Preparation and evaluation of antibacterial activity of some new 1,2,4-triazole derivatives

Article abstract of DOI:10.1002/jhet.431

(Chemical Equation Presented) Cyclizations of 1,5-diphenyl[1,2,4]triazole- 3-carbohydrazide 1 and the thiosemicarbazide 2 (derived from 1 by addition to phenylisothiocyanate) under various conditions afforded novel biheterocyclic systems. The newly prepar

Full text of DOI:10.1002/jhet.431

September 2010
Preparation and Evaluation of Antibacterial Activity of Some
New 1,2,4-Triazole Derivatives
1109
W. A. A. Arafa*
Department of Chemistry, Fayoum University, Fayoum, Egypt
*E-mail: waelarafa156@yahoo.com
Received October 21, 2009
DOI 10.1002/jhet.431
Published online 16 July 2010 in Wiley Online Library (wileyonlinelibrary.com).
Cyclizations of 1,5-diphenyl[1,2,4]triazole-3-carbohydrazide 1 and the thiosemicarbazide 2 (derived
from 1 by addition to phenylisothiocyanate) under various conditions afforded novel biheterocyclic sys-
tems. The newly prepared compounds were screened for their antibacterial activity. The results showed
that several of these compounds exhibited significant antibacterial activity.
J. Heterocyclic Chem., 47, 1109 (2010).
INTRODUCTION
[34–37]. The NH2 group of the hydrazide structure
behaves as a good nucleophile in most reactions leading
to formation of new heterocyclic rings such as 1,2,4-tri-
azoles, 1,3,4-thiadiazoles [37–39].
Many compounds consisting of five-membered hetero-
cyclic rings such as triazoles, oxazdiazoles, and thiadia-
zoles were synthesized and evaluated for their biological
activities [1–5]. During recent years, microorganisms
have increased their resistance against commonly used
antibiotics. Therefore, it was crucial to develop new
antimicrobial and antiviral compounds [6]. Studies on
1,2,4-triazole compounds have a wide range in the area
of pharmacology [7–14]. 4-Thiazolidinones are known
to possess antibacterial [15–20], antifungal [21], antivi-
ral [22,23], and antituberculosis [24] properties (Fig. 1).
4-Thiazolidinones have a novel mechanism for action
that involves the inhibition of bacterial protein synthesis
at a very early stage [25,26]. The traditional method for
the preparation of thiazolidinones includes the reaction
of an aromatic amine with substituted benzaldehyde in
the presence of mercaptoacetic acid [26,27]. In addition,
the method reported recently for the synthesis of thiazo-
lidinones involved the reaction of thiosemicarbazides
with chloroacetic acid [25]. During the recent years,
there has been a broad investigation on different classes
of thiadiazoles, many of which were found to possess an
extensive spectrum of pharmacological activities [28–
32]. Also, 1,2-pyrazole derivatives were found to pos-
sess various biological activities [33,34].
In view of these facts, hereby we report the prepara-
tion of new 1,2,4-triazoles, connected to another hetero-
cyclic ring such as 1,2,4-triazole, 1,3,4-thiadiazole, and
1,3-thiazolidin-4-one rings.
RESULTS AND DISCUSSION
The structure of the prepared compounds was eluci-
1
dated using IR, H NMR, 13C NMR, and mass spectro-
scopic methods besides elemental analyses. The path-
ways leading to the products obtained have been
depicted in Schemes 1 and 2. The key intermediate in
this study is the 1,5-diphenyl-1H-[1,2,4]triazole-3-car-
boxylic acid hydrazide 1, which was prepared according
to a previously described procedure [40].
Acid hydrazide 1 and thiosemicarbazide 2 are of con-
siderable interest as building blocks for nitrogen and/or
sulfur containing heterocyclic systems, which might
show biological activities. Therefore, our attempts are to
prepare new five-membered heterocycles of expected bi-
ological activities utilizing compounds 1 and 2 as start-
ing material.
The cyclization of relatively small and linear mole-
cules is one of the most common methods leading to the
formation of heterocyclic compounds. For example,
compounds that contain a thiosemicarbazide structure
can be considered as suitable precursors for this purpose
When compound 1 was refluxed with phenylisothio-
cyanate, the expected thiosemicarbazide 2 was obtained
in good yield. In the IR spectrum of compound 2, the
hydrazone NH stretching frequencies were observed at
3291, 3198, and 3146 cmꢀ1. The absorption band at
C
V
2010 HeteroCorporation

1110
W. A. A. Arafa
Vol 47
Figure 1
1666 cmꢀ1 is due to the presence of C¼¼O stretch. The
cmꢀ1 characteristic for C¼¼S group. Moreover, in the
1H NMR spectrum of compound 3, NH was observed as
singlet at 9.32 ppm, therefore it was proved that com-
pound 3 was found in thionic form also in DMSO. Fur-
thermore, reacting thiosemicarbazide 2 with cold con-
centrated sulfuric acid resulted in the formation of the
corresponding N-phenyl[5-(1,5-diphenyl-1H-[1,2,4]tria-
zol-3-yl)-[1,3,4]thiadiazol-2-yl]-amine 4. The reaction
involved the nucleophilic attack of the C¼¼S sulfur atom
C¼¼S stretching frequency was observed at 1373 cmꢀ1
.
Further evidence for the formation of compound 2 was
obtained by recording its mass spectrum. The mass
spectrum of 2 showed the molecular ion at m/z 414 cor-
responding to C22H18N6OS and the base peak at m/z
220. Alkaline cyclization of the thiosemicarbazide 2
using 2N sodium hydroxide solution afforded the corre-
sponding 4,10,50-triphenyl-1,4-dihydro-10H-[3,30]bi[1,2,4]
triazolyl]-5-thione 3. This reaction began with nucleo-
philic attack of thiosemicarbazide N-5 to carbonyl group
in the side chain of compound 2. The structure of com-
pound 3 was confirmed on the basis of elemental analy-
sis, IR, 1H NMR, 13C NMR, and mass spectroscopic
methods. It is interesting to note that compound 3 is
present in solid state in thionic form, C¼¼S, as indicated
by its IR spectrum, which showed the absence of
absorption in the region of 2500–2660 cmꢀ1 cited for
SH group [41] and the presence of maximum at 1455
1
on the carbonyl C of compound 2. In the H NMR spec-
trum of compound 4 the presence of only one singlet
for NH group integrated for one proton confirming the
cyclization at the side chain of triazole ring in com-
pound 2 to afford the thiadiazole 4. Also, the mass spec-
trum of compound 4 gave molecular ion peak in agree-
ment with its molecular formula. The thiazolidinone de-
rivative 5 was obtained from the treatment of thiosemi-
carbazide 2 with ethyl bromoacetate in the presence of
anhydrous sodium acetate in absolute ethanol. The first
Scheme 1
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2010
Preparation and Evaluation of Antibacterial Activity of Some
New 1,2,4-Triazole Derivatives
1111
Scheme 2
1
step of this reaction is thought to be an S-alkylation of
2. The second step is a release of ethanol to give thiazo-
lidinone 5, the IR spectrum of which showed the lactam
C¼¼O stretching band at 1708 cmꢀ1. The mass spectrum
was also in agreement with the formation of thiazolidi-
none ring. In an extension of the above reaction to
obtain another thiazolidinone derivative 6, thiosemicar-
bazide 2 undergoes a ring closure reaction, on boiling in
ethanol with monochloroacetic acid. The structure of
compound 6 was elucidated on the basis of IR, 1H
NMR, and mass spectra as well as correct elemental
analysis. The IR of 6 exhibited absorption bands at 3212
due to NH group and at 1719, 1665 cmꢀ1 because of
two carbonyl groups. H NMR of 6 revealed a singlet at
3.62 because of CH2 of thiazolidinone ring and singlet
at 9.01 ppm for NH.
Additionally, the condensation of the acid hydrazide 1
with a variety of aromatic aldehydes in ethanol leading
to the formation of compounds 7a, b. Structure elucida-
tion of the Schiff’s bases 7a, b was based on microanal-
ysis and spectral data (Tables 1 and 2). 1,5-Diphenyl-
1H-[1,2,4]triazole-3-carboxylic acid (4-oxo-2-phenyl-
thiazolidin-3-yl)-amide 8 was obtained by refluxing the
Schiff’s base 7a and thioglycolic acid in dry benzene
for 10 h using a Dean-Stark water separator. The thiazo-
lidinone 8 was characterized by IR absorption bands at
Table 1
Analytical data of the new compounds.
Elemental analyses found (calcd.)
Comp. No.
Molecular formula
mp (ꢁC)
Yield (%)
%C
%H
%N
%S
2
3
4
5
6
7a
7b
8
C22H18N6OS
C22H16N6S
C22H16N6S
215
285
179
210
202
175
187
220
195
240
225
165
80
84
85
82
78
81
88
80
68
75
82
74
63.64 (63.75)
66.77 (66.65)
66.42 (66.65)
63.61 (63.42)
63.57 (63.42)
72.12 (71.92)
72.22 (72.42)
65.55 (65.29)
72.47 (72.32)
70.06 (69.96)
66.23 (66.08)
59.63 (59.80)
4.57 (4.38)
3.86 (4.07)
4.24 (4.07)
4.24 (3.99)
3.78 (3.99)
4.51 (4.66)
5.00 (5.02)
4.24 (4.34)
4.00 (4.14)
4.82 (4.99)
4.16 (4.38)
3.71 (3.45)
20.11 (20.28)
21.33 (21.20)
21.00 (21.20)
18.35 (18.49)
18.32 (18.49)
19.13 (19.06)
18.57 (18.36)
15.59 (15.86)
18.89 (19.17)
20.11 (20.40)
20.03 (20.28)
21.52 (21.79)
7.80 (7.74)
7.86 (8.09)
8.21 (8.09)
7.22 (7.05)
7.15 (7.05)
–
–
7.33 (7.26)
C24H18N6O2S
C24H18N6O2S
C22H17N5O
C23H19N5O
C24H19N5O2S
C22H15N5O
C20H17N5O
C19H15N5O2
C16H11N5OS
9
–
10
11
12
–
–
10.14 (9.98)
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1112
W. A. A. Arafa
Vol 47
Table 2
Spectral data for the new compounds.
Comp. No.
Spectral data
2
3
IR (KBr, cmꢀ1): 3291, 3198, 3146 (NH), 1666 (C¼O), 1604 (C¼N) and 1373 (C¼S).
ms: m/z (%): 414 (Mþ, 15.30).
IR (KBr, cmꢀ1): 3129, 1598 (C¼N) and 1455 (C¼S).
1H NMR (DMSO-d6, d ppm): 7.01–7.48 (15H, m, aromatic protons) and 9.32 (1H, s, NH).
13C NMR (DMSO-d6, d ppm): arom-C: [123.8 (2CH), 125.7 (4CH), 126.1 (2CH), 128.0 (1CH),
128.8 (6CH), 135.4 (1C), 137.5 (2C)], 154.0 (C5-triazole ring), 157.4 (C3 & C03-triazole rings), 181.5 (C¼S).
ms: m/z (%): 396 (Mþ, 42.10).
4
5
IR (KBr, cmꢀ1): 3187 (NH) and 1599 (C¼N).
1H NMR (DMSO-d6, d ppm): 5.10 (1H, s, NH) and 6.91–7.33 (15H, m, aromatic protons )
ms: m/z (%): 396 (Mþ, 13.80).
IR (KBr, cmꢀ1): 3330 (NH), 1708 (C¼O), 1688 (C¼O) and 1612 (C¼N).
1H NMR (DMSO-d6, d ppm): 3.67 (2H, s, CH2 of thiazolidinone ring),
7.11–7.42 (15H, m, aromatic protons) and 9.23 (1H, s, NH).
ms: m/z (%): 454 (Mþ, 42.71)
6
IR (KBr, cmꢀ1): 3212 (NH), 1719 (C¼O), 1665 (C¼O) and 1605 (C¼N).
1H NMR (DMSO-d6, d ppm): 3.62 (2H, s, CH2 of thiazolidinone ring),
7.05–7.38 (15H, m, aromatic protons) and 9.01 (1H, s, NH).
ms: m/z (%): 454 (Mþ, 12.80)
7a
7b
8
IR (KBr, cmꢀ1): 3242 (NH), 1685 (C¼O) and 1601 (C¼N).
ms: m/z (%): 367 (Mþ, 26.37).
IR (KBr, cmꢀ1): 3222 (NH), 1680 (C¼O) and 1589 (C¼N).
ms: m/z (%): 381 (Mþ, 37.14).
IR (KBr, cmꢀ1): 3251 (NH), 1722 (C¼O) and 1704 (C¼O).
1H NMR (DMSO-d6, d ppm): 3.41 (1H, d, equatorial proton of thiazolidinone at C-5),
3.81 (1H, d, axial proton of thiazolidinone at C-5), 5.68 (1H, s, CH of thiazolidinone at C-2),
7.21–7.35 (15H, m, aromatic protons) and 9.51 (1H, s, NH).
13C NMR (DMSO-d6, d ppm): 39.2 (C5-thiazolidinone ring), 55.9 (C2-thiazolidinone ring),
arom-C: [124.5 (1CH), 125.0 (2 CH), 126.4 (3CH), 127.5 (3CH), 128.7 (6CH), 136.3 (1C),
137.4 (1C), 138.2 (1C)], 154.5 (C5-triazole ring), 158.4 (C-3 triazole ring),
165.7 (C¼O), 167.6 (C4-thiazolidinone ring).
ms: m/z (%): 441 (Mþ, 27.08).
9
IR (KBr, cmꢀ1): 1599 (C¼N).
ms: m/z (%): 365 (Mþ, 30.67).
10
IR (KBr, cmꢀ1): 1670 (C¼O).
1H NMR (DMSO-d6, d ppm):1.63, 1.81 (6H, 2s, 2CH3), 5.40 (1H, s, C4- pyrazole ring),
6.89–7.24 (10H, m, aromatic protons).
13C NMR (DMSO-d6, d ppm): 8.2 (2CH3), 109.6 (C4-pyrazole ring), arom-C: [124.3 (1CH),
125.1 (2 CH), 126.5 (2CH), 127.5 (1CH), 128.3 (4CH), 136.1 (1C), 137.7 (1C)],
149.9 (C3 & C5-pyrazole ring), 154.2 (C5-triazole ring), 159.5 (C-3 triazole ring), 169.6 (C¼O).
ms: m/z (%): 343 (Mþ, 47.51).
11
12
IR (KBr, cmꢀ1): 1728 (C¼O) and 1688 (C¼O).
1H NMR (DMSO-d6, d ppm): 2.01 (3H, s, CH3),
3.26 (2H, s, CH2 of pyrazolone ring) and 7.12–7.37 (10H, m, aromatic protons).
ms: m/z (%):345 (Mþ, 52.49).
IR (KBr, cmꢀ1): 3321 (NH).
1H NMR (DMSO-d6, d ppm) 6.99–7.50 (10H, m, aromatic protons) and 10.85 (1H, s, NH).
ms: m/z (%): 321 (Mþ, 25.47).
3251, 1722, and 1704 cmꢀ1 for NH and the two C¼¼O
phosphorous oxychloride. The IR spectrum of compound
9 showed no absorption for NH groups, this indicating
the cyclization of 1 into oxadiazole 9. Also, the mass
spectrum of 9 gave a molecular ion at m/z 365 corre-
sponding to C22H15N5O. The hydrazide 1 was allowed
to react with acetyl acetone and ethyl acetoacetate to
give dimethyl pyrazole derivative 10 and methyl pyrazo-
lone derivative 11, respectively. The IR of 11 showed
absorption band at 1728 cmꢀ1 characteristical for the
pyrazole carbonyl group. 1H NMR spectra of
1
groups, respectively. Its H NMR exhibited two signals
appearing as doublets at 3.41 ppm and 3.81 ppm
because of the nonequivalent geminal CH2 protons [42].
This splitting was not observed with derivatives 5, 6,
and 11 which may be attributed to the lacking of the
asymmetric carbon atom. A convenient method for the
preparation of oxadiazole derivative 9 was deduced
from refluxing the acid hydrazide 1 with an equimolar
amount of benzoic acid in the presence of an excess of
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

September 2010
Preparation and Evaluation of Antibacterial Activity of Some
New 1,2,4-Triazole Derivatives
1113
compounds 10 and 11 showed singlets at 1.81 ppm and
2.01 ppm assigned for the methyl groups, respectively.
The preparation of oxadiazole-2-thione 12 was achieved,
according to a reported method of Young and Wood
[43], by adopting a simple one-pot procedure that
involves reacting 1 with carbon disulfide under strong
basic conditions followed by acidification with diluted
HCl. The oxadiazole derivative 12 was characterized by
IR which showed band at 3321 cmꢀ1 attributed to NH.
bacterial study revealed that, compounds 5, 6, 7a, 7b, 8,
and 11 showed low to moderate antibacterial activities.
This result suggesting that Schiff’s base, thiazolidinone,
and pyrazole moieties play an important role in enhanc-
ing the antibacterial activities of this class of
compounds.
EXPERIMENTAL
1
The H NMR spectrum of 12 displayed the NH reso-
nance of the oxadiazole ring at 10.85 ppm.
Chemistry. Melting points were determined in open-glass
capillaries on a Stuart electric melting point apparatus and
were uncorrected. Elemental analyses were performed by the
Microanalysis center, Faculty of Science, Cairo University.
Infrared spectra were recorded on Satellite 2000 spectrometer
using KBr discs. Mass spectra were determined on GC-MS
(QP/000 EX) Shimadzu spectrometer at an ionizing voltage of
70 eV. Nuclear magnetic resonance spectra were recorded on
Varin Mercury 300 MHz spectrometer using TMS as an inter-
nal standard; chemical shifts are reported in d units. Solvents
were dried by standard procedures. Reaction progress and pu-
rity of the compounds were checked by TLC, making use of
silica gel plates (Silica gel F254 on aluminum sheets).
Preparation of thiosemicarbazide 2. A solution of 1 (0.01
mol) and equimolar amount of phenylisothiocyanate in 50 mL
of ethanol was heated under reflux for 5 h. The solid product
obtained after concentration of the solution was collected by
filtration and recrystallized from methanol to give yellow
crystals.
Preparation of 4,10,50-triphenyl-1,4-dihydro-10H-[3,30]bi
[[1,2,4]triazolyl]-5-thione 3. The thiosemicarbazide 2 (0.01
mol) in sodium hydroxide (2N, 15 mL) was refluxed for 8 h.
The solution was cooled and neutralized using diluted hydro-
chloric acid. The formed solid was collected by filtration,
washed with water, and recrystallized from methanol to give
colorless crystals.
Preparation of N-phenyl[5-(1,5-diphenyl-1H-[1,2,4]triazol-
3-yl)-[1,3,4]thiadiazol-2-yl]-amine 4. To an ice-cold stirred so-
lution of thiosemicarbazide 2 (0.01 mol) in absolute ethanol
(10 mL), concentrated sulfuric acid (10 mL) was added over a
period of 30 min, the stirring was maintained at room tempera-
ture for an additional 5 h. Then, the reaction mixture was
poured onto ice/water mixture. The solid was filtered off and
recrystallized from dioxane to give colorless crystals.
ANTIBACTERIAL STUDIES
The newly prepared compounds were screened for
their antibacterial activity against Gram positive (Bacil-
lus subtitis and Streptococci) and Gram negative (Kleb-
siella pneumoniae and Escherichia coli) bacterial strains
by the disc diffusion method [44], and the results are
summarized in Table 3. The results obtained showed
that Schiff’s bases 7a and 7b were found to be the most
active compounds against the employed microorgan-
isms; this is probably due to their ability to increase the
penetration in the bacterial cell [45]. In addition, the
substituted thiazolidinones 5, 6, and 8 as well as the
substituted pyrazole 11 exhibited promising activity
against both Gram positive and negative bacteria. How-
ever, the pyrazole derivative 10 exhibited a low activity
against Gram positive only. No satisfactory level of in-
hibition was observed with the other compounds.
CONCLUSIONS
This study reports a successful preparation and char-
acterization of new 1,2,4-triazole derivatives. The anti-
Table 3
Screening for antibacterial activity of the new compounds (diameter
zones of inhibitions in mm).
Preparation of 1,5-diphenyl-1H-[1,2,4]triazole-3-carboxylic
acid (4-oxo-3-phenyl-thiazolidin-2-ylidene)-hydrazide 5. A
mixture of 2 (0.01 mol), ethyl bromoacetate (0.01 mole), and
anhydrous sodium acetate (0.03 mol) in ethanol (30 mL) was
refluxed for 5 h. The solid product obtained after concentration
of the reaction mixture was collected by filtration and recrys-
tallized from methanol to give pale yellow crystals.
Preparation of 1,5-diphenyl-1H-[1,2,4]triazole-3-carboxylic
acid (4-oxo-2-phenylimino-thiazolidin-3-yl)-amide 6. A solu-
tion of 2 (0.01 mol) in ethanol (30 mL) was treated with
monochloroacetic acid (0.01 mol), and the reaction mixture
was refluxed for 5 h. The solid product obtained after concen-
tration of the solution was collected by filtration and recrystal-
lized from ethanol to give yellow crystals.
Gram positive
Gram negative
Comp.
No.
Bacillus
Klebsiella
Escherichia
coli
subtitis Streptococci pneumoniae
3
4
5
6
7a
7b
8
9
10
11
–
–
–
–
–
–
–
–
5
10
12
23
18
23
–
12
11
18
20
22
–
11
10
19
15
17
–
9
20
21
19
–
11
18
–
12
10
–
–
–
10
–
12
–
12
Control
(DMSO)
Preparation of 1,5-diphenyl-1H-[1,2,4]triazole-3-carboxylic
acid benzylidene-hydrazide 7a, b. A solution of 1 (0.01 mol)
and an appropriate aldehyde (0.01 mol) in ethanol (30 mL)
–
–
–
–
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DOI 10.1002/jhet

1114
W. A. A. Arafa
Vol 47
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tered and recrystallized from dioxane to give yellow crystals.
Preparation of 1,5-diphenyl-1H-[1,2,4]triazole-3-carboxylic
acid (4-oxo-2-phenyl-thiazolidin-3-yl)-amide 8. A mixture of
7a (0.01 mol) and thioglycolic acid (0.01 mol) was refluxed in
dry benzene (75 mL) for 10 h, using a Dean-Stark water sepa-
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was neutralized with cold dilute sodium bicarbonate solution.
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from ethanol to give yellow crystals.
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[1,3,4] oxadiazole-2-thione 12. To a mixture of 1 (0.01 mol)
in ethanol (50 mL) was added a solution of potassium hydrox-
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Antibacterial assay. Antibacterial screening of prepared
compounds was done by the paper disc agar diffusion method
[44] against Gram positive (B. subtitis and Streptococci) and
Gram negative (K. pneumoniae and E. coli) strains. The com-
pounds were dissolved in DMSO at a concentration of 1 mg
mLꢀ1. Antibacterial activity of DMSO against the test micro-
organisms was investigated and was found to be nil. The nutri-
ent agar medium was sterilized by autoclaving at 120ꢁC for 15
min; the Petri dishes were sterilized in hot air oven at 160ꢁC
for an hour. Into each sterilized Petri plate, about 30 mL of
molten agar medium inoculated with the respective strain of
bacteria (6 mL inoculums to 300 mL of nutrient medium) was
transferred. The plates were left at room temperature to allow
solidification. Six-millimeter diameter holes were then punched
carefully using a sterile cork borer and completely filled with
the test solutions. The plates were incubated for 24 h at 37ꢁC.
The antibacterial activity was evaluated by measuring the di-
ameter of the inhibition zone. All the experiments were carried
out in doublet, and the average value was reported. The anti-
bacterial activity results were summarized in Table 3.
2151.
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