Synthesis and neuroprotective activity of novel 1,2,4-triazine derivatives with ethyl acetate moiety against H2O2 and Aβ-induced neurotoxicity

Article abstract of DOI:10.1007/s00044-017-2003-x

A series of 5,6-diaryl-1,2,4-triazine-3-thioacetate derivatives 3a–f, 8a–d and their regioisomer 8e were synthesized. Neuroprotective activity of compounds was assessed against H₂O₂ and β-amyloid-induced toxicity in PC12 and SH-SY5Y cells respectively. Surprisingly, ethyl 2-(5-(4-chlorophenyl)-6-(4-methoxyphenyl)-3-thioxo-1,2,4-triazin-2(3H)-yl)acetate (8e) was the most potent compound in both tests with EC₅₀ of 14 μM in H₂O₂ induced apoptosis and also could increase 40% of cell viability revealed by cytometric analysis with Annexin V/PI staining. It was also shown that regioisomer 8e has more neuroprotective activity than Quercetin in β-amyloid induced toxicity. Morphologic evaluation of cells by DAPI staining and TUNEL assay showed the effectiveness of this compound to improve neurite outgrowth in neuronal cells.

Full text of DOI:10.1007/s00044-017-2003-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2003-x
ORIGINAL RESEARCH
Synthesis and neuroprotective activity of novel 1,2,4-triazine
derivatives with ethyl acetate moiety against H2O2 and Aβ-induced
neurotoxicity
Tuba Tuylu Kucukkilinc1 Kamaledin Safari Yanghagh2 Beyza Ayazgok1
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Mohammad Ali Roknipour2 Farshad Homayouni Moghadam3 Alireza Moradi4
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5
Saeed Emami5 Mohsen Amini6 Hamid Irannejad
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Received: 21 March 2017 / Accepted: 25 July 2017
© Springer Science+Business Media, LLC 2017
Abstract A series of 5,6-diaryl-1,2,4-triazine-3-thioacetate
derivatives 3a–f, 8a–d and their regioisomer 8e were syn-
thesized. Neuroprotective activity of compounds was
assessed against H2O2 and β-amyloid-induced toxicity in
PC12 and SH-SY5Y cells respectively. Surprisingly, ethyl
2-(5-(4-chlorophenyl)-6-(4-methoxyphenyl)-3-thioxo-1,2,4-
triazin-2(3H)-yl)acetate (8e) was the most potent compound
in both tests with EC50 of 14 µM in H2O2 induced apoptosis
and also could increase 40% of cell viability revealed by
cytometric analysis with Annexin V/PI staining. It was also
shown that regioisomer 8e has more neuroprotective
activity than Quercetin in β-amyloid induced toxicity.
Morphologic evaluation of cells by DAPI staining and
TUNEL assay showed the effectiveness of this compound
to improve neurite outgrowth in neuronal cells.
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Keywords Synthesis 1,2,4-triazine Alzheimer’s disease
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Apoptosis Neuroprotective activity
Introduction
Alzheimer’s disease (AD) is a complex pathologic disorder
characterized by intracellular neurofibrillary tangles and
extracellular amyloid aggregates in the CNS tend to neu-
rodegeneration, impairment to memory and cognitive defi-
cits (Butterfield 2002). Pathological deterioration in AD is
substantially associated to the overproduction of β-amyloid
(Aβ) peptide that is generated by the successive action of β
and γ-secretases on the amyloid precursor protein (APP)
(Nunan and Small 2000). Two variants of β-amyloids are
normally found in the brain of AD patients: Aβ (1–40) and
Aβ (1–42), in which β-amyloid (1–42) is the most dominant
and toxic form of β-amyloid in AD (Findeis 2007). β-
amyloid peptides are highly prone to self-aggregation and
form fibrillar plaques in neuronal cells. Accordingly, β-
amyloid aggregates in AD has been associated with over-
production of reactive oxygen species (ROS) and radicals
which damage cellular and biological macromolecules like
DNA and protein oxidation that usually leads to acute
inflammation, necrosis and eventually apoptosis (Heneka
et al. 2010; Pimplikar 2009).
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-017-2003-x) contains supplementary material,
which is available to authorized users.
* Hamid Irannejad
irannejadhamid@gmail.com
1
Department of Biochemistry, Faculty of Pharmacy, Hacettepe
University, Sihhiye, 06100 Ankara, Turkey
2
Student Research Committee, Faculty of Pharmacy, Mazandaran
University of Medical Sciences, Sari, Iran
3
Department of Cellular Biotechnology at Cell Science Research
Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
4
Department of Medicinal Chemistry, Faculty of Pharmacy and
Pharmaceutical Sciences Research Center, Shahid Sadoughi Yazd
University of Medical Sciences, Yazd, Iran
5
Department of Medicinal Chemistry, Pharmaceutical Sciences
In recent years, numerous approaches have been used to
combat AD through different mechanisms like small
molecule inhibitors of β-amyloid aggregation (Geng et al.
2011), anti-inflammatory agents (Cheng et al. 2015), inhi-
bitors of cyclooxygenase (Manev et al. 2011),
Research Center, Faculty of Pharmacy, Mazandaran University of
Medical Sciences, Sari, Iran
6
Department of Medicinal Chemistry, Drug Design & Development
Research Center, Faculty of Pharmacy, Tehran University of
Medical Sciences, Tehran, Iran

Med Chem Res
cholinesterase (Raina et al. 2008), β-secretase, and γ-
secretase (Ghosh and Osswald 2014).
Recently, we synthesized 1,2,4-triazine-3-thioacetate
derivatives (Fig. 1c) and evaluated their COX-2 inhibitory
activity and β-amyloid disaggregation ability (Dadashpour
et al. 2015). The obtained results indicated that these
compounds act as highly β-amyloid destabilizing agents,
dissolving 80–95% of β-amyloid aggregates in 24 h. Herein,
we report synthesis of some symmetrical and unsymme-
trical 5,6-diaryl-1,2,4-triazines 3a–f and 8a–e (Fig. 2) as
potential agents for treatment of AD. We evaluated them
against both H2O2 and β-amyloid induced toxicity in PC-12
(pheochromocytoma of the rat adrenal medulla) and SH-
SY5Y (human neuroblastoma) cells and the extent of cell
viability and apoptosis were assessed during 24 and 48 h of
treatment.
Triazine-based compounds have been an interesting
scaffold for design and development of anti-Alzheimer’s
drugs. Previously, some s-triazine derivatives were syn-
thesized and evaluated as inhibitors of acetylcholinesterase
(AChE) and β-amyloid aggregates (Veloso et al. 2013).
Moreover, triazine derivatives have been found to attenuate
the ROS production, caspase-3 activation and the extent of
apoptotic cell death induced by β-amyloid (1–42) (Shay-
khalishahi et al. 2010).
Particularly, investigation on 5,6-diaryl-1,2,4-triazine
derivatives against H2O2-induced apoptosis in a neuronal
cell line showed the ability of these compounds to inhibit
oxidative stress-mediated phosphorylation of MAP kinases
and improvement of cells neurite outgrowth (Khodagholi
et al. 2012a, b). Further studies by our research group on the
protective effect of the same analogs (Fig. 1a) against H2O2-
induced apoptosis in PC12 cells showed the increasing level
of stress sensing transcription factor, NF-E2 related factor 2
and NF-κB which both contribute to cell survival. We have
also showed that 5,6-diaryl-1,2,4-triazine compounds are
able to suppress the expression of cyclooxygenase-2 which
is one of the inflammatory key enzymes involved in AD
progression (Ansari et al. 2010; Irannejad et al. 2010). It
was also shown that elevated levels of glutathione perox-
idase-1, glutamylcysteine synthetase, and glutathione, as
well as superoxide dismutase and catalase affected by 5,6-
diaryl-1,2,4-triazine derivatives, increased antioxidant
capacity of cells (Khodagholi et al. 2012a, b; Khoramian
Tusi et al. 2010). Some 3-substituted-5,6-diphenyl-1,2,4-
triazine derivatives (Fig. 1b) have been also introduced as
neuroprotective agents through inhibition of cholinesterase
enzyme (Sinha et al. 2015).
Material & methods
Chemistry
All commercially available reagents were purchased from
Merck or Aldrich and used without further purification.
Column chromatography was carried out on silica gel
(230–400 mesh). Thin layer chromatography (TLC) was
conducted on silica gel F254 plates. Infrared (IR) spectra
were taken using Perkin-Elmer Fourier transform infrared
(FT-IR) (KBr disks). Mass spectra were obtained on an
Agilent 5937 Mass Selective Detector. Nuclear magnetic
resonance (NMR) spectra were recorded on a Bruker 400 or
500 MHz instruments. The chemical shifts (δ) and coupling
constants (J) are expressed in ppm and Hertz (Hz),
respectively. Elemental analyzes were carried out by ECS-
4010 (Costech International S.p.A.) elemental analyzer. The
Fig. 1 Structures of previously
reported compounds as a potent
neuroprotective agents against
LPS and H2O2-induced toxicity
on PC12 cells. b Neuroprotective
agents through cholinesterase
inhibition. c COX-2 and β-
amyloid aggregation inhibitors
Fig. 2 Structures of newly
synthesized compounds as
neuroprotective agents against
H2O2 and β-amyloid induced
toxicity

Med Chem Res
results of elemental analyzes (C, H, N) were within 0.4%
of the calculated values.
130.02, 132.25, 132.45, 139.56, 141.56, 154.07, 155.42,
168.77, 168.81. MS, m/z (%): 379.1 (M+, 38), 334.0 (24),
238.0 (27), 206.0 (100), 189.0 (34), 119.0 (83). Anal. calcd.
for C21H21N3O2S: C, 66.47; H, 5.58; N, 11.07. Found: C,
66.51; H, 5.62; N, 11.09.
General procedure for the synthesis of compounds 2a–f
To a stirred solution of appropriate 1,2-diketone 1a–f
(1 mmol) and thiosemicarbazide (1.1 mmol) in a mixture of
water and ethanol (1:1), was added a few drops of con-
centrated hydrochloric acid and resulting mixture was irra-
diated under microwave at 130 °C for 20 min. After cooling
to room temperature, it was filtered and washed with water
to give the desired product. This procedure in detail along
with structural characterization data of compounds has been
reported in our recently published study (Irannejad et al.
2014, 2015).
Ethyl 2-((5,6-bis(4-methoxyphenyl)-1,2,4-triazin-3-yl)thio)
acetate (3c) Yield 75 %; IR (KBr, cm−1) υmax: 1730
1
(C=O). H-NMR (400 MHz, CDCl3) δ (ppm): 1.28 (t, J =
7.2 Hz, 3H, CH2CH3), 3.83 (s, 3H, OCH3), 3.85 (s, 3H,
OCH3), 4.1 (s, 2H, SCH2CO), 4.23 (q, J = 7.2 Hz, 2H,
CH2CH3), 6.84 (d, J = 8.9 Hz, 2H, Ar–H), 6.91 (d, J = 8.9
Hz, 2H, Ar–H), 7.48 (d, J = 8.9 Hz, 2H, Ar–H), 7.56 (d, J
= 8.9 Hz, 2H, Ar–H). 13C-NMR (100 MHz, CDCl3) δ
(ppm): 14.15, 33.17, 55.32, 55.38, 61.87, 113.93, 114.13,
127.26, 127.84, 130.63, 131.66, 153.50, 154.71, 160.61,
161.98, 168.32, 168.84. MS, m/z (%): 411.1 (M+, 29),
280.9 (34), 238.0 (53), 206.9 (56), 191.0 (33), 162.9 (39),
135 (100). Anal. calcd. for C21H21N3O4S: C, 61.30; H,
5.14; N, 10.21. Found: C, 61.41; H, 5.23; N, 10.29.
General procedure for the synthesis of compounds 3a–f
To a stirred solution of 5,6-diaryl-1,2,4-triazine-3-thiol
derivatives 2a–f (1 mmol) in DMSO (25 ml) was added
ethyl chloroacetate (1.5 mmol) and K2CO3 (2 mmol) and it
was stirred for 10 h under nitrogen atmosphere. After
completion of the reaction, it was diluted by ethyl acetate
and washed with water three times. The organic phase was
dried over sodium sulfate and concentrated under vacuum.
Purification was performed by column chromatography.
Silica gel was used as stationary phase and eluent was
10–20% of ethyl acetate in petroleum ether.
Ethyl 2-((5,6-bis(4-fluorophenyl)-1,2,4-triazin-3-yl)thio)ac-
etate (3d) Yield 51 %, IR (KBr, cm−1) υmax: 1734 (C=O),
1602 (C=N). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.34 (t,
J = 7.12 Hz, 3H, CH2CH3), 4.11 (s, 2H, SCH2CO), 4.24 (q,
J = 7.2 Hz, 2H, CH2CH3), 7.07 (t, J = 8.5 Hz, 2H, Ar), 7.14
(t, J = 8.6 Hz, 2H, Ar), 7.50 (dd, J = 8.8 and 5.28 Hz, 2H,
Ar), 7.56 (dd, J = 8.8 and 5.28 Hz, 2H, Ar). 13C-NMR (125
MHz, CDCl3) δ (ppm): 14.11, 33.17, 61.25, 115.84 (d, J =
10.0 Hz), 116.02 (d, J = 10.0 Hz), 131.27 (d, J = 8.8 Hz),
132.09 (d, J = 8.8 Hz), 132.6 (d, J = 10.0 Hz), 153.02,
154.26, 163.6 (d, J = 250.0 Hz), 164.5 (d, J = 252.5 Hz),
168.56, 169.43. MS, m/z (%): 387.0 (M+, 22), 342.0 (19),
214.0 (100), 192.9 (28), 122.9 (46). Anal. calcd. for
C19H15F2N3O2S: C, 58.91; H, 3.90; N, 10.85. Found: C,
59.06; H, 3.98; N, 10.89.
Ethyl 2-((5,6-di(thiophen-2-yl)-1,2,4-triazin-3-yl)thio)acet-
ate (3a) Yield 86%, IR (KBr, cm−1) υmax: 1743 (C=O).
1H NMR (400 MHz, CDCl3) δ (ppm): 1.3 (t, J = 7.12 Hz,
3H, CH2CH3), 4.08 (s, 2H, SCH2CO), 4.26 (q, J = 7.12 Hz,
2H, CH2CH3), 7.02 (dd, J = 5 and 3.9 Hz, 1H), 7.14 (dd, J
= 5 and 3.6 Hz, 1H), 7.40 (dd, J = 3.6 and 1.1 Hz, 1H),
7.46 (dd, J = 3.9 and 1.0 Hz, 1H), 7.57 (dd, J = 5 and 1.1
Hz, 1H), 7.59 (dd, J = 5 and 1 Hz, 1H). 13C NMR (100
MHz, CDCl3) δ (ppm): 14.19, 33.15, 62.02, 127.69, 128.45,
129.11, 129.27, 132.51, 133.20, 136.38, 138.63, 147.07,
149.08, 168.54, 168.91. MS, m/z (%): 363.0 (M+, 25),
317.9 (9), 263.9 (7), 189.9 (100), 157.9 (12), 144.9 (23),
110.9 (44). Anal. calcd. for C15H13N3O2S3: C, 49.57; H,
3.61; N, 11.56. Found: C, 49.61; H, 3.72; N, 11.69.
Ethyl 2-((5,6-bis(4-chlorophenyl)-1,2,4-triazin-3-yl)thio)ac-
etate (3e) Yield 69%, IR (KBr, cm−1) υmax: 1734 (C=O),
1306 (C=N). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.29 (t,
J = 7.16 Hz, 3H, CH2CH3), 4.11 (s, 2H, SCH2CO), 4.24 (q,
J = 7.12 Hz, 2H, CH2CH3), 7.35 (d, J = 8.6 Hz, 2H, Ar–H),
7.38 (d, J = 8.64 Hz, 2H, Ar–H), 7.47 (d, J = 8.6 Hz, 2H,
Ar–H), 7.5 (d, J = 8.6 Hz, 2H, Ar–H). 13C-NMR (125
MHz, CDCl3) δ (ppm): 14.12, 33.17, 61.96, 129.03, 129.13,
130.60, 131.13, 133.16, 133.36, 136.11, 137.76, 152.88,
154.17, 168.48, 169.66. MS, m/z (%): 421.0 (M++2, 19),
419.0 (M+, 27), 359.9 (24), 316.1 (33), 247.9 (68), 245.9
(100), 137.0 (43), 135.0 (74). Anal. calcd. for
C19H15Cl2N3O2S: C, 54.30; H, 3.60; N, 10.00. Found: C,
54.46; H, 3.78; N, 10.12.
Ethyl 2-((5,6-di-p-tolyl-1,2,4-triazin-3-yl)thio)acetate (3b)
1
Yield 80%, IR (KBr, cm−1) υmax: 1735 (C=O). H NMR
(400 MHz, CDCl3) δ (ppm): 1.25 (t, J = 7.9 Hz, 3H,
CH2CH3), 2.38 (s, 3H, Ar–CH3), 2.39 (s, 3H, Ar–CH3),
4.11 (s, 3H, SCH2CO), 4.24 (q, J = 7.16 Hz, 2H, CH2CH3),
7.14 (d, J = 7.96 Hz, 2H, Ar–H), 7.18 (d, J = 7.88 Hz, 2H,
Ar–H), 7.42 (d, J = 7.96 Hz, 2H, Ar–H), 7.47 (d, J = 8.08
Hz, 2H, Ar–H). 13C NMR (100 MHz, CDCl3) δ (ppm):
14.15, 21.40, 21.51, 33.19, 61.91, 129.20, 129.34, 129.84,

Med Chem Res
Ethyl 2-((5,6-bis(4-bromophenyl)-1,2,4-triazin-3-yl)thio)
11.57 (s, 1H, NOH). 13C-NMR (100 MHz, DMSO-d6) δ
(ppm): 21.33, 56.16, 115.09, 125.98, 128.08, 129.57,
130.03, 131.78, 140.07, 155.23, 164.64, 193.89.
acetate (3f) Yield 76%, IR (KBr, cm−1) υmax: 1724
1
(C=O). H NMR (400 MHz, CDCl3) δ (ppm): 1.29 (t, J =
7.16 Hz, 3H, CH2CH3), 4.11 (s, 2H, SCH2CO), 4.26 (q, J
= 7.12, 2H, CH2CH3), 7.40 (d, J = 8.8 Hz, 2H, Ar–H), 7.42
(d, J = 8.8 Hz, 2H, Ar–H), 7.52 (d, J = 8.8 Hz, 2H, Ar–H),
7.54 (d, J = 8.5 Hz, 2H, Ar–H). 13C NMR (100 MHz,
CDCl3) δ (ppm): 14.16, 33.21, 62.05, 124.45, 126.27,
130.84, 131.31, 132.02, 132.11, 133.54, 133.72, 152.89,
154.31, 168.57, 169.69.MS, m/z (%): 509.8 (M++2, 15),
462.9 (22), 413.9 (18), 335.8 (48), 316.3 (32), 238.0 (23),
159.9 (54). Anal. calcd. for C19H15Br2N3O2S: C, 44.82; H,
2.97; N, 8.25. Found: C, 44.86; H, 3.08; N, 8.29.
2-(4-Fluorophenyl)-2-(hydroxyimino)-1-(4-methoxyphe-
nyl)ethanone (6c) Yield 65%; mp: 125–127 °C; IR (KBr,
1
cm−1) υmax: 1643 (C=O). H-NMR (400 MHz, DMSO-d6)
δ (ppm): 3.85 (s, 3H, OCH3), 7.1 (d, J = 8.96 Hz, 2H,
Ar–H),7.26 (t, J = 8.92 Hz, 2H, Ar–H), 7.51 (dd, J = 8.98
and 5.4 Hz, 2H, Ar–H), 7.81 (d, J = 8.92 Hz, 2H, Ar–H),
11.71 (s, 1H, NOH). 13C-NMR (100 MHz, DMSO-d6) δ
(ppm): 56.19, 115.17, 116.60 (d, J = 21.91 Hz), 127.89,
128.33 (d, J = 8.46 Hz), 131.88, 132.19 (d, J = 8.54 Hz),
154.27, 161.23 (d, J = 251.1 Hz), 164.79, 193.56.
General procedure for the preparation of compounds 5a–d
2-(4-Chlorophenyl)-2-(hydroxyimino)-1-(4-methoxyphe-
nyl)ethanone (6d) Yield 52%; mp: 120 °C; IR (KBr,
1
cm−1) υmax: 1669 (C=O). H-NMR (400 MHz, DMSO-d6)
To a stirred solution of phenylacetic acid derivative (1
mmol), anisole (1.2 mmol) and phosphoric acid (1.2 mmol)
was added trifluoroacetic acid anhydride (4 mmol) and
stirred for 5 min at room temperature. Then crushed ice was
added to the reaction mixture while stirring and the pre-
cipitate was filtered and washed with water and then pet-
roleum ether respectively. The crude product was used in
the next step without further purification.
δ (ppm): 3.84 (s, 3H, OCH3), 7.07 (d, J = 8.96 Hz, 2H,
Ar–H),7.10 (d, J = 9.0 Hz, 2H, Ar–H), 7.82 (d, J = 8. 92
Hz, 2H, Ar–H), 7.96 (d, J = 8.96 Hz, 2H, Ar–H), 11.7 (s,
1H, NOH). 13C-NMR (100 MHz, DMSO-d6) δ (ppm):
56.18, 114.24, 115.16, 116.70, 127.90, 128.37, 131.88,
133.11. 154.28, 164.78, 193.57.
General procedure for the preparation of compounds 6a–d
General procedure for the preparation of compounds 7a–d
To a stirred solution of 1-(4-methoxyphenyl)-2-aryletha-
none 5a–d (1 mmol) in methanol was added a prepared
solution of sodium (1.5 mmol) in methanol and the resulting
mixture was stirred for 5 min. Then butyl nitrite (1 mmol)
was added and stirring was continued for 30 min. Reaction
mixture was monitored by TLC. After completion of the
reaction, it was concentrated under vacuum, diluted with
water and pH was adjusted to neutral by adding HCl 10%.
Aqueous phase was extracted three times with ethyl acetate,
dried and evaporated. The residue was recrystalized from
methanol to give pure compounds 6a–d.
To a stirred solution of 2-hydroxyimino-1-(4-methox-
yphenyl)-2-arylethanone 6a–d (1 mmol) in a mixture of
water and ethanol (2:1), was added thiosemicarbazide (1.5
mmol) and five drops of conc. HCl and the resulting mix-
ture was refluxed for 4 h. After cooling of reaction mixture,
precipitate was filtered and washed with water. Crystal-
lization was performed in ethanol.
General procedure for the preparation of compounds 8a–e
To a stirred solution of 6-(4-methoxyphenyl)-5-aryl-1,2,4-
triazine-3-thiol 7a–d (1 mmol) in DMSO (25 ml) was added
ethyl chloroacetate (1.5 mmol) and K2CO3 (2 mmol) and it
was stirred for 10 h under nitrogen atmosphere. After
completion of reaction, it was diluted by ethyl acetate and
washed by water three times. The organic phase was dried
over sodium sulfate and concentrated under vacuum. Pur-
ification was performed by column chromatography. Silica
gel (230–400 mesh) was used as stationary phase and eluent
was 5% of ethyl acetate in chloroform. In this manner,
compounds 8a–d were isolated as major products.
2-(Hydroxyimino)-1-(4-methoxyphenyl)-2-phenylethanone
(6a) Yield 67%; mp: 107–109 °C; IR (KBr, cm−1) υmax
:
1638 (C=O). 1H-NMR (400 MHz, DMSO-d6) δ (ppm):
3.84 (s, 3H, OCH3), 7.11 (d, J = 9 Hz, 2H, Ar–H),
7.40–7.49 (m, 5H, Ar–H), 7.81 (d, J = 8.92 Hz, 2H, Ar–H),
11.69 (s, 1H, NOH).
2-(Hydroxyimino)-1-(4-methoxyphenyl)-2-p-tolylethanone
(6b) Yield 54%; mp: 110–111 °C; IR (KBr, cm−1) υmax
:
1667 (C=O). 1H-NMR (400 MHz, DMSO-d6) δ (ppm):
2.29 (s, 3H, Ar–CH3), 3.84 (s, 3H, OCH3), 7.09 (d, J =
8.88 Hz, 2H, Ar–H), 7.21 (d, J = 8.12 Hz, 2H, Ar–H), 7.36
(d, J = 8.2 Hz, 2H, Ar–H), 7.8 (d, J = 8.84 Hz, 2H, Ar–H),
Ethyl 2-(6-(4-methoxyphenyl)-5-phenyl-1,2,4-triazine-3-yl-
thio)acetate (8a) Yield 61%; IR (KBr, cm−1) υmax: 1741
1
(C=O). H-NMR (400 MHz, CDCl3) δ (ppm): 1.29 (t, J =

Med Chem Res
7.12 Hz, 3H, CH2CH3), 3.83 (s, 3H, OCH3), 4.11 (s, 2H,
SCH2CO), 4.22 (q, J = 7.12 Hz, 2H, CH2CH3), 6.83 (d, J
= 8.92 Hz, 2H, Ar–H), 7.39–7.43 (m, 3H, Ar–H),
7.53–7.56 (m, J = 9.0 Hz, 4H, Ar–H). 13C-NMR (100 MHz,
CDCl3) δ (ppm): 14.15, 33.19, 55.39, 61.91, 113.93,
126.91, 128.68, 129.21, 129.39, 131.83, 135.70, 153.85,
154.92, 162.10, 168.81, 168.91. MS, m/z (%): 381.1 (M+,
32), 327.0 (39), 299.0 (33), 252.9 (34), 226.9 (48), 208.0
(100), 135.0 (84). Anal. calcd. for C20H19N3O3S: C, 62.98;
H, 5.02; N, 11.02. Found: C, 63.06; H, 5.09; N, 11.11.
for C20H18ClN3O3S: C, 57.76; H, 4.36; N, 10.10. Found: C,
57.72; H, 4.29; N, 10.12.
Ethyl 2-(5-(4-chlorophenyl)-6-(4-methoxyphenyl)-3-thio-
xo-1,2,4-triazin-2(3H)-yl)acetate (8e) Yield 10%; IR
(KBr, cm−1) υmax: 1756 (C=O). 1H-NMR (400 MHz,
CDCl3) δ (ppm): 1.30 (t, J = 7.12 Hz, 3H, CH2CH3), 3.88
(s, 3H, OCH3), 4.24 (q, J = 7.16 Hz, 2H, CH2CH3), 4.67 (s,
2H, NCH2CO), 7.0 (d, J = 8.96 Hz, 2H, Ar–H), 7.32 (d, J
= 8.68 Hz, 2H, Ar–H), 7.53 (d, J = 8.64 Hz, 2H, Ar–H),
8.04 (d, J = 8.92 Hz, 2H, Ar–H). 13C-NMR (100 MHz,
CDCl3) δ(ppm):14.22, 55.60, 60.98, 71.35, 114.34, 127.52,
127.94, 129.06, 130.93, 132.28, 133.24, 136.58, 156.13,
164.87, 169.38, 191.40. MS, m/z (%): 415.0 (M+, 1), 355.1
(5), 281.0 (14), 271.0 (9), 259.0 (13), 238.0 (45), 206.0
(47). Anal. calcd. for C20H18ClN3O3S: C, 57.76; H, 4.36;
N, 10.10. Found: C, 57.82; H, 4.39; N, 10.22.
Ethyl 2-(6-(4-(methoxyphenyl)-5-p-tolyl-1,2,4-triazine-3-y-
lthio)acetate (8b) Yield 60%; IR (KBr, cm−1) υmax: 1756
1
(C=O). H-NMR (400 MHz, CDCl3) δ (ppm): 1.09 (t, J =
7.2 Hz, 3H, CH2CH3), 2.32 (s, 3H, Ar–CH3), 3.82 (s, 3H,
OCH3), 3.86 (s, 2H, SCH2CO), 4.22 (q, J = 7.16 Hz, 2H,
CH2CH3), 6.92 (d, J = 9.0 Hz, 2H, Ar–H), 7.11 (d, J = 8.0
Hz, 2H, Ar–H), 7.41 (d, J = 8.2 Hz, 2H, Ar–H), 7.89 (d, J
= 9.0 Hz, 2H, Ar–H). 13C-NMR (125 MHz, CDCl3) δ
(ppm): 14.19, 21.26, 32.52, 55.28, 61.33, 114.06, 126.11,
126.55, 129.16, 129.41, 129.61, 139.71, 154.92, 159.43,
161.89, 169.63, 171.43. MS, m/z (%): 395.1 (M+, 13),
339.2 (12), 281.0 (19), 255.7 (38), 207.0 (34), 191.1 (100).
Anal. calcd. for C21H21N3O3S: C, 63.78; H, 5.35; N, 10.63.
Found: C, 63.85; H, 5.39; N, 10.71.
In vitro stability study
The stability of compound 8e was evaluated by monitoring
its UV absorbance within 2 weeks. Different concentrations
of 8e (1, 10, 25, and 50 µM) were prepared in different
organic solutions (ethanol, chloroform, and DMF) and also
in phosphate buffer in pH = 7.4 and changes in UV spec-
trum within 2 weeks at 25 °C showed subtle variations
indicating its stability at room temperature.
Ethyl 2-(5-(4-fluorophenyl)-6-(4-methoxyphenyl)-1,2,4-tri-
azine-3-ylthio) acetate (8c) Yield 53%; IR (KBr, cm−1
)
1
υmax: 1731 (C=O). H-NMR (400 MHz, CDCl3) δ (ppm):
1.11 (t, J = 7.16 Hz, 3H, CH2CH3), 3.83 (s, 3H, OCH3),
3.87 (s, 2H, SCH2CO), 4.22 (q, J = 7.16 Hz, 2H, CH2CH3),
6.93 (d, J = 8.96 Hz, 2H, Ar–H), 7.0 (t, J = 8.72 Hz, 2H,
Ar–H), 7.5 (dd, J = 8.84 and 5.44 Hz, 2H, Ar–H), 7.88 (d,
J = 8.92 Hz, 2H, Ar–H), 13C-NMR (125 MHz, CDCl3) δ
(ppm): 14.17, 32.48, 55.29, 61.38, 114.13, 115.50 (d, J =
21 Hz), 125.48, 128.5 (d, J = 8.75 Hz), 129.35, 138.04,
153.98, 158.99, 161.74, 163.65 (d, J = 248.7 Hz), 169.45,
171.36. Anal. calcd. for C20H18FN3O3S: C, 60.14; H, 4.54;
N, 10.52. Found: C, 60.21; H, 4.59; N, 10.58.
H2O2-induced toxicity in PC12 cells
PC12 cells (pheochromocytoma of the rat adrenal medulla)
were cultured in RPMI 1640 medium supplemented with 10
% FCS, 100 units/ml penicillin and 100 µg/ml streptomycin
(All from GIBCO, Grand Island, NY, USA). After trypsi-
nization, the cells were equally seeded into 96-well culture
plates (1 × 104 cells/well). To induce neuronal differentia-
tion the following protocol was applied. The cells were
cultured in serum-free medium for 2 days, afterwards NGF
(50 ng/ml, Sigma) was added until neurite outgrowth could
be observed (Koh et al. 2003). For neuroprotectivity assay,
initially non-toxic concentration of the final compounds on
PC12 cells was determined and then differentiated PC12
cells were pretreated with different non-toxic concentrations
(1–50 µM) of compounds for 3 h. Subsequently, freshly
prepared H2O2 (final concentration 350 µM) was added to
the cells and incubated for 24 h. Induction of apoptosis was
recognized by DAPI staining. To determine the EC50
values of neuroprotection, different concentrations of the
tested compounds were evaluated for their protective
activity and the cell viability was determined. To calculate
EC50 values, four different concentrations which showed
cell viability in the range of 20–80% were chosen. EC50s
Ethyl 2-(5-(4-chlorophenyl)-6-(4-methoxyphenyl)-1,2,4-tri-
azine-3-ylthio) acetate (8d) Yield 65%; IR (KBr, cm−1
)
1
υmax: 1746 (C=O). H-NMR (400 MHz, CDCl3) δ(ppm):
1.22 (t, J = 7.12 Hz, 3H, CH2CH3), 3.86 (s, 3H, OCH3),
3.97 (s, 2H, SCH2CO), 4.25 (q, J = 7.12 Hz, 2H, CH2CH3),
7.1 (d, J = 8.96 Hz, 2H, Ar–H), 7.35 (d, J = 8.78 Hz, 2H,
Ar–H), 7.5 (d, J = 8.64 Hz, 2H, Ar–H), 8.1 (d, J = 8.96 Hz,
2H, Ar–H). 13C-NMR (100 MHz, CDCl3) δ (ppm):14.22,
33.16, 55.50, 60.90, 114.54, 127.42, 127.90, 129.26,
130.73, 132.18, 133.20, 136.68, 156.22, 164.97, 169.58,
171.27. MS, m/z (%): 415.0 (M+, 5), 355.1 (23), 281.0 (22),
271.0 (17), 259.0 (19), 237.0 (35), 207.0 (49). Anal. calcd.

Med Chem Res
were calculated using GraphPad Prism 5 (GraphPad Soft-
ware, San Diego, CA).
(%) was calculated as follows:
Aβ-induced toxicity in SH-SY5Y cells
% Cytotoxicity ¼
Compound treated LDH activity À Spontaneous LDH activity
Maximum LDH activity À Spontaneous LDH activity
 100
β-amyloid (1–42) peptide was purchased from Sigma
Aldrich. 0.1 mg peptide was dissolved in 0.02 M NaOH and
diluted in PBS pH 7.4 at a concentration of 50 µM. β-
amyloid (1–42) peptide was incubated at 37 °C for 7 days to
induce peptide aggregation (Unsal-Tan et al. 2017; Ozadali-
Sari et al. 2017).
DAPI staining to evaluate induction of apoptosis
SH-SY5Y human neuroblastoma cells were cultured in
Dulbecco’s modified Eagle medium (DMEM) supplemented
with 10% FBS and 1% antibiotics at 37 °C in %5 CO2.
Cells were differentiated into neurons with 10 μM retinoic
acid treatment for 10–15 days (Chimon et al. 2007). SH-
SY5Y cells (5000 cells/well) were treated with compounds
3a–f, 8a–e (25 µM) for 3 h prior to β-amyloid (1–42)
treatment (5 µM) and incubated for 24, 48, and 72 h. MTT
reduction and LDH release assays were performed to
evaluate cell viability. Annexin V/PI staining and TUNEL
assay were performed to evaluate effect of compound 8e on
amyloid induced apoptosis.
DAPI staining was performed to evaluate the effect of H2O2
apoptosis induction. For this purpose the differentiated
PC12 cells were cultured in 96-well plate and treated with
different doses of drugs as indicated above and stained with
the DNA-specific fluorochrome 4′,6-diamidino-2-pheny-
lindole dihydrochloride (DAPI, Sigma). DAPI was added to
the culture medium of live cells with the final concentration
of 1 µg/ml for 20 min. Due to cell membrane permeability
of apoptotic cell, DAPI penetrate into the cell and makes the
nucleus stained, while normal cell is not permeable suffi-
ciently and gets lightly stained.
Annexin V/PI staining
Inquiry of cell viability by MTT assay
Cells were harvested 24 or 48 h after treatment and stained
using Tali™ apoptosis kit (Life Technologies) as described
by the manufacterer’s instructions and evaluated using the
Tali™ Image-based Cytometer (Life Technologies). The
annexin-V positive/PI negative cells were admitted as
apoptotic, whereas the annexin V positive/PI positive cells
were identified as necrotic. Dual negative stained cells were
identified as viable cells (Koopman et al. 1994).
After 24, 48, and/or 72 h incubation, the medium was
removed, cells were washed with PBS and 10 µl of MTT [3-
(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyl-tetrazolium bro-
mide] solution (5 mg/ml, Sigma) was added to each well.
After 3.5 h of incubation the medium was removed and 150
µl DMSO was added to dissolve formazan precipitates.
Finally, optical density (OD) was measured at 560 nm
(reference at 690 nm) using microplate reader (BioTek
synergy HT) (Datki et al. 2003).
TUNEL assay
The viability (%) was calculated as follows:
Amyloid induced apoptosis in differantiated SHSY5Y cells
( 8e treatment) was detected and quantified by transferase-
mediated dUTP nick end labeling (TUNEL) assay using
Millipore ApopTag® Plus Peroxidase In situ Apoptosis Kit
(S7101). Qualitative morphometric analysis was performed
by light microscopy (Nikon TS 100F microscobe). Cells
with nuclear staining were accepted apoptotic (TUNEL-
positive) (Fried et al. 1976).
% of viability ¼
Average OD of treated wells À average OD of blank wells
 100
Average OD of control wells À average OD of blank wells
Lactate dehydrogenase (LDH) assay
The LDH assay is a method for viability via activity of
lactate dehydrogenase (LDH) released from damaged cells.
After 24, 48, and 72 h incubation, 50 µl cell culture super-
natant of each well was mixed with 50 µl substrate mixture
(6.6 mM NADH and 30 mM Sodium pyruvate in 0.2 M
Tris⋅HCl buffer, pH 7.3) and incubated for 5 min at room
temperature. Reaction was stopped by adding 50 µl 0.01 N
HCl and absorbance was read at 340 nm. The cytotoxicity
Statistical analysis
GraphPad Prism 5 Software was used to perform statistical
tests. One-way analysis of variance (one-way ANOVA)
followed by Dunnett test or two tailed t-test (where
appropriate) were used to compare the significance of
groups.

Med Chem Res
Results and discussion
Chemistry
temperature in the presence of potassium carbonate in
dimethylsulfoxide to afford compounds 3a–f. The synthetic
route for the preparation of unsymmetrical compounds 8a–e
(Scheme 2) was started by an acylation reaction of anisole
with phenylacetic acid derivatives 4a–d in the presence of
trifluoroacetic anhydride and phosphoric acid. The oximation
reaction in the alpha position of carbonyl group was per-
formed by successive use of sodium methoxide and butyl-
nitrite to give compounds 6a–d. The next two steps, triazine
ring closure and thiol-alkylation were performed in similar
conditions as described earlier for symmetrical compounds to
afford final compounds 8a–d. In the final step, the reaction
of 7a–d with ethyl chloroacetate resulted in the formation of
The synthetic routes to the final compounds 3a–f and 8a–e
are outlined in Schemes 1 and 2, respectively. The synthesis
of symmetrical ethyl 5,6-bisaryl-1,2,4-triazine-3-ylthioacetate
derivatives (3a–f) was started by 1,2-diketones 1a–f in which
triazine ring closure was performed by thiosemicarbazide in a
mixture of water and ethanol and a few drops of hydrochloric
acid under microwave irradiation (Irannejad et al. 2014,
2015). In the next step, alkylation was done at sulfur atom of
thiol group by adding ethyl chloroacetate at room
Scheme 1 Reagents and
conditions: (i)
thiosemicarbazide, HCl, H2O:
EtOH, MW, 130 °C, 20 min. (ii)
ethyl chloroacetate, K2CO3,
DMSO, rt, 10 h
Scheme 2 Reagents and
conditions: (i) Anisole, TFAA,
H3PO4, rt, 5 min; (ii) MeONa,
MeOH then BuONO, rt, 30 min;
(iii) Thiosemicarbazide, H2O,
HCl, reflux, 4 h; (iv) ethyl
chloroacetate, K2CO3, DMSO,
rt, 10 h

Med Chem Res
S-substituted derivatives 8a–d as major products which were
purified by column chromatography. Surprisingly, in the case
of 4-chloro derivative 8d, its N-substituted regioisomer 8e
was also isolated and characterized as a minor product (yield
<10%). Chemical structures of the final compounds 3a–f and
8a–e along with their melting point and quantitative yields
for the last step are summarized in Table 1.
dysfunction, DNA damage and elevated apoptotic events.
H2O2 is documented to induce apoptosis and contribute to
β-amyloid induced neuronal death (Behl et al. 1994;
Tamagno et al. 2003; Saito et al. 2001).
The ability of compounds to antagonize the oxidative
insult was evaluated in H2O2-treated PC12 cells by MTT
assay. The EC50 values (concentration of compounds in
which 50% of cells are viable in the presence of H2O2) were
determined for compounds as shown in Table 3. All com-
pounds showed significant neuroprotective activity against
H2O2-induced toxicity with EC50 values ranging from 14 to
30 µM. Compounds 8e and 3e were found to be the most
neuroprotective agents with EC50 values of 14.44 and 17.86
µM, respectively. Quercetin as a well-known neuroprotec-
tive agent was used as a reference drug for comparison and
could protect PC12 cells against H2O2 with EC50 of 8 µM.
There is marginal difference between EC50 values of
unsymmetrical compounds 8a–d (20–23 µM) but higher
variations in symmetrical ones (17–23 µM) which could be
mostly due to the presence of two new atoms in each
compound structure in symmetrical ones.
In the symmetrical compounds, 3e with chlorine-
substitution on both phenyl rings was shown to be the
most potent one in H2O2-induced toxicity assay on PC12
cells similar to unsymmetrical compounds in which the
chlorine-substituted derivatives 8d and its regioisomer 8e
were the best ones in this group (Table 3).
Morphological assessment was used to indicate the
apoptotic state of cells treated with H2O2 or neuroprotective
agents. As illustrated in Fig. 4, normal cells differentiate in
from of neurite outgrowth while apoptotic cells have DNA
condensation and fragmented parts identified by blue stains
after DAPI staining. The neuroprotective effect of com-
pound 8e against H2O2-induced apoptosis in PC12 cells was
investigated by DAPI staining. Pretreatment with com-
pound 8e (20 µM) could substantially reduce apoptotic
markers as DNA condensation and fragmentation compared
to H2O2 treated group (Fig. 5).
1
The FT-IR, MS, H and 13C NMR spectra were appro-
priately confirmed the chemical structure of final compounds
3a–f and 8a–e (the NMR spectra of some representative
compounds are in Supplementary material). In the FT-IR
spectra of these compounds, the characteristic band
appearing in the range of 1724–1756 cm−1 confirms exis-
1
tence of the carbonyl group of ester moiety. In the HNMR
spectra, ethyl group of ester function shows a triplet and
quartet at about 1.2 and 4.2 ppm, respectively while
methylene group of acetate side chain appears at about
3.8–4.1 ppm as singlet form. Moreover, aromatic protons for
the two para-substituted aromatic or the two thienyl rings
could be easily assigned in the range of 6.8–8.0 ppm. A
minor product was also isolated in the preparation of com-
pound 8d and its chemical structure was identified as N-
substituted regioisomer 8e by means of 1H, 13C, and HSQC
NMR spectra. Accordingly, the chemical shift of methylene
protons of compound 8e was seen to be desheilded to 4.6
ppm. Inspection of HSQC spectrum of compound 8e reveals
chemical shift of the side chain methylene carbon of 8e to be
71.35 ppm, 40 units more than the same carbon atom for
other S-substituted compounds (Fig. 3, Table 2).
Further confirmation of this possible structure is based on
the unexpected chemical shift of C3-carbon atom of triazine
ring at 191.40 ppm that is 20 units of ppm more than the
same atom on the other S-substituted final compounds and
highly favors the possible ex35istence of a thione group on
the C3-carbon atom of triazine ring of regioisomer 8e.
Therefore, these observations have truly confirmed the
linkage of methylene side chain to the nitrogen atom of
triazine ring. Occurrence of N- over S-alkylation on 1,2,4-
triazine-3(2H)-thione ring system has been reported in a
previous study but with no detail structural information and
discussion about synthesized compounds (Mullick et al.
2009). In the case of 8a–c and under the current experi-
mental protocol, some traces of by-products were observed
by TLC but not as much that could be isolated and char-
acterized. Probably, some change in experimental procedure
would be inevitable to obtain N-alkylated regioisomers of
8a–c and also to increase the production yield of 8e.
Neuroprotective activity against β-amyloid induced
toxicity in SH-SY5Y cells
Amyloid peptide (1–42) accumulation has been proposed to
have significant role in the pathogenesis of AD and inci-
dence of neurotoxicity (Novitskaya et al. 2006). SH-SY5Y
cells are neuroblastoma cells that differentiate into neuronal
cells by retinoic acid treatment (Dwane et al. 2013).
To evaluate the neuroprotective effects of compounds
against β-amyloid (1–42) peptide induced cytotoxicity, SH-
SY5Y cells were pretreated with compounds 3a–f and 8a–e
(25 µM) and cell viability was determined by MTT assay.
The data is expressed in Fig. 6 as percent of cell viability
compared to normal cells (100% viability) at 24 h.
Neuroprotective activity against H2O2-induced toxicity
in PC12 cells
β-amyloid induced neurotoxicity involves several compo-
nents as increased oxidative stress, mitochondrial

Med Chem Res

Med Chem Res
Fig. 3 1H and 13CNMR
Chemical shifts of the methylene
group of acetate side chain and
also C3 carbon atom of triazine
ring in compounds 3a–f and 8a–
d compared to the regioisomer
1
8e. HNMR chemical shifts are
in red and 13CNMR ones
represented in blue(color figure
online)
Table 2 1H, 13C chemical shift data of compound 8e provided by
Table 3 Neuroprotective activity of the target compounds on H2O2-
HSQC spectrum
induced toxicity on PC12 cells
Compounds
Neuroprotective
activity (EC50
SEM, µM)
3a
21.13 1.05
23.32 1.89
19.7 2.41
30.04 2.78
17.86 2.24
19.56 1.2
23.13 2.56
22.74 3.69
21.20 1.53
20.70 1.23
14.44 0.85
8.18 1.45
3b
3c
3d
3e
Atom no.
13C (δ, ppm)
1H (δ, ppm), Mult.
3f
8a
1
2
3
4
5
6
7
8
14.22
1.3, t
8b
60.98
4.24, q
4.67, s
3.88, s
7.0, d
8c
71.35
8d
55.60
8e
114.34
132.28
129.06
127.94
Quercetin
8.04, d
7.32, d
7.53, d
regioisomer 8e. Therefore, 8e was the most potent com-
pound in both studies.
β-amyloid reduced cell viability to 51% in the absence of
any neuroprotective agent compared to the normal cell
(100%). In general, all compounds could increase cell via-
bility compared to β-amyloid treated group with the
exception of 3c, 3e,and 8c. Interestingly, the most effective
compound was again found to be 8e; pretreatment at 25 µM
for 3 h increased significantly cell viability to 65% (P <
0.01) compared to β-amyloid treated cells (51%).
However, results on β-amyloid induced toxicity in SH-
SY5Y cells showed controversy especially in symmetrical
compounds in which chlorine-substituted derivative 3e was
the weakest but 3f with bromine substituent owns the
highest potency in this group. This incomparable result
among symmetrical compounds may be attributed to the
kind of cell used in this study. In the unsymmetrical ones
there is a relevant order of variation in potency so that
electron-withdrawing substituents like fluoro and chloro
derivatives have more potency than compounds 8a and 8b.
Similarly to the results obtained in H2O2-induced toxicity
on PC12 cells, the best compound was again found to be
Neuroprotective effect of compound 8e was further stu-
died in detail by MTT and LDH assays at 24–48 h and
compared to Quercetin (25 µM). Mitochondrial activity
reduction was significantly attenuated 24% (after 24 and 48
h of treatment) compared to amyloid treated cells and
8–12% compared to Quercetin-pretreated cells at 24 and 48
h, respectively. This shows that the neuroprotective activity
of compound 8e is higher than Quercetin at the same con-
centration and in the presence of β-amyloid. LDH release
was not changed at any time points, pointing anti-apoptotic
effect of compound 8e (Fig. 7). LDH assay is an experiment
to show necrotic cell death. A key signature for necrotic
cells is the permeabilization of the plasma membrane and
release of LDH enzyme. On the contrary, in apoptotic death
mechanisms cell membrane is mostly intact and any change
in LDH release is observed. Figure 7 shows that β-amyloid
1–42 treatment induces apoptosis rather than necrosis.
We further examined the anti-apoptotic role of the most
potent neuroprotective compound 8e against amyloid induced
cytotoxicity via Annexin V/PI staining and TUNEL assay.

Med Chem Res
Fig. 4 Left undifferentiated PC12 cells; Middle Differentiated PC12 cells showing neurite outgrowth; Right Apoptotic cells stained with DAPI
(intense blue) in H2O2-treated group, DNA condensation and fragmentation is obvious in apoptotic cells (marked by arrows) (color figure online)
Fig. 5 Morphological
assessment of cells using DAPI
staining. Differentiated PC12
cells were treated with 8e (20
µM) followed by exposure to
H2O2. Left compound 8e treated
group in the presence of H2O2.
Right H2O2-treated group, DNA
condensation and fragmentation
is obvious in apoptotic cells
measure apoptosis. During apoptosis, PS is transferred from
the inner or cytoplasmic face of the plasma membrane to the
cell surface. Annexin V has a strong, Ca2+-dependent
affinity for PS and therefore can be used as a probe for
detecting apoptosis.
100
80
Propidium iodide (PI) in combination with Annexin V is
widely used to determine if cells are viable, apoptotic, or
necrotic through differences in plasma membrane integrity
and permeability. The Annexin V/PI protocol is a com-
monly used method for studying apoptotic cells. The ability
of PI to enter a cell is dependent upon the integrity of the
membrane; PI does not stain live or early apoptotic cells due
to the presence of an intact plasma membrane. In late
apoptotic and necrotic cells, disruption of the integrity of
plasma and nuclear membranes allows PI to pass through
the membranes, intercalate into nucleic acids, and display
red fluorescence.
Apoptosis is a cellular mechanism for programmed cell
death. Early apoptotic events are characterized by pyknotic
nuclei and membrane blebbing while late apoptosis may be
determined by disintegrated nucleus membrane (Yakovlev
and Faden 2004).
**
60
40
+ β-amyloid 1-42 (5 μM)
Fig. 6 Effects of pretreatment with test compounds on Aβ-induced
cell death at 24 h. Values are means SEM (n = 3). (**P < 0.01,
compared to amyloid treated group)
Cytometric analysis with Annexin V/Propidium iodide
staining
To evaluate the effect of compound 8e on β-amyloid
(1–42) peptide induced apoptosis (5 µM) for 24 and 48 h,
differentiated SH-SY5Y cells with or without 8e pretreat-
ment (25 µM for 3 h) were evaluated by Tali apoptosis kit
and the results are presented in Fig. 8. Interestingly, com-
pound 8e significantly increased (P < 0.005) cell viability
Studies of cellular apoptosis have been significantly
impacted since the introduction of cytometry-based meth-
ods. Phosphatidylserine (PS) residues is normally absent on
the plasma membrane but its appearance on cell surface is
an early sign of apoptosis, and can be used to detect and

Med Chem Res
A
A
*
100
90
80
70
60
50
100
80
60
40
20
0
Amyloid 1-42, 5 μM
8e +amyloid treated-24h
amyloid treated-24h
Quercetin(25 μM) +Amyloid
1-42 (5 μM)
8e ( 25 μM) +Amyloid 1-42
(5 μM)
*
*
*
B
100
80
60
40
20
0
8e+amyloid treated-48h
amyloid treated-48h
B
Amyloid 1-42, 5 μM
120
100
80
Quarcetin(25 μM) +Amyloid
1-42 (5 μM)
*
8e ( 25 μM) +Amyloid 1-42
(5 μM)
60
Fig. 8 Evaluation of neuroprotective effect of 8e (25 µM) pretreatment
for 3 h on β-amyloid peptide 1–42 induced apoptosis (5 µM) for 24 h
(a) and 48 h (b); Values are means SEM (n = 3). (*, p < 0.005 vs.
only amyloid treated by two tailed t-test)
determined at 24 h, 8e pretreatment significantly decreased
(P < 0.005) necrosis from 25 to 9% at 48 h.
Fig. 7 Protective effects of 8e on β-amyloid induced cell death.
Mitochondrial activity reduction and LDH release. a 24 h b 48 h.
Values are means SEM (n = 3). (*P < 0.05, compared to amyloid
treated group)
Morphological evaluation with TUNEL assay
from 40 to 80% and 67 to 89% at 24 and 48 h compared to
β-amyloid treated cells, respectively.
Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-
End Labeling (TUNEL) assay has been widely used to
detect apoptotic cells that undergo extensive DNA degra-
dation during the late stages of apoptosis. The assay relies
on the presence of nicks in the DNA which can be identified
by terminal deoxynucleotidyl transferase (TdT), an enzyme
that catalyzes the addition of dUTPs that are secondarily
labeled with a marker to the blunt ends of double-stranded
DNA breaks. It may also label cells that have suffered
severe DNA damage.
Tunel assay was carried out to assess the morphological
changes of cell death in vitro. As shown in Fig. 9, β-
amyloid (1–42) treatment induced apoptosis at 24 h (Fig.
9a) that is followed by necrosis at 48 h (Fig. 9c) in which
apoptotic cells are characterized by condensed chromatin,
disintegration of cell membrane and accumulated cells.
Pretreatment with 8e for 3 h remarkably reduced cell
death after 24 h (Fig. 9b) and late-apoptosis after 48 h (Fig.
9d) respectively. As depicted in Fig. 9b, cells have normal
appearance in the presence of compound 8e at 24 h but late-
Moreover, compound 8e attenuated late-apoptosis from
42 to 6% (P < 0.005) and 7 to 1% at 24 and 48 h respec-
tively compared to amyloid treated cells. Similarly, apop-
tosis was reduced from 12 to 4% at 24 h, although no
substantial changes were evident in apoptotic percentage of
8e treated cells at 48 h. Decrease in late apoptosis and cell
death at 48 h would be late to observe amyloid induced
apoptosis. These findings were compatible with recent
reports demonstrating β-amyloid induced neuronal apopto-
sis even at 6 h (Zhu et al. 2014).
Necrosis is an unprogrammed type of cell death due to
the loss of membrane integrity which is followed by cell
lysis. Necrosis is usually induced in neuronal cells with
exposure to toxins and reactive oxygen species (Ziegler and
Groscurth 2004). With the release of the cellular compo-
nents and increased reactive species, acute inflammation
usually occurs in response to necrotic cell death. Although a
statistically negligible increase in necrotic cells was

Med Chem Res
Fig. 9 TUNEL staining of
differentiated SH-SY5Y cells
after 24 h exposure to a β-
amyloid peptide 1–42 (5 µM) b
pretreatment with 8e (25 µM) for
3 h + β-amyloid peptide 1–42
(5 µM). After 48 h exposure to c
β-amyloid peptide 1–42 (5 µM)
d pretreatment with 8e (25 µM)
for 3 h + β-amyloid peptide
1–42 (5 µM). Necrotic cells
showed membrane
A
B
Chromaꢀne
condensaꢀon
disintegration and diffused
chromatin. Apoptotic cells
showed membrane blebbing and
chromatine condensation
Late apoptosis
Viable cells
Late apoptosis
D
C
Apoptoꢀc cells
Chromaꢀne
condensaꢀon
Membrane
disintegraꢀon
Necroꢀc cells
Membrane blebbing
apoptosis is obvious at 48 h due to the beta-amyloid induced
neurotoxicity.
Table 4 Predicted logP of parent compounds, logS and logD (pH 7.4)
values of hydrolyzed compounds (acidic form)
Based on our previous research in the past few years,
5,6-diaryl-1,2,4-triazine compounds have shown to be dual
Cyclooxygenase and β-amyloid aggregation inhibitors and
hence explains the mechanism behind ability of these
compounds to reduce cell toxicity probably through dual
mechanism of action (Dadashpour et al. 2015).
Compd
logP
logS (acidic form)
logD (acidic form)
3a
3b
3c
3d
3e
3f
3.7
5.1
3.8
4.4
5.3
5.6
3.9
4.5
4.1
4.5
4.4
−1.6
−3.1
−1.9
−2.3
−3.3
−3.9
−2.0
−2.5
−2.0
−2.6
−2.3
0.4
1.6
0.1
0.6
1.6
2.0
0.4
0.9
0.4
0.9
0.6
Prediction of solubility and lipophilicity
8a
8b
8c
8d
8e
Lipophilicity and solubility are two major issues which
have to be evaluated especially for CNS active drug can-
didates. The predicted values of these two physicochemical
parameters for compounds 3a–f and 8a–e are calculated and
represented in Table 4. As shown, most of the logP values
are less than 5 except compounds 3e and 3f. Therefore,
according to the Lipinski’s rule of 5, most of compounds
would have good oral bioavailability and permeability
across biologic membranes. However compounds 3a–f and
8a–e have an ester group and therefore could be considered
as prodrugs, which helps them to permeate easily through
membranes. On the other hand, one might think of low
solubility of compounds 3a–f and 8a–e due to their high
lipophilic character. Indeed, after passage into membranes
and being exposed to body fluids, hydrolyzation of ester
Calculated by ChemAxon
function and conversion to acidic group is inevitable in vivo
in the presence of various and abundant amounts of esterase
enzymes in the body. Actually, free acidic form of com-
pounds would have acceptable solubility as predicted in
Table 4. Since more than 80% of drugs on the market have
an estimated logS value greater than −4, shows that our
synthesized compounds have admissible solubility. Calcu-
lated logD of compounds in the range of 0.1–2.0 truly
confirms
a good balance between lipophilicity and

Med Chem Res
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Conclusion
In order to find new agents with beneficial effect against
AD, we introduced some 5,6-diaryl-1,2,4-triazine deriva-
tives and evaluated them against H2O2− and β-amyliod
induced toxicity in PC12 and SH-SY5Y cell lines respec-
tively. Interestingly, all compounds showed remarkable
neuroprotective effect in PC12 cell line. Particularly, the N-
substituted regioisomer 8e was the most potent compound
with EC50 of 14 µM in H2O2 induced toxicity assay and was
also the most potent one against toxic effect of β-amyloid
peptide. Further morphological evaluation revealed the
ability of this compound to prevent apoptosis and DNA
fragmentation in treated cells. Eventually, compound 8e
could be considered as a novel neuroprotective agent in
developing anti-Alzheimer’s leading drugs and further stu-
dies would be enlightening the underlying mechanisms
involved in the neuroprotective activity of regioisomer 8e in
future. Furthermore, it demands more effort to synthesize
new and various substituted derivatives of N-alkylated
regioisomer 8e and evaluate their neuroprotective ability in
the future.
Acknowledgements This research was supported by grant from the
Research Council of Mazandaran University of Medical Sciences. This
study is related to the Pharm.D thesis of K. Safari Yanghagh and M.A.
Roknipour.
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
Khodagholi F, Khoramian Tusi S, Zeighamy Alamdary S, Amini M,
Ansari N (2012b) 3-Thiomethyl-5,6-(dimethoxyphenyl)-1,2,4-
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and activation of Nrf2 signaling by 1,2,4-triazine derivatives,
protects neuron-like PC12 cells against apoptosis. Apoptosis
15:738–751
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