Novel 1,2,4-Triazolo[1,5- a ]pyridines and their fused ring systems attenuate oxidative stress and prolong lifespan of caenorhabiditis elegans

Article abstract of DOI:10.1021/jm2014315

In this paper we report the synthesis of some novel 1,2,4-triazolo[1,5-a] pyridine and azolotriazolopyridine ring systems. The products were screened for various types of activity like antibacterial, antifungal, and antioxidative activity. Compound 13 was found to pose an antioxidative activity. In addition, this compound was found to extend the life span of Caenorhabiditis elegans under standard laboratory conditions and reduces both heat and chemical induced oxidative stress in C. elegans in a dose-dependent manner. Furthermore, treatment of worms with compound 13 was found to significantly attenuate the formation of advanced glycation end products and malondialdehyde in a dose-dependent manner.

Full text of DOI:10.1021/jm2014315

Article
pubs.acs.org/jmc
Novel 1,2,4-Triazolo[1,5-a]pyridines and Their Fused Ring Systems
Attenuate Oxidative Stress and Prolong Lifespan of Caenorhabiditis
elegans
Ramadan Ahmed Mekheimer,*,†,§ Ahmed Amir Radwan Sayed,‡,§ and Eltaib Ali Ahmed∥
†Department of Chemistry, Faculty of Science for Girls, King Abdulaziz University, Jeddah, P.O. Box 50918, Jeddah 21533,
Saudi Arabia
‡Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah, P.O. Box 80203, Jeddah 215089, Saudi Arabia
§Department of Chemistry, Faculty of Science, El-Minia University, El-Minia 61519, Egypt
∥Department of Chemistry, Faculty of Education, AlFashir University, AlFashir 125, Sudan
ABSTRACT: In this paper we report the synthesis of some novel 1,2,4-
triazolo[1,5-a]pyridine and azolotriazolopyridine ring systems. The
products were screened for various types of activity like antibacterial,
antifungal, and antioxidative activity. Compound 13 was found to pose an
antioxidative activity. In addition, this compound was found to extend the
life span of Caenorhabiditis elegans under standard laboratory conditions
and reduces both heat and chemical induced oxidative stress in C. elegans in a dose-dependent manner. Furthermore, treatment
of worms with compound 13 was found to significantly attenuate the formation of advanced glycation end products and
malondialdehyde in a dose-dependent manner.
and naringenin improved the antioxidant status of wild-type C.
elegans, which results in life span extension.
1. INTRODUCTION
Aging is associated with increased incidence of a range of
diseases, some of which are pathogenetically linked to elevated
levels of reactive oxygen species (ROS).1 The mitochondrial
free radical theory of aging2 proposes an association between
ROS generated during mitochondrial respiration (mtROS, the
principal source of ROS in cells), accumulation of oxidative
damage in mitochondrial DNA (mtDNA), and occurrence of
mutations in the mitochondrial genome. These molecular changes
lead to damaged or misfolded proteins, mitochondrial dysfunction,
decline of cellular and tissue functions, enhanced formation of
advanced glycation end products (AGE), and shortened life span.
ROS generated due to mitochondrial dysfunction are thought to
further induce mtDNA mutations, thus contributing to a vicious
cycle of aging.3−5 The extent of ROS formation and oxidative
damage to mtDNA is inversely correlated with longevity across
species.6 Moreover, several studies have demonstrated an
association between oxidative stress, mtDNA mutations, AGE
formation, and age-related organ dysfunction.4−7
Antioxidants may play an important role in preventing free
radical damage associated with aging by interfering directly in
the generation of radicals or by scavenging them. Previously,
Brown et al.,8 Bartholome et al.,9 and Zhang et al.10 have been
indicated that antioxidants like Epigallocatechin gallate and
α-lipoic acid have the ability to attenuate oxidative stress and
prolong life span of wild-type Caenorhabditis elegans (C. elegans)
under both standard and induced stress conditions. Recently,
Wilson et al.11 have demonstrated that proanthocyanidin, a
potent antioxidant, reduced oxidative stress and extend the life
We designed a study to determine whether the new
synthesized 1,2,4-triazol[1,5-a]pyridine derivatives can prolong
lifespan in a whole organism. For these studies, we required an
organism with relatively short lifespan that could be assayed
reproducibly and robustly and for which the genetic and
environmental factors affecting lifespan were well-defined. The
experimental organism that could best accommodate these
requirements was the nematode, C. elegans, which has become a
popular model for studying aging and longevity due to its short
2−3-week lifespan, rapid generation time, and experimental
flexibility.13
1,2,4-Triazolo[1,5-a]pyridines constituted an important class
of heterocyclic compounds, which are of considerable interest
due to their uses as active ingredients in antihypertensive, bro-
nchodilatory, antiinflammatory, analgesic, and positive ino-
tropic agents.14−16
Isoxazoles, pyrroles, and pyrazoles are well-known examples
of heteroaromatic organic compounds associated with diverse
biological and pharmacological properties. Isoxazoles constitute
an important family of five-membered hetrocycles in view
of their use in many natural products syntheses17,18 and
occurrence in pharmaceutical agents such as COX-2 inhibitor
Bextra.19 The pyrrole skeleton is of great importance to chemists
as well as biologists, as it is found naturally in plants and in
animal cell constituents.20 Pyrazole derivatives are synthetic
targets of utmost importance in the pharmaceutical industry
span of wild-type C. elegans. In 2012, Grunz and his co-
Received: October 22, 2011
Published: April 17, 2012
̈
workers12 have proved that myricetin, quercetin, kaempferol,
© 2012 American Chemical Society
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because the pyrazole ring has been known as an important
framework in a large number of drugs.21−25 Moreover, recent
reports display the pyrazole chemotype as the structural motif
of a number of highly potent inhibitors of coagulation factor
Xa26 and antitumor CDK.27
the intense azide absorption at 2130 cm−1 in the IR spectrum).
The structure of 5 is supported by analytical and spectral data.
Thus, the IR spectrum of 5 revealed the absence of the azido
group and the presence of absorption band at 3210 cm−1,
1
attributed to the NH group. While in the H NMR spectrum,
Considering the above very interesting pharmacological
properties and as a part of our studies on the design of new
routes for the synthesis of novel heterocyclic ring systems
containing the 1,2,4-triazole skeleton, with potential pharma-
ceutical activities,28 we have now designed and synthesized
some novel functionalized 1,2,4-triazolo[1,5-a]pyridines and their
fused tricyclic ring systems, with important heterocycles such as
pyrrole, isoxazole, and pyrazole. The products were screened for
various types of activity like antibacterial, antifungal, and anti-
oxidative activity. Compound 13 was found to pose an anti-
oxidative activity. In addition, this compound was found to extend
the life span of C. elegans under standard laboratory conditions
and reduces both heat and chemical induced oxidative stress in C.
elegans in a dose-dependent manner. Furthermore, treatment of
worms with compound 13 was found to significantly attenuate the
formation of advanced glycation end products and malondialde-
hyde in a dose-dependent manner.
the pyrrole NH proton resonates as a singlet at δ = 12.65 ppm.
The signal attributable to the H-6 aromatic proton of the
pyrrole ring is found at δ = 7.33 ppm as singlet besides the set
of signals due to phenyl and ethyl protons. Additionally, its
structure was fully supported by 13C NMR, which was com-
patible with the assigned structure. Furthermore, in its mass
spectrum, this product has the molecular ion m/z 407 (M+, 88)
and other peaks (see Experimental Section), confirming its
presumed structure. Analytical data are in accordance with the
proposed structure for compound 5. A mechanism for the
transformation of 4 into 5 could involve a well-known cycliza-
tion of a singlet nitrene.30,31
Next, we have examined the azidation of the chloroaldehyde
1 with sodium azide with a view to synthesize the interesting
isoxazolotriazolopyridine 7 as a novel tricyclic system. Thus,
when compound 1 was reacted with sodium azide in acetone
for one hour at room temperature, the new 7-ethylthio-4-oxo-8-
phenyl-4,8-dihydro-isoxazolo[3,4-d]-1,2,4-triazolo[1,5-a]-
pyridine-9-carbonitrile (7) was directly obtained without
isolation of the azide intermediate 6 (see Scheme 2). To our
2. RESULTS AND DISCUSSION
2.1. Chemistry. 7-Chloro-6-formyl-1,2,4-triazolo[1,5-a]-
pyridine derivative 129 served as a starting point for studying
all the reactions leading to the construction of novel linearly
tricyclic systems incorporating pyrrole, isoxazole, and pyrazole
nucleus in addition to the triazolopyridine moiety as illustrated
in Schemes 1−3. Scheme 1, shows the first synthesis of the
Scheme 2
Scheme 1
knowledge, compound 7 is the first example of the linear
isoxazolotriazolopyridine ring system.
Attention was next turned to synthesize the azido compound
9 possessing the azomethine moiety, as another cyclization
partner, at the 6-position, which was expected to rearrange with
loss of nitrogen to the novel pyrazolotriazolopyridines 10. Thus,
the chloroaldehyde 1 was reacted with hydroxylamine in MeOH
at reflux temperature for one hour to generate the oxime 8. The
7-azido-triazolopyridine 9 was readily formed from the 7-chloro
derivative 8 by displacement with azide ion (Scheme 3). The azide
Scheme 3
hitherto unknown ethyl 9-cyano-2-ethylthio-5-oxo-1-phenyl-
5,8-dihydro-1H-pyrrolo[2,3-d]-1,2,4-triazolo[1,5-a]pyridine-7-
carboxylate (5) in which 7-chloro-6-ethoxycarbonylvinyl-1,2,4-
triazolo[1,5-a]pyridines 3 was prepared, as described previously
by Mekheimer et al.,29 by treatment of compound 1 with an
equimolar amount of the stabilized yield 2 in CHCl3 at room
temperature. Azidation of 3 with sodium azide in DMF at
room temperature gave the corresponding 7-azido-triazolopyr-
idine derivative 4. Ring closure to the hitherto unknown ethyl
9-cyano-2-ethylthio-5-oxo-1-phenyl-5,8-dihydro-1H-pyrrolo-
[2,3-d]-1,2,4-triazolo[1,5-a]pyridine-7-carboxylate (5) could
be achieved by thermolysis of the azide 4 in refluxing bromo-
benzene for one hour, resulting in loss of nitrogen and
subsequent intramolecular cyclization (the reaction may be
monitored by the evolution of nitrogen or by disappearance of
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Scheme 4
9 underwent smooth thermal decomposition in refluxing acetone
to afford the novel 7-ethylhio-2-hydroxy-4-oxo-8-phenyl-4,8-
dihydro-2H-pyrazolo[3,4-d]-1,2,4-triazolo[1,5-a]pyridine-9-carbon-
itrile (10) in good yield. A mechanism for the formation of the
isoxazolotriazolopyridines 7 and pyrazolotriazolopyridines 10
could be explained in the terms of neighboring group attack on
the azido moiety with concurrent loss of nitrogen without transient
formation of the nitrene, as previously reported.31−34
Scheme 5
To construct new derivatives of the interesting tricyclic
systems of type 10, we attempted thermal intramolecular cycliza-
tion of the amino compound 11, utilizing the azido compound 9
as a good starting material for this purpose. Reduction of the
azide moiety 9 with Na2S2O4 gave the corresponding 7-amino-
1,2,4-triazolo[1,5-a]pyridines 11. We envisaged that the thermal
cyclization of 11 in bromobenzene might afford the hitherto
unknown tricyclic pyrazolotriazolopyridines 12. Interestingly,
however, an unexpected new compound, 7-amino-1,2,4-triazolo-
[1,5-a]pyridine-6,8-dicarbonitriles 13, was obtained (Scheme 4).
Variation of solvents, temperature, and reaction time gave no
the desire pyrazolotriazolopyridine derivative 12. To obtain
unequivocal evidence for the structure, compound 13 could
also be obtained by an alternative route: dehydration of oxime
8 with POCl3 afforded the corresponding 7-chloro-1,2,4-
triazolo[1,5-a]pyridine-6,8-dicarbonitriles 14. Reacting chloro
compound 14 with excess of sodium azide in DMF at room
temperature gave the azido compound 15. Reduction of 15
with Na2S2O4 in MeOH/H2O mixture at room temperature
afforded the 7-amino-1,2,4-triazolo[1,5-a]pyridine derivative 13
(Scheme 4).
In an attempt to synthesize new derivatives of the interesting
pyrazolotriazolopyridine of type 18, we further examined the
thermal cyclization of the oximes 17a,b, which prepared, as
depicted in Scheme 5, by refluxing chloro compound 8 with an
excess of alkylamines 16a,b in DMF for 2 h. We first tried the
cyclization of 17a,b under various conditions for extended
periods. However, when only starting material was recovered,
we attempted the cyclization in a more basic and higher boiling
point solvent such as DMF. However, the thermal cyclization of
17a,b in boiling DMF for 15 h also failed to yield the tricyclic
compound 18, instead forming 7-alkyl-amino-1,2,4-triazolo[1,5-
a]pyridines 19a,b as a new derivative of 1,2,4-triazolo[1,5-a]-
pyridine (Scheme 5).
2.2. Biological Activity. To study the effect of the com-
pounds 5, 8, 13, and 17b on the lifespan of wild type C. elegans,
N2 worms were cultivated on agar plates under standard
laboratory conditions at 20 °C either in the absence (control)
or presence of different concentrations of these compounds
(500 nM, 50 nM and 1 μM). A significantly prolonged lifespan
of wild-type C. elegans under standard laboratory conditions in a
dose-dependent manner was obtained with compound 13 (P =
0.012 for 1 μM, 0.034 for 500 nM, and 0.065 for 50 nM) as
indicated in Figure 1. Compounds 5, 8, and 17b do not affect
the lifespan of the wild-type animals under standard laboratory
conditions (data not shown). The life span extending effect of
all concentrations of compound 13 was still lower than the
effect of proanthocyanidin.
In C. elegans, there is a good correlation between stress
resistance and lifespan because most genetic mutants with a
longevity phenotype have increased resistance to a variety of
acute stressors like oxidative stress35 and heat.36 For this reason,
acute stressors have been successfully used as surrogates to
identify new gerontogenes in genetic screens37 and it was
discussed whether such a surrogate assay might also be helpful
in identifying compounds that affect lifespan.38
One possible explanation for the beneficial effects of 1,2,4-
triazolo[1,5-a]pyridine compounds on aging in C. elegans is
that these compounds may able to increase cellular stress resist-
ance. To test this possibility, we examined stress resistance of 5,
8, 13 and 17b treated animals. Resistance to oxidative stress
was examined by exposing animals to paraquat, an intracellular
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Figure 1. Survival of C. elegans on agar cultures of N2 worms in absence of treatment (control) and in presence of the indicated concentrations of 13
or 67 μg/mL proanthocycanidin. The curves represent the means of the survival curves from six individual experiments. Values represent the mean
from three independent experiments, each including 100 worms.
free-radical-generating compound. Compound 13 treatment
significantly increased survival time in the presence of paraquat
(P = 0.011 for 1 μM, 0.04 for 500 nM, and 0.057 for 50 nM) as
indicated in Figure 2. Compounds 5, 8, and 17b have no effect
on the paraquat induced oxidative stress.
Figure 3. Average survival rates of C. elegans. Treatment with 13
improved thermotolerance. Fractional survival at 35 °C for seven-day-
old N2 worms or worms treated with indicated treatments (500 nM,
50 nM, and 1 μM) of 13. This is the average of four independent
experiment with an average of 30 individual animals (7 days old) used
per experiment. Error bars are the standard error of the mean.
*Denotes a significant difference from N2 (Control) strain. p < 0.05.
Oxidative stress results in the formation of reactive oxygen
species (ROS). ROS have the ability to react with proteins and
macromolecules leading to formation of advanced glycation end
Figure 2. Kaplan−Meier curve. Treatment with 13 significantly
improved survival rates in the presence of 40 μM paraquat of seven-
products (AGEs), e.g., carboxymethyllysine (CML). CML plays
a major role in decreasing lifespan of C. elegans.4,39 To test the
degree of CML formation in worms cultivated under standard
laboratory conditions at 20 °C in the absence (control) and pre-
sence of different concentrations of compound 13, CML con-
centration was determined using ELISA. The data in Figure 4
showed that treatment of worms with compound 13 significantly
decreased CML formation in wild-type animals in a dose-
dependent manner (P < 0.05).
Lipid peroxidation has been and remains one of the most
widely used indicators of oxidant/free radical formation in vitro
and in vivo. Reactive oxygen species (ROS) play a central role
in the formation of active aldehydes, e.g., glyoxal, methyl
glyoxal, and 3-deoxyglucosone. The role of 13 attenuating lipid
peroxidation was tested by measuring the concentration of
malondialdehyde (MDA) in the control and 13-treated animals.
Compound 13 was found to significantly attenuate MDA forma-
tion in treated animals. The effect was also dose dependent, as
shown in Figure 5.
day-old N2 worms. Worms were either left untreated (control) or
treated with different concentrations of 13. Three independent
experiments with an average of 30 individual animals (7 days old)
were used per experiment.
In addition, thermotolerance was increased significantly (P <
0.01) as a result of compound 13 treatment in a dose-
dependent manner. In wild-type animals, treatment with 13 was
correlated with a 1.4, 1.8, 2, and 2.5-fold increase in 16 h
survival at 35 °C, respectively, as shown in Figure 3.
In this study, we found such a correlation between stress
resistance and lifespan because compound 13 conferred an in-
creased resistance in a surrogate assay using heat and paraquat
as well as an extension of lifespan. In our hands, heat and
paraquat were the suited surrogate because the worms died
within a few hours under conditions of an acute lethal thermal
and oxidative stress and the bacteria acting as a source of food
were not affected by both. The thermal and paraquat stress
caused an increase of ROS accumulation in the worms and
therefore it is likely that the death of the worms was at least
partially due to oxidative stress.
In the present study, we found that different concentrations
of compound 13 significantly extended lifespan of C. elegans
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be taken into account, including enhanced expression of anti-
oxidant genes like superoxide dismutase, catalase, and others. It
is also thinkable that compound 13 influences mitochondrial
free radical generation by stabilization of the complex or
reduced AGE modification, to which our data point.
Yet what other pathways might be influenced we do not
know because these data are preliminary. The importance of these
effects on mammals could not be judged yet, but many pathways
in C. elegans are very similar to mammal and human systems.
Proanthocyanidin was selected as a positive control in this
study because of its well-known antioxidant effect.
3. CONCLUSION
Figure 4. CML ELISA for seven-day-old worms in absence of
treatment (control) or in presence of different concentration of 13 and
67 μg/mL proanthocyanidin as indicatred. 13 significantly attenuated
CML formation in a dose-dependent manner. Vertical bars represent
SEM from three independent experiments. *P < 0.05, **P = 0.01.
In conclusion, we have developed for the first time an efficient
synthesis of previously unreported 1,2,4-triazolo[1,5-a]pyridine
derivatives and some of their fused linear tricyclic ring systems
and studied their biological activity. Compound 13 was found
to extend lifespan of wild-type C. elegans under standard
laboratory conditions. Lifespan extension of compound 13 in
the nematode C. elegans might be attributed to its direct ROS
scavenging activity and indirect free radical-scavenging activity
through up-regulating stress-resistance-associated genes such as
SOD, hsp, daf 2, clk, mev, and skn-1. These interesting findings
highlight the therapeutic efficacy of compound 13 as an
antioxidant and the possibility of its use as an antioxidant drug.
Further studies using different mutants of C. elegans are
required to examine the exact mechanism of compound 13
action and the optimal dose required.
4. EXPERIMENTAL SECTION
4.1. Chemistry. Melting points were determined on a Gallenkamp
melting point apparatus and were uncorrected. 1H and 13C NMR
1
Figure 5. MDA contents in the seven-day-old worms in absence of
treatment (control) or in presence of 500 nM, 50 nM, and 1 μM of 13
and 67 μg/mL proanthocyanidin. Vertical bars represent SEM from
five replicate experiments. *Denotes a significant difference from N2/
control strain (P < 0.05).
spectra were recorded on Bruker-DPX-300 (300 MHz for H NMR
1
and 75 MHz for 13C NMR) and Bruker ARX500 (500.1 MHz for H
NMR and 125.8 MHz for 13C NMR) spectrometers with DMSO-d6 as
solvent, and the chemical shifts were expressed in δ (ppm) values with
trimethylsilane as an internal reference (s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet, and br = broad peak). IR spectra
were recorded in KBr disks using a Schimadzu 470 spectrophotometer.
Mass spectra were measured on a Shimadzu GCMS-QP2010 mass
spectrometer. Microanalyses were performed at the Microanalytical
Data Unit, Cairo University, and results were within 0.4% of the calcu-
lated values. Both the analytical and spectroscopic data confirmed ≥95%
purity of the all prepared compounds.
and increased their stress resistance against both heat and
paraquate induced stress under standard culture conditions.
These effects were found to be dose-dependent as shown in
Figures 1, 2, and 3.
Compound 13 was also found to significantly modulate the
formation of AGEs, in particular CML in the wild-type animals
under standard laboratory conditions at 20 °C in a dose-dependent
manner as shown in Figure 5. These observations indicated that
glycosylation of proteins in the treated animals was significantly
attenuated as a result of compound 13 supplementation.
In all trials, the effect of all concentrations of compound 13
was found to be lower than the effect of positive control
proanthocyanidin.
Attenuation of AGE formation resulted in lifespan extension
and improved stress resistance of the wild-type C. elegans under
standard laboratory conditions. The obtained data were in line
with previous work.4,5,7−12,40,41 This suggests an antioxidant
power of 13 or its ability to activate the antioxidant enzymes of
the animals. This was also supported with the obtained data from
lipid peroxidation. Compound 13 has the ability to attenuate lipid
peroxidation resulting in the obtained concentration of MDA.
Attenuation of lipid peroxidation results in attenuation of AGE
formation and reduces oxidative stress.
7-Azido-6-ethoxycarbonylvinyl-2-ethylthio-1,5-dihydro-5-oxo-1-
phenyl-1,2,4-triazol[1,5-a]pyridine-8-carbonitrile (4). Sodium azide
(0.060 g, 0.932 mmol) was added to a solution of 3 (0.20 g, 0.466
mmol) in DMF (5 mL), and the mixture was stirred for 3 h at room
temperature (25 °C). Then the reaction mixture was poured into H2O
and the precipitated solid product was collected by filtration, washed
well with H2O, dried, and recrystallized from CHCl3 to give
compound 4 (0.190 g, 94%) as yellowish crystals: mp 164−166 °C
(dec.). 1H NMR (DMSO-d6) δ 1.25 (t, 3H, J = 7.2 Hz, CH3), 1.41 (t,
3H, J = 7.2 Hz, CH3), 3.30 (q, 2H, J = 7.2 Hz, CH2), 4.17 (q, 2H, J = 7
Hz, CH2), 7.15 (d, 1H, J = 16 Hz, CH), 7.69 (m, 5Harom), 7.82 (d, 1H,
J = 16 Hz, CH). 13C NMR (DMSO-d6) δ 14.1 (2CH3), 26.3 (CH2),
59.8 (CH2), 67.0 (C-8), 103.4 (Cβ), 110.2 (CN), 117.9 (C-6), 128.7,
129.8, 130.4, 132.0 (Ar−C), 134.8 (Cα), 145.8 (C-7), 147.9 (C-2),
152.6 (C-8a), 155.7 (CO, amide), 167.2 (CO, ester). IR (KBr) ν 3100
(arom CH), 2950 (aliph CH), 2220 (CN), 2130 (N3), 1710 (CO,
ester), 1680 (CO, amide) cm−1. MS: m/z (%) = 407 (M+− N2, 92).
Anal. Calcd for C20H17N7O3S (435.46): C, 55.16; H, 3.93; N, 22.52; S,
7.36. Found: C, 55.33; H, 4.12; N, 22.67; S, 7.19.
Ethyl 9-Cyano-2-ethylthio-5-oxo-1-phenyl-5,8-dihydro-1H-
pyrrolo[2,3-d]-1,2,4-triazolo[1,5-a]pyridine-7-carboxylate (5). A solution
of azide compound 4 (0.10 g, 0.229 mmol) in bromobenzene (5 mL)
Further studies must show which is the exact mechanism
leading to the effects described above. Several pathways must
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was refluxed for one hour. After concentration and cooling to room
temperature, a small amount of EtOH was added. The resulting
solid product was collected by filtration, dried, and recrystallized from
EtOH to give compound 5 (0.085 g, 90%) as buff crystals: mp 316−
318 °C. 1H NMR (DMSO-d6) δ 1.32 (t, 3H, J = 7 Hz, CH3), 1.40 (t,
3H, J = 7 Hz, CH3), 3.26 (q, 2H, J = 7 Hz, CH2), 4.28 (q, 2H, J =
7 Hz, CH2), 7.33 (s, 1H, pyrrole CH), 7.62−7.72 (m, 5Harom), 12.65
(s, 1H, NH). 13C NMR (DMSO-d6) δ 14.2 (CH3), 14.2 (CH3) 26.0
(CH2), 56.6 (C-9), 60.2 (CH2), 108.5 (C-5a), 111.8 (C-6), 112.0
(CN), 125.8 (C-7), 129.0, 129.7, 130.8, 131.5 (Ar−C), 139.3 (C-8a),
146.5 (C-2), 151.0 (CO), 153.8 (C-9a), 159.7 (CO). IR (KBr) ν 3210
(NH), 2965 (aliph CH), 2210 (CN), 1700 (CO, ester), 1670 (CO,
amide) cm−1. MS: m/z (%) = 408 (M+ + 1, 20). Anal. Calcd for
C20H17N5O3S (407.45): C, 58.96; H, 4.21; N, 17.19; S, 7.87. Found:
C, 59.07; H, 4.09; N, 17.28; S, 7.96.
7-Ethylthio-4-oxo-8-phenyl-4,8-dihydro-isoxazolo[3,4-d]-1,2,4-
triazolo[1,5-a]pyridine-9-carbonitrile (7). Sodium azide (0.037 g,
0.569 mmol) was added to a solution of compound 1 (0.20 g, 0.557
mmol) in acetone (10 mL), and the reaction mixture was stirred for
one hour at room temperature (25 °C). Then it was poured into H2O,
and the precipitated solid product was collected by filtration, washed
well with H2O, dried, and recrystallized from EtOH to give compound
7 (0.160 g, 85%) as yellow crystals: mp 245−246 °C (dec.). 1H NMR
(DMSO-d6) δ 1.40 (t, 3H, J = 7.2 Hz, CH3), 3.26 (q, 2H, J = 7.2 Hz,
CH2), 7.61−7.70 (m, 5Harom), 10.26 (s, 1H, isoxazole CH). 13C NMR
(DMSO-d6) δ 14.1 (CH3), 26.0 (CH2), 51.5 (C-9), 109.4 (C-3a),
111.8 (CN), 128.8, 129.9, 130.3, 131.8 (Ar−C), 149.9 (C-7a), 150.3
(C-9a), 155.3 (CO), 155.5 (C-8a), 164.8 (C-3). IR(KBr) ν 3100
(arom CH), 2928 (aliph CH), 2224 (CN), 1710 (CO) cm−1. MS: m/z
(%) = 338 (M+ + 1, 18). Anal. Calcd for C16H11N5O2S (337.36): C,
56.96; H, 3.29; N, 20.76; S, 9.50. Found: C, 56.83; H, 3.40; N, 20.89;
S, 9.39.
7-Chloro-2-ethylthio-6-[(hydroxyimino)methyl]-5-oxo-1-phenyl-
1,5-dihydro-1,2,4-triazolo[1,5-a]pyridine-8-carbonitrile (8). A mix-
ture of compound 1 (0.10 g, 0.279 mmol) and hydroxylamine
hydrochloride (0.038 g, 0.547 mmol) in MeOH (5 mL) was refluxed
for one hour, during which a yellow solid product separated out. After
cooling to room temperature, the resulting solid product was collected
by filtration, washed with MeOH, dried, and recrystallized from DMF to
give compound 8 (0.080 g, 77%) as yellow crystals: mp 271−272 °C.
1H NMR (DMSO-d6) δ 1.4 (t, 3H, J = 7 Hz, CH3), 3.30 (q, 2H,
J = 7 Hz, CH2), 7.64−7.72 (m, 5Harom), 8.17 (s, 1H, CH), 11.52 (s,
1H, OH). 13C NMR (DMSO-d6) δ 14.1 (CH3), 26.2 (CH2), 72.1 (C-8),
109.0 (C-6), 111.7 (CN), 128.7, 129.8, 130.0, 131.9 (Ar−C), 142.4
(C-2), 142.7 (oxime-carbon), 145.7 (C-7), 151.7 (C-8a), 155.7 (CO).
IR (KBr) ν 3296 (OH), 3050 (arom CH), 2944 (aliph CH), 2220
(CN), 1692 (CO) cm−1. MS: m/z (%) = 375 (M+, 16). Anal. Calcd
for C16H12ClN5O2S (373.82): C, 51.41; H, 3.24; Cl, 9.48; N, 18.73; S,
8.58. Found: C, 51.53; H, 3.13; Cl, 9.64; N, 18.61; S, 8.72.
7-Azido-2-ethylthio-6-[(hydroxyimino)methyl]-5-oxo-1-phenyl-
1,5-dihydro-1,2,4-triazolo[1,5-a]pyridine-8-carbonitrile (9). To a solu-
tion of compound 8 (0.20 g, 0.535 mmol) in DMF (5 mL), NaN3
(0.07 g, 1.07 mmol) was added. The reaction mixture was stirred for
3 h at room temperature (25 °C), and then it was poured into H2O.
The precipitated solid product was collected by filtration, washed well
with H2O, dried, and recrystallized from CHCl3 to give compound 9
(0.170 g, 84%) as colorless crystals: mp 162−164 °C (dec.). 1H NMR
(DMSO-d6) δ 1.38 (t, 3H, J = 7 Hz, CH3), 3.27 (q, 2H, J = 7 Hz,
CH2), 7.67 (m, 5Harom), 8.18 (s, 1H, CH), 11.51 (s, 1H, OH). 13C
NMR (DMSO-d6) δ 14.3 (CH3), 26.3 (CH2), 66.7 (C-8), 103.1 (C-6),
111.3 (CN), 128.8, 129.9, 130.1, 132.0 (Ar−C), 142.2 (C-2), 145.9
(oxime-carbon), 147.0 (C-7), 153.3 (C-8a), 155.7 (CO). IR (KBr) ν
3250 (OH), 2210 (CN), 2120 (N3), 1670 (CO) cm−1. MS: m/z (%) =
381 (M+ + 1, 13). Anal. Calcd for C16H12N8O2S (380.38): C, 50.52; H,
3.18; N, 29.46; S, 8.43. Found: C, 50.45; H, 3.27; N, 29.33; S, 8.57.
7-Ethylthio-2-hydroxy-4-oxo-8-phenyl-4,8-dihydro-2H-pyrazolo-
[3,4-d]-1,2,4-triazolo[1,5-a]pyridine-9-carbonitrile (10). A solution of
azide compound 9 (0.20 g, 0.526 mmol) in dry acetone (7 mL) was
refluxed for 12 h. After concentration and cooling to room tem-
perature, the reaction mixture was poured into H2O. The obtained
precipitate was collected by filtration, washed with H2O, dried, and
recrystallized from EtOH to give compound 10 (0.160 g, 86%) as
yellow crystals: mp 242−243 °C. 1H NMR (DMSO-d6) δ 1.37 (t, 3H,
J = 7 Hz, CH3), 3.23 (q, 2H, J = 7 Hz, CH2), 7.58−7.93 (m, 5Harom),
8.68 (s, 1H, pyrazole CH), 13.81 (s, 1H, OH). 13C NMR (DMSO-d6)
δ 15.0 (CH3), 26.7 (CH2), 56.9 (C-9), 105.0 (C-3a), 113.6 (CN),
122.5 (C-9a), 129.8, 130.5, 131.6, 132.2 (Ar−C), 144.4 (C-3), 147.9
(C-7), 154.5 (CO), 163.0 (C-8a). IR (KBr) ν 3550 (OH), 3100, 3056
(arom CH), 2928 (aliph CH), 2210 (CN), 1689 (CO) cm−1. MS: m/z
(%) = 353 (M+ + 1, 21); Anal. Calcd for C16H12N6O2S (352.37): C,
54.54; H, 3.43; N, 23.85; S, 9.10. Found: C, 54.67; H, 3.59; N, 23.72;
S, 9.23.
7-Amino-2-ethylthio-6-[(hydroxyimino)methyl]-5-oxo-1-phenyl-
1,5-dihydro-1,2,4-triazolo[1,5-a]pyridine-8-carbonitrile (11). So-
dium dithionite (0.6 g, 3.45 mmol) was added portionwise to a
stirred suspension of 9 (0.30 g, 0.788 mmol) in a 2:1 MeOH−H2O
(15 mL) mixture. Stirring was maintained at room temperature for 5
h; during this period of time, a solid product was formed. Then, the
reaction mixture was poured into H2O. The resulting solid product
was collected by filtration, washed well with H2O, dried, and recry-
stallized from CHCl3 to give compound 11 (0.20 g, 72%) as yellowish
crystals: mp 282−284 °C (dec.). 1H NMR (DMSO-d6) δ 1.37 (t, 3H,
J = 7.2 Hz, CH3), 3.28 (q, 2H, J = 7.2 Hz, CH2), 7.65 (m, 7H, 5Harom
+
NH2), 8.45 (s, 1H, CH), 10.71 (s, 1H, OH). 13C NMR (DMSO-d6)
δ 14.2 (CH3), 26.3 (CH2), 73.2 (C-8), 102.1 (C-6), 111.4 (CN),
128.7, 129.7, 130.1, 131.9 (Ar−C), 142.2 (C-2), 142.6 (oxime-
carbon), 148.7 (C-7), 151.4 (C-8a), 155.6 (CO). IR (KBr) ν 3392
(OH), 3289, 3184 (NH2), 3054 (arom CH), 2912 (aliph CH), 2210
(CN), 1650 (CO) cm−1. MS: m/z (%) = 355 (M+ + 1, 29). Anal.
Calcd for C16H14N6O2S (354.39): C, 54.23; H, 3.98; N, 23.71; S, 9.05.
Found: C, 54.39; H, 4.11; N, 23.62; S, 8.87.
7-Amino-2-ethylthio-1,5-dihydro-5-oxo-1-phenyl-1,2,4-triazolo-
[1,5-a]pyridine-6,8-dicarbonitrile (13). Route A. A solution of
compound 11 (0.110 g, 0.310 mmol) in bromobenzene (5 mL)
was refluxed for 3 h. After cooling to room temperature, the
mixture was evaporated to dryness in vacuum. The remaining
oily residue was triturated with H2O. The resulting solid product
was collected by filtration, washed with H2O, and dried.
Route B. To a stirred suspension of compound 15 (0.30 g, 0.789
mmol) in a methanol (10 mL)−water (5 mL) mixture, sodium
dithionite (0.6 g, 3.45 mmol) was added portionwise. Stirring was
maintained at room temperature for 1.5 h; during this period of time, a
solid product was formed. Then, the reaction mixture was poured into
H2O. The resulting solid product was collected by filtration, washed
well with H2O, dried, and recrystallized from DMF to give compound
13 [0.10 g, 96% (route A); 0.240 g, 86% (route B)] as yellow crystals:
mp 324−326 °C. 1H NMR (DMSO-d6) δ 1.35 (t, 3H, J = 7 Hz, CH3),
3.20 (q, 2H, J = 7 Hz, CH2), 7.25 (s, 2H, NH2), 7.59−7.67 (m,
5Harom). 13C NMR (DMSO-d6) δ 14.3 (CH3), 26.2 (CH2), 60.0 (C-6),
71.9 (C-8), 111.5 (CN), 116.0 (CN), 128.9, 129.8, 130.3, 131.9 (Ar−
C), 148.0 (C-2), 153.9 (CO), 154.2 (C-8a), 159.0 (C-7). IR (KBr) ν
3360, 3230 (NH2), 3050 (arom CH), 2900 (aliph CH), 2210 (CN),
1680 (CO) cm−1. MS: m/z (%) = 337 (M+ + 1, 30). Anal. Calcd for
C16H12N6OS (336.37): C, 57.13; H, 3.60; N, 24.98; S, 9.53. Found: C,
57.29; H, 3.74; N, 24.79; S, 9.44.
7-Chloro-2-ethylthio-1,5-dihydro-5-oxo-1-phenyl-1,2,4-triazolo-
[1,5-a]pyridine-6,8-dicarbonitrile (14). Compound 8 (0.20 g,
0.535 mmol) in POCl3 (3 mL) was refluxed for one hour. The excess
of POCl3 was evaporated under reduced pressure. The remaining residue
was triturated with cold H2O. The resulting solid product was neutralized
with a dilute KOH and collected by filtration, washed well with H2O,
dried, and recrystallized from EtOH to give compound 14 (0.180 g, 95%)
as buff crystals: mp 261−263 °C. 1H NMR (DMSO-d6) δ 1.40 (t, 3H, J =
7.3 Hz, CH3), 3.31 (q, 2H, J = 7.3 Hz, CH2), 7.67−7.73 (m, 5Harom). 13
C
NMR (DMSO-d6) δ 14.2 (CH3), 26.5 (CH2), 75.0 (C-8), 91.1 (C-6),
110.4 (CN), 114.2 (CN), 128.6, 129.5, 130.2, 132.5 (Ar−C), 147.4 (C-2),
150.4 (C-7), 151.8 (CO), 157.1 (C-8a). IR (KBr) ν 3050 (arom CH),
2980, 2930 (aliph CH), 2210 (CN), 1675 (CO) cm−1. MS: m/z (%) =
357 (M+, 30). Anal. Calcd for C16H10ClN5OS (355.80): C, 54.01; H,
4174
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2.83; Cl, 9.96; N, 19.68; S, 9.01. Found: C, 53.93; H, 3.03; Cl, 9.85; N,
19.79; S, 8.92.
(CH2), 26.2 (CH2), 30.6 (CH2), 42.8 (CH2), 59.8 (C-6), 71.8 (C-8),
111.4 (CN), 115.9 (CN), 128.8, 129.7, 130.2, 131.8 (Ar−C), 147.9
(C-2), 153.8 (CO), 154.1 (C-8a), 158.9 (C-7). IR (KBr) ν 3312
(NH), 3050 (arom CH), 2960, 2928, 2864 (aliph CH), 2210 (CN),
1654 (CO) cm−1. MS: m/z (%) = 393 (M+ + 1, 17), 392 (M+, 64).
Anal. Calcd for C20H20N6OS (392.48): C, 61.20; H, 5.14; N, 21.41; S,
8.17. Found: C, 61.13; H, 5.31; N, 21.51; S, 8.29.
7-Azido-2-ethylthio-1,5-dihydro-5-oxo-1-phenyl-1,2,4-triazolo-
[1,5-a]pyridine-6,8-dicarbonitrile (15). NaN3 (0.055 g, 0.846 mmol)
was added to a solution of chloro compound 14 (0.150 g, 0.422
mmol) in DMF (5 mL). The reaction mixture was stirred for 4 h at
room temperature (25 °C), and then it was poured into H2O. The
precipitated solid product was collected by filtration, washed well with
H2O, dried, and recrystallized from MeOH to give compound 15
(0.110 g, 72%) as colorless crystals: mp 190−191 °C (dec.). 1H NMR
(DMSO-d6) δ 1.40 (t, 3H, J = 7.16 Hz, CH3), 3.30 (q, 2H, J = 7.28 Hz,
CH2), 7.68 (m, 5Harom). 13C NMR (DMSO-d6) δ 14.1 (CH3), 26.4
(CH2), 67.4 (C-8), 82.4 (C-6), 109.4 (CN), 113.1 (CN), 128.5, 129.6,
129.9, 132.2 (Ar−C), 147.3 (C-2), 152.7 (C-7), 153.7 (CO), 156.5
(C-8a). IR (KBr) ν 3050 (arom CH), 2985, 2930 (aliph CH), 2130
(N3), 2210 (CN), 1683 (CO) cm−1. MS: m/z (%) = 362 (M+, 9).
Anal. Calcd for C16H10N8OS (362.37): C, 53.03; H, 2.78; N, 30.92; S,
8.85. Found: C, 53.24; H, 2.89; N, 31.05; S, 8.97.
7-Alkylamino-2-ethylthio-6-[(hydroxyimino)-methyl]-5-oxo-1-
phenyl-1,5-dihydro-1,2,4-triazolo[1,5-a]pyridine-8-carbonitriles
17a,b: General Procedure. A mixture of compound 8 (0.150 g, 0.40
mmol) and alkylamines 16a,b (0.116 g, 1.6 mmol) in DMF (5 mL)
was refluxed for 2 h until TLC showed the disappearance of the
starting compounds. After cooling to room temperature, the mixture
was evaporated to dryness in vacuum. The remaining oily residue was
triturated with H2O. The precipitated solid product was collected by
filtration, washed with water, dried, and finally recrystallized from
MeOH to give compounds 17a,b.
7-Isobutylamino-2-ethylthio-5-oxo-1-phenyl-1,5-dihydro-1,2,4-
triazolo[1,5-a]pyridine-6,8-dicarbonitrile (19b). Yield: (0.160 g,
1
84%) as brownish crystals: mp 286−287 °C. H NMR (DMSO-d6)
δ 0.87 (d, 6H, J = 7 Hz, 2CH3), 1.38 (t, 3H, J = 7 Hz, CH3), 1.92 (m,
1H, Haliph), 3.23 (q, 2H, J = 7 Hz, CH2), 3.40 (t, 2H, J = 7 Hz, CH2),
7.13 (t, 1H, J = 5 Hz, NH), 7.68 (m, 5H arom). 13C NMR (DMSO-d6)
δ 14.2 (CH3), 19.5 (2CH3), 26.2 (CH2), 28.8 (aliph CH), 52.7 (CH2),
59.8 (C-6), 71.8 (C-8), 111.4 (CN), 115.8 (CN), 128.8, 129.8, 130.2,
131.8 (Ar−C), 147.9 (C-2), 153.8 (CO), 154.0 (C-8a), 158.9 (C-7).
IR (KBr) ν 3328 (NH), 3050 (arom CH), 2944 (aliph CH), 2210
(CN), 1664 (CO) cm−1. MS: m/z (%) = 393 (M+ + 1, 80), 392 (M+,
71); Anal. Calcd for C20H20N6OS (392.48): C, 61.20; H, 5.14; N,
21.41; S, 8.17. Found: C, 61.34; H, 5.05; N, 21.58; S, 8.02.
4.2. Biology. Caenorhabditis elegans Strains Used, Maintenance
and Culture Conditions. Strains were maintained and manipu-
lated under standard conditions.42,43 The strain used in this
study was Bristol N2 (wild type). To obtain age-synchronized
nematodes, self-fertilizing hermaphrodites (3 days old) were
left to lay eggs and afterward removed to set the eggs in
synchrony. After hatching and reaching adulthood, adult worms
were transferred to new plates every day during the egg-laying
period to obtain large amounts of age-synchronous worms and
young adult worms were transferred to NGM agar plates
containing 40 μM 5-fluorodesoxyuridine (Sigma-Aldrich,
Germany)42 to prevent eggs from hatching.39
Lifespan Assay. The lifespan assay was conducted at 20 °C as des-
cribed previously.43,44 The worms were observed and counted daily in
the absence (control) or presence of different concentrations of com-
pounds 5, 8, 13, and 17b (50 nM, 500 nM, 5 μM) and 67 μg/mL of
proanthocyanidin (as a positive control antioxidant) to determine whether
they were dead or alive. They were prodded gently with the tip of a
platinum thin wire, and when they no longer responded, they were
considered to be dead. Dead worms were removed from the plates.
The number of worms for each analysis was approximately 100. For all
lifespans, experiments and assays were repeated at least three times.
Thermotolerance Assay. Thermotolerance assay was performed
with hermaphrodites on adult day 7, after the majority of egg-laying
had ceased. Animals were transferred onto 3 cm NGM agar plates
supplemented as indicated and then incubated at 35 °C for 16 h45 for
N2 strain in the presence and absence of different concentrations of
compounds 5, 8, 13, and 17b.
Survival was scored as the number of animals responsive to gentle
touch as a fraction of the original number of animals on the plate.
Animals that had died from desiccation on the sides of the plate were
censored.
Oxidative Stress Resistance Assay. Paraquat assay was performed
with 30 worms per trial at 20 °C. To assay paraquat sensitivity, seven-
day-old adult worms46 were placed overnight on agar plates containing
40 μM 5′-fluorodeoxyuridine (FUDR), and 40 mM paraquat39 in the
presence and absence of different concentrations of compounds 5, 8,
13, and 17b. Viability was assayed over a 20 h period.
Isolation of C. elegans Protein Extracts. A large number of worms
was incubated with different concentrations compounds 5, 8, 13, and
17b and 67 μg/mL proanthocyanidin or without treatment (control)
for 7 days in the presence of 40 μM FUDR to prevent production of
progeny. Subsequent to the incubation times, worms were harvested
from plates using M9 buffer and washed free of bacteria by sucrose
flotation and suspended in a 2-fold volume of homogenization buffer
(0.05 M Tris-HCl, pH 7.9, 25% glycerol, 0.1 mM EDTA, 0.32 M
NH4SO4) containing a protease inhibitor tablet (Roche, Germany). The
lysates were homogenized on ice using Polytron homogenizer. The
homogenates were centrifuged at 12000 rpm for 5 min and the super-
natants assayed for protein concentration (Pierce, Rockford, USA).
7-Butylamino-2-ethylthio-6-[(hydroxyimino)-methyl]-5-oxo-1-
phenyl-1,5-dihydro-1,2,4-triazolo[1,5-a]pyridine-8-carbonitrile
1
(17a). Yield: 0.145 g (88%) as yellow crystals: mp 242−244 °C. H
NMR (DMSO-d6) δ 0.85 (t, 3H, J = 7.3 Hz, CH3), 1.29 (m, 2H,
CH2), 1.34 (t, 3H, J = 7.3 Hz, CH3), 1.52 (m, 2H, CH2), 3.20 (q, 2H,
J = 7.3 Hz, CH2), 3.52 (q, 2H, J = 7.3 Hz, CH2), 7.56−7.67 (m,
5Harom), 8.47 (s, 1H, CHNOH), 9.06 (t, 1H, J = 5 Hz, NH), 10.78
(s, 1H, OH). 13C NMR (DMSO-d6) δ 13.6 (CH3), 14.4 (CH3), 19.3
(CH2), 26.2 (CH2), 31.8 (CH2), 44.3 (CH2), 58.1 (C-8), 90.2 (C-6),
114.4 (CN), 128.9, 129.6, 130.8, 131.5 (Ar−C), 147.3 (oxime-
carbon), 147.8 (C-2), 153.6 (CO), 153.9 (C-8a), 154.2 (C-7). IR
(KBr) ν 3400 (OH), 3200 (NH), 3056 (arom CH), 2960 (aliph CH),
2210 (CN), 1650 (CO) cm−1. MS: m/z (%) = 411 (M+ + 1, 12), 410
(M+, 12). Anal. Calcd for C20H22N6O2S (410.49): C, 58.52; H, 5.40;
N, 20.47; S, 7.81. Found: C, 58.66; H, 5.29; N, 20.33; S, 7.94.
7-Isobutylamino-2-ethylthio-6-[(hydroxyimino)methyl]-5-oxo-1-
phenyl-1,5-dihydro-1,2,4-triazolo[1,5-a]pyridine-8-carbonitrile
1
(17b). Yield: (0.160 g, 97%) as yellow crystals: mp 222−224 °C. H
NMR (DMSO-d6) δ 0.92 (d, 6H, J = 7 Hz, 2CH3), 1.39 (t, 3H, J =
7 Hz, CH3), 1.81 (m, 1H, Haliph), 3.26 (q, 2H, J = 7 Hz, CH2), 3.38 (t,
2H, J = 7 Hz, CH2), 7.65 (m, 5Harom), 8.54 (s, 1H, CHNOH), 9.17
(t, 1H, J = 5 Hz, NH,), 10.82 (s, 1H, OH). 13C NMR (DMSO-d6) δ
14.2 (CH3), 19.4 (2CH3), 26.3 (CH2), 28.8 (aliph CH), 52.8 (CH2),
58.0 (C-8), 90.2 (C-6), 114.3 (CN), 128.9, 129.6, 130.7, 131.5 (Ar−C),
147.3 (oxime-carbon), 147.7 (C-2), 153.6 (CO), 154.0 (C-8a), 154.2
(C-7). IR (KBr) ν 3350 (OH), 3312 (NH), 3050 (arom CH), 2944,
2912, 2880 (aliph CH), 2210 (CN), 1650 (CO) cm−1. Anal. Calcd for
C20H22N6O2S (410.49): C, 58.52; H, 5.40; N, 20.47; S, 7.81. Found: C,
58.46; H, 5.56; N, 20.62; S, 7.64.
7-Alkylamino-2-ethylthio-5-oxo-1-phenyl-1,5-dihydro-1,2,4-tria-
zolo[1,5-a]pyridine-6,8-dicarbonitriles 19a,b: General Procedure. A
solution of compounds 17a,b (0.20 g, 0.487 mmol) in DMF (5 mL)
was refluxed for 15 h (TLC control). After concentration and cooling
to room temperature, H2O was added and the resulting solid product
was collected by filtration, washed with H2O, dried, and recrystallized
from MeOH to give compounds 19a,b.
7-Butylamino-2-ethylthio-5-oxo-1-phenyl-1,5-dihydro-1,2,4-
triazolo[1,5-a]pyridine-6,8-dicarbonitrile (19a). Yield: (0.180 g,
1
94%) as brownish crystals: mp 244−246 °C. H NMR (DMSO-d6)
δ 0.87 (t, 3H, J = 7.3 Hz, CH3), 1.28 (m, 2H, CH2), 1.35 (t, 3H, J =
7.3 Hz, CH3), 1.55 (m, 2H, CH2), 3.23 (q, 2H, J = 7.3 Hz, CH2,), 3.57
(q, 2H, J = 7.3 Hz, CH2), 7.11 (t, 1H, J = 5 Hz, NH), 7.68 (m,
5Harom). 13C NMR (DMSO-d6) δ 13.4 (CH3), 14.2 (CH3), 18.1
4175
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ELISA of N-ε-(Carboxymethyl)lysine-modified proteins. N-ε-
(Carboxymethyl)lysine (CML) concentrations were determined by a
competitive enzyme-linked immunosorbent assay (Roche Diagnostics,
Germany) using streptavidin-coated microtiter plates as described.40,47
Biotinylated bovine serum albumin, which was glycated with glucose
over 3 weeks, bound to a mouse monoclonal antibody specific for the
CML epitope, which was labeled with horseradish peroxidase. ABTS:
2,2-azino-di-[3-ethylbenzthiazolinesulfonate]diammonium salt
(ABTS) plus sodium perborate served as a substrate for the indicator
enzyme. ABTS is a trademark of a member of the Roche group. CML-
modified proteins from the sample compete for the antibody binding
site. As a standard, the monomeric epitope N-carboxy-methylamino-
caproate was used, yielding results as ng of CML. The limit of detection
was 5 mg CML/mL.
Lipid Peroxidation. Lipid peroxidation was determined by
measuring malondialdehyde (MDA), an index of lipid peroxidation
according to the method used in previous work4,5,11 using 1,1′,3,3′-
tetramethoxypropane as standard. In brief, 8.1% SDS was added to the
worm lysate (prepared as described above) and incubated for 10 min
at room temperature, followed by boiling with 20% acetic acid and
0.6% thiobarbituric acid for 60 min in a water bath. On cooling, a
mixture of butanol:pyridine (15:1 v/v) was added and centrifuged at
1200 rpm for 5 min. Absorbance of the upper colored layer was
measured at 532 nm, and the concentration of MDA was expressed in
terms of nmol/g protein. Butylhydroxy toluene (0.01%) was added to
each assay mixture in order to prevent undesirable auto-oxidation of
the sample during the assay.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +2 086 2345978. E-mail: rmekh@yahoo.com.
■
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
We are thankful to Prof. Dr. Volker Jager and Dr. Peter Fischer,
■
̈
Institute fur Organische Chemie, Pfaffenwaldring 55, Uni-
̈
versitat Stuttgart, D-70569 Stuttgart, Germany, for recording
̈
NMR spectra (1H and 13C NMR). Also, we are thankful to Dr.
Michael Morcos, Internal Medicine I, Faculty of Medicine,
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characterization of novel substituted pyrrole, thiophene and furans.
ARKIVOC 2008, xii, 17−29.
Luprecht-Karls University, Heidelberg, Germany, for the
̈
provision of laboratory facilities and valuable discussion.
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ABBREVIATIONS USED
ROS; reactive oxygen species; AGE; advanced glycation end
products; C. elegans; Caenorhabditis elegans
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