Synthesis, antibacterial, antielastase, antiurease and antioxidant activities of new methoxy substitued bis-1,2,4-triazole derivatives

Article abstract of DOI:10.3109/14756366.2011.631536

The methoxy substitued two novel bis triazole-schiff bases (6 a-b) were synthesized with 4-amino-3,5-diethyl-4H-1,2,4-triazole and various bis-aldehydes. Their amine derivatives prepared by reduced with NaBH4 (5 a-b). The obtained products 6 a-b and 7 a-b were identified by FT-IR, H-NMR, ¹³C-NMR. The bis triazole-schiff bases and amine derivatives were tested for antimicrobial activity using the agar diffusion technique against 11 bacteria. The synthesized compounds (6 a-b and 7 a-b) were screened for their antielastase, antiurease and antioxidant activities. The resuts showed that the synthesized compounds (6 a-b and 7 a-b) had effective antielastase and antiurease activities.

Full text of DOI:10.3109/14756366.2011.631536

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(1): 72–77
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.631536
RESEARCH ARTICLE
Synthesis, antibacterial, antielastase, antiurease and
antioxidant activities of new methoxy substitued
bis-1,2,4-triazole derivatives
Bahar Bilgin Sokmen1, Nurhan Gumrukcuoglu2, Serpil Ugras3, Halil Ibrahim Ugras1, and
Refiye Yanardag4
1Department of Chemistry, Faculty of Arts and Sciences, Giresun University, Giresun, Turkey, 2Vocational School of Health
Sciences, Karadeniz Technical University, Trabzon, Turkey, 3Department of Biology, Faculty of Arts and Sciences, Giresun
University, Giresun, Turkey, and 4Department of Chemistry Faculty of Engineering, Istanbul University, Istanbul, Turkey
Abstract
The methoxy substitued two novel bis triazole-schiff bases (6 a–b) were synthesized with 4-amino-3,5-diethyl-
4H-1,2,4-triazole and various bis-aldehydes. Their amine derivatives prepared by reduced with NaBH4 (5 a–b). The
1
obtained products 6 a–b and 7 a–b were identified by FT-IR, H-NMR, 13C-NMR. The bis triazole-schiff bases and
amine derivatives were tested for antimicrobial activity using the agar diffusion technique against 11 bacteria. The
synthesized compounds (6 a–b and 7 a–b) were screened for their antielastase, antiurease and antioxidant activities.
The resuts showed that the synthesized compounds (6 a–b and 7 a–b) had effective antielastase and antiurease
activities.
Keywords: Bis triazole-schiff bases, synthesis, enzyme inhibition activity
Introduction
cells5. Neutrophil activation by soluble or particulate
stimuli leads to generation of reactive oxygen species
(ROS) through the oxidative metabolism, which involves
activation of the NADPH oxidase enzymatic complex and
increase in cellular oxygen consumption6. In addition,
these cells release proteolytic enzymes, such as elastase
and cathepsins, that act as microbicidal agents, degrade
the extracellular matrix and contribute to cellular migra-
tion at the inflammatory site7. is potent serine protein-
ase is capable of digesting a panoply of matrix proteins and
is involved in numerous inflammatory respiratory diseases
including emphysema, cystic fibrosis, chronic obstructive
pulmonary disease, pulmonary fibrosis and asthma8.
A variety of ureases (E.C.3.5.1.5) are found in bacteria,
fungi, higher plants and in soil as soil enzymes. Medically,
bacterial ureases are important virulence factors impli-
cated in the pathogenesis of many clinical conditions
such as pyelonephritis, hepatic coma, peptic ulcerationn
and the formation of injection-induced urinary stones9
Triazole derivatives have been reported to have pharma-
cological, insecticidal, fungicidal, and herbicidal activi-
ties1. In addition, it was reported that compounds having
triazole moieties, such as Vorozole (1), Letrozole (2) and
Anastrozole (3), (in Scheme 1) have been used as non-
steroidal aromatase inhibitors in medicine for treating
breast cancer2. 1,2,4-triazoles are also important in diverse
field’s chemistry has been owing to their biological activi-
ties3. Schiff bases comprise a group of both cyclic and acy-
clic chemical compounds containing -C=N- moieties. ey
are made from the condensation of an amine and a mol-
ecule bearing an active carbonyl function. e presence of
potentially donating atoms in their structure makes these
molecules an important class of ionophore, which are
widely used in metal ion complexation studies4.
Neutrophils, which are the most abundant leukocytes
in the circulation, constitute the first line of defense
against microorganisms, virally infected cells and tumor
Address for Correspondence: Prof. Bahar Bilgin Sokmen, Department of Chemistry, Faculty of Arts and Sciences, Giresun University,
Giresun, Turkey. Tel: +904542161255. Fax: +904542164518. E-mail:baharsokmen@yahoo.com
(Received 05 August 2011; revised 10 October 2011; accepted 10 October 2011)
72

Synthesis of methoxy substitued bis-1,2,4-triazole derivatives 73
Scheme 1
and stomach cancer10. In agriculture, high urease activity
causes significant environmental and economic prob-
lems by releasing abnormally large amounts of ammo-
nia into the atmosphere during urea fertilization. is
further induces damage to germinating seeds, seedling
and young plants primarily by depriving them of their
nutrition by the essential nutritient and secondarily by
ammonia toxicity, increasing the pH of the soil. In the
near past, a number of compounds have been proposed
as urease inhibitors to reduce environmental problems
and enhance the uptake of urea nitrogen by plants.
Antioxidants are extensively studied for their capac-
ity to protect organisms and cells from damage that is
induced by oxidative stress. Nowadays antioxidants
arouse researchers’ interest in both medical plants and
synthetic compounds. Synthetic antioxidants, such as
butylated hydroxyanisole (BHA), butylated hydroxytolu-
ene (BHT), and tert-butylhydroquinone (TBHQ), have
been widely used in the food industry to prevent oxida-
tive deterioration, but BHA and BHT are suspected of
being responsible for liver damage and carcinogenesis11.
Scientists in various disciplines have become more inter-
ested in new compounds, either synthesized or obtained
from natural sources that could provide active compo-
nents to prevent or reduce the impact of oxidative stres
on cells. Exogenous chemicals and endogenous meta-
bolic processes in human body or in food system might
produce highly reactive free radicals, especially oxygen
derived radicals, which are capable of oxidizing biomol-
ecules, resulting in cell death and tissue damage. ROS
and free radicals are known to induce lipid peroxidation,
the damage of lipids, proteins and nucleic acids in cells.
In addition, there is much evidence that these molecules
may be related to ageing and diseases, such as cancer,
atherosclerosis, rheumatoid arthritis and emphysema12.
In this study, we have synthesized some new methoxy
substitued bis-1,2,4-triazole derivatives. eir antioxi-
dant activity were assessed by various in vitro assays and
compared to the activities of synthetic standard antioxi-
dant compound. Moreover, the newly synthesized com-
pounds were screened as urease and elastase inhibitors
and antimicrobial activities.
(Barnstead-Electrothermal 9200, Iowa USA ); uncor-
rected. IR Spectra: solns. in KBr pellets. with a Perkin-
Elmer 100 FT-IR spectrometer (Cambridge, England).
1H- and 13C-NMR spectra: 200 (50) MHz Varian spec-
trometer (Danbury, CT); δ in ppm; Me4Si as the internal
standard. Mass spectra: Agilent 6230 TOF (ESI-MS) (CA,
USA). Antioxidant activities of samples were determined
in a spectrophotometer (UV-1240, Shimadzu, Japan).
Synthesis of bis-aldehydes 1a–b and amino
compound 3
Bis-aldehydes (4a–b)13–15 and 4-Amino-3,5-diethyl-4-
H-1,2,4-triazole (5)15 were prepared by using literature
procedures.
Synthesis of bis-schiff bases 6a–b
e corresponding bis-aldehyde (0.01 mol) was added
to a solution of compound 5 (0.005mol) in glacial acetic
acid (20 mL) and the mixture was refluxed for 16 h. After
cooling, the mixture was poured into a beaker containing
ice–water (100 mL). e precipitate formed was filtered.
After drying in vacuo, the product was recrystallized
from 1:2 benzene:petroleum ether to give the desired
compound.
Synthesis of reduced compounds 7a–b
e corresponding compound 6a–b (0.005 mol) was dis-
solved in dried methanol (50 mL) and NaBH4 (0.01 mol)
was added in small portions to this solution. e mixture
was refluxed for 20 min and then allowed to cool. After
evaporation at 30–35°C under reduced pressure, the
solid residue was washed with cold water. After drying
in vacuo, the solid product was recrystallized from an
appropriate solvent (1:1 ethanol:water, unless otherwise
noted) to afford the desired compound.
N,N′-(2,2′-(2,2′-oxybis(ethane-2,1-diyl)bis(oxy))bis(4-
methoxy-2,1-phenylene))bis (methylene) bis(3,5-diethyl-
4H-1,2,4-triazol-4-amine) (7a). Yield (1.80g, 71.43%); m.p.
116–117°C; IR: 3374 (NH), 1594 (C=N), 1261 (C-O), 640–
1
738cm−1 (aromatic ring); H-NMR (DMSO-d6) δ (ppm):
1.08 (t, 12H, CH3), 1.78–1.92 (m, 4H, OCH2), 2,42 (g, 8H,
CH2), 3.63 (d, 4H, NH-CH2), 3.67 (s, 6H, OCH3), 3.94–4.06
(m, 4H, OCH2), 6.19 (t, 2H, NH), Ar-H: [6.64 (d, 2H), 7.00–
7.10 (m, 4H)]; 13C-NMR (DMSO-d6) δ (ppm): 155.00 (4C,
triazole C3, C5), Ar-C: [148.18 (2C), 130.45 (2C), 129.25
(2CH), 128.65 (4CH), 121.52 (2C), 115.08 (4C)], 68.20 (2C,
OCH2), 58.01 (2C, NH-CH2), 55.34 (2C, OCH3), 24.12 (4C,
CH2), 18.11 (2C, OCH2), 11.13 (4C, CH3). ESI-MS(TOF)
(M+H)+:623.3116, Anal. Calc. For (C32H46N8O5):622.7904.
Materials and methods
General
All chemicals and solvents are commercially avail-
able and were used after distillation or treatment
with drying agents. Mp: cap. Melting-point apparatus
© 2013 Informa UK, Ltd.

74 B. B. Sokmen et al.
Antibacterial activity was measured using the standard
methodofdiffusiondiscplatesonagar16.Elastaseactivitywas
examined by using N-succinyl-Ala-Ala-Ala-p-nitroanilide
(STANA) as a substrate and by the measuring the release
of p-nitroaniline at 410nm17. Urease inbitory activity was
determined according to Van Slyke and Archilbald18. e
DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging
activity of the triazole derivatives was measured according
to the procedure described by Brand-Williams et al19. e
ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid)diammonium salt) radical scavenging activity of the
triazole derivatives was measured according to the pro-
cedure described by Arnao et al20. e reducing power of
the of the triazole derivatives was measured according to
the method of Oyaizu21. For the reducing ability of triazole
derivatives, the cupric ions reducing power capacity was
also used22 with slight modifications.
of propionic acid with hydrazine using the published meth-
ods shown in Scheme 315. Finally reactions of compounds
1 and 3 afforded the desired compounds 6 (Scheme 3). In
general, reduction of imine type compounds is possible,
but attempts to reduce imines such as 6 may also lead to a
reduction of the heterocyclic ring. For this reason, the selec-
tive reduction of the imino group present in compounds 6
without affecting the heterocyclic ring was another aim of
the study. us, a general and convenient method using
NaBH4 as a selective reducing agent was employed for the
synthesis in good yields of the corresponding bis amino
triazole compounds 7 (Scheme 3).
In the IR spectra of compounds 6, the characteris-
tic C=N absorption bands appeared at around 1605–
1607 cm−1. e 1H-NMR signals for the -N=CH group were
observed between δ 8.73–8.76 ppm. e 13C-NMR signals
for the−N=CH- group were recorded at δ 163–165 ppm.
Reduced compounds 7 showed IR absorption bands
1
around 3228–3230 cm−1 (υNH). e H-NMR signals for
Statistical analysis
the -NH-CH2- group of these compounds were observed
as a doublet at around δ 3.58–3.63 ppm and the proton
signals of−NH- groups were recorded as a triplet or strong
singlet between at δ 6.20 ppm. In the 13C-NMR spectra,
the triazole C3 and C5 of the bis-schiff base derivatives 6
were observed between δ 150–152 ppm and the triazole
C3 and C5 signals of the reduced compounds 7 were
observed between δ 155 ppm.
Results were expressed as mean standart deviation
(SD) of triplicate analyses. Statistical comparisons were
performed with Student’s t-test. Differences were con-
sidered significant at p < 0.05.
Results and discussion
e syntheses of bis triazole-schiff bases 6 were accom-
plished according to the reactions shown in Schemes 2
and 3. First, bis-aldehydes 4 were synthesized using a pub-
lished method13,14, as indicated in Scheme 2. 3,5-di-ethyl-4-
amino-4H-1,2,4-triazole 5 was obtained from the reaction
Antibacterial activity was measured using the standard
method of diffusion disc plates on agar, but unfortunately,
a few ligands compounds do show biological activ-
ity against the studied microorganisms. 6a compound
has a moderate activity against Enterococcus faecalis,
Scheme 2
Scheme 3
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of methoxy substitued bis-1,2,4-triazole derivatives 75
Table 1. Antimicrobial screening data for the synthesized compounds (6a–b,7a–b).
Zone of inhibition*
Microorganisms
6a
—
12
—
—
12
—
—
12
—
—
—
6b
—
—
—
—
—
—
—
—
—
—
—
7a
—
—
—
—
—
—
—
—
—
—
—
7b
—
12
—
—
—
—
—
—
—
—
—
C**
3
1
2
3
4
5
6
7
8
9
Enterobacter cloaceae (ATCC 13047)
Enterococcus faecalis (ATCC 29212)
Salmonella typhiminium (ATCC 14028)
Escherichia coli (ATCC 25922)
—
8
3
Staphylococcus epidermitidis (ATCC 13047)
Proteus vulgaris (ATCC 13315)
8
29
10
23
—
8
Yersinia pseudotuberculosis (ATCC 911)
Staphylococcus aureus (ATCC 25923)
Pseudomonas aeroginosa (ATCC 27853)
10 Klebsiella pneumonia (ATCC 13883)
11 Bacillus subtilis (ATCC 16633)
*Millimeter values.
—
**Positive control, sefpotec 200mg was used. DMSO has no values for negative control.
Staphylococcus epidermitidis and Staphylococcus aureus.
Antibacterial activity results were given in Table 1.
e inhibition effect of elastase activity is shown in
Table 2. In this study, elastase inhibitor activity of triazole
derivatives was found to increase dose dependently. e
inhibition was increased with increasing triazole concen-
tration. Triazole derivatives exhibited good elatase inhib-
itor activity. A high elastase inhibition (62.69 0.73%) was
seen in 1 µg/mL at 6a. Lower IC50 values indicate higher
enzyme inhibitor activity. A low elastase inhibition
(56.07 0.15 %) was seen in 1 µg/mL at 7a. Compound
6a proved to be the most potent showing an enzyme
inhibitory activity with an IC50 = 0.00095 µg/mL (Table 2).
Previous studies have shown that the attachment of vari-
ous leaving groups (halogen, carboxylate, heterocyclic
sulfide, sulfone, methoxy) to the thiazole and triazole
compound yields highly potent inhibitors of human leu-
kocyte elastase23.
e inhibition effect of urease activity is shown in
Table 2. We found that all concentrations exerted inhibi-
tor effects on urease activity in a dose dependent man-
ner. e inhibition was increased with increasing triazole
concentration. All the traizole derivatives exhibited good
urease inhibition activity. e compound 6a proved to be
most potent showing an enzyme inhibition activity with
an IC50 = 0.99 0.077 μg/mL. e least active compound
7a had an IC50 = 3.05 2.17 μg/mL. e activity of the rest
of the compounds falls in the range 1.15–1.41 μg/mL
(Table 2). Since all the synthesized triazoles exhibited
promising urease inhibitory activity, this may be due to
their basic skeleton.
Table 2. e elastase and urease inhibitory activity of triazole
derivatives.
Triazole
derivatives
concentration
(µg/mL)
Elastase
IC50 (µg/mL)*
0.00095 2.12×10−5
Urease IC50
(µg/mL)*
Compounds
6a
0.001
0.01
0.1
0.99 0.077
1.15 0.028
3.05 2.17
1.41 0.035
1
6b
0.001
0.01
0.1
0.45 0.042
1
7a
0.001
0.01
0.1
0.00099 7.07×10−6
0.41 0.021
1
7b
0.001
0.01
0.1
1
*Mean SD.
ability of various samples. DPPH decreases significantly
upon exposure to proton radical scavenger24. e DPPH
free radical scavenging activities of triazole derivatives
and BHT are presented in Table 3. For each compound,
different concentrations (500–2000 µg/mL) were
prepared. e DPPH scavenging activities of triazole
derivatives were between 13.65–28.63 at 500 µg/mL and
between 26.17–53.66 % at 2000 µg/mL. In this study, BHT
showed a high radical scavenging ability. At 500–2000
µg/mL, the radical scavenging of BHT were between
79.18–92.34%. IC50 value is the effective concentration
to inhibit 50% of DPPH radicals. A lower IC50 value is
resulted in a stronger DPPH radical scavenging activity,
with regard to IC50 values. BHT (315.75 3.66 µg/mL) and
6b (1782.51 56.09 µg/mL) had the highest radical scav-
enging abilities, whereas 6a (5809.65 1639.21 µg/mL)
had a lowest radical scavenging ability (Table 3). IC50
In the present study, antioxidant and radical scaveng-
ing effects of the synthesized triazol compounds (6a–b,
7a–b) were determined in vitro with different bioanalyti-
cal methodologies. e antioxidant and radical scaveng-
ing activities of the compounds were compared with BHT.
ese comparisons were performed using in vitro tests
including DPPH, ABTS and reducing power (Fe+3→Fe+2
biotransformation and cuprac assay).
DPPH is a free radical compound that has been
widely used to determine the free radical scavenging
© 2013 Informa UK, Ltd.

76 B. B. Sokmen et al.
Table 3. DPPH and ABTS radical scavenging activity of triazole derivatives.
Triazole derivatives
Triazole derivatives
Compounds
concentration (µg/mL)
DPPH IC50 (µg/mL)*
concentration (µg/mL)
ABTS IC50 (µg/mL)*
6a
500
5809.65 1639.21
250
500
8734.22 1115.86
1000
750
1500
2000
500
1000
6b
7a
1782.51 56.09
1977.04 90.76
250
500
4771.55 283.03
5261.73 271.84
1000
1500
2000
500
750
1000
250
500
1000
1500
2000
500
750
1000
7b
2705.19 109.05
315.75 3.66
250
500
3274.88 180.54
288.14 20
1000
1500
750
1000
2000
500
BHT
250
500
1000
750
1500
2000
1000
*Mean SD.
values, scavenging abilities on DDPH radicals, were
significantly different (p < 0.05) from the IC50 values
obtained for BHT. e presence of electrone donating
methoxy substituent in the phenolic compounds is
known to increase the stability of the free radical and
hence the antioxidant activity25. us, the compound
6b bearing a methoxy group (electron donating group)
showed high DPPH activity.
Table 3 showed the ABTS radical scavenging activity of
triazole derivatives compared with BHT. All tested com-
pounds showed some degree of ABTS radical scavenging
activity. e scavenging effect of triazole compounds and
BHT on ABTS decreased in the order: BHT > 7b > 6b > 7a
> 6a at the concentration in 1000 µg/mL.
e reducing power of prepared triazole derivatives,
which may serve as a significant reflection of the antioxi-
dant activity, was determined using the iron (III) to iron
(II) reduction assay. In this assay, the yellow colour of
the test solution changes to various shades of green and
blue, depending on the reducing power of compounds.
e presence of reductants in the solution causes the
reduction of the Fe+3/ferricyanide complex to the fer-
rous form. erefore, the Fe+2 ion can be monitored by
measurement of the formation of Perl’s Prussian blue at
700 nm. Table S1 showed the reducing power of triazole
derivatives compared with BHT. All tested compounds
showed some degree of reducing power; in the triazole
derivatives, the reducing power followed the following
order; BHT > 7b > 6b > 7a > 6a.
In this study, presence of methyl group and type of sub-
stitution affected decrease in reducing power. Similarly, the
triazole derivatives showed marked cupric ion (Cu+2) reduc-
ing ability. Cupric ions (Cu+2) reducing ability of triazole
derivatives is shown in Table 4. Cupric ions (Cu+2) reduc-
ing capability of triazole derivatives by cuprac method was
found to be concentration-dependent (25–100 µg/mL).
Cupric ions (Cu+2) reducing power of triazole derivatives
and BHT at the same concentration (100 µg/mL) exhibited
the following order: BHT > 6b=7b >7a > 6a.
In this study, the results showed that the synthesized
new methoxy substitued bis-1,2,4-triazole derivatives
had antiurease and antielastase activities. For reason,
new methoxy substitued bis-1,2,4-triazole derivatives
may be considered as a main elastase and urease inhibi-
tory. erefore, these compounds could be used as a
source of antielastase and antiurease in pharmaceutical,
cosmetic and agriculture industries.
Declaration of interest
e authors declare no conflicts of interest.
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