182
M.K. Bharty et al. / Journal of Molecular Structure 1130 (2017) 181e193
1
,2,4-triazole and their substituted derivatives are of current in-
the surface of the MHA plates seeded with test bacteria and the
plates were incubated in a B. O. D. incubator (Caltan-152, Narang
Scientific Works, New Delhi, India) for 24 h at 37 ± 2 C. The in-
hibition zones around each disc were measured after 24 h of in-
cubation. Commercial antibacterial drugs streptomycin sulphate
and neomycin sulphate (Himedia) were used in same concentration
terest as bridging ligands between transition metal ions for the
preparation of coordination polymers, and a wide variety of poly-
nuclear complexes. Recently, substituted 1,2,4-triazoles have
attracted more attention than the unsubstituted one, because of the
presence of more complex coordination modes for the formation of
new coordination complexes [15]. Some trinuclear complexes of Ni
ꢀ
of 5e50 mg/disc to compare effectiveness of the test compounds.
(
II), Cu(II) and Fe(II) with similar triazoles have been reported
which contain anionic coligands such as thiocynate/perchlorate
16e19] but no such trinuclear complexes of triazole are known
2.3.1. Determination of inhibitory concentration (IC50
Inhibitory concentration 50 of compound 2, 3, Ni(OAc)
and Ni(Cl) $6H O were determined using broth dilution method
with MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay. The test compounds were dissolved in DMSO to a
final concentration of 8 mg/ml. The test compounds were diluted in
)
[
2 2
$4H O,
which contain thenoyl trifluroacetone as the secondary ligand. A
survey of literature shows that a few papers are available on the
syntheses and spectral characterization of metal complexes of
2
2
1,2,4-triazoles, but scanty of information is available on their mixed
ligand or ternary complexes. Therefore, it will be of great interest to
investigate the mixed ligand complexes of 1,2,4-triazole ligand and
to compare the mode of bonding and structure of the complexes. In
view of this, we have prepared and characterized the polymeric
Cd(II) complex and trinuclear and mononuclear Ni(II) complexes of
various concentrations (2, 4, 8,16, 32, 64, 128 mg/mL) in 5 ml culture
tubes containing 4 ml Mueller-Hinton broth. One drop of expo-
nentially growing culture of test bacterium was inoculated in each
ꢀ
concentration of compounds and incubated at 37 ± 2 C for 24 h. A
control was prepared using test bacterium and equal volume of the
5
-methyl-4-phenyl-1,2,4-triazole-3-thione containing ethylenedi-
DMSO (64
ml) in Mueller-Hinton broth without test compounds.
amine/thenoyltrifluoroacetone as a secondary ligand.
After incubation period, 400
mL MTT solution (5 mg/ml) prepared in
phosphate saline buffer (PBS) was added in each vial and incubated
ꢀ
2
. Experimental section
at 37 ± 2 C for one hour. The absorbance was observed at 600 nm.
Minimum concentration of compounds which were able to inhibit
2.1. Chemicals and starting materials
growth of half of the test bacterium is considered as IC50.
Commercial reagents were used without further purification
2.4. Synthesis
and all experiments were carried out in open atmosphere. Ethyl
acetate, (SD Fine Chemicals), phenyl isothiocyanate (Sigma Aldrich)
and NaOH (Qualigens) were used as received. All the synthetic
manipulations were carried out in open atmosphere and at room
temperature. The solvents were dried and distilled before use,
following the standard procedure.
2.4.1. Synthesis of 5-methyl-4-phenyl-1,2,4-triazole-3-thione
Acetic acid hydrazide was prepared by the literature method as
described earlier [22]. 1-Acetyl-4-phenyl-3-thiosemicarbazide
(Hpmt) was prepared by refluxing equimolar amounts of acetic
acid hydrazide (0.741 g, 10 mmol) and phenylisothiocyanate (1.35 g,
ꢀ
1
0 mmol) in ethanol at 70 C for 3h. The white precipitate obtained
2.2. Physical measurements
on cooling the reaction mixture was filtered off, washed with
ethanol followed by water and air dried. Yield: 70%.
Carbon, hydrogen, nitrogen and sulfur contents were estimated
5-Methyl-4-phenyl-1,2,4-triazole-3-thione (Hmptt) was syn-
thesized by dissolving the freshly prepared 1-acetyl-4-phenyl-3-
thiosemicarbazide (1.06 g, 5 mmol) in an aqueous NaOH (10%,
10 mL) and refluxing the solution for 3 h. The solution was cooled in
ice and acidified with conc. HCl. The white precipitate so obtained
was filtered, washed with water, air dried and recrystallized from
on a CHN Model CE-440 Analyzer and on an Elementar Vario EL III
1
13
Carlo Erba 1108. H and C NMR spectra were recorded in DMSO-d
6
on a JEOL AL300 FT-NMR spectrometer using TMS as internal
reference. Electronic spectra were recorded on a SHIMADZU 1700
UVeVis spectro-photometer. The fluorescent data were collected at
room temperature with a Varian CARYECLIPSE spectrophotometer
in DMSO solution. Thermogravimetric curves of complexes were
recorded using a Perkin Elmer-STA 6000 thermal analyzer, TA In-
ꢀ
ethanol. Yield: 58%; m.p.220 C. Anal. Found: C ¼ 56.70, H ¼ 4.85,
N ¼ 21.75, S ¼ 16.55 (%) Anal. Calcd for C
9
H
9
N
3
S (191.00) C ¼ 56.54,
1
H ¼ 4.71, N ¼ 21.98, S ¼ 16.75%. IR (
n
cm- , KBr):
n
(NH) 3053,
(C]S) 1001. H NMR (CDCl ppm): 12.28
). C NMR (CDCl
ppm): 168.16 (C]S), 149.55 (C]N), 127.71e133.37 (phenyl ring
carbons), 12.03 (CH carbon).
n(C]
ꢀ
ꢁ1
1
struments with heating rate of 15 min and a platinum sample
holder.
N) 1580;
n
(NeN) 1017s;
n
3
; d
13
(NH), 7.26e7.57 (m, 5H, phenyl ring), 2.21 (s, CH
d
3
3
;
2
.3. Antibacterial tests
3
Five human bacterial pathogens Salmonella typhi (MTCC 3216),
Shigella flexneri (ATCC 12022), Staphylococcus aureus (ATCC 25323),
Aeromonas hydrophila (ATCC 7966) and Enterococcus faecalis (ATCC
2.4.2. Synthesis of complex 1
Complex 1 was synthesized by the reaction of 5-methyl-4-
phenyl-1,2,4-triazole-3-thione (0.191 g, 1 mmol) in MeOHeCH CN
(10 mL) and methanolic solution of Cd(OAc) $2H O (0.130 g,
3
2
5923) were used to test the antibacterial activity of complexes 2
2
2
and 3. The antibacterial assay was done according to Kirby-Bauer
Disk Diffusion the method with some slight modifications [20,21].
The test compounds were dissolved in DMSO to a final concen-
tration of 5 mg/mL. Sterilized Whatman no. 1 filter paper discs
0.5 mmol). The reaction mixture was stirred for 2 h at room tem-
perature. The resulting brown precipitate was filtered off and
washed thoroughly with methanol. The precipitate was dissolved
in chloroform-methanol solution (20 mL) of thenoyltrifluroacetone
(2.0 mL), yielding a light yellow solution was filtered off and kept
for crystallization. The single crystals of complex 1 suitable for X-
ray analyses were obtained by slow evaporation of the above so-
(
5 mm) were impregnated with different volume (1, 2, 4, 6, 8 and
L) of compounds to get a final concentration of 5, 10, 20, 30, 40
and 50 g per disc. Sterilized paper disc loaded with the 10 L of
10 m
m
m
ꢀ
DMSO was taken as a control. The bacterial test pathogens were
spread on fresh Mueller Hinton Agar (MHA) plates with the help of
cotton swabs to form an even lawn of the test bacteria. The filter
paper disc impregnated with the test compound were placed on
lution over a period of 10 days. Yield: 50%; m.p.325 C. Anal. Found:
C ¼ 43.60, H ¼ 3.35, N ¼ 17.20, S ¼ 12.70%. Anal. Calcd for
C
18
H
16CdN
6
S
2
(492.89), C ¼ 43.82, H ¼ 3.24, N ¼ 17.04, S ¼ 12.98%.
1
IR (n cm- , KBr): n(C]N) 1595; n(CeN) 1541; n(NeN) 1038s; n(C]S)