Design and synthesis of donepezil analogues as dual AChE and BACE-1 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2018.06.031

Multi-target-directed ligands (MTDLs) centered on β-secretase 1 (BACE-1) inhibition are emerging as innovative therapeutics in addressing the complexity of neurodegenerative diseases. A new series of donepezil analogues was designed, synthesized and evaluated as MTDLs against neurodegenerative diseases. Profiling of donepezil, a potent acetylcholinesterase (hAChE) inhibitor, into BACE-1 inhibition was achieved through introduction of backbone amide linkers to the designed compounds which are capable of hydrogen-bonding with BACE-1 catalytic site. In vitro assays and molecular modeling studies revealed the dual mode of action of compounds 4–6 against hAChE and BACE-1. Notably, compound 4 displayed potent hAChE inhibition (IC₅₀ value of 4.11 nM) and BACE-1 inhibition (IC₅₀ value of 18.3 nM) in comparison to donepezil (IC₅₀ values of 6.21 and 194 nM against hAChE and BACE-1, respectively). Moreover, 4 revealed potential metal chelating property, low toxicity on SH-SY5Y neuroblastoma cells and ability to cross the blood–brain barrier (BBB) in PAMPA-BBB assay which renders 4 a potential lead for further optimization of novel small ligands for the treatment of Alzheimer's disease.

Full text of DOI:10.1016/j.bioorg.2018.06.031

Accepted Manuscript
Design and Synthesis of Donepezil Analogues as Dual AChE and BACE-1 in-
hibitors
Moustafa T. Gabr, Mohammed S. Abdel-Raziq
PII:
S0045-2068(18)30412-7
DOI:
https://doi.org/10.1016/j.bioorg.2018.06.031
YBIOO 2411
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
27 April 2018
20 June 2018
23 June 2018
Please cite this article as: M.T. Gabr, M.S. Abdel-Raziq, Design and Synthesis of Donepezil Analogues as Dual
AChE and BACE-1 inhibitors, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.06.031
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Design and Synthesis of Donepezil Analogues as Dual AChE and
BACE-1 inhibitors
a,b
c,d
Moustafa T. Gabr * and Mohammed S. Abdel-Raziq
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
b
Department of Chemistry, University of Iowa, Iowa City, Iowa 52242, USA
c
Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
d
School of Chemistry and Molecular Biosciences, University of Queensland, St Lucia 4072, Queensland, Australia

Corresponding author: Tel.: +1 3193599500; E-mail address: gabr2003@gmail.com
Abstract: Multi-target-directed ligands (MTDLs) centered on β-secretase 1 (BACE-1)
inhibition are emerging as innovative therapeutics in addressing the complexity of
neurodegenerative diseases. A new series of donepezil analogues was designed, synthesized and
evaluated as MTDLs against neurodegenerative diseases. Profiling of donepezil, a potent
acetylcholinesterase (hAChE) inhibitor, into BACE-1 inhibition was achieved through
introduction of backbone amide linkers to the designed compounds which are capable of
hydrogen-bonding with BACE-1 catalytic site. In vitro assays and molecular modeling studies
revealed the dual mode of action of compounds 4-6 against hAChE and BACE-1. Notably,
compound 4 displayed potent hAChE inhibition (IC50 value of 4.11 nM) and BACE-1 inhibition
(
IC50 value of 18.3 nM) in comparison to donepezil (IC50 values of 6.21 and 194 nM against
hAChE and BACE-1, respectively). Moreover, 4 revealed potential metal chelating property, low
toxicity on SH-SY5Y neuroblastoma cells and ability to cross the blood-brain barrier (BBB) in
PAMPA-BBB assay which renders 4 a potential lead for further optimization of novel small
ligands for the treatment of Alzheimer's disease.
Keywords: Alzheimer’s disease, Acetylcholinesterase, β-Secretase 1, Donepezil analogues,
Permeability
1
. Introduction
Alzheimer's disease (AD), a progressive neurodegenerative disease, is the most common cause
of dementia in elderly people [1]. It is estimated that approximately one-third of people over 85
years of age are affected by AD. The dysfunction in basal forebrain cholinergic system is
considered a hallmark of AD that is directly associated with memory impairment and cognitive
deficit [2]. Consequently, the inhibition of acetylcholinesterase (AChE), an enzyme that
catalyzes acetylcholine hydrolysis emerged as a valuable approach in the treatment of AD by
improving the cholinergic functions in AD patients [3]. AChE inhibitors are still the only FDA-
approved drugs as palliative therapeutics in the early symptomatic stage of AD [4]. Blocking the
production of amyloid-β (Aβ) plaques is an alternative therapeutic approach for AD through
the inhibition of β-secretase 1 (BACE-1) [5]. Extracellular cleavage of the amyloid precursor
-
1 -

protein (APP) by BACE-1 is the limiting step in the deposition of Aβ plaques in the brain
which contributes significantly in the pathogenesis of AD [5]. The current complexity of AD
therapy as well as poor efficacy of lead BACE-1 and AChE inhibitors have directed research
towards identification of multi-target-directed ligands (MTDLs) as potential therapeutics of
AD [6].
Donepezil, a potent and selective hAChE inhibitor, is a FDA-approved drug for treatment of
mild to severe dementia in AD [7-9]. The potency and safety of donepezil have directed
tremendous research efforts with the aim of designing donepezil analogues for AD therapy [10-
1
2]. The X-ray crystal structure of donepezil with hAChE (PDB code: 4EY7) identified two
main sites for receptor-ligand interactions: the catalytic anionic site (CAS) featuring Trp86,
Tyr130, Tyr133, Phe 338 residues and the peripheral anionic site (PAS) including Trp286,
Tyr341, Asp74 residues [13-16]. Analysis of donepezil binding to hAChE revelaed dual
interaction of donepezil with both CAS and PAS [17-19]. The indanone moiety of donepezil
stacks against Trp286 residue in PAS, while the benzyl ring features π–π interaction with Trp 86
residue in CAS (Figure 1). Moreover, the carbonyl group of the indanone moiety is involved in
hydrogen bonding with Phe295 residue.
Figure 1. X-ray crystal structure of recombinant human acetylcholinesterase in complex with donepezil (PDB ID:
4
EY7). (A) 3D binding pose. (B) 2D binding pose.
Structure-based design of hAChE inhibitors relying on donepezil as a lead shared the
structural features displayed in Figure 2. The general structure of hAChE inhibitors revealed a
methoxy-substituted aromatic ring that interacts with PAS of hAChE , a basic center that binds to
CAS and a linker between these two fragments such as CH
2
, CONH and CONHCH [20-23].
2
-
2 -

Figure 2. General structure of hAChE inhibitors based on donepezil.
Structure identification of BACE-1 with polypeptide inhibitors (PDB code: 1M4H) by X-ray
diffraction supported structure-based approaches for BACE-1 inhibitors [24,25]. The Asp32 and
Asp228 residues in BACE-1 play a key role in catalyzing the hydrolysis of amide bonds of APP,
which are identified as catalytic sites of BACE-1 [26]. The isatin-based BACE-1 inhibitor (I,
Figure 3), identified using a virtual high-throughput screening approach, displayed potent
inhibition of BACE-1 with IC50 value of 2.4 µM through hydrogen-bonding interactions with the
catalytic aspartate residues [26]. This design strategy was further extended recently to identify
BACE-1-based MTDLs through introduction of connecting amide bonds to isoquinoline
derivatives to enable hydrogen bond formation with BACE-1 active site [27]. In the aim of
development of potent MTDLs, the pharmacophoric features displayed by hAChE inhibitors
were further tuned to incorporate other functional groups that can inhibit BACE-1. In this
context, the multi-targeted coumarin derivative (II, Figure 3) exhibited dual AChE and BACE-1
submicromolar inhibitory potency [28]. Moreover, donepezil analogue (III, Figure 3) emerged as
a potential lead for dual AChE/BACE-1 inhibitors owing to the structural rigidity caused by the
double bond on the indanone moiety [29]. Development of MTDLs centered on BACE-1 is
limited owing to the poor pharmacokinetic profile and CNS penetration ability of the lead
compounds as well as the complexity of obtaining a balanced inhibitory profile against multiple
targets [25, 30]. Based on these findings, research strategies have focused on development of
hybrid molecules characherized by similarity to known AD drugs [31-33]. In this study, we
report the synthesis and evaluation of donepezil analogues that maintain the general structural
features required for hAChE inhibition (Figure 2). In addition, backbone amide linkers are
introduced to the donepezil tempelate to maximize BACE-1 inhibition through hydrogen-
bonding interactions with the catalytic aspartate residues based on previous findings [26, 27].
Moreover, amide linkers are proposed to introduce metal chelating ability to the designed ligands
which represents a vital therapeutic approach for AD. The effectiveness of the design strategy
was further validated via evaluation of analogues lacking the backbone amide bonds. Molecular
modeling studies were employed to investigate the dual interaction mechanism of the new
compounds with hAChE and BACE-1. In addition, the ability of the new compounds identified
in this study to cross the blood-brain barrier (BBB) and their effect on viability of SH-SY5Y
neuroblastoma cells were further evaluated.
-
3 -

Figure 3. Chemical structures of compounds I-III.
2
. Results and Discussion
2.1. Chemistry
A general approach for the synthesis of the designed compounds is outlined in Scheme 1. As
shown in Scheme 1, the starting isonipecotic acid 1 reacted with benzoyl chloride 2 under
microwave irradiation to afford compound 3 in a good yield. Further coupling of 3 with
substituted benzylamines under microwave irradiation proceeded smoothly to furnish target
compounds 4-6 in serviceable yields. Moreover, refluxing 4-6 and lithium aluminum hydride in
tetrahydrofuran yielded compounds 7-9 (Scheme 1).
Scheme 1.
2.2. Dual hAChE and BACE-1 inhibitory activities
The biological effect of the synthesized compounds 4-9 has been assessed for their inhibitory
activities exerted on the in vitro hAChE and BACE-1 activities. To test the preliminary
inhibition power, the hAChE and BACE-1 activities were measured in the absence or in the
presence of 0.1 μM of compounds 4-9 and donepezil (Figure 4 and 5). The results reported in
-
4 -

Figure 4 revealed that compound 4 exhibited the greatest inhibitory activity on hAChE which
was greater than that exhibited by donepezil. In addition, compounds 5 and 6 demonstrated
comparable potency to donepezil in terms of inhibitory activity on hAChE. Such finding clearly
demonstrate the importance of the 3,4-dimethoxy substitution pattern of 7 on the hAChE
inhibitory effect of the tested compounds. Moreover, compounds 7-9 revealed generally lower
activity as hAChE inhibitors which reveals that the amide bonds play a key role in the interaction
with the target receptor. In Table 1, the inhibitory potency of 4-9 on hAChE is expressed as IC50
,
which represents the concentration of inhibitor required to decrease the enzyme activity by 50%.
Compound 4, the most active member of this study displayed IC50 value of 4.11 nM in
comparison to IC50 value of 6.21 nM for donepezil.
The effect of 4-9 on BACE-1 activity was compared to that exhibited by donepezil (Figure 5).
Interestingly, compounds 4-6 exhibited potent BACE-1 inhibitory activity at 0.1 μM in contrast
to the effect of donepezil. It is noteworthy to mention that the comparable potency of 4-6 as
BACE-1 inhibitors suggests that the substitution pattern of the methoxy groups on the benzyl
moiety has no influence on potential interaction with BACE-1. However, compounds 7-9 lacking
the backbone amide linkers displayed negligible BACE-1 inhibitory activity at 0.1 μM in
agreement with our proposed hypothesis. The IC50 values of 4-6 as BACE-1 were in the range of
1
8.3-19.1 nM which further confirmed their potencty in comparison to IC50 value of 194 nM for
donepezil. These results further validate our hypothesis of tailoring donepezil template to BACE-
inhibitory activity through the introduction of backbone amide linkers in 4-6. Compound 4
1
displayed better dual activity and lower IC50 values against both hAChE and BACE-1 than the
FDA-approved donepezil.
Figure 4. AChE activity determined in the presence of donepezil and compounds 4-9 (0.1 μM). Negative control
represents AChE activity in the absence of the tested compounds. The data represents the average of three different
#
#
determinations with indication of the standard error, (**p < 0.05, vs control; ***p < 0.01 vs. control; p < 0.05, vs
#
##
donepezil; p < 0.01 vs. donepezil (ANOVA)).
-
5 -

Figure 5. BACE-1 activity determined in the presence of donepezil and compounds 4-9 (0.1 μM). Negative control
represents BACE-1 activity in the absence of the tested compounds. The data represents the average of three
#
#
different determinations with indication of the standard error. (**p < 0.05, vs control; ***p < 0.01 vs. control; p <
###
0
.05, vs donepezil; p < 0.01 vs. donepezil (ANOVA)).
Table 1. IC50 values (nM) of compounds 4-9 for their AChE and BACE-1 inhibitory activity.
Comp.
AChE
IC50 (nM)
BACE-1a
IC50 (nM)
a
4
5
6
7
8
9
4.11 ± 0.12
8.46 ± 0.23
9.78 ± 0.14
145 ± 1.42
157 ± 2.43
165 ± 1.75
6.21 ± 0.04
18.3 ± 0.17
18.5 ± 0.13
19.1 ± 0.06
243 ± 1.13
277 ± 2.11
253 ± 1.62
194 ± 1.24
15.1 ± 0.06
Donepezil
BACE-1 inhibitor IV
b
Nd
Bold values represent best results.
Data are shown as mean values ± standard deviation (n=3); Nd; not determined.
a
b
2.3. In vitro cytotoxicity testing
The effect of compounds 4-6 on cell viability was assessed in SH-SY5Y neuroblastoma
cells. Compounds 4-6 did not affect SH-SY5Y cells viability, in comparison with the untreated
controls and rotenone as a positive control (Figure 6). Further screening at 10-dose level revealed
that 4-6 possessed IC50 values of >50 μM against SH-SY5Y neuroblastoma cells. These results
demonstrate the potential utility of these compounds as leads for development of therapeutics for
neurological diseases.
-
6 -

Figure 6. The effect of donepezil, compounds 4-6 and rotenone on SH-SY5Y cells viability at three different
concentrations. As controls, RPMI medium in the absence or in the presence of 0.1% DMSO, were used.
2.4. Molecular modeling
In order to get an insight into the dual interaction mechanism of 4 with hAChE and BACE-1, a
molecular docking study based on the X-ray crystal structures of hAChE (PDB code 4EY7) and
BACE-1 (PDB code 1M4H) was carried out. The results of the docking study using hAChE
(
PDB code 4EY7) as shown in Figures 7A & 7B indicated that compound 4 covered the binding
gorge in a satisfactory orientation comparable to that of donepezil resulting in potent hAChE
inhibitory activity. The dimethoxy substituted benzyl moiety occupied the PAS of hAChE
interacting by π–π stacking with Trp286 and Tyr341 residues. Similarly to donepezil, Phe295
residue of hAChE was involved in hydrogen bonding with the amide bond of 4. In addition, the
piperidinyl moiety established hydrophobic interactions with Phe297, Phe338 and Tyr124
residues in the CAS of hAChE (Figure 7).
-
7 -

Figure 7. (A) 3D Interaction of compound 4 with the binding site of hAChE (PDB code 4EY7). The atoms are
colored as following: red for oxygen atoms, blue for nitrogen atoms, white for hydrogen atoms and cyan for carbon
atoms. (B) 2D Interaction of compound 4 with the binding site of hAChE (PDB code 4EY7). Dashed lines represent
hydrogen bonds. Hydrophobic interactions are shown by green solid lines.
Moreover, compound 4 displayed preferential binding to BACE-1 (PDB code 1M4H) with
−
1
estimated binding energy of −18.76 kcal mol which comes in good agreement with in vitro
BACE-1 inhibitory assay. The detailed analysis of the binding interaction is displayed in the 2D
binding mode of 4 in Figure 8. The piperidinyl moiety of 4 is stretched into a pocket of the target
enzyme revealing interactions with Gly11, Ile110 and Gln12 residues. The dimethoxy substituted
benzyl moiety of 4 was involved in π–π stacking with Tyr71 and Tyr198 residues. In addition,
the amide linkers in 4 displayed hydrogen bonding with Asp228, Thr232 and Thr72 residues of
the catalytic site of BACE-1. In addition, the binding mode of donepezil to BACE-1 (PDB code
1
M4H) was examined. The 2D binding interaction of donepezil displayed in Figure 9 revealed
interaction of donepezil with hydrogen bonding to Thr232 and Thr72 residues. Lacking
hydrogen bonding interaction with Asp228 residue for donepezil further validates its key role in
maximizing the in vitro potency of compound 4 against BACE-1 in comparison to donepezil.
Similarly to compound 4, donepezil displayed π–π stacking with Tyr71 and Tyr198 residues.
Taking into consideration the analysis of the docking results and in vitro enzymatic assays, it was
confirmed that compound 4 was a dual inhibitor of hAChE and BACE-1.
-
8 -

Figure 8. 2D Interaction of compound 4 with the active site of BACE-1 (PDB code 1M4H). Dashed lines represent
hydrogen bonds. Hydrophobic interactions are shown by green solid lines.
Figure 9. 2D Interaction of donepezil with the active site of BACE-1 (PDB code 1M4H). Dashed lines represent
hydrogen bonds. Hydrophobic interactions are shown by green solid lines.
2.5. In vitro blood-brain barrier permeability
In the process of development of therapeutics for neurological diseases, the ability of a drug to
permeate the blood–brain barrier (BBB) is a crucial property for central nervous system (CNS)
drugs. The parallel artificial membrane permeability assay for BBB (PAMPA-BBB) is a high
throughput technique used to evaluate the ability of small molecules to transverse the BBB
expressed as effective permeability (P
comparing the P values of compounds 4-6 to that of caffeine as a low passive permeability drug
and donepezil as a high passive permeability drug. Based on the P values obtained (Table 2),
compounds 4-6 showed high passive permeability with P values ranging from 20.13 to 22.97
e
, cm/s) [34]. Assay validation was performed by
e
e
e
-6
-6
×
10 cm/s which is comparable to P value of donepezil 23.35 ×10 cm/s. Moreover, the
e
lipophilicity of a drug is considered a critical parameter that affects its pharmacokinetic
properties. Compounds with moderate lipophilicity (Log D7.4 0–3) possess a good balance
between solubility and permeability required for oral absorption and cell membrane permeation
in cell-based assays [35]. In general, CNS drugs possess (Log D7.4 ~2) which is optimal for BBB
penetration and low metabolic liability. To examine the likelihood of success of the investigated
compounds 4-6, their Log D7.4 were experimentally determined using shake flask method (Table
2
). Compounds 4-6 displayed optimal (Log D7.4 ~2) required for CNS drugs which renders them
potential leads for further development as AD therapeutics.
−
6
−1
Table 2. PAMPA-BBB permeability (P
e
× 10 cm s ) of 4-6 and controls expressed as P , their
e
lipophilicities at pH 7.4 (Log D7.4) and their predictive penetration to the CNS.
Comp.
P
e
LogD7.4
Predicition of
CNS penetration
−6
−1
(
× 10 cm s )
4
5
6
20.13
22.43
22.97
23.35
2.24
2.27
2.43
2.49
2.71
-0.63
High
High
High
High
Low
Donepezil
Caffeine
-
9 -

2.6. Metal chelating ability
2
+
2+
Numerous studies reported higher levels of biometals such as Cu and Zn in an AD brain
compared to healthy brain [36-38]. These results suggested that destruction of the balance of
metal ions in CNS is an underlying cause of neurodegenerative diseases. Indeed, several lead
compounds with metal-chelating property have displayed promising potential as AD therapeutics
[
39-41]. These findings directed us to postulate that incorporation of amide linkers in the
designed compounds could introduce metal-chelating property that would be useful in the design
of MTDLs for AD treatment. The metal chelating property of compound 4 was examined by
recording its UV-visible absorption spectra with wavelength ranging from 200 to 500 nm in the
2
+
presence of increasing concentrations of Cu . The differential absorption spectra obtained upon
2
+
incremental addition of Cu (5-50 μM) to 4 revealed remarkable hyperchromic effect which
2
+
accounts for potential interaction between 4 and Cu (Figure 10). These results indicate that 4
2
+
could effectively chelate Cu , and thereby could serve as a metal chelator in treating AD. The
metal chelating property of 4 is attributed to the presence of the amide bonds as well as the
dimethoxy groups.
Figure 10. Differential UV-vis absorption spectra of of compound 4 (25 μM) in methanol in the presence of
increasing concentrations of CuCl
absorption spectra from the spectra of the free ligand and CuCl
2
(5-50 μM). The differential spectra were obtained by substraction of the mixture
at the corresponding concentration.
2
3. Conclusion
There is a pressing need to develop MTDLs for neurodegenerative diseases that combine
structural requirements for potency with the strict need of tolerable toxicity and BBB
permeation. In a summary, donepezil analogues are introduced in this study as dual hAChE and
BACE-1 inhibitors which are considered promising MTDLs as therapeutics for neurodegerative
diseases. Profiling the donepezil template with potent hAChE inhibiton to exert BACE-1
inhibition was achieved through introduction of backbone amide bonds to compounds 4-6 which
-
10 -

are capable of hydrogen-bonding to the active site of BACE-1. In preliminary biological
screening, compounds 4-6 maintained excellent hAChE inhibitory activity comparable to the
FDA-approved drug, donepezil. In addition, 4-6 exhibited significantly superior BACE-1
inhibition compared to donepezil. The corresponding analogues 7-9 lacking the amide linkers
displayed minimal inhibitory activity against BACE-1 which demonstrated the key role of the
amide bonds in the interaction with the target receptor. The optimal candidate compound 4,
exhibited IC50 values of 4.11 and 18.3 nM against hAChE and BACE-1, respectively. Moreover,
4
displayed potential biometal-chelating ability, high permeability in PAMPA-BBB assay and no
effect on the viability of SH-SY5Y neuroblastoma cells in its effective concentration. Molecular
modeling studies further revealed the dual binding capability of 4 to the active sites of hAChE
and BACE-1. The lead compounds identified in this investigation proved to be promising
scaffolds for the development of new potent multitarget compounds for neurological diseases.
4
. Experimental
4
.1. Chemistry
All commercially available starting materials, reagents, and solvents were used as supplied,
unless otherwise stated. Reported yields are isolated yields. Purification of all final products
was accomplished by silica gel flash column chromatography. Chloroform : methanol or hexane
1
13
:
ethyl acetate were used as elution solvents. Proton ( H) and carbon ( C) NMR were collected
1
13
on Bruker NMR spectrometer at 400 MHz for H and 100 MHz for C. Chemical shifts ( ) are
reported in parts-per million (ppm) relative to residual undeuterated solvent. Melting points
were recorded using a capillary melting point apparatus and are uncorrected. High resolution
mass spectra were obtained using electron spray ionization (ESI). Purity (>95%) of compounds
4
-9 was confirmed by analytical HPLC.
4
.1.1. 1-Benzoylpiperidine-4-carboxylic acid (3)
A mixture of isonipecotic acid 1 (129 mg, 1.0 mmol), benzoyl chloride 2 (140 mg, 1.0 mmol)
and Et N (0.32 mL, 2.5 mmol) in THF (5 mL) was stirred under microwave irradiation (120 W)
3
at 70 °C for 15 min. The reaction mixture was acidified using 1M HCl (10 mL) and extracted
with dichloromethane (3× 25 mL). The combined organic layers were dried over anhydrous
Na
2
SO , filtered, and concentrated under reduced pressure. The crude product was purified by
4
flash column chromatography using 5% ethyl acetate in hexane, followed by 80% ethyl acetate
1
in hexane as eluent to yield 3 (94%) as white solid. Mp 136-138°C. H NMR (400 MHz,
CDCl
3
)
1.71-1.85 (m, 3H), 2.03-2.06 (m, 1H), 2.59 (tt, 1H, J = 10.4, 4.1 Hz), 3.04-3.12 (m,
1
3
2
H), 3.72-3.76 (m, 1H), 4.48-4.52 (m, 1H), 7.36-7.44 (m, 5H), 11.16 (br s, 1H). C NMR (100
MHz, CDCl
3
)
40.4, 41.4, 46.8, 126.7, 128.3, 129.6, 135.3, 170.7, 178.3. HRMS (ESI): calcd
[M-H] , 232.0973; found, 232.0979.
-
for C13
H14NO
3
4
.1.2. 1-Benzoyl-N-(3,4-dimethoxybenzyl)piperidine-4-carboxamide (4)
To a solution of compound 3 (233 mg, 1.0 mmol) in MeCN (4 mL), PyBroP (466 mg, 1.0
mmol) and Et N (0.32 mL, 2.5 mmol) were added and stirred at room temperature for 10 mins.
3
A solution of 3,4-dimethoxybenzylamine (167 mg, 1.0 mmol) in MeCN (3 mL) was then added
and the reaction mixture stirred under microwave irradiation (80 W) at 90 °C for 20 min. The
-
11 -

solvent was removed under reduced pressure, and the crude mixture was suspended in H
mL) and extracted with dichloromethane (2× 20 mL). The combined organic layers were dried
over anhydrous Na SO , filtered, and concentrated under reduced pressure. The crude product
was purified by flash column chromatography using 10% ethyl acetate in hexane, followed by
0% ethyl acetate in hexane as eluent to yield 4 (91%) as yellowish white solid. Mp 177-179°C.
2
O (20
2
4
5
1
H NMR (400 MHz, CDCl
3
)
1.63-1.81 (m, 3H), 2.27-2.33 (m, 2H), 2.71-2.86 (m, 2H), 3.67-
3
.71 (m, 1H), 3.77 (s, 3H), 3.78 (s, 3H), 4.25 (d, 2H, J = 5.4 Hz), 4.52-4.56 (m, 1H), 6.68-6.75
1
3
(
m, 3H), 7.10-7.19 (m, 2H), 7.25-7.35 (m, 3H). C NMR (100 MHz, CDCl
3
) 41.3, 42.5, 42.8,
4
1
6.8, 55.5, 55.6, 110.9, 119.6, 126.4, 127.9, 128.2, 128.7, 129.4, 135.6, 148.0, 148.7, 170.0,
+
73.7. HRMS (ESI): calcd for C22
.1.3. 1-Benzoyl-N-(2-methoxybenzyl)piperidine-4-carboxamide (5)
Using the procedure given for the preparation of 4, coupling of 3 (233 mg, 1.0 mmol) and 2-
H
27
N
2
O
4
[M+H] , 383.1970; found, 383.1967.
4
methoxybenzylamine (137 mg, 1.0 mmol) gave 5 (87%) as pale yellow solid after purification
by flash column chromatography using 40% ethyl acetate in hexane as eluent. Mp 152-153°C.
1
H NMR (400 MHz, CDCl
3
)
1.52-1.75 (m, 4H), 2.26-2.32 (m, 1H), 2.31-2.81 (m, 2H), 3.61-
3
.66 (m, 1H), 3.70 (s, 3H), 4.28 (d, 2H, J = 5.6 Hz), 4.46-4.51 (m, 1H), 6.74-6.81 (m, 2H), 6.90
1
3
(
t, 1H, J = 5.7 Hz), 7.08-7.16 (m, 2H), 7.22-7.32 (m, 5H). C NMR (100 MHz, CDCl
3
) 38.1,
3
1
8.2, 42.2, 46.7, 54.8, 109.8, 120.0, 125.9, 126.2, 128.0, 128.1, 128.3, 129.1, 135.6, 156.8,
+
69.8, 173.6. HRMS (ESI): calcd for C21
H
25
N
2
3
O [M+H] , 353.1865; found, 353.1871.
4.1.4. 1-Benzoyl-N-(4-methoxybenzyl)piperidine-4-carboxamide (6)
Using the procedure given for the preparation of 4, coupling of 3 (233 mg, 1.0 mmol) and 4-
methoxybenzylamine (137 mg, 1.0 mmol) gave 6 (82%) as yellowish white solid after
purification by flash column chromatography using 50% ethyl acetate in hexane as eluent. Mp
1
1
2
1
88-191°C. H NMR (400 MHz, CDCl
3
) 1.62-1.82 (m, 4H), 2.27-2.32 (m, 1H), 2.82-2.87 (m,
H), 3.65-3.69 (m, 1H), 3.73 (s, 3H), 4.26 (d, 2H, J = 5.7 Hz), 4.52-4.57 (m, 1H), 6.50 (br s,
H), 6.79 (d, 2H, J = 8.4 Hz), 7.10 (d, 2H, J = 8.4 Hz), 7.24-7.36 (m, 5H). C NMR (100 MHz,
1
3
CDCl
3
)
41.3, 42.5, 42.7, 47.0, 55.1, 113.8, 126.5, 128.3, 128.8, 129.5, 130.3, 135.8, 158.7,
+
1
70.2, 173.7. HRMS (ESI): calcd for C21
H
25
N
2
3
O [M+H] , 353.1865; found, 353.1869.
4
.1.5. 1-(1-Benzylpiperidin-4-yl)-N-(3,4-dimethoxybenzyl)methanamine (7)
To a solution of 4 (382 mg, 1.0 mmol) in THF (8 mL), LiAlH
added at 0°C. The reaction mixture was refluxed for 6 hrs, then it was cooled to room
temperature and quenched with H O (10 mL) and extracted with dichloromethane (2× 10 mL).
The combined organic layers were dried over anhydrous Na SO , filtered, and concentrated
under reduced pressure. The crude product was purified by flash column chromatography using
4
(189 mg, 5.0 mmol) was
2
2
4
1
3
0% ethyl acetate in hexane to yield 7 (76%) as yellow solid. Mp 144-146°C. H NMR (400
MHz, CDCl 1.21-1.28 (m, 3H), 1.65-1.70 (m, 2H), 1.89-1.94 (m, 2H), 2.48 (d, 2H, J = 6.7
Hz), 2.83-2.87 (m, 2H), 3.45 (s, 2H), 3.69 (s, 2H), 3.83 (s, 3H), 3.85 (s, 3H), 6.77-6.80 (m, 2H),
3
)
1
3
6
.86 (s, 1H), 7.23-7.31 (m, 5H). C NMR (100 MHz, CDCl
3
)
30.5, 36.0, 53.5, 53.7, 55.2,
-
12 -

5
5.7, 55.8, 63.3, 110.8, 111.1, 119.9, 126.7, 128.0, 129.0, 133.0, 138.4, 147.8, 148.8. HRMS
+
(
ESI): calcd for C22
H
31
N
2
O
2
[M+H] , 355.2385; found, 355.2388.
4.1.6. 1-(1-Benzylpiperidin-4-yl)-N-(2-methoxybenzyl)methanamine (8)
Using the procedure given for the preparation of 7, compound 5 (352 mg, 1.0 mmol) and
LiAlH (189 mg, 5.0 mmol) gave 8 (69%) as yellowish white solid after purification by flash
column chromatography using 40% ethyl acetate in hexane as eluent. Mp 135-136°C. H NMR
400 MHz, CDCl ) 1.26-1.34 (m, 2H), 1.48-1.53 (m, 1H), 1.70-1.75 (m, 2H), 1.94-1.99 (m,
H), 2.49-2.54 (m, 2H), 2.90 (d, 2H, J = 6.5 Hz), 3.50 (s, 2H), 3.80 (s, 2H), 3.83 (s, 3H), 6.84-
4
1
(
3
2
1
3
6
.98 (m, 2H), 7.23-7.35 (m, 7H). C NMR (100 MHz, CDCl
3
) 30.4, 36.0, 49.3, 53.3, 53.6,
5
5.0, 63.3, 110.0, 120.2, 122.4, 126.7, 127.9, 128.0, 129.0, 129.5, 138.5, 157.4. HRMS (ESI):
+
calcd for C21
H
29
2
N O [M+H] , 325.2279; found, 325.2284.
4.1.7. 1-(1-Benzylpiperidin-4-yl)-N-(4-methoxybenzyl)methanamine (9)
Using the procedure given for the preparation of 7, compound 6 (352 mg, 1.0 mmol) and
LiAlH (189 mg, 5.0 mmol) gave 9 (71%) as yellowish white solid after purification by flash
column chromatography using 50% ethyl acetate in hexane as eluent. Mp 150-151°C. H NMR
400 MHz, CDCl ) 1.25-1.35 (m, 2H), 1.49-1.53 (m, 1H), 1.70-1.74 (m, 2H), 1.94-2.01 (m,
H), 2.53 (d, 2H, J = 6.7 Hz), 2.90-2.93 (m, 2H), 3.51 (s, 2H), 3.74 (s, 2H), 3.82 (s, 3H), 6.89
4
1
(
3
2
1
3
(
d, 2H, J = 8.5 Hz), 7.26 (d, 2H, J = 8.5 Hz), 7.31-7.37 (m, 5H). C NMR (100 MHz, CDCl
3
)
3
0.5, 36.1, 53.4, 53.6, 55.1, 55.2, 63.4, 113.6, 126.7, 128.0, 129.0, 129.1, 132.6, 138.5, 158.4.
+
HRMS (ESI): calcd for C21
H
29
2
N O [M+H] , 325.2279; found, 325.2282.
4.2. Biological Testing
4.2.1. Dual hAChE and BACE-1 inhibitory activities
Inhibitory activity of the synthesized compounds against AChE was measured using the
spectrophotometric Ellman’s method
Aldrich. Acetylthiocholine (ATC) and 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB) were
purchased from Sigma Aldrich and stock solutions of 0.01 M in H O were prepared. Enzymatic
Human recombinant AChE was obtained from Sigma
2
hydrolysis of ATC was monitored by UV-visible absorption at 412 nm in the presence and
absence of various concentrations of inhibitors at 25 °C in 0.1 M potassium phosphate, pH 7.0.
To determine the IC50 value, six different concentrations of each compound were used to obtain
enzyme activities between 5% and 95%. All reactions were performed in triplicate. The IC50
values were calculated using nonlinear regression (GraphPad Prism 5 (GraphPad Software, San
Diego, CA, USA, 5.00)) by plotting the residual enzyme activities against the applied inhibitor
concentration.
All the compounds were tested as BACE-1 inhibitors using a PanVera’s BACE-1 fluorescence
resonance energy transfer (FRET) assay kit
The analyses were carried out according to
the supplied protocol. The wavelength was optimized for 553 nm excitation and 576 nm for
emission monitoring. Stock solutions of the tested compounds were prepared in DMSO and
further diluted with assay buffer (50 mM sodium acetate; pH 4.5). 10 µL of BACE-1 substrate
-
13 -

(
750 nM) was mixed with 10 µL of test compound (or assay buffer; i.e., blank sample), then 10
µL of BACE-1 (1 Unit/mL) was added to start the reaction. After 60 min of incubation at 25 °C,
0 µL of 2.5 M sodium acetate was added to stop the reaction. The emission signal was
monitored at 576 nm. Inhibition percentage was calculated from [1 − (S60 − S )] ×
00, where S and S60 are the fluorescence intensities of the test sample (enzyme, substrate, test
compound) at the beginning of the reaction and after 60 min, respectively, while C and C60 are
1
0
)/(C60 − C
0
1
0
0
the analogical fluorescence intensities of the blank sample (enzyme, substrate, buffer). Each
compound was analyzed in triplicate. BACE-1 Inhibitor IV (Cayman Chemical) was used as a
reference compound. ANOVA (analysis of variance) assay was conducted to evaluate the
difference between the experiment and control groups. Significance was set at p < 0.05.
4.2.2.
SH-SY5Y neuroblastoma cells were grown in Roswell Park Memorial Institute medium (RPMI)
supplemented with 2 mM L-glutamine, 100 UI/mL penicillin, 100 µg/mL streptomycin and 10%
v/v) fetal bovine serum (FBS). Cells were maintained in culture dishes at 37 °C in a saturated
humidity atmosphere containing 95% air and 5% CO . Cells were seeded at an initial density of
04 cells/cm in culture dishes. At a confluence of 80%, cells were detached by trypsin (0.25%)
treatment for 2 min, suspended in fresh medium without FBS and transferred to 96-multiwell (1 ×
(
2
2
1
4
1
0 cells/well). After 24 h from seeding, cells were incubated in the absence or in the presence of
the 3 different concentrations (50, 150 and 450 nM) of compounds 4-6, donepezil and rotenone.
The viability of the cells was evaluated after 48 h treatment as mitochondrial activity using the
MTT assay [45-47]. Briefly, after the treatment the medium was removed and cells were incubated
with 100 μl MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) (0.5 mg/ml) for
1
h. After that, the solution was removed, the formazan formed solubilized in100 μl of 0.1N HCl
in 90 % (v/v) 2-propanol and the absorbance measured at 570 nm using a microplate reader
BioRad 680, USA). Results were expressed as absorbance values at 570 nm of the treated cells
vs. control cells.
(
The 2D structures of compound 4 and donepezil were built and converted into the 3D using
vLife MDS 3.0 software. The 3D structures were then energetically minimized up to the rms
gradient of 0.01 using the CHARMM22 force field. All conformers were then energetically
minimized up to the rms gradient of 0.01. Molegro software was used for molecular docking
studies and Lead IT software was used to generate 2D binding poses of compound 4 and
donepezil.
In vitro blood-brain barrier permeability
The ability of the investigated compounds to penetrate into blood−brain barrier was evaluated
using a parallel artificial membrane permeation assay (PAMPA) for blood−brain barrier according
to the method established by Di et al.
Donepezil and caffeine were used as reference
compounds in the PAMA assay. The donor microplate (96-wellfilter plate, PVDF membrane) and
the acceptor microplate (indented 96-well plate) were both obtained from Millipore. The acceptor
9
6-well microplate was filled with 300 μL of PBS/EtOH (7:3), and the filter membrane was
-
14 -

impregnated with 10mL of PBL in dodecane (20 mg/mL). The compound was dissolved in DMSO
at a concentration of 5 mg/mL followed diluting 50-fold with a mixture of PBS/EtOH (7:3) to give
a final concentration of 100 μg/mL. After that, 200 μL of diluted solution and 300 μL of
PBS/EtOH (7:3) were added to the donor wells. The donor filter plate was placed on the acceptor
plate to make the underside of filter membrane in contact with buffer solution. After leaving this
sandwich assembly for 16 h at 25 °C, the donor plate was removed, and the concentrations of
tested compound in the acceptor, donor and reference wells were measured with a UV plate
reader. Each sample was analyzed three independent runs in four wells. Log D7.4 values were
obtained using shake flask method as previously reported
.
Metal chelation property
The study of metal chelation was performed in methanol at 25 °C using SHIMADZU UV-vis
spectrophotometer with wavelength ranging from 200 to 500 nm. The absorption spectra of
compound 4 (25 μM, final concentration) alone or in the presence of CuCl (5-50 μM) for 30
2
min in methanol were recorded in quartz cells.
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-
17 -

Highlights




Donepezil analogues as multi-target-directed ligands
Dual inhibition of AChE and BACE-1 in the nanomolar range
Molecular modeling reveals dual mechanism against AChE and BACE-1
Compound 4 shows potential biometal chelation ability and high BBB
permeability
Graphical abstract
Profiling donepezil template to dual AChE and BACE-1 inhibition
-
18 -