Multi-target inhibitors against Alzheimer disease derived from 3-hydrazinyl 1,2,4-triazine scaffold containing pendant phenoxy methyl-1,2,3-triazole: Design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bioorg.2018.11.038

Alzheimer's disease (AD) is a complex neurological disorder with diverse underlying pathological processes. Several lines of evidence suggest that BACE1 is a key enzyme in the pathogenesis of AD and its inhibition is of particular importance in AD treatme

Full text of DOI:10.1016/j.bioorg.2018.11.038

Accepted Manuscript
Multi-target inhibitors of Alzheimer disease derived from 3-hydrazinyl 1,2,4-
triazine scaffold containing pendant phenoxy methyl- 1,2,3-triazole: Design,
synthesis and biological evaluation
Mahnaz Yazdani, Najmeh Edraki, Rashid Badri, Mehdi Khoshneviszadeh, Aida
Iraji, Omidreza Firuzi
PII:
S0045-2068(18)31120-9
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.038
YBIOO 2646
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 October 2018
22 November 2018
23 November 2018
Please cite this article as: M. Yazdani, N. Edraki, R. Badri, M. Khoshneviszadeh, A. Iraji, O. Firuzi, Multi-target
inhibitors of Alzheimer disease derived from 3-hydrazinyl 1,2,4- triazine scaffold containing pendant phenoxy
methyl- 1,2,3-triazole: Design, synthesis and biological evaluation, Bioorganic Chemistry (2018), doi: https://
doi.org/10.1016/j.bioorg.2018.11.038
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Multi-target inhibitors of Alzheimer disease derived from 3-hydrazinyl 1,2,4- triazine
scaffold containing pendant phenoxy methyl- 1,2,3-triazole: Design, synthesis and
biological evaluation
Mahnaz Yazdania,b, Najmeh Edrakic*, Rashid Badrib, Mehdi Khoshneviszadehc, Aida Irajic,
Omidreza Firuzic
a Department of Chemistry, Khozestan Science and Research Branch, Islamic Azad University, Ahvaz,
Iran.
b Department of Chemistry, Ahvaz Branch, Islamic Azad University, Ahvaz, Iran.
c Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences,
Shiraz, Iran.
*Corresponding author:
Najmeh Edraki, PhrmD, PhD
Medicinal and Natural Products Chemistry Research Center, Shiraz university of Medical Sciences,
Shiraz, Iran
Tel: +98 713 230 3872; Fax: +98 713 230 2225
E-mail: edrakin@sums.ac.ir, najmeh_edraki@yahoo.com
1

Abstract
Alzheimer's disease (AD) is a complex neurological disorder with diverse underlying
pathological processes. Several lines of evidence suggest that BACE1 is a key enzyme in the
pathogenesis of AD and its inhibition is of particular importance in AD treatment. Ten new 3-
hydrazinyl-1,2,4-triazine bearing pendant aryl phenoxy methyl-1,2,3-triazole as multifunctional
ligands against AD. We show that compounds containing Cl and NO2 groups at the para position
of the phenyl ring, namely compounds 7c (IC50= 8.55 ± 3.37 µM) and 7d (IC50= 11.42 ± 2.01
µM), demonstrate promising BACE1 inhibitory potential. Furthermore, we assessed the
neuroprotective activities of 7c and 7d derivatives in the PC12 neuronal cell line, which showed
moderate protection against amyloid β peptide toxicity. In addition, compound 7d demonstrated
metal chelating activity and moderate antioxidant potential (IC50= 44.42 ± 7.33 µM). Molecular
docking studies of these molecules revealed high-affinity binding to several amino acids of
BACE1, which are essential for efficient inhibition. These results demonstrate that 1,2,4-triazine
derivatives bearing an aryl phenoxy methyl-1,2,3-triazole have promising properties as
therapeutic agents for AD.
2

Graphical Abstract
Key words:
Alzheimer's disease, BACE1, antioxidant, metal chelator, 1,2,4- triazine, phenoxy methyl-1,2,3-
triazole, neuroprotection
3

1. Introduction
Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder. It is the sixth leading
cause of death in the United States and poses a significant economic burden [1]. The pathology
of AD is not fully understood, but diverse factors including the formation of amyloid plaques,
neurofibrillary tangles consisting of hyperphosphorylated tau protein, neuroinflammation,
oxidative stress and dyshomeostasis of biometals are associated with AD [2-4]. Currently, there
is no definitive disease-modifying treatment available for AD and therapeutic approaches only
slow down neurodegeneration [5, 6]. Therefore, there is an urgent need to discover novel
therapeutic methods that target the core pathology of the disease.
Recent evidence suggests that inhibition of BACE1, a type I transmembrane aspartic protease
may be a promising approach to treat AD [7]. So far, many studies have examined peptide
inhibitors of this enzyme [8, 9]. However, peptide drugs or closely related analogs may face
several impediments such as the inability to penetrate the blood-brain barrier, low bioavailability
and susceptibility to efflux by p-glycoprotein [7]. To overcome these problems, inhibitors with a
small and non-peptide chemical structure have been designed and synthesized without the
limitations of peptide inhibitors [10].
In addition to BACE1 inhibition, chelation therapy as a novel method of therapeutic intervention
has been investigated in many CNS disorders including AD, Parkinson disease and multiple
sclerosis [11]. This may be in part explained by the association between increased deposits of
biometal cations to changes that lead to degeneration. The protective effects of metal chelators
are based on trapping Fe, Cu, Zn ions [12-14].
4

Furthermore, oxidative stress has been also considered as a crucial contributing factor in the
pathogenesis of AD [15, 16]. Generation of reactive oxygen species (ROS) and reactive nitrogen
species (RNS) exacerbate neuronal and brain damage [17]. In this context, antioxidants would be
of therapeutic value to AD.
In light of the notions mentioned above and to continue our efforts to achieve novel anti-
Alzheimer agents [18-20], we have focused on the synthesis, biological evaluation and
computational analysis of a novel class of 3-hydrazynyl 1,2,4-triazine. We evaluated the
synthesized compounds for their BACE1 inhibition, metal chelation, antioxidant and
neuroprotective potential.
2. Results and discussion
2.1 Analogue-based pharmacophore design
Acylguanidine has been developed as a BACE1 inhibitor with IC50 of 3.7 µM. The X-ray
structure of compound A with BACE1 demonstrated the key H bond interactions with the
catalytic dyad. It seems that polar groups required for the enzyme's active site inhibition and
improve BACE1 inhibition [21]. Recently, we characterized a series of 2- thiophene-2-yl-1,2,4-
triazine B scaffolds as potent BACE1 inhibitors, with antioxidant and metal chelating potential.
Structure-activity relationship (SAR) of this scaffold demonstrates that heteroaromatic moieties
bearing H-bond donating atoms would enhance the BACE1 inhibitory potential (IC50 of 0.69 µM
for the best compound) and improve antioxidant capacity (IC50 of 38.09 µM for the mentioned
compound) [22]. The metal chelatory activity of Fe2+, Fe3+, Zn2+, Cu2+ may be achieved by
introducing triazole in the structure. Consequently, We used compound B as the starting point for
further modification and optimization to target metal-Aβ species in AD [23]. We have also
5

previously reported novel series of amino methylene triazole C with BACE1 inhibitotion, metal
chelation and neuroprotective potential [24].
Hence, in the present work, we used a molecular hybridization approach to design novel dual
acting agents with BACE1 inhibition, neuroprotective and metal chelating properties. We
assumed that replacing the thiophen groups (compound B) with phenyl ones would improve the
compounds’ accessibility to the BACE1 active site, while maintaining the hydrazinile triazine
linker for key H-bound interactions with the catalytic residue of the active site. The other
structural modification involved the replacement of the amino methylene triazole (compound C)
with oxo methylene triazole. Specifically, to increase favorable interactions, the nitrogen atom of
the amino methylene linker was replaced with an oxygen atom. Moreover, to improve the metal
chelating ability of the resulting compounds, we functionalized the triazole-ring at the C-4
position of the phenyl group. Different aryl substitutes were introduced at the triazole motif to
better construct the Structure-activity relationship (SAR) of the designed scaffold.
6

Fig. 1. Design strategy of target compounds as BACE1 inhibitor and metal chelating agent.
2.2 Chemistry
The synthetic route to target compounds 7a–7j is represented in Scheme 1. Reaction of benzil
and hydrazinecarbothioamide in refluxing acetic acid resulted in compound 1 [25]. In the next
step, the reaction of compound 1 and iodomethane in the presence of NaOH in EtOH at 25 ºC
resulted in the desired 3-(methylthio)-5,6-diphenyl-1,2,4-triazine 2 [22]. Reaction of compound 2
with hydrazine hydrate refluxing EtOH gave 3-hydrazineyl-5, 6-diphenyl-1,2,4-triazine 3 [16]. 4-
(prop-2-yn-1-yloxy)benzaldehyde 4 was afforded via the reaction of 4-hydroxyaldehyde and 3-
bromoprop-1-yne in the presence of K2CO3 in DMF at 80 ºC. Further reaction of compound 4
and 3 in EtOH gave (E)-5,6-diphenyl-3-(2-(4-(prop-2-yn-1-yloxy)benzylidene)hydrazineyl)-
1,2,4-triazine 5. Different substituted (bromomethyl)benzene derivatives were reacted with
7

sodium azide in the presence of triethylamine in THF and H2O at 70 ºC .After around 30 min,
intermediate compound 6a-j was added to the mixture of reaction in the presence of catalytic
amount of CuSO4 . 5H2O (10 mol%) and sodium (2R)-2-[(1S)-12-dihydroxyethyl]-4-hydroxy-5-
oxo-2H-furan-3-olate (25 mol%). The resulting mixture was stirred at 50 ºC and completion of
the reaction was monitored by using TLC to give compounds 7a-7j. The chemical structure and
some physical properties of synthesized derivatives are presented in Table 1.
N
N
H2N
H2N
N
N
2
N
N
O
O
N
NH
N
N
a
b
c
NH2
SH
S
S
N
H
1
4
3
N
N
O
O
OH
d
N
e
H
N
H
3
C
O
N
Br
C
H
O
5
Aryl
N
N
N
N
O
N
N
Ar
f
g
N
NaN3
ArCH2Br
N N NH
6a-j
5
+
+
N
H
7a-7j
Scheme 1: Reagent: a) CH3COOH, EtOH, 2h, Reflux. b) NaOH, CH3I, EtOH, 4h, 25º C . c)
NH2NH2.H2O, EtOH,12h, Reflux. d) K2CO3, DMF, 80ºc, 18 h. e) EtOH, 70 º C. f) TEA, THF/water, 70 º
C. g) CuSO4 . 5H2O (10 mol%) and sodium (2R)-2-[(1S)-12-dihydroxyethyl]-4-hydroxy-5-oxo-
2H-furan-3-olate (25 mol%), at 50 ºC.
8

Table 1: Chemical structures and physical properties of the synthesized compounds 7a-7j.
Ar
N
N
N
N
N
O
N
N
N
H
Compound
7a
Ar
M.p. (°C)
Yield (%)
235~241
91
H
7b
7c
7d
7e
7f
248~251
228 ~233
228 ~231
227~ 229
232 ~ 237
240 ~ 237
230 ~ 234
96
93
84
76
88
95
94
Br
Cl
NO2
Me
F
7g
7h
Cl
Cl
9

Cl
7i
7j
241 ~ 245
240 ~ 243
87
85
O
N
O
2.3 In vitro inhibition of BACE1 enzyme
All synthesized compounds 7a–7j were screened for their BACE1 inhibitory activity using an in
vitro FRET-based technique. In an attempt to find potent BACE1 inhibitors, we synthesized a
focused library of (E)-3-(2-(4-((1H-1,2,3-triazol-4-yl)methoxy)benzylidene)hydrazinyl)-5,6-
diphenyl-1,2,4-triazine derivatives. The BACE1 inhibitory activities of these compounds are
displayed in Table 2. OM99-2 (Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe) was used as a reference
BACE1 inhibitor.
Notably, the unsubstituted phenyl derivative 7a had considerable inhibitory effect against
BACE1 (IC50ꢀ=ꢀ18.77ꢀμM, 58.32% inhibition at 30ꢀμM).
To investigate the effect of substituted moiety on phenyl ring, different groups were
introduced at para position of pendant phenyl. It was found that introduction of bulky
electron withdrawing substituted group such as Cl (7c) and Br (7b) resulted in increased
inhibitory potential of compounds; particularly 7c showed an IC50 value of 8.55ꢀμM with
79.17% inhibition at 30ꢀμM. on the other hand 7f bearing pendant fluorobenzyl
demonstrate less inhibitory potential (16.81% inhibition at 30ꢀμM) compared to 7c and
7b.
10

The promising activity of 7c as the most potent compound led us to shift our focus
toward finding the suitable position for the Cl group on the phenyl ring. Cl was
substituted at various position of the pendant phenyl. 3-clorophenyl (7i) (37.04%
inhibition at 30ꢀμM) and 3,4 dicholoro phenyl (7h) (42.16% inhibition at 30ꢀμM)
exhibited lower inhibitory activity in comparison with 7c.
Subsequently, the inhibitory effect of electron donating groups was further evaluated.
Introduction of a methyl group (7e) diminished the activity (30.04% inhibition at 30ꢀμM)
compared to an unsubstantiated one (7a). In order to confirm the necessity of optimum
bulkiness, the methyl group was replaced with t-butyl at 4 position. These results clearly
depicted that t-butyl contributed to a higher BACE1 inhibitory activity with
IC50ꢀ=ꢀ24.58ꢀμM (63.59% inhibition at 30ꢀμM)
With the promising results on the BACE1 inhibitory activity of our nitro substituted
derivatives as hydrogen bond interacting motif [22], the potency of nitro phenyl
derivatives was investigated . The outcome implies the superior inhibitory activity of the
nitro group at para position of pendant phenyl (7d) (IC50ꢀ=ꢀ11.42ꢀμM, 77.08% inhibition
at 30ꢀμM).
 Based on enzymatic inhibitory activity, 7j containing propylisoindoline fragment with
IC50ꢀ=ꢀ18.08ꢀμM (67.09% inhibition at 30ꢀμM) emerged as the other interesting inhibitor
of the series, producing a substantial improvement of BACE1 inhibition potency.
11

Table 2. Results of BACE1 inhibitory and antioxidant effects of the synthesized compounds
Ar
N
N
N
N
N
O
N
N
N
H
% Inhibition at
30 µM ( ± SE) a
% Inhibition at
10 µM ( ± SE) a
Compoun
d
Ar
IC50 µM ( ± SE)
a
IC50 µM ( ± SE)a
BACE1 Assay
DPPH assay
7a
7b
7c
7d
7e
18.77 ± 2.68
24.09 ± 3.36
8.55 ± 3.37
11.42 ± 2.01
--
58.32 ± 6.40
29.98 ± 5.62
33.37 ± 7.98
58.94 ± 11.56
45.54 ± 6.43
15.23 ± 3.12
>50
H
Br
54.09 ± 4.40
79.17 ± 5.01
77.08 ± 8.88
30.04 ± 7.37
>50
>50
Cl
44.42 ± 7.33
NO2
Me
>50
12

7f
--
16.81 ± 5.55
63.59 ± 10.14
42.16 ± 2.72
37.04 ± 3.14
11.44 ± 13.67
29.98 ± 7.69
37.72 ± 1.50
28.49 ± 4.75
>50
>50
>50
>50
F
7g
24.58 ± 5.90
Cl
--
--
Cl
7h
7i
Cl
O
N
7j
18.05 ± 4.30
67.09 ± 8.74
35.51 ± 4.76
>50
O
OM99-2b
14.7 ( ± 2.8)
(nM)
Quercetinc
4.62 ( ± 2.26)
a Data presented here are the mean ± S.E.M. of three to six independent experiments
b OM99-2 was used as positive control.
d Quercetin was used as a standard positive control agent.
2.4 DPPH radical scavenging activity
The in vitro antioxidant activity of the synthesized compounds was tested by the 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical scavenging assay as a widely accepted method to evaluate the
antioxidant effect. A screen was performed at the concentrations of 30, 100 and 300 μM and
quercetin was used as the reference compound. Amongst the compounds screened for DPPH
13

radical scavenging activity, 7d containing a nitro motif showed a modest antioxidant effect with
an IC50 value of 44.42 μM.
2.5 Neuroprotective activity against Aβ25-35-induced cell death in PC12 neurons
The cleavage of amyloid precursor protein (APP) generates amyloid β-peptide (Aβ), which can
be toxic to neuronal cells. Aβ may also induce the production of reactive oxygen species (ROS)
and has a central role in the pathogenesis of AD [26]. To test the neuronal protective activity of
our two selected compounds against Aβ 25-35-induced damage, we used the MTT assay to
examine toxicity in PC12 neuronal cell line. Our results show that compounds 7c and 7d have
moderate neuroprotective activity at 5ꢀμM (10% protection for 7c and 14% protection for 7d).
2.6 Metal chelation
The ability of the synthesized compounds in chelating metals would be an added advantage in
the treatment of AD. To test the metal-chelating effect of our compounds, we used compound 7d
as an example to chelate biometals such as Zn2+ and Fe2+ in wavelengths ranging from 200 to
700 nm . As shown in Fig. 2, after the addition of either FeSO4 or ZnSO4 to a solution of 7d,
there was no obvious optical shift; however, the dramatic increase in absorbance indicated a
possible interaction between these biometals and the ligand. This suggests that compound 7d is
able to chelate biometals.
To determine the stoichiometry of compound 7d in complex with the biometals mentioned
above, we used the mole ratio method by preparing solutions of compound 7d (20 µM) and
metal ions ranging from 0-60 µM. The UV spectra were recorded and the absorbance change at
328 nm was plotted against the mole fraction of compound 7d, resulting in two straight lines. As
14

shown in Fig. 3, the absorbance linearly increased initially and then plateaued. The two straight
lines intersected at a mole fraction of 1, indicating a 1:1 stoichiometry for the 7d-Fe+2 and 7d-
Zn+2 complexes.
Fig.2 The UV spectrum of compound 7d alone or in the presence of, FeSO4
1
0.95
0.9
1.1
1
0.85
0.8
0.75
0.7
0.65
0.6
0.9
0.8
0.7
0.6
0
1
2
3
0
0.5
1
1.5
2
2.5
Mole fraction of Zn+2/7d
Mole fraction of Fe+2/7d
Fig. 3 Determination of the stoichiometry of complex-Fe+2 and Zn+2 using molar ratio method
through titrating the methanol solution of compound 7d with ascending amounts of FeSO4 and
ZnSO4
15

2.7 Docking study
We next sought to confirm the in vitro enzymatic inhibition results of our synthesized
compounds against BACE1 by performing molecular docking studies. First, the ligand of Co-
crystallized 1W51 was re-docked to the binding site of the protein structure to validate the
docking protocol. The RMSD value of re-docking in the binding cavity of the enzyme was 1.46.
Based on the standard and accepted RMSD value (less than 2.00 Å), it was concluded that the
docking protocol is acceptable for the analysis of binding modalities of the investigated
compounds. (See the supplementary data Table 3 for all docked results). In almost all active
structure, hydrazine linker depicted hydrogen bonds interactions with Asp32 and 228 crucial for
the efficient inhibition of BACE1. The bi-phenyl forms hydrophobic interaction with residues in
S'2 pocket and different pendant (hetero) aryl oriented toward S2 pocket
Our docking study showed that compound 7d had the lowest binding energy of -13.63 kcal
mol−1. The lowest energy pose of 7d demonstrated hydrogen bond interaction with the side chain
residues Asp32, Asp228, Asn233 and Arg235 within the active site (Fig. 4). Strong hydrogen
bond interactions were found with two key catalytic residues: I) the NH group of hydrazine
linker and the oxygen atom of Asp228 II) N atom of hydrazine linker accepted hydrogen from
OH group of Asp32. Three hydrogen bond interactions were observed between Asn233 and
Arg235 with the nitro group attached at the para position of the pendant phenyl. Moreover, the
triazine ring showed П-П stacking interaction with Tyr71of the S1 sub-pocket. In fact, the
interaction with Tyr71 is the key element for the open and closed conformations of BACE1[27].
Binding modes of another promising compound, 7c (Fig. 5), showed that 7c was also involved in
H-bond interaction between hydrazine linker and Asp228 and Asp32. Furthermore, the triazine
ring was accommodated in the S1 pocket and П-П stacking interaction was observed with Tyr71.
16

Fig. 4 3D docking pose showing interaction for compound 7d (blue) in the binding site of
BACE1.
Fig. 5 3D docking pose showing interaction for compound 7c (grey) in the binding site of
BACE1.
17

3. Conclusion
Here, we report the design and synthesis of a new series of 3-hydrazynyl 1,2 4-triazine
derivatives as multitarget structures for managing AD. Some of our synthesized compounds were
strong inhibitors of BACE1 activity. The key structural feature that enabled BACE1 inhibition
was the presence of electron withdrawing groups at the para position of the phenyl ring. More
specifically, derivatives containing Cl and NO2 in the para position of the phenyl ring (7c, 7d,
respectively) showed higher inhibitory activity compared to the unsubstituted analog 7a. On the
contrary, the replacement of the Cl by F and CH3 groups reduced the inhibitory activity.
Molecular docking analysis of compounds 7c and 7d showed that the high BACE1 inhibitory
potential may be in part due to the hydrogen bond interactions with Asp32, Asp228 within
catalytic cavity of the BACE1 active site. The triazine ring was accommodated in the S1 pocket
and stabled via pi-pi stacking interactions with Tyr71. The bi-phenyl moiety occupied the S'2
pocket and different pendant (hetero) aryl group oriented toward the S2 pocket. Furthermore, our
in vitro neuroprotective assay revealed that compounds 7c and 7d can protect cells from toxic
Aβ25–35 species to various degrees. Additionally, the metal chelatory properties of the most potent
compound (7d) was confirmed using the mole ratio method. Antioxidant potential of all
compounds was tested by the DPPH assay and the results showed that only compound 7d can be
regarded as antioxidant with an IC50 value of 44.42 μM. In addition, the summarized SAR
provided insights for the further development of specific BACE1 inhibitors. More specifically,
7c and 7d can be relevant starting compounds to achieve therapeutic agents against AD.
18

4. Materials and methods
4.1. Chemistry
All reagents were purchased from the suppliers (Sigma-Aldrich, Fluka and Merck) without
further purification. Reaction progress was observed by thin layer chromatographyon on the
glass-backed silica gel sheets (Silica Gel 60 GF254) and visualized under UV light (254 nm).
Melting points were taken on Thermo Scientific Electrothermal digital apparatus (Thermo Fisher
1
Scientific Inc.). H NMR (300 or 500 MHz) and 13C NMR (100 or 125 MHz) spectra were
recorded on a Bruker AV- 300 or 500 (300 or 500 MHz) spectrometer at ambient temperature.
FT- NMR spectrometer are expressed in (δ, ppm) from the solvent resonance (CDCl3, DMSO-d6
2.50 ppm). Mass spectra were obtained on SHIMADZU LC-MS 2010 spectrometer. The infrared
(IR) spectra were run as KBr disk on Perki -Elmer Spectrum RXI FTIR spectrophotometer.
4.1.1. Synthesis of 5,6-diphenyl-1,2,4-triazine-3-thiol (1)
Benzyl (37 mmol) was added to 60 ml of acetic acid and the mixture was heated to about 100° C
with stirring. Hydrazinecarbothioamide (75.04 mmol) was added and the mixture was refluxed
for about 2 h. After cooling, the solid was filtered and washed with cold acetic acid and water.
1
Orange solid; Yield: 88%; mp: 234-236º C, H NMR (300 MHz, DMSO-d6) δH (ppm): 7.34 (m,
10H, Ar-H), 15.18 (s, 1H, S-H).
4.1.2. Synthesis of 3-(methylthio)-5, 6-diphenyl-1,2,4-triazine (2)
Sodium hydroxid (20 mmol) was dissolved in 60 ml of ethanol at 100° C. The basic solution was
cooled to room temperature and 5, 6-diphenyl-1,2,4-triazine-3-thiol (20mmol) was added and
stirred for 15 min. Afterward, iodomethane (47 mmol) was added drop wise to the reaction and
stirred at room temperature for 4 h. After cooling, the precipitated solid was separated and
19

recrystallized from ethanol to give pure compound 3. Yellow solid; Yield: 90%, mp: 154-157 º
C, 1H NMR (300 MHz, DMSO-d6) δH (ppm): 2.71 (s, 3H, S-CH3), 7.40-7.92 (m, 10H, Ar-H).
4.1.3. Synthesis of 3-hydrazineyl-5, 6-diphenyl-1,2,4-triazine (3)
A mixture of 3-(methylthio)-5, 6-diphenyl-1,2,4-triazine (11.8 mmol) and NH2 NH2.H2O (3 mL,
96 mmol) in 30 ml ethanol was refluxed for 12 h. After cooling, the precipitated solid was
separated and washed with cold ethanol and recrystallized from ethanol to give 3-hydrazineyl-
1
5,6-diphenyl-1,2,4-triazine. Dark yellow solid; Yield: 83%, mp: 182-185º C, H NMR (300
MHz, DMSO-d6) δH (ppm): 4.49 (s, 1H, N-H), 6.70 (s, 2H, NH2), 7.34 (m, 10H, Ar-H).
4.1.4. Synthesis of 4-(prop-2-yn-1-yloxy) benzaldehyde (4)
4-Hydroxybenzaldehyde (20 mmol), 3-bromoprop-1-yne (20 mmol) and K2CO3 (0.6 mmol) were
dissolved in DMF (30 mL). The reaction mixture was mixed at 80º C for 18 h. After cooling, the
precipitate was filtered and washed with cold water and n-hexan, dried and give pure 4-(prop-2-
yn-1-yloxy) benzaldehyde . Gray solid; Yield : 76%, mp:153-159º C ,1H NMR (300 MHz,
DMSO-d6) δH (ppm): 3.64 (s, 1H, ≡C-H), 4.89 (s, 2H, O-CH2), 7.09 (d, 2H, J=8.3 Hz, Ar-H),
7.82 (d, 2H, J=8.3 Hz, Ar-H), 8.65 (s, 1H, CHO).
4.1.5. Synthesis of (E)-5,6-diphenyl-3-(2-(4-(prop-2-yn-1-yloxy)benzylidene)hydrazineyl)-1,2,4-
triazine (5)
A mixture of 3-hydrazineyl-5,6-diphenyl-1,2,4-triazine (12 mmol) and 4-(prop-2-yn-1-yloxy)
benzaldehyde (12 mmol) in ethanol (40 mL) was refluxed for 5 h. After cooling, the precipitated
solid was separated and recrystallized from diethyl ether to give pure compound (E)-5,6-
diphenyl-3-(2-(4-(prop-2-yn-1-yloxy)benzylidene)hydrazineyl)-1,2,4-triazine. Yellow solid:
Yield 85%; mp: 211-214° C, IR (KBr, cm-1) ν: 1080, 1173, 1243, 1509, 1614, 2888, 3214, 3432.
20

1H NMR (500 MHz, DMSO-d6) δH (ppm): 3.63 (s, 1H, ≡C-H), 4.86 (s, 2H, CH2), 7.06 (d, 2H,
J=8.8 Hz, Ar-H), 7.37-7.49 (m, 10H, Ar-H), 7.68 (d, 2H, J=8.8 Hz, Ar-H), 8.24 (s, 1H, N=C-H),
11.87 (s, 1H, N-H),13C NMR (125 MHz, DMSO-d6) δc (ppm): 56.0, 78.9, 79.5, 115.7, 128.5,
128.7, 128.9, 129.4, 129.9, 130.7, 136.4, 136.6, 144.2, 151.1, 156.9, 158.7. MS m/z (%): 405
(M+) (5), 248 (30), 178 (100).
4.1.6.
Synthesis
of
substituted
derivative
of
(E)-3-(2-(4-((1H-1,2,3-triazol-4-
yl)methoxy)benzylidene)hydrazinyl)-5,6-diphenyl-1,2,4-triazine (6a-j)
Different derivatives of 5ai were prepared via the reaction of proper alkyl or aryl halide (1.1
mmol) with sodium azide(1 mmol) in the presence of triethylamine in THF/water. After around
30 min, (E)-5,6-diphenyl-3-(2-(4-(prop-2-yn-1-yloxy)benzylidene)hydrazineyl)-1,2,4-triazine
(5) (1 mmol) in absolute THF (5 mL) was added to the solution in the presence of CuSO4 . 5H2O
(10 mol%) and sodium (2R)-2-[(1S)-12-dihydroxyethyl]-4-hydroxy-5-oxo-2H-furan-3-olate (25
mol%), at 50° C and stirred at 70º C for 48 h. After completion of the reaction (checked by using
TLC), the solvent was evaporated under vacuum and the residue was recrystallized from
ethylacetate and n-hexan.
4.1.7. Synthesis of (E)-3-(2-(4-((1-benzyl-1H-1,2,3-triazol-4yl)methoxy) benzylidene)
hydrazineyl) -5,6-diphenyl-1,2,4-triazine (7a):
White solid: Yield 91%; mp: 235~241° C, IR (KBr, cm-1) ν : 1081, 1168, 1239, 1500, 1610,
1
3032, 3435. H NMR (500 MHz, DMSO-d6) δH (ppm): 5.20 (s, 2H, N-CH2-Ph), 5.62 (s, 2H,
CH2-O-Ph), 7.11 (d, 2H, J=8.5 Hz, Ar-H), 7.32-7.49 (m, 15H, Ar-H), 7.67 (d, 2H, J=8.5 Hz, Ar-
H), 8.24 (s, 1H, N=CH), 8.32 (s, 1H, C=CH-N), 11.86 (s, 1H, N-H), 13C NMR (125 MHz,
DMSO-d6) δc (ppm): 53.3, 61.6, 115.6, 125.3, 125.4, 128.1, 128.4, 128.6, 128.7, 129.2, 129.5,
129.9, 130.7, 136.5, 136.6, 139.7, 143.2, 144.0, 151.1, 156.8, 159.6. LC- MS (m/z):538 [M+].
21

4.1.8. Synthesis of (E)-3-(2-(4-((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-
yl)methoxy)benzylidene)hydrazinyl)-5,6-diphenyl-1,2,4-triazine (7b):
Yellow solid: Yield %96; mp: 248~251° C, IR (KBr, cm-1) ν: 1068, 1081, 1167, 1237, 1508,
1610, 2923, 3228 . 1H NMR (500 MHz, DMSO-d6) δH (ppm): 5.20 (s, 2H, N-CH2-Ph), 5.61 (s,
2H, CH2-O-Ph), 7.11 (d, 2H, J=8.3 HZ, Ar-H), 7.28 (d, 2H, J=7.5 Hz, Ar-H), 7.37-7.49 (m, 10H,
Ar-H), 7.58 (d, 2H, J=7.5 Hz, Ar-H), 7.67 (d, 2H, J=8.3 Hz, Ar-H), 8.24 (s, 1H, N=C-H), 8.32
(s, 1H, C=CH-N), 11.86 (s, 1H, N-H),13C NMR (125 MHz, DMSO-d6) δc (ppm): 52.6, 84.5,
115.6, 121.9, 125.3, 126.8, 128.2, 128.6, 128.7, 128.8, 129.5, 129.9, 130.7, 131.3, 132.2, 135.7,
136.5, 136.6, 138.4, 143.3, 156.8. LC- MS (m/z):616 [M+].
4.1.9. Synthesis of (E)-3-(2-(4-((1-(4-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy) benzylidene)
hydrazineyl)-5,6-diphenyl-1,2,4-triazine (7c):
White solid: Yield %93, mp: 228 ~233° C, IR (KBr, cm-1) ν: 1057, 1082, 1168, 1240, 1507,
1
1610, 3000, 3429. H NMR (500 MHz, DMSO-d6) δH (ppm): 5.20 (s, 2H, N-CH2-Ph), 5.63 (s,
2H, CH2-O-Ph), 7.11 (d, 2H, J=8.0 Hz, Ar-H), 7.35-7.48 (m, 14H, Ar-H), 7.67 (d, 2H, J=8.0 Hz,
13
Ar-H), 8.24 (s, 1H, N=C-H), 8.33 (s,1H, C=CH-N), 11.86 (s, 1H, N-H), C NMR (125 MHz,
DMSO-d6) δc (ppm): 52.5, 61.6, 115.6, 128.6, 128.7, 128.8, 129.0, 129.2, 129.4, 129.5, 129.9,
130.4, 133.4, 135.5, 136.5, 136.7, 143.3, 144.3, 146.6, 156.8, 159.6 . LC- MS (m/z): 573 [M+].
4.1.10. Synthesis of (E)-3-(2-(4-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methoxy) benzylidene)
hydrazineyl)-5,6-diphenyl-1,2,4-triazine (7d):
Yellow solid: Yield %84; mp: 228 ~ 231° C, IR (KBr, cm-1) ν: 1080, 1170, 1239, 1347, 1508,
1610, 2924, 3430 .1H NMR (500 MHz, DMSO-d6) δH (ppm): 5.22 (s, 2H, N-CH2-Ph), 5.82 (s,
2H, CH2-O-Ph), 7.11 (d, 2H, J=8.0 Hz, Ar-H), 7.37-7.55 (m, 14H, Ar-H), 7.67 (d, 2H, J=8.0 Hz,
Ar-H), 8.24 (s, 1H, C=CH-N), 8.39 (s, 1H, N=C-H), 11.86 (s, 1H, N-H), 13C NMR (125 MHz,
22

DMSO-d6) δc(ppm): 52.4, 61.6, 115.6, 124.4, 125.7, 128.2, 128.6, 128.7, 128.8, 129.5, 129.5,
129.9, 130.7, 136.0, 136.6, 143.4, 143.8, 144.3, 147.7, 156.8, 159.5. LC- MS (m/z): 583 [M+].
4.1.11.
Synthesis
of
(E)-3-(2-(4-((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methoxy)
benzylidene) hydrazineyl)-5,6-diphenyl-1,2,4-triazine (7e):
Yellow solid, Yield; %76, mp: 227~ 229 ° C, IR (KBr, cm-1) ν: 1074, 1123, 1215, 1520, 1711,
2400, 2927, 3406. 1H NMR (300 MHz, DMSO-d6) δH (ppm): 1.35 (s, 3H, CH3), 5.19 (s, 2H, N-
CH2-Ph), 5.62 (s, 2H, CH2-O-Ph), 7.10 (d, 2H, J=8.4 Hz, Ar-H), 7.20-7.47 (m, 14H, Ar-H), 7.66
(d, 2H, J=8.4 Hz, Ar-H), 8.23 (s,1H, C=CH-N), 8.31 (s, 1H, N=C-H), 11.85 (s, 1H, N-H), LC-
MS (m/z): 552 [M+].
4.1.12.
Synthesis
of
(E)-3-(2-(4-((1-(4-fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)
benzylidene) hydrazineyl)-5,6-diphenyl-1,2,4-triazine (7f):
Yellow solid: Yield %88; mp: 232 ~ 237° C, IR (KBr, cm-1) ν: 1123, 1168, 1240, 1509, 1608,
1
2869, 3214. H NMR (500 MHz, DMSO-d6) δH (ppm): 5.20 (s, 2H, N-CH2-Ph), 5.61 (s, 2H,
CH2-O-Ph), 7.11 (d, 2H, J=8.5 Hz, Ar-H), 7.20-7.49 (m, 14H, Ar-H), 7.67 (d, 2H, J=8.5 Hz, Ar-
H), 8.24 (s, 1H, N=C-H), 8.31 (s,1H, C=CH-N), 11.83 (s, 1H, N-H), 13C NMR (125 MHz,
DMSO-d6) δc (ppm): 52.5, 61.6, 115.5, 125.2, 128.2, 128.6, 128.7, 129.5, 129.9, 130.8, 130.8,
132.7, 136.5, 136.7, 143.3, 144.3, 151.1, 156.8, 159.6, 161.4, 163.3. LC- MS (m/z): 556 [M+].
4.1.13. Synthesis of (E)-3-(2-(4-((1-(4-(tert-butyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)
benzylidene)hydrazineyl)-5,6-diphenyl-1,2,4-triazine (7g):
Yellow solid: Yield %95; mp: 237 ~ 240° C, IR (KBr, cm-1) ν: 1080, 1168, 1243, 1500, 1608,
2950, 2961, 3482 .1H NMR (500 MHz, DMSO-d6) δH (ppm): 1.25 (s, 9H, -C (CH3)3), 5.19 (s,
2H, N-CH2-Ph), 5.57 (s, 2H, CH2-O-Ph), 7.11 (d, 2H, J=8.5 Hz, Ar-H), 7.25 (d, J=8.0 Hz, 2H,
23

Ar-H), 7.37-7.46 (m, 10H, Ar-H), 7.48 (d, J=8.0 Hz, 2H, Ar-H), 7.67 (d, 2H, J=8.5 Hz, Ar-H),
8.24 (s, 1H, C=CH-N), 8.30 (s, 1H, N=C-H), 11.86 (s, 1H, N-H), 13C NMR (125 MHz, DMSO-
d6) δc (ppm):31.5, 34.7, 53.0, 61.6, 115.6, 125.2, 126.0, 128.1, 128.2, 128.6, 128.7, 128.8, 129.5,
129.9, 130.7, 133.5, 136.6, 143.2, 143.7, 144.3, 147.6, 151.1, 156.8, 159.6. LC- MS (m/z): 594
[M+].
4.1.14. Synthesis of (E)-3-(2-(4-((1-(3,4-dichlorobenzyl)-1H-1,2,3-triazol-4-yl) methoxy)
benzylidene) hydrazineyl)-5,6-diphenyl-1,2,4-triazine (7h):
Yellow solid: Yield % 94; mp: 230 ~ 234° C, IR (KBr, cm-1) ν: 1049, 1082, 1185, 1360, 1504,
1735, 3045, 3148 . 1H NMR (500 MHz, DMSO-d6) δH (ppm): 5.21 (s, 2H, N-CH2-Ph), 5.65 (s,
2H, CH2-O-Ph), 7.11 (d, 2H, J=8.3 Hz, Ar-H), 7.30-7.66 (m, 12H, Ar-H), 7.67 (d, 2H, J=8.3 Hz,
Ar-H), 7.95 (s,1H, Ar-H), 8.24 (s, 1H, N=C-H), 8.36 (s, 1H, C=CH-N), 11.86 (s, 1H, N-H), 13
C
NMR (125 MHz, DMSO-d6) δc (ppm): 79.7, 81.9, 115.6, 117.3, 125.4, 128.2, 128.6, 128.7,
128.9, 129.5, 129.9, 130.6, 131.4, 131.5, 131.7, 136.5, 137.4, 137.9, 151.6, 152.4, 153.9, 159.5,
162.8. LC- MS (m/z): 606 [M+].
4.1.15.
Synthesis
of
(E)-3-(2-(4-((1-(3-chlorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)
benzylidene) hydrazineyl)-5,6-diphenyl-1,2,4-triazine (7i):
Yellow solid, Yield; %87, mp: 241 ~ 245° C , IR (KBr, cm-1) ν: 1029, 1080, 1168, 1235, 1507,
1610, 2870, 3214. 1H NMR (300 MHz, DMSO-d6) δH (ppm): 5.21 (s, 2H, N-CH2-Ph), 5.65 (s,
2H, CH2-O-Ph), 7.10 (d, 2H, J=7.2 Hz, Ar-H), 7.38-7.45 (m, 14H, Ar-H), 7.66 (d, 2H, J=7.2 Hz,
Ar-H), 8.24 (s,1H, C=CH-N), 8.36 (s, 1H, N=C-H), 11.84 (s, 1H, N-H),13C NMR (125 MHz,
DMSO-d6) δc (ppm): 52.5, 61.7, 115.6, 125.4, 127.2, 128.2, 128.3, 128.9, 128.6, 128.7, 129.5,
24

129.9, 130.7, 131.2, 133.8, 136.5, 136.7, 138.9, 143.3, 144.3, 151.1, 156.8, 159.5. LC- MS (m/z):
572 [M+].
4.1.16. Synthesis of (E)-2-(3-(4-((4-((2-(5,6-diphenyl-1,2,4-triazin-3-
yl)hydrazono)methyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)propyl)isoindoline-1,3-dione (7j):
Yellow solid, Yield; %85, mp: 240 ~ 243° C, IR (KBr, cm-1) ν : 1080, 1168, 1245, 1508, 1607,
1711, 1771, 2926, 3461. 1H NMR (500 MHz, DMSO-d6) δH (ppm): 2.19 (p, 2H, CH2-CH2-CH2),
3.63 (t, 2H,CO-N-CH2-CH2), 4.45 (t, 2H, N=N-N-CH2-CH2), 5.18 (s, 2H, N-CH2-Ph), 7.11 (d,
2H, J=8.0 Hz, Ar-H), 7.37-7.49 (m, 10H, Ar-H), 7.67 (d, 2H, J=8.0 Hz, Ar-H), 7.84-7.86 (m,
4H, Ar-H), 8.23 (s,1H, C=CH-N), 8.26 (s, 1H, N=C-H), 11.85(s, 1H, N-H), 13C NMR (125
MHz, DMSO-d6) δc (ppm): 23.0, 63.1, 65.3, 76.2, 112.9, 115.5, 118.2, 123.5, 125.2, 128.6,
128.7, 129.5, 129.7, 129.9, 132.2, 134.8, 137.1, 144.7, 145.5, 147.0, 151.2, 156.5, 168.4. LC-
MS (m/z): 635 [M+].
4.2. BACE1 activity evaluations
The BACE1 FRET (fluorescence resonance energy transfer) assay kit was purchased from
Invitrogen (former Pan Vera corporation, Madison, WI). Enzyme inhibition assay was carried
out according to the manufacturer’s instructions; the enzyme (purified baculovirus-expressed
BACE1), substrate (Rh-EVNLDAEFK-Quencher) and test compounds (in DMSO) at different
concentrations were diluted in the reaction buffer (50 mM sodium acetate, pH 4.5) to make 3X
working solutions. The assay was carried out in a black 96-well microplate with a final volume
of 30 µL consisting of equal amounts of 3X enzyme, substrate and test compound. The final
concentration of DMSO was less than 6% (v/v). The reaction mixtures were incubated at 25 ºC
for 90 min in the dark and then 10 µL of the stop solution (2.5 M sodium acetate) was added to
25

stop the reaction. Finally, the fluorescent intensity of the enzymatic product was measured using
a multiwell spectrofluorometer instrument (BMG LABTECH, Polar star, Germany) capable of
measuring at 544 nm excitation and 590 nm emission wavelengths [28]. Each experiment was
repeated 4–5 times and the percentage of enzyme inhibition for each concentration of test
compound was considered from the difference between the maximum enzyme activity wells
(containing substrate plus enzyme) and baseline wells (containing substrate). CurveExpert
software version 1.34 for Windows was used to calculate IC50 values from the concentration
inhibition curves [20].
4.3. Assessment of antioxidant activity
The in vitro antioxidant activity of the synthesized compounds were tested by 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical scavenging activity assay and using Quercetin as the reference
compound. A screening was performed at the concentration of 10, 30 and 100 μM. The free
radical scavenging activity of all synthesized compounds was determined by the DPPH assay.
Several concentrations of the tested compounds dissolved in DMSO were added to DPPH
solution in methanol (110 µM). The mixtures were left in the dark for 30 min and the absorbance
of the solutions was measured at 517 nm by an UV/visible spectrophotometer. Measurements
were carried out in triplicate for each experiment. The percentage of reduced DPPH was
calculated by the following equation: (%) = 100 - [(Abs (control)- Abs (sample))/Abs (control).
4.4. Assessment of neuroprotective activity against Aβ
Neuroprotective activity following exposure to Aβ and synthetic compounds was estimated using
the MTT reduction assay. PC12 neuronal cells were plated in rat-tail collagen coated 96-well
microplates at density of 5* 105 cells/ml (100 ml in each well) and incubated for 48 h to adhere
at 37 °C in humidified air containing 5% CO2. Three to four different concentrations of test
26

compounds were added to each well in triplicate and incubated for 3 h. Ten µl of human Aβ25-35
was then added at a final concentration of 5 µM. At the end of the incubation time (24 h), 90 µl
of the medium was replaced with 20 µL of 0.5 mg/ml MTT dissolved in RPMI containing phenol
red and incubated for 2 h at 37 °C. Afterwards, formazan crystals were solubilized in 200 µl
DMSO. The optical density was measured at 570 nm with background correction at 655 nm
using a Bio-Rad microplate reader (Model 680, Bio-Rad). Stock solution of Aβ25-35 was prepared
by dissolution in distilled water to a 0.5 mM concentration [24]. Tested compounds were all first
dissolved in DMSO and then diluted in the growth medium. For maintenance, two-thirds of the
growth medium were changed every 4 days and the cells were sub-cultured once a week.
4.5. Assessment of metal-chelating effect
The metal chelation assay was carried out using a dual-beam UV–vis spectrophotometer Agilent
100 with 1 cm quartz cells. To investigate the metal binding ability of compound 7d the UV
absorption was recorded in the absence and presence of ZnSO4, FeSO4 with a wavelength
ranging from 200 to 700ꢀnm after incubating for 30ꢀminꢀat room temperature. The molar ratio
method was performed to determine the stoichiometry of the complex. Final concentrations of
the tested compound were 20ꢀμM and the final concentrations of the metal ions ranging from 0–
80 µM. Final volume of the reaction mixture was 1ꢀml. The UV spectra were normalized to the
blank. The difference UV–Vis spectra were examined to investigate the ratio of 6d /metals in the
complexes [22, 24].
4.6. In silico studies
For molecular docking studies, 3D crystal structure of BACE1 (1w51) was retrieved from
protein Data Bank. Water molecules and the cognate ligand were removed from the receptor and
the hydrogen atoms were added and non-polar hydrogens were merged into related atoms of the
27

receptor using ADT. The structures of the compounds were drawn using Chem3D Ultra 8.0
software. The energy minimizations were done by the semi-empirical MM+ of HyperChem
software [29]. The compounds were docked into the active site of proteins with default
parameters. 100 runs for each ligand; 27000 as maximum number of generations; 2500000 as
maximum number of energy evaluations; 0.02 as rate of gene mutation and 0.8 as rate of
crossover. The grid box was set with 50, 50, and 50 points in the x, y, and z directions,
respectively, with the default grid spacing of 0.375 Å. All other options were set as default. Top-
scored conformation was recorded for each compound and used for further analysis.
Acknowledgements
The authors wish to thank the financial support of the Ahvaz branch, Islamic Azad University
and National Institute for Medical Research Development, NIMAD (Grant No. 957334). We also
thank Vice-Chancellor for Research, Shiraz University of Medical Sciences, Shiraz, Iran. We
thank Alireza Edraki from Umass Medical School, MA, USA for thorough editing of this
manuscript.
28

References
[1] 2018 Alzheimer's disease facts and figures, Alzheimer's & Dementia 14(3) (2018) 367-429.
[2] R.J. Castellani, G. Perry, The complexities of the pathology–pathogenesis relationship in
Alzheimer disease, Biochemical Pharmacology 88(4) (2014) 671-676.
[3] M.G. Savelieff, S. Lee, Y. Liu, M.H. Lim, Untangling amyloid-β, tau, and metals in
Alzheimer’s disease, ACS chemical biology 8(5) (2013) 856-865.
[4] L. Mucke, D.J. Selkoe, Neurotoxicity of amyloid β-protein: synaptic and network
dysfunction, Cold Spring Harbor perspectives in medicine 2(7) (2012) a006338.
[5] Y.-H. Liu, B. Giunta, H.-D. Zhou, J. Tan, Y.-J. Wang, Immunotherapy for Alzheimer
disease—the challenge of adverse effects, Nature Reviews Neurology 8(8) (2012) 465.
[6] H. Stower, Searching for Alzheimer’s disease therapies, Nature Medicine 24(7) (2018) 894-
897.
[7] Y. Hamada, Y. Kiso, Discovery of BACE1 Inhibitors for the Treatment of Alzheimer’s
Disease, Quantitative Structure-activity Relationship, InTech2017.
[8] W.J. Lukiw, Amyloid beta (Aβ) peptide modulators and other current treatment strategies for
Alzheimer’s disease (AD), Expert opinion on emerging drugs 17(1) (2012) 43-60.
[9] J.L. Cummings, R. Doody, C. Clark, Disease-modifying therapies for Alzheimer disease
Challenges to early intervention, Neurology 69(16) (2007) 1622-1634.
[10] G. Probst, Y.-z. Xu, Small-molecule BACE1 inhibitors: a patent literature review (2006 –
2011), Expert Opinion on Therapeutic Patents 22(5) (2012) 511-540.
[11] D. Amantea, Editorial overview: Neurosciences: Brain and immunity: new targets for
neuroprotection, Current opinion in pharmacology 26 (2016) v.
[12] A. Robert, Y. Liu, M. Nguyen, B. Meunier, Regulation of copper and iron homeostasis by
metal chelators: A possible chemotherapy for Alzheimer’s disease, Accounts of chemical
research 48(5) (2015) 1332-1339.
[13] A. Kumar, C.M. Nisha, C. Silakari, I. Sharma, K. Anusha, N. Gupta, P. Nair, T. Tripathi, A.
Kumar, Current and novel therapeutic molecules and targets in Alzheimer's disease, Journal of
the Formosan Medical Association 115(1) (2016) 3-10.
[14] M. Nguyen, A. Robert, A. Sournia‐Saquet, L. Vendier, B. Meunier, Characterization of new
specific copper chelators as potential drugs for the treatment of Alzheimer’s disease, Chemistry-
A European Journal 20(22) (2014) 6771-6785.
[15] D. Praticò, Oxidative stress hypothesis in Alzheimer’s disease: a reappraisal, Trends in
pharmacological sciences 29(12) (2008) 609-615.
[16] W.R. Markesbery, Oxidative stress hypothesis in Alzheimer's disease, Free Radical Biology
and Medicine 23(1) (1997) 134-147.
[17] E. Candelario-Jalil, Injury and repair mechanisms in ischemic stroke: considerations for the
development of novel neurotherapeutics, Curr Opin Investig Drugs 10(7) (2009) 644-654.
[18] N. Edraki, O. Firuzi, A. Foroumadi, R. Miri, A. Madadkar-Sobhani, M. Khoshneviszadeh,
A. Shafiee, Phenylimino-2H-chromen-3-carboxamide derivatives as novel small molecule
inhibitors of β-secretase (BACE1), Bioorganic & medicinal chemistry 21(8) (2013) 2396-2412.
[19] N. Edraki, O. Firuzi, Y. Fatahi, M. Mahdavi, M. Asadi, S. Emami, K. Divsalar, R. Miri, A.
Iraji, M. Khoshneviszadeh, L. Firoozpour, A. Shafiee, A. Foroumadi, N-(2-(Piperazin-1-
yl)phenyl)arylamide Derivatives as β-Secretase (BACE1) Inhibitors: Simple Synthesis by Ugi
29