Cyclization of some carbothioamide derivatives containing antipyrine and triazole moieties and investigation of their antimicrobial activities

Article abstract of DOI:10.1016/j.ejmech.2010.07.018

Acetohydrazide derivative containing both antipyrine and triazole nuclei (5) was obtained starting from ethyl hydrazinecarboxylate derivative (2) and 4-aminoantipyrine (1) by three steps. The treatment of compound 5 with CS ₂ afforded the conve

Full text of DOI:10.1016/j.ejmech.2010.07.018

European Journal of Medicinal Chemistry 45 (2010) 4726e4732
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Cyclization of some carbothioamide derivatives containing antipyrine and triazole
moieties and investigation of their antimicrobial activities
,
b
Hacer Bayrak a, Ahmet Demirbas a, Neslihan Demirbas a , Sengül Alpay Karaoglu
*
a Karadeniz Technical University, Department of Chemistry 61080 Trabzon, Turkey
b Rize University, Department of Biology, 53100 Rize, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Acetohydrazide derivative containing both antipyrine and triazole nuclei (5) was obtained starting from
ethyl hydrazinecarboxylate derivative (2) and 4-aminoantipyrine (1) by three steps. The treatment of
compound 5 with CS2 afforded the conversion of hydrazide function into 5-mercapto-1,3,4-oxadiazole
ring leading to the formation of 7. Then, 7 gave the product containing triazolotriazine moiety (9) by the
reaction with hydrazine hydrate. The synthesis of the compounds incorporating the 1,3,4-thiadiazole
(10aec), 1,2,4-triazole (11aec) or 1,3-thiazole (12, 13) nucleus as third heterocycle was performed by the
acidic or basic treatment of compounds 6aec which were obtained from the reaction of 5 with several
isothiocyanates, or by the condensation of 6a with two different phenacyl bromides, respectively.
The antimicrobial activity study revealed that all the compounds showed good activities except 3e5.
Ó 2010 Published by Elsevier Masson SAS.
Received 1 March 2010
Received in revised form
9 July 2010
Accepted 12 July 2010
Available online 19 August 2010
Keywords:
1H-pyrazole
1,3,4-Oxadiazole
1,2,4-Triazole
1,3,4-Thiadiazole
1,3-Thiazole
Antimicrobial activity
1. Introduction
the antimicrobial resistance. Beside the development of completely
new agents possessing chemical characteristics that clearly differ
The treatment of infectious diseases still remains an important
and challenging problem due to a combination of factors including
emerging infectious diseases and the increasing number of multi-
drug resistant microbial pathogens. In spite of wide range of anti-
microbial drugs with different mechanisms of action used to treat
with microbial infections either alone or in combination and also
the existence many compounds used in different phases of clinic
trials, microbial infections have been becoming a worldwide
problem. There is already evidence that antimicrobial resistance
associated with an increase in mortality. The problem with clini-
cally used drugs is not only the increasing microbial resistance, but
also they are accompanied by toxic side effects that are often dose
limiting [1e5]. Moreover, the long term use of several drugs to treat
microbial infections may cause serious health problems, especially
in patients with impaired liver or kidney functions.
from those of existing ones, there is another approach containing to
combine two or more pharmacophores into a single molecule.
Therefore, a single molecule containing more than one pharmaco-
phore, each with different mode of action, could be beneficial for the
treatment of microbial infectious. These merged pharmacophore
may be addressing the active site of different targets and offer the
possibility to overcome drug resistance. In addition, this approach
can also reduce unwanted side effects [6e11].
Since the discovery of antipyrine as the first pyrazolin-5-one
derivative used in the treatment of pain and inflammation, a great
deal has been devoted for the synthesis of pyrazole derivatives as
potent anti-inflammatory, analgesic, antipyretic and antimicrobial
agents [12e17]. Furthermore, the synthesis of 1,2,4-triazole deriv-
atives possessing such diverse pharmacological properties as
antimicrobial [18e21], anti-inflammatory [22], analgesic [23],
antitumorial [24], antihypertensive [25], anticonvulsant and anti-
viral activities [26] has been attracting widespread attention. With
regarding to antimicrobial activity, triazole moiety resembles
structurally similar to imidazole molecule. Although triazole and
imidazole nuclei act by the same mechanism of action, triazoles
have been possess advantages over imidazoles, which have slow
metabolic rate, oral bioavailability, and less effect on human sterol
To search and synthesize of combinational chemotherapeutic
drugs with different mechanisms of action and with low side effects
constitute an important part of the methods that aims to overcome
* Corresponding author. Tel.: þ90 462 3772600; fax: þ90 4623253196.
E-mail address: ndemirbas651@ktu.edu.tr (N. Demirbas).
0223-5234/$ e see front matter Ó 2010 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2010.07.018

H. Bayrak et al. / European Journal of Medicinal Chemistry 45 (2010) 4726e4732
4727
synthesis. For these reasons imidazoles have slowly being replaced
by triazole molecules. It was reported that incorporation of various
halo substituents into the heterocyclic ring systems augments
biological activities considerably. Furthermore, introduction
a chloro-substituted benzene moiety into triazole scaffold results in
increase of broad spectrum of antimicrobial activity due to the
ability of halogen to act as polar hydrogen or hydroxy mimic.
Substitution of hydrogen by halogen atom has been a strategy in
designing molecules having biological activity [27].
The treatment of compound 5 with carbon disulfide in the
presence of KOH resulted in the formation of 4-(1,5-dimethyl-3-
oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-5-(4-chlorobenzyl)-2-
[(5-sulfanyl-1,3,4-oxadiazol-2-yl)methyl]-2,4-dihydro-3H-1,2,
4-triazole-3-one (7). Then, compound 7 was concerted to 1,5-
dimethyl-4-{7-(4-chlorobenzyl)-3-thioxo-2,10-dihydro-3H,6H-bis
[1,2,4]triazolo[5,1-c:30,40-f][1,2,4]triazin-6-yl}-2-phenyl-1,2-dihyd-
ro-3H-pyrazol-3-one (9) by the treatment with hydrazine hydrate
via the formation of compound 8 that was not isolated (Fig. 1).
Another pharmacophore heterocycle, 1,3,4-thiadiazole nucleus
has been incorporated into some therapeutically important drugs,
such as Acetazolam and Cefazolin [28e30]. In addition to their well
documented potential antimicrobial activities, thiadiazole ring
system has occupied a unique position in the design and synthesis
of novel biologically active agents with remarkable analgesic and
anti-inflammatory activities [31e39].
3. Results and discussion
The main aim of the present study is to synthesize and inves-
tigate the antimicrobial activities of new antipyrine derivatives
containing also 1,2,4-triazole, 1,3,4-thiadiazole, 1,3,4-oxadiazole
and/or 1,3-thiazole ring in the same structure.
Divers pharmacological properties of the compounds containing
antipyrine, triazole or thiadiazole moieties have prompted the
medicinal chemists to design and synthesize hybrid molecules
incorporating antipyrine and triazole scaffolds in a single molecule
[40]. Moreover, the compounds including both 1,2,4-triazole and
1,3,4-thiadiazole moieties in their structures have been obtained as
antimicrobial compounds in our laboratory [41e44].
Keeping this observation in view and in continuation of our
study on the synthesis of biologically active compounds, this paper
has presented the synthesis of new antipyrine derivatives incor-
porating different pharmacophores as hybrid molecules possessing
antimicrobial activity.
Synthesis of the intermediate and target compounds was per-
formed according to the reactions outlined in Fig. 1. The starting
compound ethyl 2-[2-(4-chlorophenyl)-1-ethoxyethylidene] hydra-
zinecarboxylate (2) was prepared following a previously reported
literature procedure [45].
In the present study, compound 4 was obtained by two methods
in good yields. The 1H NMR spectrum of compound 4 exhibited
additional signals derived from ester group were observed at
1.21 ppm (eOCH2CH3) and 4.18 (eOCH2CH3) ppm integrating for
three protons and two protons, respectively. This group resonated
at 13.88 and 61.08 ppm in the 13C NMR spectrum. When compound
4 was converted to the corresponding hydrazide (5) the signals
derived from ester group disappeared in the 1H NMR spectrum of 5,
instead, new signals representing hydrazide structure appeared at
4.28 (eNHNH2) and 9.31 (eNHNH2) ppm (controlled by changing
with D2O) integrating for two protons and one proton, respectively.
The IR spectra of compounds 4 and 5 showed an additional peak
at 1753 (for ester) or 1732 (for hydrazide) cmꢀ1 due to exocyclic-
carbonyl function originated from ester or hydrazide structure
beside the endocyclic-carbonyl peak at the position 3 of 1,2,4-tri-
azole ring.
2. Chemistry
The treatment of 4-aminoantipyrine (1) with ethyl 2-[2-(4-
chlorophenyl)-1-ethoxyethylidene]hydrazinecarboxylate
(2)
resulted in the formation of 5-(4-chlorobenzyl)-4-(1,5-dimethyl-3-
oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-yl)-2,4-dihydro-3H-1,2,
4-triazole-3-one (3). The reaction of compound 3 with ethyl
bromoacetate in the presence of sodium ethoxide produced ethyl
[4-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-3-
(4-chlorobenzyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]acetate
(4). Then, this ester (4) was converted to the corresponding
hydrazide derivative, 2-[4-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-il)-3-(4-chlorobenzyl)-5-okso-4,5-dihydro-
1H-1,2,4-triazole-1-yl]acetohydrazide (5) by the reaction of 4 with
hydrazine hydrate. 2-{[4-(1,5-Dimethyl-3-oxo-5-phenyl-2,3-dihy-
dro-1H-pyrazole-4-yl)-3-(4-chlorobenzyl)-5-oxo-4,5-dihydro-1H-1,
2,4-triazole-1-yl]acetyl}-N-(aryl)hydrazinecarbotiyoamides (6aec)
were obtained by the reaction of compound 5 with phenylisothio
cyanate (for 6a), benzylisothiocyanate (for 6b) or 4-flouropheny
lisothiocyanate (for 6c).
Thetreatmentofcompounds6aecwithcoldconcentratedsulfuric
acid caused to the conversion of carbothioamide structure into 1,3,
4-thiadiazol rings; thus, 4-(2-phenyl-1,5-dimethyl-3-oxo-2,3-dihy-
dro-1H-pyrazol-4-yl)-5-(4-chlorobenzyl)-2-{[5-(alkylamino)-1,3,4-
thiadiazol-2-yl]methyl}-2,4-dihydro-3H-1,2,4-triazol-3-ones (10aec)
was obtained. On the other hand, the cyclisation of the same
intermediates, 6aec, in the presence of 2N NaOH produced
4-(2-phenyl-1,5-dimethyl-3-oxo-2,3-dihydro-1H-pyrazol-4-yl)-5-(4-
chlorobenzyl)-2-[(4-alkyl-5-mercapto-4H-1,2,4-triazol-3-yl)methyl]-
2,4-dihydro-3H-1,2,4-triazol-3-ones (11aec). The synthesis of
2-[3-(4-chlorobenzyl)-4-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-
1H-pyrazol-4-yl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]-N0-[4-
(4-chloro or 4-nitrophenyl)-3-phenyl-1,3-thiazol-2(3H)-ylidene]
acetohydrazides (12, 13) was performed by the reaction of
compound 6a with 4-chloro or 4-nitrophenacyl bromide.
As different from compound 5, 1H and 13C NMR spectra of
compounds 6aec exhibited additional signals due to carbothioa-
mide moiety at the related chemical shift values. In addition,
compounds 6aec gave relatively stable molecular ion peak in the
Mass spectra.
With the conversion of compound 5 into 4-(1,5-dimethyl-3-oxo-2-
phenyl-2,3-dihydro-1H-pyrazol-4-yl)-5-(4-chlorobenzyl)-2-[(5-sulf-
anyl-1,3,4-oxadiazol-2-yl)methyl]-2,4-dihydro-3H-1,2,4-triazole-3-
one (7), the eNHeNH2 signals disappeared; instead, new signal
belonging to eSH proton appeared at 14.73 ppm as singlet in the 1H
NMR spectrum of compound 7. Furthermore, the IR spectrum of
compound 7 showed no signals belonging to eNHNH2 group; while
new signal belonging to eSH proton was recorded at 2549 cmꢀ1. The
C-2 and C-5 carbon atoms of 1,3,4-oxadiazole ring resonated at
133.87 (C-2) and 160.01 (C-5), respectively, in the 13C NMR spectrum
of compound 7. Moreover, the elemental analysis of compound 7 was
consistent with the assigned structure.
In the IR spectrum of compound 9, only one signal at 1681 cmꢀ1
appeared due to carbonyl function. Moreover, in the 1H NMR
spectrum of compound 9, no signal derived from eSH group was
recorded, instead, new signals due to eNH group was detected at
9.37 ppm (exchangeable with D2O).
In the IR spectra of compounds 10aec, the eNHe stretching
band appeared at 3032e3036 cmꢀ1. The eNHe proton resonated at
about 10.39e10.42 ppm in the 1H NMR spectrum of compounds
10aec. The IR spectra of compounds 11aec displayed eSH
stretching bands at 2857e2923 cmꢀ1. Moreover, in the 1H NMR
spectra of compounds 11aec, additional signal due to eSH group

4728
H. Bayrak et al. / European Journal of Medicinal Chemistry 45 (2010) 4726e4732
O
O
O
Cl
N
NNH
NH
N
Cl
Cl
NH2
OEt
N
N
NHNH2
N
N
OEt
O
N
O
O
O
N
N
O
i
N
N
2
Cl
ii
O
O
N
N
N
N
N
5
1
3
4
N
N
iii
iv
Cl
O
S
SH
O
N
N
Cl
NHNH
NHR
O
N
N
N
O
O
N
N
N
O
O
Cl
N
N
NHN
N
N
N
7
S
O
N
X
v
ii
N
N
6a-c
O
Cl
SH
N
N
N
N
N
NH2
O
N
12: X= -Cl
13: X= -NO2
O
N
N
vi
vii
8
N
N
N
N
N
Cl
NH
Cl
SH
NHR
N
S
N
N
N
N
N
N
N
R
S
N
Cl
O
N
O
N
N
N
N
O
O
O
N
N
N
N
N
,
6
,
10 11
O
R
a
-C6H5
N
N
b
c
-CH2C6H5
-C6H4F(4-)
10a-c
11a-c
9
Fig. 1. Reactions and conditions: i: BrCH2COOEt, ii: H2NNH2, iii: KOH/CS2, iv: RNCS, v: C6H4COCH2X, vi: H2SO4, vii: NaOH.
was observed at 14.04e14.01 ppm (controlled with changing by
D2O), while the eNHe signals disappeared.
Escherichia coli (Ec) Enterobacter aerogenes (Ea), Yersinia pseudo-
tuberculosis (Yp) and Gram negative bacillus, Pseudomonas aerugi-
nosa (Pa), whereas the compounds (10aec) displayed completely
inactivity towards Staphylococcus aureus (Sa), Enterococcus faecalis
(Ef) which are Gram positive cocci, and Bacillus cereus (Bc) that is
Gram positive spore bacillus. When 1,3,4-thiadiazole nucleus
replaced by 1,2,4-triazole ring in the structure of compounds
11aec, good antimicrobial and antitubercular activities were
observed on the test microorganisms except Candida albicans (Ca),
Candida tropicalis (Ct), Aspergillus niger (An) and Saccharomyces
cerevisiae (Sc). Compound 12 that was obtained from the conden-
sation of 6a with 4-chlorophenacyl bromide displayed good
activities against Ec, Yp and Ms. On the other hand, the replacement
of chlorine atom in 12 by a bulky nitro group leading to 13 resulted
in good activities towards all the test microorganisms except Ca, Ct,
Sc and An.
The IR and 1H NMR spectra of compounds 12 and 13 showed no
signal representing eSH group.
All the compounds have been tested for their antimicrobial
activities. The results are presented in Table 1. All compounds,
except 3e5, exhibited activity against Mycobacterium smegmatis
(Ms) that is atipic tuberculosis factor, whereas none of them was
found to have antifungal activity.
According to the obtained results, it can be concluded that the
conversion of carbothioamide moiety into 5-mercapto-1,3,4-oxa-
diazole nucleus in compound 7, and further ring fusion into
compound 9 afforded an increase in the antimicrobial and antitu-
bercular activity. On the other hand, the cyclization of linear car-
bothioamide side change of 6aec into 1,3,4-thiadiazole ring caused
to an increase in the activity of 10aec against enteric bacteria,

H. Bayrak et al. / European Journal of Medicinal Chemistry 45 (2010) 4726e4732
4729
Yield 78%, m.p. 270e271 ꢂC. IR (KBr,
y
, cmꢀ1): 3094 (NH),1724and
Table 1
Screening for antimicrobial activity of the compounds (mm).
1651 (C]O),1623 (C]N); Anal. Calcd (%) for C20H18N5O2Cl: C, 60.69;
H, 4.58; N, 17.69. Found: C, 60.68; H, 4.61; N, 17.49. 1H NMR (DMSO-
No
Microorganisms and inhibition zone (mm)
d6, d ppm): 1.80 (3H, s, CH3), 3.09 (3H, s, CH3), 3.80 (2H, AB system,
Ec
Ea
Yp
Pa
Sa
Ef
Bc
Ms
Ca
Ct
Sc
An
CH2), 7.13 (2H, d, arH, J ¼ 8.3 Hz), 7.30e7.50 (5H, m, arH), 7.59 (2H, t,
3
4
5
6a
6b
6c
7
9
10a
10b
10c
11a
11b
11c
12
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
arH, J ¼ 7.8 Hz), 11.78 (1H, s, NH); 13C NMR (DMSO-d6,
d ppm): 9.67
e
e
e
e
e
e
e
e
(CH3), 31.25 (CH2), 34.74 (CH3), 100.78 (C-antipyrine), arC:[124.62
(2CH), 127.10 (CH), 128.11 (2CH), 129.06 (2CH), 130.25 (2CH), 133.96
(2C), 160.11 (C)], 131.23 (antipyrine C), 146.72 (triazole C-5), 152.59
(antipyrine C]O), 153.97 (triazole C-3), MS: m/z (%) 102 (20), 215
(100), 216 (19), 316 (13), 396 (Mþ, 78), 398 (28), 418 (20).
e
e
e
e
e
e
e
e
10
15
15
35
35
25
25
20
25
35
35
15
15
10
e
8
8
8
8
8
8
28
28
e
10
10
10
30
28
e
10
15
15
38
40
25
25
25
34
33
30
10
25
10
8
10
10
30
28
25
25
25
30
30
30
12
30
18
10
10
30
28
35
30
20
30
30
28
e
10
10
30
30
e
30
30
25
25
25
25
30
30
e
e
e
e
4.1.2. Ethyl [4-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-3-(4-chlorobenzyl) -5-oxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl]acetate (4)
Method 1: Compound 3 (10 mmol) was refluxed with 1 equiv. of
sodium in absolute ethanol for 2 h. Then, ethyl bromoacetate
(10 mmol) was added and refluxed for an additional 8 h. After
evaporating the solvent under reduced pressure, a solid appeared.
The crude product was recrystallized from ethanol/water (1:2) to
afford compound 4.
e
e
e
25
30
30
e
20
28
28
e
30
30
30
e
13
e
20
18
30
35
18
10
20
15
Amp.
Strep.
Flu
10
35
25
25
>25
ND
Ec: Escherichia coli ATCC 25922, Ea: Enterobacter aeroginosa ATCC 13048, Yp: Yersinia
pseudotuberculosis ATCC 911, Pa: Pseudomonas aeruginosa ATCC 43288, Sa: Staphy-
lococcus aureus ATCC 25923, Ef: Enterococcus faecalis ATCC 29212, Bc: Bacillus cereus
702 Roma, Ms: Mycobacterium smegmatis ATCC607, Ca: Candida albicans ATCC
60193, Ct: Candida tropicalis ATCC 13803, Saccharomyces cerevisiae RSKK 251, An:
Aspergillus niger RSKK 4017, Amp.: Ampisilin, Flu.: Flukonazol, (ꢀ): Aktivite yok, ND:
Not detected.
Method 2: To a solution of compound 3 (10 mmol) in ethanol,
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 crude
product was recrystallized from ethanol/water (1:2) to afford
compound 4. Yield 59%, m.p. 167e168 ꢂC. IR (KBr, , cmꢀ1): 1753,
y
4. Experimental
1724 and 1662 (3C]O),1592 (C]N),1207 (CeO); Anal. Calcd (%) for
C24H24N5O4Cl: C, 59.81; H, 5.02; N, 14.53.Found: C, 59.81; H, 5.00;
4.1. Chemistry
N, 14.28. 1H NMR (DMSO-d6,
d
ppm): 1.21 (3H, t, CH3, J ¼ 7.0 Hz),
1.82 (3H, s, CH3), 3.11 (3H, s, CH3), 3.89 (2H, AB system, CH2), 4.18
(2H, q, CH2, J ¼ 7.3 Hz), 4.63 (2H, s, CH2), 7.10 (2H, d, arH, J ¼ 8.3 Hz),
7.32e7.42 (5H, m, arH), 7.57 (2H, t, arH, J ¼ 7.5 Hz); 13C NMR
All the chemicals were purchased from Fluka Chemie AG Buchs
(Switzerland) are used without further purification. Melting points
of the synthesized compounds are determined in open capillaries
on a Büchi B-540 melting point apparatus and are uncorrected and
are uncorrected. Reactions are monitored by thin-layer chroma-
tography (TLC) on silica gel 60 F254 aluminium sheets. The mobile
phase was ethanol and ethyl acetate (1:1) and detection was made
using UV light. IR spectra are recorded as potassium bromide
pellets using a PerkineElmer 1600 series FTIR spectrometer. 1H
NMR and 13C NMR spectra were recorded on BRUKER AVENE II
400 MHz NMR Spectrometer (Chemical shift in ppm down field
from TMS as an internal reference). 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 for compounds 3, 6aec and 11a at a Quattro LC-MS
(70 eV) Instrument.
(DMSO-d6, d ppm): 9.71 (CH3), 13.88 (CH3), 31.05 (CH2), 34.72 (CH3),
46.46 (CH2), 61.08 (CH2), 100.85 (antipyrine C), arC:[124.92 (2CH),
127.36 (CH), 128.30 (2CH), 129.21 (2CH), 130.32 (2CH), 131.48 (C),
133.72 (C), 133.86 (C)], 146.36 (triazole C-5), 152.35 (antipyrine C),
152.88 (triazole C-3), 159.88 (antipyrine C]O), 167.69 (C]O).
4.1.3. 2-[4-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-
4-il)-3-(4-chlorobenzyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazole-1-yl]
acetohydrazide (5)
A solution of compound 4 (10 mmol) in ethanol was refluxed
with hydrazine hydrate (30 mmol) for 5 h (controlled with TLC).
After cooling it to room temperature, acetone was added to the
mixture and was kept overnight in cold. The resulting solid sepa-
rated was collected by filtration and recrystallized from ethanol to
afford the desired product. Yield 96%, m.p. 267e268 ꢂC. IR (KBr,
y,
cmꢀ1): 3190 and 3093 (NHeNH2), 1732 and 1655 (3C]O), 1596
(C]N); Anal. Calcd (%) for C22H22N7O3Cl: C, 56.47; H, 4.74; N, 20.95.
4.1.1. 5-(4-Chlorobenzyl)-4-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazole-4-yl)-2,4-dihydro-3H-1,2,4-triazole-3-one (3)
Method 1: A mixture of compound 1 (10 mmol) and 4-amino-
antipyrin (10 mmol) was heated at 110e120 ꢂC in an oil bath for 2 h.
Then, n-butyl acetate-diethyl ether (1:2) was added and the reac-
tion mixture was kept overnight in cold. The solid formed was
filtered off and recrystallized several times from ethyl acetate to
afford the target compound.
Method 2: The solution of compound 1 (10 mmol) in water was
refluxed with 4-aminoantipyrin (10 mmol) for 5 h (controlled with
TLC). The solvent was evaporated under reduced pressure and an
oily product was obtained. n-Butyl acetate-diethyl ether (1:2) was
added into it and was kept overnight in cold. The solid formed was
filtered off and recrystallized several times from ethyl acetate to
afford the target compound.
Found: C, 56.39; H, 4.78; N, 20.93. 1H NMR (DMSO-d6,
d ppm): 1.78
(3H, s, CH3), 3.06 (3H, s, CH3), 3.37 (2H, s, CH2), 3.78 (2H, AB system,
CH2), 4.28 (2H, s, NH), 7.10 (2H, d, arH, J ¼ 8.2 Hz), 7.23e7.42 (5H, m,
arH), 7.56 (2H, t, arH, J ¼ 8.2 Hz), 9.31 (1H, s, NH); 13C NMR (DMSO-
d6,
d ppm): 10.56 (CH3), 32.14 (CH2), 35.62 (CH3), 39.27e40.94
(DMSO-d6þCH2), 101.61 (antipyrine C), arC:[125.51 (CH), 125.66
(CH), 128.01 (2CH), 129.04 (2CH), 129.99 (2CH), 131.18 (CH), 132.14
(C), 134.58 (C), 134.77 (C)], 147.65 (triazole C-5), 153.51 (antipyrine
C), 154.88 (triazole C-3), 160.99 (C]O), 166.56 (antipyrine C]O).
4.1.4. General method for the synthesis of compounds 6aec
A mixture of compound 5 (10 mmol) and phenylisothiocyanate
(for 6a), benzylisothiosyanate (for 6b) or 4-fluorophenylisothiosya
nate (for 6c) (10 mmol) was allowed to reflux in ethanol for 4 h.

4730
H. Bayrak et al. / European Journal of Medicinal Chemistry 45 (2010) 4726e4732
After evaporating the solvent under reduced pressure an oily
product appeared; acetone was added into it and kept overnight in
cold. This crude product was filtered off and recrystallized from
ethanol to afford the desired products.
4.1.5. 4-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-
yl)-5-(4-chlorobenzyl)-2-[(5-sulfanyl-1,3,4-oxadiazol-2-yl)methyl]-
2,4-dihydro-3H-1,2,4-triazole-3-one (7)
Compound 5 (10 mmol) and CS2 (0.6 mL, 10 mmol) were added
to a solution of KOH (0.56 g, 10 mmol) in 50 mL H2O and 50 mL
ethanol and the 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
4.1.4.1. 2-{[4-(1,5-Dimetyl-3-oxo-5-phenyl-2,3-dihydro-1H-pyr-
azole-4-il)-3-methyl-5-okso-4,5-dihydro-1H-1,2,4-triazole-1-yl]
acetyl}-N-phenylhydrazinecarbothioamide (6a). Yield 95%, m.p.
165e166 ꢂC. IR (KBr,
y
, cmꢀ1): 3232 (3NH), 1704 and 1651 (3C]O),
desired compound. Yield 88%, m.p. 232e233 ꢂC. IR (KBr, , cmꢀ1):
y
1615 (C]N), 1191 (C]S); Anal. Calcd. (%) for C29H27O3N8SCl: C,
2549 (SH), 1722 and 1640 (2C]O), 1615, 1588 and 1491 (3C]N);
57.75; H, 4.51; N, 18.50. Found: C, 57.69; H, 4.54; N, 18.46. 1H NMR
Anal. Calcd (%) for C23H20N7O3SCl: C, 54.17; H, 3.95; N, 19.31. Found:
(DMSO-d6,
d
ppm): 1.84 (3H, s, CH3), 3.10 (3H, s, CH3), 3.82 (2H, AB
C, 54.23; H, 3.90; N, 19.28; 1H NMR (DMSO-d6,
d ppm): 1.85 (3H, s,
system, CH2), 4.58 (2H, s, CH2), 7.11e7.21 (3H, m, arH), 7.34e7.52
(8H, m, arH), 7.56e7.60 (3H, m, arH), 9.69 (1H, s, NH), 9.78 (1H, s,
CH3), 3.13 (3H, s, CH3), 3.82 (2H, AB system, CH2), 5.15 (2H, s, CH2),
7.15 (2H, d, arH, J ¼ 8.2 Hz), 7.24e7.42 (5H, m, arH), 7.57 (2H, t, arH,
NH), 10.38 (1H, s, NH); 13C NMR (DMSO-d6,
d
ppm): 9.81 (CH3),
J ¼ 7.8 Hz), 14.73 (1H, brs, SH); 13C NMR (DMSO-d6,
d ppm): 9.91
31.06 (CH3), 34.76 (CH2), 38.05e40.58 (DMSO-d6 þ CH2), 100.57
(antipyrine C), arC: [124.90 (2CH), 125.21 (2CH), 127.36 (2CH),
128.01 (2CH), 128.29 (2CH), 129.23 (2CH), 130.40 (2CH), 131.45 (C),
133.74 (C), 133.89 (C), 138.84 (C)], 146.10 (triazole C-5), 152.48
(antipyrine C), 153.19 (triazole C-3), 159.93 (antipyrine C]O),
166.401 (C]O), 181.98 (C]S); MS m/z (%): 176 (19), 229 (17), 357
(10), 468 (44), 490 (20), 532 (17), 585 (27), 603 (Mþ, 56), 625 (100),
627 (45), 648 (19).
(CH3), 31.30 (CH2), 34.80 (CH3), 38.10e40.60 (DMSO-d6 þ CH2),
100.13 (antipyrine C), arC: [125.40 (2CH), 127.86 (CH), 128.55 (2CH),
129.50 (2CH), 130.62 (2CH), 131.77 (C), 133.62 (2C)], 133.87 (oxa-
diazole C-2), 147.51 (triazole C-5), 152.32 (antipyrine C), 152.76
(triazole C-3), 159.09 (antipyrine C]O), 160.01 (oxadiazole C-5).
4.1.6. 1,5-Dimethyl-4-{7-(4-chlorobenzyl)-3-thioxo-2,10-dihydro-
3H,6H-bis[1,2,4]triazolo [5,1-c:30,40-f][1,2,4]triazin-6-yl}-2-phenyl-
1,2-dihydro-3H-pyrazole-3-one (9)
4.1.4.2. 2-{[4-(1,5-Dimethyl-3-oxo-5-phenyl-2,3-dihydro-1H-pyr-
azole-4-yl)-3-(4-chlorobenzyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazole-
1-yl]acetyl}-N-benzylhydrazinecarbothioamide (6b). Yield 87%, m.p.
To the solution of compound 7 (10 mmol) in ethanol hydrazine
hydrate (30 mmol) was added and the reaction mixture was
refluxed for 4 h. On cooling it overnight in cold, a solid obtained.
The crude product was filtered off, washed with H2O and recrys-
tallized from ethanol to afford the desired compound. Yield 75%,
140e141 ꢂC. IR (KBr, , cmꢀ1): 3288 and 3164 (3NH), 1708 and 1652
y
(3C]O), 1548 (C]N), 1189 (C]S); Anal. Calcd. (%) for
C30H29N8O3SCl: C, 58.39; N, 18.16; H, 4.74. Found: C, 58.37; N, 18.15;
m.p. 219e220 ꢂC. IR (KBr, , cmꢀ1): 3195 (NH), 1681 (C]O), 1607,
y
H, 4.88. 1H NMR (DMSO-d6,
d
ppm): 1.83 (3H, s, CH3), 3.09 (3H, s,
1590, 1547 and 1489 (4C]N); Anal. Calcd (%) for C23H20N9OSCl: C,
CH3), 3.79 (2H, AB system, CH2), 4.51 (2H, s, CH2), 4.76 (2H, s, CH2),
7.10 (3H, d, arH, J ¼ 8.6 Hz), 7.14e7.44 (9H, m, arH), 7.59 (2H, t, arH,
J ¼ 7.5 Hz), 8.58 (1H, s, NH), 9.49 (1H, s, NH), 10.18 (1H, s, NH); 13C
54.60; H, 3.98; N, 24.91. Found: C, 54.57; H, 3.90; N, 24.94; 1H NMR
(DMSO-d6,
CH2), 3.58 (2H, s, CH2), 7.20e7.38 (10H, m, arH), 7.40e7.56 (4H, m,
arH), 9.37 (1H, s, NH); 13C NMR (DMSO-d6,
ppm): 11.38 (CH3),
d ppm): 2.01 (3H, s, CH3), 2.98 (3H, s, CH3), 3.35 (2H, s,
NMR (DMSO-d6,
d
ppm): 9.83 (CH3), 31.17 (CH2), 34.76 (CH3),
d
38.49e40.17 (DMSO-d6þCH2), 46.49 (CH2), 100.61 (antipyrine C),
arC: [124.89 (2CH), 126.55 (CH), 126.81 (CH), 127.36 (2CH), 127.98
(2CH), 128.28 (2CH), 129.23 (2CH), 130.38 (2CH), 131.46 (C), 133.74
(C), 133.88 (C), 139.05 (C)], 146.06 (triazole C-5), 152.45 (antipyrine
C), 153.17 (triazole C-3), 159.93 (antipyrine C]O), 166.40 (C]O),
181.98 (C]S); MS m/z (%): 116.78 (29), 116.90 (25), 124.70 (22),
203.67 (16), 203.98 (22), 228.94 (28), 270.32 (14), 337.97 (16),
356.08 (20), 357.15 (29), 358.09 (15), 467.94 (33), 468.19 (53),
507.99 (21), 530.25 (23), 532.26 (20), 617.21 (Mþ, 64), 639. 03 (91),
639.16 (100), 641.11 (M þ 1 þ Na, 51), 642.30 (19).
36.08 (CH3), 38.27e40.77 (DMSO-d6 þ CH2), 41.69 (CH2), 106.84
(antipyrine C), arC: [124.76 (2CH), 127.50 (2CH), 128.89 (2CH),
129.88 (2CH þ C), 131.36 (CH þ C), 135.11 (C)], 135.55 (antipyrine C),
152.82 (triazole C-5), 158.01 (triazole C-3), 162.43 (triazole C-5),
163.45 (antipyrine C]O), 170.94 (C]S).
4.1.7. General method for the synthesis of compounds 10aec
A mixture of the corresponding carbothioamide 6aec (10 mmol)
and concentrated sulfuric acid (64 mmol) was stirred in an ice bath
for 15 min. Then, the mixture was allowed to reach 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, washed with
water and recrystallized from ethanol to afford the desired product.
4.1.4.3. 2-{[4-(1,5-Dimethyl-3-oxo-5-phenyl-2,3-dihydro-1H-pyr-
azole-4-yl)-3-(4-chlorobenzyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazole-
1-yl]acetyl}-N-(4-fluorophenyl) hydrazinecarbothioamide (6c). Yield
83%, m.p. 139e140 ꢂC. IR (KBr, , cmꢀ1): 3233 (3NH), 1710 and 1657
y
(3C]O), 1590 (C]N), 1213 (C]S); Anal. Calcd. (%) for
4.1.7.1. 4-(2-Phenyl-1,5-dimethyl-3-oxo-2,3-dihydro-1H-pyrazole-4-
yl)-5-(4-chlorobenzyl)-2-{[5-(phenylamino)-1,3,4-thiadiazol-2-yl]
methyl}-2,4-dihydro-3H-1,2,4-triazole-3-one (10a). Yield 89%, m.p.
C29H26N8O3SFCl: C, 56.08; N, 18.04; H, 4.22. Found C, 56.19; N,
18.00; H, 4.28. 1H NMR (DMSO-d6,
d ppm): 1.86 (3H, s, CH3), 3.11
(3H, s, CH3), 3.79 (2H, AB system, CH2), 4.58 (2H, s, CH2), 7.16e7,21
(4H, m, arH), 7.26e7.53 (6H, m, arH), 7.57 (2H, t, arH, J ¼ 7.8 Hz),
229e230 ꢂC. IR (KBr, , cmꢀ1): 3032 (NH), 1720 and 1681 (2C]O);
y
C29H25O2N8SCl: C, 59.53; N,19.15; H, 4.31. Found C, 59.48; N,19.16; H,
9.79 (2H, brs, 2NH), 10.39 (1H, s, NH); 13C NMR (DMSO-d6,
d
ppm):
4.26; 1H NMR (DMSO-d6,
d
ppm): 1.84 (3H, s, CH3), 3.13 (3H, s, CH3),
9.84 (CH3), 31.14 (CH2), 34.77 (CH3), 38.48e40.16 (DMSO-d6þCH2),
100.61 (antipyrine C), arC: [114.49 (2CH), 114.94 (2CH), 124.93
(2CH), 127.39 (CH), 128.31 (2CH), 129.25 (2CH), 130.43 (2CH),
131.50 (C), 133.74 (C), 133.91 (C), 135.19 (2C)], 146.14 (triazole C-5),
152.48 (antipyrine C), 153.24 (triazole C-3), 159.98 (antipyrine C]
O), 162.02 (C]O), 166.49 (C]S); MS m/z (%): 107.09 (100), 115.01
(50), 122.94 (41), 124.76 (19), 152.55 (24), 154.81 (11), 178.64 (10),
229.76 (10), 468.07 (21), 490.14 (23), 508.12 (23), 603.07 (13),
621.11 (Mþ, 33).
3.88(2H, ABSystem,CH2),5.23(2H,s,CH2),7.10(3H, d,arH,J¼ 8.3Hz),
7.37e7.45 (6H, m, arH), 7.52e7.56 (5H, m, arH), 10.42 (1H, s, NH).
4.1.7.2. 4-(2-Phenyl-1,5-dimethyl-3-oxo-2,3-dihydro-1H-pyrazole-4-
yl)-5-(4-chlorobenzyl)-2-{[5-benzylamino)-1,3,4-thiadiazol-2-yl]
methyl}-2,4-dihydro-3H-1,2,4-triazole-3-on (10b). Yield 80%, m.p.
212e213 ꢂC. IR (KBr, , cmꢀ1): 3034 (NH), 1709 and 1641 (2C]O);
y
C30H27O2N8SCl: C, 60.14; N, 18.70; H, 4.54. Found C, 59.99; N, 18.72;
H, 4.59; 1H NMR (DMSO-d6,
d
ppm): 1.81 (3H, s, CH3), 3.18 (3H, s,

H. Bayrak et al. / European Journal of Medicinal Chemistry 45 (2010) 4726e4732
4731
CH3), 3.87 (2H, AB System, CH2), 5.12 (2H, s, CH2), 5.28 (2H, s, CH2),
7.08 (3H, d, arH, J ¼ 8.3 Hz), 7.37e7.45 (6H, m, arH), 7.50e7.55 (5H,
m, arH), 10.39 (1H, s, NH).
J ¼ 8.2 Hz), 14.01 (1H, s, SH); 13C NMR (DMSO-d6,
d
ppm): 10.65
(CH3), 36.27 (CH3), 39.54e41.98 (DMSO-d6þCH2), 48.12 (CH2),
101.76 (antipyrine C), arC: [127.51 (2CH), 127.98 (2CH), 129.71
(2CH), 129.89 (2CH), 130.33 (2CH), 131.02 (CH), 131.53 (CH), 131.80
(CH), 132.63 (CH), 133.52 (C), 135.45 (C), 135.60 (C)], 149.31 (triazole
C-5), 150.03 (triazole C-3), 153.92 (antipyrine C), 154.75 (triazole
C-3), 160.66 (antipyrine C]O), 169.87 (C]S).
4.1.7.3. 4-(2-Phenyl-1,5-dimethyl-3-oxo-2,3-dihydro-1H-pyrazole-4-
yl)-5-(4-chlorobenzyl)-2-{[5-(4-fluorophenylamino)-1,3,4-thiadiazol
-2-yl]methyl}-2,4-dihydro-3H-1,2,4-triazole-3-on (10c). Yield 59%,
m.p. 237e239 ꢂC. IR (KBr, , cmꢀ1): 3036 (NH),1709 and 1640 (2C]O);
y
C29H24O2N8SClF: C, 57.76; N,18.58; H, 4.01. Found C, 57.71; N,18.62; H,
4.1.9. General method for the synthesis of compounds 12 and 13
4-Chlorophenacylbromide (10 mmol) (for 12) or 4-nitro-
phenacyl bromide (10 mmol) (for 13) and sodium acetate (16.4 g
200 mmol) were added to the solution of compound 6 in ethanol
and the reaction mixture was allowed to reflux for 8 h. Then, the
mixture was cooled to room temperature, poured into ice-cold
water while stirring and left overnight in cold. The formed solid
was filtered, washed with water three times and recrystallized from
water to afford the pure compounds.
4.07; 1H NMR (DMSO-d6,
d
ppm): 1.83 (3H, s, CH3), 3.14 (3H, s, CH3),
3.88 (2H, ABSystem, CH2), 5.24 (2H, s, CH2), 7.07 (3H, d, arH, J¼ 8.3 Hz),
7.35e7.44 (6H, m, arH), 7.50e7.53 (5H, m, arH), 10.42 (1H, s, NH).
4.1.8. General method for the synthesis of compounds 11aec
A solution of corresponding carbothioamide 6aec (10 mmol) in
ethanol/water (1:1) was refluxed in the presence of 2 N NaOH for
3 h then, the resulting solution was cooled to room temperature
and acidified to pH 3e4 with 37%HCl. The precipitate formed was
filtered off, washed with water and recrystallized from ethanol/
water (1:1) to afford the desired compound.
4.1.9.1. 2-[3-(4-Chlorobenzyl)-4-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-yl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]-
N0-[4-(4-chlorophenyl)-3-phenyl-1,3-thiazol-2(3H)-ylidene]acetohy-
4.1.8.1. 4-(2-Phenyl-1,5-dimethyl-3-oxo-2,3-dihydro-1H-pyrazol-4-
yl)-5-(4-chlorobenzyl)-2-[(4-phenyl-5-mercapto-4H-1,2,4-triazol-3-
yl)methyl]-2,4-dihydro-3H-1,2,4-triazol-3-one (11a). Yield 76%, m.p.
drazide (12). Yield 79%, m.p.180e181 ꢂC. IR (KBr, , cmꢀ1): 3126 (NH),
y
1719 and 1673 (3C]O), 1603 (C]N); Anal. Calcd. (%) for
C37H30N8O3SCl2: C, 60.25; N, 15.19; H, 4.10. Found C, 60.19; N, 15.13; H,
247e248 ꢂC. IR (KBr,
y
, cmꢀ1): 2857 (SH), 1703 and 1681 (2C]O),
4.12. 1H NMR (DMSO-d6,
d ppm): 1.60 (3H, s, CH3, 3.06 (3H, s, CH3),
1594, 1492 and 1423 (3C]N); C29H25O2N8SCl: C, 59.53; N, 19.15; H,
3.60e3.90 (4H, m, 2CH2) 4.11 (1H, s, CH), 6.98e7.21 (5H, m, arH),
4.31. Found C, 59.49; N, 19.18; H, 4.28. 1H NMR (DMSO-d6,
d
ppm):
7.23e7.79 (10H, m, arH), 7.98e8.17 (3H, m, arH),10.68 (1H, m, NH); 13C
1.59 (3H, s, CH3), 3.06 (3H, s, CH3), 3.63 (2H, AB system, CH2), 4.93
NMR (DMSO-d6, d ppm): 9.81 (CH3), 31.21 (CH2), 34.69 (CH3),
(2H, AB system, CH2), 7.04 (2H, d, arH, J ¼ 8.3 Hz), 7.29e7.61 (11H, m,
38.48e40.59 (DMSO-d6þCH2), 100.29 (antipyrine C), 116.90 (CH), arC:
[125.08 (2CH), 125.19 (2CH), 126.56 (2CH), 127.54 (2CH), 128.36 (2CH),
128.94 (2CH),129.07 (CH),129.34 (CH),129.67 (CH),129.92 (CH),130.31
(CH), 130.44 (CH), 131.60 (C), 132.17 (2C), 133.65 (C), 133.84 (C), 138.70
(C)], 146.44 (C), 146.97 (triazole C-5), 151.78 (triazole C-3), 152.21
(antipyrine C), 152.30 (N]C), 155.07 (antipyrine C]O), 159.84 (C]O).
arH),14.04 (1H, s, SH);13C NMR (DMSO-d6,
dppm): 10.51 (CH3), 31.85
(CH2), 35.44 (CH3), 38.84e41.35 (DMSO-d6þCH2), 100.96 (antipy-
rine C), arC: [125.74 (2CH),128.19 (3CH),129.08 (3CH),130.03 (3CH),
131.13 (3CH), 132.30 (C), 133.67 (C), 134.36 (C), 134.58 (C)], 147.28
(triazole C-5), 148.03 (triazole C-3), 152.45 (antipyrine C), 152.96
(triazole C-3), 160.51 (antipyrine C]O), 169.23 (C]S); MS m/z (%):
121 (41), 123 (44), 148 (41), 171 (22), 188 (41), 189 (19), 197 (16), 211
(13), 229 (28), 263 (22), 273 (16), 344 (16), 357 (16), 447 (11), 585
(Mþ, 66), 607 (100), 609 (44), 610 (19), 629 (16), 663 (19).
4.1.9.2. 2-[3-(4-Nitrobenzyl)-4-(1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-yl)-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl]-
N0-[4-(4-nitrophenyl)-3-phenyl-1,3-thiazol-2(3H)-ylidene]acetohy-
drazide (13). Yield 81%, m.p. 192e193 ꢂC. IR (KBr, , cmꢀ1): 3230
y
4.1.8.2. 4-(2-Phenyl-1,5-dimethyl-3-oxo-2,3-dihydro-1H-pyrazol-4-
yl)-5-(4-chlorobenzyl)-2-[(4-benzyl-5-mercapto-4H-1,2,4-triazol-3-
yl)methyl]-2,4-dihydro-3H-1,2,4-triazol-3-one (11b). Yield 87%, m.p.
(NH), 1701 and 1643 (3C]O), 1592 (C]N), 1519 and 1351 (NO2);
Anal. Calcd (%) for C37H30N9O5SCl: C: 59.39, N: 16.87, H: 4.01. Found:
C: 59.33, N: 16.81, H: 3.97; 1H NMR (DMSO-d6,
d ppm): 1.79 (3H, s,
131e132 ꢂC. IR (KBr,
y
, cmꢀ1): 2923 (SH), 1701 and 1685 (2C]O),
CH3), 3.09 (3H, s, CH3), 3.38 (2H, s, DMSO þ CH2), 3.75 (2H, brs, CH2),
4.40 (1H, brs, CH), 6.91e6.95 (2H, d, arH, J ¼ 8.1 Hz), 7.05e7.22 (3H,
m, arH), 7.34e7.46 (7H, m, arH), 7.56 (2H, t, arH, J ¼ 7.5 Hz), 7.99 (2H,
d, arH, J ¼ 9.1 Hz), 8.25 (2H, d, arH, J ¼ 8.1 Hz), 10.04 (1H, s, NH).
1592, 1490 and 1455 (3C]N); C30H27O2N8SCl: C, 60.14; N, 18.70; H,
4.54. Found C, 60.09; N, 18.68; H, 4.58. 1H NMR (DMSO-d6,
d
ppm):
1.62 (3H, s, CH3), 3.05 (3H, s, CH3), 3.78 (2H, AB system, CH2), 4.92
(2H, AB system, CH2), 5.25 (2H, s, CH2), 7.05 (2H, d, arH, J ¼ 8.2 Hz),
7.17 (2H, d, arH, J ¼ 7.4 Hz), 7.21e7.43 (8H, m, arH), 7.52 (2H, t, arH,
5. Antimicrobial activity
J ¼ 8.2 Hz), 14.02 (1H, s, SH); 13C NMR (DMSO-d6,
d ppm): 11.65
(CH3), 33.26 (CH2), 36.54 (CH3), 39.79e42.30 (DMSO-d6þCH2),
48.09 (CH2), 101.84 (antipyrine C), arC: [127.55 (2CH), 128.54 (2CH),
129.84 (2CH), 130.05 (2CH), 130.46 (2CH), 130.82 (CH), 131.53 (CH),
131.82 (CH), 132.60 (CH), 133.74 (C), 135.44 (C), 135.59 (C), 137.13
(C)], 149.29 (triazole C-5), 149.91 (triazole C-3), 153.93 (antipyrine
C), 154.87 (triazole C-3), 160.51 (antipyrine C]O), 169.90 (C]S).
5.1. Antimicrobial activity assessment
All test microorganisms were obtained from the Hifzissihha
Institute of Refik Saydam (Ankara, Turkey) and were as follows:
E. coli ATCC35218, E. aerogenes ATCC13048, Y. pseudotuberculosis
ATCC911, P. aeruginosa ATCC43288, S. aureus ATCC25923, E. faecalis
ATCC29212, B. cereus 709 Roma, M. smegmatis ATCC607, C. albicans
ATCC60193, C. tropicalis ATCC 13803, A. niger RSKK 4017 and
S. cerevisiae RSKK 251. All the newly synthesized compounds were
weighed and dissolved in dimethylsulphoxide to prepare extract
4.1.8.3. 4-(2-Phenyl-1,5-dimethyl-3-oxo-2,3-dihydro-1H-pyrazol-4-
yl)-5-(4-chlorobenzyl)-2-{[4-(4-fluorophenyl)-5-mercapto-4H-1,2,4-
triazol-3-yl]methyl}-2,4-dihydro-3H-1,2,4-triazol-3-one (11c). Yield
77%, m.p. 157e158 ꢂC. IR (KBr,
y
, cmꢀ1): 2857 (SH), 1707 and 1682
stock solution of 5.000 mg/mL.
(2C]O),1596, 1492 and 1448 (3C]N); C29H24O2N8SClF: C, 57.76; N,
18.58; H, 4.01. Found C, 57.84; N, 18.51; H, 4.08. 1H NMR (DMSO-d6,
5.1.1. Agar-well diffusion method
d
ppm): 1.66 (3H, s, CH3), 3.07 (3H, s, CH3), 3.75 (2H, AB system,
Screening test using agar-well diffusion method [46] as adapted
earlier [47] was used for all newly synthesized compounds. Each
microorganism was suspended in Mueller Hinton (MH) (Difco,
CH2), 5.00 (2H, AB system, CH2), 7.08 (2H, d, arH, J ¼ 8.2 Hz), 7.22
(2H, d, arH, J ¼ 8.3 Hz), 7.24e7.46 (7H, m, arH), 7.54 (2H, t, arH,

4732
H. Bayrak et al. / European Journal of Medicinal Chemistry 45 (2010) 4726e4732
Detroit, MI) broth and diluted 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 incubated for 18 h at 35 ꢂC. Antimicrobial
activity was evaluated by measuring the zone of inhibition against
[18] B.S. Holla, R. Gonsalves, S. Shenoy, Il Farmaco 53 (1998) 574e578.
[19] S. Ersan, S. Nacak, R. Berkem, Il Farmaco 53 (1998) 773e776.
[20] H. Yüksek, A. Demirbas¸ , A. Ikizler, C.B. Johansson, C. Çelik, A.A. Ikizler, Arzn.-
Forsh. Drug Res. 47 (1997) 405e409.
[21] A.A. Ikizler, F. Uçar, N. Demirbas, I. Yasa, A. Ikizler, T. Genzer, Indian J. Het.
Chem. 61 (1999) 271e274.
[22] B. Tozkoparan, N. Gökhan, G. Aktay, E. Yes¸ ilada, M. Ertan, Eur. J. Med. Chem. 34
(2000) 743e750.
[23] G. Turan-Zitouni, Z.A. Kaplancikli, K. Erol, F.S. Kilic, IlFarmaco54(1999)218e223.
[24] N. Demirbas, R. Ugurluoglu, A. Demirbas, Bioorg. Med. Chem. 10 (2002)
3717e3723.
[25] H. Emilsson, H. Salender, J. Gaarder, Eur. J. Med. Chem. Chim. Ther. 21 (1985)
333e338.
[26] M. Kritsanida, A. Mouroutsou, P. Marakos, N. Pouli, S. Papakonstantinou-
Garoufalias, C. Pannecouque, M. Witvouw, E. De Clercq, Il Farmaco 57 (2002)
253e257.
the test organism. Ampicillin (10
mg), Streptomisin (10 mg) and
fluconazole (5 g) were standard drugs. Dimethyl sulfoxide and
m
ethanol were used as solvent controls. The antimicrobial activity
results are summarized in Table 1.
[27] P.K. Sahoo, E.R. Sharma, E.P. Pattanayak, Med. Chem. Res. 19 (2010) 127e135.
[28] C. Temperini, A. Cecchi, A. Scozzafava, C.T. Supuran, Bioorg. Med. Chem. 17
(2009) 1214e1221.
Acknowledgement
[29] S.S. Castle, Compr. Pharm. Ref (2008) 1e5.
This work was supported by the Scientific and Technological
Research Council of Turkey (TUBITAK, Project no: 107T333).
[30] A.S. Crucq, C. Slegers, V. Deridder, B. Tilquin, Talanta 52 (2000) 873e877.
[31] P.X. Franklin, A.D. Pillai, P.D. Rathod, S. Yerande, M. Nivsarkar, H. Padh,
K.K. Vasu, V. Sudarsanam, Eur. J. Med. Chem. 43 (2008) 129e134.
[32] A.A. Geronikaki, A.A. Lagunin, D.I. Hadjipavlou-Litina, P.T. Eleftheriou,
D.A. Filimonov, V.V. Poroikov, I. Alam, A.K. Saxena, J. Med. Chem. 51 (2008)
1601e1609.
References
[33] S. Schenone, C. Brullo, O. Bruno, F. Bondavalli, A. Ranise, W. Filippelli,
B. Rinaldi, A. Capuano, G. Falcone, Bioorg. Med. Chem. 14 (2006) 1698e1705.
[34] U. Salgın-Goksen, N. Gokhan-Kelekc, O. Goktas, Y. Koysal, E. Kılıc, S. Isık,
G. Aktay, M. Ozalp, Bioorg. Med. Chem. 15 (2007) 5738e5751.
[35] C.L. Liu, Z.M. Li, B. Zhong, J. Flour. Chem. 125 (2004) 1287e1290.
[36] A. Cukurovali, I. Yilmaz, S. Gur, C. Kazaz, Eur. J. Med. Chem. 41 (2006)
201e207.
[1] R.K. Rawal, Y.S. Phabhakar, S.B. Kati, E. De Clercq, Bioorg. Med. Chem. 13
(2005) 6771e6776.
[2] C.G. Bonde, N.J. Gaikwad, Bioorg. Med. Chem. 12 (2004) 2151e2161.
[3] R.K. Rawal, R. Tripathi, S.B. Kati, C. Pannecouque, E. De Clercq, Bioorg. Med.
Chem. 15 (2007) 1725e1731.
[4] P.P. Dixit, V.J. Patil, P.S. Nair, S. Jain, N. Sinha, S.K. Arora, Eur. J. Med. Chem. 41
(2006) 423e428.
[37] A.M. Mahran, N.A. Hassan, Arch. Pharm. Res. 29 (2006) 46e49.
[38] C.J. Chen, B.A. Song, S. Yang, G.F. Xu, P.S. Bhadury, L.H. Jin, D.Y. Hu, Q.Z. Li,
F. Liu, W. Xue, P. Lu, Z. Chen, Bioorg. Med. Chem. 15 (2007) 3981e3989.
[39] P. Karegoudar, M.S. Karthikeyan, D.J. Prasad, M. Mahalinga, B.S. Holla,
N.S. Kumari, Eur. J. Med. Chem. 43 (2008) 261e267.
[5] Z.A. Kaplancikli, G. Turan-Zitouni, A. Özdemir, G. Revial, Eur. J. Med. Chem. 43
(2008) 155e159.
[6] V.R. Solomon, C. Hua, H. Lee, Bioorg. Med. Chem. 18 (2010) 1563e1572.
[7] C. Hu, V.R. Solomon, G. Ulibarri, H. Lee, Bioorg. Med. Chem. 16 (2008) 7888e7893.
[8] C. Hu, V.R. Solomon, P. Cano, H. Lee, Eur. J. Med. Chem. 45 (2010) 705e709.
[9] V.V. Kouznetsov, A. Gomez-Barrio, Eur. J. Med. Chem. 44 (2009) 3091e3113.
[10] J. Adamec, R. Beckert, D. Weiss, V. Klimesova, K. Waisser, U. Mollmann,
J. Kaustova, V. Buchta, Bioorg. Med. Chem. 15 (2007) 2898e2906.
[11] B. Meunier, Acc. Chem. Res. 41 (2008) 69e72.
[40] G. Turan-Zitouni, M. Sıvacı, F.S. Kılıc, K. Erol, Eur. J. Med. Chem. 36 (2001)
685e689.
ꢀ
[41] H. Bayrak, A. Demirbas¸ , S. Alpay-Karaoglu, N. Demirbas¸ , Eur. J. Med. Chem. 44
(2009) 1057e1066.
[42] H. Bayrak, A. Demirbas, N. Demirbas, S. Alpay-Karaoglu, Eur. J. Med. Chem. 44
[12] S.A.F. Rostom, I.M. El-Ashmawy, H.A. Abd El Razik, M.H. Badr, H.M.A. Ashour,
Bioorg. Med. Chem. 17 (2009) 882e895.
(2009) 4362e4366.
[43] A. Demirbas, D. Sahin, N. Demirbas, S. Alpay-Karaoglu, Eur. J. Med. Chem. 44
[13] A.A. Bekhit, T. Abdel-Azneim, Bioorg. Med. Chem. 12 (2004) 1935e1945.
[14] E. Banoglu, Ç. Akoglu, S. Ünlü, E. Küpeli, E. Yesilada, M.F. Sahin, Arch. Pharm.
337 (2004) 7e14.
(2009) 2896e2903.
[44] N. Demirbas, S. Alpay-Karaoglu, A. Demirbas, K. Sancak, Eur. J. Med. Chem. 39
(2004) 793e804.
[45] R. Milcent, C. Redeuilh, J. Het. Chem. 16 (1979) 403e407.
[15] S.C. Jain, J. Sinha, S. Bhagat, W. Errington, C.E. Olsen, Synth. Commun. 33
(2003) 563e577.
[46] C. Perez, M. Pauli, P. Bazerque, Acta Biologia Med. Experimentalis 15 (1990)
[16] A. Gürsoy, S. Demirayak, G. Capan, K. Erol, K. Vural, Eur. J. Med. Chem. 35
(2000) 359e364.
113e115.
[47] I. Ahmad, Z. Mehmood, F. Mohammed, J. Ethnopharmacology 62 (1998)
[17] S. Bondock, R. Rabie, H.A. Etman, A.A. Fadda, Eur. J. Med. Chem. 43 (2008)
2122e2129.
183e193.