Synthesis and in vitro evaluation of novel 1,2,4-triazine derivatives as neuroprotective agents

Article abstract of DOI:10.1016/j.bmc.2010.04.097

The role of novel triazine derivatives against oxidative stress exerted by hydrogen peroxide on differentiated rat pheochromocytoma (PC12) cell line was examined and a consistent protection from H₂O₂-induced cell death, associated with a marked reduction in caspase-3 activation, was observed. Moreover, activation of NF-κB, a known regulator of a host of genes that involves in specific stress and inflammatory responses by H₂O₂, was greatly impaired by triazine pretreatment in differentiated PC12 cells. Neuroprotective effect of such compounds may represent a promising approach for treatment of neurodegenerative diseases.

Full text of DOI:10.1016/j.bmc.2010.04.097

Bioorganic & Medicinal Chemistry 18 (2010) 4224–4230
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and in vitro evaluation of novel 1,2,4-triazine derivatives
as neuroprotective agents
Hamid Irannejad a, Mohsen Amini a, Fariba Khodagholi b, Niloufar Ansari a,b, Solaleh Khoramian Tusi b,
Mohammad Sharifzadeh c, Abbas Shafiee d,
*
a Department of Medicinal Chemistry, Faculty of Pharmacy, and Drug Design & Development Research Center, Tehran University of Medical Sciences, Tehran 14176, Iran
b Neuroscience Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
c Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14176, Iran
d Department of Medicinal Chemistry, Faculty of Pharmacy, and Pharmaceutical Sciences Research center, Tehran University of Medical Sciences, Tehran 14176, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
The role of novel triazine derivatives against oxidative stress exerted by hydrogen peroxide on differen-
tiated rat pheochromocytoma (PC12) cell line was examined and a consistent protection from H2O2-
induced cell death, associated with a marked reduction in caspase-3 activation, was observed. Moreover,
Received 3 March 2010
Revised 29 April 2010
Accepted 30 April 2010
Available online 8 May 2010
activation of NF-jB, a known regulator of a host of genes that involves in specific stress and inflammatory
responses by H2O2, was greatly impaired by triazine pretreatment in differentiated PC12 cells. Neuropro-
tective effect of such compounds may represent a promising approach for treatment of neurodegenera-
tive diseases.
Keywords:
Alzheimer’s disease
Oxidative stress
PC12 cells
Ó 2010 Elsevier Ltd. All rights reserved.
1,2,4-Triazine
1. Introduction
against Ab-induced toxicity.10 Kutuk and Basaga have also reported
that aspirin prevents H2O2-induced apoptosis in HeLa cells through
Oxidative stress-induced cell damage has long been implicated
both in the physiological process of aging and in a variety of neu-
rodegenerative diseases such as Alzheimer’s disease (AD).1,2 Oxida-
tive damage mediated by reactive oxygen species (ROS) which can
be generated by cell lysis, oxidative burst or in the presence of an
excess of free transition metals, can attack proteins, deoxynucleic
acid and lipid membranes; hence, disrupt cellular function and
integrity. Hydrogen peroxide (H2O2), one of the main reactive oxy-
gen species, is produced during the redox process and is considered
as a messenger in intracellular signaling cascades.3 In addition, it is
well known that H2O2 can cause lipid peroxidation and DNA dam-
age and induce apoptosis in many different cell types.4,5 In this
NF-j
B inactivation.11
1,2,4-Triazine nucleus is a prominent structural core system
present in numerous biologically active compounds, possessing a
wide range of biological activities. Many investigations revealed
the anticancer potential of triazine derivatives.12 Triazine deriva-
tives have been also introduced as anti-inflammatory,13,14 radical
scavenger,15 b-sheet breaker,16 and antifungal17 agents. 5,6-Diphe-
nyl-1,2,4-triazine-3-thiol is one of the triazine derivatives that its
anti-inflammatory action has been reported by Saxena et al.18
In the present investigation, some of the novel triazine deriva-
tives were synthesized and evaluated for their neuroprotective
effect.
pathway, NF-
j
j
B is the first transcription factor reported to respond
B, which is a redox-sensitive transcription factor,
directly.6 NF-
2. Results and discussion
regulates expression of genes that are involved in cellular differen-
tiation, proliferation, apoptosis, oxidative response, inflammation,
and immune response.7 Its activation is evident by many distinct
stimuli including bacterial lipopolysaccharide, oxidative stress,
and amyloid beta (Ab).8 Several evidences have shown that NF-
2.1. Synthesis of 1,2,4-triazine derivatives
The synthetic pathway for the preparation of triazine deriva-
tives (5) is outlined in Scheme 1. 5,6-Diaryl-1,2,4-triazine-3-thiol
(4) was prepared by cyclization of compound 3 with thiosemicar-
bazide as reported previously.18 The method for the preparation of
benzil20 was used for the preparation of 1,2-bis(4-chlrophenyl)eth-
anedione (3). Compound 2 was prepared as reported.19 Alkylation
of compound 4 was carried out by using alkyl iodides in the
j
B activation may act as a pro-apoptotic factor,9 and reduction in
NF-jB activation protect PC12 cells and hippocampal neurons
* Corresponding author. Tel.: +98 21 66406757; fax: +98 21 66461178.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.097

H. Irannejad et al. / Bioorg. Med. Chem. 18 (2010) 4224–4230
4225
O
O
O
CHO
OH
b
a
R
R
R
1
R
R
2
3
c
R
R
S
N
R'
H (a)
Cl (b)
SH
N
d
N
N
N
N
R
CH3 (1)
R
C2H5 (2)
5
n-C3H7 (3)
n-C4H9 (4)
4
Scheme 1. Synthesis pathway for the preparation of triazine derivatives. Reagents and conditions: (a) NaCN, EtOH, reflux, 2 h; (b) CuSO4, pyridine, 80 °C;
(c) thiosemicarbazide, H2O/dioxane (2:1), reflux, 2–5 h; (d) alkyl iodide, TEA, CH3OH, 2 h.
presence of triethylamine in methanol to give compounds 5a(1–4)
and 5b(1–4).
atoms substituted on thiol group and the neuroprotective effect of
compounds. In other words, increase in the length of thioalkyl moi-
ety, from methyl to n-butyl, decreases protective effect of com-
pounds against H2O2-induced cell death in both series (a and b).
However, 5b derivatives possess significant advantages over its
5a counterparts. A possible explanation for this difference could
be due to optimum lipophilicity of 5b in comparison with 5a series.
Comparison of the activity of two triazine series showed that b ser-
ies was more active than their homologous a series where H group
is substituted by chlorine in 4-position of phenyl ring. All of the
compounds showed parallel activity except 5a-2. The most active
compound was 5b-1, in which thioalkyl group in 3-position of tri-
azine ring was the smallest alkyl and the lipophilicity of the com-
pound was increased by chlorine substitution.
2.2. Protection against oxidative stress-induced cell death in
PC12 neurons
The present investigation was aimed to determine the protec-
tive role of triazine derivatives 5 in oxidative stress-induced cell
death. The PC12 cell line is a useful and widespread model for
studying neuronal differentiation in sympathetic neurons and
other neurobiochemical and neurobiological events.21 Therefore,
differentiated PC12 cells were chosen as in vitro models and
H2O2 as an oxidative agent.22 Differentiated PC12 cells seeded in
96-well plates were treated with different concentrations (0.5–
20
l
M) of compounds and were exposed to H2O2 3 h later. After
The neuroprotective effect of these compounds was further con-
firmed by morphological observations (Fig. 1E). After incubation
with 50 ng/ml NGF for 6 days, the PC12 cells displayed a neuronal
phenotype that resembled to sympathetic neurons. While the
H2O2-treated cells showed heterogeneity in their shape and were
mostly detached from the cultured plate surface, the 5b-1 pre-
treated cells showed remarkable resistance to damaging effects
of H2O2 and this was evident through their almost uniform neuro-
nal shape, being mostly attached to the plate surface, and restora-
tion of networks that were formed between neurons after
differentiating by NGF (Fig. 1E). On the contrary, 5a-3 and 5a-4
treated cells were completely damaged.
24 h, the cells were assayed for cell viability. Figure 1B showed that
H2O2 significantly reduced cell viability to 25%, compared to con-
trol, whereas pretreatment with 5a-1 and 5a-2 (10 lM) protected
neurons against H2O2-induced cell death (47% and 54%, compared
to control, respectively). However, synergistic effects with H2O2
were observed for 5a-3 and 5a-4 in reducing cell viability. Preincu-
bation with 5b(1–4) increased cell viability to 83, 40, 45 and 50%,
compared to control, respectively. Similar results were obtained
from undifferentiated PC12 cells. Undifferentiated PC12 cells
seeded in 96-well plates were treated with different concentra-
tions (0.5–20 lM) of compounds and were exposed to H2O2 3 h la-
ter. After 24 h, the cells were assayed for cell viability. Figure 1D
showed that H2O2 significantly reduced cell viability to 29%, com-
For further investigation, apoptosis assays and western blot
analysis were performed to determine their mechanism of action,
pared to control, whereas pretreatment with 5a(1–2) (10
tected neurons against H2O2-induced cell death up to 53% and 59%,
l
M) pro-
at their best protective concentration (10
stress-induced cell death.
lM), against oxidative
respectively, compared to control. Preincubation with 5b(1–4)
(10
lM) increased cell viability to 88%, 58%, 60% and 63%, respec-
2.3. Morphological evaluation of apoptosis
tively, compared to control.
Compounds 5a(1–2) and 5b(1–4) alone did not show any toxic-
ity effect at 0.5–10 lM in both undifferentiated and differentiated
PC12 cells after 24 h (Fig. 1A and C).
According to the chemical structure of compounds, it seems
that there is an inverse correlation between the number of carbon
Acridine orange/ethidium bromide (AO/EB) staining discrimi-
nates live cells from dead ones on the basis of membrane integrity.
AO is a cell-permeable nucleic acid selective cationic dye which is
taken up by both viable and nonviable cells and emits green fluo-
rescence if intercalates into double stranded nucleic acid (DNA). EB

4226
H. Irannejad et al. / Bioorg. Med. Chem. 18 (2010) 4224–4230
Differentiated PC12 cells
120
100
80
60
40
20
0
0.5 μM
1 μM
5 μM
10 μM
15 μM
20 μM
A
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
##
##
##
5a-1
5a-2
5a-3
5a-4
5b-1
5b-2
5b-3
5b-4
Control
Differentiated PC12 cells
100
80
60
40
20
0
B
0.5 μM
1 μM
5 μM
10 μM
15 μM
20 μM
##
##
##
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
***
#
#
#
#
#
#
#
#
Undifferentiated PC12 cells
120
100
80
60
40
20
0
C
0.5 μM
1 μM
5 μM
#
#
#
#
#
#
10 μM
15 μM
20 μM
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
##
##
##
5a-1
5a-2
5a-3
5a-4
5b-1
5b-2
5b-3
5b-4
Control
Undifferentiated PC12 cells
100
80
60
40
20
0
0.5 μM
1 μM
D
##
##
##
5 μM
10 μM
15 μM
20 μM
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
#
***
#
#
#
#
#
#
#
#
Figure 1. Effect of triazine derivatives on cell viability and morphology. Differentiated and/or undifferentiated PC12 cells were treated with different concentrations (0.5–
20 M) of compounds for 3 h. After 24 h, cell viability was determined by MTT assay in the absence (A and C) and/or presence (B and D) of H2O2. (E) Morphological evaluation
l
of differentiated PC12 cells after 24 h exposure to H2O2 in the presence of triazine derivatives (10 lM). Viability was calculated as the percentage of living cells in treated
cultures compared to those in control cultures. Each value represents the mean S.D (n = 3). *Significantly different from untreated cells. #Significantly different from H2O2-
treated cells. (B) Morphological evaluation of PC12 cells.
intercalates and stains DNA, providing a red–orange fluorescent.
Although it does not stain healthy cells, it can be used to identify
cells that are in the early or final stages of apoptosis which have
much more permeable membranes. The result obtained from AO/
EB double staining is represented in Figure 2. Four types of cells
could be distinguished according to the fluorescence emission
and the morphological aspect of chromatin condensation in the
stained nuclei: (1) Viable cells which have uniform bright green

H. Irannejad et al. / Bioorg. Med. Chem. 18 (2010) 4224–4230
4227
Fig. 1 (continued)
Figure 2. Morphological evaluation of cells, using AO/EB double staining. Differentiated PC12 cells were exposed to triazine derivatives (10
lM) for 3 h followed by exposure
to 150 lM H2O2 for 24 h. The morphological patterns of apoptotic cells are described in the text.

4228
H. Irannejad et al. / Bioorg. Med. Chem. 18 (2010) 4224–4230
nuclei with organized structure. (2) Early apoptotic cells (which
still have intact membranes but have started to undergo DNA
cleavage) have green nuclei, but perinuclear chromatin condensa-
tion is visible as bright green patches or fragments. (3) Late apop-
totic cells which have orange to red nuclei with condensed or
fragmented chromatin. (4) Necrotic cells which have uniformly
red nuclei. Quantitative analysis of the stained cells indicated that
pretreatment of the cells with 5a-1 and 5a-2 has significantly de-
creased the extent of cell apoptosis to about 35–45% from 70% ob-
served among the cells exposed solely to H2O2. Preincubation of
the cells with 5b-1 prevented the extent of apoptosis to about
20%, while 5b(2–4) pretreatment decreased apoptotic proportion
to about 40–50%. The difference observed between MTT results
and the proportion of apoptosis was obtained from AO/EB staining
test shows the involvement of necrosis in this process.
2.5. Inhibition of NF-jB nuclear translocation in PC12 cells
Although many signals and metabolic events are important in
the regulation of cell death, the intracellular redox level, in partic-
ular, has been shown to play a critical role. Redox regulation is an
interesting and important issue which tightly associates with oxi-
dative stress. Redox-sensitive NF-
tive stress and regulates the expression of many cellular signaling
molecules.25 The link between oxidative stress and NF-
B was
established mainly from the inhibition of NF- B activation by cel-
lular oxidants.26,27 NF-
B is normally located in the cytosol, bound
and inhibited by I- B. Following oxidative stress, NF- B is released,
jB is increased in states of oxida-
j
j
j
j
j
translocates to the nucleus and activates transcription of down-
stream inflammatory mediators. Since H2O2 was first suggested
as an endogenous NF-
azine derivatives on NF-
found that in those cells exposed to H2O2, the level of NF-
j
B activator, investigation of the effect of tri-
B inactivation was of our interest. We
B in nu-
j
2.4. Inhibition of caspase-3 activation in neuron-like PC12 cells
j
cleus in comparison to control was increased 6.02-fold, as deter-
mined by western blot (Fig. 4). This increase was prevented by
5a-1 and 5a-2 by about 1.79-and 1.56-fold, compared to H2O2-
treated cells, respectively. Interestingly, 5b-1, 5b-2, 5b-3, and 5b-
4 treatments resulted in 4.04-, 2.00-, 1.89-, and 1.78-fold reduction
Another important finding, which confirmed the protective ef-
fect of these derivatives, was the data obtained from western blot
analysis of procaspase-3 and caspase-3. The progress of apoptosis
is regulated by a series of signal cascades. Caspases are cysteine-
dependent enzymes that play central role in the induction, trans-
duction and amplification of intracellular apoptotic signals and
previous studies have demonstrated efficacy of caspase inhibitors
in preventing H2O2-induced apoptosis in PC12 cells.23,24 To verify
whether these triazine derivatives interfere apoptosis via sup-
pressing caspase-3, the level of cleaved caspase-3 was measured
by western blot analysis in the presence of compounds. As shown
in Figure 3, H2O2-induced the appearance of cleaved active cas-
pase-3, showing the involvement of caspase-3 in H2O2-induced cell
death in PC12 neurons. In the cells pretreated with 5a(1–2), band
of cleaved (active) caspase-3 was weaker compared to H2O2-trea-
ted cells by about 1.91- and 1.64-fold, respectively. Caspase-3 level
was decreased to 4.59-, 2.00-, 1.92-, and 1.83-fold, compared to
H2O2-treated cells by 5b-1, 5b-2, 5b-3, and 5b-4, respectively,
demonstrating the ability of these compounds to suppress oxida-
tive stress-induced apoptosis in differentiated PC12 cells.
of NF-jB, respectively, compared to H2O2. This inhibition is associ-
ated with the prevention of cell death induced by H2O2.
2.6. Inhibition of H2O2-induced ROS generation
Many studies have shown that oxidative stress is a major cause
of cellular injuries in a variety of human diseases including neuro-
degenerative disorders. ROS such as hydrogen peroxide, superox-
ide anion, and hydroxyl radical readily damage biological
molecules, which can ultimately lead to apoptotic or necrotic cell
death.5,4 Thus, removal of excess ROS or suppression of their gen-
eration may be effective in preventing oxidative cell death.
ROS levels produced by H2O2 were determined based on DCF
fluorescence. The fluorescence intensity of DCF in cells treated with
H2O2 (150
lM) was increased by 5.07-fold compared to untreated
control cells. Pretreatment of the cells with 5a(1–2) (10
lM) pre-
vented the accumulation of ROS by 1.50- and 1.62-fold, respec-
A
A
35 kDa
17 kDa
Procaspase-3
Caspase-3
β-actin
NF-κB
65 kDa
45 kDa
β-actin
45 kDa
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Procaspase-3
Caspase-3
1.4
B
##
B
**
1.2
1
**
#
#
#
#
#
#
#
#
#
0.8
0.6
0.4
0.2
0
#
#
#
#
#
#
##
**
##
Figure 4. Western blot analysis to measure the effects of triazine derivatives on the
nuclear levels of NF- B in PC12 cells. (A) Differentiated PC12 cells were pretreated
with compounds for 3 h and then exposed to H2O2 for 24 h. Twenty micrograms of
nuclear proteins were separated on SDS–PAGE, western blotted, probed with anti-
Figure 3. Caspase-3 activity in PC12 cells pretreated with triazine derivatives.
Differentiated PC12 cells were pretreated with triazine derivatives for 3 h and then
exposed to H2O2 for 24 h. Twenty micrograms of proteins were separated on SDS–
PAGE, western blotted, probed with anti-caspase antibody and reprobed with anti-
b-actin antibody. (One representative western blot was shown; n = 3). (B) The
densities of corresponding bands were measured and the ratio was calculated. The
j
NF-
j
B
antibody and reprobed with anti-b-actin antibody. (One representative
B bands were measured
western blot was shown; n = 3). (B) The densities of NF-
j
and the ratio was calculated. The median of three independent experiments is
*
*
median of three independent experiments is shown. Significantly different from
shown. Significantly different from untreated cells. #Significantly different from
untreated cells. #Significantly different from H2O2-treated cells.
H2O2-treated cells.

H. Irannejad et al. / Bioorg. Med. Chem. 18 (2010) 4224–4230
4229
350
300
250
200
150
100
50
added and the mixture was stirred for 2 h at room temperature.
The solvent was removed under reduced pressure. Water (20 ml)
and dichloromethane (50 ml) were added to the mixture. The or-
ganic phase was separated, dried (sodium sulfate), filtered and
evaporated under reduced pressure and the residue was crystal-
lized from methanol–water to yield 3-thioalkyl-5,6-diaryl-1,2,4-
triazines. The spectroscopic and analytical data of compounds are
as follows:
***
#
#
#
#
#
##
0
4.2.1.1. 3-Thiomethyl-5,6-diphenyl-1,2,4-triazine (5a-1). Mp: 89–
91 °C, 1H NMR (CDCl3) d: 2.70 (s, 3H, SCH3), 7.30–7.45 (m, 6H),
7.50–7.58 (m, 4H). Mass: m/z (%), 279 (M+, 7), 178 (25), 176
(100), 100 (5), 85 (15), Anal. Calcd for C16H13N3S: C, 68.79; H,
4.69; N, 15.04. Found: C, 68.57; H, 4.73; N, 14.96.
Figure 5. Inhibitory effect of compounds on accumulation of intracellular ROS.
DifferentiatedPC12cellswereincubated3 hwithtriazinederivatives(10 M)andthen
l
exposed to H2O2 for 24 h. Intracellular levels of reactive oxygen species were measured
with DCFH-DA. The median of three independent experiments is shown. *Significantly
different from control cells. #Significantly different from H2O2-treated cells.
4.2.1.2. 3-Thioethyl-5,6-diphenyl-1,2,4-triazine (5a-2). Mp: 70–
72 °C, 1H NMR (CDCl3) d: 1.52 (t, J = 7.5 Hz, 3H, SCH2–CH3), 3.37 (q,
J = 7.5 Hz, 2H, SCH2–CH3), 7.28–7.48 (m, 6H), 7.55–7.62 (m, 4H).
Mass: m/z (%), 293 (M+, 5), 178 (100), 176 (30), Anal. Calcd for
C17H15N3S: C, 69.59; H, 5.15; N, 14.32. Found: C, 69.48; H, 4.99;
N, 14.58.
tively, compared to H2O2-treated cells (Fig. 5). Preincubation of the
cells with 5b(1–4) (10 lM) reduced intracellular ROS by 2.26-,
1.46-, 1.53-, and 1.60-fold, respectively, compared to H2O2-treated
cells. It seems that in this signaling pathway ROS act as second
messengers. Because, ROS have been associated with neurodegen-
erative disease and recent studies have demonstrated that ROS,
over a narrow concentration range, might function as second mes-
sengers in cell-signaling pathways.3 Several data have shown that
4.2.1.3.
3-Thio-n-propyl-5,6-diphenyl-1,2,4-triazine
(5a-
3). Mp: 69–71 °C, 1H NMR (CDCl3) d: 1.10 (t, J = 7.4 Hz, 3H,
SCH2–CH2–CH3), 1.88–1.98 (m, 2H, SCH2–CH2–CH3), 3.31 (q,
J = 7.5 Hz, 2H, SCH2–CH2–CH3), 7.25–7.47 (m, 6H), 7.48–7.60 (m,
4H). Mass: m/z (%), 307 (M+, 8), 264 (12), 178 (20), 176 (100),
152 (5), 105 (37), 77 (19), Anal. Calcd for C18H17N3S: C, 70.33; H,
5.57; N, 13.67. Found: C, 70.51; H, 5.65; N, 13.75.
ROS activates NF-
protease, can be activated by ROS. So the suppressive effect of
our compounds on translocation of NF- B and also the expression
of active forms of caspase-3 shows that these triazine derivatives
suppress NF- B activating via suppression of ROS production.
j
B.28 In addition, caspase-3 a family of cysteine
j
j
4.2.1.4. 3-Thio-n-butyl-5,6-diphenyl-1,2,4-triazine (5a-4). Mp:
45–47 °C, 1H NMR (CDCl3) d: 0.81 (t, J = 7.3 Hz, 3H, SCH2–CH2–
CH2–CH3), 1.28–1.38 (m, 2H, SCH2–CH2–CH2–CH3), 1.78–1.96 (m,
2H, SCH2–CH2–CH2–CH3), 3.40 (q, J = 7.5 Hz, 2H, SCH2–CH2–CH2–
CH3), 7.34–7.48 (m, 6H), 7.51–7.59 (m, 4H). Mass: m/z (%), 321
(M+, 10), 264 (7), 178 (35), 176 (100), 149 (5), 85 (15), Anal. Calcd
for C19H19N3S: C, 70.99; H, 5.96; N, 13.07. Found: C, 71.09; H, 5.77;
N, 12.85.
3. Conclusion
In summary, our results clearly indicate that triazine derivatives
prevent the H2O2-induced cell death in differentiated PC12 cells. It
seems that in this cytoprotection, inhibitions of caspase-3 and NF-
jB via suppression of ROS production are the main factors which
protect neurons against oxidative stress-induced apoptosis. How-
ever, the complete molecular milieu that links all these events need
to be elucidated. A further study of the detailed mechanisms is now
in progress in our laboratory that will pave the way to exploiting pre-
ventive or therapeutic strategies for neurodegenerative diseases.
4.2.1.5. 3-Thiomethyl-5,6-(4-dicholrophenyl)-1,2,4-triazine (5b-
1). Mp: 137–139, °C 1H NMR (CDCl3) d: 2.70 (s, 3H, SCH3), 7.34 (d,
J = 9.0 Hz, 2H), 7.37 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 9.0 Hz, 2H), 7.50
(d, J = 8.5 Hz, 2H). Mass: m/z (%), 347 (M+, 17), 245 (100), 176 (45),
123 (15), 63 (42), Anal. Calcd for C16H11Cl2N3S: C, 55.09; H, 3.18; N,
12.05. Found: C, 55.39; H, 3.24; N, 11.89.
4. Experimental
4.2.1.6. 3-Thioethyl-5,6-(4-dichlorophenyl)-1,2,4-triazine (5b-
2). Mp: 115–117 °C, 1H NMR (CDCl3) d: 1.48 (t, J = 7.4 Hz, 3H,
SCH2–CH3), 3.39 (q, J = 7.4 Hz, 2H, SCH2–CH3), 7.38 (d, J = 9.0 Hz,
2H), 7.40 (d, J = 8.5 Hz, 2H), 7.45 (d, J = 9.0 Hz, 2H), 7.50 (d,
J = 8.5 Hz, 2H). Mass: m/z (%), 361 (M+, 9), 247 (65), 245 (100),
209 (5), 175 (20), Anal. Calcd for C17H13Cl2N3S: C, 56.36; H, 3.62;
N, 11.60. Found: C, 56.45; H, 3.78; N, 11.75.
4.1. Instrumentation and chemicals
Melting points were determined with a Reichert-Jung hot-stage
microscope and are uncorrected. 1H NMR (500 MHz) spectra were
recorded on a Brucker spectrometer using CDCl3 or DMSO-d6 as
solvent. Chemical shifts (d) are reported in ppm relative to TMS
as internal standard. Infrared spectra were acquired on a Nicolet
Magna 550-FT spectrometer. Mass spectra were obtained with a
Finnigan Mat TSQ-70 spectrometer. Elemental microanalyses were
within 0.4% of the theoretical values for C, H, and N.
4.2.1.7. 3-Thio-n-propyl-5,6-(4-dichlorophenyl)-1,2,4-triazine
(5b-3). Mp: 88–90 °C, 1H NMR (CDCl3) d: 1.15 (t, J = 7.5 Hz, 3H,
SCH2–CH2–CH3), 1.88–1.95 (m, 2H, SCH2–CH2–CH3), 3.30 (q,
J = 7.5 Hz, 2H, SCH2–CH2–CH3), 7.30 (d, J = 9.1 Hz, 2H), 7.37 (d,
J = 8.3 Hz, 2H), 7.45 (d, J = 9.1 Hz, 2H), 7.48 (d, J = 8.3 Hz, 2H). Mass:
m/z (%), 375 (M+, 5), 333 (9), 248 (64) 246 (100), 176 (35), Anal.
Calcd for C18H15Cl2N3S: C, 57.45; H, 4.02; N, 11.17. Found: C,
57.29; H, 4.19; N, 10.87.
Antibodies directed against caspase-3, NF-jB, and b-actin were
obtained from Cell Signaling Company. All the other reagents, un-
less otherwise stated, were from Sigma–Aldrich (St. Louis, MO).
4.2. Synthesis
4.2.1. General procedures for preparation of triazine derivatives
(5)
To a stirring solution of compound 4 (1 mmol) in methanol
(50 ml), alkyl iodide (1.2 mmol), and triethylamine (1 ml) were
4.2.1.8. 3-Thio-n-butyl-5,6-(4-dicholorophenyl)-1,2,4-triazine
(5b-4). Mp: 90–91 °C, 1H NMR (CDCl3) d: 0.80 (t, J = 7.5 Hz, 3H,
SCH2–CH2–CH2–CH3), 1.31–1.40 (m, 2H, SCH2–CH2–CH2–CH3),

4230
H. Irannejad et al. / Bioorg. Med. Chem. 18 (2010) 4224–4230
1.78–1.98 (m, 2H, SCH2–CH2–CH2–CH3), 3.40 (q, J = 7.4 Hz, 2H,
SCH2–CH2–CH2–CH3), 7.31 (d, J = 9.0 Hz, 2H), 7.38 (d, J = 8.5 Hz,
2H), 7.45 (d, J = 9.0 Hz, 2H), 7.50 (d, J = 8.3 Hz, 2H). Mass: m/z (%),
389 (M+, 42), 332 (30), 247 (98), 208 (18), 176 (100), 100 (5), 85
(15), Anal. Calcd for C19H17Cl2N3S: C, 58.46; H, 4.39; N, 10.76.
Found: C, 58.47; H, 4.23; N, 10.96.
this purpose, the DCFH-DA solution (10 lM) was added to the
suspension of differentiated PC12 cells (1 ꢀ 106/ml) and the mix-
ture was incubated at 37 °C for 1 h. The cells were then washed
twice with PBS and finally, the fluorescence intensity was mea-
sured by Varian-spectrofluorometer, model Cary Eclipse with
excitation and emission wavelengths of 485 nm and 530 nm,
respectively.
4.3. Cell culture, differentiation, and treatment conditions
4.8. Data analysis
Rat pheochromocytoma (PC12) cells obtained from Pasteur
Institute (Tehran, Iran) were grown in Dulbecco’s modified Eagle’s
medium (DMEM) (Sigma, Aldrich), supplemented with 10% fetal
bovine serum and 1% antibiotic mixture comprising penicillin–
streptomycin, in a humidified atmosphere at 37 °C with 5% CO2.
Growth medium was changed three times a week. The cells were
differentiated by treating with nerve growth factor (NGF) (50 ng/
ml) every other day for 6 days.
All data are represented as the mean S.D. Comparison be-
tween groups was made by one-way analysis of variance (ANOVA)
followed by a specific post-hoc test to analyze the difference. The
statistical significances were achieved when P <0.05 (* P <0.05,
**P <0.01, and ***P <0.001).
Acknowledgments
Differentiated PC12 cells were pretreated with different con-
centrations (0.5–20
bated with H2O2 (150
of compounds, for an additional 24 h.
l
M) of triazine derivatives for 3 h and incu-
M.A. and F.K. thank National Elite Fund, Iran, for the award of
Young Scientist Research Fellowship. This work was supported
partially by Shahid Beheshti University of Medical Sciences Re-
search Funds, Tehran University of Medical Sciences Research
Council and INSF (Iran National Sciences Foundation). The
authors thank Fatemeh Shaerzadeh for her excellent technical
assistance.
lM), as an oxidative agent, in the presence
4.4. Measurement of cell viability
Cell viability was determined by the conventional MTT (3-[4,5-
dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction
assay. The dark blue formazan crystals formed in intact cells were
solubilized in dimethyl sulfoxide (DMSO) and the absorbance was
measured at 550 nm. Results were expressed as the percentages
of reduced MTT, assuming the absorbance of control cells as 100%.
References and notes
1. Götz, M. E.; Künig, G.; Riederer, P.; Youdim, M. B. Pharmacol. Ther. 1994, 63,
37.
2. Butterfield, D. A.; Drake, J.; Pocernich, C. B.; Castegna, A. Trends Mol. Med. 2001,
7, 548.
3. Behl, C.; Davis, J. B.; Lesley, R.; Schubert, D. Cell 1994, 77, 817.
4. Halliwell, B.; Aruoma, O. I. FEBS Lett. 1991, 281, 9.
5. Yoshikawa, A.; Saito, Y.; Maruyama, K. Biochem. Biophys. Res. Commun. 2006,
344, 394.
6. Zingarelli, B.; Sheehan, M.; Wong, H. R. Crit. Care Med. 2003, 31, 105.
7. Janssen-Heininger, Y. M.; Poynter, M. E.; Baeuerle, P. A. Free Radical Biol. Med.
2000, 28, 1317.
8. Akama, K. T.; Van Eldik, L. J. J. Biol. Chem. 2000, 275, 7918.
9. Karin, M.; Lin, A. Nat. Immunol. 2002, 3, 221.
10. Jang, J. H.; Surh, Y. J. Free Radical Biol. Med. 2005, 38, 1604.
11. Kutuk, O.; Basaga, H. Free Radical Res. 2003, 37, 1267.
12. Bekircan, O.; Küxük, M.; Kahveci, B.; Kolayli, S. Arch. Pharm. (Weinheim) 2005,
338, 365.
13. Misra, U.; Hitkari, A.; Saxena, A. K.; Gurtu, S.; Shanker, K. Eur. J. Med. Chem.
1996, 31, 629.
14. Brzozowski, Z.; Saczewski, F. Eur. J. Med. Chem. 2002, 37, 709.
15. Iwashita, A.; Maemoto, T.; Nakada, H.; Shima, I.; Matsuoka, N.; Hisajima, H. J.
Pharmacol. Exp. Ther. 2003, 307, 961.
4.5. AO/EB double staining
Apoptosis was determined morphologically after staining the
cells with AO/EB followed by fluorescence microscopy inspection.
Briefly, differentiated PC12 cells were seeded in a 96-well plate
and were treated with triazine derivatives (10
lM) followed by
adding H2O2 (150 M). After 24 h, the cells were harvested and
l
washed three times with PBS and were adjusted to a density of
106 cells/ml of PBS. AO/EB solution (1:1 v/v) was added to the cell
suspension in a final concentration of 100 lg/ml. The cellular mor-
phology was evaluated by fluorescence microscope (Zeiss, Ger-
many). All experiments were repeated three times and the
number of stained cells was counted in 10 randomly selected
fields.
16. Kim, W.; Kim, Y.; Min, J.; Kim, D. J.; Chang, Y. T.; Hecht, M. H. ACS Chem. Biol.
2006, 1, 461.
17. Hunt, J. C. A.; Briggs, E.; Clarke, E. D.; Whittingham, W. G. Bioorg. Med. Chem.
Lett. 2007, 17, 5222.
4.6. Western blot analysis
The total proteins were electrophoresed in 12% SDS–PAGE gel,
transferred to polyvinylidene fluoride membranes and probed with
specific antibodies. Immunoreactive polypeptides were detected
by chemiluminescence using enhanced ElectroChemiLumines-
cence (ECL) reagents (Amersham Bioscience, USA) and subsequent
autoradiography. Quantification of the results was performed by
densitometric scan of films. Data analysis was done by Image.J.
Protein concentrations were determined according to the Brad-
ford’s method.29 A standard plot was generated by using bovine
serum albumin. Nuclear and cytoplasmic proteins were isolated
as described by Kutuk and Basaga.11
18. Saxena, S.; Verma, M.; Saxena, A. K.; Shanker, K. Arzneim.-Forsch./Drug Res.
1994, 44, 766.
19. Adams, R.; Marvel, C. S. Org. Synth. Coll. 1941, 1, 94.
20. Clark, H. T.; Dreger, E. E. Org. Synth. Coll. 1941, 1, 87.
21. Guroff, G. In Cell Culture in Neurosciences; Bottenstein, J. E., Ed.; Plenum Press:
New York, 1985; pp 245–272.
22. Tamango, E.; Bardini, P.; Obbili, A.; Vitali, A.; Borghi, R.; Zaccheo, D. J. Neurobiol.
Dis. 2002, 10, 279.
23. Miller, D. K. Semin. Immunol. 1997, 9, 35.
24. Chen, L.; Liu, L.; Yin, J.; Luo, Y.; Huang, S. Int. J. Biochem. Cell Biol. 2009, 41,
1284.
25. Zingarelli, B. Crit. Care Med. 2005, 33, 414.
26. Eftekharzadeh, B.; Khodagholi, F.; Abdi, A.; Maghsoudi, N. Carbohydr. Polym.
2010, 79, 1063.
27. Guzhova, I. V.; Darieva, Z. A.; Melo, A. R.; Margulis, B. A. Cell Stress Chaperones
1997, 2, 132.
4.7. Measurement of intracellular ROS
28. Ruiz-Ramos, R.; Lopez-Carrillo, L.; Rios-Perez, A. D.; De Vizcaya-Ruíz, A.;
Cebrian, M. E. Mutat. Res. 2009, 674, 109.
The fluorescent probe 20,70-dichlorofluorescein diacetate (DCF-
DA) was used to monitor intracellular accumulation of ROS. For
29. Bradford, M. M. Anal. Biochem. 1976, 72, 248.