Synthesis and in-vitro antimicrobial activity of new 1,2,4-triazoles

Article abstract of DOI:10.1211/0022357011775307

We have described the synthesis of new 1,2,4-triazoles and have evaluated their antimicrobial profile. Antitubercular activity was determined in triplicate using the Lowenstein-Jensen medium. A loopful of Mycobacterium tuberculosis suspension was inoculated on the surface of each Lowenstein-Jensen media containing the test compounds (100, 10 or 1 μg mL^-1). To evaluate in-vitro antibacterial activity, compounds (50, 5 or 0.5 μg) were evaluated against B. subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus typhi by the disc diffusion method. To evaluate antifungal activity Sabourauds Dextrose agar medium was used. Some of the compounds (5, 0.5 or 0.05 μg mL^-1) were screened for activity against Aspergillus niger 88 and Aspergillus niger 90 and others were screened for activity against T. rubrum TR1, T. rubrum R6, T. rubrum R7 and T. mentagrophyte M1, using the cup plate method. Our results show that the triazoles with a pyrazine moiety at position 3 were more active as antitubercular and antifungal agents compared with the triazoles which had a pyrazine moiety at position 4 of the molecule.

Full text of DOI:10.1211/0022357011775307

Journal of
Pharmacy and Pharmacology
JPP 2001, 53: 267–272
# 2001 The Authors
Received September 20, 2000
Accepted October 3, 2000
ISSN 0022-3573
Synthesis and in-vitro antimicrobial activity of new
1,2,4-triazoles
A. R. Bhat, G. Varadaraj Bhat and G. Gautham Shenoy
Abstract
We have described the synthesis of new 1,2,4-triazoles and have evaluated their antimicrobial
profile. Antitubercular activity was determined in triplicate using the Lowenstein-Jensen
medium. A loopful of Mycobacterium tuberculosis suspension was inoculated on the surface of
each Lowenstein-Jensen media containing the test compounds (100, 10 or 1 µg mL 1). To
−
evaluate in-vitro antibacterial activity, compounds (50, 5 or 0.5 µg) were evaluated against B.
subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus
typhi by the disc diffusion method. To evaluate antifungal activity Sabourauds Dextrose agar
medium was used. Some of the compounds (5, 0.5 or 0.05 µg mL 1) were screened for activity
−
against Aspergillus niger 88 and Aspergillus niger 90 and others were screened for activity
against T. rubrum TR1, T. rubrum R6, T. rubrum R7 and T. mentagrophyte M1, using the cup
plate method. Our results show that the triazoles with a pyrazine moiety at position 3 were
more active as antitubercular and antifungal agents compared with the triazoles which had a
pyrazine moiety at position 4 of the molecule.
Introduction
With the introduction of streptomycin, aminosalicylic acid and isoniazid, decline in
the tuberculosis mortality rate has been observed in developed countries. However,
no such downward trend has been seen in most of the developing countries and
tuberculosis has emerged to be one of the leading causes of death of the population.
Drugs such as isoniazid, rifampicin, pyrazinamide, streptomycin and ethambutol
are the first line of drugs used in the management of tuberculosis. Second-line drugs
include ethionamide, aminosalicylic acid, cycloserine, amikacin and capreomycin.
Currently tuberculosis is treated with combinations of drugs, mainly to ensure that
the mutants resistant to a single drug do not emerge. According to a WHO report
(1998), nearly 3 million people die due to tuberculosis and approximately 8–10
million new cases are reported every year. The emergence of AIDS and multi-drug
resistant mutants have contributed to the resurgence of tuberculosis. Therefore
there is a need for new antitubercular drugs. Encouraged by results of earlier work
on triazoles (Udupi & Bhat 1996), we have synthesized 1,2,4-triazoles containing
a pyrazine moiety and investigated the in-vitro antitubercular, antibacterial and
antifungal activities of these synthesized compounds.
College of Pharmaceutical
Sciences, Manipal-576 119, India
A. R. Bhat, G. Varadaraj Bhat,
G. Gautham Shenoy
Correspondence: G. Gautham
Shenoy, College of
Pharmaceutical Sciences,
Manipal-576 119, India.
267

268
A. R. Bhat et al
S
CH3OH/H+
CS2
ArCONHNHCS–K+
ArCONHNH2
ArCOOH
NH2NH2
KOH
S
CS2
N
N
N
N
CONHNHCS–K+
CONHNH2
KOH
S
N
N
N
CONHNH2
ArCONHNHCS–K+
+
Ar
SH
N
N
N
N
HN-CO
1a–j
S
CONHNHCS–K+
N
N
N
N
N
N
+
ArCONHNH2
SH
N
HNCO-Ar
2a–p
Figure 1 Scheme for the synthesis of 1a–j and 2a–p.
(99%, 1 mL, 0.02 mol) was added and the reaction
mixture was refluxed for a further 10 h. Excess methanol
was removed under reduced pressure and the reaction
mixture was poured into 200 mL ice-cold water. The
acid hydrazide obtained was purified by crystallization
from ethanol (50%). The melting point and percentage
yield were in agreement with reported values (Udupi
1995).
Materials and Methods
Chemical procedures
Melting points were determined on a Toshniwal ap-
paratus in an open capillary and were uncorrected. IR
spectra (KBr) were recorded on a Perkin Elmer 599-B
spectrophotometer and NMR spectra (DMSO-d6) on a
Varian EM 390 spectrophotometer. Purity of the com-
pounds was checked by thin layer chromatography.
Synthesis of 3-aryl 4-N-[pyrazin-2-carboxamido]-1,2,4-
triazole-5-thiol (1a–j; Figure 1)
To a solution of aromatic acid hydrazide (0.1 mol) and
Preparation of acid hydrazides
A solution of aromatic acid (0.01 mol) in methanol potassium hydroxide (7.0 g, 0.125 mol) in 10 mL etha-
(50 mL) containing a few drops of conc. sulphuric acid nol, carbon disulphide (7.6 g, 0.1 mol) was added drop-
was heated under reflux for 15 h. Hydrazine hydrate wise and the reaction mixture was stirred for 2 h. The

269
Synthesis and antimicrobial activity of new 1,2,4-triazoles
Table 1 Physical properties of compounds 1a–j.
Compound Ar
mp (mC) Yield Elemental analysis
(%)
Calculated (%)
Found (%)
C
H
N
C
H
N
1a
1b
1c
1d
1e
1f
Phenyl
154–156 59
204–206 65
140–142 69
100–102 62
174–176 66
102–104 60
260–262 68
220–222 65
106–108 72
104–106 65
52.35 3.36 28.18
48.16 3.01 32.77
46.98 2.71 25.30
45.48 2.62 28.57
45.04 2.95 26.27
52.63 4.09 24.56
52.14 3.40 28.06
48.02 2.98 32.56
46.74 2.78 25.08
45.20 2.50 28.38
44.90 2.84 26.04
52.46 3.94 24.50
4-Pyridyl
2-Chlorophenyl
4-Nitrophenyl
4-Nitrophenoxymethyl
4-Methylphenoxymethyl
3-Methylphenoxymethyl
2-Methylphenoxymethyl
4-Chlorophenoxymethyl
2-Chloro 5-
1g
1h
1i
1j
47.80 3.45 22.31
47.62 3.20 22.02
methylphenoxymethyl
precipitated potassium β-acyl dithiocarbazinate was re- (0.1 mol) and potassium hydroxide (7.0 g, 0.125 mol) in
moved by filtration, washed with cold methanol and 10 mL ethanol, and the reaction mixture was stirred for
used for the next step directly. A mixture of potassium- 2 h. The precipitated potassium β-acyl dithiocarbazi-
β-acyl dithiocarbazinate (0.01 mol) and pyrazin-2-car- nate was removed by filtration, washed with cold meth-
boxylic acid hydrazide (0.01 mol) was heated on an oil anol and used for the next step directly. A mixture of
bath at 160–180mC for 6 h. The product was dissolved potassium β-acyl dithiocarbazinate (0.01 mol) and aro-
in 25 mL water and the solution acidified with conc. matic acid hydrazide (0.01 mol) was heated on an oil
hydrochloric acid. The precipitated triazole was filtered bath at 160–180mC for 8 h. The product was dissolved
and purified by crystallization from ethanol. The physi- in 25 mL water and the solution acidified with conc.
cal properties of compounds 1a–j are detailed in Table 1. hydrochloric acid. The precipitated triazole was filtered
and purified by crystallization from ethanol. The physi-
cal properties of compounds 2a–p are detailed in
Table 2.
3-phenyl-4-N-[pyrazin-2-carboxamido]-1,2,4-triazole-5-
thiol (1a). Yield 59%; mp 154–156mC (ethanol).
Anal: C, H, N (C13H10N6OS). H NMR-DMSO-d6 δ:
1
4-N(phenyl carboxamido)-3-(pyrazin-2-yl)-1, 2, 4-tri-
azole-5-thiol (2a). Yield 72%; mp 268–270mC (etha-
nol). Anal: C, H, N (C13H10N6OS). H NMR-DMSO-
3.0–4.5(s, 1H, NH), 7.6–9.3 (m, 8H, ArH and pyrazinyl).
1
−
Mass: 298 (molecular ion). IR (KBr, cm ) 1600 (C ꢀ
1
N), 1700 (C ꢀ O), 720 (Ar), 3140 (NH), 1390 (C ꢀ S).
d6 δ: 3.0–4.5 (s, 1H, NH), 7.6–9.2 (m, 8H, ArH and
1
pyrazinyl). Mass: 298 (molecular ion). IR (KBr, cm )
−
3-(2-chloro 5-methyl-phenoxymethyl)-4-N(pyrazin-2-
carboxamido)-1,2,4-triazole-5-thiol (1j). Yield 65%;
1600 (C ꢀ N), 1700 (C ꢀ O), 720 (Ar), 3140 (NH), 1390
(C ꢀ S).
mp 104–106mC (ethanol). Anal: C, H,
N
1
(C15H13N6O2SCl). H NMR-DMSO-d6 δ: 2.32 (s, 3H,
CH3), 4.66 (s, 2H -OCH2) 6.8–9.2 (m, 6H, ArH and
pyrazinyl), 10.35 (s, 1H, NH), 10.78 (s, 1H, SH). IR
Evaluation of in-vitro antitubercular activity
Antitubercular activity was determined in triplicate
using the Lowenstein-Jensen medium (Cruickshank et
al 1975). Stock solutions of the test compounds were
prepared in dimethylsulfoxide (DMSO) at 1000, 100
1
−
(KBr, cm ) 1660 (C ꢀ N), 1700 (C ꢀ O), 820 (Ar), 3200
(NH), 1380 (C ꢀ S).
1
−
Synthesis of 4-N(aryl carboxamido)-3-(pyrazin-2-yl)-1, and 10 µg mL . Solution of the test compound (0.8 mL)
2,4-triazole-5-thiol (2a–p; Figure 1) was added to 7.2 mL of the Lowenstein-Jensen media
Carbon disulphide (7.6 g, 0.1 mol) was added dropwise taken in sterile McCartney bottles to achieve a final
1
to a solution of pyrazin-2-carboxylic acid hydrazide concentration of 100, 10 or 1 µg mL . Controls received
−

270
A. R. Bhat et al
Table 2 Physical properties of compounds 2a–p.
Compound Ar
mp (mC) Yield Elemental analysis
(%)
Calculated (%)
Found (%)
C
H
N
C
H
N
2a
2b
2c
2d
2e
2f
Phenyl
268–270 72
108–110 68
122–124 64
274–276 70
164–166 75
130–132 77
256–358 68
144–146 74
266–268 76
124–126 80
212–214 78
190–192 75
124–126 80
248–250 82
228–230 71
52.35 3.36 28.18
52.24 3.42 28.00
4-Chlorophenyl
4-Methylphenyl
53.85 3.85 26.92
49.68 3.18 26.75
49.84 3.51 31.30
53.50 3.50 26.70
49.46 3.02 26.58
49.63 3.40 31.06
4-Hydroxyphenyl
4-Aminophenyl
2-Chlorophenyl
2g
2h
2i
4-Nitrophenyl
45.48 2.62 28.57
45.32 2.40 28.35
2-Hydroxyphenyl
4-Pyridyl
48.16 3.01 32.77
51.21 3.65 25.60
48.00 2.81 32.49
50.84 4.00 24.98
2j
Phenoxymethyl
2k
2l
4-Chlorophenoxymethyl
2-Chlorophenoxymethyl
4-Methylphenoxymethyl
3-Methylphenoxymethyl
2-Methylphenoxymethyl
46.34 3.04 23.26
46.50 3.00 23.50
2m
2n
2o
2p
52.63 4.09 24.56
50.27 3.91 23.46
52.80 4.50 25.00
50.30 4.00 23.70
4-Methoxyphenoxymethyl 120–122 74
Table 3 Antitubercular activity of compounds 1a–i and 2a–p.
0.8 mL DMSO. Streptomycin was used as the reference
standard. The bottles were inspissated at 75–80mC for
2 h for three successive days for solidification and sterili-
zation. A sweep from H37 Rv strain culture of Myco-
bacterium tuberculosis was discharged with the help of
22 S. W. Nichrome wire loop of 3-mm external diameter
into a sterile bijou bottle containing six 3-mm glass
beads and 4 mL sterile distilled water. The bottle was
shaken with the aid of a mechanical stirrer. Using a
27 S. W. G. Nichrome wire loop of 3-mm external di-
ameter, a loopful of the suspension was inoculated on
the surface of each Lowenstein-Jensen media containing
the test compounds. All the bottles were incubated at
37mC for six weeks at the end of which the readings were
taken.
1
1
1
−
−
−
Compound
100 µg mL
10 µg mL
1 µg mL
1a
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
j
j
j
j
j
j
j
j
j
j
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
j
j
j
j
j
j
j
j
j
j
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
j
j
1b
1c
1d
1e
1f
1g
1h
1i
2a
2b
2c
2d
2e
2f
2g
Evaluation of in-vitro antibacterial activity
2h
2i
Compounds 1a–j were evaluated for in-vitro anti-
bacterial activity against B. subtilis, Escherichia coli,
Pseudomonas aeruginosa, Staphylococcus aureus, S.
typhi by disc diffusion method (Stokes & Waterworth
1972). Stock solutions of the compounds were prepared
in DMSO and sterile paper discs (6-mm diameter) were
impregnated with the stock solution of the test com-
pounds to obtain a concentration of 50, 5 or 0.5 µg of
the test compounds per disc. Antibacterial screening
was performed in triplicate on Muller-Hinton agar and
the antibacterial activity profile was evaluated.
2j
2k
2l
2m
2n
2o
2p
Streptomycin
Control
Growth present, j; growth absent, k.

271
Synthesis and antimicrobial activity of new 1,2,4-triazoles
Table 4 Antifungal activity profile of compounds 1a–i and 2a–p.
Compound A. niger 88
1
A. niger 90
1
T. rubrum TR1
1
T. rubrum T6
1
T. rubrum T7
1
T. mentagrophyte
1
−
−
−
−
−
−
(µg mL
)
(µg mL
)
(µg mL
)
(µg mL
)
(µg mL
)
(µg mL
)
5.0 0.5 0. 05
5.0 0.5 0.05
5.0 0.5 0.05
5.0 0.5 0.05
5.0 0.5 0.05
5.0 0.5 0.05
1a
1b
1c
1d
1e
1f
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
k
j
j
k
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
k
j
j
k
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
k
j
j
k
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
k
j
j
k
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
k
j
j
k
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
j
k
j
j
k
j
1g
1h
1i
2a
2b
2c
2d
2e
2f
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
2g
2h
2i
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
k
2j
2k
2l
2m
2n
2o
2p
k
j
k
k
k
k
k
k
k
k
k
k
k
Growth present, j; growth absent, k.
Evaluation of the in-vitro antifungal activity
having a pyrazine moiety at position 4. The nature of
the functional group on the benzene ring did not affect
the antitubercular activity of the compounds.
Sabourauds Dextrose agar medium was used for the
antifungal screening. Compounds 1a–i and 2a–i were
screened for activity against Aspergillus niger 88 and
Aspergillus niger 90 by the cup plate method (Clayton
Compounds 1a–j did not exhibit antibacterial activity
against B. subtilis, E. coli, P. aeruginosa, S. aureus or S.
typhi even at 50 µg\disc. Compounds 2l and 2o were
found to be active against Aspergillus niger 88 and
1
−
1978) at 5, 0.5 or 0.05 µg mL . Compounds 2a–c, 2i–l
and 2o were screened for activity against T. rubrum
TR1, T. rubrum R6, T. rubrum R7 and T. mentagrophyte
M1 by the cup plate method in triplicate at 5, 0.5 or
1
−
Aspergillus niger 90 at 0.05 µg mL (Table 4). Com-
pounds 2a, 2b, 2i–l and 2o were found to be active
against T. rubrum TR1, T. rubrum R6, T. rubrum R7,
1
−
0.05 µg mL .
1
and T. mentagrophyte M1 at 0.05 µg mL .
−
Results and Discussion
References
Compounds 1a–i and 2a–p were evaluated for in-vitro
antitubercular activity against Mycobacterium tubercu-
losis H37 Rv strain. A good correlation between the
structure and the antitubercular activity was observed
(Table 3). Compounds having a pyrazine moiety in the
3 position were foundto bemore active thanthe triazoles
Clayton, Y. M. (1978) Yeasts and other fungi. In: Phillips, I.,
Reeves, D. S., Williams, J. D., Wise, R. (eds) Laboratory
Methods in Antimicrobial Chemotherapy. pp 122–123
Cruickshank, R., Duguid, J. P., Marmion, B. P., Swain, R. H.
A. (1975) Mycobacteria. In: Medical Microbiology. 12th
edn, vol. 2, Churchill Livingstone, New York, pp 204–205

272
A. R. Bhat et al
Stokes, E. J., Waterworth, P. M. (1972) Antibiotic sensitivity Udupi, R. H., Bhat, A. R. (1996) Synthesis of 4-(N-pyridyl
tests by diffusion methods. In: Association of Clinical
Pathologists Broadsheet p. 55
carboxamido)-5-mercapto-3-substituted 1, 2, 4-triazoles for
possible antitubercular activity. Indian J. Heterocyclic
Chem. 1: 41–44
Udupi, R. H. (1995) Studies on the Synthesis of Substituted
Azetidinones, Quinazolinones and Related Compounds for World Health Organisation (1998) The Double Burden:
Possible Antitubercular Activity and Other Pharmacologi-
cal Profiles. Ph.D. Thesis, Mangalore University, India, pp
233–234
Emerging Epidemics and Persistent Problems. The World
Health Report, World Health Organisation, Geneva, pp
21–23