Synthesis, characterization and antifungal evaluation of 5-substituted-4-amino-1,2,4-triazole-3-thioesters

Article abstract of DOI:10.3390/molecules16021297

A series of 5-substituted-4-amino-1,2,4-triazole-3-thioesters was synthesized by converting variously substituted organic acids successively into the corresponding esters, hydrazides, 5-substituted-1,3,4-oxadiazole-2-thiols, 5-substituted-1,2,4-triazole-2-thiols and 5-substituted-1,3,4-oxadiazole-2- thioesters. Finally the target compounds were obtained by refluxing 5-substituted-1,3,4-oxadiazole-2-thioesters in the presence of hydrazine hydrate and absolute alcohol. The structures of the synthesized compounds were established by physicochemical and spectroscopic methods. The synthesized compounds were evaluated for their in vitro antifungal activity. Some of the evaluated compounds possessed significant antifungal activity as compared to a terbinafine standard.

Full text of DOI:10.3390/molecules16021297

Molecules 2011, 16, 1297-1309; doi:10.3390/molecules16021297
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis, Characterization and Antifungal Evaluation of
5-Substituted-4-Amino-1,2,4-Triazole-3-Thioesters
Aurangzeb Hasan *, Noel Francis Thomas and Shelly Gapil
Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur-50603, Malaysia
* Author to whom correspondence should be addressed: E-Mail: flavonoids@hotmail.com;
Tel.: +603 7967 5165; Fax: +603 7967 4193.
Received: 14 December 2010 / Accepted: 1 December 2010 / Published: 28 January 2011
Abstract: A series of 5-substituted-4-amino-1,2,4-triazole-3-thioesters was synthesized by
converting variously substituted organic acids successively into the corresponding esters,
hydrazides, 5-substituted-1,3,4-oxadiazole-2-thiols, 5-substituted-1,2,4-triazole-2-thiols
and 5-substituted-1,3,4-oxadiazole-2-thioesters. Finally the target compounds were
obtained by refluxing 5-substituted-1,3,4-oxadiazole-2-thioesters in the presence of
hydrazine hydrate and absolute alcohol. The structures of the synthesized compounds were
established by physicochemical and spectroscopic methods. The synthesized compounds
were evaluated for their in vitro antifungal activity. Some of the evaluated compounds
possessed significant antifungal activity as compared to a terbinafine standard.
Keywords: triazolethioesters; oxadiazolethioesters; oxadiazoles; triazoles; in vitro
antifungal activity
1. Introduction
1,2,4-Triazoles and their derivatives constitute an important class of organic compounds with
diverse agricultural, industrial and biological activities [1-3] including anti-microbial [4,5] sedative,
anti-convulsant [6] and anti-inflammatory properties [7], and consequently, the synthesis of these
heterocycles has received considerable attention in recent years [8-14]. In this work we report the
synthesis of some new 5-substituted-4-amino-1,2,4-triazole-3-thioesters through the intermolecular

Molecules 2011, 16
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cyclization of variably substituted acid hydrazides to the corresponding 5-substituted-1,3,4-
oxadiazoles-2-thioles, 5-substituted-1,2,4-triazoles-2-thioles and 5-substituted-1,3,4-oxadiazoles-2-thioesters.
2. Results and Discussion
Nine 5-substituted-4-amino-1,2,4-triazole-3-thioesters were obtained in 66-83% yield by converting
variously substituted organic acids Ra-i to the corresponding esters 1a-i and acid hydrazides 2a-i by
reaction with methanol and hydrazine hydrate, respectively. The hydrazides were converted to 5-
substituted-1,3,4-oxadiazole-2-thiols 3a-i and 5-substituted-1,2,4-triazole-2-thiols 4a-i as described in
[15,16]. 5-Substituted-1,3,4-oxadiazole-2-thiols and 5-substituted-1,2,4-triazole-2-thiols were next
converted to 5-substituted-1,3,4-oxadiazole-2-thioesters 5a-i using potassium hydroxide and acid
chlorides and finally to 5-substituted-4-amino-1,2,4-triazole-3-thioesters 6a-i by refluxing the 5-
substituted-1,3,4-oxadiazole-2-thioesters in the presence of hydrazine hydrate and absolute ethanol, as
outlined in Scheme 1.
Scheme 1. Synthesis of 5-substitued-4-amino-1,2,4-triazole-3-thioesters.
+
O
O
O
Me OH/ H
OH
80 %
NH
2
NH
2
R
R
NH NH
2
R
OMe
1
1
1
Aci d
Ra-i
Est er
1a-i
Aci d hydr azi de
2a-i
i
CS
2
ii
KOH
N
N
N
N
NH
NH
80 %
2
2
R
N
SH
1
R
O
SH
1
NH
2
5- Substit ut ed- 4- a mi no-
1, 2, 4-tri azol e- 3-t hi ol
4a-i
5- Substit ut ed- 1, 3, 4-
oxadi azol e- 2-t hi ol
3a-i
i
KOH
ii R COCl
2
N
N
N
N
O
NH NH
2
2
O
R
N
S
C R
2
S
C R
2
R
O
1
1
NH
2
Thi oest er of 5-substit ut ed
4- a mi no- 1, 2, 4-tri azol e
Thi oest er of 5-substit ut ed-
1, 3, 4- oxadi azol e
6a-i
5a-i
Cl
,
R1
=
,
,
,
O2N
Cl
NO2
NO2
NO2
,
,
,
H3CO
O
H3CO
OCH3
OCH3
,
R2
=
CH3(CH2)5CH2
CH3(CH2)4CH2
,
,
,
CH3CH2CH2
CH3(CH2)6CH2

Molecules 2011, 16
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Mass spectral data support the proposed structures. Scheme 2 shows the fragmentation pattern for
5-(4-methoxybenzyl)-4-amino-1,2,4-triazole-2-ylnapthalene-1-carbothioate (6a) which serves as an
example for all the compounds mutatis mutandis. The mass spectrum showed various characteristic
peaks. A peak at m/z 390 was assigned to the molecular ion. A peak at m/z 155 (the base peak) was due
to the naphthoyl fragment and a characteristic peak at m/z 121 was attributed to the tropylium ion
produced by the loss of triazole-3-thioester moiety.
Scheme 2. Mass fragmentation pattern of thioester 6a.
+
OCH3
m/z = 121 (65%)
+
N
O
C
N
N
O
C
S
S
N
N
NH2
+
NH2
m/z = 243 (50%)
N
N
O
C
CH3O
CH2
S
N
NH2
m/z = 390 (45%)
+
CH3O
CH2
C
N
N
-N2H2
m/z = 147 (60%)
N
N
CH3O
CH2
S
N
+
N
O
NH2
CH2
S C
+
O
C
m/z = 360 (35%)
CH3O
m/z = 155 (100%)
-CO
+
m/z = 127 (55%)
The FTIR spectrum showed absorption bands at 1,518 cm–1, 3,204 cm–1, 1,684 cm–1 and 1,575 cm-1
which were assigned to C=N, N–H, C=O (ester), C–O and C=C (aromatic), respectively. The 1H-NMR
spectrum of 6a showed characteristic signals at 6.90 to 7.85 ppm which were assigned to the aromatic
protons. A signal at 2.50 ppm was assigned to the methylene proton. The singlet at 3.56 and 3.83 ppm
were assigned to the methoxy and amino protons, respectively.
The 13C-NMR spectrum of 6a showed characteristic peaks at 120.5-134.5 ppm which were assigned
to the aromatic carbon atoms. A peak at 180.01 ppm was attributed to the carbonyl carbon atom. The
peaks at 166.04 and 175.10 ppm were assigned to the carbon atoms of the triazole moiety. The peaks at
13
30.6 and 55.9 ppm were assigned to the methylene and methoxy carbon atoms. The significant C-
NMR spectra signals are listed in Table 2 and are in agreement with the proposed structure. On the
basis of the combined physical and spectral data compound 6a was assigned the following structure,

Molecules 2011, 16
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corresponding
(Figure 1):
to
5-(4-methoxybenzyl)-4-amino-1,2,4-triazole-3-ylnaphthalene-1-carbothioate
Figure 1. Structure of Compound (6a).
N
N
O
C
CH3O
CH2
S
N
NH2
Similarly, the other members of the series of 5-substituted-4-amino-1,2,4-triazole-3-thioester 6b-i
were synthesized by the ring opening reaction due to the nucleophilic attack of hydrazine on the ring
1
carbon of the 5-substituted-1,3,4-oxadiazole-2-thioesters, followed by dehydration. The H-NMR and
13C-NMR, of the 5-substituted-1,3,4-oxadiazole-2-thioesters are outlined in Tables 1 and
2 respectively.
Table 1. 1H-NMR spectral data of 5-substitued-4-amino-1,2,4-triazole-3-thioesters 6a-i.
Protons [1H-(ppm), Multiplicity, J(Hz)]
Comp.
6a
Ar–H
NH2 Other protons
H-1
7.72
H-2
8.03
H-3
H-4
H-1
H-2
H-3
H-4
H-5
8.01
7.73
7.76
1H,m
7.42
1H,m
7.90
1H,m
7.88
1H,m
7.55
4.32
–
1H,dd 1H,dd 1H,dd 1H,dd
1.2,4.6 2.5,7.2 2.4,7.1 2.3,4.4
1H,m 2H,s
7.60
7.77
7.04
7.45
7.88
1H,m
7.79
1H,m
7.62
1H,m
8.25
1H,m
7.21
1H,m 2H,s
3.95
6b
1H,dd 1H,dd 1H,dd 1H,dd
3.1,4.2 3.2,4.1 2.5,6.6 4.2,3.1
–
–
7.65
7.59
7.69
7.98
7.45
4.12
7.48
1H,m
7.32
1H,m
7.22
1H,m
7.97
1H,m
6c
1H,dd 1H,dd 1H,dd 1H,dd 1H,dd
3.1,6.7 1H,m 2.5,4.7 3.1,6.8 2.8,6.5
2H,s
2.88
4H,t
3.2
7.22
1H,dd
2.4,6.7
7.22
1H,dd
2.4,6.7
7.12
1H,m
7.08
1H,m
4.33
2H,s
6d
–
–
–
–
–
2H,t
2.1,1.5
10H,m
2.90
4H,t
2.8
2.50
2H,t
3.2,1.52
10H,m
2.50
7.97
1H,dd 1H,dd
2.4,6.5 3.1,4.5
7.58
7.14
1H,m
7.47
1H,m
7.12
1H,m
7.67
1H,m
7.77
1H,m
7.67
1H,m
4.16
2H,s
6e
6f
–
–
7.47
7.27
6.97
7.47
3.94
2H,s
1H,dd 1H,dd 1H,dd 1H,dd
3.2,6.2 2.1,7.2 3.2,7.3 3.4,6.2
–
–
–
–
7.76
1H,dd 1H,dd
2.4,6.2 3.5,6.5
7.42
6.50
1H,m
6.87
1H,m
6.50
1H,m
6.87
1H,m
7.73
1H,m
7.46
1H,m
7.55
3.83
1H,m 2H,s
2H,s
3.56
3H,s
6g
2.5
6.52
1H,m
6.77
1H,m
7.14
1H,m
7.02
1H,m
4.45
2H,s
2H,t
3.2,1.60
12H,m
6h
6i
–
–
–
–
–
7.12
7.27
7.12
7.77
1H,m
7.67
1H,m
7.85
1H,m
7.58
1H,m
7.82
1H,m
7.25
4.26
1H,m 2H,s
1H,dd 1H,dd 1H,dd
2.3,6.4 3.2,7.4 2.3,6.4
–

Molecules 2011, 16
Table 2. 13C-NMR spectral data of 5-substitued-4-amino-1,2,4-triazole-3-thioesters 6a-i.
1301
13C-(ppm), Compounds
Carbon
No.
6a
6b
6c
6d
6e
6f
6g
6h
6i
1
2
129.41
128.40
116.52
115.52
116.31
130.43
160.42
171.40
182.31
135.61
127.12
120.43
122.41
125.32
126.32
128.43
129.42
130.21
129.66
–
128.40
127.41
155.50
116.62
118.62
127.42
162.61
174.65
180.37
136.23
129.12
122.31
125.71
128.81
127.50
130.64
132.80
126.21
129.44
–
130.05
126.41
116.24
115.24
116.37
126.21
166.04
176.23
184.32
134.05
130.73
126.32
125.61
126.67
130.73
–
28.36
30.18
127.21
126.33
127.41
116.24
118.44
116.26
166.23
175.90
182.64
45.46
33.67
26.34
25.80
20.21
18.44
22.37
–
30.33
32.71
130.43
118.23
117.23
115.48
118.23
125.63
159.21
170.62
183.47
134.24
128.40
129.65
126.31
130.52
127.43
–
130.06
127.25
152.44
127.25
126.31
117.21
161.37
170.68
182.04
54.34
44.31
33.39
24.36
25.62
20.22
–
30.60
130.46
125.34
116.73
118.62
116.73
125.23
159.68
168.77
184.66
53.62
44.02
34.71
29.01
25.72
25.69
20.02
18.33
–
133.06
127.45
153.44
125.21
127.40
117.67
163.23
177.45
181.08
136.38
127.21
116.90
126.64
128.03
125.64
–
55.90
3
130.02
127.05
116.65
158.03
166.04
175.10
180.01
116.65
127.05
134.50
127.12
125.62
120.50
119.42
129.31
131.61
130.71
123.63
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
In the in vitro antifungal bioassay, the compounds 6a, 6b, 6d and 6g were found to have significant
antifungal activities against the four tested fungal strains (Table 3). In particular 6a and 6g had high
inhibitory effects on the growth of Aspergillus. flavus, Mucor species, Aspergillus niger and Aspergillus
fumigatus. A structure activity relationship comparison shows that the presence of naphthyl group at

Molecules 2011, 16
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the 3-position of the triazole-3-thioester enhances the activity of the compounds. It is interesting to
note that a similar group is also present in the structure of the reference compound terbinafine .
Table 3. Antifungal activity of 5-substituted-4-amino-1,2,4-triazole-3-thioesters.
Fungal strains/ Inhibition (%)
Structure of 5-substituted-4-amino-
Sample No.
1,2,4-triazole-thioester
Aspergillus
fumigatus
Aspergillus
flavus
Mucor
species
Aspergillus
niger
N
N
O
C
O2N
S
N
6a
6b
96.22
84.12
87.66
74.79
81.20
97.20
NH2
N
N
O
S
C
N
100.00
100.00
NH2
O2N
N
N
O
S
C
6c
6d
6e
69.00
81.00
71.00
80.90
78.36
45.89
75.20
59.19
69.99
87.00
93.60
73.00
N
NH2
N
N
O
C
CH2 CH2
S
S
C7H15
N
NH2
N
N
O
C
CH2 CH2
N
NH2
N
N
N
O
C
S
C6H13
6f
27.00
15.58
24.39
75.00
NH2
O2N
N
N
S
O
CH3O
CH2
C
N
6g
6h
6i
100.00
54.00
50.00
88.64
21.22
19.48
98.20
20.00
51.99
88.00
52.00
88.00
NH2
N
N
O
C
S
C8H17
N
NH2
N
N
O
C
S
N
NH2
O2N
Terbinafine
standard drug
100.00
90.00
110.80
98.40
The antifungal assay results also show that the activity of the compounds 6c, 6e, 6f, 6h and 6i is
relatively low as compared to other members of the series due to the absence of this group in these

Molecules 2011, 16
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structures. It is therefore concluded that the potential pharmacophore in the triazole-3-thioesters is
probably naphthalene-1-carbothioate.
3. Experimental
3.1. General
Melting points of the synthesized compounds were recorded on Gallenkamp digital melting point
apparatus MFB-595-101M in open-end capillary tubes and are uncorrected. Thin layer
chromatography was carried out on pre-coated silica gel plates (0.2 mm, E. Merck, 20 × 20 cm,
60F254). FTIR spectra were recorded on a Bio-Red Merlin Spectrophotometer using KBr discs.1H-
NMR (300 MHz) and 13C-NMR (75.43 MHz) spectra were recorded on Bruker AM-250 Spectrometer
in DMSO and CDCl3 solutions using TMS as internal standard. EIMS were recorded on Agilent VG:
70 SE Mass Spectrometer.
3.2. Procedure for the preparation of methyl-4-nitrobenzoate (1a)
4-Nitrobenzoic acid (5 g, 0.029 mol) was taken in a 250 mL round bottom flask fitted with a reflux
condenser and a calcium chloride guard tube. Absolute methyl alcohol (25 mL) and few drops of
concentrated sulphuric acid were added and the reaction mixture was refluxed for 4 hours. The reflux
time was monitored by thin layer chromatography (tlc) (silica; ethyl acetate-pet. ether, 1:2). After the
completion of the reaction the excess of alcohol was distilled off on a rotary evaporator. The residue
was poured into 250 mL of water contained in a separating funnel. Dichloromethane (20 mL) was
added to the separating funnel and the mixture was shaken vigorously. The solution was allowed to
stand and the methyl-4-nitrobenzoate in the dichloromethane separated and settled at the bottom of the
separating funnel. The lower layer was carefully separated and the upper aqueous layer was rejected.
The methyl-4-nitrobenzoate was returned to the funnel and shaken with a strong solution of sodium
bicarbonate until all the free acid was removed. Methyl-4-nitrobenzoate was washed once with water
and dried by pouring into a small dry conical flask containing 2 g of anhydrous magnesium sulphate. It
was shaken for 5 minutes and allowed to stand for one hour. The methyl-4-nitrobenzoate solution was
filtered through a small fluted filter paper into a distillation flask. The flask was fitted with 360°
thermometer, a condenser and a receiving flask. Dichloromethane was distilled off at 40 °C and the
solid methyl-4-nitrobenzoate was obtained from the flask. Ethanol was used as solvent for
recrystallization of the ester.
3.3. Procedure for the preparation of 4-nitrobenzoic acid hydrazide (2a)
Methyl-4-nitrobenzoate ester (7 g, 0.041 mol) was dissolved in absolute ethanol (40 mL) and taken
in a flask fitted with a reflux condenser and a calcium chloride guard tube. Hydrazine hydrate (80%,
13 mL) was added and the reaction mixture was refluxed for eight hours. The reflux time was
monitored by tlc as before. After the completion of the reaction, the excess hydrazine was distilled off.
The crude solid was collected, washed with water and recrystallized from 30% aqueous ethanol.

Molecules 2011, 16
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3.4. Procedure for the preparation of 5-(4-nitrophenyl)-1,3,4-oxadiazole-2-thiol (3a)
4-Nitrobenzoic hydrazide (7 g, 0.038 mol) was dissolved in absolute ethanol (40 mL) in a
250 mL flask. Carbon disulfide (2 mL, 0.034 mol) was then added to the solution followed by the
addition of a solution of potassium hydroxide (1.2 g, 0.019 mol) in water (20 mL). The reaction
mixture was thoroughly stirred and refluxed. It was initially yellow which turned to green and then
light yellow with the progress of the reaction. Evolution of hydrogen sulfide gas was observed during
each reaction. After completion of the reaction, excess of ethanol was removed under reduced
pressure. The mixture was diluted with distilled water (200 mL) and acidified with 4N hydrochloric
acid to pH 2-3. It was then filtered, washed with diethyl ether and recrysallized from ethanol.
3.5. Procedure for the preparation of 5-(4-nitrophenyl)-4-amino-1,2,4-triazole-3-thiol (4a)
5-(4-Nitrophenyl)-1,3,4-oxadiazole-2-thiol (4 g, 0.017 mol) and 80% hydrazine hydrate (6 mL,
0.124 mol) in absolute ethanol (25) were refluxed in a 250 mL flask fitted with a condenser and a
guard tube. After the completion of the reaction the solvent and excess of hydrazine hydrate were
removed under reduced pressure using rotary evaporator. The residue was washed with diethyl ether
and recrystallized from ethanol.
3.6. Procedure for the preparation of 5-(4-methoxybenzyl)-1,3,4-oxadiazole-2-ylnaphthalene-1-
carbothioate (5a)
5-(4-Methoxybenzyl)-1,3,4-oxadiazole-2-thiol (5 g, 0.021 mol) was dissolved in absolute ethanol
(30 mL) in a 250 mL flask. Potassium hydroxide (1 g, 0.178 mol) solution in water (20 mL) was added
to the solution. After one hour reflux 1-naphthoyl chloride (4 g, 0.021 mol) was added to the reaction
mixture. The reaction mixture was thoroughly stirred and refluxed for six hours. After completion of
the reaction, the excess of ethanol in the reaction mixture was under reduced pressure. The mixture left
behind was diluted with distilled water (200 mL) and then filtered. The crude product was dried in an
oven and recrystallized from ethanol.
3.7. Preparation for the preparation of 5-(4-methoxybenzyl)-4-amino-1,2,4-triazole-3-ylnaphthalene-
1-carbothioate (6a).
5-(4-Methoxybenzyl)-1,3,4-oxadiazole-2-ylnaphthalene-1-carbothioate (5 g, 0.013 mol) and 80%
hydrazine hydrate (8 mL, 0.165 mol) in absolute ethanol (30 mL) were refluxed in a 250 mL flask
fitted with a condenser and a guard tube. After completion of the reaction, the solvent and excess
hydrazine hydrate were removed under reduced pressure. The residue was washed with diethyl ether
and recrystallized from ethanol.
Purification of all the synthesized compounds was achieved by recrystallization and purity of each
compound was monitored by thin layer (tlc) and gas (gc) chromatography.

Molecules 2011, 16
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5-(4-Nitrophenyl)-4-amino-1,2,4-triazole-3-ylnaphthalene-1-carbothioate (6a). 5-(4-Nitrophenyl)-
1,3,4-oxadiazole-2-yl naphthalene-1-carbothioate (3.0 g; 8 mmol), NH2-NH2 (5.10 g; 103 mmol) were
reacted according to the general procedure. Color light brown; yield: 80% (2.40 g); recrystallization
from EtOH; m.p. 168-170 °C; Molecular formula C19H13O3N5S; Mol. Wt. 191. FTIR (KBr, cm-1):
1
3250 (N-H), 1557 (C=C)Ar, 1633 (C=O), 1517 (C=N); H-NMR (DMSO-d6, δ, ppm): 7.72 (dd, 1H,
J = 1.2, 4.6 Hz, ArH-1), 8.03 (dd, 1H, J = 2.5, 7.2 Hz, Ar H-2) 8.01 (dd, 1H, J = 2.4, 7.1 Hz, A H-3),
7.73 (dd, 1H, J = 2.3, 4.4 Hz, Ar H-4), 7.76 (m, 1H, Ar H-1´ ),7.42 (m, 1H, Ar H-2´ ), 7.90 (m, 1H, Ar
13
H-3´ ), 7.88 (m, 1H, Ar H-4´ ), 7.55 (m, 1H, Ar H-5´ ), 4.32 (s, 2H, NH2); C-NMR (DMSO-d6, δ,
ppm): 129.41 (C-1), 128.40 (C-2), 116.52 (C-3), 115.52 (C-4), 116.31 (C-5), 130.43 (C-6), 160.42 (C-
7), 171.40 (C-8), 182.31 (C-9), 135.61 (C-10), 127.12 (C-11), 120.43 (C-12), 122.41(C-13), 125.32
(C-14), 126.32 (C-15), 128.43 (C-16), 129.42 (C-17), 130.21 (C-18), 129.66 (C-19); MS (m/z) 391
(40%), (M+), 361 (35%), 243 (50%), 155 (100%; loss of naphthoyl fragment), 148 (60%), 136 (65%),
127 (55%; loss of naphthoyl fragment – CO).
5-(3-Nitrophenyl)-4-amino-1,2,4-triazole-3-ylnaphthalene-1-carbothioate (6b). 5-(3-Nitrophenyl)-
1,3,4-oxadiazole-2-ylnaphthalene-1-carbothioate (4.5 g; 12 mmol), NH2-NH2 (6.20 g; 124 mmol) were
reacted according to the general procedure. Color light yellow; yield: 83% (3.80 g); recrystallization
from EtOH; m.p. 155-157 °C; Molecular formula C19H13O3N5S; Mol. Wt. 191. FTIR (KBr, cm-1):
1
3202 (N-H), 1566 (C=C)Ar, 1655 (C=O), 1519 (C=N); H-NMR (DMSO-d6, δ, ppm): 7.60 (dd, 1H,
J = 3.1, 4.2 Hz, ArH-1), 7.77 (dd, 1H, J = 3.2, 4.1 Hz, Ar H-2), 7.04 (dd, 1H, J = 2.5, 6.6Hz, A H-3),
7.45 (dd, 1H, J = 4.2, 3.1 Hz, Ar H-4), 7.88 (m, 1H, Ar H-1´), 7.79 (m, 1H, Ar H-2´ ), 7.62 (m, 1H, Ar
13
H-3´ ), 8.25 (m, 1H, Ar H-4´ ), 7.21 (m, 1H, Ar H-5´ ), 3.95 (s, 2H, NH2); C-NMR (DMSO-d6, δ,
ppm): 128.40 (C-1), 127.41 (C-2), 155.50 (C-3), 116.62 (C-4), 118.62 (C-5), 127.42 (C-6), 162.61 (C-
7), 174.65 (C-8), 180.37 (C-9), 136.23(C-10), 129.12 (C-11),122.31 (C-12), 125.71(C-13), 128.81 (C-
14), 127.50 (C-15), 130.64 (C-16), 132.80 (C-17), 126.21 (C-18), 129.44 (C-19); MS (m/z) 391 (40%),
(M+), 361 (35%), 243 (50%), 155 (100%; loss of naphthoyl fragment) 148 (60%), 136 (65%), 127
(55%; loss of naphthoyl fragment - CO).
5-Phenyl-4-amino-1,2,4-oxadiazole-3-ylbenzothioate (6c). 5-Phenyl-1,3,4-oxadiazole-2-yl benzo-
thioate (4.0 g; 14 mmol), NH2-NH2 (5.60 g; 113 mmol) were reacted according to the general
procedure. Color red; yield: 78% (3.10 g); recrystallization from EtOH; m.p. 214-215 °C; Molecular
formula C15H12ON4S; Mol. Wt. 296. FTIR (KBr, cm-1): 3212 (N-H), 1574 (C=C)Ar, 1664 (C=O), 1525
(C=N); 1H-NMR (DMSO-d6, δ, ppm): 7.48 (m, 1H, ArH-1) 732 (m, 1H, Ar H-2), 7.22 (m, 1H, A H-3)
7.97 (m, 1H, Ar H-4), 7.65 (dd, 1H, J = 3.1, 6.7 Hz, Ar H-1´ ), 7.59 (dd, 1H, J = 3.2, 4.4 Hz, Ar H-2´),
7.69 (dd, 1H, J = 2.5, 4.7 Hz, Ar H-3´ ), 7.90 (dd, 1H, J = 3.1, 6.8 Hz, Ar H-4´ ), 7.45 (dd, 1H, J = 2.8,
6.5 Hz, Ar H-5´ ), 4.12 (s, 2H, NH2); 13C-NMR (DMSO-d6, δ, ppm): 130.05 (C-1), 126.41 (C-2),
116.24 (C-3), 115.24 (C-4), 116.37 (C-5), 126.21 (C-6), 166.04 (C-7), 176.23 (C-8), 184.32 (C-9),
134.05 (C-10), 130.73 (C-11),126.32 (C-12), 125.61 (C-13), 126.67 (C-14), 130.73 (C-15); MS (m/z)
296 (42%) (M+), 266 (35%), 193 (50%), 103 (62%),105 (100%; loss of acylium ion fragment), 77
(52%; loss of phenyl cation fragment).

Molecules 2011, 16
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5-Phenethyl-4-amino-1,2,4-triiazole-3-yloctanethioate (6d). 5-Phenethyl-1,3,4-oxadiazole-2-yl octane-
thioate (6.0 g; 18 mmol), NH2-NH2 (8.20 g; 165 mmol) were reacted according to the general
procedure. Color orange; yield: 74% (4.40 g); recrystallization from EtOH; m.p. 179-180 °C;
Molecular formula C18H26ON4S; Mol. Wt. 346. FTIR (KBr, cm-1): 3210 (N-H), 1520 (C=C)Ar, 1660
(C=O), 1582 (C=N); 1H-NMR (DMSO-d6, δ, ppm):; 1HNMR (DMSO-d6) δ (ppm): 7.12 (m, 1H, ArH-
1), 7.22 (dd, 1H, J = 2.4, 6.7 Hz, ArH-2), 7.08 (m, 1H, ArH-3), 7.22 (dd, 1H, J = 2.4, 6.7 Hz, ArH-2),
4.33 (s, 2H, NH2), 2.88, 3.2, 1.5 (t, 4H, t, 2H, m, 10 H other protons) 2.50, 3.2, 1.52 (t, 2H, m, 10H,
other protons); 13C-NMR (DMSO-d6, δ, ppm): 28.36 (C-1), 30.18 (C-2), 127.21 (C-3), 126.33 (C-4),
127.41 (C-5), 116.24 (C-6), 118.44 (C-7), 116.26 (C-8), 166.23 (C-9), 175.90(C-10), 182.64 (C-
11),45.46 (C-12), 33.67(C-13), 26.34 (C-14), 25.80 (C-15), 20.21 (C-16), 18.44 (C-17), 22.37 (C-18);
MS (m/z) 346 (40%) (M+), 316 (36%), 215 (50%), 131 (60%), 127 (100%), 99 (55%).
5-Phenethyl-4-amino-1,2,4-triazole-3-ylbenzothioate (6e). 5-Phenethyl-1,3,4-oxadiazole-2-yl benzo-
thioate (5.0 g; 16 mmol), NH2-NH2 (7.20 g; 144 mmol) were reacted according to the general
procedure. Color light yellow; yield: 80% (4.00 g); recrystallization from EtOH; m.p. 158-159 °C;
Molecular formula C17H16ON4S; Mol. Wt. 324. FTIR (KBr, cm-1): 3195 (N-H), 1509 (C=C)Ar, 1673
1
(C=O), 1567 (C=N); H-NMR (DMSO-d6, δ, ppm): 7.14 (m, 1H, ArH-1), 7.47 (dd, 1H, J = 2.1,
7.2 Hz, Ar H-2), 7.12 (m, 1H, ArH-3), 7.67 (m, 1H, Ar H-4), 7.97 dd, 1H, J = 2.4, 6.5 Hz, ArH-1´),
7.58 (dd, 1H J = 3.1, 4.5 Hz, Ar-H-2´), 7.77 (m, 1H ArH-3´), 7.67 (m, 1H ArH-4´) 4.16 (s, 2H, NH2),
2.90 (t, 4H other protons); 13C-NMR (DMSO-d6, δ, ppm): 30.33 (C-1), 32.71 (C-2), 130.43 (C-3),
118.23 (C-4), 117.23 (C-5), 115.48 (C-6), 118.23 (C-7), 125.63 (C-8), 159.21 (C-9), 170.62 (C-10),
183.47 (C-11),134.24 (C-12), 128.40 (C-13), 129.65 (C-14), 126.31 (C-15), 130.52 (C-16), 127.43 (C-
17); MS (m/z) 324 (45%) (M+), 294 (35%), 193 (50%), 131 (62%), 105 (100%; loss of acylium ion
fragment), 77 (52%; loss of phenyl cation fragment).
5-(3-Nitrophenyl)-4-amino-1,2,4-triazole-3-ylheptanethioate (6f). 5-(3-Nitrophenyl)-1,3,4-oxadiazole-
2-ylheptanethioate (3.0g; 8 mmol), NH2-NH2 (5.10 g; 103 mmol) were reacted according to the general
procedure. Color golden; yield: 76% (2.30 g); recrystallization from EtOH; m.p. 148-150 °C;
Molecular formula C15H19O3N5S; Mol. Wt. 349. FTIR (KBr, cm-1): 3140 (N-H), 1570 (C=C)Ar, 1667
1
(C=O), 1514 (C=N); H-NMR (DMSO-d6, δ, ppm): 7.47 (dd, 1H, J = 3.2, 6.2 Hz, ArH-1) ,7.27 (dd,
1H, J = 2.1, 7.2 Hz, Ar H-2), 6.97 (dd, 1H, J = 3.2, 7.3 Hz, Ar H-3), 7.47 (dd, 1H, J = 3.4, 6.2 Hz, Ar
H-4), 3.94 (s, 2H, NH2), 2.50, 3.2, 1.52 (t, 2H m, 10H, other protons); 13C-NMR (DMSO-d6, δ, ppm):
130.06 (C-1), 127.25 (C-2), 152.44 (C-3), 127.25 (C-4), 126.31 (C-5), 117.21 (C-6), 161.37 (C-7),
170.68 (C-8), 182.04 (C-9), 54.34 (C-10), 44.31 (C-11),33.39 (C-12), 24.36 (C-13), 25.62 (C-14),
20.22 (C-15); MS (m/z) 349 (40%) (M+), 319 (37%), 201 (50%), 148 (60%),113 (100%), 85 (55%).
5-(4-Methoxybenzyl)-4-amino-1,2,4-triazole-3-ylnaphthalene-1-carbothioate (6g). 5-(4-Methoxy-
benzyl)-1,3,4-oxadiazole-2-yl naphthalene-1-carbothioate (5.0 g; 13 mmol), NH2-NH2 (8.20 g;
165 mmol) were reacted according to the general procedure. Color reddish brown; yield: 82% (4.10 g);
recrystallization from EtOH; m.p. 166-167 °C; Molecular formula C21H18O2N4S; Mol. Wt. 390. FTIR
1
(KBr, cm-1): 3204 (N-H), 1575 (C=C)Ar, 1684 (C=O), 1518 (C=N); H-NMR (DMSO-d6, δ, ppm):
6.50 (m, 1H, ArH-1), 6.87 (m, 1H, Ar H-2), 6.50 (m, 1H, A H-3), 6.87 (m, 1H, Ar H-4), 7.56 (dd, 1H,

Molecules 2011, 16
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J = 2.4, 6.2 Hz, Ar H-1´ ), 7.42 (dd, 1H, J = 3.5, 6.5 Hz, Ar H-2´ ), 7.73 (m, 1H, Ar H-3´ ), 7.46 (m,
1H, Ar H-4´ ), 7.55 (m, 1H, Ar H-5´ ), 3.83 (s, 2H, NH2), 2.50, (s, 2H, methylene protons) 3.56 (s, 3H,
methyl protons); 13C-NMR (DMSO-d6, δ, ppm): 30.60 (C-1), 55.90 (C-2), 130.02 (C-3), 127.05 (C-4),
116.65 (C-5), 158.03 (C-6), 166.04 (C-7), 175.10 (C-8), 180.01 (C-9), 116.65 (C-10), 127.05 (C-
11),134.50 (C-12), 127.12 (C-13), 125.62 (C-14), 120.50 (C-15), 119.42 (C-16), 129.31 (C-17), 131.61
(C-18), 130.71 (C-19), 123.63 (C-20); MS (m/z) 390 (40%) (M+), 360 (37%), 243 (50%), 147 (60%),
155 (100%; loss of naphthoyl fragment) 148 (60%), 136 (65%), 127 (55%; loss of naphthoyl fragment
- CO) 121 (65%; loss of tropylium ion).
5-Phenethyl-4-amino-1,2,4-triazole-3-ylnonanethioate
(6h).
5-Phenethyl-1,3,4-oxadiazole-2-yl
nonane-thioate (4.0 g; 12 mmol), NH2-NH2 (5.60 g; 113 mmol) were reacted according to the general
procedure. Color yellow; yield: 78% (3.10 g); recrystallization from EtOH; m.p. 138-139 °C;
Molecular formula C17H24ON4S; Mol. Wt. 332. FTIR (KBr, cm-1): 3260 (N-H), 1512 (C=C)Ar, 1652
1
(C=O), 1590 (C=N); H-NMR (DMSO-d6, δ, ppm): 6.52.(m, 1H, ArH-1), 6.77 (m, 1H, Ar H-2), 7.14
(m, 1H, A H-3), 7.02 (m, 1H, Ar H-4), 4.45 (s, 2H, NH2), 2.50, 3.2, 1.60 (t, 2H, m, 12H other protons);
13C-NMR (DMSO-d6, δ, ppm): 130.46 (C-1), 125.34 (C-2), 116.73 (C-3), 118.62 (C-4), 116.73 (C-5),
125.23 (C-6), 159.68 (C-7), 168.77 (C-8), 184.66 (C-9), 53.62 (C-10), 44.02 (C-11),34.71 (C-12),
29.01 (C-13), 25.72 (C-14), 25.69 (C-15), 20.02 (C-16), 18.33 (C-17). MS (m/z) 332 (40%) (M+), 302
(37%), 229 (52%), 141 (100%), 113 (50%), 103 (58%)
5-(3-Nitrophenyl)-4-amino-1,2,4-triazole-3-ylbenzothioate (6i). 5-(3-Nitrophenyl)-1,3,4-oxadiazole-2-
ylbenzothioate (3.0 g; 9 mmol), NH2-NH2 (5.10 g; 103 mmol) were reacted according to the general
procedure. Color brown; yield: 66% (2.10 g); recrystallization from EtOH; m.p. 138-139 °C;
Molecular formula C15H11O3N5S; Mol. Wt. 341. FTIR (KBr, cm-1): 3318 (N-H), 1510 (C=C)Ar, 1625
1
(C=O), 1587 (C=N); H-NMR (DMSO-d6, δ, ppm): 7.12 (dd, 1H, J = 3.2, 6.4 Hz, ArH-1),7.27 (dd,
1H, J = 3.2, 7.4 Hz, Ar H-2), 7.12 (dd, 1H, J = 3.2, 6.4 Hz, A H-3), 7.77 (m, 1H, Ar H-4), 7.67 (m, 1H,
Ar H-1´), 7.85 (m, 1H, Ar H-2´), 7.58 (m, 1H, Ar H-3´), 7.82 (m, 1H, Ar H-4´), 7.25 (m, 1H, Ar H-5´),
13
4.26 (s, 2H, NH2); C-NMR (DMSO-d6, δ, ppm): 133.06 (C-1), 127.45 (C-2), 153.44 (C-3), 125.21
(C-4), 127.40 (C-5), 117.67 (C-6), 163.23 (C-7), 177.45 (C-8), 181.08 (C-9), 136.38 (C-10), 127.21
(C-11),116.90 (C-12), 126.64 (C-13), 128.03 (C-14), 125.64 (C-15); MS (m/z) 341 (42%) (M+), 311
(35%), 193 (50%), 148 (60%), 105 (100%; loss of acylium ion fragment), 77 (52%; loss of phenyl
cation fragment).
3.8. Antifungal evaluation
The synthesized compounds were tested by agar tube dilution method [17] for their in vitro
fungicidal activity. Terbinafine (200 g/mL) was used as positive control. All experiments were done
in three replicates. Four fungal strains: Aspergillus flavus, Mucor species, Aspergillus niger and
Aspergillus fumigatus were used. All fungal strains were grown on 6.5% SDA (Sabouraud dextrose
agar, pH 5.7) at 28 °C and preserved at 4 °C in a refrigerator. One hundred mm slants with sterilized
SDA were prepared by adding each compound in 200 g/mL concentration. Terbinafine (200 g/mL)
was used as standard drug while DMSO was used as negative control. Each slant was inoculated with

Molecules 2011, 16
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4 mm piece of respective fungal strain and incubated at 28 °C for 7-10 days. Fungal growth was
compared with negative control to get the %age inhibition.
4. Conclusions
Nine 5-substitued-1,3,4-oxadiazole-2-thioesters derivatives 5a-i containing different groups and
obtained via 5-substituted-1,3,4-oxadiazole-2-thiols using potassium hydroxide and acid chloride were
subjected to reactions with hydrazine hydrate to furnish nine new 5-substituted-4-amino-1,2,4-triazole-
1
3-thioesters 6a-i.Their structures were confirmed by infrared, H- and 13C -NMR and mass
spectrometric analysis. In the in vitro antifungal bioassay, compounds 6a, 6b, 6d and 6g were found to
have significant antifungal activities against the four tested fungal strains: Aspergillus flavus, Mucor
species, Aspergillu niger and Aspergillu. fumigatus Structure activity relationship comparison shows
that the presence of naphthyl group at 3-position of the triazole-3-thioester enhances the activity of the
compounds, probably because a similar group is also present in the structure of the reference
compound terbinafine.
Acknowledgements
The financial support for the research project No. RG095/10AFR from the Universiti Malaya
Research Grant (UMRG), University of Malaya is highly appreciated.
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© 2011 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).