Synthesis and antimicrobial activities of some new 1-(5-phenylamino-[1,3,4] thiadiazol-2-yl)methyl-5-oxo-[1,2,4]triazole and 1-(4-phenyl-5-thioxo-[1,2,4] triazol-3-yl)methyl-5-oxo- [1,2,4]triazole derivatives

Article abstract of DOI:10.1016/j.ejmech.2004.06.007

Acetic acid ethyl esters containing 5-oxo-[1,2,4]triazole ring (2) were synthesized by the condensation of compounds 1a-f with ethyl bromoacetate in basic media. The reaction of compounds 2a-f with hydrazine hydrate led to the formation of acid hydrazides

Full text of DOI:10.1016/j.ejmech.2004.06.007

European Journal of Medicinal Chemistry 39 (2004) 793–804
www.elsevier.com/locate/ejmech
Original article
Synthesis and antimicrobial activities of some new
1-(5-phenylamino-[1,3,4]thiadiazol-2-yl)methyl-5-oxo-[1,2,4]triazole
and 1-(4-phenyl-5-thioxo-[1,2,4]triazol-3-yl)methyl-5-oxo-
[1,2,4]triazole derivatives
Neslihan Demirbas a,*, Sengül Alpay Karaoglu b, Ahmet Demirbas a, Kemal Sancak a
a Department of Chemistry, Karadeniz Technical University, 61080 Trabzon, Turkey
b Department of Biology, Karadeniz Technical University, 53100 Rize, Turkey
Received 2 March 2004; received in revised form 11 June 2004; accepted 14 June 2004
Available online 10 August 2004
Abstract
Acetic acid ethyl esters containing 5-oxo-[1,2,4]triazole ring (2) were synthesized by the condensation of compounds 1a–f with ethyl
bromoacetate in basic media. The reaction of compounds 2a–f with hydrazine hydrate led to the formation of acid hydrazides (3a–f). The
treatment of compounds 3 with two divers aromatic aldehydes resulted in the formation of arylidene hydrazides as cis–trans conformers
(4a,c,e,f, 5a,e,f). The thiosemicarbazide derivatives (6a,c,d,f) were afforded by the reaction of corresponding compounds 3 with phenyl-
isothiocyanate. The treatment of compounds 6a,c,d,f with sulfuric acidic caused the conversion of side-chain of compounds 6a,c,d,f into
1,3,4-thiadiazol ring; thus, compounds 7a,c,d,f were obtained. On the other hand, the cyclization of compounds 6a,c,d,f in the presence of 2 N
NaOH resulted in the formation of compounds 8a,c,d,f containing two [1,2,4]triazole rings which are linked to each other via a methylene
bridge. Compounds 4a, f, 5a, 7a, d, f, 8a and d have shown antimicrobial activity against one or more microorganism, but no antifungal
activity has been observed against yeast like fungi. Also inhibitory effect on mycelial growth by compounds 4e, 7d and 8f has been observed.
Compounds 4c and 5f were found to possess antitumor active towards breast cancer.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Antimicrobial activity; Triazoles; Antitumor activity
1. Introduction
well as their antimicrobial effects [15]. Beside this, although
there are antimicrobial agents having different structures are
frequently used in treatment of microbial infections, there is
increasing resistance to these drugs [15]. To overcome the
development of drug resistance it is crucial to synthesize a
new class of antibiotics possessing different chemical prop-
erties from those of used commonly. 5-Oxo-[1,2,4]triazole
ring containing a side chain that has thiosemicarbazide struc-
ture is an ideal heterocyclic for this purpose.
During the last few decades, a considerable attention has
been devoted to the synthesis of [1,2,4]triazole derivatives
possessing such diverse pharmacological properties as anti-
microbial [1–4], antiinflammatory [5], analgesic [6], antitu-
morial [7], antihypertensive [8], anticonvulsant and antiviral
activities [9]. In addition, [1,3,4]thiadiazoles exhibit divers
biological activities possibly due to the presence of =N–C–S
moiety [10]. Moreover, some biheterocyclic compounds in-
corporating [1,3,4]thiadiazole or [1,2,4]triazole ring have
been produced as antimicrobial agents [11–14].
The cyclization of the compounds having thiosemicarba-
zide structure has shown to be an excellent strategy for the
synthesis of [1,2,4]triazole, [1,3,4]thiadiazole, [1,3,4]oxa-
diazole and (or) [1,3,4]triazine derivatives; for instance, the
cyclization of N,N′-disubstituted thiosemicarbazides re-
sulted in the formation of [1,3,4]thiadiazoles in acidic media
[5,16]. On the other hand, the same thiosemicarbazides un-
Some of azole derivatives used as common antibiotics
such as Amphotericin B posses a toxic effect on humans as
* Corresponding author. Tel.: +90-462-3772600; fax: +90-462-3253196.
E-mail address: neslihan@ktu.edu.tr (N. Demirbas).
0223-5234/$ - see front matter © 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.ejmech.2004.06.007

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N. Demirbas et al. / European Journal of Medicinal Chemistry 39 (2004) 793–804
Scheme 1. Synthetic pathway for the preparation of compounds 2–8.
derwent a cyclization to yield 1,2,4-triazole derivatives in the
presence of NaOH, [16–18].
[1,2,4]triazole ring to bromine-bearing C atom of ethyl bro-
moacetate. Compounds 2a, b and f were prepared as
precursors of some biological active 5-oxo-[1,2,4]triazole
derivatives previously [19]. Compounds 2c–e were obtained
for the first time in this present study. Compounds 3a–f were
produced from the reaction of compounds 2a–f with hydra-
zine hydrate. Although the acid hydrazides contain two
amino groups in their structures, only the hydrazide–NH2
reacted with aldehydes in ethanol leading to the formation of
compounds 4a,c,e,f and 5a,e,f.
In recent years, various antitumor drugs have been devel-
oped for the treatment of cancer. Among these, some
[1,2,4]triazole derivatives incorporating Shiff Base structure
were synthesized as antitumor agents in our laboratory [7].
However, cancer is still a major health problem because of
the insufficiency of the conventional methods.
In view of these facts, the aim of this present study is to
obtain [1,2,4]triazole derivatives incorporating Shiff Base
structure that possess antitumor activity and the synthesis of
[1,2,4]triazole derivatives involving another [1,2,4]triazole
or [1,3,4]thiadiazole ring, which is linked to 5-oxo-
[1,2,4]triazole ring via a methylene group (Scheme 1) as
antibacterial agents.
So far, the compounds containing imine bond have been
synthesized intensively with various reasons, the major of
which is their biological activities [7,20–22]. In addition, the
compounds having arylidene–hydrazide structure may exist
as E/Z geometrical isomers about –C=N double bond and
cis–trans amide conformers (Scheme 2) [19,22–24].Accord-
ing to the literature [22,24], the compounds containing imine
2. Chemistry
bond are present in higher percentage in dimethyl-D sulfox-
6
ide solution in the form of geometrical E isomer about –C=N
double bond. The Z isomer can be stabilized in less polar
In the first part of the study, compounds 2a–f were synthe-
sized via the nucleophylic attack of N-1 on 5-oxo-

N. Demirbas et al. / European Journal of Medicinal Chemistry 39 (2004) 793–804
795
Scheme 2. E/Z geometrical isomers and cis–trans conformers of compounds 4a,c,d,f and 5a,e,f.
solvents by an intramolecular hydrogen bond. In the present
(–NCH2), 4.10 ppm (–OCH2CH3) and 1.09–1.17 ppm
(–OCH2CH3) ppm integrating for two proton, two proton and
three proton, respectively. In the 13C NMR spectra of these
compounds, the signals belonging to the same groups were
recorded at 46.43, 60.93, and 13.86–14.95 ppm, respectively.
study, the spectral data were obtained in dimethyl-D sulfox-
6
ide solution and no signal belonging to Z isomer was ob-
served. On the other hand, the cis–trans conformers of E
isomer were present in the dimethyl-D sulfoxide solution of
6
1
compounds 4a,c,e,f 4 and 5a,e,f.
The H NMR spectra of compounds 3a–f displayed no
The key intermediate thiosemicarbazides (6a,c,d,f) were
obtained from the reaction of compounds 3a,c,d,f with phe-
nyl isothiocyanate in reasonable good yields.
signals belonging to OCH2CH3 group; instead, new signals
derived from hydrazide structure appeared between 4.28–
4.46 ppm (–NHNH2) and 9.18–9.29 ppm (–NHNH2) inte-
grating for two proton and one proton, respectively (con-
trolled by changing with D2O). In addition, the signals
derived from –NH2 group at position-4 on 5-oxo-
[1,2,4]triazole ring resonated between 5.24 and 5.52 ppm
integrating for two proton (exchangeable with D2O). The IR
spectra of acid hydrazides (3a–f) showed an additional peak
at 1651–1682 cm–1 due to exocyclic-carbonyl function de-
rived from hydrazide structure beside the endocyclic-
carbonyl peak at position-5 of [1,2,4]triazole ring.
Compounds 7a,c,d,f were prepared by the treatment of
thiosemicarbazides with concentrated sulfuric acid and the
1
structures of these compounds confirmed by IR, H NMR,
13C NMR, mass spectral data (for compound 7f), and el-
emental analyses.
Compounds 8a,c,d,f were afforded by three different
ways, all of which contained thiosemicarbazides (6a,c,d,f) as
intermediates. According to the literature [13–15] it is ex-
pected that the treatment of thiosemicarbazides with aqueous
NaOH resulted in a cyclization leading to the formation of
5-thioxo-[1,2,4]triazole ring. Beside this method, in the
present study compounds 8a,c,d,f were prepared by heating
of the corresponding compounds 6 in an oil bath upon 130 °C
for 2 h. In the third method, compound 8f was synthesized by
refluxing of 6f in ethanol for 8 h. The yields of these three
methods are very close to each other. These results show that
the effect of heating on thiosemicarbazides (6a,c,d,f) causes
a cyclization resulting in the formation of 5-thioxo-
[1,2,4]triazole ring, but not [1,3,4]thiadiazole ring.
1
In the H NMR and 13C NMR spectra of compounds
4a,c,e,f the signals belonging to benzylidene group were
observed at aromatic region while the signals belonging to
–NHNH2 disappeared. The 1H NMR spectra of compounds
5a,e,f displayed three different singlets due to 2,4-
dichlorobenzylidene group between 7.50 and 8.55 ppm each
1
integrating for one proton. In the H NMR spectra of com-
pounds 4a,c,e,f and 5a,e,f two sets of signals each belonging
to the –NH group of cis and trans conformers were observed
between 11.16–11.92 and 11.72–11.97 ppm; the ratio of
trans–cis in each case were calculated by using NMR data.
The upfield lines of –NH protons were assigned to cis con-
former of the amide structure and downfield lines of the
protons of the same group to trans- conformer of the amide
structure [21]. Moreover, the 1H NMR spectra of compound
4e showed additional two singlets derived from –N=CH
group at 8.01 and 8.21 each representing cis and trans con-
formers; the 13C signals of the same conformers appeared at
144.15 and 144.20 ppm. In addition, the 1H NMR spectra of
compound 4f displayed two singlets belonging to –NH2
It is interesting to note that compounds 8a,c,d,f are
present in solid state in –C=S form as indicated by their IR spectra
cm–1 for
cm–1 characteristic
(absence of absorption in the region of 2500–2660
–SH stretching and presence of two absorption maxima at 1342–1320
of –C=S group in these type of compounds
[11]). The –C=S form is present also in dimethyl sulfoxide by
suggested NMR spectral data.
3. Results and discussion
1
In the H NMR spectra of compounds 2c–f additional
signals derived from ester group were observed at 4.52

796
N. Demirbas et al. / European Journal of Medicinal Chemistry 39 (2004) 793–804
Table 1
Screening for antimicrobial activity of the selected compounds
Compound Solvent
No.
Microorganisms and inhibition zone (mm)
Ec
+++
+
Pa
++
–
Yp
–
Kp
+++
+
Ef
–
–
–
–
–
–
+
–
–
–
–
–
–
Sa
–
Bs
+++
+
Ca
–
–
–
–
–
–
–
–
–
+
–
–
–
Ct
–
+
–
–
–
–
–
+
–
+
–
–
–
Cg
–
–
–
–
–
–
–
–
–
–
–
–
–
4a
DMSO
4e
4f
5a
5e
–
++
–
–
+
–
+++
+++
–
–
+++
–
++
–
–
–
++
–
–
–
7a
+++
++
–
+++
+
–
+++
+++
++
–
++
++
–
+++
++
–
7d
7f
Ethanol
DMSO
+
–
+
8a
8c
8d
8f
With etha-
nol
+
–
–
++
–
+
–
–
–
–
–
–
–
–
–
++
–
+
–
–
–
–
–
–
–
+
+
++
+
With
DMSO
Ceftazi-
dime
Triflucan
–
–
–
–
–
–
–
–
–
–
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
Results were interpreted in terms of the diameter of the inhibition zone: (–): <5.5 mm; (+): 5.5–10 mm; (++): 11–16 mm; (+++): ≥17 mm.
Ec: Escherichia coli ATCC 35218, Pa: Pseudomonas aeruginosa ATCC 10145,Yp: Yersinia pseudotuberculosis ATCC 911, Kp: Klebsiella pneumoniae ATCC
13883, Ef: Enterococcus faecalisATCC 29212, Sa: Staphylococcus aureusATCC 25923, Bs: Bacillus subtilisATCC 6633, Ca: Candida albicansATCC 60193,
Ct: Candida tropicalis ATCC 13803, Cg: Candida glabrata
group at 5.58 and 5.61 ppm. In the 13C NMR spectra of
compounds 4a,c,e,f and 5a,e,f –NCH2, triazole-C-3 and
triazole-C-5 signals were recorded as two sets between
46.14–46.72 and 46.17–46.57 ppm (–NCH2), 144.25–
144.77 and 144.28–146.94 ppm (triazole-C-3), 153.03–
154.46 and 153.44–163.25 ppm (triazole-C-5) as the result of
formation of cis and trans conformers. Furthermore, in the
13C NMR spectra of compounds 5e and f, the signals belong-
ing to exocyclic-C=O group derived from each cis and trans
conformers were recorded at 168.22 and 168.30 ppm. The
13C NMR values of triazole-C-3 and triazole-C-5 are consis-
tent with literature [7,25]. Besides, compound 5a gave a
molecular ion peak consistent with its formulae.
compounds 6a,c,d,f. In the 13C NMR spectra of compounds
7a,c,d,f no signal due to –C=S group were observed, while
the signal derived from this group have resonated between
168.20 and 171.96 ppm in the spectra of compounds 8a,c,d,f.
Beside these, the melting points highly increased when com-
pounds 6 were converted to corresponding products (7 and
8). Elemental analyses are consistent with the assigned struc-
tures for compounds 7a,c,d,f and 8a,c,d,f. The mass spectra
of the cyclization products (7f, 8a,c,d,f) showed molecular
ion peaks which were consistent with their molecular formu-
lae.
Compounds 4a, 5a, 7a,d,f, 8a and d showed antibacterial
activity against one and/or more test microorganisms
(Table 1). Compounds 4a, 5a and 7a, which contain a methyl
The elemental analyses of compounds 6a,c,d,f are consis-
tent with the assigned structures. The IR spectra of com-
pounds 6a,c,d,f showed an –C=S absorption between
1309 and 1341 cm–1; the 13C signal of this group were
observed at 180.37–181.03 ppm. In the 1H NMR spectra of
these compounds, the –NH2 signal derived from hydrazide
structure disappeared while the signal belonging to –NH2
group on 1,2,4-triazole ring was observed between 5.30 and
group on position
3
of 5-oxo-[1,2,4]triazole or
[1,3,4]thiadiazol-2-yl-5-oxo-[1,2,4]triazole ring were found
to be effective on E. coliATCC 35218, Pseudomonas aerugi-
nosa ATCC 10145, K. pneumonia ATCC 13883 and Bacillus
subtilis ATCC 6633. No antimicrobial activity was observed
on the yeast like fungi (Table 1). The compounds including a
phenyl group on position 3 of 5-oxo-[1,2,4]triazole ring (4f)
or [1,3,4]thiadiazol-2-yl-5-oxo-[1,2,4]triazole ring (7f)
showed activity against Klebsiella pneumoniae ATCC
13883 and lower activity against P. aeruginosaATCC 10145.
Compounds 7d which has a benzyl group on position 3 and
consists of [1,3,4]thiadiazol-2-yl-5-oxo-[1,2,4]triazole ring
exhibited activity towards K. pneumoniae ATCC 13883,
Escherichia coli ATCC 35218, Staphylococcus aureus
ATCC 25923 and B. subtilis ATCC 6633, while the com-
pound having a benzyl group at position 3 of 5-thioxo-
[1,2,4]triazol-2-yl-5-oxo-[1,2,4]triazole ring (8d) was found
1
5.36 ppm. In addition, the H NMR spectra of compounds
6a,c,d,f displayed three singlets due to three different –NH
groups around 9.60, 9.70 and 10.30 ppm (exchangeable with
D2O) each integrating for one proton. The aromatic protons
derived from phenyl group resonated between 7.18 and
7.54 ppm.
In the 1H NMR spectra of compounds 7a,c,d,f and
8a,c,d,f the presence of only one signal for –NH group
integrating for one proton (exchangeable with D2O) con-
firmed the cyclization at the side chain of triazole ring in

N. Demirbas et al. / European Journal of Medicinal Chemistry 39 (2004) 793–804
797
Table 2
Table 4
Effects of test compounds on mould growth (mm)
The GI50 values of compounds 4c and 5f towards several tumor cell line a
(µM)
Compound no.
Penicillum spp.
Aspergillus spp.
Panel/cell line
Leukemia
CCRF-CEM
MOLT-4
4c
5f
4a
4e
4f
5a
5e
7a
7d
7f
8a
8c
8d
8f
20
11
ND
16
ND
16
12
20
18
18
20
10
17
20
17
7
86.2
53.2
45.2
43.7
14.6
ND
12
ND
10
8
>50.0
RPMI-8226
SR
″
″
Non-small cell lung cancer
A549/ATCC
EKVX
51.8
>50.0
″
13
12
13
11
9
>50.0
95.8
b
HOP-62
27.4
25.5
24.9
>50.0
13.1
21.8
35.9
HOP-92
NCI-H226
NCI-H23
-
23.4
25.5
34.1
-
Triflucan
9
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
HCC-2998
HCT-116
Untreated control
17
ND: Not detected.
to be effective only against S. aureus ATCC 25923. Com-
pound 4e, which has a p-chlorobenzyl group on position
3 showed moderate activity against S. aureus ATCC
25923 and lower activity against B. subtilis ATCC 6633,
while the other compound (5e) including p-chlorobenzyl
group on position 3 exhibited no activity. Among the com-
pounds containing a phenyl group at position 3, compounds
4f and 7f showed activity on K. pneumoniae ATCC 13883,
while 8f incorporating both 5-oxo- and 5-thioxo-
[1,2,4]triazole rings exhibited no activity on test microorgan-
isms. Compound 8c having an n-propyl group on position
3 of 5-thioxo-[1,2,4]triazol-3-yl-5-oxo-[1,2,4]triazole struc-
ture showed only lower activity against Candida albicans
ATCC 60193 and Candida tropicalis ATCC 13803. Inhibi-
tory effect on mycelial growth has been especially observed
by compounds 4e, 7d and 8f (Table 2).
According to the data listed in Tables 3 and 4, compounds
4c and 5f both have potentials to reduce the growth of MCF7
cell line. Especially, compound 5f having a GI50 value of
1.87 µM seems to be effective towards breast cancer cell
lines. It has been reported that compounds having triazole
moieties such as vorozole, letrozole and anastrozole appear
to be very effective aromatase inhibitors in terms for prevent-
ing breast cancer [26–28]. It is known that [1,2,4]triazole
moieties interact strongly with the heme iron and aromatic
substituents in the active site of aromatase [29]. It can be
speculated that the tested Shiff Bases (4c, 5f) possessing a
[1,2,4]triazole ring inhibit the MCF7 cell line by possibly
interacting with the aromatase. Although both the tested
compounds contain such a 1,2,4-triazole ring, the reason for
5f being most effective against MCF7 cell line might be due
to a phenyl group at position 3 of the [1,2,4]triazole ring
21.1
b
>50.0
19.3
Breast cancer
MCF7
43.3
24.9
1.87
43.1
26.6
>50.0
NCI/ADR-RES
MDA-MB-231/ATCC 33.6
MDA-MB-435
CNS cancer
SF-268
56.9
b
34.1
30.6
18.7
SF-295
77.5
34.3
U251
Melanoma
LOX IMIV
MALME-3M
SK-MEL-2
UACC-62
Ovarian cancer
IGROVI
51.4
65.7
b
>50.0
22.3
45.3
49.7
92.3
53.2
23.8
31.0
30.7
49.9
56.3
13.0
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
SK-OV-3
Renal cancer
786-0
>50.0
22.4
>50.0
>50.0
11.8
56.4
23.7
23.7
22.9
25.5
22.5
>50.0
19.6
20.0
49.2
29.7
25.5
Achn
Cakı-1
Sn12c
Tk10
UO-31
Prostate cancer
PC-3
54.0
27.7
a GI50: Molar concentration for 50% growth inhibition.
b The GI50value ≥100 µM.
Table 3
Antitumor screening data for the selected Shiff Bases
Compound no.
Number assigned by NCI
Growth percentage of tumor cell
MCF7
NCI-H460
SF-268
94
4c
5f
D-729281
D-729282
5
1
96
86
76

798
N. Demirbas et al. / European Journal of Medicinal Chemistry 39 (2004) 793–804
seems to fit to the active site of aromatase. This proposal has
to be elucidated in detail with [1,2,4]triazole derivatives
having aromatic substituents. Finally, our present data sug-
gest that compound 5f should be a potent therapeutic agent
for the treatment of breast cancer.
cm–1), 3330–3212 (–NH2), 1747 (ester –C=O), 1700 (triaz-
ole –C=O), 1578 (–C=N), 1226 (–C–O); 1H NMR (DMSO-
d6) d (ppm) 1.09 (t, J = 6.0 Hz, –OCH2CH3), 3.89 (s, benzyl–
CH2), 4.13 (q, J = 6.0 Hz, –OCH2CH3), 4.52 (s, –NCH2),
5.38 (s, –NH2), 7.10–7.25 (m, 5H, ar-H); 13C NMR (DMSO-
d6) d (ppm) 14.95 (–OCH2CH3), 30.14 (benzyl–CH2), 46.43
(NCH2), 60.93 (–OCH2CH3), ar C: [126.42 (CH), 128.26
(4CH), 135.92 (C)], 145.92 (triazole-C-3), 151.15 (triazole-
C-5), 167.85 (exocyclic-C=O).
4. Experimental
4.1. Chemistry
4.1.1.3.
4-Amino-3-p-chlorobenzyl-5-oxo-4,5-dihydro-
[1,2,4]tria-
Melting points were determined on a Gallenkamp melting
point apparatus and are uncorrected. 1H NMR and 13C NMR
spectra were recorded on a Varian-Mercury 200 MHz spec-
trometer. The IR spectra were measured as potassium bro-
mide pellets using a Perkin-Elmer 1600 series FTIR spec-
trometer. Mass spectra were obtained at a Quattro LC-MS
(70 eV) Instrument. Combustion analysis was performed on
a Carlo Erba 1106 elemental analyzer.All the chemicals were
obtained from Fluka ChemieAG Buchs (Switzerland). Com-
pounds 1a–f, 2a, b and f were synthesized by a published
method [19,30]. Melting points and IR data of known com-
pounds are consistent with the ones reported previously
[19,30].
zol-1-yl-acetic acid ethyl ester (2e). Recrystallization from
ethanol–water (1:2), (yield: 55.30%), m.p. 156–157 °C.
Analysis (Calc/found %): for C13H15O3N4Cl C: 50.25/51.03,
H: 4.86/4.89, N: 18.03/17.98; IR (KBr) (m, cm–1), 3390–3225
(–NH2), 1736 (ester –C=O), 1714 (triazole –C=O), 1582
(–C=N), 1233 (–C–O); 1H NMR (DMSO-d6) d (ppm) 1.17 (t,
J = 6.0 Hz, –OCH2CH3), 3.90 (s, benzyl–CH2), 4.10 (q,
J = 6.0 Hz, –OCH2CH3), 4.52 (s, –NCH2), 5.17 (s, NH2),
7.28 (d, J = 8.0 Hz, 2H, ar-H), 7.30 (d, J = 8.0 Hz, 2H, ar H);
13C NMR (DMSO-d6) d (ppm) 13.86 (–OCH2CH3), 29.55
(benzyl–CH2), 46.43 (NCH2), 60.94 (–OCH2CH3), ar C:
[128.22 (2CH), 130.53 (2CH), 131.10 (C), 134.63 (C)],
146.91 (triazole-C-3), 153.10 (triazole-C-5), 167.85
(exocyclic-C=O).
4.1.1. General method for the synthesis of compounds 2
The corresponding 3-alkyl-4-amino-5-oxo-4,5-dihydro-
[1,2,4]triazole (1) (0.01 mol) was refluxed with equivalent
amount of natrium in absolute ethanol for 2 h. Then, ethyl
bromoacetate (0.01 mol) was added and refluxed for an
additional 5 h. After evaporating at 35–40 °C under reduced
pressure, a solid appeared. This was recrystallized from an
appropriate solvent to afford the desired compound.
4.1.2. General method for the synthesis of compounds 3
A solution of the corresponding compound 2 (0.01 mol) in
n-butanol was refluxed with hydrazine hydrate (0.025 mol)
for 4 h. After cooling to room temperature, a white solid
appeared. This was recrystallized from an appropriate sol-
vent to afford the desired product.
4.1.1.1. 4-Amino-5-oxo-3-n-propyl-4,5-dihydro-[1,2,4]tri-
azol-1-yl-acetic acid ethyl ester (2c). Recrystallization from
ethyl acetate/petroleum ether (1:2), (yield: 62.30%), m.p.
88–89 °C. Analysis (Calc/found %): for C9H16O3N4 C:
47.35/47.91, H: 7.06/7.37, N: 24.54/24.32; IR (KBr) (m,
cm–1), 3314–3215 (–NH2), 1751 (ester –C=O), 1708 (triaz-
ole –C=O), 1591 (–C=N), 1216 (–C–O); 1H NMR (DMSO-
d6) d (ppm) 1.11 (t, J = 5.8 Hz, –CH2CH2CH3), 1.18 (t,
J = 6.5 Hz, –OCH2CH3), 1.65–1.78 (m, –CH2CH2CH3,),
2.45 (q, J = 6.5 Hz, –CH2CH2CH3), 4.10 (q, J = 5.8 Hz,
–OCH2CH3), 4.54 (s, –NCH2), 5.23 (s, –NH2); 13C NMR
4.1.2.1.
4-Amino-3-methyl-5-oxo-4,5-dihydro-[1,2,4]tri-
azol-1-yl-acetic acid hydrazide (3a). Recrystallization from
ethanol (yield: 67.12%), m.p. 190 °C. Analysis (Calc/found
%): for C5H10O2N6 C: 32.25/32.55, H: 5.41/5.36, N:
45.14/44.79.18; IR (KBr) (m, cm–1), 3357–3211 (NH +
2NH2), 1716 (triazole –C=O), 1682 (hydrazide –C=O), 1500
1
(–C=N); H NMR (DMSO-d6) d 2.09 (s, –CH3), 4.27 (s,
–NCH2), 4.32 (s, –NHNH2), 5.24 (–NH2), 9.18 (s,
–NHNH2); 13C NMR (DMSO-d6) d 16.11 (–CH3), 51.34
(–NCH2), 150.55 (triazole C-3), 158.72 (triazole C-5),
171.58 (exocyclic-C=O).
(DMSO-d6)
(–OCH2CH3),
d
(ppm) 13.82 (–CH2CH2CH3), 14.74
19.01 (–CH2CH2CH3), 27.13
4.1.2.2. 4-Amino-3-ethyl-5-oxo-4,5-dihydro-[1,2,4]triazol-
1-yl-acetic acid hydrazide (3b). Recrystallization from etha-
nol (yield: 68.21%), m.p. 170–171 °C. Analysis (Calc/found
%): for C6H12O2N6 C: 35.99/36.25, H: 6.04/6.09, N:
41.98/41.15; IR (KBr) (m, cm–1), 3284–3173 (NH + 2NH2),
1705 (triazole –C=O), 1666 (hydrazide –C=O), 1547
(–CH2CH2CH3), 46.54 (NCH2), 61.17 (–OCH2CH3), 146.54
(triazole-C-3), 151.85 (triazole-C-5), 167.25 (exocyclic-
C=O).
4.1.1.2.
4-Amino-3-benzyl-5-oxo-4,5-dihydro-[1,2,4]tri-
1
azol-1-yl-acetic acid ethyl ester (2d). Recrystallization from
ethyl acetate/petroleum ether (1:2), (yield: 62.30%), m.p.
105–106 °C. Analysis (Calc/found %): for C13H16O3N4 C:
56.51/56.45, H: 5.84/5.87, N: 20.28/19.98; IR (KBr) (m,
(–C=N); H NMR (DMSO-d6) d (ppm) 1.29 (t, J = 5.8 Hz,
–CH2CH3), 2.47 (q, J = 5.8 Hz, –CH2CH3), 4.20 (s, –NCH2),
4.28 (s, –NHNH2), 5.24 (s, –NH2), 9.19 (s, –NHNH2); 13C
NMR (DMSO-d6) d (ppm) 10.19 (–CH2CH3), 18.22

N. Demirbas et al. / European Journal of Medicinal Chemistry 39 (2004) 793–804
799
(–CH2CH3), 38.75 (–NCH2), 149.52 (triazole-C-3), 154.10
(triazole-C-5), 166.25 (exocyclic-C=O).
(C), 128.16 (2CH), 128.26 (2CH), 128.57 (CH)], 144.30
(triazole-C-3), 153.92 (triazole-C-5), 165.78 (exocyclic-
C=O).
4.1.2.3. 4-Amino-3-n-propyl-5-oxo-4,5-dihydro-[1,2,4]tri-
azol-1-yl-acetic acid hydrazide (3c). Recrystallization from
ethanol (yield: 68.21%), m.p. 144–145 °C. Analysis
(Calc/found %): for C7H14O2N6 C: 39.24/39.25, H:
6.59/6.18, N: 39.23/39.35; IR (KBr) (m, cm–1), 3286–3164
(NH + 2NH2), 1717 (triazole –C=O), 1653 (hydrazide
–C=O), 1547 (–C=N); 1H NMR (DMSO-d6) d (ppm) 1.04 (t,
J = 6.0 Hz, –CH2CH2CH3), 1.60–1.72 (m, –CH2CH2CH3,),
2.40 (q, J = 6.0 Hz, –CH2CH2CH3), 4.33 (s, –NCH2), 4.41 (s,
–NHNH2), 5.26 (s, –NH2), 9.32 (s, –NHNH2); 13C NMR
4.1.3. General method for the synthesis of compounds 4
and 5
A solution of the corresponding compound 3 (0.01 mol) in
ethanol was refluxed with appropriate aldehyde (0.01 mol)
for 3 h. After cooling to room temperature, a white solid
appeared. This crude product was recrystallized from an
appropriate solvent to afford the desired product.
4.1.3.1.
4-Amino-3-methyl-5-oxo-4,5-dihydro-[1,2,4]tri-
(DMSO-d6)
d
(ppm) 13.43 (–CH2CH2CH3), 18.81
azol-1-yl acetic acid benzylidene-hydrazide (4a). Recrystal-
lization from dimethyl sulfoxide–water (1:1) (yield:
87.41%), m.p. 246–247 °C the ratio of trans–cis: 60/40.
Analysis (Calc/found %): for C12H14O2N6 C: 52.54/52.81,
H: 5.12/5.17, N: 30.64/30.19; IR (KBr) (m, cm–1), 3330–3225
(NH + NH2), 1722 (triazole C=O), 1694 (hydrazide –C=O),
1594 (–C=N), 1410 (–C=N); 1H NMR (DMSO-d6) d (ppm)
2.23 (s, –CH3), 4.92 (s, –NCH2), 5.44 (s, –NH2), 7.54–7.60
(m, 3H, ar-H), 7.70–7.85 (m, 2H, ar H), 8.10 (s, –N=CH),
11.76 and 11.80 (s, NH, cis and trans conformers); 13C NMR
(DMSO-d6) d (ppm) 10.55 (–CH3), 46.14 and 46.17 (–NCH2,
cis and trans conformers); ar C: [126.82 (2CH), 127.13
(CH), 129.88 (2CH), 130.07 (C)], 144.16 (N=CH),
144.77 and 144.80 (triazole-C-3, cis and trans conformers),
154.41 and 153.43 (triazole-C-5, cis and trans conformers),
168.05 (exocyclic-C=O).
(–CH2CH2CH3), 26.23 (–CH2CH2CH3), 46.19 (–NCH2),
147.52 (triazole-C-3), 153.95 (triazole-C-5), 165.93
(exocyclic-C=O).
4.1.2.4.
4-Amino-3-benzyl-5-oxo-4,5-dihydro-[1,2,4]tri-
azol-1-yl-acetic acid hydrazide (3d). Recrystallization from
ethanol (yield: 78.29%), m.p. 196–196.5 °C. Analysis
(Calc/found %): for C11H14O2N6 C: 50.37/50.27, H:
5.38/5.23, N: 32.05/32.15; IR (KBr) (m, cm–1), 3302–3173
(NH + 2NH2), 1714 (triazole –C=O), 1651 (hydrazide
–C=O), 1548 (–C=N); 1H NMR (DMSO-d6) d (ppm) 3.87 (s,
benzyl–CH2), 4.21 (s, –NCH2), 4.27 (s, –NHNH2), 5.29 (s,
–NH2), 7.10–7.32 (m, 5H, ar-H), 9.20 (s, –NHNH2); 13C
NMR (DMSO-d6) d (ppm) 30.21 (benzyl–CH2), 46.26
(–NCH2), ar C: [126.50 (CH), 128.51 (2CH), 128.89 (2CH),
136.01 (C)], 147.00 (triazole-C-3), 153.46 (triazole-C-5),
166.09 (exocyclic-C=O).
4.1.3.2.
4-Amino-3-p-chlorobenzyl-5-oxo-4,5-dihydro-
[1,2,4]triazol-1-yl acetic acid benzylidene-hydrazide
(4c). Recrystallization from dimethyl sulfoxide–water (1:1)
(yield: 83.38%), m.p. 174–175 °C the ratio of trans–cis:
60/40. Analysis (Calc/found %): for C14H18O2N6 C:
55.61/55.81, H: 6.00/6.17, N: 27.80/27.59; IR (KBr) (m,
cm–1), 3330–3219 (NH + NH2), 1720 (triazole C=O), 1694
(hydrazide C=O), 1599 (C=N), 1443 (C=N); 1H NMR
(DMSO-d6) d (ppm) 1.17 (t, J = 6.0 Hz, –CH2CH2CH3),
1.65–1.72 (m, –CH2CH2CH3,), 2.44 (q, J = 6.0 Hz,
–CH2CH2CH3), 4.94 (s, –NCH2), 5.40 (s, –NH2), 7.50–7.62
(m, 3H, ar-H), 7.71–7.78 (m, 2H, ar H), 8.10 (s, –N=CH),
11.70 and 11.78 (s, NH, cis and trans conformers); 13C NMR
4.1.2.5.
4-Amino-3-p-chlorobenzyl-5-oxo-4,5-dihydro-
[1,2,4]triazol-1-yl-acetic acid hydrazide (3e). Recrystalli-
zation from ethanol (yield: 72.58%), m.p. 208–209 °C.
Analysis (Calc/found %): for C11H13O2N6Cl C: 44.52/44.97,
H: 4.41/4.47, N: 28.32/28.20; IR (KBr) (m, cm–1), 3303–3167
(NH + 2NH2), 1726 (triazole –C=O), 1658 (hydrazide
–C=O), 1569 (–C=N); 1H NMR (DMSO-d6) d (ppm) 3.87 (s,
benzyl–CH2), 4.22(s, –NCH2), 4.33 (s, –NHNH2), 5.28 (s,
–NH2), 7.31 (d, J =8.0 Hz, 2H, ar-H), 7.38 (d, J =8.0 Hz, 2H,
ar H,), 9.20 (s, –NHNH2); 13C NMR (DMSO-d6) d (ppm)
29.60 (benzyl–CH2), 46.10 (–NCH2), ar C: [128.19 (2CH),
130.58 (2CH), 131.10 (C), 134.74 (C)], 146.10 (triazole-C-
3), 153.22 (triazole-C-5), 165.92 (exocyclic-C=O).
(DMSO-d6)
d
(ppm) 13.67 (–CH2CH2CH3), 19.03
(–CH2CH2CH3), 26.21 (–CH2CH2CH3), 46.14 and 46.18
(–NCH2, cis and trans conformers); ar C: [126.91 (2CH),
127.22 (CH), 129.79 (2CH), 130.13 (C)], 144.24 (–N=CH),
144.78 and 144.82 (triazole-C-3, cis and trans conformers),
154.46 and 153.48 (triazole-C-5, cis and trans conformers),
168.02 (exocyclic-C=O).
4.1.2.6.
4-Amino-3-phenyl-5-oxo-4,5-dihydro-[1,2,4]tri-
azol-1-yl-acetic acid hydrazide (3f). Recrystallization from
ethanol (yield: 72.58%), m.p. 202–203 °C. Analysis
(Calc/found %): for C10H12O2N6 C: 48.38/48.94, H:
4.87/4.87, N: 33.85/33.10; IR (KBr) (m, cm–1), 3296–3245
(NH + 2NH2), 1704 (triazole –C=O), 1658 (hydrazide
–C=O), 1542 (–C=N); 1H NMR (DMSO-d6) d (ppm) 4.37 (s,
–NCH2), 4.46 (s, –NHNH2), 5.52 (s, –NH2), 7.40–7.58 (m,
4.1.3.3.
4-Amino-3-p-chlorobenzyl-5-oxo-4,5-dihydro-
[1,2,4]triazol-1-yl acetic acid benzylidene-hydrazide
(4e). Recrystallization from dimethyl sulfoxide–water (1:1)
(yield: 88.59%), m.p. 246–247 °C; the ratio of trans–cis:
60/40. Analysis (Calc/found %): for C18H17O2N6Cl C:
3H, ar-H), 7.60–8.10 (m, 2H, ar H), 9.29 (s, –NHNH2); 13
NMR (DMSO-d6) d (ppm) 46.61 (–NCH2), ar C: [127.46
C

800
N. Demirbas et al. / European Journal of Medicinal Chemistry 39 (2004) 793–804
56.18/56.41, H: 4.45/4.47, N: 21.83/21.69; IR (KBr) (m,
4.1.3.6.
[1,2,4]triazol-1-yl
4-Amino-3-p-chlorobenzyl-5-oxo-4,5-dihydro-
acetic acid 2,4-dichloro-
cm–1), 3447–3204 (NH + NH2), 1719 (triazole C=O), 1676
1
benzylidenehydrazide (5e). Recrystallization from dimethyl
sulfoxide–water (1:1) (yield: 80.26%), m.p. 266–267 °C; the
ratio of trans–cis: 61/39. Analysis (Calc/found %): for
C18H15O2N6Cl3 C: 47.65/48.10, H: 3.33/3.57, N:
18.52/18.47; IR (KBr) (m, cm–1), 3342–3325 (NH + NH2),
1702 (triazole –C=O), 1699 (hydrazide –C=O), 1587
(hydrazide –C=O), 1575 (–C=N), 1489 (–C=N); H NMR
(DMSO-d6) d (ppm) 3.92 (s, benzyl–CH2), 4.86 (s, –NCH2),
5.38 (s, –NH2), 7.20–7.35 (m, 5H, ar-H), 7.38 (d, J = 8.0 Hz,
2H, ar H), 7.42 (d, J = 8.0 Hz, 2H, ar H), 8.01 and 8.21 (s,
–N=CH, cis and trans conformers), 11.68 and 11.72 (s, –NH,
cis and trans conformers); 13C NMR (DMSO-d6) d (ppm)
29.64 (benzyl–CH2), 46.43 and 46.50 (–NCH2, cis and trans
conformer), ar C: [127.09 (CH), 128.49 (2CH), 128.96
(2CH), 130.15 (CH), 130.84 (2CH), 131.50 (C), 134.05 (C),
135.05 (C), 138.21 (CH)], 144.15 and 144.20 (–N=CH, cis
and trans conformers), 146.41 and 146.85 (triazole-C-3, cis
and trans conformers), 153.61 and 153.63 (triazole-C-5, cis
and trans conformers), 168.02 (exocyclic-C=O).
1
(–C=N), 1489 (–C=N); H NMR (DMSO-d6) d (ppm) 3.95
(s, benzyl–CH2), 4.95 (s, –NCH2), 5.40 (s, –NH2), 7.30 (d,
J = 6.0 Hz, 2H, ar-H), 7.38 (s, 2H, ar H), 7.68 (bs, 1H, ar H),
8.10 (bs, 1H, ar H), 8.38 (bs, 1H, ar H), 8.55 (s, –N=CH),
11.90 and 11.95 (s, –NH, cis and trans conformer), 13C NMR
(DMSO-d6) d (ppm) 29.66 (benzyl–CH2), 46.50 and 46.55
(–NCH2, cis and trans conformer), ar C: [127.87 (CH),
128.27 (3CH), 129.26 (CH), 130.19 (C), 130.62 (2CH),
131.28 (C), 133.60 (C), 134.81 (C), 134.94 (C)], 138.94
(–N=CH), 146.57 and 146.83 (triazole-C-3, cis and trans
conformers), 153.61 and 163.22 (triazole-C-5, cis and trans
conformers), 168.22 and 168.35 (exocyclic-C=O, cis and
trans conformers).
4.1.3.4.
4-Amino-3-phenyl-5-oxo-4,5-dihydro-[1,2,4]tri-
azol-1-yl acetic acid benzylidene-hydrazide (4f). Recrystal-
lization from dimethyl sulfoxide–water (1:1) (yield:
85.59%), m.p. 263–264 °C; the ratio of trans–cis: 62/38.
Analysis (Calc/found %): for C17H16O2N6 C: 60.70/60.43,
H: 4.80/4.87, N: 24.99/24.64; IR (KBr) (m, cm–1), 3317–3109
(NH + NH2), 1722 (triazole –C=O), 1692 (hydrazide –C=O),
1623 (–C=N), 1544 (–C=N); 1H NMR (DMSO-d6) d (ppm)
5.00 (s, –NCH2), 5.58 and 5.61 (s, –NH2, cis and trans
conformers), 7.40–7.55 (m, 5H, ar-H), 7.73 (bs, 2H, ar H),
8.05 (bs, 3H, ar H), 8.22 (s, –N=CH), 11.70 and 11.74 (s,
–NH, cis and trans conformer); 13C NMR (DMSO-d6) d
(ppm) 46.72 and 46.80 (–NCH2, cis and trans conformer), ar
C: [126.58 (C), 126.92 (2CH), 127.11 (2CH), 127.46 (3CH),
128.35 (2CH), 128.78 (CH), 133.82 (C)], 144.16 (N=CH),
144.25 and 144.28 (triazol-C-3, cis and trans conformer),
153.03 and 153.05 (triazole-C-5, cis and trans conformer),
167.92 (exocyclic-C=O).
4.1.3.7.
4-Amino-5-oxo-3-phenyl-4,5-dihydro-[1,2,4]tri-
azol-1-yl acetic acid 2,4-dichloro-benzylidene-hydrazide
(5f). Recrystallization from dimethyl sulfoxide–water (1:1)
(yield: 80.38%), m.p. 272–273 °C; the ratio of trans–cis:
60/40. Analysis (Calc/found %): for C17H14O2N6Cl2 C:
50.39/50.61, H: 3.48/3.57, N: 20.74/20.47; IR (KBr) (m,
cm–1), 3343–3319 (NH + NH2), 1701 (triazole –C=O), 1696
1
(hydrazide –C=O), 1587 (–C=N), 1492 (–C=N); H NMR
(DMSO-d6) d (ppm) 4.98 (s, –NCH2), 5.34 (s, –NH2), 7.39
(m, 3H, ar-H), 7.46 (m, 2H, ar H), 7.62 (bs, 1H, ar H), 8.21
(bs, 1H, ar H), 8.41 (bs, 1H, ar H), 8.58 (s, –N=CH),
11.92 and 11.97 (s, –NH, cis and trans conformer), 13C NMR
(DMSO-d6) d (ppm) 46.51 and 46.57 (–NCH2, cis and trans
conformer), ar C: [127.94 (CH), 128.36 (3CH), 129.63 (CH),
130.38 (C), 130.64 (2CH), 131.28 (C), 133.66 (C), 134.82
(C), 134.94 (C)], 138.94 (–N=CH), 146.57 and 146.80
(triazole-C-3, cis and trans conformers), 154.21 and 163.25
(triazole-C-5, cis and trans conformers), 168.22 and 168.30
(exocyclic-C=O, cis and trans conformers).
4.1.3.5.
4-Amino-3-methyl-5-oxo-4,5-dihydro-[1,2,4]tri-
azol-1-yl acetic acid 2,4-dichloro-benzylidene-hydrazide
(5a). Recrystallization from dimethyl sulfoxide–water (1:1)
(yield: 74.39%), m.p. 251–252 °C; the ratio of trans–cis:
61/39. Analysis (Calc/found %): for C12H12O2N6Cl2 C:
41.99/42.47, H: 3.52/3.63, N: 24.48/24.55; IR (KBr) (m,
cm–1), 3382–3240 (NH + NH2), 1717 (triazole C=O), 1691
4.1.4. General method for the synthesis
of thiosemicarbazides (6)
1
(hydrazide –C=O), 1587 (–C=N), 1465 (–C=N); H NMR
(DMSO-d6) d (ppm) 2.33 (s, –CH3), 4.85 (s, –NCH2), 5.34 (s,
–NH2), 7.50 (d, J = 6.0 Hz, 1H, ar-H), 7.71 (s, 1H, ar H), 8.08
(d, J = 6.0 Hz, 1H, ar H), 8.55 (s, –N=CH), 11.90 and 11.95
(s, –NH, cis and trans conformers); 13C NMR (DMSO-d6) d
(ppm) 10.60 (–CH3), 46.19 and 46.25 (–NCH2, cis and trans
conformers), ar C: [128.16 (CH), 129.60 (CH), 130.19 (C),
133.57 (C), 134.19 (C), 139.50 (CH)], 144.62 (–N=CH),
144.89 and 146.94 (triazole-C-3, cis and trans conformers),
153.80 and 155.84 (triazole-C-5 cis and trans conformers),
168.32 (exocyclic-C=O); (EI MS, 70 eV) m/z (%) 343
(M+,6), 341 (13), 339 (25), 325 (21), 255 (19), 111 (46), 96
(49), 95 (70), 80 (100), 64 (38), 62 (56).
A mixture of corresponding acid hydrazide (3) (0.01 mol)
and phenyl isothiocyanate (0.015 mol) was refluxed in etha-
nol for 2 h. The solution was cooled and a white solid
appeared. This was filtered and recrystallized from an appro-
priate solvent to afford the desired product.
4.1.4.1. 1-(4-Amino-3-methyl-5-oxo-4,5-dihydro-[1,2,4]tri-
azol-1-yl)acetyl-4-phenyl thio-semicarbazide (6a). Recrys-
tallization from ethanol (yield: 82.56%), m.p. 179–180 °C.
Analysis (Calc/found %): for C12H15O2N7S C: 44.85/44.50,
H: 4.70/4.77, N: 30.51/30.57; IR (KBr) (m, cm–1), 3375–3298
(3NH + NH2), 1717 (triazole –C=O), 1696 (exocyclic-C=O),

N. Demirbas et al. / European Journal of Medicinal Chemistry 39 (2004) 793–804
801
1551 (–C=N), 1309 (–C=S); 1H NMR (DMSO-d6) d (ppm)
2.10 (s, –CH3), 4.40 (s, –NCH2), 5.31 (s, –NH2), 7.18–7.21
(m, 1H, ar-H), 7.31–7.44 (m, 4H, ar H), 9.66 (s, –NH), 9.75
(s, –NH), 10.30 (s, –NH); 13C NMR (DMSO-d6) d (ppm)
10.57 (–CH3), 46.44 (–NCH2), ar C: [125.25 (CH), 128.04
(4CH), 138.37 (C)], 145.15 (triazole-C-3), 153.30 (triazole-
C-5), 166.56 (exocyclic-C=O), 180.55 (–C=S).
4.1.5. General method for the synthesis of compounds 7
A mixture of the corresponding thiosemicarbazide (6)
(0.01 mol) in cold concentrated sulfuric acid (28 ml) was
stirred for 10 min. Then, the mixture was allowed 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 an appropriate solvent to
afford the desired product.
4.1.4.2. 1-(3-n-Propyl-4-amino-4,5-dihydro-1H-1,2,4-tri-
azol-5-on-1yl)acetyl-4-phenyl thio-semicarbazide (6c). Re-
crystallization from isobutyl acetate (yield: 66.48%), m.p.
140–141 °C. Analysis (Calc/found %): for C14H19O2N7S C:
48.12/48.19, H: 5.48/5.91, N: 28.06/28.00; IR (KBr) (m,
cm–1), 3389–3310 (3NH + NH2), 1717 (triazole –C=O),
1696 (exocyclic-C=O), 1549 (–C=N), 1309 (–C=S); 1H
NMR (DMSO-d6) d (ppm) 1.08 (t, J = 6.0 Hz,
–CH2CH2CH3), 1.62–1.71 (m, –CH2CH2CH3,), 2.42 (q,
J = 6.0 Hz, –CH2CH2CH3), 4.41 (s, –NCH2), 5.30 (s, –NH2),
7.19–7.24 (m, 2H, ar-H), 7.30–7.42 (m, 3H, ar H), 9.66 (s,
–NH), 9.72 (s, –NH), 10.38 (s, –NH); 13C NMR (DMSO-d6)
d (ppm) 13.49 (–CH2CH2CH3), 18.78 (–CH2CH2CH3),
26.57 (–CH2CH2CH3), 46.44 (–NCH2), ar C: [125.20 (2CH),
128.94 (2CH), 129.10 (CH), 137.97 (C)], 145.15 (triazole-C-
3), 153.33 (triazole-C-5), 167.05 (exocyclic-C=O), 180.37
(–C=S).
4.1.5.1. 1-(5-Phenylamino-1,3,4-thiadiazol-2-yl)methyl-4-
amino-3-methyl-5-oxo-4,5-dihydro-[1,2,4]triazole (7a). Re-
crystallization from ethanol (yield: 78.34%), m.p. 227–
228 °C. Analysis (Calc/found %): C12H13ON7S C:
47.51/48.03.92, H: 4.31/4.29, N: 32.32/32.19; IR (KBr) (m,
cm–1), 3316–3140 (NH + NH2), 1699(–C=O), 1573 (–C=N),
1498 (–C=N); 1H NMR (DMSO-d6) d (ppm) 2.12 (s, –CH3),
5.11 (s, –NCH2), 5.34 (s, –NH2), 7.00 (d, J = 8 Hz, 2H, ar-H),
7.30 (t, J = 6.4 Hz, 1H, ar H), 7.60 (t, J1= 8 Hz, J2= 6.4 Hz,
2H, ar H), 10.37 (s, –NH); 13C NMR (DMSO-d6) d (ppm)
10.56 (–CH3), 43.60 (–NCH2), ar C: [117.29 (2CH), 121.86
(CH), 129.00 (2CH), 140.34 (C)], 145.88 (triazole-C-3),
152.49 (triazole-C-5), 154.46 (thiadiazol-C-2), 165.27
(thiadiazol-C-5).
4.1.5.2. 1-(5-Phenylamino-1,3,4-thiadiazol-2-yl)methyl-4-
amino-5-oxo-3-n-propyl-4,5-dihydro-[1,2,4]triazole (7c).
Recrystallization from ethanol (yield: 86.63%), m.p. 165–
166 °C. Analysis (Calc/found %): C14H17ON7S C:
50.74/50.18, H: 5.17/5.35, N: 29.58/28.81; IR (KBr) (m,
cm–1), 3316–3125 (NH + NH2), 1695(–C=O), 1581 (–C=N),
1496 (–C=N); 1H NMR (DMSO-d6) d (ppm) 1.06 (t,
J = 6.0 Hz, –CH2CH2CH3), 1.61–1.70 (m, J = 6.0 Hz,
–CH2CH2CH3), 2.44 (t, J = 6.0 Hz, –CH2CH2CH3), 4.92 (s,
–NCH2), 5.36 (s, –NH2), 7.11 (bs, 2H, ar-H), 7.26 (bs, 3H, ar
H), 10.68 (s, –NH); 13C NMR (DMSO-d6) d (ppm) 13.92
4.1.4.3. 1-(4-Amino-3-benzyl-5-oxo-4,5-dihydro-[1,2,4]tri-
azol-1-yl)acetyl-4-phenyl thio-semicarbazide (6d). Recrys-
tallization from ethanol (yield: 68.14%), m.p. 110–111 °C.
Analysis (Calc/found %): for C18H19O2N7S C: 54.39/55.06,
H: 4.81/4.97, N: 24.66/24.45; IR (KBr) (m, cm–1), 3389–3308
(3NH + NH2), 1715 (triazole –C=O), 1695 (exocyclic-C=O),
1557 (–C=N), 1315 (–C=S); 1H NMR (DMSO-d6) d (ppm)
3.84 (s, benzyl–CH2), 4.41 (s, –NCH2), 5.36 (s, –NH2),
7.10–7.28 (m, 5H, ar-H), 7.32–7.40 (m, 3H, ar H), 7.49–7.54
(m, 2H, ar-H), 9.46 (s, –NH), 9.95 (s, –NH), 10.76 (s, –NH);
13C NMR (DMSO-d6) d (ppm) 30.47 (benzyl–CH2), 46.48
(–NCH2), ar C: [125.35 (2CH), 126.58 (2CH), 126.94 (CH),
128.79 (CH), 128.94 (2CH), 129.10 (2CH), 133.25 (C),
137.97 (C)], 147.15 (triazole-C-3), 153.33 (triazole-C-5),
166.75 (exocyclic-C=O), 180.12 (–C=S).
(–CH2CH2CH3),
19.04
(–CH2CH2CH3),
31.07
(–CH2CH2CH3), 45.13 (–NCH2), ar C: [121.01 (2CH),
121.75 (CH), 128.13 (2CH), 133.35 (C)], 146.11 (triazole-C-
3), 152.87 (triazole-C-5), 154.11 (thiadizole-C-2), 165.19
(thiadizole-C-5).
4.1.5.3. 1-(5-Phenylamino-1,3,4-thiadiazol-2-yl)methyl-4-
amino-3-benzyl-5-oxo-4,5-dihydro-[1,2,4]triazole (7d). Re-
crystallization from ethanol (yield: 80.25%), m.p. 155–
186 °C. Analysis (Calc/found %): C18H17N7S C:
56.97/57.44, H: 4.51/4.79, N: 25.84/26.17; IR (KBr) (m,
cm–1), 3286–3200 (NH + NH2), 1712(–C=O), 1599 (–C=N),
1550 (–C=N); 1H NMR (DMSO-d6) d (ppm) 3.81 (s, benzyl–
CH2), 5.05 (s, –NCH2), 5.29 (s, –NH2), 6.91 (t, J = 6.0 Hz,
1H, ar H), 7.19–7.30 (m, 7H, ar H), 7.49 (d, J = 8.0 Hz, 2H,
ar-H), 10.29 (s, –NH); 13C NMR (DMSO-d6) d (ppm), 30 21
(benzyl–CH2), 43.81 (–NCH2), ar C: [117.32 (2CH), 121.91
(CH), 126.58 (CH), 128.34 (2CH), 128.64 (2CH), 129.02
(2CH), 135.59 (C), 140.37 (C)], 147.80 (triazole-C-3),
152.62 (triazole-C-5), 154.44 (thiadizole-C-2), 165.31
(thiadizole-C-5).
4.1.4.4. 1-(4-Amino-3-phenyl-5-oxo-4,5-dihydro-[1,2,4]tri-
azol-1-yl)acetyl-4-phenyl thio-sem-carbazide (6f). Recrys-
tallization from ethanol (yield: 76.54%), m.p. 215–216 °C.
Analysis (Calc/found %): for C17H17O2N7S C: 53.25/53.92,
H: 4.46/4.69, N: 25.57/25.39; IR (KBr) (m, cm–1), 3295–3208
(3NH + NH2), 1705 (triazole –C=O), 1680 (exocyclic-C=O),
1
1548 (–C=N), 1341(–C=S); H NMR (DMSO-d6) d (ppm)
4.34 (s, –NCH2), 5.35 (s, –NH2), 7.42–7.58 (m, 5H, ar-H),
7.72 (bs, 3H, ar H), 8.02–8.18 (bs, 2H, ar-H), 9.38 (s, –NH),
9.96 (s, –NH), 11.26 (s, –NH); 13C NMR (DMSO-d6) d
(ppm) 46.78 (–NCH2), ar C: [126.58 (C), 126.94 (2CH),
127.13 (2CH), 127.61 (3CH), 128.35 (2CH), 128.74 (CH),
133.85 (C)], 148.07 (triazole-C-3), 154.39 (triazole-C-5),
167.45 (exocyclic-C=O), 181.03 (–C=S).

802
N. Demirbas et al. / European Journal of Medicinal Chemistry 39 (2004) 793–804
4.1.5.4. 1-(5-Phenylamino-1,3,4-thiadiazol-2-yl)methyl-4-
amino-3-phenyl-5-oxo-4,5-dihydro-[1,2,4]triazole (7f). Re-
crystallization from ethanol–water (1:1) (yield: 87.52%),
m.p. 222–223 °C. Analysis (Calc/found %): C17H15ON7S C:
55.87/56.05, H: 4.13/4.19, N: 26.83/26.76; IR (KBr) (m,
cm–1), 3318–3266 (NH + NH2), 1713(–C=O), 1556 (–C=N),
1508 (–C=N); 1H NMR (DMSO-d6) d (ppm) 5.28 (s,
–NCH2), 5.60 (s, –NH2), 7.01 (t, J = 6.0 Hz, 1H, ar H), 7.33
(t, J = 8.0 Hz, 2H, ar H), 7.51–7.60 (m, 5H, ar-H), 7.89 (bs,
2H, ar H), 10.38 (s, –NH); 13C NMR (DMSO-d6) d (ppm),
44.01 (–NCH2), ar C: [117.30 (2CH), 120.88 (CH), 126.21
(C),127.53 (2CH), 128.28 (2CH), 128.99 (2CH), 130.06
(CH), 140.31 (C)], 144.40 (triazole-C-3), 153.00 (triazole-C-
5), 154.13 (thiadizole-C-2), 165.33 (thiadizole-C-5); (EI
MS, 70 eV) m/z (%) 365 (M+,56), 359 (25), 290 (18), 266.61
(28), 250 (18), 249 (100), 190 (44), 177 (34), 100 (44), 90
(44), 79 (48), 74 (38), 61 (20), 58.29 (80).
4.1.6.2.
1-(4-phenyl-5-thioxo-[1,2,4]triazol-3-yl)methyl-
4-amino-5-oxo-3-n-propyl-4,5-dihydro-[1,2,4]triazole
(8c). Recrystallization from ethanol–ethyl acetate (1:1)
(yield: 78.20%), m.p. 215–216 °C. Analysis (Calc/found %):
C14H17ON7S C: 50.74/50.56, H: 5.17/5.48, N: 29.58/29.32;
IR (KBr) (m, cm–1), 3304–3117 (NH + NH2), 1695(–C=O),
1573 (–C=N), 1491 (–C=N), 1362 (C=S); 1H NMR (DMSO-
d6) d (ppm) 0.93 (t, J = 5.9 Hz, –CH2CH2CH3), 1.53 (hex,
J = 5.9 Hz, –CH2CH2CH3), 2.37 (t, J = 5.9 Hz,
–CH2CH2CH3), 4.83 (s, –NCH2), 5.07 (s, –NH2), 7.47 (bs,
2H, ar-H), 7.51 (bs, 3H, ar H), 12.97 (s, –NH); 13C NMR
(DMSO-d6)
d
(ppm) 17.16 (–CH2CH2CH3), 22.53
(–CH2CH2CH3), 29.81 (–CH2CH2CH3), 44.03 (–NCH2), ar
C: [131.22 (2CH), 132.80 (2CH), 133.12 (CH), 136.48 (C)],
151.37 (triazole-C-3), 151.87 (5-thioxo-triazole-C-3),
156.03 (triazol-C-5), 171.96 (5-thioxo-triazol-C-5); (EI MS,
70 eV) m/z (%) 331 (M+ 56), 254 (17), 249 (19), 215 (14),
176 (13), 143 (14), 79 (100), 64 (19), 63 (16).
4.1.6. General method for the synthesis of compounds 8
4.1.6.1. Method A. A solution of corresponding thiosemicar-
bazide (6) (0.01 mol) in 2 N NaOH were refluxed for 3 h. The
resulting solution was cooled to room temperature and acidi-
fied to pH 3–4 with 37% HCl. The precipitate formed was
filtered, washed with water and recrystallized from an appro-
priate solvent to afford the desired compound.
4.1.6.3. 1-(4-phenyl-5-thioxo-[1,2,4]triazol-3-yl)methyl-4-
amino-3-benzyl5-oxo-4,5-dihydro-[1,2,4]triazole (8d). Re-
crystallization from dimethyl sulfoxide–water (1:1) (yield:
81.12%), m.p. 294–295 °C. Analysis (Calc/found %):
C18H17ON7S C: 56.97/57.28, H: 4.51/4.83, N: 25.84/26.03;
IR (KBr) (m, cm–1), 3357–3173 (NH + NH2), 1694(–C=O),
1577 (–C=N), 1473 (–C=N), 1384 (C=S); 1H NMR (DMSO-
d6) d (ppm) 3.78 (s, benzyl–CH2), 4.81 (s, –NCH2), 5.10 (s,
–NH2), 7.23–7.29 (m, 4H, ar-H), 7.39–7.43 (m, 6H, ar H),
13.95 (s, –NH); 13C NMR (DMSO-d6) d (ppm) 30.09 (ben-
zyl–CH2), 40.36 (–NCH2), ar C: [126.36 (CH), 127.54
(2CH), 128.37 (2CH), 128.73 (2CH), 129.09 (2CH), 129.47
(CH), 132.71 (C), 135.61 (C)], 147.46 (triazole-C-3), 147.59
(5-thioxo-triazole-C-3), 152.29 (triazol-C-5), 168.29 (5-
thioxo-triazol-C-5); (EI MS, 70 eV) m/z (%) 379 (M+ 38),
249 (100), 246 (14), 228 (14), 135 (24), 108 (31), 100 (34),
88 (35), 86 (53), 79 (44), 63 (30).
4.1.6.2. Method B. The corresponding thiosemicarbazide (6)
was heated in an oil bath at 130 °C for 2 h. The white solid
formed recrystallized from an appropriate solvent to afford
the compounds 8.
4.1.6.3. Method C (only for 8f). A mixture of corresponding
acid hydrazide (3f) (0.01 mol) and phenyl isothiocyanate
(0.015 mol) in ethanol was refluxed for 8 h. The solution was
cooled and a white solid appeared. This was filtered and
recrystallized from dimethyl sulfoxide–water (1:1) to afford
the desired product. (Yield: 72.68%).
4.1.6.4 1-. (4-phenyl-5-thioxo-[1,2,4]triazol-3-yl)methyl-4-
amino-
4.1.6.1. 1-(4-phenyl-5-thioxo-[1,2,4]triazol-3-yl)methyl-4-
amino-3-methyl-5-oxo-4,5-dihydro-[1,2,4]triazole (8a). Re-
crystallization from dimethyl sulfoxide (yield: 76.39%), m.p.
240–241 °C. Analysis (Calc/found %): C12H13ON7S C:
47.51/48.03.92, H: 4.31/4.29, N: 32.32/32.19; IR (KBr) (m,
cm–1), 3317–3223 (NH + NH2), 1698 (–C=O), 1602 (–C=N),
1573 (–C=N), 1343 (–C=S); 1H NMR (DMSO-d6) d (ppm)
2.00 (s, –CH3), 4.78 (s, –NCH2), 5.05 (s, –NH2), 7.20–7.30
5-oxo-3-phenyl-4,5-dihydro-[1,2,4]triazole (8f). Recrystal-
lization from dimethyl sulfoxide–water (1:1) (yield:
81.12%), m.p. 292–293 °C. Analysis (Calc/found %):
C17H15ON7S C: 55.87/55.89, H: 4.13/4.29, N: 26.83/26.39;
IR (KBr) (m, cm–1), 3344–3145 (NH + NH2), 1721(–C=O),
1587 (–C=N), 1497 (–C=N), 1320 (C=S); 1H NMR (DMSO-
d6) d (ppm) 4.97 (s, –NCH2), 5.34 (s, –NH2), 7.34 (bs, 2H,
ar-H), 7.48 (bs, 6H, ar H), 7.83 (bs, 2H, ar H), 14.00 (s, –NH);
13C NMR (DMSO-d6) d (ppm) 40.77 (–NCH2), ar C: [126.
(C), 127.56 (4CH), 128.25 (2CH), 129.15 (2CH), 129.47
(CH), 129.99 (CH), 132.80 (C)], 145.07 (triazole-C-3),
147.51 (5-thioxo-triazole-C-3), 152.61 (triazole-C-5),
168.28 (5-thioxo-triazole-C-5); (EI MS, 70 eV) m/z (%) 365
(M+ 39), 249 (21), 177 (13), 79 (38), 64 (80), 63 (100).
(m, 2H, ar-H), 7.40–7.50 (m, 3H, ar H), 13.95 (s, –NH); 13
C
NMR (DMSO-d6) d (ppm) 10.36 (–CH3), 46.78 (–NCH2), ar
C: [127.51 (2CH), 129.06 (2CH), 129.38 (CH), 132.71 (C)],
145.49 (5-thioxo-triazole-C-2), 147.68 (triazole-C-3),
152.11 (triazole-C-5), 168.20 (5-thioxo-triazole-C-5); (EI
MS, 70 eV) m/z (%) 303 (M+14), 249 (100), 205 (66), 102
(20), 101 (37), 79 (41), 60 (22).

N. Demirbas et al. / European Journal of Medicinal Chemistry 39 (2004) 793–804
803
4.2. Biological activity studies
added at a single concentration and culture incubated for
48 h. End-point determinations were made with alamar blue.
The screening results are summarized in Table 3. Results for
each test agents are reported as the percent of growth of the
treated cells when compared to the untreated control cells.
Compounds (4c and 5f) which reduce the growth any one of
the cell lines to approximately 32% or less are passed on for
evaluation in the full panel of 60 cell lines derived from
human solid tumors (brain, breast, colon, leukemia, lung,
melanoma, ovarian and renal) over a 5-log dose range. Com-
pound 4c and 5f was tested at a minimum of five concentra-
tions at 10-fold dilutions. A 48 h continuous drug exposure
protocol was used and a sulforhodamin B (SRB) protein
assay was used to estimate cell viability or growth. The
screening results of compounds 4c and 5f towards several
tumor cell line are presented in Table 4 as GI50 values. As
seen in Table 3, compounds 4c and 5f are effective towards
breast cancer (MCF7).
4.2.1. Antimicrobial activity
All test microorganisms except for Penicillium spp. and
Aspergillus spp. were obtained from the Hifzissihha Institute
of Refik Saydam (Ankara, Turkey) and are as follows; E. coli
ATCC 35218, P. aeruginosa ATCC 10145, Yersinia
pseudotuberculosis ATCC 911, K. pneumoniae ATCC
13883, Enterococcus faecalis ATCC 29212, S. aureus ATCC
25923, B. subtilis ATCC 6633, C. albicans ATCC 60193, C.
tropicalis ATCC 13803, Candida glabrata. Penicillum spp.
and Aspergillus spp. were isolated from soil. All the newly
synthesized compounds except 4c and 5f were weighed and
dissolved in dimethylsulphoxide (DMSO) and 95% ethanol
to prepare extract stock solution of 1 mg/ml.
4.2.1.1. Agar well diffusion method. Simple susceptibility
screening test using agar-well diffusion method [31] as
adapted earlier [32] was used. Each microorganism was
suspended in Brain Heart Infusion (BHI) (Difco, Detroit, MI)
broth and diluted to 106 colony forming unit (cfu) per ml.
They were “flood-inoculated” onto the surface of BHI agar
and Sabouraud Dextrose Agar (SDA) (Difco, Detriot, MI)
and then dried. For C. albicans, C. tropicalis, C. glabrata,
Penicillum spp. and Aspergillus spp., SDA were used. Five-
millimeter diameter wells were cut from the agar using a
sterile cork-borer, and 250–5000 µg/50 µl of the chemical
substances were 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. Ceftazidime (Fortum) (10 µg) and Triflucan (5 µg)
were standard drugs. Ethanol and dimethylsulphoxide were
used as solved control. Results were interpreted in terms of
the diameter of the inhibition zone: (–): <5.5 mm; (+): 5.5–
10 mm; (++): 11–16 mm; (+++): ≥17 mm. The results are
shown in Table 1.
Acknowledgments
The authors thank to Dr. S. Guner (Karadeniz Technical
University, Department of Chemistry) and Dr. R.S. Aslan
(Karadeniz Technical University, Department of Foreign lan-
guage and Literature) for critical reading this manuscript.
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