahydrofuran, morpholine (for 8a) (0.87 mL,
0.01 mol), or thiomorpholine (for 8b) (0.94 mL, 0.01 mol
was added in the presence of formaldehyde (37 %,
3.72 mL, 0.05 mol) and the mixture was stirred at room
temperature for 4 h. After evaporating the solvent under
reduced pressure, a solid appeared. The crude product was
recrystallized from ethanol (for 8a) or from dimethyl
sulfoxide–water (1:3) (for 8b) to yield the target
compounds.
General method for the synthesis of compounds 7a and
7b To a solution of corresponding compound 15 (3.11 g,
0.01 mol) in tetrahydrofuran, morpholine (for 7a) (0.87 mL,
0.01 mol), or thiomorpholine (for 7b) (0.94 mL, 0.01 mol
was added in the presence of formaldehyde (37 %, 3.72 mL,
0.05 mol) and the mixture was stirred at room temperature
for 4 h. After evaporating the solvent under reduced pres-
sure, a solid appeared.
4-{[1H-Indol-3-ylmethylene]amino}-2-(morpholin-4-ylme-
thyl)-5-pyridin-3-yl-2,4-dihydro-3H-1,2,4-triazole-3-thione
(8a) Recrystallization from EtOH to give yellow solid;
yield 74 %; m.p. 195–197 °C. IR (KBr) νmax: 3106, 1523,
1
1208, 1127 cm−1; H NMR (DMSO-d6): δ = 2.49 (s, 4H,
4-{[(4-Methoxyphenyl)methylene]amino}-2-(morpholin-4-
ylmethyl)-5-pyridin-3-yl-2,4-dihydro-3H-1,2,4-triazole-3-
thione (7a) Recrystallization from ethonol–water (1:3) to
give white solid; yield 84 %; m.p. 150–151 °C; IR (KBr)
N-2CH2), 3.55 (brs, 4H, O-2CH2), 5.57 (s, 2H, CH2), 7.26
(brs, 3H, arH), 7.62–7.72 (m, 2H, arH), 7.98 (s, 2H, arH),
8.36 (d, 2H, arH, J = 7 Hz), 8.91 (s, 1H, N=CH), 11.08 (s,
1H, NH); 13C NMR (DMSO-d6): δ = 44.45 (N-2CH2), 64.91
(O-2CH2), 70.17 (CH2), 115.11 (CH, N=CH), 118.11,
119.98, 121.53, 123.74, 126.80, 130.41, 136.18, 140.59,
151.57, 154.20 (aromatic carbons), 161.81 (triazol C-3),
163.91 (triazol C-5); EI MS m/z (%): 485.36 (38), 457.58
([M-1+K]+, 16), 439.37 ([M+2+H2O]+, 20), 417.28 ([M-2]+,
38), 416.35 (100); anal. calcd. for C21H21N7OS: C, 60.12;
H, 5.05; N, 23.27 %. Found: C, 60.48; H, 5.35; N, 23.58 %.
1
νmax: 1513, 1200, 1089 cm−1; H NMR (DMSO-d6): δ =
2.77 (s, 4H, N-2CH2), 3.58 (s, 4H, O-2CH2), 3.85 (s, 3H,
CH3), 5.18 (s, 2H, CH2), 7.11 (d, 2H, arH, J = 8.2 Hz), 7.59
(brs, 1H, arH), 7.87 (d, 2H, arH, J = 8.6 Hz), 8.26 (d, 1H,
arH, J = 7.8 Hz), 8.72 (s, 1H, arH), 9.04 (s, 1H, arH), 9.53
(s, 1H, N=CH). 13C NMR (DMSO-d6): δ = 50.98
(N-2CH2), 56.31 (CH3), 66.80 (O-2CH2), 69.92 (CH2),
115.52 (CH, N = CH), 122.39, 124.48, 124.82, 131.64,
136.75, 146.07, 149.49, 152.13, 168.37 (aromatic carbons),
163.93 (triazol C-3), 164.11 (triazol C-5); EI MS m/z (%):
412.34 ([M+2]+, 25), 411.34 ([M+1]+, 100), 312.23 (56),
179.02 (50), 128.22 (53); anal. calcd. for C20H22N6O2S: C,
4-{[1H-Indol-3-ylmethylene]amino}-5-pyridin-3-yl-2-(thio-
morpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
(8b) Recrystallization from dimethyl sulfoxide–water
(1:3) to give yellow solid; yield 71 %, m.p. 175–177 °C.

Med Chem Res
IR (KBr) νmax: 3233, 1522, 1207 cm−1; H NMR (DMSO-
d6): δ = 2.59 (brs, 4H, N-2CH2+ DMSO-d6), 3.44 (brs, 4H,
S-2CH2+H2O), 5.62 (brs, 2H, CH2), 7.32 (brs, 3H, arH),
7.56 (brs, 2H, arH), 8.00 (brs, 2H, arH), 8.39 (brs, 2H, arH),
8.93 (brs, 1H, N=CH), 10.58 (s, 1H, NH); 13C NMR
(DMSO-d6): δ = 28.91 (N-2CH2), 53.82 (S-2CH2), 71.27
(CH2), 115.33 (CH, N=CH), 118.92, 120.85, 123.33,
126.61, 129.75, 133.55, 138.18, 143.29 , 150.07, 153.31
(aromatic carbons), 160.86 (triazol C-3), 163.25 (triazol C-
5); EI MS m/z (%): 480.42 (28), 475.66 ([M+K]+, 22),
460.14 (22), 438.62 ([M+2]+, 22), 432.61 (56), 402.27
(100); anal. calcd. for C21H21N7S2: C, 57.91; H, 4.86; N,
22.51 %. Found: C, 58.27; H, 5.00; N, 22.61 %.
incubated with 5 µL of the test compounds at room tem-
perature for 15 min in microtiter plates. The production of
ammonia was measured by indophenol method and used to
determine the urease inhibitory activity. The phenol reagent
(45 µL, 1 % w/v phenol, and 0.005 % w/v sodium nitro-
prusside) and alkali reagent (70 µL, 0.5 % w/v sodium
hydroxide, and 0.1 % v/v NaOCl) were added to each well
and the increasing absorbance at 625 nm was measured after
20 min, using a microplate reader (Molecular Device,
USA). The percentage inhibition was calculated from the
formula 100—(ODtestwell/ODcontrol) × 100. Thiourea was
used as the standard inhibitor. In order to calculate IC50
values, different concentrations (250–0.114 µg/mL) of
synthesized compounds and standard were assayed at the
same reaction conditions (Weatherburn, 1967). The
obtained positive results were presented in Table 2.
1
Antimicrobial activity assessment
All bacterial and yeast strains were obtained from the Hif-
zissihha Institute of Refik Saydam (Ankara, Turkey) and were
as follows: Escherichia coli ATCC35218, Yersinia pseudo-
tuberculosis) ATCC911, Pseudomonas aeruginosa ATCC
27853, Enterococcus faecalis ATCC 29212, Staphylococcus
aureus ATCC 25923, B. cereus 709 ROMA, Ms: Myco-
bacterium smegmatis ATCC607, C. albicans ATCC 60193,
Sc: Saccharomyces cerevisiae RSKK 251. All the newly
synthesized compounds were dissolved in DMSO and ethanol
to prepare chemicals of stock solution of 10 mg/1 mL.
Pancreatic lipase inhibition assay
The inhibitory effects of those compounds were evaluated
against porcine pancreatic lipase (PPL) (obtained from
Applichem, Germany) (15 ng/mL). Lipase activity assays
were done according to Kurihara et al. (2003). The lipase
activity was measured using 4-methylumbelliferyl oleate
(4-MU oleate) as a substrate. Briefly, compounds were
mixed with PPL 1:3 (v/v) and incubated for 30 min. The
microtiter plates containing 50 µL 0.1 mM 4-MU oleate, 25
µL diluted compound-lipase solution, 25 µL dH2O and
assay buffer (13 mM Tris–HCl, 150 mM NaCl, and 1.3 mM
CaCl2, pH 8.0) were incubated at 37 °C for 20 min. The
amount of 4-methylumbelliferone released by the lipase was
measured by using a spectrofluorometer (SpectraMax M5,
Molecular Devices) at an excitation wavelength of 355 nm
and an emission wavelength of 460 nm. The inhibitory
activity of these compounds and Orlistat (Xenical, Hoff-
man, La Roche, Segrate, Italy), an inhibitor control of
pancreatic lipase, were measured at various concentration.
Residual activities were calculated by comparing to a con-
trol without inhibitor. The assays were done in triplicate.
The IC50 values were determined as the concentration of a
compound that gave 50 % inhibition of maximal activity.
The obtained positive results were presented in Table 3.
Agar-well diffusion method
Simple susceptibility screening test using agar-well diffu-
sion method as adapted earlier (Ahmad et al., 1998) was
used. Each microorganism was suspended in Mueller Hin-
ton (MH) (Difco, Detroit, MI) broth and diluted approxi-
mately to 106 colony forming unit (cfu)/mL. They were
“flood-inoculated” onto the surface of MH agar and
Sabouraud Dextrose Agar (SDA) (Difco, Detriot, MI) and
then dried. For C. albicans and C. tropicalis, SDA were
used. Five-millimeter diameter wells were cut from the agar
using a sterile cork-borer, and 50 mL of the extract sub-
stances was delivered into the wells. The plates were
incubated for 18 h at 35 °C. Antimicrobial activity was
evaluated by measuring the zone of inhibition against the
test organism. Ampicillin (10 mg) and Fluconazole (5 mg)
were used as standard drugs. Dimethyl sulfoxide and
ethanol were used as solvent controls. The antimicrobial
activity results are summarized in Table 1. There are only
positive results was placed on the table.
Acknowledgments This project was supported by Artvin Çoruh
University, BAP, Turkey (Ref, No. 2014.F11.02.04), Karadeniz
Technical University (Ref. 8623 and 8663) and is gratefully
acknowledged. The author thanks to Serdar Ulker for performing the
antimicrobial screening studies.
Urease inhibition assay
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