New thiophene-1,2,4-triazole-5(3)-ones: Highly bioactive thiosemicarbazides, structures of Schiff bases and triazole-thiols

Article abstract of DOI:10.1016/j.ejmech.2014.01.014

Key compound 2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4- tiazole-1-yl) acetohydrazide (3) was synthesized by reacting hydrazine hydrate with ethyl-2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-tiazole- 1yl)acetate (2), obtai

Full text of DOI:10.1016/j.ejmech.2014.01.014

Accepted Manuscript
New thiophene-1,2,4-triazole-5(3)-ones: highly bioactive thiosemicarbazides,
structures of schiff bases and triazole-thiols
Yasemin Ünver, Kemal Sancak, Fatih Çelik, Emrah Birinci, Murat Küçük, Serkan
Soylu, Nesibe Arslan Burnaz
PII:
S0223-5234(14)00045-2
10.1016/j.ejmech.2014.01.014
EJMECH 6667
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 April 2013
Revised Date: 30 December 2013
Accepted Date: 6 January 2014
Please cite this article as: Y. Ünver, K. Sancak, F. Çelik, E. Birinci, M. Küçük, S. Soylu, N.A. Burnaz,
New thiophene-1,2,4-triazole-5(3)-ones: highly bioactive thiosemicarbazides, structures of schiff bases
and triazole-thiols, European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.01.014.
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ACCEPTED MANUSCRIPT
S
O
100
90
80
70
60
50
40
30
20
10
0
H
N
N
O
NHNH
N
Ar
88.2
84.4
NHN
C
H
R
SH
N
N
83.4
N
N
N
N
N
O
O
Ar
N
O
N
S
N
69.0
S
S
NH2
NH2
(a-d)
NH2
6 (a-e)
(a-d)
4
5
O
NHN
C
H
R
N
N
O
N
N
S
R
C
H
10.0
2
crystal structure of 4d
7(a-e)
3.1
1.9
3
1.5
1.6
1.1
1.2
0.5
.
7d 6d 5a 5b 5c
5d 4a
4b 4c 4d
Active Compounds (all at 10 mg/mL)

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New thiophene-1,2,4-triazole-5(3)-ones: highly bioactive
thiosemicarbazides, structures of schiff bases and triazole-thiols
Yasemin Ünvera,*, Kemal Sancaka ,Fatih Çelika, Emrah Birincia,Murat Küçüka, Serkan Soylub, Nesibe
Arslan Burnazc
aDepartment of Chemistry, Faculty of Sciences, Karadeniz Technical University,61080 Trabzon,
Turkey
bGiresun University, Faculty of Arts and Sciences, Department of Physics, 28100 Giresun, Turkey
cDepartment of Nutrition and Dietetics, School of Health, Gümüşhane University, 29100 Gümüşhane,
Turkey
ABSTRACT
Key compound 2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-tiazole-
1-yl) acetohydrazide (3) was synthesized by reacting hydrazine hydrate with ethyl-2-(4-
amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-tiazole-1yl)acetate (2), obtained in
basic media from 4-amino-5-(thiophene-2-ylmethyl)-2H-1,2,4-triazole-3(4H)-one (1).
Compound 3 was converted to thiosemicarbazide derivatives (4a-d) and Schiff base
derivatives 6a-e and 7a-e. The treatment of compound 4 with NaOH gave 4-amino-2-((4-(4-
aryl)-5-mercapto-4H-1,2,4-triazole-3-yl)methyl)-5-(thiophene-2-ylmethyl)-2H-1,2,4-triazole-
1
3(4H)-ones (5a-d). All newly compounds, well characterized by elemental analyses, IR, H
NMR, 13C NMR and mass spectral studies were tested for their antioxidant and antimicrobial
activities. Thiosemicarbazide derivatives (4a-d) were highly active in two antioxidant tests
with 69.0-88.2% DPPH• scavenging and 503-1257 ꢀM TEAC values, while the others
showed lower or no activity. The results of the two antioxidant tests correlated well.
Moreover, Thiosemicarbazide derivatives (4a-d) also showed antibacterial activity against S.
aureus, B. cereus, and M. smegmatis. Thiosemicarbazide group deserves attention in the
synthesis of bioactive compounds.
Keywords: Schiff base, Thiosemicarbazide, 1,2,4-triazole/thiol, Antioxidant, Antimicrobial
1

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NOTE FOR GRAPHICALABSTRACT
Twenty novel triazole derivatives were synthesized. Compounds 4a-d possessing triazole and
thiosemicarbazide exhibited both antioxidant and antimicrobial activities. Crystal structures of
2 and 4d were determined.
2

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1. Introduction
Heterocyclic compounds that possess 1,2,4-triazole ring have relevance to the
pharmacological properties. These properties are anti-inflammatory, antihypertensive
fungicidal, antioxidant, antimicrobial and antiviral activities [1-5]. In addition to this kind of
relevance, it was reported that Schiff bases that were originated from triazole compounds
possessed some biological activities [6].
It has been stated that 1,2,4,-triazole-3-thiones possess biological activities which cover
antimicrobial [7-10], antithyroid [11], hypoglycemic [12] and antidepressant [13,14].
Heterocyclic derivatives containing sulfur possess essential biological properties
including anti-inflammatory [15], analgesic [16], antidepressant [17], antimicrobial [18] and
anticonvulsant activities [19-22]. Antiepileptic drugs (AEDs) including tiagabine [19],
etizolam [21], and brotizolam [22] contain thiophene moiety in their structures as active
pharmacophore. Moreover, it has been stated that the presence of thiophene ring in its
structure gives the higher activity of sodium phethenylate [20]. Furthermore, anticonvulsant
activity of the compounds which contain hydrazones and thiosemicarbazones with different
types of substitution has been well recorded. [23-27].
Antioxidants have capacity to protect organisms and cells from damage caused by
oxidative stress during metabolism. For this reason, the synthetic compounds are extensively
studied for their antioxidant activities using different methodologies. The search for active
components that prevent or reduce the impact of oxidative stress on cells is a quite
contemporary field. Exogenous chemicals involved in food systems and endogenous
compounds involved in metabolic processes in human body produce highly reactive free
radicals, particularly oxygen derived ones. They have the potential to oxidize biomolecules
and cause cell death, consequently causing tissue damage. It is known that free radical
oxidative processes also play a significant pathological role in causing many human diseases
together with aging [28-29].
Thiosemicarbazide derivatives were reported to have stronger inhibitory effects on LP
levels and more substantial scavenger effects on DPPH radical than those of their cyclic
counterparts namely, 1,3,4-thiadiazoles and 2H-1,2,4-triazole-3(4H)-thiones [30].
Therefore, in this study, we proposed the synthesis of a new series of hybrid molecules
which possess triazole-schiff base, triazole-triazol/tiol, triazole-thiosemicarbazide including
thiophene for more efficacious biologically activities.
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2. Chemistry
The synthesis of compounds 2,3,4,5,6 and 7 was carried out according to the steps
shown in Scheme 1. Compound 1 with ethyl bromoacetate by refluxing in absolute ethanol in
presence of sodium ethoxide afforded the ethyl-2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-
4,5-dihydro-1,2,4-triazole-1-yl) acetate (2) in good yield. Then, the compound 2 was
converted to the corresponding compound, 2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-
dihydro-1,2,4-tiazol-1-yl)acetohydrazide (3) via the reaction with hydrazine hydrate. The
treatment of acetohydrazide derivative (3) with several aromatic aldehydes (1 mol and 2 mol)
in ethanol gave the Schiff bases 6a-e and 7a-e respectively. The reaction of compound 3
with 4-arylisothiocyanates produced 1-(2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-
dihydro-1,2,4-triazole-1-yl)acetyl)-4-(4-aryl)thiosemicarbazides (4a-d).
The treatment of
compound 4 with 2N NaOH caused the conversion of carbothioamide structure into 1,3,4-
thiadiazole ring; thus, 4-amino-2-((4-(4-aryl)-5-mercapto-4H-1,2,4-triazole-3-yl)methyl)-5-
(thiophene-2-ylmethyl)-2H-1,2,4-triazole-3(4H)-ones (5a-d) were obtained . The structures
1
were confirmed by IR, H NMR, 13C NMR, elemental analyses. In addition, X-ray for
compounds 2 and 4d is presented in this paper.
3. Results and discussion
3.1. Synthesis
The main goal of the present study was to synthesize and investigate the antioxidant and
antimicrobial activities of new thiophene-1,2,4- triazole-containing biologically active groups.
Synthesis of the intermediate and target compounds was performed according to the reactions
outlined in Scheme 1.
Following a previously reported literature procedure was performed, the starting
compound 4-amino-5-thiophene-2-ylmethyl 2,4-dihydro[1,2,4]triazole-3-one (1) was prepared
[6]. 4-amino-5-thiophene-2-ylmethyl 2,4-dihydro[1,2,4]triazole-3-one (1) with ethyl
bromoacetate by refluxing in absolute ethanol in the presence of sodium ethoxide afforded the
ethyl-2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-triazole-1-yl)acetate (2)
1
in good yield. The H and 13C-NMR spectra of compound 2 exhibited additional signals
originated from the –CH2CO2Et group at the related chemical shift values.
The treatment of compound 2 resulted in the formation of hydrazide derivative 3 in good
yield, and this was employed as key intermediate for synthesis of the target compounds. The
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1H-NMR spectrum of compound 3 did not display any signal belonging to the –OCH2CH3
group; instead, new signals originating from the hydrazide structure appeared at 4.06 ppm (-
NHNH2) and 9.21 ppm (-NHNH2) integrating for 2 protons and 1 proton, respectively
(controlled by changing with D2O).
With the help of the reaction of isonicotinic acid hydrazides with 2-(4-amino-5-oxo-3-
(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-tiazole-1-yl) acetohydrazide (3) phenyl/substitute
phenyl thiosemicarbazides (4a-d) were obtained. In the 1H-NMR spectra of compounds 4a-d,
additional NH- signal originating from thiosemicarbazide structures were observed at 9.18-
9.68, 9.65-9.83 and 9.30-10.35 ppm, while the signal due to –NH2 group of hydrazide
structures did not appear. Additional signals belonging to the phenyl rings were observed in
1
the aromatic region in the H and 13C-NMR spectra of compounds 4a-d. Moreover, -C=S
groups resonated at 181.32-181.64 ppm in the 13 C-NMR spectra of compounds 4a-d.
When compounds 4a-d were converted to 1,2,4-triazole-3-thiols (5) in basic media, the
signal observed at 13.60-13.97 ppm in the 1H-NMR spectra of compounds 5a-d was attributed
to –SH group, but -NH peak disappeared.
The synthesis of different Schiff base derivatives of compounds 6a-e and 7a-e were
carried out by the reaction of compound 3 with several aromatic aldehydes (1 and 2 mol) in
oil bath. Owing to the aromatic ring originated from aldehyde moiety at aromatic region, the
1H and 13C-NMR spectra of compounds 6a-e and 7a-e displayed additional signals, while the
signal of –NH2 group of hydrazide (for compound 6a-e) and triazole –NH2 (for compound 6a-
e and 7a-e) structure did not appear.
3.2. Biological Activities
3.2.1. Antioxidant Activities
Compounds 2-7 were tested for their antioxidant activity based on the two methods most
widely used in literature [30,32]. The first method applied was DPPH• scavenging, which
may have disadvantage of dependence on the shape of the compound tested because the
compound may have sterical hindrance to reach the radical site of DPPH•. DPPH method
utilizes both single electron transfer (SET) and hydrogen atom transfer (HAT) reactions with
antioxidants. The method is also known to have relatively slow kinetics. There are examples
in which the test samples are inactive in DPPH• scavenging test, while they show high
antioxidant activity in other test methods. Therefore, ferric reducing/antioxidant power
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(FRAP) assay to measure antioxidant activity was also utilized. FRAP method utilizes only
SET reaction mechanism and is widely used for determining antioxidant capacities of various
samples including food, biological and synthetics.
DPPH radical scavenging activities were determined as %scavenging through 1 h
incubation period, during which absorbance at 517 nm decreases as the active compound
scavenges DPPH• present in the medium. The data is provided in Figure 1 for kinetic
behavior of the reaction between only the active 12 compounds and DPPH•, and in Figure 2
for their comparison with the values only at 60 min incubation time. Because compounds 4a-
d were much more active in the pretests, their concentration was 10 ꢀg/mL, while the
concentration of the other compounds tested were 200 ꢀg/mL.
The starting compounds 2 and 3, all the thiosemicarbazides (4a-d), and triazole-thiols
showed DPPH• radical scavenging activity at the test concentrations. Among the Schiff bases
only 6d and 7d were slightly active and the others showed no activity at 200 ꢀg/mL. With the
DPPH scavenging compounds, especially highly active 4a-d, the reaction appeared to be slow
and continue even after 60 min incubation though at much reduced rates.
The comparatively high DPPH• scavenging activities of compounds 4a-d can better be
observed if the percentage scavenging values of the other active compounds (2, 3, 5a-d, 6d,
7d) were divided by 20, as the concentrations of 4a-d were diluted 20 fold to see the kinetic
behavior during 60 minute incubation. Therefore, the activities of 4a-d were at least 20 fold
higher than all other compounds. The activity order in the thiosemicarbazides based on the
substituents was –CH3 > –Br > –H > –F. However, the activity order in the triazole-thiols
based on the substituents was different as –H > –F > –CH3 > –Br, reflecting the importance of
main structure in determination of the effect of substituent on DPPH• scavenging activity. The
presence of N-H group on thiosemicarbazone moiety may provide hydrogen atom to DPPH•
radical as stated by Nguyen et al. [33], which makes the compound a radical itself and reacts
with other DPPH• radicals increasing the total antiradical capacity, and thus scavenging more
radicals. The radical electron may delocalize towards the aromatic ring so as to stabilize the
radical preventing it from self-destruction. Electron donating -CH3 group attached to the
aromatic ring (compound 4d) resulted in highest antiradical activity in the thiosemicarbazide
series probably by stabilizing the thiosemicarbazide radical, as opposed to the electron
withdrawing group -F (compound 4c) that reduced the antiradical activity. The presence of
furan structure in Schiff bases appear to play a crucial role in antiradical activity as only the
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furan derivatives (6d and 7d) were active. Compounds 2 and 3 were only slightly active in
DPPH• test.
With FRAP assay the sample compounds were tested for their abilities to reduce
iron(III) to iron(II) ions, and the absorbance of TPTZ-Fe2+ complex was measured at 595 nm
increasing with higher activity antioxidants. The absorbances measured for the samples were
converted to Trolox equivalent antioxidant capacity (TEAC) values obtained from the
absorbance – [Trolox] calibration graph, and the ꢀM TEAC values are given in Figure 3.
FRAP values show a similar trend with DPPH• scavenging results indicating higher activity
with thiosemicarbazides (4a-d) and much lower activity with triazole-thiols (5a-d) and Schiff
bases (6-7). The starting compound 2 also showed low activity as with DPPH• scavenging
test. However, compound 3 was highly active in FRAP test as opposed to low activity in
DPPH• scavenging test, which may be an indication of structural dependence of the
antioxidation reaction in the latter case. The activity order for the thiosemicarbazides based on
the substituents in compounds 4a-d was same with DPPH• scavenging test as –CH3 > –Br > –
H > –F, reflecting the role of electron withdrawing and donating substituents as decreasing
and increasing antioxidant capacity.
The results of the correlation of the two antioxidant tests are plotted (Figure 4), and a
relatively good correlation was observed (R2=0.83). The differences seen between the results
of the two antioxidant methods are probably due to the differences between the reaction
mechanisms and in dependence on the reaction conditions and sterical issues in the case of
DPPH• test. The results of the antioxidant tests clearly show that thiosemicarbazide group
cause a radical increase in antioxidant capacity, and its conversion to triazole thiol reduced the
activity, which may be attributed to the disappearance of hydrogen atom on nitrogen of
thiosemicarbazone structure. In many of the earlier antioxidant studies in the literature in
which thiosemicarbazide structures appear as intermediate compounds towards the synthesis
of triazoles or other products, thiosemicarbazides have not been tested for their biological
activities including antioxidant activity [34,35]. However, as evident from our findings that
the testing of their antioxidant activities must be reconsidered and the new structures to be
included in the future literature must certainly be evaluated for their antioxidant activities.
3.2.2. Antimicrobial Activities
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The compounds were tested against three Gram (-) bacteria (E. coli, Y. pseudotuberculosis,
and P. auroginosa), four Gram (+) bacteria (S. aureus, E. fecalis, L. monocytogenes, and B.
cereus), the mycobacterial model organism M. smegmatis, the yeast-like fungus C. albicans,
and the yeast S. cerevisiae. Besides the activity of compounds 2 and 3 against M. smegmatis,
activity was observed with only thiosemicarbazide derivatives at test concentrations and
against only three microorganisms, two Gram (+) bacteria and the mycobacterium (Table 1).
Thiosemicarbazide derivatives, being highly active in antioxidant tests, showed moderate
activity, though all other derivatives were inactive. The reason for the Gram (-) bacteria to be
resistant to all the compounds tested may be the relatively more impermeant structure of the
cell wall including the more closely spaced peptidoglycan and the extra lipopolysaccharide
layers.
4. Conclusion
In this study, the synthesis of some new 1,2,4-triazole-Schiff base, 1,2,4-triazole-
thiosemicarbazide and 1,2,4-triazole-triazole/thiol compounds possessing thiophene ring was
reported. The antioxidant studies revealed that all the compounds were active in FRAP assay,
while thiosemicarbazide derivatives (4a-d) were very active and triazole-thiol derivatives (5a-
d) and Schiff bases 6d and 7d showed low activity in DPPH• scavenging assay.
Thiosemicarbazide derivatives also showed antibacterial activity against three
microorganisms. As being potential pharmacophore, thiosemicarbazide groups in these types
of compounds should be considered for the synthesis of lead compounds in search of active
compounds against concomitant oxidative stress and bacterial infections.
5. Experimental
5.1. Chemistry
The 1H-, and 13C-Nuclear Magnetic Resonance spectra were recorded on a Varian-Mercury 200
MHz spectrometer, where TMS as an internal standard and DMSO-d6 as solvent are used. IR
spectrum was recorded on a Perkin-Elmer Spectrum one FT-IR spectrometer
(resolution 4) in
KBr pellets. For elemental analysis, combustion/gas chromatography method was used. Elemental
analyses was performed on a Hewlett-Packard 185 CHN analyzer; their values agreed with the
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calculated ones. The MS spectrum was measured with an Micromass Quattro LC-MS/MS
spectrometer with methanol as solvent. M.p. was measured on an electrothermal apparatus and are
uncorrected. Compound 1 was prepared by the way reported earlier [6].
5.1.1. Synthesis of the compound 2
0.1 mol metallic sodium was added absolute alcohol and mixed until dissolved in room
temperature. Then 0.1 mol 4-amino-5-(thiophene-2-ylmethyl)-2H-1,2,4-triazole-3(4H)-one
was added this mixture and heated under reflux till observed the formation of salt. After that
this mixture was cooled and 0.1 mol ethyl 2-bromoacetate was added in this mixture. Then
mixture was heated under reflux for 6h. The reaction mixture was cooled refrigerator. White
crude product was collapsed and recrystallized with water.
5.1.1.1.
Ethyl-2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-triazole-1-yl)
o
acetate (2). Yield: 78.02%, m.p.119-120 C. IR (KBr, cm-1): 3278-3216 (NH2), 1752 (ester
1
C=O), 1716 (C=O), 1667 (C=N), 1206 (C-O) ; H NMR (200 MHz, DMSO-d6) δ: 1.17 (t,
3H,CH3), 4.05-4.16 (m, 4H, O-CH2+N-CH2), 4.52 (s, 2H, tyf-CH2), 5.40 (s, 2H, NH2),
13
thiophen H [ 6.96 (s, 2H), 7.37 (bs, 1H)]; C NMR (50MHz, DMSO-d6) δ : 15.60 (CH3),
25.98 (tyf-CH2), 47.90 (N-CH2), 61.95 (O-CH2), Tyf-C [124.77 (CH), 126.13 (CH), 126.57
(CH), 137.95(C)], 146.10 (C=N), 153.10 (C=O), 169.51 (ester C=O); LC-MS (m/z): 284.09
(M+,70%); Anal. Calcd for C11H14N4O3S: C, 46.80; H, 5.00; N, 19.85; Found: C,46.88;
H,5.01; N,19.90.
5.1.2.Synthesis of the compound 3
0.1 mol ethyl 2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-triazole-1-
yl)acetate and 0.1 mol hydrazine hydrate was mixed in butan-1-ol, then was heated under
reflux for 6 h. At the end of the reaction, white solid product was collapsed and recrystallized
with water.
5.1.2.1.
2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-triazole-1-yl)aceto
hydrazide (3). Yield: 85.50%, m.p.196-197oC. IR (KBr, cm-1): 3417-3338 (NH2), 3300-(NH),
1
1716 (trz C=O), 1650 (C=O), 1571 (C=N), 1524 (C=C); H NMR (200 MHz, DMSO-d6) δ:
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4.20 (s, 2H, N-CH2), 4.26 (s, 2H, tyf-CH2), 5.32 (s, 2H, N-NH2), 4.06 (s, 2H, NH-NH2),
thiophen H [ 6.96 (d, 2H), 7.37 (d, 1H)], 9.21 (s, 1H, NH); 13C NMR (50MHz, DMSO-d6) δ
: 25.66 (tyf-CH2), 47.10 (N-CH2), Tyf-C [125.77 (CH), 127.21 (CH), 127.57 (CH),
137.99(C)], 146.99 (C=N), 154.10 (trz C=O), 166.61 (C=O); LC-MS (m/z): 268.99 (M+2,
100%);Anal. Calcd for C9H12N6O2S: C, 40.29; H, 4.51; N, 31.32; Found: C,40.91; H, 4.76;
N,31.01.
5.1.3. .General method for the synthesis of compounds 4
0.1
mol
2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-triazole-1-yl)
acetohydrazide (3) was mixed with various isothiocyanates in ethanol. Then this mixture was
heated under reflux for 6h. At the and of the reaction,crude product collapsed. The precipitate
formed was filtered off and purified by recrystallization from ethanol and DMF.
5.1.3.1.
1-(2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-triazole-1-
o
yl)acetyl)-4-phenylthiosemicarbazide (4a). Yield:80.25%, m.p.177-178 C. IR (KBr, cm-1) :
3413-3320 (NH2), 3170 (NH), 3120 (NH), 1719 (C=0), 1705 (C=O), 1637 (C=N), 1597
1
(C=C), 1214 (C=S); H NMR (200 MHz, DMSO-d6) δ: 4.10 (s, 2H, N-CH2), 4.50 (s, 2H,
tyf-CH2), 5.41 (s, 2H, NH2), thiophen+ Arom. H [ 6.97 (bs, 2H), 7.20 (s, 1H), 7.40 (bs, 5H) ],
9.68 (s, 1H, NH), 9.77 (s, 1H, NH), 10.32 (s, 1H, NH); 13C NMR (50MHz, DMSO-d6) δ
(ppm): 25.69 (tyf-CH2), 47.41 (N-CH2), thiophen C [125.85 (CH), 127.30 (CH), 127.50 (CH),
137.82 (C)], Arom. C [127.96 (2CH), 128.83 (2CH), 137.41 (C),139.67 (C)], 147.24 (C=N),
154.14 (C=O), 167.23 (C=O), 181.42 (C=S); LC-MS (m/z): 403.98 (M+,95%); Anal Calcd for
C16H17N7O2S2: C, 47.63; H, 4.25; N, 24.30, Found: C, 48.55; H, 5.23; N, 25.26.
5.1.3.2.
1-(2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-triazole-1-
o
yl)acetyl)-4-(4-bromophenyl)thiosemicarbazide (4b). Yield:89.56%, m.p. 188-189 C. IR
(KBr, cm-1): 3415-3329 (NH2), 3251 (NH), 3221 (NH), 1713 (C=O), 1697 (C=O), 1675
1
(C=N), 1587 (C=C), 1225 (C=S) ; H NMR (200 MHz, DMSO-d6) δ: 4.11 (s, 2H, N-CH2),
4.50 (s, 2H, tyf-CH2), 5.41 (s, 2H,NH2 ), thiophene + Arom. H [ 6.97 (s, 2H), 7.41- 7.52 (m,
5H) ], 9.70 (s, 1H, NH), 9.82 (s, 1H, NH), 10.35 (s, 1H, NH); 13C NMR (50MHz, DMSO-d6)
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δ (ppm): 25.68 (tyf-CH2), 47.41 (N-CH2), thiophene C [125.82 (CH), 127.29 (CH), 127.61
(CH), 137.79 (C)], Arom. C [131.67 (4CH), 139.08 (2C) ], 147.24 (C=N), 154.11 (trz-C=O),
167.21 (C=O), 181.32 (C=S); LC-MS (m/z): 483.85 (M+1,65%); Anal Calcd for
C16H16BrN7O2S2: C, 39.84; H, 3.34; N, 20.33, Found: C, 41.00; H, 4.20; N, 21.39.
5.1.3.3.
1-(2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-triazole-1-
yl)acetyl)-4-(4-fluorophenyl)thiosemicarbazide (4c). Yield:78.85%, m.p.180-181 oC. IR (KBr,
cm-1) : 3416-3345 (NH2), 3231 (NH), 3211 (NH), 1708 (C=O), 1686 (C=O), 1609 (C=N),
1
1570 (C=N), 1215 (C=S); H NMR (200 MHz, DMSO-d6) δ: 3.62 (s, 2H, N-CH2), 4.00 (s,
2H, tyf-CH2), 4.91 (s, 2H,NH2 ), thiophene + Arom. H [ 6.49 (s, 2H), 6.66- 6.74 (m, 2H),6.92
13
(bs,3H)], 9.83 (s, 1H, NH), 9.30 (s, 1H, NH), 9.18 (s, 1H, NH); C NMR (50MHz, DMSO-
d6) δ (ppm): 25.66 (tyf-CH2), 47.35 (N-CH2), thiophene C [125.82 (CH), 127.27 (CH), 127.60
(CH), 137.82 (C)], Arom. C [115.27 (2CH), 115.72 (2CH), 135.96 (C), 136.01 (C)], 147.19
(C=N), 154.11 (C=O), 167.18 (C=O), 181.64 (C=S); LC-MS (m/z): 421.98 (M+,100%); Anal
Calcd for C16H16FN7O2S2: C, 45.60; H, 3.83; N, 23.26, Found: C, 46.45; H, 5.01; N, 24.30.
5.1.3.4.
1-(2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-triazole-1-
o
yl)acetyl)-4-p-tolylthiosemicarbazide (4d). Yield:89.34%, m.p.180-181 C. IR (KBr, cm-1)
νmax: 3410-3340 (NH2), 3226 (NH), 3210 (NH), 1705 (C=O), 1695 (C=O), 1673 (C=N),
1581 (C=C), 1225 (C=S) ; 1H NMR (200 MHz, DMSO-d6) δ: 2.27 (s, 3H, CH3), 4.08 (s, 2H,
N-CH2), 4.47 (s, 2H, tyf-CH2), 5.37 (s, 2H,NH2 ), thiophene + Arom.H [ 6.95 (s, 2H), 7.11-
7.37 (m, 5H)], 9.58 (s, 1H, NH), 9.65 (s, 1H, NH), 10.26 (s, 1H, NH); 13C NMR (50MHz,
DMSO-d6) δ (ppm): 21.27 (CH3), 25.65 (tyf-CH2), 47.31 (N-CH2), thiophene C [125.83 (CH),
127.28 (CH), 127.60 (CH), 137.82 (C)], Arom. C [126.62 (2CH), 129.28 (2CH), 135.18 (C),
137.06 (C)], 147.19 (C=N), 154.11 (C=O), 167.22 (C=O), 181.42 (C=S); LC-MS (m/z):
417.99 (M+,100%); Anal Calcd for C17H19N7O2S2: C, 48.90; H, 4.59; N, 23.48, Found: C,
50.21; H, 5.78; N, 24.60.
5.1.4. General method for the synthesis of compounds 5
11

ACCEPTED MANUSCRIPT
Sodium hydroxide (2N, 50 mL) solution was added 0.1 mol isothiocyanato and heated
under reflux for 3 h. Then this mixture was poured in ice-water mixture. After that,
concentrated hydrogen chloride ( %37 ) was added drop by drop this mixture and white crude
products was filtered and dried.
5.1.4.1. 4-amino-2-((5-mercapto-4-phenyl-4H-1,2,4-triazole-3-yl)methyl)-5-(thiophene-2-yl
o
methyl)-2H-1,2,4-triazole-3(4H)-one (5a). Yield:90.78%, m.p.288-289 C. IR (KBr, cm-1) :
1
3281-3176 (NH2), 1696 (C=O), 1581 (C=N), 1535 (C=C),1214 (C=S); H NMR (200 MHz,
DMSO-d6) δ: 3.99 (s, 2H, N-CH2), 4.81 (s, 2H, tyf-CH2 ), 5.14 (s, 2H, NH2 ),
13
thiophene+Arom.H [ 6.95 (s, 1H), 7.23-7.42 (m, 7H)], 13.95 (s, 1H, SH); C NMR (50MHz,
DMSO-d6) δ (ppm): 25.50 (tyf-CH2), 47.53 (N-CH2), thiophene C [125.91 (CH), 127.34
(CH), 127.62 (CH), 137.66 (C)], Arom. C [128.28 (2CH), 129.85 (2CH),130.21 (CH), 133.43
(C)], 147.58 (C=N), 148.31 (C=N), 152.88 (C=N), 169.07 (C=O); LC-MS (m/z): 385.96
(M+,100%); Anal Calcd for C16H15N7OS2: C, 49.85; H, 3.92; N, 25.44, Found: C, 51.34; H,
4.89; N, 26.33.
5.1.4.2.
4-amino-2-((4-(4-bromophenyl)-5-mercapto-4H-1,2,4-triazole-3-yl)methyl)-5-
(thiophene-2-ylmethyl)-2H-1,2,4-triazole-3(4H)-one (5b). Yield:80.68%, m.p.199-200 oC. IR
1
(KBr, cm-1) : 3413-3312 (NH2), 1697 (C=O), 1577 (C=N), 1510 (C=C), 1225 (C=S) ; H
NMR (200 MHz, DMSO-d6) δ: 3.99 (s, 2H, N-CH2), 4.82 (s, 2H, tyf-CH2 ), 5.18 (s, 2H,NH2
), thiophene+ Arom. H [ 6.94 (s, 2H), 7.20 (d, 2H), 7.39 (s, 1H), 7.58 (s, 2H)], 13.98 (s, 1H,
13
SH); C NMR (50MHz, DMSO-d6) δ (ppm): 25.53 (tyf-CH2), 49.30 (N-CH2), thiophene C
[125.93 (CH), 127.35 (CH), 127.65 (CH), 137.63 (C)], Arom. C [130.70 (2CH), 132.72
(2CH), 123.04 (C),133.61 (C)], 147.72 (C=N), 149.55 (C=N), 152.95 (C=N), 168.90 (C=O;
LC-MS (m/z): 463.26 (M+,100%);Anal Calcd for C16H14BrN7OS2: C, 41.38; H, 3.04; N,
21.11, Found: C, 42.50; H, 4.11; N, 22.40.
12

ACCEPTED MANUSCRIPT
5.1.4.3.
4-amino-2-((4-(4-fluorophenyl)-5-mercapto-4H-1,2,4-triazole-3-yl)methyl)-5-
o
(thiophene-2-ylmethyl)-2H-1,2,4-triazole-3(4H)-one (5c). Yield:90.56%, m.p.133-134 C. IR
(KBr, cm-1) : 3314-3194 (NH2), 1697 (C=O), 1579 (C=N), 1509 (C=C), 1233 (C=S); 1H
NMR (200 MHz, DMSO-d6) δ: 3.99 (s, 2H, N-CH2), 4.80 (s, 2H, tyf-CH2 ), 5.19 (s, 2H,NH2
), thiophene + Arom.H [ 6.94 (s, 2H), 7.22-7.25 (m, 4H), 7.40 (s, 1H) ], 13.97 (s, 1H, SH); 13C
NMR (50MHz, DMSO-d6) δ (ppm): 25.51 (tyf-CH2), 47.39 (N-CH2), thiophene C [125.93
(CH), 127.32 (CH), 127.63 (CH), 137.71 (C)], Arom. C [116.65 (2CH), 117.11 (2CH), 129.78
(C), 129.83 (C)], 147.66 (C=N), 148.29 (C=N), 152.91 (C=N), 169.31 (C=O); LC-MS
(m/z): 403.95 (M+,60%); Anal Calcd for C16H14FN7OS2: C, 47.63; H, 3.50; F,4.71; N, 24.30;
O,3.97; S,15.90. Found: C, 48.50; H, 2.39; N, 25.67.
5.1.4.4. 4-amino-2-((5-mercapto-4-p-tolyl-4H-1,2,4-triazole-3-yl)methyl)-5-(thiophene-2-yl
o
methyl)-2H-1,2,4-triazole-3(4H)-one (5d). Yield:92.45%, m.p.208-209 C. IR (KBr, cm-1) :
1
3269-3129 (NH2), 1734 (C=O), 1577 (C=N), 1517 (C=C), 1225 (C=S); H NMR (200 MHz,
DMSO-d6) δ: 2.30 (s, 3H, CH3), 3.99 (s, 2H, N-CH2), 4.63 (s, 2H, tyf-CH2 ), 5.12 (s,
2H,NH2 ), thiophene + Arom. H [ 6.96 (d, 4H), 7.09 (d, 2H), 7.38 (s, 1H) ], 13.60 (s, 1H, SH);
13C NMR (50MHz, DMSO-d6) δ (ppm): 21.32 (CH3), 25.36 (tyf-CH2), 47.44 (N-CH2),
thiophene C [125.91 (CH), 127.47 (CH), 127.77 (CH), 137.26 (C)], Arom. C [127.90 (2CH),
130.67 (2CH), 139.23 (C), 132.02 (C)], 147.57 (C=N), 147.76 (C=N), 153.05 (C=N),
168.00 ( C=O); LC-MS (m/z): 399.98 (M+,100%); Anal Calcd for C17H17N7OS2: C, 51.11; H,
4.29; N, 24.54, Found: C, 52.20; H, 3.70; N, 24.90.
5.1.5. General procedure for the synthesis of the compounds 6
0.1 mol 2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-triazol-1-yl)
acetohydrazide and 0.1 mol various aldehydes in ethonol was heated under reflux for 3 h.
After three h., crude product collapsed in the reaction medium and recrystllized with DMF
and alcohol.
13

ACCEPTED MANUSCRIPT
5.1.5.1.
2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-tiazole-1-yl-
o
N’(thiophene-2-ylmethylene)acetohydrazide (6a). Yield: 67.73%, m.p.244-245 C. IR (KBr,
cm-1): 3461- 3414 (NH2), 3320 (NH), 1719 (trz C=O), 1696 (C=O), 1617 (C=N), 1565
(C=C); 1H NMR (200 MHz, DMSO-d6) δ: 4.09 (s, 2H, tyf-CH2), 4.40-4.73 (m, 2H, N-CH2),
5.39 (s, 2H, N-NH2), thiophene +Arom. H [ 6.96 (bs, 2H), 7.10- 7.12 (m,2H), 7.38-7.43 (m,
2H), 7.64 (s, 1H)], 8.16-8.39 (d, 1H, N=CH), 11.65 (s, 1H, NH); 13C NMR (50MHz, DMSO-
d6) δ : 25.69 (tyf-CH2), 46.92 (N-CH2), thiophene+Arom. C [125.81 (CH), 127.22 (CH),
127.60 (CH), 128.63(CH), 129.38 (CH), 131.40 (CH), 137.93 (C), 139.26(C)], 143.18
(N=CH), 147.21 (C=N), 154.08 (trz C=O), 168.28 (C=O); LC-MS (m/z): 357.01 (M+,100%);
Anal. Calcd for C14H14N6O2S2: C, 46.40; H, 3.89; N, 23.19; Found: C, 47.23; H, 4.56; N,
23.21.
5.1.5.2. 2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-triazole-1-yl-N’(furan-
o
2-yl methylene)acetohydrazide (6b). Yield: 78.02%, m.p.140-141 C. IR (KBr, cm-1): 3448-
1
3325 (NH2), 3093 (NH), 1720 (trz C=O), 1698 (C=O), 1627 (C=N), 1567 (C=C); H NMR
(200 MHz, DMSO-d6) δ: 3.79 (s, 3H, O-CH3), 4. 08 (s, 2H, tyf-CH2), 4.74 (bs, 2H, N-CH2),
5.35 (s, 2H, trz-NH2), thiophen+Arom. H [ 6.60 (bs, 2H), 6.90-6.95 (m, 3H), 7.35-7.38 (m,
13
1H), 7.80 (s, 1H)], 8.05 (bs, 1H, N=CH), 11.58 (bs, 1H, NH); C NMR (50MHz, DMSO-d6)
δ : 27.86 (tyf-CH2), 49.89 (N-CH2), thiophene+Arom. C [127.96 (CH), 129.37 (CH), 129.76
(CH), 140.10 (C)], Arom-C [115.03 (CH), 116.56 (CH), 116.90 (CH), 149.34 (C)], 139.91
(N=CH), 151.93 (C=N), 156.44 (trz C=O), 170.65 (C=O); LC-MS (m/z): 387.01 (M+,100%);
Anal. Calcd for C14H14N6O3S: C, 48.55; H, 4.07; N, 24.26, Found: C, 49.92; H, 5.01; N,
22.90.
5.1.5.3.
2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-triazole-1-yl)-N'-
benzylidene acetohydrazide (6c). Yield:89.12%, m.p.246-247 oC, IR (KBr, cm-1): 3429-3210
1
(NH2), 3131 (NH), 1719 (trz C=O), 1694 (C=O), 1670 (C=N), 1622 (C=C); H NMR (200
MHz, DMSO-d6) δ: 4.10 (s, 2H, tyf-CH2), 4.84 (bs, 2H, N-CH2), 5.39 (s, 2H, trz-NH2),
thiophene+Arom. H [ 6.96 (s, 2H), 7.40 (bs, 4H), 7.69 (bs, 2H)], 7.99-8.19 (m, 1H, N=CH),
11.68 (bs, 1H, NH); 13C NMR (50MHz, DMSO-d6) δ (ppm): 25.69 (tyf-CH2), 47.69 (N-
CH2), thiophene C [125.80 (CH), 127.20 (CH), 127.60 (CH), 138.02 (C)], Arom. C [127.81
(CH), 129.51 (2CH), 130.69 (2CH), 134.61 (C)], 147.95 (N=CH), 147.15 (C=N), 154.31 (trz
14

ACCEPTED MANUSCRIPT
C=O), 168.68 (C=O); LC-MS (m/z): 362.97 (M+,90%); Anal. Calcd for C16H16N6O2S: C,
53.92; H, 4.52; N, 23.58, Found: C, 53.10; H, 3.62; N, 22.40.
5.1.5.4. N'-(4-methoxybenzylidene)-2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-
o
1,2,4-triazole-1-yl)acetohydrazide (6d). Yield: 78.00%, m.p.232-233 C. IR (KBr, cm-1) :
1
3414-3320 (NH2), 3217 (NH), 1709 (trz C=O), 1687 (C=O), 1607 (C=N), 1572 (C=C); H
NMR (200 MHz, DMSO-d6) δ: 4.10 (s, 2H, tyf-CH2), 4.82 (bs, 2H, N-CH2), 5.38 (s, 2H,
NH2), ), thiophene + Arom.H [ 6.97 (bs, 4H), 7.39 (bs, 1H), 7.63 (bs, 2H)], 8.14 (bs, 1H,
13
N=CH), 11.52 (bs, 1H, NH); C NMR (50MHz, DMSO-d6) δ : 25.68 (tyf-CH2), 47.68 (N-
CH2), 55.97 (O-CH3), thiophene C [127.18(CH), 127.24 (CH), 129.24 (CH), 138.03 (C)],
Arom. C [114.96 (2CH), 125.79 (2CH), 147.15 (C), 161.39 (C)], 144.60-147.86 (N=CH),
146.89 (C=N), 154.29 (trz C=O), 168.44 (C=O); LC-MS (m/z): 346.99 (M+,100%);Anal.
Calcd for C17H18N6O3S: C, 52.84; H, 4.70; N, 21.75, Found: C, 53.02; H, 4.85; N, 22.90.
5.1.5.5.
2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-triazole-1-yl)-N'-
o
(pyridin-3-ylmethylene)acetohydrazid (6e).Yield:67.88%, m.p.201-202 C, IR (KBr, cm-1) :
1
3337-3187 (NH2), 3091 (NH), 1721 (trz C=O), 1674 (C=O), 1607 (C=N), 1573 (C=C); H
NMR (200 MHz, DMSO-d6) δ: 4.09 (s, 2H, tyf-CH2), 4.86 (bs, 2H, N-CH2), 5.38 (s, 2H,
NH2), thiophene + Arom.H [ 6.96 (s, 2H), 7.39- 7.44 (m, 2H), 8.13 (d, 1H), 8.59 (s, 1H), 8.85
(s, 1H)], 8.24 (bs, 1H, N=CH), 11.81 (s, 1H, NH); 13CNMR (50MHz, DMSO-d6) δ (ppm):
25.68 (tyf-CH2), 47.69 (N-CH2), thiophene C [125.80 (CH), 127.19 (CH), 127.58 (CH),
137.93 (C)], Arom. C [124.65 (CH), 134.24 (CH), 149.28 (CH),151.25 (CH), 138.04 (C)],
145.31 (N=CH), 147.20 (C=N), 154.28 (trz C=O), 168.93 (C=O); LC-MS (m/z): 358.00
(M+,100%); Anal. Calcd for C15H15N7O2S: C, 50.41; H, 4.23; N, 27.43, Found: C, 51.65; H,
4.90; N, 28.56.
5.1.6. General procedure for the synthesis of the compounds 7
0.1 mol 2-(4-amino-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-1,2,4-triazole-1-yl)
acetohydrazides (4a-d) and 0.2 mol different aldehydes in oil bath dry to dry was heated for 2
h. at 150 0C. At the and of the reaction, substance trituared with ethylacetete and was filtered
and recrystallized with DMF and alcohol.
15

ACCEPTED MANUSCRIPT
5.1.6.1. 2-(5-oxo-3-(thiophene-2-ylmethyl)-4-(thiophene-2-ylmethyleneamino)-4,5-dihydro-
1,2,4-triazole-1-yl)-N’-(thiophen-2-ylmethylene)acetohydrazide
(7a).
Yield:
56.20%,
m.p.228-229 oC. IR (KBr, cm-1) : 3467 (NH), 1706 (trz C=O), 1686 (C=O), 1597 (C=N), 1525
1
(C=C); H NMR (200 MHz, DMSO-d6) δ: 4.24 (s, 2H, tyf-CH2), 4.82 (bs, 2H, N-CH2), ),
thiophene+Arom. H [ 6.83-7.45 (m, 4H), 7.68- 7.97 (m, 5H)], 8.43 (bs, 1H, N=CH), 9.82 (bs,
13
1H, N=CH), 11.74 (bs, 1H, NH); C NMR (50MHz, DMSO-d6) δ : 26.20 (tyf-CH2), 47.61
(N-CH2), thiophene+Arom. C [126.26 (CH), 127.45 (CH), 128.61 (CH), 129.06 (CH), 129.46
(CH), 129.89 (CH), 131.51 (CH), 132.11 (CH), 135.00 (CH), 137.53 (C), 138.51 (C),
139.18(C)], 143.45 (N=CH), 144.88 (C=N), 149.64 (N=CH), 150.78 (trz C=O), 167.88
(C=O); LC-MS (m/z): 445.01 (M+,100%); Anal. Calcd for C19H16N6O2S3: C, 49.98; H, 3.53;
N, 18.41, Found: C, 50.24; H, 2.90; N, 18.96.
5.1.6.2.
N’-(furan-2-ylmethylene)-2-(4-(furan-2-ylmethyleneamino)-5-oxo-3-(thiophene-2-
o
ylmethyl)-4,5-dihydro-1,2,4-triazole-1-yl)acetohydrazide (7b).Yield:60.20%, m.p.160-161 C.
1
IR (KBr, cm-1) : 3417 (NH), 1708 (trz C=O), 1690 (C=O), 1637 (C=N), 1613 (C=C); H
NMR (200 MHz, DMSO-d6) δ: 2.71 (s, 3H, OCH3), 2.86 (s, 3H,OCH3), 4.21 (s, 2H, tyf-
CH2), 4.80 (bs, 2H, N-CH2), Thiophene + Arom-H [ 6.58 (bs, 1H), 6.68 (s, 1H), 6.91-6.95
(m, 3H), 7.16 (s, 1H), 7.31-7.33 (m, 1H), 7.73 (bs, 1H), 7.86 (s, 1H)], 8.03 (bs,1H, N=CH),
9.46 (s, 1H, N=CH), 11.69 (bs, 1H, NH); 13C NMR (50MHz, DMSO-d6) δ (ppm): 26.06 (tyf-
CH2), 47.57 (N-CH2), thiophene C [126.19 (CH), 127.60 (CH), 128.20 (CH), 137.30 (C)],
Arom. C [112.59 (CH), 113.39 (CH), 114.54 (CH), 114.86 (CH), 118.82 (CH), 119.52 (CH),
148.87 (C), 149.57 (C)], 138.02 (N=CH), 149.71 (C=N), 143.70 (N=CH), 150.78 (trzC=O),
168.09 (C=O); LC-MS (m/z): 505.06 (M+,100%); Anal. Calcd for C19H16N6O4S: C, 53.77; H,
3.80; N, 19.80, Found: C, 54.67; H, 4.27; N, 21.10.
5.1.6.3. N'-benzylidene-2-(4-(benzylideneamino)-5-oxo-3-(thiophene-2-ylmethyl)-4,5-dihydro-
1,2,4-triazole-1-yl)acetohydrazide (7c). Yield:59.87%, m.p.250-251oC. IR (KBr, cm-1): 3414
1
(NH), 1708 (trz C=O), 1691 (C=O), 1637 (C=N), 1613 (C=C); H NMR (200 MHz, DMSO-
d6) δ: 4.33 (s, 2H, tyf-CH2), 4.94 (bs, 2H, N-CH2), thiophene + Arom.H [ 6.90-7.02 (m,
2H), 7.41- 7.54 (m, 7H), 7.70 (bs, 2H), 7.87 (bs, 2H)], 8.21 (bs,1H, N=CH), 9.70 (s, 1H,
13
N=CH), 11.74 (bs, 1H, NH); C NMR (50MHz, DMSO-d6) δ (ppm): 26.20 (tyf-CH2), 48.89
(N-CH2), thiophene C [125.80 (CH), 127.19 (CH), 127.65 (CH), 138.05 (C)], Arom. C
16

ACCEPTED MANUSCRIPT
[127.84 (2CH), 127.89 (2CH), 129.53 (4CH),130.72 (CH), 130.81 (CH), 134.61 (C), 134.65
(C)], 148.01 (N=CH), 143.90 (C=N), 149.85 (N=CH), 150.79 (trz C=O), 168.35 (C=O); LC-
MS (m/z): 456.96 (M+,100%); Anal. Calcd for C23H20N6O2S: C, 62.15; H, 4.54; N, 18.91,
Found: C, 63.21; H, 3.96; N, 20.01.
5.1.6.4. N'-(4-methoxybenzylidene)-2-(4-(4-methoxybenzylideneamino)-5-oxo-3-(thiophene-2-
ylmethyl)-4,5-dihydro-1,2,4-triazole-1-yl)acetohydrazide (7d). Yield: 50.23%, m.p.260-261
1
oC. IR (KBr, cm-1) : 3198 (NH), 1705 (trz C=O), 1687 (C=O), 1606 (C=N), 1587 (C=C) ; H
NMR (200 MHz, DMSO-d6) δ: 4.29 (s, 2H, tyf-CH2), 4.89 (d, 2H, N-CH2), thiophene +
Arom.H [ 6.94-7.08 (m, 5H), 7.35- 7.38 (m, 1H), 7.64 (d, 2H), 7.82 (d, 2H), 7.93 (s, 1H)],
13
8.14 (bs, 1H, N=CH), 9.59 (s, 1H, N=CH), 11.58 (bs, 1H, NH); C NMR (50MHz, DMSO-
d6) δ : 26.25 (tyf-CH2), 47.60 (N-CH2), 56.13 (O-CH2), thiophene C [126.11 (CH), 127.42
(CH), 127.59 (CH), 137.82 (C)], Arom. C[114.96 (2CH), 115.22 (2CH),129.29 (2CH),130.43
(2CH), 126.43 (C),127.17 (C), 161.41 (C), 161.61 (C)], 148.01 (N=CH), 145.18 (C=N),
150.92 (trz C=O), 154.57 ( N=CH), 168.08 (C=O). LC-MS (m/z): 424.98 (M+,100%); Anal.
Calcd for C25H24N6O4S: C, 59.51; H, 4.79; N, 16.66, Found: C, 58.61; H, 5.90; N, 17.23.
5.1.6.5.
2-(5-oxo-4-(pyridin-3-ylmethyleneamino)-3-(thiophene-2-ylmethyl)-4,5-dihydro-
1,2,4-triazole-1-yl)-N'-(pyridin-3-ylmethylene)acetohydrazide (7e). Yield:45.56%, m.p.215-
216 oC. IR (KBr, cm-1) : 3096 (NH), 1716 (trz C=O), 1703 (C=O), 1614 (C=N), 1587 (C=C);
1H NMR (200 MHz, DMSO-d6) δ: 4.39 (s, 2H, tyf-CH2), 5.01 (bs, 2H, N-CH2), thiophene +
Arom.H [ 6.94-7.15 (m, 2H), 7.45- 7.62 (m, 3H), 8.25-8.38 (m, 2H), 8.45-9.11 (m, 4H) ], 8.23
( 1H, N=CH), 9.79 (s, 1H, N=CH), 11.82 (s, 1H, NH); 13C NMR (50MHz, DMSO-d6) δ
(ppm): 26.14 (tyf-CH2), 47.59 (N-CH2), thiophene C [126.21 (CH), 127.54 (CH), 127.65
(CH), 137.53 (C)], Arom. C [124.59 (CH), 124.84 (CH), 129.98 (C), 130.50 (C), 134.27
(CH),135.09 (CH), 150.17 (CH), 151.28 (CH), 151.76 (CH), 152.80 (CH)], 145.53 (N=CH),
145.33 (C=N), 149.30 (N=CH), 150.70 (trz C=O), 168.52 (C=O); LC-MS (m/z): 447.04 (M+,
100%); Anal. Calcd for C21H18N8O2S: C, 56.49; H, 4.06; N, 25.10, Found: C, 57.54; H, 5.11;
N, 26.16.
5.2. Antioxidant Activity Tests
17

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Different antioxidant test methods with varying strategies or chemistries are simultaneously
used in antioxidant activity determinations since the results of different assays appear in many
cases to be different due to differences in reaction mechanisms and kinetics, the effects of
solvents, sterical sensitivities, and varying effects of temperature, pH, and matrix
composition. Two of the most widely used antioxidant test methods were used in the current
study. DPPH• radical scavenging assay has been developed and used widely for various
samples including synthetic compounds [33]. The second method used was ferric
reducing/antioxidant power (FRAP) assay [34,35], which has found application in many
investigations with synthetic organics. The results of the two tests were comparatively
evaluated.
5.2.1. DPPH• Scavenging Test
One of the most widely used spectrophotometric test methods, DPPH• radical scavenging test
relies on the absorbance change of the radical when deactivated by antioxidants, which easily
observable with naked eye as color changes from purple to yellow. DPPH• radical (2,2-
diphenyl-1-picrylhydrazyl), a stable solid, was used in 50 ꢀM final test concentration in
methanol. The method developed by Cuendet et al was used with some modification [36]. The
synthesized compounds were dissolved in ethanol-DMSO (3:2) at 200 ꢀg/mL concentration.
The working test concentrations were determined with a pretest observation, and all the
compounds were tested at 200 ꢀg/mL fixed concentration except thiosemicarbazides
(compounds 4a-d) which showed much higher activity in pretests and therefore diluted 20
fold to 10 ꢀg/mL as test concentration. Equal volumes (750 ꢀL) of DPPH• and sample
solutions were mixed, vortexed, and incubated for a 60 min period at room temperature. The
tests were made in duplicates. The absorbances were measured at 517 nm at 0.5, 20, 40, and
60 min incubation times and used for the calculation of percent DPPH• scavenging using the
following equation:
%DPPH• Scavenging = [ ( Absinitial – Absfinal ) / Absinitial ] x 100
The absorbance of the reagent blanks at the end of incubation was used as Absinitial. The
absorbance of each sample blank, having solvent instead of DPPH•, was subtracted from the
absorbance measured for the sample to obtain Absfinal. The percent DPPH scavenging values
as a function of time were plotted in Figure 1, and the percent DPPH scavenging values at 60
18

ACCEPTED MANUSCRIPT
min incubation time are given in Figure 2 for the activity comparison of the compounds
synthesized.
5.2.2. Ferric reducing/antioxidant power (FRAP) Test
First developed as antioxidant test method by using FeSO4 solutions [37], FRAP test method
was later improved by using TPTZ (2,4,6-tris(2-pyridyl)-s-triazine) as ferrous ions
complexing agent [38]. FRAP activities indicate total reducing potential of samples. The
method utilizes the measurement of absorbance at 595 nm caused by Fe2+ - TPTZ complex.
To overcome expected solubility problems of the compounds when the sample
solutions are mixed with FRAP reagent, the original method [38] was modified to contain
methanol in 3:2 ratio in water as reagent solvent.
Fresh FRAP reagent was prepared by combining 300 mM acetate buffer (pH 3.6), 10
mM TPTZ in 40 mM HCl, and 20 mM FeCl3.6H2O in 10:1:1 ratio, respectively. Calibration
curve was prepared by using methanolic Trolox solutions at five different concentrations
(1000 – 500 – 250 – 125 – 62.5 ꢀM). The compound solutions of 200 ꢀg/mL concentration
and Trolox solutions (100 ꢀL) were mixed with FRAP reagent (3 mL), vortexed, and
incubated for 20 min. at room temperature. The absorbance was read at 595 nm against
water. Reagent and sample blanks were also tested, and the sum of the absorbance of these
two measurements was subtracted from the absorbance mean obtained by triplicate
measurements with each sample. FRAP activities were expressed as Trolox Equivalent
Antioxidant Capacity (TEAC, ꢀM), calculated as the Trolox concentration from the graph
corresponding to the absorbance observed with sample. Higher TEAC values means higher
FRAP and thus higher antioxidant capacity.
5.3. Antimicrobial activity
5.3.1. Antimicrobial activity assessment
19

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All test microorganisms were obtained from the Hifzissihha Institute of Refik Saydam
(Ankara, Turkey) and were as follows: Ec: Escherichia coli ATCC 25922, Yp: Yersinia
pseudotuberculosis ATCC 911, Pa: Pseudomonas auroginosa ATCC 10145, Sa:
Staphylococcus aureus ATCC 25923, Ef: Enterococcus fecalis ATCC 29212, Li: Listeria
monocytogenes ATCC 43251, Bc: Bacillus cereus 709 Roma, Ms: Mycobacterium smegmatis
ATCC607, Ca: Candida albicans ATCC 60193, Sc: Saccharomyces cerevisiae RSKK 251.
All the newly synthesized compounds were dissolved in DMSO to prepare stock solutions.
5.3.2. Minimum inhibition concentration
The agar dilution method, described by Vanden Berghe and Vietinck [39] was used for the
antibacterial screening with slight modifications. Instead of 96 well microtitre plates 24 well
tissue culture (Corning) plates were used. The crude extracts were dissolved in 70% ethanol
and physiological Tris buffer (Amresco 0826-500G) 1:4, and mixed with an equal amount of
3% agar solution at 45 °C to a final concentration of 50, 25, 12.5, and 6.25 µg of sample
compound/mL. From the solutions, 400 µL was transferred into each well of the tissue
culture (Corning) plate. After solidification each well was inoculated with 10 µL of freshly
prepared bacterial suspension of 108 bacteria/mL and incubated at 37 °C for 24 h. Ampicillin
(10 ꢀg) and Fluconasole (5 ꢀg) were standard antibacterial and antifungal drugs, respectively.
Dimethylsulphoxide (DMSO) was used as solvent control. The smallest concentration with
which the growth of test microorganism was totally inhibited is reported as minimum
inhibitory concentration (MIC, ꢀg/mL) value (Table 1).
6. Crystal data for the compounds 2 and 4d
The crystallographic data were collected on a CCD system (Oxford Xcalibur Eos
Mova) with a graphite monochromatized Mo Kα radiation (λ = 0.71073 Å). The crystals were
mounted with grease on the top of a glass fiber. Data collection: CrysAlis PRO CCD [40]; cell
refinement: CrysAlis PRO CCD; data reduction: CrysAlis PRO RED [41]. Cell constants and
orientation matrix for data collection were obtained by least-squares refinement of the
diffraction data in the range of 6.48 - 55.44° for 1 and 7 - 57.9° for 2. Using Olex2 [42], the
structures were solved with the ShelXS [43] structure solution program using Direct Methods
20

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and refined with the ShelXL [44] refinement package using F2 by Least Squares
minimization. Molecular figures were prepared by using ORTEP III [45] and OLEX 2. All
non-H atoms were refined anisotropically. The H atoms were placed in geometrically
idealized positions. The crystal data and structure refinement details are given in Table 2.
Selected bond lengths and bond angles are listed in Table 3.
6.1. Crystallographic study for 2 and 4d
Both of the compounds crystallizes in the P21/c space group and in a monoclinic
crystal system, with one isolated molecule in the asymmetric unit cell. ORTEP III diagram of
2 and 4d with the atom numbering scheme is shown in Figure 5 and Figure 6 respectively.
While the molecular structure of 2 contains two five membered 1,2,4-triazole and thiophene
rings linked through a methylene group, compound 4d additionally has a aromatic ring.
Dihedral angles between the rings are 69.08(3)º for 2 and 65.17(1)º for 4d. The 1,2,4-triazole
ring in the compound 4d reveals a tendency of somewhat differences of some ring bonds in
comparison with 1,2,4-triazole ring in compound 4d (Table 3). All the bond lengths along
the 1,2,4-triazole ring changes between 1.358 and 1.390 Å for 2. Also, the N3-N4 bond
lengths of 2 (1.396(3) Å) is shorter than that in 4d (1.402(3)Å). The obtained results indicate a
conjugation between the π-system of the 1,2,4-triazole ring and the 4-amino group for
compound 2. The C1–O1 bond length is practically the same as the both of the molecules
[1.226(4) Å for 2 and 1.221(3) Å for 4d] and within the values observed for a C=O double
bond.
For compound 2, the 4-amino group of 1,2,4-triazole ring is hydrogen bonded to the
carbonyl O atom of symmetry related molecule by intermolecular hydrogen bond (Figure 7, 8
and Table 4). N4-H4a…O1i hydrogen bonds with an
graph set notation (Fig. 7) [43].
The 4-amino group also hydrogen bonded to O3 atom as shown in Figure 7. The molecules
are further interconnected into a 2D network by C8-H8b…N4ii hydrogen bonds on the (100)
plane (Figure 8, symmetry codes as in Table 4).
Inspection of molecular packing reveals that there are non-classical hydrogen bonds (N-
H…X; X=N, O, S) in the crystal structure of 4d (Table 4) which link the molecules to form
three-dimensional network. N7—H7···O1ii and N6—H6···S2iii hydrogen bonds link the
molecules with
and
graph set notation respectively [45], along the b axis
(Figure 9). On the other hand, The non-classical hydrogen bond formed by N5 and
21

ACCEPTED MANUSCRIPT
carboxylate O2 atoms are resulted in a chain along the c axis with C(4) notation (Figure 10).
All these separate chains come together to form a 2D framework on the (100) plane as shown
in Figure 11.
Acknowledgement
This work was supported by grants from Karadeniz Technical University (BAP, Project no:8764)
7. Supplementary data
Full crystallographic data in CIF format for 2 and 4d have been deposited with the Cambridge
Crystallographic Data Centre (CCDC No. 914599 for 2 and CCDC No. 915002 for 4d). Copies of the
data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge, CB2 1EZ,
U.K; Fax: +44(0)-1223-336033; or email: deposit@ccdc.cam.ac.uk.
22

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CAPTIONS
Scheme 1. Synthetic pathway for the preparation of target compounds 2,3,4,5,6 and 7
Fig. 1. %DPPH scavenging of the compounds as a function of time (highly active compounds
4a-d were tested at 10 ꢀg/mL, while the others were at 200 ꢀg/mL concentration,
only the active compounds are shown).
Fig. 2. %DPPH scavenging of the compounds at 60 min incubation time (highly active
compounds 4a-d were tested at 10 ꢀg/mL, while the others were at 200 ꢀg/mL
concentration, only the active compounds are shown).
Fig. 3. FRAP test results of all the synthesized compounds at 200 ꢀg/mL concentration as ꢀM
TEAC (Trolox equivalent antioxidant capacity) values obtained from [Trolox]-
absorbance calibration graph.
Fig. 4. The correlation graph of the results of FRAP test (TEAC values) and DPPH•
scavenging test (percentage scavenging of DPPH• at 60 min incubation time).
Fig. 5. The molecular structure of the compound 2, with the atom-numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
Fig. 6. The molecular structure of the compound 4d, with the atom-numbering scheme.
Displacement ellipsoids are drawn at the 50% probability level.
Fig. 7. A wiev of N4-H4a…O1# hydrogen bond with an
compound 2. Symmetry code #:1-x, -y, 2-z.
graph set notation for
Fig. 8. Hydrogen bonds interaction and packing diagram along the c axis for compound
2. Hydrogen atoms omitted for clarity except atoms H4a,H4b and H8b.
Fig. 9. A view of N7—H7···O1ii and N6—H6···S2iii hydrogen bonds for compound 4d, with
and
graph set notation respectively. Symmetry codes #:-x, -y, 1-z; $: -
x, -1-y, 1-z.
Fig. 10. A view of the N5-H5...O2# interaction resulted in a chain along the c axis with C(4)
notation for the structure 4d. Symmetry codes #:#:-x, -y, 1-z; $: -x, -1-y, 1-z.
Fig. 11. Hydrogen bonds interaction and packing diagram along the c axis for compound
4d. Hydrogen atoms omitted for clarity except atoms H5, H6, H7 and H4b.
26

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Table 1. Screening for antimicrobial activity of the compounds
Compound
No
Stock
Concentration
µg/mL
Microorganismsa and MIC values (µg/mL)
Ec Yp Pa
Sa
Ef Li
Bc
Ms Ca Sc
2
3
31800
4500
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
g
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
g
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
g
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
g
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
g
-
-
994
994
-
-
-
-
-
4a
20200
7600
2525
5050 1262
-
-
4b
950
950
950
-
-
4c
19300
12900
16000
14000
12500
10800
23100
7800
2412
-
-
-
-
1612
-
-
-
4d
3225
-
-
5a
-
-
-
5b
1750
1750 1750
-
-
5c
-
-
-
-
-
-
-
-
5d
-
-
6a
-
-
-
-
-
6b
-
-
-
-
-
6c
8900
-
-
-
-
-
6d
3600
-
-
-
-
-
6e
4800
-
-
-
-
-
7a
18300
7900
-
-
-
-
-
7b
-
-
-
-
-
7c
7600
-
-
-
-
-
7d
8600
-
-
-
-
-
7e
12500
-
-
-
-
-
DMSO
Amp.b
g
ng
g
ng
g
ng
g
nt
g
nt
at 100
at 100
ng ng ng
nt nt nt
ng ng
nt nt
Flu.c
ng ng
nt
nt
nt
a
Ec: Escherichia coli ATCC 25922, Yp: Yersinia pseudotuberculosis ATCC 911, Pa:
Pseudomonas auroginosa ATCC 10145, Sa: Staphylococcus aureus ATCC 25923, Ef:
Enterococcus fecalis ATCC 29212, Li: Listeria monocytogenes ATCC 43251, Bc: Bacillus
cereus 709 Roma, Ms: Mycobacterium smegmatis ATCC607, Ca: Candida albicans ATCC
60193, Sc: Saccharomyces cerevisiae RSKK 251.
b
Amp.: Ampicillin
Flu.: Fluconazole
c
symbols: (-): no activity at test concentrations, g: growth was observed, ng: no growth (total
inhibition) was observed, nt: not tested.
27

ACCEPTED MANUSCRIPT
Table 2. Crystal data and structure refinement for 2 and 4d.
Compound 2
C11H14N4O3S
282.32
Compound 4d
Empirical formula
C17H19N7O2S2
417.51
Formula weight
Temperature/K
293(2)
293(2)
Crystal system
monoclinic
P21/c
monoclinic
P21/c
Space group
a/Å
11.7829(4)
14.525(4)
7.9468(2)
90.00
19.6992(7)
11.6204(4)
8.8498(4)
90.00
b/Å
c/Å
α/°
β/°
97.7(3)
92.438(4)
90.00
γ/°
90.00
Volume/Å3
Z
1347.8(4)
4
2024.00(14)
4
ρ
calcmg/mm3
1.391
1.370
m/mm-1
0.250
0.291
F(000)
592.0
872.0
Crystal size/mm3
2Θ range for data collection
Index ranges
0.28 × 0.25 × 0.18
6.48 to 55.44°
0.3 × 0.25 × 0.2
7 to 57.9°
-8 ≤ h ≤ 15, -14 ≤ k ≤ 17, -10 ≤ -25 ≤ h ≤ 11, -8 ≤ k ≤ 14, -11 ≤ l ≤
l ≤ 10
12
Reflections collected
5280
8153
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
2711[R(int) = 0.0123]
2711/0/181
4609[R(int) = 0.0301]
4609/0/255
1.074
1.018
Final R indexes [I>=2σ (I)]
Final R indexes [all data]
Largest diff. peak/hole / e Å-3
R1 = 0.0662, wR2 = 0.2027
R1 = 0.0737, wR2 = 0.2123
0.62/-0.71
R1 = 0.0664, wR2 = 0.1449
R1 = 0.1138, wR2 = 0.1684
0.42/-0.34
28