Multifunctional iminochromene-2H-carboxamide derivatives containing different aminomethylene triazole with BACE1 inhibitory, neuroprotective and metal chelating properties targeting Alzheimer's disease

Article abstract of DOI:10.1016/j.ejmech.2017.09.057

Alzheimer's disease (AD) is a neurodegenerative disorder known for the presence of amyloid beta plaques resulting from the sequential action of β-secretase and γ-secretase on amyloid precursor protein. We developed and synthesized, through click reactions, a new family of iminochromene carboxamides containing different aminomethylene triazole. The BACE1 inhibition, neuroprotective capacity and metal chelation of these derivatives make them ideal candidates against AD. Most of the synthesized compounds were shown to have potent BACE1 inhibitory activity in a FRET assay, with an IC₅₀ value of 2.2 μM for the most potent compound. Moreover, molecular modeling evaluation of these BACE1 inhibitors demonstrates the vital role of the amine and amide linkers through hydrogen bond interactions with key amino acids in the BACE1 active site. Our in vitro neuroprotective evaluations in PC12 neuronal cells of Aβ-induced neuroprotection demonstrated promising activity for most of the compounds as neuroprotective agents. Based on our findings, we propose that introduction of a phthalimide substitute on the triazole ring shown to be interesting multifunctional lead compound worthy of further study.

Full text of DOI:10.1016/j.ejmech.2017.09.057

Accepted Manuscript
Multifunctional iminochromene-2H-carboxamide derivatives containing different
aminomethylene triazole with BACE1 inhibition, neuroprotective and metal chelating
properties targeting Alzheimer's disease
Aida Iraji, Omidreza Firuzi, Mehdi Khoshneviszadeh, Marjan Tavakkoli, Mohammad
Mahdavi, Hamid Nadri, Najmeh Edraki, Ramin Miri
PII:
S0223-5234(17)30772-9
10.1016/j.ejmech.2017.09.057
EJMECH 9772
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 May 2017
Revised Date: 25 September 2017
Accepted Date: 27 September 2017
Please cite this article as: A. Iraji, O. Firuzi, M. Khoshneviszadeh, M. Tavakkoli, M. Mahdavi, H. Nadri,
N. Edraki, R. Miri, Multifunctional iminochromene-2H-carboxamide derivatives containing different
aminomethylene triazole with BACE1 inhibition, neuroprotective and metal chelating properties
targeting Alzheimer's disease, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.09.057.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Multifunctional iminochromene-2H-carboxamide derivatives containing different aminomethylene
triazole with BACE1 inhibition, neuroprotective and metal chelating properties
Targeting Alzheimer’s disease
Aida Irajia, Omidreza Firuzia, Mehdi Khoshneviszadeha,b, Marjan Tavakkolia, Mohammad Mahdavic,
Hamid Nadrid, Najmeh Edrakia*, Ramin Miria*
aMedicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences,
Shiraz, Iran.
bDepartment of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences,
Shiraz, Iran.
cEndocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences
Institute, Tehran University of Medical Sciences, Tehran, Iran.
dDepartment of Medicinal Chemistry, Faculty of Pharmacy, Shahid Sadoughi University of Medical
Sciences, Yazd, Iran.
*Corresponding authors:
Ramin Miri, PhrmD, PhD
Medicinal and Natural Products Chemistry Research Center, Shiraz university of Medical Sciences,
Shiraz, Iran
Tel: +98 713 230 3872; Fax: +98 713 230 222
E-mail: ramin.miri.15@gmail.com
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
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Abstract
Alzheimer's disease (AD) is a neurodegenerative disorder known for the presence of amyloid beta plaques
resulting from the sequential action of -secretase and -secretase on amyloid precursor protein. We
β
γ
developed and synthesized, through click reactions, a new family of iminochromene carboxamides
containing aminomethylene triazole. The BACE1 inhibition, neuroprotective capacity and metal chelation
of these derivatives make them ideal candidates against AD. Most of the synthesized compounds were
shown to have potent BACE1 inhibitory activity in a FRET assay, with an IC50 value of 2.2 µM for the
most potent compound. Moreover, molecular modeling evaluation of these BACE1 inhibitors
demonstrates the vital role of the amine and amide linkers through hydrogen bond interactions with key
amino acids in the BACE1 active site. Our in vitro neuroprotective evaluations in PC12 neuronal cells of
Aβ-induced neuroprotection demonstrated promising activity for most of the compounds as
neuroprotective agents. Based on our findings, we propose that introduction of a phthalimide substitute on
the triazole ring shown to be interesting multifunctional lead compound worthy of further study.
.
Keywords
Click reaction, BACE1 inhibitor, Metal chelator, Molecular modeling, Neuroprotective assay, Synthesis.
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1. Introduction
Alzheimer's disease (AD) is a neurodegenerative disorder and the most common cause of dementia in
individuals above the age of 65 [1]. AD is generally characterized by malfunctioning of neurons followed
by gradual loss of neurons. The hallmarks of AD are an accumulation of amyloid beta (A
neurons and neurofibrillary tangles of hyperphosphorylated tau protein within the cells [2]. The
amyloidogenic pathway triggered from the breakdown of amyloid precursor protein (APP) by -secretase
(BACE1) followed by -secretase cleavage [3], results in the formation of A peptides, which are prone
to polymerization into pathological fibrils and other biophysical forms. Various factors, such as mutations
of the APP gene, cellular prions [4], hypoxia [5], and environment, may increase -cleavage relative to α-
β) around
β
γ
β
β
cleavage, which in turn increases the accumulation of Aβ [6, 7]. Based on the above findings, BACE1, a
transmembrane aspartic protease comprising two key aspartate residues (Asp32 and Asp228) in the large
hydrophobic cleft of the active site, has served as an attractive target for the development of inhibitors
against AD [8, 9]. Some reported BACE1 inhibitors are peptidic and pseudopeptidic modified molecules
[10] such as hydroxyethylene (HE), hydroxyethyleneamine (HEA), carbinamine, acylguanidine,
aminoimidazole, and aminoquinazoline [9, 11] designed based on the parent structure of APP.
Unfortunately, the peptidic inhibitors of BACE1 have poor physicochemical and pharmaceutical
properties, such as insufficient oral bioavailability, short serum half-life, or low blood–brain barrier
(BBB) penetration. As a consequence, there is an ongoing demand for small molecule BACE1 inhibitor
drugs [12].
Many cellular processes in the brain, particularly neuronal activity, require metal ions. Cells have adopted
complex mechanisms for maintaining metal ion homeostasis. Disruptions in any of these mechanisms
could alter the ionic balance and result in several neurodegenerative disorders including AD [13].
Evidence suggests that bio metals have two distinct roles in the pathophysiology of AD: (i) aggregation of
A
β
peptide, and (ii) production of reactive oxygen species induced by A
β [13-15]. Excess metal ions have
been found in A plaques of AD patients’ brains [16]. To overcome these neurological disorders, a
β
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number of therapeutic strategies aimed at ameliorating or inhibiting Aβ aggregation and induced ROS
generation, including the use of metal chelators, are currently being investigated for their potential
application in AD therapy [17-19].
The toxic effects of A
mediated neuronal death [20, 21] and apoptosis [22, 23]. Recently, efforts have focused on designing new
compounds with the potential to protect neural cells from the side effects of A [24, 25].
β have been broadly examined. Aβ has been reported to induce oxidative stress-
β
Garino and colleagues reported a new series of compounds containing two fused aromatic rings
(naphthyl, coumarin) coupled to aryl piperazine. These compounds demonstrate significant BACE1
inhibition in enzymatic and cell based assays (Scheme 1, compound 1 and 2) [26, 27]. Further structural
modifications to identify more potent BACE1 inhibitors led us to imino-2H-chromene and phenylimino-
2H-chromen carboxamide moiety coupled to aryl piperazine derivatives as promising BACE1 inhibitors
with high potency against BACE1 and cellular production of Aβ in N2a-APPswe cells (Scheme 1,
compound 3) [28]. Following our ongoing research program on finding potent anti-Alzheimer agents [29-
31], we aimed to design a novel multi-targeted structure based on our previously reported scaffold,
(Scheme 1, compound 3) [28] with the purpose of optimizing physichochemical properties and anti-
Alzheimer potential of this scaffold. To this end, nine candidates of iminochromene-2H-carboxamide
derivatives containing different aminomethylene triazole were selected and synthesized based on
molecular docking analysis of a small library of designed backbone and further evaluated for their
BACE1 inhibitory potential. Protection of neuronal PC12 cells against Aβ-induced damage was examined
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay. Furthermore,
metal chelating activity of some compounds was evaluated using a ferrozine-based assay.
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Scheme 1 Molecular hybridization and analogue-based design of imino-2H-chromene carboxamide bearing
aminomethylene triazole pendant as novel anti-AD agents
2. Results and discussion
2.1 Molecular design and docking study
Designing strategy was carried out in three steps
1. Replacement of the piperazine pendant with aminomethylene triazole: metal therapy is the
preferred medical treatment to reduce the toxic effects of metals. Chelators bind to excess metal
ions to form complex structures, which are easily excreted from the body. Jiaranaikulwanitch et al.
reported tryptoline and tryptamine triazole derivatives as promising metal chelating agent. The
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metal chelating capability of their compounds could be attributed to the ione pair of electrons on
the nitrogen atom in the amino ethylene linker as well as the nitrogen atom in the triazole ring
(Scheme 1, compound 4) [20]. In the other study 1,2,3-triazole pyridine were reported as
bifunctional agent for targeting metal–Aβ species in AD [15]. In this regard, the piperazine linker
of previous scaffolds (compounds 1-3) was replaced with the aminomethylene triazole pendant
with the aim of introducing a metal chelating moiety and facilitating the interactions with the key
catalytic residues of BACE1 active site (Asp32, Asp228). The docking results confirmed that the
amine linker demonstrated H-bond interaction with Asp32 in most derivatives. Additional
hydrophobic interaction occurs due to the interactions between the amino methylene triazine motif
of the ligand and flap loop residues.
2. Imino-2H-chromen carboxamide containing amide linker (Scheme 2, Y part): arylcarboxamide
part was selected based on the molecular docking study of five different fused (hetero) aromatic
rings which was previously reported as potential BACE1 inhibitors. Imino-2H-chromen
demonstrated favorable pose and oriented toward the S2 pocket of the active site (Table S1). In
comparison with the other examined (hetero)aromatic groups, the imino-2H-chromen mostly
oriented in the manner that the amide linker of the backbone could involve in H-bound
interactions with residues such as Asp228, Gly230 and Thr231. As it is depicted in table S2,
comparison the physicochemical properties of the designed scaffold 5 (LogP = 3.8 with molecular
refractivity = 129.6) with the potent derivatives of our previous study [28] (LogP ranging from 5.3
to 7.7 with molecular refractivity ranging from 146.8 to 179.8), demonstrated the superior
physicochemical characteristic of newly designed scaffold. These findings prompted us to select
compound 5 as promising lead for further optimization and modification. Moreover, the in vitro
results of our previous study demonstrated that although phenyliminochromene derivatives
demonstrated superior BACE1 inhibition in enzymatic assay, they were weakly effective in the
cell-based assay. This observation might be attributed to the poor physicochemical properties
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(such as the low solubility in the culture media and high lipophilicity) of phenyliminochrone
derivatives that might compromise its efficiency in the cellular model and hinder their access to
their intracellular target. Furthermore, based on computational analysis inappropriate orientation
of phenyl fragment in phenyliminochromene derivatives may hinder access and prevent
interaction with the Asp228 of the enzyme's active site. The other two heteroaromatic moiety
named indole and naphthyl cannot even occupy favorable pockets (Table S1).
3. Aryl or alkyl motif as X substitute: computational approaches provided a starting point in the
development of new chemical entities. Docking study was performed owing to have limited
resources for synthesis, enzymatic and cell based assay. Various substitutions (100 motif) were
placed at X positions of the triazole pendant to find the best derivatives for synthesis (Scheme 4).
Overall, the X-substituted moieties of the constructed library were selected based on the essential
requirement for effective binding interaction with BACE1 active site (especially S'2 pocket),
feasibility of synthesis and accessibility to chemical reagents. The X substitute attached to the
triazole ring is mainly oriented toward the S'2 of active site. It seems that introducing aromatic
moieties bearing the appropriate hydrogen bond establishing groups into the triazole ring that
facilitates both hydrophobic and hydrogen-bond interaction with the surrounding amino acids of
S'2 pocket, may improve ligand-enzyme interactions. The docking poses of the nine selected
derivatives for synthesis are depicted in Figure 1. (see the supplementary data Table S3 for all
docked structure).
Overall, candidates were selected based on (i) H-bound interactions with the key catalytic residues of
BACE1 active site (Asp32, Asp228), (ii) stacking interaction with the amino acids of the flap pocket,
specially Tyr71 and Trp76 (iii) H-bound interactions with the Gly34 and/or Gly230 (iv) Occupation of a
key pocket within the active site such as S2 and S'2 (v) and the lowest binding free energies (
desired orientation
∆Gb) in the
.
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Scheme 2. Schematic representation of molecular design and docking study
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8a
8b
8c
8d
8e
8f
8g
8h
8i
Figure 1. Binding orientation and interaction of of all synthetic compounds in the BACE1 active site. Hydrogen
bonds are depicted as green lines. Stacking interactions are demonstrated as purple dashed lines
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2.2 Chemistry
To evaluate the influence of aminomethylene triazole-containing iminochromene-2H-carboxamides on
BACE1 activity and neuroprotection, the top 9 derivatives were synthesized. 1-Bromo-2-nitro-N-(prop-2-
yn-1-yl)aniline was obtained following a nucleophile substitution of 4-dibromo-2-nitrobenzene with
propagylamine (Scheme 3)
.
Scheme 3 Protocol, reagents and conditions for the synthesis intermediates 4a-i. (a) TEA, DMF, reflux, 24 h;
(b)NaN3, TEA t-BuOH/water, rt; (c) CuSO4.5H2O, sodium ascorbate, rt. (d) Na2S2O6, Na2CO3, MeOH/water, 70 °C,
3–4 h
.
Various triazole derivatives were prepared by click reaction between the previously prepared molecule
and the appropriate aromatic compound (Scheme 3). As it is depicted in Scheme 3d, reduction of nitro
group of compounds 3a–i was performed using aqueous solution of sodium carbonate and sodium
dithionate in very good yield. Cyanoacetylation of synthesized amines was done with the reaction of 2-
cyanoacetyl-3,5-dimethyl pyrazole intermediate with 4a-i (Scheme 4). To synthesize the final products,
salicylaldehyde was reacted with 7a-i derivatives through Knoevenagel condensation (Scheme 4)
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Scheme 4 Synthesis of imino-2H-chromen-3-carboxamide derivatives 8a-i. (e) neat, rt; (f) water, 2 h, rt. (g) toluene,
reflux (10 min), rt (overnight); (h) piperidine, MeOH, 50°C, 7 h.
2.3 BACE1 enzymatic assay
The BACE1 inhibitory activity of each compound was tested at multiple concentrations and IC50 values
were calculated (Table 1). The results of the in vitro BACE1 inhibition experiments were in agreement
with the molecular docking and computational analysis. According to the obtained data, bromo-phenyl
iminochromene-2H-carboxamide derivatives containing various aminomethylene triazole scaffolds
demonstrated moderate to strong inhibitory potential against BACE1 enzyme at 10 and 50 µM
concentrations.
a)
Substitution of phthalimide on amino methylene triazole plays a significant role in BACE1
inhibition. Compound 8b containing a phthalimide pendant on amino methylene triazole was the most
potent derivative with IC50 value of 2.2 ± 0.5 µM. Elongation of the ethylene linker between the
phthalimide group and triazole ring partially reduced the inhibitory potential of compound 8c (IC50 = 6.8
± 0.9 µM).
b)
Based on the enzyme inhibition comparison of 8d and 8e with 8a, it could be assumed that
introduction of small lipophilic groups such as methoxy or methyl substitutes into the amino methylene
triazole linker may not significantly alter the potency of compounds against BACE1.
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c)
Compound 8i bearing 4-bromophenyl moiety inhibited BACE1 with an IC50 of 6.4 ± 2.4 µM and
could be considered as another potent derivative against BACE1. Compound 8g bearing a 3,4 di-
chlorophenyl moiety demonstrated superior BACE1 inhibitory potential (IC50 = 7.5 ± 1.4 µM) over a 2,3
di-chlorophenyl containing counterpart (IC50 = 15.6 ± 7.4 µM). It suggests that the positioning of such
electron withdrawing substituted groups might affect the ligand-BACE1 binding interaction.
Table 1. BACE1 inhibitory activity of test compounds (8a-i) assessed by BACE1-FRET assay
% Inhibition at 50
µM a/b
% Inhibition at 10
µM a/b
a
Compounds
8a
Structure
IC50
-
25.2 (±0.1)
12.1 (±0.4)
8b
8c
2.2 (±0.5) (µM)
6.8 (±0.9) (µM)
84.2 (±2.1)
83.5 (±2.3)
77.6 (±5.9)
55.7 (±3.3)
-
-
-
34.5 (±5.5)
52.4 (±3.3)
45.9 (±6.3)
10.0 (±0.3)
38.3 (±4.2)
21.3 (±4.8)
8d
8e
8f
8g
7.5 (±1.4) (µM)
60.8 (±2.0)
52.0 (±3.4)
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8h
15.6 (±7.4) (µM)
62.0 (±4.6)
47.8 (±3.4)
72.3 (±18.3)
8i
6.4 (±2.4) (µM)
14.7 (±2.8) (nM)
100.0 (±7.2)
OM99-2 c
a Presented data are the mean (± S.E.M.) of three to five independent determinations.
b % Inhibition were calculated at four different concentrations (50, 10, 1 and 0.1 µM). However, just 50 and 10 µM were reported
c OM99-2 was used as a positive control.
In general, as depicted in Figure 1 H-bound interaction of aminomethylene with Asp32 and amide linker
with Asp228 were observed in most cases which confirm the BACE1 inhibitory of synthesize
compounds. By way of illustration, molecular docking study of 8b and BACE1 active site (Figure 1-8b).
Two key residues of the binding site Asp32 and Asp228 are involved in hydrogen bond interactions with
the amino methylene triazole linker and amide linker, respectively. The additional hydrogen bond
interaction with Gly230 as a second important amino acid was observed through the amide linker.
stacking interaction was observed between Tyr71 and the bromophenyl ring. The phthalimide moiety
establishes favorable stacking interaction with side chain of Thr76.
2.4 Protection of neuronal PC12 cells exposed to A 25-35
The toxic effects of A have been broadly examined. Recently, efforts have focused on designing new
compounds in the hope to protect neural cells against the side effects of A [24, 25]. To our knowledge,
π-π
π-π
β
β
β
there has been no research published on the neuroprotective properties of iminochromene derivatives. In
order to investigate the neuroprotective role of our synthesized compounds, PC12 cells were treated by
these derivatives and then challenged with A
β25–35. In the absence of any synthesized derivatives, the
cells that were treated with A showed 43.4 ± 2.2 % viability compared to control untreated cells (Table
β
2). Caffeic acid as a positive control protected the PC12 cells with an IC50 value of 75.8 ± 11.3 µM.
Significant protection was observed with compounds 8b and 8c containing phthalimide motifs with IC50
values of 7.9 µM and 6.8 µM, respectively. The percentage of protection of PC12 cells challenged with
A
β
25–35 with 10 µM of compounds 8b and 8c were 78.8 and 67.8 %, respectively. The cytotoxicity of
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synthesized derivatives against PC12 cells was also tested. The compounds did not show any cytotoxicity
up to the concentration of 100 µM.
Table 2. Inhibition of Aβ induced neurotoxicity in PC12 neuronal cells by synthesized derivatives.
% Protection at % Protection at
Compounds IC50 (µM) a
25 µMa
10 µMa
8a
8b
8c
8d
8e
8f
-
ND
ND
7.9 (±1.7)
100
78.8 (±18.1)
67.8 (±10.4)
28.4 (±2.4)
ND
6.8 (±1.0)
NDb
-
-
-
22.0 (±0.4)
19.2 (±9.1)
22.2 (±9.9)
13.1 (±9.9)
8g
8h
8i
90.5 (±32.2)
18.9 (±4. 9)
37.8 (±9.2)
49.8 (±12.6)
57.2 (±14.5)
37.5 (±6.9)
21. 8 (±7.3)
23.6 (±10.5)
16.4 (±7.3)
Caffeic acidc
75.8 (±11.3)
-
-
a Protection against Aβ-induced PC12 cell death was measured with MTT assay. The data are expressed as the mean (± S.E.M.)
of three to five independent experiments.
b The % protection was not measured at 25 µM, but it was 83. 8 ± 7. 7 µM at 15 µM.
c Caffeic acid was used as a positive control.
2.5 Metal chelating effect
Results of metal chelating assay of the most potent compounds are demonstrated in Table 3. Ferrozine
forms a complex with ferrous ions and produces a magenta color. In the presence of chelating agents,
formation of ferrous-ferrozine complex is blocked and as a result, absorbance is decreased. Compound 8i
showed acceptable biometal-chelating ability compared to EDTA as a well-known chelator [32].
Table 3. Metal chelating activity of synthesized compounds determined by a colorimetric method.
Percent of Fe2+ chelating
Code
at 400 µmol/La
8b
8c
10.2 ± 1.3
12.8 ± 1.4
45.2 ± 2.1
96.4 ± 5.3
8i
EDTAb
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a Values represent the mean of three independent experiments ± S.E.M..
b EDTA was used as a positive control.
2.6 Iron-chelating properties
The ability of 8i to chelate Fe+2 as biometals was studied by UV-VIS spectroscopy using mole ratio
method. In this method the total molar concentration of Fe(II) was continuously varied, while the molar
concentration of the tested ligand was kept constant throughout the series of samples [33]. According to
Figure 2A the UV absorption of compound 8i in methanol demonstrated three sharp picks in 353, 302 and
293 nm. As shown in Figure 2A, the addition of Fe+2 to 8i does not lead to a red or blue shift of
wavelength but increased the maximum absorbance intensity initially at fix amount of 8i and then
plateaued. Figure 2B indicates that under the experimental conditions the stoichiometry for the Fe+2-8i
complexes was 1.5, suggesting a complex with 3:2 [Fe+2-8i] molar ratio.
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Figure 2. (A) UV spectrum of compound 8i (final concentration was 20 µM) alone or in the presence of
Fe(II) (the final concentration of Fe+2 ranged from 0 µM to 80 µM). (B) Determination of the stoichiometry
of complex Fe+2-8i using molar ratio method through titrating the methanol solution of compound 8i with
ascending amounts of Fe(II)
.
2.7 Physicochemical parameters prediction
All the compounds were predicted by ADMET SAR to have moderate Blood-Brain Barrier (BBB)
permeability (Table 4).
Table 4. Summary of physicochemical properties of synthesized compounds.
BBB penetration a
H- bond
Compound
H- bond donorb
Molar Refractivityb
acceptorb
%
CNS activity
+
8a
8b
8c
8d
8e
8f
0.9
4
5
4
4
5
4
4
4
4
7
129.6
147.5
147.7
134.4
136.2
128.9
137.2
142.1
129.1
0.5
0.5
0.6
0.8
0.9
0.9
0.9
11
10
8
+
+
+
+
8
7
+
+
8g
8h
8i
7
7
+
+
7
0.9
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a http://lmmd.ecust.edu.cn:8000/predict/
b http://www.scfbio-iitd.res.in/software/drugdesign/lipinski.jsp
3. Conclusion
In conclusion, fragment based and hybrid design strategies were used to design a novel series of anti-
Alzheimer agents with multiple modes of action including BACE1 inhibition, neuroprotection, and metal
chelation. Considering the results of the present study, we conclude that bromo phenyl iminochromene-
2H-carboxamides containing aminomethylene triazole pendants could be potentially useful scaffold for
the discovery of anti-Alzheimer agents. Based on the BACE1 molecular docking, the amide and
aminomethylene linkers of the designed compounds will maintain their critical roles of hydrogen bond
interactions with the catalytic dyad in the most derivatives. Generally, the triazole pendant oriented
toward the flap pocket and, this moiety was synthesized via a click reaction as a metal chelating fragment
and developed via a diverse library of anti-Alzheimer agents. In addition, imino-2H-chromen of the
designed scaffold would be indispensable for efficacy, solubility and BBB penetration of the scaffold.
Moreover, these derivatives showed significant neuroprotective activity. Among them, compound 8b and
8c had the highest level of neuronal protection which decreases IC50 to at least ten orders of magnitude in
comparison with positive control. Most of the derivatives also showed interesting BACE1 inhibitory
potency. Among the studied compounds, 8b and 8c demonstrated good inhibitory effect against BACE1
.
The affinity of some synthesized compounds for ferrous ions as nontoxic chelating ligands proposes them
as potential agents for chelation therapy as well. The compounds 8b and 8c would provide a promising
lead for further optimization in targeting Alzheimer's disease.
4. Material and Method
4.1 Molecular docking
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To select the best BACE1 inhibitor candidates for synthesis, a library of small molecules with various
motifs at position Y and then X were drawn using HyperChem and energetically minimized with MM+
force field. The Gastiger charges and torsional degrees of freedom were assigned to the generated PDB
files. The X-ray crystallographic structures were retrieved from the Brookhaven protein data bank
(http://www.rcsb.org/). Virtual screening of designed library was done using Autodock 4.2 and Auto
Dock Tools 1.5.4 (ADT). Water molecules and the cognate ligand were removed from the receptor. All
hydrogens were properly added to the receptor PDB and non-polar hydrogens were merged into related
atoms of the receptor using ADT. Kollman charges were also assigned. The grid maps were calculated
with AutoGrid (part of the AutoDock package). The created three-dimensional grids were 60*60*60 (x, y,
z) with a grid spacing of 0.375 Å and the cubic grids were centered on the binding site of native ligand.
Lamarckian genetic algorithm (LGA) was applied to model the interaction/binding between the ligand
and the BACE1 active site. For Lamarckian GA, 27,000 maximum generations, a gene mutation rate of
0.02, population size of 100 and a cross over rate of 0.8 were used [34]. Scoring function of AD 4.2
includes five energy terms: van der Walls term, hydrogen binding term, electrostatic term, desolvation
term and the term for loss of torsional entropy of ligand upon binding. In the actual docking simulation of
a compound in the docking library, the empirical scoring function of the original Auto Dock program was
employed [35].
4.1.1 Selection of Pdb code
In order to find a suitable x-ray crystallographic structure of BACE1 for virtual study, thirteen pdb
structures were chosen based on the similarity of their cognate ligands with our designed structure.
Among the 13 different pdb structures, 1w51 was selected because of its optimal self- docking results
(free energies of -9.5 kcal/mol, RMSD 0.6, resolution 1.7Å and number in cluster) The results of self-
docking are summarized in Table S4.
4.2 Chemistry
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1
All the reagents were purchased from commercial sources and used without further purification. H and
13C NMR spectra were determined by a Bruker FT-300 MHz spectrometer in CDCl3 or DMSO-d6. All the
chemical shifts were reported as (δ) values (ppm) against tetra methyl silane as an internal standard.
CHNOS analysis was performed using the ECS4010 from Costech Company. The MS spectra were
recorded using Agilent 7000-3Q mass spectrometer at an electron impact mode with an ionization voltage
of 70 eV. The IR spectra were obtained on a FT-IR Perkin–Elmer Precisely system spectrophotometer
(potassium bromide disks). Melting points were determined on a Kofler hot stage apparatus and are
uncorrected. Merck silica gel 60 F254 plates were used for analytical TLC. Preparative thin-layer
chromatography was done with prepared glass-backed plates (20*20 cm2, 500 µ) using silica gel
(MerkKieselgel60 HF254, Art. 7739). Preparative column chromatography was carried out on silica gel
(230–400 mesh, G60 Merck).
General procedures
Synthesis of 4-bromo-2-nitro-N-(prop-2-yn-1-yl)aniline (1)
The alkyne derivative 1 was synthesized in DMF under reflux condition for 24 hours by the reaction of
1,4-dibromo-2-nitrobenzene (10 mmol) with propagylamine (20 mmol) in presence of a catalytic amount
of triethylamine. The product was purified by flash chromatography on silica gel, using 10% ethyl acetate
in petroleum ether as the eluent, affording the product as an orange powder. Yield 54%; MP: 80–84 °C;
MS: m/z(%) 256 ((M+2)+, 41), 254 (M+, 41), 209 (40), 207 (40), 185 (45), 183 (100), 181 (62), 171 (22),
169 (22), 129 (45), 128 (62), 102 (68), 90 (22), 75 (30), 63 (37); 1H NMR (CDCl3, 300 MHz): δH (ppm) =
2.24 (s, 1H, alkyn-H ), 4.04-4.06 (dd, 2H, J= 3, 5.7 Hz, CH2), 6.81-6.84 (d, 2H, J= 9.0 Hz, Ar-H), 7.48-
7.52 (dd, 1H, J= 2.4, 9 Hz, Ar-H), 8. 033 (s, 1H, NH), 8.263–8.271 (d, 1H, J= 2.4 Hz, Ar-H).
Synthesis of aryl substituted derivatives of 4-bromo-N-((1-methyl-1H-1,2,3-triazol-4-yl)methyl)-2-
nitroaniline (3a-i)
19

ACCEPTED MANUSCRIPT
Different derivatives of intermediates 2a-i were prepared in situ via reaction of the appropriate alkyl or
halide (7 mmol) with sodium azide (7 mmol) in the presence of triethylamine in t-BuOH/water. After
around 30 minutes, 4-bromo-2-nitro-N-(prop-2-yn-1-yl)aniline (6.5 mmol), CuSO4.5H2O (10 mol%) and
sodium ascorbate (25 mol%) were added to 2a-i. [36, 37]. The mixture was stirred at room temperature
for 48 h and an orange precipitate was produced. Produced solids were washed with water several times.
Further purification was performed using column chromatography with a decreasing ratio of petroleum
ether to ethyl acetate (90:10 to 50:50) to produce the desired products 3a-i.
N-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-4-bromo-2-nitroaniline (3a)
Benzyl bromide (7 mmol) and NaN3 (6.9 mmol) in the presence of triethylamine in t-BuOH/water was
stirred in room temperature for 30 minutes and then 4-bromo-2-nitro-N-(prop-2-yn-1-yl)aniline 1,
CuSO4.5H2O (10 mol%) and sodium ascorbate (25 mol%) were added. The mixture was stirred at room
temperature for 48 h and an orange participate was produced. The resulted precipitate was filtered off,
washed with water and diethyl ether and recrystallized from ethanol. Yield 62%; MP: 189–191 °C; MS:
m/z (%) 389 ((M+2)+, 5), 387 (M+, 5), 343 (18), 341 (18), 104 (8), 91 (100), 77 (6), 65 (14); 1H NMR
(DMSO-d6, 300 MHz): δH (ppm) = 4.69 (s, 2H, CH2 ), 5.57 (s, 2H, CH2), 7.07–7.10 (d, 1H , J= 9.3 Hz,
Ar-H), 7.29- 7.36 (m, 5H, Ar-H), 7.60-7.63 (dd, 1H, J= 2.1, 9.3 Hz, Ar-H), 7.85 (s, 1H, Ar-H), 8.16–8.17
(d, 2H, J=2.1 Hz, Ar-H).
Synthesis of aryl substituted derivatives of 4-bromo-N1-((1-methyl-1H-1,2,3-triazol-4-
yl)methyl)benzene-1,2-diamine (4a-i)
The nitro compound 3a–i (6.7 mmol) was dissolved in methanol (25 ml). The metabolic solution of nitro
compound was added drop wise (using dropping funnel) to an aqueous solution of sodium carbonate (6.5
g, 61.3 mmol) and sodium dithionite (13 g, 74.4 mmol) containing 100 ml water at 70°C. When all of the
nitro starting material was added to mixture, the mixture was stirred at 70°C for 2 h to complete the
reaction (Checked by TLC). To remove methanol of mixture, it was evaporated under reduced pressure
20

ACCEPTED MANUSCRIPT
and the resultant aqueous mixture was extracted with ethyl acetate, and dried with Na2SO4, filtered, and
concentrated. The resulting product was further purified using preparative thin layer chromatography
chloroform/EtOH (90:10).
N1-((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)-4-bromobenzene-1,2-diamine (4a)
Brown oily product; Yield 87%; MS: m/z (%) 359 ((M+2)+, 80), 357 (M+, 80), 187 (33), 144 (40), 118
(10), 91 (100), 65 (15); 1H NMR (CDCl3, 300 MHz): δH (ppm)= 3.41 (brs, 2H, NH2), 4.23 (s, 2H, CH2),
5.35 (s, 2H, CH2), 6.36– 6.39 (d, 1H, J= 6.0 Hz, Ar-H), 6.67–6.73 (m, 2H, Ar-H), 7.11-7.13 (brm, 2H,
Ar-H), 7.245 (s, 6H, Ar-H).Synthesis of 3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxopropanenitrile (6)
2-cyano acetohydrazide (compound 5) was synthesized by the reaction of hydrazine hydrate and ethyl 2-
cyanoacetate as previously described (yield: 97%) [38]. An equimolar amount of compound 5 was reacted
with acetyl acetone in the presence of a catalytic amount of HCl in water (15 mL). The mixture was
stirred at room temperature for two hours. The white precipitate (scheme 4, compound 6) was filtered off,
washed with cold water and subsequently dried in an oven. Yield 91%; MS: m/z (%) 163 (M+, 46), 135
(23), 121 (8), 95 (100), 81 (11.5), 68 (10), 54 (5), 39 (12); 1H NMR (CDCl3, 400MHz) δH (ppm): 2.23 (s,
3H, CH3), 2.55 (s, 3H, CH3), 4.29 (s, 2H, CH2-CN), 6.03 (s, 1H, Pyrazole-H).
Synthesis of aryl substitiated derivatives of N-(5-bromo-2-(((1-methyl-1H-1,2,3-triazol-4-
yl)methyl)amino)phenyl)-2-cyanoacetamide (7a-i)
The 3-(3,5-dimethyl-1H-pyrazol-1-yl)-3-oxopropanenitrile 6 (1.2 g, 7 mmol) was added to a solution of
different aryl substituted derivatives of 4-bromo-N1-((1-methyl-1H-1,2,3-triazol-4-yl)methyl)benzene-
1,2-diamine 4a-i in toluene (10 ml). The mixture was refluxed for 30 min and then stirred at room
temperature overnight. The resulting precipitate was filtered off, washed with Et2O and further purified by
thin layer chromatography using chloroform/EtOH (97:3) to afford compound 7a–i as pure solid product.
Synthesis
of
N-(2-(((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)amino)-5-bromophenyl)-2
cyanoacetamide (7a)
21

ACCEPTED MANUSCRIPT
Brown powder; Yield 51%; MS: m/z (%) 426 ((M+2)+, 7) , 424 (M+, 7), 408 (20), 406 (20), , 383 (7), 144
(13), 115 (6), 104 (6), 91 (100); 1H NMR (CDCl3, 300 MHz) δH (ppm) = 4.25 (s, 2H, CH2), 5.35 (s, 2H,
CH2), 5.38 (s, 2H, CH2), 7.03-7.05 (d, 1H, J= 7.5 Hz, Ar-H), 7.29-7.33 (m, 2H, Ar-H), 7.41-7.43 (m, 4H,
Ar-H), 7.48-7.52 (m, 1H, Ar-H), 7.8103 (s, 1H, Ar-H).
Synthesis
of
different
derivatives
of
N-(5-bromo-2-(((1-methyl-1H-1,2,3-triazol-4-
yl)methyl)amino)phenyl)-2-imino-2H-chromene-3-carboxamide (8a-i)
An equimolar mixture of salicylaldehyde (5 mmol,), aryl substituted analogues of N-(5-bromo-2-(((1-
methyl-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)-2-cyanoacetamide (7a-i) (5 mmol), methanol (10 ml)
and a catalytic amount of piperdine (0.1 ml) was stirred at 50 °C for 6–7 hours. After cooling, the yellow
precipitate was filtered, washed with Et2O and subsequently dried under reduced pressure. The resulting
residue was further purified using thin layer chromatography using chloroform/EtOH (85:15) to afford
yellow precipitate.
Synthesis of N-(2-(((1-benzyl-1H-1,2,3-triazol-4-yl)methyl)amino)-5-bromophenyl)-2-imino-2H-
chromene-3-carboxamide (8a)
Pale yellow powder; MP 210–216°C; Yield 76%; IR (CDCl3, cm−1) νmax = 3683 (NH), 3620 (NH), 3584
(NH), 3019 (CH), 1710 (C=O; C=N), 1260 (C-N), 1216 (C-O); MS: m/z (%) 532 ((M+2)+, 1.5), 530 (M+,
1.5), 515 (25), 513(25), 394 (45), 392 (45), 367 (10), 342 (23), 340 (23), 312 (15), 314 (15), 212 (21), 144
(17), 120 (23), 106 (40), 91 (100), 77 (21); 1H NMR (DMSO-d6, 300MHz) δH (ppm): 5.66 (s, 2H, CH2),
5.92 (s, 2H, CH2), 7.14-7.16 (d, 1H, J= 8.7 Hz, Ar-H), 7.32-7.42 (m, 7H, Ar-H), 7.58-7.64 (m, 1H, Ar-H),
7.70-7.77 (m, 2H, Ar-H), 7.84-7.87 (d, 1H, J= 8.7 Hz, Ar-H), 8.33-8.38 (m, 3H, Ar-H), 9.093 (s, 1H, CH
(iminochromene)); 13C NMR (DMSO-d6,75 MHz) δC (ppm): 51.4, 78.1, 108.1, 114.2, 115.8, 118.9, 120.0,
120.5, 121.4, 123.0, 124.3, 125.7, 126.9, 128.1, 130.2, 131.7, 133.3, 135.7, 138.5, 140.1, 141.4, 145.7,
149.3, 153.7, 156.3, 160.4; C27H23BrN6O3: C 57.97, H 4.14, N 15.02. Found: C 57.92, H 4.14, N 15. 00 ;
C26H21BrN6O2: C 58.99, H 4.00, N 15.87. Found: C 58.93, H 4.05, N 15. 90.
22

ACCEPTED MANUSCRIPT
Synthesis of N-(5-bromo-2-(((1-(2-(1,3-dioxoisoindolin-2-yl)ethyl)-1H-1,2,3-triazol-4-yl)methyl)
amino) phenyl)-2-imino-2H-chromene-3-carboxamide (8b)
Yellow powder ;Yield 86%; MP 238–242°C; IR (CDCl3, cm−1) νmax = 3690 (NH), 3650 (NH), 3615 (NH),
3019 (CH), 1710 (C=O; C=N), 1261 (C-N), 1216 (C-O); MS: m/z (%) 613((M+2)+, 0.5), 611 (M+, 0.5),
598 (20), 596 (20), 411 (22), 409 (22), 394 (100), 392 (100), 342 (30), 340 (30), 314 (16.5), 174 (28), 160
(40), 146 (24), 104 (10); 1H NMR (DMSO-d6, 300MHz) δH (ppm): 3.73-3.75 (t, 2H, J= 6.0 Hz, CH2),
4.00-4.02 (t, 2H, J=6.0, CH2), 5.59 (s, 2H, CH2), 6.73-6.76 (d, 1H, J= 7.4 Hz, Ar-H), 7.23-7.30 (m, 4H,
Ar-H), 7.55-7.58 (m, 3H, Ar-H), 7.89-8.01 (m, 5H, Ar-H), 8.53 (s, 1H, NH (iminochromene)), 9.20 (s,
1H, CH (iminochromene)), 12.26 (s, 1H, NHCO); 13C NMR (DMSO-d6,75 MHz) δC (ppm): 52.43, 55.21,
81.19, 107.06, 109.81, 112.48, 114.22, 115.59, 117.2, 120.8, 122.2, 123.7, 126.8, 128.8, 129.5, 130.6,
132.1, 133.7, 135.1, 138.4, 140.6, 141.7, 143.3, 146.9, 149.9, 152.3, 154.6, 156.3, 161.2; C29H22BrN7O4:
C 56.87, H 3.62, N 16.01. Found: C 56.92, H 3.60, N 15. 94.
Synthesis of N-(5-bromo-2-(((1-(3-(1,3-dioxoisoindolin-2-yl)propyl)-1H-1,2,3-triazol-4-yl)methyl)
amino) phenyl) -2-imino-2H-chromene-3-carboxamide (8c)
Yellow powder; Yield 84%; MP 230–235°C; IR (CDCl3, cm−1) νmax = 3684 (NH), 3610 (NH), 3584 (NH),
3019 (CH), 1710 (C=O; C=N), 1261 (C-N), 1216 (C-O); MS: m/z (%) 627 ((M+2)+, 0.1), 625 (M+, 0.1),
530 (50), 359 (36), 357 (36), 339 (40), 337 (40), 324 (27.4), 322 (28), 316 (20), 314 (20),172 (100), 145
(70), 118 (60), 89 (50); 1H NMR (DMSO-d6, 300MHz) δH (ppm): 2.145 (brm, 2H, CH2), 3.56 (brm, 2H,
CH2), 3.70 (brm, 2H, CH2), 5.58 (s, 2H, CH2), 6.73-6.75 (d, 1H, J= 7.0 Hz, Ar-H), 7.23-7.29 (m, 4H, Ar-
H), 7.55-7.58 (m, 3H, Ar-H), 7.84 (s, 4H, Ar-H), 7.99 (s, 1H, Ar-H), 8.52 (s, 1H, NH (iminochromene)),
9.19 (s, 1H, CH (iminochromene)), 12.27 (s, 1H, NHCO); 13C NMR (DMSO-d6, 75 MHz) δC (ppm): 32.3,
52.4, 52.8, 80.9, 108.2, 110.3, 114.5, 115.4, 118.9, 121.0, 123.4, 124.7, 126.2, 126.9, 128.5, 130.5, 132.0,
133.7, 134.7, 136.1, 138.4, 140.7, 141.7, 146.2, 150.5, 153.9, 156.1, 160.7, 163.7, 165.6; C30H24BrN7O4:
C 57.52, H 3.86, N 15.65. Found: C 57.49, H 3.89, N 15. 67.
23

ACCEPTED MANUSCRIPT
Synthesis of N-(5-bromo-2-(((1-(4-methoxybenzyl)-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)-2-
imino-2H-chromene-3-carboxamide (8d)
Yellow powder; Yield 84%; MP 234–237°C; IR (CDCl3, cm−1) νmax = 3670 (NH), 3583 (NH), 3401 (NH),
3018 (CH), 1622 (C=O; C=N), 1218 (C-N), 1216 (C-O),1053 (C-O); MS: m/z (%) 544 ((M+2)+, 0.7), 542
(M+, 0.7), 427 (32), 392 (30), 342 (30), 340 (30), 323 (21), 321 (21), 211 (17), 121 (100), 91 (35), 77
(22), 63 (16); 1H NMR (DMSO-d6, 300MHz) δH (ppm): 3.72 (s, 3H, CH3), 5.40 (s, 2H, CH2), 5.55 (s, 2H,
CH2), 6.83-6.86 (d, 2H, J= 8.4 Hz, Ar-H), 7.14-7.16 (d, 2H, J= 8.4 Hz, Ar-H), 7.44-7.56 (m, 4H, Ar-H),
7.68-7.77 (m, 3H, Ar-H), 7.85-7.90 (m, 2H, Ar-H), 8.00 (s, 1H, NH (iminochromene)), 8.51 (s, 1H, CH
(iminochromene)); 13C NMR (DMSO-d6,75 MHz) δC (ppm): 59.3, 76.0, 80.7, 108.3, 112.9, 116.8, 122.2,
122.5, 123.4, 123.6, 125.2, 127.4, 128.5, 130.4, 130.8, 130.9, 134.1, 134.4, 140.0, 145.0, 149.1, 152.3,
154.5, 158.4, 159.1, 161.8, 167.3; C27H23BrN6O3: C 57.97, H 4.14, N 15.02. Found: C 57.92, H 4.14, N
15. 00.
Synthesis of N-(5-bromo-2-(((1-(4-methylbenzyl)-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)-2-
imino-2H-chromene-3-carboxamide (8e)
Yellow powder; Yield 81%; MP 215–219°C; IR (CDCl3, cm−1) νmax = 3671 (NH), 3584 (NH), 3404 (NH),
3020 (CH), 1710 (C=O; C=N), 1363 (C-N), 1216 (C-O); MS: m/z (%) 543 ((M+2)+, 2), 541 (M+, 2), 527
(10), 525 (10), 394 (100), 394 (100) 342 (36), 340 (36), 314 (14), 312 (14), 143 (14), 105 (57), 91 (50),
1
77 (15); H NMR (DMSO-d6, 300MHz) δH (ppm): 1.23 (s, 3H, CH3), 4.38 (s, 2H, CH2), 5.57 (s, 2H,
CH2), 6.73-6.76 (d, 1H, J= 8.1 Hz, Ar-H), 7.08-7.17 (m, 3H, Ar-H), 7.20 -7.24 (m, 4H, Ar-H), 7.31-7.33
(m, 2H, Ar-H), 7.35-7.56 (d, 1H, J= 8.1, Ar-H), 7.72-7.74 (d, 1H, J= 7.2 Hz, Ar-H), 7.90 (s, 1H, Ar-H),
8.03 (s, 1H, NH (iminochromene)) , 8.30 (s, 1H, CH (iminochromene); 13C NMR (DMSO-d6,75 MHz) δC
(ppm): 31.0, 52.4, 79.5, 105.5, 113.5, 115.9, 116.1, 116.2, 119.5, 123.1, 124.6, 124.8, 125.5, 130.0, 130.6,
131.5, 131.8, 132.7, 132.8 133.0, 133.4, 143.6, 146.6, 153.6, 154.1, 159.8, 160.6; C27H23BrN6O2: C 59.68,
H 4.27, N 15.47. Found: C 59.61, H 4.34, N 15. 53.
24

ACCEPTED MANUSCRIPT
Synthesis of N-(5-bromo-2-(((1-(4-fluorobenzyl) -1H-1,2,3-triazol-4-yl)methyl)amino )phenyl)-2-
imino-2H-chromene-3-carboxamide (8f)
Yellow powder; Yield 83%; MP 235–240°C; IR (CDCl3, cm−1) νmax = 3683 (NH), 3585 (NH), 3406 (NH),
3020 (CH), 1710 (C=O; C=N), 1363 (C-N), 1216 (C-O); MS: m/z (%) 548 ((M+2)+, 4), 546 (M+, 4), 401
(6), 387 (15), 385 (15), 342 (11), 340 (11), 292 (9), 290 (9), 171 (29), 143(31), 109 (100), 77 (10); 1H
NMR (DMSO-d6, 300MHz) δH (ppm): 4.383 (s, 2H, CH2), 5.57 (s, 2H, CH2), 6.73-6.76 (d, 1H, J= 7.8 Hz,
Ar-H), 7.08-7.18 (m, 2H, Ar-H), 7.21 (s, 1H, Ar-H), 7.22-7.25 (m, 4H, Ar-H), 7.31-7.34 (m, 2H, Ar-H),
7.51-7.56 (m, 1H, Ar-H), 7.30-7.75 (s, 1H, Ar-H), 7.90 (s, 1H, Ar-H), 8.04 (s, 1H, NH (iminochromene)),
8.313 (s, 1H, CH (iminochromene). C26H20BrFN6O2: C 57.05, H 3.68, N 15.35. Found: C 56.99, H 3.71,
N 15. 35.
Synthesis of N-(5-bromo-2-(((1-(3,4-dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)amino) phenyl)-2-
imino-2H-chromene-3-carboxamide (8g)
Yellow powder; Yield 72%; MP 227–230°C; IR (CDCl3, cm−1) νmax = 3681 (NH), 3584 (NH), 3407 (NH),
3020 (CH), 1710 (C=O; C=N), 1363 (C-N), 1216 (C-O); MS: m/z (%) 601 ((M+4)+, 0.5), 599 ((M+2)+ 1),
597 (M+, 0.5), 581 (8), 579 (5.2), 502 (2.7), 500 (6.7), 498 (4.1), 392 (100), 390 (100), 342 (40), 340 (40),
314 (13), 312 (13), 290 (8), 288 (8), 159 (50), 143 (17), 115 (12), 89 (23), 63 (14); 1H NMR (DMSO,
300MHz) δH (ppm): 4.39 (s, 2H, CH2), 5.54 (s, 2H, CH2), 6.66-6.70 (d, 1H, J= 13.0 Hz, Ar-H), 7.01-7.14
(m, 5H, Ar-H), 7.30-7.37 (m, 5H, Ar-H), 7.77-7.82 (m, 2H, Ar-H), 8.81 (s, 1H, CH (iminochromene));
C26H19BrCl2N6O2: C 52.20, H 3.20, N 14.05. Found: C 52.16, H 3.23, N 14. 06.
Synthesis of N-(5-bromo-2-(((1-(2,3-dichlorobenzyl)-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)-2-
imino-2H-chromene-3-carboxamide (8h)
Yellow powder; Yield 73%; MP 229–232°C; IR (CDCl3, cm−1) νmax = 3683 (NH), 3586 (NH), 3406 (NH),
3020 (CH), 1710 (C=O; C=N), 1362 (C-N), 1216 (C-O); MS: m/z (%) 601 ((M+4)+, 1), 599 ((M+2)+ 2),
597 (M+, 1), 581 (10), , 392 (100), 390 (100), , 342 (50), 340 (50), 314 (20), 312 (20), 290 (18), 288 (18),
25

ACCEPTED MANUSCRIPT
1
173 (40), 159 (80), 143 (27), 89 (32), 63 (24); H NMR (DMSO-d6, 300MHz) δH (ppm): 4.41 (s, 2H,
CH2), 5.56 (s, 2H, CH2), 6.67-6.71 (d, 1H, J= 12.0 Hz, Ar-H), 7.03-7.19 (m, 5H, Ar-H), 7.32-7.41 ( m,
5H, Ar-H), 7.81-7.86 (t, 2H, J= 8.0 Hz, Ar-H), 8.44 (s, 1H, CH (iminochromene)); 13C NMR (DMSO-
d6,75 MHz) δC (ppm): 57.3, 80.9, 109.6, 112.9, 115.2, 117.1, 117.4, 123.2, 123.3, 124.5, 125.5, 127.6,
128.8, 129.4, 131.6, 132.7, 134.2, 136.3, 143.2, 148.6, 152.5, 155.7, 158.8, 159.1, 162.5, 165.2;
C26H19BrCl2N6O2: C 52.20, H 3.20, N 14.05. Found: C 52.15, H 3.25, N 13. 98.
Synthesis of N-(5-bromo-2-(((1-(4-bromobenzyl)-1H-1,2,3-triazol-4-yl)methyl)amino)phenyl)-2-
imino-2H-chromene-3-carboxamide (8i)
Yellow powder; Yield 91%; MP 241–243°C; IR (CDCl3, cm−1) νmax = 3684 (NH), 3620 (NH), 3527 (NH),
3019 (CH), 1710 (C=O; C=N), 1452 (C-N), 1216 (C-O); MS: m/z (%) 610 ((M+4)+, 0.3), 608 ((M+2)+
0.7), 606 (M+, 0.3), 541 (11), 539 (12), 537 (12), 512 (13), 510 (27), 508 (14), 463 (28), 447 (26), 396
(16), 392 (16), 342 (51), 340 (51), 290 (44), 288 (44), 249 (20), 171 (100), 143 (46), 115 (19), 90 (38); 1H
NMR (CDCl3, 300MHz) δH (ppm): 4.43 (s, 2H, CH2), 5.38 (s, 2H, CH2), 6.51-6.54 (d, 1H, J= 8.4 Hz, Ar-
H), 7.03-7.20 (m, 5H, Ar-H), 7.38-7.47 (m, 7H, Ar-H), 7.76 (s, 1H, NH (iminochromene)), 8.44 (s, 1H,
CH (iminochromene)), 12.09 (s, 1H, NHCO); 13C NMR (DMSO-d6,75 MHz) δC (ppm): 52.4, 80.7, 108.3,
114.5, 115.4, 118.9, 119.4, 120.9, 121.7, 123.4, 124.7, 126.1, 126.7, 128.5, 130.5, 132.0, 133.7, 136.0,
138.2, 140.5, 141.8, 146.1, 150.2, 153.9, 156.1, 160.7; C26H20Br2N6O2: C 51.34, H 3.31, N 13.82. Found:
C 51.30, H 3.20, N 13. 81.
4.3 BACE1 enzymatic assay
The BACE1 enzyme inhibition assay was carried out using a FRET (Forster resonance energy transfer)
kit, from Invitrogen (former Pan Vera corporation, Madison, WI) according to the manufacturer
instructions. BACE1 (purified baculovirus-expressed enzyme) was diluted with the assay buffer (50 mM
sodium acetate, pH 4.5) to make a 3X working solution of 1 Unit/ml. The peptide substrate (Rh-
EVNLDAEFK-Quencher) was also diluted with the same assay buffer to provide the 3X stock solution
26

ACCEPTED MANUSCRIPT
(750 nM). The inhibitor stock solutions in DMSO were diluted with buffer to provide 3X solution of test
compounds at different concentrations. The 3X solution of BACE1 enzyme (10 µl) and each inhibitor
sample (10 µl) were placed in 96-well plates and gently mixed. The substrate 3X solution (10 µl) was
then added to this mixture in each well to start the reaction at the final reaction volume of 30 µl (the final
concentration of DMSO in each sample and control wells was equal or less than 4%). The reaction
mixtures were incubated at 25 °C for 90 min in the dark and then the reaction was stopped by adding 10
µl of 2.5 mM sodium acetate. Fluorescence was monitored at 544 nm (excitation wave length) and 590
nm (emission wavelength). OM99-2 was used as a reference inhibitor compound. A multi-well
spectrofluorometer instrument (BMG LABTECH, Polar star, Germany) was used for measurements. IC50
values were calculated with Curve Expert software version 1.34 for Windows. Each experiment was
repeated for three to five times. All data were presented as mean ± S.E.M.
4.4 Protection of neuronal PC12 cells against Aβ-induced damage
PC12 (rat pheochromocytoma) cells were a generous gift from Professor Lloyd A. Greene (Department of
Pathology and Cell Biology, Columbia University, New York, NY). Cells were grown in monolayer
culture on collagen-coated plates at 37ºC in humidified air containing 5% CO2. Two-thirds of the growth
medium was changed every 3-4 days and the cells were sub-cultured once a week. The ability of the
synthesized derivatives in protecting neuronal PC12 cells against Aβ25–35 induced damage was examined
by the MTT assay. Stock solution of Aβ25-35 was prepared by dissolution in distilled water to a 0.5 mM
concentration. Prior to use, Aβ was diluted in media at a concentration of 55 µM. PC12 cells at a density
of 5 × 105 cells/ml (100 µl in each well) were seeded in rat-tail collagen-coated 96-well plates and
incubated for 48 h to adhere at 37ºC. Synthesized derivatives at various concentrations were added in
triplicate and incubated for 3 hours. Human Aβ25–35 was then added at a final concentration of 5 µM. After
24 h, 90 µL the medium was taken out and 20 µL of MTT (0.5 mg/ml dissolved in RPMI containing
phenol red) was added and incubated for an additional 2 h at 37ºC. Afterwards, formazan crystals were
solubilized in 200 µl DMSO. The optical density was measured at 570 nm with background correction at
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655 nm using a Bio-Rad microplate reader (Model 680, Bio-Rad). Each experiment was repeated 3–5
times. The percentage of protection was calculated for each compounds based on the equation: Protection
of cells (%) = [(A0 – Ab) / (100- Ab)] * 100
A0 is the viability% of the cell in the presence of A
presence of A regarded as 0% protection
4.5 Chelating activity of ferrous (Fe2+) ion
β and derivatives, Ab is the viability % of cells in the
β
All synthesized compounds were tested for their metal chelating effect using spectroscopic
measurements. The chelating effect of the synthesized compounds on ferrous ions was estimated by the
method of Dinis with slight modifications [39, 40]. Briefly, 100ꢀµL of each test sample (4 mM) was added
to 100
µL of 0.6 mM FeCl2. The volume of this solution was increased to 900
µL with methanol. The
reaction was initiated by the addition of 100
µ
L of 5 mM ferrozine into the mixture, which was then left
at room temperature for 10 min and followed by measurement of the absorbance at 562 nm [41].
Methanol, Fe2+ and ferrozine were used as a control and methanol was used as a sample blank.
4.6 Metal binding studies
The metal binding studies were carried out in a Agilent UV- 2450 spectrophotometer in a 1 cm quartz
cuvette using mole ratio method [42]. To investigate the metal binding ability of compound, the UV
absorption of the 8i (dissolved in DMSO and diluted in MeOH) as best synthetic metal chelator in the
absence or presence of FeSO4 was recorded with wavelength ranging from 200 to 800 nm after incubating
for 30 min at room temperature [43]. All agents were dissolved in absolute methanol, and the final
concentrations of the 8i were 20 µ µM. The
M and the final concentration of Fe+2 ranged from 0 to 80
stoichiometry of the Fe+2-8i complex was determined by titrating the methanol solution of tested
compound with ascending of FeSO4.
4.7 Physicochemical Parameters
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