Synthesis of some new 1,2,4-triazoles, their Mannich and Schiff bases and evaluation of their antimicrobial activities

Article abstract of DOI:10.1016/j.ejmech.2008.06.019

4-Phenyl-5-pyridin-4-yl-4H-1,2,4-triazole-3-thiol (3) was obtained in basic media via the formation of 2-isonicotinoyl-N-phenylhydrazinecarbothioamide (2), and converted to some alkylated derivatives (4a,b) and Mannich base derivatives (5a-c). 2-[(4-Pheny

Full text of DOI:10.1016/j.ejmech.2008.06.019

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European Journal of Medicinal Chemistry 44 (2009) 1057e1066
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Original article
Synthesis of some new 1,2,4-triazoles, their Mannich and Schiff
bases and evaluation of their antimicrobial activities
Hacer Bayrak a, Ahmet Demirbas a, Sengul Alpay Karaoglu b, Neslihan Demirbas a,
*
¨
a Karadeniz Technical University, Department of Chemistry, 61080 Trabzon, Turkey
b Rize University, Department of Biology, 53100 Rize, Turkey
Received 10 March 2008; received in revised form 19 June 2008; accepted 20 June 2008
Available online 26 June 2008
Abstract
4-Phenyl-5-pyridin-4-yl-4H-1,2,4-triazole-3-thiol (3) was obtained in basic media via the formation of 2-isonicotinoyl-N-phenylhydrazine-
carbothioamide (2), and converted to some alkylated derivatives (4a,b) and Mannich base derivatives (5aec). 2-[(4-Phenyl-5-pyridin-4-yl-
4H-1,2,4-triazol-3-yl)thio]acetohydrazide (7) that was obtained by using compound 3 as precursor in two steps was converted to thiosemicar-
bazide derivative (8), Schiff base derivatives (9) and 5-{[(4-phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)thio]methyl}-1,3,4-oxadiazole-2-thiol
(10). Moreover, 5-{[(4-phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)thio]methyl}-3-{[(2-morpholin-4-ylethyl)amino]methyl}-1,3,4-oxadiazole-
2(3H )-thione (11) was synthesized via reaction of compound 10 with 2-(4-morpholino)ethylamine. The treatment of compound 8 with
NaOH gave 4-(4-methylphenyl)-5-{[(4-phenyl-5-pyridine-4-yl-4H-1,2,4-triazol-3-yl)thio]methyl}-4H-1,2,4-triazole-3-thiol (12), while the
acidic treatment of compound 8 afforded 5-{[(4-phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)thio]methyl}-2(4-methylphenyl)-amino-1,3,4-thia-
diazole (14). N-Methyl derivative of compound 14 and a Mannich base derivative of compound 12 were synthesized from the reactions of these
precursors with methyl iodide and methyl piperazine, respectively.
All newly synthesized compounds were screened for their antimicrobial activities. The antimicrobial activity study revealed that all the com-
pounds screened showed good or moderate activity except compounds 3, 5c, 7, 9c, 9e, 9g, 9h, 11, and 13.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Isonicotinic acid hydrazide; 1,2,4-Triazole; 1,3,4-Oxadiazole; 1,3,4-Thiadiazole; Antimicrobial activity
1. Introduction
1,2,4-triazole ring, besides bi- and polyheterocyclic com-
pounds that contain triazole ring or triazole-fused compounds
[5e13]. For instance, some bi-heterocyclic compounds,
consisting of 1,2,4-triazole and 1,3,4-thiadiazole rings or two
1,2,4-triazole rings, have been synthesized by us as antimicro-
bial compounds [14].
Literature survey reveals that piperazine or morpholine ring
is important for antimicrobial activity [15e18]. For instance,
Linezolid, Eperezolid, AZD2563 and Itraconazole, which are
currently important antibiotics used for the treatment of micro-
bial infections, contain a piperazine or morpholine ring in their
structures. They also contain an azole ring such as
oxazolidinone or 1,2,4-triazole ring [19,20]. Other important
chemotherapeutics, such as Vorozole, Letrozole and Anastro-
zole that consist of substituted 1,2,4-triazole ring, are currently
being used for the treatment of breast cancer[21].
In the past 25 years, the incidence of microbial infection has
increased on alarming levels over the world as a result of anti-
microbial resistance. A growing number of immuno-compro-
mised patients are as a result of cancer chemotherapy, organ
transplantation and HIV infection which are the major factors
contributing to this increase. The health problem demands to
search and synthesize a new class of antimicrobial compounds
effective against pathogenic microorganisms that developed
resistance to the antibiotics used in the current regimen [1e4].
The therapeutic importance of 1,2,4-triazoles is well docu-
mented. Among these, there are simple molecules including
* Corresponding author. Tel.: þ90 462 3772600; fax: þ90 462 3253196.
E-mail address: neslihan@ktu.edu.tr (N. Demirbas).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.06.019

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H. Bayrak et al. / European Journal of Medicinal Chemistry 44 (2009) 1057e1066
Our efforts for the synthesis of compounds possessing
compounds 4a and 4b were obtained. On the other hand, the
reactions of the same 1,2,4-triazole-3-thiol (3) with several
amines in the presence of formaldehyde solution afforded
the corresponding Mannich base derivatives (5aec) incorpo-
rating piperazine or morpholine ring. The reaction of 4-
phenyl-5-pyridin-4-yl-4H-1,2,4-triazole-3-thiol (3) with ethyl
bromoacetate in the presence of sodium ethoxide or triethyl-
amine produced ethyl [(4-phenyl-5-pyridin-4-yl-4H-1,2,4-
triazol-3-yl)thio]acetate (6). Then, this ester (6) was converted
to the corresponding hydrazide derivative, 2-[(4-phenyl-5-pyr-
idin-4-yl-4H-1,2,4-triazol-3-yl)thio]acetohydrazide (7) via the
reaction with hydrazine hydrate. The treatment of 7 with CS2
in the presence of KOH resulted in the formation of 5-{[(4-
phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)thio]methyl}-1,3,
4-oxadiazole-2-thiol (10), which was converted to the corre-
sponding Mannich base derivative (11) by using 2-(4-morpho-
lino)ethylamine in the presence of formaldehyde solution.
The treatment of acetohydrazide derivative (7) with several
aldehydes gave N0-arylmethylene-2-[(4-phenyl-5-pyridin-4-yl-
4H-1,2,4-triazol-3-yl)thio]acetohydrazides (9aeh). The com-
pounds having arylideneehydrazide structure may exist as
E/Z geometrical isomers about eC]N double bond and as
cis/trans amide conformers (Scheme 3) [25,26]. According
antitumor activity led us to the discovery of several Schiff
base derivatives of 1,2,4-triazol-5-ones [14,22e24]. Among
these, some Schiff base derivatives of acetic acid hydrazides
containing 1,2,4-triazol-5-one ring have displayed antitumoral
activity only against breast cancer, while 2-phenyl ethylidena-
mino and 2-phenyl ethylamino derivatives of 4-amino-
1,2,4-triazol-5-ones have been found to be effective towards
non-small cell lung cancer, CNC and breast cancer [5,22,23].
In view of these facts, the aim of the present study is to
obtain 1,2,4-triazole derivatives incorporating Schiff base
and Mannich base structures (Schemes 1 and 2) as antibacte-
rial agents.
2. Chemistry
1-Isonicotinoyl-4-phenyl thiosemicarbazide (2) was ob-
tained by the reaction of isonicotinic acid hydrazide with
phenylisothiocyanate. The cyclization of compound 2 in the
presence of 2 N NaOH resulted in the formation of 4-phenyl-
5-pyridin-4-yl-4H-1,2,4-triazole-3-thiol (3). The alkylation of
1,2,4-triazole-3-thiol (3) was performed by the reaction with
methyl iodide or isobutyl bromide in basic media, thus
N
N
RX
R: 4a:CH3, 4b: i-bu
SR
N
S
R
5
N
O
NHNH2
N
N
O
NHNH C
N
NCH3
a:
NHPh
NaOH
SH
N
PhNCS
b:
c:
4
NH(CH2)2
N
O
N
R
N
N
2
N
N
HCHO,
amine
1
N
N
N
N
3
S
N
S
N
N
EtO-
N
BrCH2CO2Et
5
N
N
O
S
C
N
OEt
N
9
R
a:
OCH3
F
6
b:
c:
N
N
N
N
OH
OH
H2NNH2
N
SH
S
N
O
O
N
N
HO
Cl
O
N
O
H2NCH2CH2
HCHO
N
d:
e:
C
CS2, KOH
S
N
10
NHNH2
N
NH
N
N
N
N
S
N
Cl
S
7
O
N
N
CH3C6H4NCS
N
RCHO
f:
11
N
N
N
N
O
O
g:
h:
S
S
N
S
C
N
C
NHNH C
CHR
NHN
N
N
N
NH
CH3
8
9
Scheme 1. Synthetic pathway for the preparation of compounds 2e11.

H. Bayrak et al. / European Journal of Medicinal Chemistry 44 (2009) 1057e1066
1059
N
N
O
S
S
N
C
NHNH C
N
NH
CH3
8
H2SO4
NaOH
N
N
N
N
N
N
N
N
S
N
SH
S
N
N
NH
S
N
N
CH3
14
CH3I
12
CH3
HCHO
methyl piperazyne
N
N
N
N
CH3
N
N
CH3
S
N
N N
N
N
N
S
N
S
N
S
N
N
CH3
15
13
CH3
Scheme 2. Synthetic pathway for the preparation of compounds 12e15.
to the literature [26], the compounds containing imine bond
are present in higher percentage in dimethyl-d6 sulfoxide solu-
tion in the form of geometrical E isomer about eC]N double
bond. The Z isomer can be stabilized in less polar solvents by
an intramolecular hydrogen bond. In the present study, the
spectral data were obtained in dimethyl-d6 sulfoxide solution
and no signal belonging to Z isomer was observed. On the
other hand, the cis/trans conformers of E isomer were present
in the dimethyl-d6 sulfoxide solution of compounds 9aeh.
The reaction of compound 7 with 4-methylphenylisothiocya-
nate produced N-(4-methylphenyl)-2-{[(4-phenyl-5-pyridin-4-
yl-4H-1,2,4-triazol-3-yl)thio]acetyl} hydrazinecarbothioamide
(8). The treatment of compound 8 with sulfuric acid caused the
conversion of carbothioamide structure into 1,3,4-thiadiazole
ring; thus, 5-{[(4-phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)th-
io]methyl}-2(4-methylphenyl)-amino-1,3,4-thiadiazole (14) was
obtained. On the other hand, the cyclization of the same com-
pound (8) in the presence of 2 N NaOH resulted in the formation
of 4-(4-methylphenyl)-5-{[(4-phenyl-5-pyridine-4-yl-4H-1,2,4-
triazol-3-yl)thio]methyl}-4H-1,2,4-triazole-3-thiol (12), which
was converted to 5-{[(4-phenyl-5-pyridine-4-yl-4H-1,2,4-tria-
zol-3-yl)thio]methyl}-4-(4-methylphenyl)-2-[(4-methylpipera-
zin-1-yl)methyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (13) by
Mannich reaction. Compound 14 was condensed with methyl io-
dide in the presence of sodium ethoxide, thus, 5-{[(4-phenyl-5-
pyridin-4-yl-4H-1,2,4-triazol-3-yl)thio]methyl}-2(4-methylphen
yl)-2-methylamino-1,3,4-thiadiazole (15) was obtained.
H
N
H
C
3. Results and discussion
H
N
R
C
G
G
C
O
N
R
C
O
N
H
1
In the H NMR spectra of compound 2, additional eNHe
N
N
signals (controlled with D2O) derived from thiosemicarbazide
structure were observed at 9.65, 9.75 and 10.30 ppm, while the
signal due to eNH2 group of hydrazide structure did not
appear. Additional signals belonging to phenyl ring were ob-
SCH2
trans, E
trans, Z
N
G=
N
H
R
C
1
served in the aromatic region in the H and 13C NMR spectra
H
N
H
O
N
O
C
N
N
H
C
R
of compound 2. Moreover, eC]S group resonated at
180.55 ppm in the 13C NMR spectra of compound 2. When
compound 2 was converted to 4-phenyl-5-pyridin-4-yl-4H-
1,2,4-triazole-3-thiol (3) in basic media, eNHe peaks disap-
peared, while new signal due to eSH group was observed at
C
G
G
cis, E
cis, Z
Scheme 3. E/Z Geometrical isomers and cis/trans conformers of compounds
9aeh.

1060
H. Bayrak et al. / European Journal of Medicinal Chemistry 44 (2009) 1057e1066
1
14.36 ppm (controlled with D2O) in the H NMR spectra of
compound 3. It is interesting to note that thiocarbonyl com-
pounds are present in their thionethiol tautomeric forms in
In addition, eOCH3 group of compound 9a resonated at
3.84 ppm integrating three protons as a singlet in the 1H
NMR spectrum, while this group was observed at 54.84 ppm
in the 13C NMR spectrum. Moreover, the signals derived
from one eOH group in compound 9c and two eOH groups
in compound 9d were recorded at 8.65 ppm integrating two
protons and 10.91 and 10.58 ppm integrating one proton, re-
spectively (controlled by changing D2O). The eOH group of
compound 9d was seen as two different singlets due to the ex-
istence of cis/trans-amide conformers.
1
solution as indicated by their IR and H NMR spectra [14].
These tautomeric forms are also present in dimethyl sulfoxide
as suggested by NMR spectral data. When compound 3 was
converted to its alkylated derivatives (4a and 4b), the eSH
signal disappeared, instead, new signals that originated from
methyl or isobutyl groups were observed in the H and 13C
1
1
NMR spectra of compounds 4a and 4b. The H NMR spectra
of compounds 5aec displayed additional signals due to
eCH2e group at 4.68e5.22 ppm integrating two protons
besides the peaks belonging to methyl piperazine, piperazine
or 2-(4-morpholino)ethylamine moiety, which are used as
amine components in these Mannich reactions; these groups
resonated between 2.42 and 5.70 ppm. The carbon signals of
these groups were recorded between 46.55 and 53.70 ppm.
Due to less solubility of compounds 5a and 5c in DMSO-d6,
13C NMR spectrum could not be recorded.
Compound 8 was obtained by a way analogous to the
synthesis of compound 2 in 85% yield, and its structure was
confirmed on the basis of IR, NMR and mass spectroscopic
methods and elemental analysis. The treatment of compound
8 with sulfuric acid and sodium hydroxide produced 4-
(4-methylphenyl)-5-{[(4-phenyl-5-pyridine-4-yl-4H-1,2,4-triazol-
3-yl)thio]methyl}-4H-1,2,4-triazole-3-thiol (12) and 5-{[(4-
phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)thio]methyl}-2(4-
methylphenyl)-amino-1,3,4-thiadiazole (14), respectively.
These compounds displayed IR, 1H and 13C NMR spectra
and elemental analyses consistent with the assigned structures.
The eSH proton is acidic enough and some substitution re-
action could be achieved on this group in the presence of
a base [27,28]. In the present study, compound 6, acetic acid
ester derivative of compound 3, was obtained by two methods
1
In the H NMR spectrum of compound 12, additional signal
due to eSH group appeared at 13.89 ppm (controlled by
changing D2O), while the eNHe signals disappeared.
Moreover, the IR spectrum of compound 12 displayed an e
SH stretching band at 2732 cmꢀ1. The eNHe group in com-
1
in good yields. In the H NMR spectrum of compound 6, ad-
ditional signals derived from ester group were observed at 1.29
(eOCH2CH3), 4.13 (eSCH2) and 4.22 (eOCH2CH3) ppm in-
tegrating for three protons, two protons and two protons, re-
spectively. In the 13C NMR spectrum of the compound, the
signals belonging to the same groups were recorded at
14.05, 34.43 and 62.13 ppm, respectively.
The 1H NMR spectrum of compound 7 displayed no signals
belonging to eOCH2CH3 group; instead, new signals derived
from hydrazide structure appeared at 4.32 (eNHNH2) and
9.31 (eNHNH2) ppm integrating for two protons and one pro-
ton, respectively (controlled by changing D2O).
1
pound 14 resonated at 10.30 ppm in the H NMR spectrum.
The eNHe stretching band was observed at 3187 cmꢀ1 in
the IR spectra. When compound 14 was converted to be an
alkylated derivative (15), the signal disappeared; instead,
new signal belonging to methyl protons appeared at
1
2.83 ppm as singlet in the H NMR spectrum of compound
15. The elemental analysis of compound 15 is consistent
with the assigned structure.
The synthesis of compound 13 was carried out by a Man-
nich reaction containing compound 12 as a carbonyl compo-
nent, methyl piperazine as an amine component and
The IR spectra of compounds 6 and 7 showed a peak at
1735 and 1673 cmꢀ1 due to carbonyl function derived from es-
ter or hydrazide structure, respectively.
1
formaldehyde. In the H NMR spectrum of compound 13,
the signal derived from eSH group disappeared, instead,
The synthesis of eight different Schiff base derivatives of
compound 7 was carried out by the reaction of compound 7
with several aromatic aldehydes in ethanol in the presence
new signals due to methyl piperazine moiety were detected
1
in the H and 13C NMR spectrum of compound 13.
1
of catalytic amount of H2SO4. The H and 13C NMR spectra
4-Phenyl-5-pyridin-4-yl-4H-1,2,4-triazole-3-thiol (3) dis-
played no antimicrobial activity, whereas its alkylated deriv-
atives and Mannich bases except 5c, displayed good
activities against all tested microorganisms except Candida
tropicalis and Candida albicans. Ethyl [(4-phenyl-5-pyri-
din-4-yl-4H-1,2,4-triazol-3-yl)thio]acetate (6) showed mod-
erate antimicrobial activities towards C. tropicalis and
C. albicans, while 2-[(4-phenyl-5-pyridin-4-yl-4H-1,2,4-tria-
zol-3-yl)thio]acetohydrazide (7) showed no activity against
all bacterial and yeast strains. N-(4-Methylphenyl)-2-{[(4-
phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)thio]acetyl} hy-
drazinecarbothioamide (8) indicated slight activity against
C. tropicalis (Ct.) and C. albicans (Ca.). The conversion of
hydrazide structure in compound 7 to 1,3,4-oxadiazole ring
of 5-{[(4-phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)thio]-
methyl}-1,3,4-oxadiazole-2-thiol (10) caused slight antimicrobial
of compounds 9aeh displayed additional signals due to the ar-
omatic ring derived from aldehyde moiety at aromatic region,
while the signal belonging to eNH2 group of hydrazide struc-
ture did not appear.
1
In the H NMR spectra of compounds 9aeh two sets of
signals each belonging to the eSCH2 group, eN]CH group
and eNH group of cis- and trans-conformers were observed
between 4.11 and 4.60, 7.98 and 8.44 and 11.68 and 12.14 ppm,
respectively. The upfield lines of eSCH2, eN]CH, and eNH
protons were assigned to cis-conformer of the amide structure
and downfield lines of the protons of the same group to trans-
conformer of the amide structure [29]. In the 13C NMR spectra
of compounds 9aeh, the eN]CH signals belonging to individ-
ual cis/trans conformers were observed as two sets between
112.21 and 119.85 ppm.

H. Bayrak et al. / European Journal of Medicinal Chemistry 44 (2009) 1057e1066
1061
activities against Staphylococcus aureus, Bacillus cereus, Ct.
and Ca. 5-{[(4-Phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)
thio]methyl}-3-{[(2-morpholin-4-ylethyl) amino]methyl}-1,
3,4-oxadiazole-2(3H )-thione (11), which is the Mannich
base derivative of compound 10, demonstrated slight activity
only towards Escherichia coli. The conversion of compound
8 to compound 12 caused no important antimicrobial
activity. Similarly, 5-{[(4-phenyl-5-pyridine-4-yl-4H-1,2,4-
triazol-3-yl)thio]methyl}-4-(4-methylphenyl)-2-[(4-methylpi
perazin-1-yl)methyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione
(13) displayed no activity. On the contrary to the activity of com-
pound 12, compound 14, which was obtained from the reaction
of compound 8with H2SO4, indicated good activitiestowards all
bacterial strains except C. tropicalis and C. albicans. The meth-
ylated derivative of compound 14, 5-{[(4-phenyl-
5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)thio]methyl}-2(4-methyl-
phenyl)-2-methylamino-1,3,4-thiadiazole (15), also showed
good activity except against Ct. and Ca. Among N0-(arylmethy-
lene)-2-[(4-phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)thio]
acetohydrazides (9aeh), which are the Schiff bases of com-
pound 7, compounds 9a, 9b, 9d and 9f displayed good activi-
ties against the tested microorganisms except Ct. and Ca.,
while 9c, 9e, 9g and 9h showed no activity.
(compound 2) or dimethyl sulfoxide/water (1:2) to afford the
desired product.
5.1.1.1.
2-Isonicotinoyl-N-phenylhydrazinecarbothioamide
(2). Yield 90%, m.p. 120 ꢁC. IR (KBr, n, cmꢀ1): 3375 and
3298 (3NH), 1717 (C]O), 1309 (C]S); Anal. Calcd (%)
for C13H12N4OS: C, 57.34; H, 4.44; N, 20.57. Found: C,
57.37; H, 4.48; N, 20.52; 1H NMR (DMSO-d6, d ppm):
7.17e7.22 (2H, m, arH), 7.30e7.44 (5H, m, arH), 8.43e
8.79 (2H, m, arH), 9.65 (1H, s, NH), 9.75 (1H, s, NH),
10.30 (1H, s, NH); 13C NMR (DMSO-d6, d ppm): arC:
[125.25 (CH), 128.04 (4CH), 127.89 (2CH), 129.46 (2CH),
133.84 (C), 138.62 (C)], 166.56 (C]O), 180.55 (C]S).
5.1.1.2. N-(4-Methylphenyl)-2-{[(4-phenyl-5-pyridin-4-yl-4H-
1,2,4-triazol-3-yl)thio]acetyl} hydrazinecarbothioamide (8). Yield
85%, m.p. 122 ꢁC. IR (KBr, n, cmꢀ1): 3216 and 3156 (3NH),
1720 (C]O), 1602 and 1551 (2C]N), 1187 (C]S); Anal.
Calcd (%) for C23H21N7OS2: C, 58.08; H, 4.45; N, 20.62. Found:
C, 58.11; H, 4.48; N, 20.64; 1H NMR (DMSO-d6, d ppm): 2.14
(3H, s, CH3), 3.92 (2H, s, SeCH2), 7.26e7.35 (2H, dd, j ¼ 6 Hz,
arH), 7.42e7.46 (2H, m, arH), 7.69e7.72 (5H, m, arH), 8.54e
8.62 (4H, d, j ¼ 5 Hz, arH), 9.31 (1H, s, NH), 9.56 (1H, s,
NH), 10.67 (1H, s, NH); 13C NMR (DMSO-d6, d ppm): 21.44
(CH3), 34.00 (SeCH2), arC: [121.12 (2CH), 121.33 (2CH),
127.83 (2CH), 130.22 (2CH), 131.53 (2CH), 132.92 (C),
134.41 (C), 135.15 (C), 138.67 (C), 147.17 (3CH)], 151.41 (tri-
azole C-3), 154.73 (triazole C-5), 166.38 (C]O), 180.79
(C]S); MS: m/z (%) 118.68 (22), 148.76 (27), 254.93 (13),
302.94 (13), 316.97 (13), 334.65 (81), 476.13 (M þ 1, 11),
530.10 (11).
4. Conclusions
This study reports the synthesis of some heterocyclic com-
pounds containing 1,2,4-triazole, 1,3,4-oxadiazole, 1,3,4-thia-
diazole and/or morpholine, piperazine rings. The antimicrobial
activity study revealed that all the compounds screened
showed good and moderate antimicrobial activities, except
compounds 3, 5c, 7, 9c, 9e, 9g, 9h, 11, and 13.
Among compounds 9feh, the compound which contains
a pyridinyl ring in their structures displayed diverse antimicro-
bial activities due to the position of nitrogen atom in the
heterocyclic ring.
5.1.2. General method for the synthesis of compounds
3 and 12
A solution of corresponding carbothioamide 2 (for com-
pound 3) or 8 (for compound 12) (10 mmol) in 2 N NaOH was
refluxed for 3 h. The resulting solution was cooled to room tem-
perature and acidified to pH 3e4 with 37% HCl. The precipitate
formed was filtered, washed with water and recrystallized from
ethanol/water (1:1) to afford the desired compounds.
5. Experimental
5.1. Chemistry
Melting points were determined on a Buchi B-540 melting
¨
5.1.2.1. Synthesis of 4-phenyl-5-pyridin-4-yl-4H-1,2,4-triazole-
3-thiol (3). Yield 83%, m.p. 195e200 ꢁC. IR (KBr, n, cmꢀ1):
2652 (SH), 1326 (C]S), 1608 and 1569 (2C]N); Anal.
Calcd (%) for C13H10N4S: C, 61.40; H, 3.96; N, 22.03. Found:
1
point apparatus and are uncorrected. H and 13C NMR spectra
were recorded on a Varian-Mercury 200 MHz spectrometer.
The IR spectra were measured as potassium bromide pellets us-
ing a PerkineElmer 1600 series FTIR spectrometer. Combustion
analysis was performed on a Costech Elemental Combustion
System CHNS-O elemental analyzer. All the chemicals were ob-
tained from Fluka Chemie AG Buchs (Switzerland).
1
C, 61.49; H, 4.01; N, 21.98; H NMR (DMSO-d6, d ppm):
7.8e7.21 (2H, m, arH), 7.39e7.42 (2H, m, arH), 7.49e7.52
(3H, m, arH), 8.45e8.60 (2H, m, arH), 14.36 (1H, s, SH);
13C NMR (DMSO-d6, d ppm): arC: [121.74 (2CH), 128.49
(2CH), 129.36 (2CH), 129.63 (CH), 133.00 (C), 133.97 (C),
149.90 (2CH)], 148.24 (triazole C-3), 169.02 (triazole C-5).
5.1.1. General method for the synthesis of compounds
2 and 8
A mixture of corresponding compound 1 or 7 (10 mmol)
and phenylisothiocyanate (for compound 2) or 4-methylpheny-
lisothiocyanate (for compound 8) (15 mmol) was refluxed in
ethanol for 4 h. The solution was cooled and a white solid ap-
peared. This was filtered and recrystallized from ethanol
5.1.2.2. 4-(4-Methylphenyl)-5-{[(4-phenyl-5-pyridine-4-yl-4H-
1,2,4-triazol-3-yl)thio]methyl}-4H-1,2,4-triazole-3-thiol
(12). Yield 48%, m.p. 276 ꢁC. IR (KBr, n, cmꢀ1): 2732 (SH),
1607, 1516, 1495, 1439 (4C]N), 1237 (C]S); Anal. Calcd
(%) for C23H19N7S2: C, 60.37; H, 4.19; N, 21.43. Found: C,

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H. Bayrak et al. / European Journal of Medicinal Chemistry 44 (2009) 1057e1066
1
60.44; H, 4.26; N, 21.32; H NMR (DMSO-d6, d ppm): 2.36
(3H, s, CH3), 4.20 (2H, s, CH2), 7.16e7.52 (7H, m, arH),
7.49e7.53 (2H, m, arH), 8.02e8.09 (2H, m, arH), 8.57e
8.62 (2H, m, arH), 13.89 (1H, s, SH); 13C NMR (DMSO-d6,
d ppm): 26.84 (CH3), 34.99 (CH2), arC: [121.72 (2CH),
126.95 (2CH), 127.91 (2CH), 128.32 (2CH), 129.35 (2CH),
132.52 (CH), 134.69 (C), 136.89 (C), 137.91 (C), 139.56
(C), 151.84 (2CH)], 149.07 (triazole C-3), 149.12 (triazole
C-3), 151.46 (triazole C-5, CeSH), 159.38 (triazole C-5).
152.38 (thiadiazole C-2), 156.44 (triazole C-3 and thiadiazole
C-5), 166.02 (triazole C-5).
5.1.4. General method for the synthesis of compounds 5, 11
and 13
To a solution of corresponding compound 3, 10 or 12
(10 mmol) in dimethyl formamide, formaldehyde (37%,
1.55 mL) and amine (10 mmol) were added and the mixture
was stirred at room temperature for 2.5 h. Then, excess amount
of pure water was added to this solution and the mixture was
kept overnight in cold. The resulting solid separated was col-
lected by filtration, washed with water, recrystallized from di-
methyl sulfoxide/water (1:2) to yield the title compounds.
5.1.3. General method for the synthesis of compounds
4 and 15
To a solution of compound 3 (for compounds 4a and 4b) or
14 (for compound 15) (10 mmol) in absolute ethanol, 1 equiv.
of sodium was added and the mixture was stirred at room tem-
perature for 30 min. Then, methyl iodide (for compounds 4a
and 15) or isobutyl bromide (for compound 4b) (20 mmol)
was added and refluxed for 4 h. After evaporating the solvent
under reduced pressure a solid appeared. The solid was recyr-
stallized from ethanol/water (1:1) to obtain target compound.
5.1.4.1. 2-[(4-Methylpiperazin-1-yl)aminomethyl]-4-phenyl-5-
pyridin-4-yl-2,4-dihydro-3H-1,2,4-triazole-3-thione
(5a).
Yield 93%, m.p. 170e174 ꢁC. IR (KBr, n, cmꢀ1): 1602
(C]N), 1326 (C]S); Anal. Calcd (%) for C19H22N6S: C,
62.27; H, 6.05; N, 22.93. Found: C, 62.31; H, 6.11; N,
22.99; 1H NMR (DMSO-d6, d ppm): 2.16 (3H, s, CH3),
2.47e2.58 (8H, t, j ¼ 6 Hz, 4CH2), 5.25 (2H, s, CH2), 7.26
(2H, br s, arH), 7.38e7.70 (5H, m, arH), 8.59 (2H, br s, arH).
5.1.3.1. 3-Methylthio-4-phenyl-5-pyridin-4-yl-4H-1,2,4-tria-
zole (4a). Yield 46%, m.p. 168e170 ꢁC. IR (KBr, n, cmꢀ1):
1655 and 1600 (2C]N); Anal. Calcd (%) for C14H12N4S:
C, 62.66; H, 4.51; N, 20.88. Found: C, 62.72; H, 4.57; N,
20.81; 1H NMR (DMSO-d6, d ppm): 2.74 (3H, s, CH3),
7.05e7.40 (4H, m, arH), 7.50e7.62 (3H, m, arH), 8.44e
8.61 (2H, m, arH); 13C NMR (DMSO-d6, d ppm): 14.22
(CH3), arC: [121.38 (2CH), 127.03 (2CH), 127.24 (CH),
130.45 (2CH), 133.95 (C), 133.99 (C), 150.12 (2CH)], 152.1
(triazole C-3), 171.0 (triazole C-5).
5.1.4.2. 4-Phenyl-2-{[(2-morpholin-4-ylethyl)amino]methyl}-
5-pyridin-4-yl-2,4-dihydro-3H-1,2,4-triazole-3-thione
(5b).
Yield 89%, m.p. 191e193 ꢁC. IR (KBr, n, cmꢀ1): 3051
(NH), 1680 (C]N), 1279 (C]S); Anal. Calcd (%) for
C20H24N6OS: C, 62.11; H, 6.10; N, 21.20. Found: C, 62.05;
1
H, 6.17; N, 21.23; H NMR (DMSO-d6, d ppm): 2.42 (4H,
br s, 2CH2), 3.38 (4H, br s, 2CH2), 3.92 (2H, br s, CH2),
4.68 (2H, s, CH2), 5.43e5.70 (2H, m, CH2), 7.25e7.38 (3H,
m, arH), 7.43e7.60 (4H, m, arH), 8.52e8.62 (2H, m, arH),
14.37 (1H, br s, NH).
5.1.3.2. 3-Isobutylthio-4-phenyl-5-pyridin-4-yl-4H-1,2,4-tria-
zole (4b). Yield 54%, m.p. 182e184 ꢁC. IR (KBr, n, cmꢀ1):
1600 and 1498 (2C]N); Anal. Calcd (%) for C17H18N4S:
C, 65.78; H, 5.84; N, 18.05. Found: C, 65.81; H, 5.89; N,
17.99; 1H NMR (DMSO-d6, d ppm): 0.88e1.08 (6H, d,
j ¼ 6 Hz, 2CH3), 1.80e2.19 (1H, m, CH), 3.00e3.22 (2H, d,
j ¼ 7 Hz, CH2), 7.28e7.34 (2H, m, arH), 7.43e7.58 (2H, m,
arH), 7.61e7.74 (3H, m, arH), 8.57e8.62 (2H, d, j ¼ 4 Hz,
arH); 13C NMR (DMSO-d6, d ppm): 21.82 (2CH3), 27.90
(CH), 40.05 (CH2), arC: [121.82 (2CH), 127.86 (2CH),
130.08 (2CH), 130.15 (CH), 150.03 (2CH), 133.88 (C),
134.05 (C)], 151.99 (triazole C-3), 153.34 (triazole C-5).
5.1.4.3. 4-Phenyl-5-pyridin-4-yl-2-{[(4-{[(4-phenyl-5-pyridine-
4-yl-3-thioxo-2,4-dihydro-3H-1,2,4-triazol-2-yl)methyl]amino}
piperazin-1-yl)amino]methyl}-2,4-dihydro-3H-1,2,4-triazole-
3-thione (5c). Yield 85%, m.p. 245e247 ꢁC. IR (KBr, n,
cmꢀ1): 1600, 1498 (2C]N), 1325 (C]S); Anal. Calcd (%)
for C32H30N10S2: C, 62.11; H, 4.89; N, 22.64. Found: C,
1
62.07; H, 4.95; N, 22.59; H NMR (DMSO-d6, d ppm): 2.54
(8H, br s, 4CH2), 5.21 (4H, br s, 2CH2), 7.14 (4H, d,
j ¼ 5 Hz, arH), 7.38e7.79 (10H, m, arH), 8.72 (4H, d,
j ¼ 5 Hz, arH).
5.1.3.3. 5-{[(4-Phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)th-
io]methyl}-2(4-methylphenyl)-2-methylamino-1,3,4-thiadiazole
(15). Yield 54%, m.p. 231 ꢁC. IR (KBr, n, cmꢀ1): 3183 (NH),
1688 and 1600 (4C]N); Anal. Calcd (%) for C24H21N7S2: C,
61.12; H, 4.49; N, 20.79. Found: C, 61.14; H, 4.53; N, 20.88;
1H NMR (DMSO-d6, d ppm): 2.18 (3H, s, CH3), 3.88 (3H, s,
CH3), 4.71 (2H, s, SeCH2), 7.12e7.17 (2H, d, j ¼ 8 Hz, arH),
7.30 (2H, br s, arH), 7.46e7.55 (5H, m, arH), 7.55e7.62 (4H,
m, arH); 13C NMR (DMSO-d6, d ppm): 20.25 (CH3), 32.45
(SeCH2), 34.91 (NeCH3), arC: [121.13 (2CH), 123.76
(2CH), 125.46 (3CH), 128.15 (2CH), 131.48 (2CH), 136.13
(C), 136.61 (C), 137.19 (C), 142.44 (C), 147.21 (2CH)],
5.1.4.4. 5-{[(4-Phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)th-
io]methyl}-3-{[(2-morpholin-4-ylethyl)amino]methyl}-1,3,4-ox
adiazole-2(3H )-thione (11). Yield 45%, m.p. 288 ꢁC. IR
(KBr, n, cmꢀ1): 3211 (NH), 1603, 1506 (2C]N), 1265
(C]S); Anal. Calcd (%) for C23H26N8O2S2: C, 54.10; H,
5.13; N, 21.94. Found: C, 54.19; H, 5.13; N, 21.56; 1H
NMR (DMSO-d6, d ppm): 2.52 (4H, br s, 2CH2), 2.78 (2H,
br s, CH2), 2.85 (2H, br s, CH2), 3.41 (4H, br s, 2CH2), 4.18
(2H, s, CH2), 7.20 (2H, s, NCH2NH), 7.38e7.63 (7H, m,
arH), 8.60 (2H, br s, arH), 14.40 (1H, br s, NH); 13C NMR
(DMSO-d6, d ppm): 31.61 (CH2), 49.67 (2NHeCH2), 52.77
(2NeCH2), 54.67 (NeCH2), 63.00 (NeCH2eN), 67.56 (Oe

H. Bayrak et al. / European Journal of Medicinal Chemistry 44 (2009) 1057e1066
1063
CH2), arC: [121.72 (2CH), 129.09 (2CH), 129.78 (2CH),
129.92 (CH), 133.54 (C), 133.89 (C), 150.23 (2CH)], 149.19
(triazole C-3), 152.90 (oxadiazole C-2), 166.65 (triazole C-
5), 172.77 (oxadiazole C-5).
and 1600 (2C]N), 3321 and 3241 (NHeNH2); Anal. Calcd
(%) for C15H14N6OS: C, 55.20; H, 4.32; N, 25.75. Found:
C, 55.28; H, 4.28; N, 25.69; H NMR (DMSO-d6, d ppm):
3.90 (2H, s, SeCH2), 4.32 (2H, s, NH2), 7.23e7.38 (2H, dd,
j ¼ 6 Hz, arH), 7.44e7.48 (2H, m, arH), 7.68e7.74 (3H, m,
arH), 8.54e8.62 (2H, d, j ¼ 5 Hz, arH), 9.31 (1H, s, NH);
13C NMR (DMSO-d6, d ppm): 34.00 (CH2), arC: [122.02
(CH), 127.86 (2CH), 130.05 (2CH), 130.57 (2CH), 133.02
(C), 138.09 (C), 150.06 (2CH)], 152.10 (triazole C-3),
153.30 (triazole C-5), 165.84 (C]O).
1
5.1.4.5. 5-{[(4-Phenyl-5-pyridine-4-yl-4H-1,2,4-triazol-3-yl)th-
io]methyl}-4-(4-methyl phenyl)-2-[(4-methylpiperazin-1-yl)me
thyl]-2,4-dihydro-3H-1,2,4-triazole-3-thione (13). Yield 66%,
m.p. 283 ꢁC. IR (KBr, n, cmꢀ1): 2735 (SH), 1609, 1518,
1498 and 1442 (4C]N); 1609, 1518 and 1494 (3C]N),
1235 (C]S); Anal. Calcd (%) for C29H31N9S2: C, 61.13; H,
5.48; N, 22.13. Found: C, 61.27; H, 5.50; N, 22.29; 1H
NMR (DMSO-d6, d ppm): 2.39 (3H, s, CH3), 2.42 (3H, s,
CH3), 2.55e2.63 (8H, t, j ¼ 6 Hz, 4CH2,), 4.23 (2H, s, CH2),
4.58 (2H, s, CH2), 7.18e7.58 (7H, m, arH), 8.63e8.67 (2H,
m, arH), 7.55e7.59 (2H, m, arH), 8.11e8.14 (2H, m, arH);
13C NMR (DMSO-d6, d ppm): 26.15 (CH3), 35.09 (CH2),
45.62 (CH3), 52.14 (2NeCH2), 53.20 (2NeCH2), 66.03
(CH2), arC: [121.66 (2CH), 127.09 (2CH), 127.94 (2CH),
128.67 (2CH), 129.42 (2CH), 132.60 (CH), 134.78 (C),
136.98 (C), 137.96 (C), 139.51 (C), 151.71 (2CH)], 149.23
(triazole C-3, second ring), 149.32 (triazole C-3), 151.51 (tri-
azole C-5, CeSH), 159.51 (triazole C-5).
5.1.7. General method for the synthesis of compound 9ae
9h
A solution of the corresponding compound 7 (10 mmol) in ab-
solute ethanol was refluxed with appropriate aldehyde (10 mmol)
for 3 h. After cooling the mixture to room temperature, a white
solid appeared. This crude product was recrystallized from di-
methyl sulfoxide/water (1:2) to afford the desired product.
5.1.7.1. N0-[(4-Methoxyphenyl)methylene]-2-[(4-phenyl-5-pyri-
din-4-yl-4H-1,2,4-triazol-3-yl)thio]acetohydrazide (9a). Yield
97%, m.p. 224e226 ꢁC. IR (KBr, n, cmꢀ1): 3189 (NH),
1689 (C]O), 1618 and 1604 (2C]N); Anal. Calcd (%) for
C23H20N6O2S: C, 62.15; H, 4.54; N, 18.91. Found: C, 62.18;
1
5.1.5. Ethyl [(4-phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)
thio]acetate (6)
H, 4.50; N, 19.01; H NMR (DMSO-d6, d ppm): 3.84 (3H,
s, CH3), 4.58 and 4.18 (2H, s, SeCH2, trans/cis conformers),
6.93e7.02 (2H, d, j ¼ 8 Hz, arH), 7.29e7.31 (2H, m, arH),
7.42e7.78 (7H, m, arH), 8.19 and 7.98 (1H, s, N]CH,
trans/cis conformers), 8.57e8.62 (2H, d, j ¼ 5 Hz, arH),
11.72 and 11.68 (1H, s, NH, trans/cis conformers); 13C
NMR (DMSO-d6, d ppm): 34.48 (CH2), 54.81 (CH3), 113.82
and 113.36 (N]CH, trans/cis conformers), arC: [120.99
(2CH), 122.34 (C), 125.23 (C), 127.06 (3CH), 127.97
(2CH), 129.71 (3CH), 133.34 (C), 143.26 (CH), 149.59
(2CH), 160.24 (C)], 147.67 (triazole C-3), 152.48 (triazole
C-5), 167.64 (C]O).
Method 1: the corresponding compound 3 (10 mmol) was re-
fluxed with 1 equiv. of sodium in absolute ethanol for 2 h. Then,
ethyl bromoacetate (10 mmol) was added and refluxed for an ad-
ditional 3 h. After evaporating the solvent under reduced pressure,
a solid appeared. The solid was recrystallized from ethanol/water
(1:2) to afford compound 6. Yield 75%, m.p. 119e121 ꢁC.
Method 2: to a solution of compound 3 (10 mmol) in etha-
nol, ethyl bromoacetate (10 mmol) was added and the mixture
was stirred at room temperature for 1 h and refluxed for 4 h in
the presence of triethylamine (10 mmol). Then, the solvent was
removed under reduced pressure and a solid obtained. The
solid was recrystallized from ethanol/water (1:2) to afford com-
pound 6. Yield 71%, m.p. 119e121 ꢁC. IR (KBr, n, cmꢀ1):
1735 (C]O), 1599 and 1499 (2C]N), 1182 (CeO); Anal.
Calcd (%) for C17H16N4O2S: C, 59.98; H, 4.74; N, 16.46.
5.1.7.2. N0-[(4-Fluorophenyl)methylene]-2-[(4-phenyl-5-pyri-
din-4-yl-4H-1,2,4-triazol-3-yl)thio]-acetohydrazide (9b).
Yield 94%, m.p. 231e232 ꢁC. IR (KBr, n, cmꢀ1): 3186 (NH),
1681 (C]O), 1616 and 1600 (2C]N); Anal. Calcd (%) for
C22H17FN6OS: C, 61.10; H, 3.96; N, 19.43. Found: C, 61.16;
1
Found: C, 60.02; H, 4.79; N, 16.50; H NMR (DMSO-d6,
1
d ppm): 1.29 (3H, s, j ¼ 7 Hz, CH3), 4.13 (2H, s, SeCH2),
4.22 (2H, q, j ¼ 7 Hz, CH2), 7.26e7.35 (5H, m, arH), 7.54e
7.63 (4H, m, arH); 13C NMR (DMSO-d6, d ppm): 14.05
(CH3), 34.43 (SeCH2), 62.13 (OeCH2), arC: [126.30 (C),
127.08 (3CH), 130.38 (3CH), 130.61 (3CH), 150.12 (C)],
152.2 (triazole C-3), 161.80 (triazole C-5), 169.17 (C]O).
H, 4.00; N, 19.46; H NMR (DMSO-d6, d ppm): 4.49 and
4.18 (2H, s, SeCH2, trans/cis conformers), 7.22e7.38 (4H, m,
arH), 7.51e7.79 (7H, m, arH), 8.22 and 8.12 (1H, s, N]CH,
trans/cis conformers), 8.63 (2H, br s, arH), 11.97 and 11.75
(1H, s, NH, trans/cis conformers); 13C NMR (DMSO-d6,
d ppm): 34.44 (CH2), 115.67 and 115.24 (N]CH, trans/cis con-
formers), arC: [121.13 (2CH), 127.10 (3CH), 128.67 (2CH),
129.75 (2CH), 130.05 (CH), 132.90 (C), 133.34 (C), 142.26
(CH), 149.63 (2CH), 163.27 (C), 164.89 (C)], 149.86 (triazole
C-3), 151.71 (triazole C-5), 167.92 (C]O).
5.1.6. 2-[(4-Phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-
yl)thio]acetohydrazide (7)
A solution of the corresponding compound 6 (10 mmol) in
n-butanol was refluxed with hydrazine hydrate (25 mmol) for
4 h. After cooling it to room temperature, a white solid ap-
peared. The solid was recrystallized from dimethyl sulfox-
ide/water (1:1) to afford the desired product. Yield 98%,
m.p. 239e241 ꢁC. IR (KBr, n, cmꢀ1): 1673 (C]O), 1619
5.1.7.3. N0-[(3,4-Dihydroxyphenyl)methylene]-2-[(4-phenyl-5-
pyridin-4-yl-4H-1,2,4-triazol-3-yl)-thio]acetohydrazide (9c).
Yield 89%, m.p. 243e245 ꢁC. IR (KBr, n, cmꢀ1): 3193
(NH), 1688 (C]O), 1622 and 1609 (2C]N); Anal. Calcd

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H. Bayrak et al. / European Journal of Medicinal Chemistry 44 (2009) 1057e1066
(%) for C22H18N6O3S: C, 59.18; H, 4.06; N, 18.82. Found: C,
1
m, arH), 7.39e7.72 (9H, m, arH), 7.84e7.98 (1H, m, arH),
8.22 and 8.07 (1H, s, N]CH, trans/cis conformers), 8.57e
8.70 (2H, m, arH), 12.07 and 11.92 (1H, s, NH, trans/cis con-
formers); 13C NMR (DMSO-d6, d ppm): 34.65 (CH2), 119.87
and 119.69 (N]CH, trans/cis conformers), arC: [120.13
(3CH), 123.46 (CH), 127.76 (CH), 131.69 (2CH), 132.35
(CH), 133.16 (C), 134.70 (C), 136.04 (CH), 144.13 (CH),
147.42 (CH), 148.85 (CH), 149.96 (CH), 152.12 (C)], 153.35
(triazole C-3), 164.31 (triazole C-5), 168.51 (C]O).
59.12; H, 4.09; N, 18.79; H NMR (DMSO-d6, d ppm): 4.54
and 4.11 (2H, s, SeCH2, trans/cis conformers), 6.65e6.88
(2H, m, arH), 6.81e6.98 (2H, m, arH), 7.18e7.38 (3H, m,
arH), 7.42e7.63 (3H, m, arH), 8.12 and 7.84 (s, N]CH
trans/cis conformers), 8.65 (2H, br s, 2OH), 9.37e9.58 (2H,
m, arH), 11.59 and 11.50 (1H, s, NH, trans/cis conformers);
13C NMR (DMSO-d6, d ppm): 35.05 (CH2), 112.42 and
112.21 (N]CH, trans/cis conformers), arC: [111.31 (CH),
120.17 (2CH), 120.48 (2CH), 121.37 (CH), 125.08 (C),
125.20 (C), 127.32 (2CH), 130.04 (CH), 130.32 (CH),
133.07 (C), 133.16 (C), 133.89 (C), 144.53 (CH), 149.60
(CH)], 152.82 (triazole C-3), 162.44 (triazole C-5), 167.59
(C]O).
5.1.7.7. 2-[(4-Phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)thio]-
N0-[pyridin-3-ylmethylene]-acetohydrazide (9g). Yield 91%,
m.p. 186e188 ꢁC. IR (KBr, n, cmꢀ1): 3184 (NH), 1689
(C]O), 1618 and 1602 (2C]N); Anal. Calcd (%) for
C21H17N7OS: C, 60.71; H, 4.12; N, 23.60. Found: C, 60.68;
1
5.1.7.4. N0-[(2-Hydroxyphenyl)methylene]-2-[(4-phenyl-5-pyr-
H, 4.15; N, 23.64; H NMR (DMSO-d6, d ppm): 4.62 and
idin-4-yl-4H-1,2,4-triazol-3-yl)-thio]acetohydrazide
(9d).
4.42 (2H, s, SeCH2, trans/cis conformers), 7.21e7.29 (4H,
m, arH), 7.42e7.69 (6H, m, arH), 8.21 and 8.07 (1H, s,
N]CH, trans/cis conformers), 8.53e8.68 (3H, m, arH),
12.11 and 11.91 (1H, s, NH, trans/cis conformers); 13C NMR
(DMSO-d6, d ppm): 34.61 (CH2), 119.85 and 119.68
(N]CH, trans/cis conformers), arC: [121.36 (CH), 124.31
(CH), 127.44 (3CH), 130.11 (2CH), 130.34 (CH), 132.68 (C),
133.83 (C), 137.23 (CH), 143.77 (CH), 147.11 (CH), 149.16
(CH), 149.91 (CH), 152.21 (C)], 152.75 (triazole C-3), 164.03
(triazole C-5), 168.55 (C]O).
Yield 95%, m.p. 235e237 ꢁC. IR (KBr, n, cmꢀ1): 3189
(NH), 1686 (C]O), 1620 and 1606 (2C]N); Anal. Calcd
(%) for C22H18N6O2S: C, 61.38; H, 4.21; N, 19.52. Found:
1
C, 61.42; H, 4.25; N, 19.60; H NMR (DMSO-d6, d ppm):
4.65 and 4.22 (2H, s, CH2, trans/cis conformers), 6.87e7.05
(3H, m, arH), 7.28e7.44 (4H, m, arH), 7.52e7.71 (6H, m,
arH), 8.44 and 8.33 (1H, s, N]CH, trans/cis conformers),
10.91 and 10.58 (1H, s, ar-OH, trans/cis conformers), 12.32
and 11.61 (1H, s, CeNH, trans/cis conformers); 13C NMR
(DMSO-d6, d ppm): 34.89 and 34.44 (CH2, trans/cis con-
formers), 116.22 and 116.03 (N]CH trans/cis conformers),
arC: [119.29 (2CH), 126.04 (CH), 127.40 (2CH), 128.97
(CH), 130.14 (CH), 130.42 (CH), 131.20 (CH), 131.42
(CH), 141.15 (CH), 146.98 (CH), 149.98 (CH), 152.71 (C),
156.30 (C), 157.13 (C), 157.13 (C)], 152.07 (triazole C-3),
163.01 (triazole C-5), 167.95 (C]O).
5.1.7.8. 2-[(4-Phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)thio]-
N0-[pyridin-4-ylmethylene]-acetohydrazide (9h). Yield 87%,
m.p. 205e207 ꢁC. IR (KBr, n, cmꢀ1): 3189 (NH), 1687
(C]O), 1616 and 1600 (2C]N); Anal. Calcd (%) for
C21H17N7OS: C, 60.71; H, 4.12; N, 23.60. Found: C, 60.68;
1
H, 4.15; N, 23.64; H NMR (DMSO-d6, d ppm): 4.60 and
4.41 (2H, s, SeCH2, trans/cis conformers), 7.24e7.31 (4H,
m, arH), 7.42e7.69 (5H, m, arH), 8.21 and 8.12 (1H, s,
N]CH, trans/cis conformers), 8.51e8.59 (4H, m, arH),
12.10 and 11.90 (1H, s, NH, trans/cis conformers); 13C NMR
(DMSO-d6, d ppm): 33.91 (CH2), 119.82 and 119.67
(N]CH, trans/cis conformers), arC: [120.39 (2CH), 122.71
(CH), 125.83 (2CH), 129.17 (2CH), 130.41 (CH), 133.11 (C),
133.71 (C), 136.81 (CH), 144.13 (CH), 147.16 (CH), 148.31
(CH), 149.96 (CH), 150.21 (C)], 153.04 (triazole C-3), 164.78
(triazole C-5), 168.49 (C]O).
5.1.7.5. N0-[(2,6-Dichlorophenyl)methylene]-2-[(4-phenyl-5-
pyridin-4-yl-4H-1,2,4-triazol-3-yl)-thio]acetohydrazide (9e).
Yield 98%, m.p. 265e267 ꢁC. IR (KBr, n, cmꢀ1): 3186
(NH), 1686 (C]O), 1618 and 1600 (2C]N); Anal. Calcd
(%) for C22H16Cl2N6OS: C, 54.66; H, 3.34; N, 17.39. Found:
1
C, 54.68; H, 3.37; N, 17.41; H NMR (DMSO-d6, d ppm):
4.50 and 4.19 (2H, s, SeCH2, trans/cis conformers), 7.21e
7.37 (2H, m, arH), 7.39e7.70 (8H, m, arH), 8.41 and 8.22
(1H, s, N]CH, trans/cis conformers), 8.57 (2H, br s, arH),
12.14 and 11.99 (1H, s, NH, trans/cis conformers); 13C
NMR (DMSO-d6, d ppm): 34.52 (CH2), 115.92 and 115.68
(N]CH, trans/cis conformers), arC: [121.52 (2CH), 127.17
(3CH), 128.90 (2CH), 130.12 (2CH), 133.19 (C), 133.22
(C), 142.51 (CH), 149.72 (2CH), 150.23 (C), 165.37 (2C)],
150.49 (triazole C-3), 152.68 (triazole C-5), 167.79 (C]O).
5.1.8. 5-{[(4-Phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-
yl)thio]methyl}-1,3,4-oxadiazole-2-thiol (10)
Corresponding compound 7 (10 mmol) and CS2 (0.60 mL,
10 mmol) were added to a solution of KOH (0.56 g, 10 mol)
in 50 mL H2O and 50 mL ethanol. The reaction mixture was
refluxed for 3 h. Then, the reaction content was acidified
with conc. HCl. The precipitate was filtered off, washed
with H2O and recrystallized from ethanol to afford the desired
compound. Yield 57%, m.p. 293 ꢁC. IR (KBr, n, cmꢀ1): 2565
(SH), 1617 and 1596 (2C]N); Anal. Calcd (%) for
C16H12N6OS2: C, 52.16; H, 3.28; N, 22.81. Found: C, 52.20;
5.1.7.6. 2-[(4-Phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-yl)thio]-
N0-[pyridin-2-ylmethylene]-acetohydrazide (9f). Yield 89%,
m.p. 180e183 ꢁC. IR (KBr, n, cmꢀ1): 3187 (NH), 1691
(C]O), 1622 and 1606 (2C]N); Anal. Calcd (%) for
C21H17N7OS: C, 60.71; H, 4.12; N, 23.60. Found: C, 60.68;
1
1
H, 4.15; N, 23.64; H NMR (DMSO-d6, d ppm): 4.58 and
4.27 (2H, s, SeCH2, trans/cis conformers), 7.21e7.32 (1H,
H, 3.31; N, 22.78; H NMR (DMSO-d6, d ppm): 4.60 (2H,
s, CH2), 7.25e7.35 (2H, m, arH), 7.48e7.72 (5H, m, arH),

H. Bayrak et al. / European Journal of Medicinal Chemistry 44 (2009) 1057e1066
1065
8.60 (2H, br s, arH), 14.60 (1H, br s, SH); 13C NMR (DMSO-
d6, d ppm): 26.00 (CH2), arC: [121.99 (2CH), 127.34 (2CH),
130.04 (2CH), 130.44 (CH), 132.93 (C), 133.59 (C), 149.80
(2CH)], 150.99 (triazole C-3), 152.48 (oxadiazole C-2),
160.02 (triazole C-5), 172.36 (oxadiazole C-5).
d6, d ppm): 20.26 (CH3), 30.55 (CH2), arC: [117.41 (2CH),
127.43 (2CH), 129.40 (2CH), 130.13 (2CH), 130.51 (3CH),
130.78 (CH), 133.10 (C), 133.59 (C), 138.02 (C), 150.06
(CH), 151.99 (C)], 152.54 (thiadiazole C-2), 155.37 (triazole
C-3 and thiadiazole C-5), 165.47 (triazole C-5).
5.2. Antimicrobial activity
5.1.9. 5-{[(4-Phenyl-5-pyridin-4-yl-4H-1,2,4-triazol-3-
yl)thio]methyl}-2(4-methylphenyl)-amino-1,3,4-thiadiazole (14)
A mixture of the corresponding carbothioamide (8)
(10 mmol) in cold concentrated sulfuric acid (28 mL) was
stirred for 10 min. Then, the mixture was allowed reach to
cool to room temperature. After stirring for an additional
30 min, the resulting solution was poured into ice-cold water
and made alkaline to pH 8 with ammonia. The precipitated
product was filtered and recrystallized from ethanol to afford
the desired product. Yield 63%, m.p. 211e214 ꢁC. IR (KBr,
n, cmꢀ1): 3187 (NH), 1696 and 1604 (4C]N); Anal. Calcd
(%) for C23H19N7S2: C, 60.37; H, 4.19; N, 21.43. Found: C,
5.2.1. Antimicrobial activity assessment
All bacterial and yeast strains were obtained from the Hif-
zissihha Institute of Refik Saydam (Ankara, Turkey) and were
as follows: E. coli ATCC 25922, Y. pseudotuberculosis ATCC
911, Pseudomonas aeruginosa ATCC 27853, Enterococcus
faecalis ATCC 29212, S. aureus ATCC 25923, B. cereus 709
ROMA, C. tropicalis ATCC 13803 and C. albicans ATCC
60193. All the newly synthesized compounds were dissolved
in dimethyl sulfoxide (DMSO) and ethanol to prepare chemi-
cals of stock solution of 10 mg/1 mL.
1
60.41; H, 4.21; N, 21.39; H NMR (DMSO-d6, d ppm): 2.22
5.2.1.1. Agar-well diffusion method. Simple susceptibility
screening test using agar-well diffusion method [30] as adap-
ted earlier [31] was used. Each microorganism was suspended
in Mueller Hinton (MH) (Difco, Detroit, MI) broth and di-
luted approximately 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
substances was delivered into the wells. The plates were in-
cubated for 18 h at 35 ꢁC. Antimicrobial activity was evalu-
ated 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 re-
sults are summarized in Table 1.
(3H, s, CH3), 4.72 (2H, s, CH2), 10.30 (1H, s, NH), 7.16e
7.19 (2H, d, j ¼ 8 Hz, arH), 7.34 (2H, br s, arH), 7.48e7.53
(5H, m, arH), 7.58e7.69 (4H, m, arH); 13C NMR (DMSO-
Table 1
Screening for antimicrobial activity of compounds (mm)
Compound no.
Microorganisms and inhibition zone (mm)
Ec.
Yp.
Pa.
Ef.
Sa.
Bc.
Ct.
Ca.
3a
e
e
e
e
e
e
e
9
e
9
4aa
4ba
5aa
5ba
5ca
30
25
24
20
e
30
30
24
22
e
30
30
30
14
e
30
30
28
16
e
25
25
24
21
e
30
30
23
15
e
8
8
e
e
e
13
e
7
e
e
e
13
e
7
6a
e
e
e
e
e
e
7a
e
e
e
e
e
e
e
e
e
e
e
e
8a
9aa
9ba
9ca
24
30
e
30
e
30
25
e
30
e
30
38
e
30
e
28
30
e
30
e
28
30
e
25
e
25
23
e
25
e
e
e
e
e
e
e
e
e
6
e
e
e
e
e
e
e
e
6
Acknowledgement
9da
9ea
9fa
42
8
30
7
40
e
20
e
24
e
20
e
This project was supported by Karadeniz Technical Univer-
sity, BAP, Turkey (ref. no. 2005.111.002.3) and is gratefully
acknowledged.
9ga
9ha
10b
11b
12b
13b
14b
15b
Ethanol
DMSO
Amp.
Flu.
e
e
e
e
e
e
e
e
e
e
8
6
6
e
e
e
e
e
15
e
e
e
e
6
e
e
e
e
e
11
e
e
e
e
e
e
11
e
e
References
e
e
e
e
e
31
28
e
22
16
e
35
15
e
20
7
25
22
e
20
17
e
¨
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