Vol. 23, No. 5, 2012
Hanif et al.
855
Introduction
inhibition studies, antioxidant and antibacterial activities of
5-substituted-4-amino-1,2,4-triazole and 3,6-disubstituted-
1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazoles.
Urease (urea amidohydrolase, E.C. 3.5.1.5) is
an enzyme that catalyzes the hydrolysis of urea to
ammonia and carbamate, which is the final step of nitrogen
metabolism in living organisms.1,2 Carbamate rapidly and
spontaneously decomposes, yielding a second molecule
of ammonia. These reactions may cause significant
increase in pH and are responsible for negative effects of
urease activity in human health and agriculture. Urease is
responsible for urinary tract and gastrointestinal infections,3
possibly causing severe diseases such as peptic ulcers and
stomach cancer as in the case of Helicobacter pylori.4
Ureases are also involved in the development of urolithiasis,
pyelonephritis, hepatic encephalopathy, hepatic coma and
urinary catheter encrustation.5
The efficiency of soil nitrogen fertilization with
urea (the most used fertilizer worldwide) decreases due
to ammonia volatilization and root damage caused by
increased soil pH.6 Control of the activity of urease through
the use of inhibitors could counteract these negative effects.
Reactive oxygen species (ROS), which are capable
of causing damage to DNA, have been associated with
carcinogenesis, coronary heart diseases and many other
health problems related to advancing age.7,8 In low
concentrations, synthetic antioxidants are also in use
for many industrial processes, e.g. inhibition of radical
formation for preventing premature polymerization during
processing, storage and transportation of unsaturated
monomers. They exert their effects by scavenging or
preventing the generation of ROS,9 protecting the formation
of free radicals and retarding the progress of many
chronic diseases,10 including cancer, neurodegenerative,
inflammation and cardiovascular diseases.11
Recently, 5-substituted-4-amino-1,2,4-triazole-
3-thiones and their fused triazolothiadiazole derivatives
have received considerable attention owing to their
synthetic and potent pharmacological importance.
Heterocyclic compounds bearing 1,2,4-triazole nuclei and
their triazolothiadiazole derivatives have been reported to
show a broad spectrum of pharmacological properties such
as antimicrobial,12 anti-inflammatory,13 anticonvulsant,14
anticancer,15 antitubercular16 and antitumor activities.17
Literature survey reveals that 5-substituted-4-amino-
1,2,4-triazoles and their 3,6-disubstituted-1,2,4-triazolo-
[3,4-b]-1,3,4-thiadiazole derivatives have not been paid
much attention for their enzymatic inhibition studies.
Hence, the present studies were undertaken to explore
the urease inhibition potential of 5-substituted-4-amino-
1,2,4-triazoles and their fused triazolothiadiazole
derivatives. In this work, we report the synthesis, urease
Experimental
All the common solvents and chemicals were of
analytical grade or purified by distillation. The purity
of the synthesized compounds was ascertained by thin
layer chromatography (TLC) and the Rf values were
determined by employing pre-coated silica gel aluminum
plates (Kieselgel 60 F254 from Merck (Germany)), using
petroleum ether:EtOAc (8:2) as an eluent. The spots
were visualized under an UV lamp. Melting points (mp)
were determined on a Stuart melting point apparatus
(SMP3) and are uncorrected. The infrared (IR) spectra were
recorded on a Bruker OpticsAlpha FTIR spectrophotometer.
Nuclear magnetic spectra (NMR) spectra were recorded
on a Bruker Avance 300 MHz spectrometer with TMS
(tetramethylsilane) as internal standard. The multiplicities
were expressed as s = singlet, d = doublet, t = triplet,
q = quartet, dt = doublet of triplets. Mass spectra (MS)
were recorded on Agilent Technologies 6890N gas
chromatograph and an inert mass selective detector 5973
mass spectrometer. The elemental analysis was performed
on Leco CHNS-932 ElementalAnalyzer, Leco Corporation
(USA).
General procedure for synthesis of substituted aromatic
esters (2a-e) and aromatic acid hydrazides (3a-e)
Substituted aromatic acids (1a-e) were esterified (2a-e)
by refluxing in methanol and in the presence of catalytic
amount of sulfuric acid. Substituted aromatic esters (2a-e)
were converted into their corresponding acid hydrazides
3 by refluxing in hydrazine hydrate in methanol through
literature procedure.18-20
General procedure for synthesis of 3-substituted 4-amino-
5-aryl-3H-1,2,4-triazole-3-thiones (5a-e)
Aryl hydrazide 3 (0.125 mol) was added to a solution
of potassium hydroxide (0.125 mol) in dry methanol
(50 mL) and cooled in ice. Carbon disulfide (0.125 mol)
was then added in small portions with constant stirring.
The solid product of potassium dithiocarbazinate 4 formed
was filtered, washed with chilled diethyl ether and dried.
It was directly used for next step without purification. The
above potassium dithiocarbazinate 4 was taken in water
(20 mL), and hydrazine hydrate (0.250 mol) was added
followed by reflux for 4-5 h. The reaction mixture turned