Multipotent AChE and BACE-1 inhibitors for the treatment of Alzheimer's disease: Design, synthesis and bio-analysis of 7-amino-1,4-dihydro-2H-isoquilin-3-one derivates

Article abstract of DOI:10.1016/j.ejmech.2017.07.006

In this paper, the preparation of a new class of multi-target-directed ligands (MTDLs) based on a 7-amino-1,4-dihydro-2H-isoquilin-3-one, whose lead (compound I) showed promising properties in acetylcholinesterase (AChE) inhibitory activity [1], is described. The results of in vitro activities and molecular docking demonstrated that the target molecule (compounds 10a-n) with three parts of aromatic moieties and appropriate structural length can interact with aromatic residues in catalytic active site (CAS), peripheral anionic site (PAS) and the channel of AChE. And the introduce of connecting amide bonds, enables the target molecules provide sufficient hydrogen bond donors and acceptors to interact with the catalytic site of BACE-1. Notably, compound 10d exerted excellent AChE inhibition (IC₅₀ = 18.93 ± 1.02 pM, 181-fold more inhibitory effect compared with donepezil), BACE-1 inhibition (97.68 ± 8.01% at 20 μM), and good metal chelating property, which can be chosen as lead compound for further optimization of novel small ligand for the treatment of Alzheimer's disease.

Full text of DOI:10.1016/j.ejmech.2017.07.006

Accepted Manuscript
Multipotent AChE and BACE-1 inhibitors for the treatment of Alzheimer's disease:
Design, synthesis and bio-analysis of 7-amino-1,4-dihydro-2H-isoquilin-3-one
derivates
Xiong-jie Zhao, Yu-ren Jiang, Dong Guo, Da-min Gong, Yao Zhu, You-chao Deng
PII:
S0223-5234(17)30528-7
10.1016/j.ejmech.2017.07.006
EJMECH 9569
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 July 2015
Revised Date: 3 July 2017
Accepted Date: 4 July 2017
Please cite this article as: X.-j. Zhao, Y.-r. Jiang, D. Guo, D.-m. Gong, Y. Zhu, Y.-c. Deng, Multipotent
AChE and BACE-1 inhibitors for the treatment of Alzheimer's disease: Design, synthesis and bio-
analysis of 7-amino-1,4-dihydro-2H-isoquilin-3-one derivates, European Journal of Medicinal Chemistry
(2017), doi: 10.1016/j.ejmech.2017.07.006.
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ACCEPTED MANUSCRIPT
Multipotent AChE and BACE-1 Inhibitors for the
Treatment of Alzheimer’s Disease: Design,
Synthesis and Bio-Analysis of
7-amino-1,4-dihydro-2H-isoquilin-3-one Derivates
Xiong-jie Zhao, Yu-ren Jiang*, Dong Guo, Da-min Gong, Yao Zhu, You-chao Deng
Department of Pharmaceutical Engineering, College of Chemistry and Chemical Engineering,
Central South University, Changsha 410083, P.R.China
Key words: Alzheimer’s disease; Acetylcholinesterase; β-secretase; Metal Chelators; Inhibitor
*Corresponding author. Telephone: +86-731-88887895. Fax: +86-731-88859988. E-mail:
jiangyr@mail.csu.edu.cn
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ABSTRACT
In this paper, the preparation of a new class of multi-target-directed ligands (MTDLs) based
on a 7-amino-1,4-dihydro-2H-isoquilin-3-one, whose lead (compound I) showed promising
properties in acetylcholinesterase (AChE) inhibitory activity[1], is described. The results of in
vitro activities and molecular docking demonstrated that the target molecule (compounds 10a-n)
with three parts of aromatic moieties and appropriate structural length can interact with aromatic
residues in catalytic active site (CAS), peripheral anionic site (PAS) and the channel of AChE.
And the introduce of connecting amide bonds, enables the target molecules provide sufficient
hydrogen bond donors and acceptors to interact with the catalytic site of BACE-1. Notably,
compound 10d exerted excellent AChE inhibition (IC50=18.93±1.02 pM, 181-fold more
inhibitory effect compared with donepezil), BACE-1 inhibition (97.68±8.01% at 20 µM), and
good metal chelating property, which can be chosen as lead compound for further optimization
of novel small ligand for the treatment of Alzheimer’s disease.
GRAPHIC ABSTRACT
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1. INTRODUCTION
Alzheimer’s disease (AD) is the most common form of aging progressive neuro-degenerative
disorder associated with memory impairment and cognitive deficit. Despite decades of intensive
study, the specific cause of AD remains enigmatic[2]. According to different hypotheses,
researchers proposed different strategies to design suited single functional compounds. Based on
the cholinergic hypothesis, the inhibition of acetylcholinesterase (AChE), an enzyme that
catalyzes acetylcholine hydrolysis, increases the levels of acetylcholine by inhibiting the
breakdown of ACh in the brain and thus improves cholinergic functions in AD patients[3]. To
block the formation of amyloid β-protein (Aβ) plaques, one approach is to inhibit the β-secretase
(BACE-1), an aspartyl protease, which is an attractive therapeutic target since it plays a key role
in the rate-limiting step of Aβ generation[4]. The other method is to enhance Aβ clearance in the
AD brain on the basis of amyloid cascade hypothesis[5,6]. To restrain the abnormal
posttranslational modifications, abundant strategies have been attempted, either to inhibit
serine/threonine kinases, or to accelerate the dephosphorylation of hyper-phosphorylated tau
protein[7]. Besides mentioned above, according to the metal ion hypothesis[8,9], modulation of
biometals (Fe2+,Cu2+, and Zn2+) in the brain represents an additional rational approach for the
treatment of AD.
Because the pathogenesis of AD is complicated and related to the abnormality and dysfunction
of multiple systems, there has been no ideal drug for preventing and treating AD. The goal of
current treatments is to correct neuronal dysfunction[10]. Current treatment of AD focuses on
symptomatic aspects of the pathology and is based on drugs increasing cholinergic
neurotransmission by inhibiting AChE, like donepezil, rivastigmine, or galantamine, despite of
only delaying cognitive decline by typically one year. Therefore, the limited efficiency of
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singular AChE inhibitors has led to quests for more causative pathogenic targets[5]. As
β-secretase is proposed to play a key role in the pathogenesis of AD, the role of β-secretase
inhibition for AD therapy has received increasing attention from the medicinal chemistry in
recent years[11]. But most of singular BACE-1 inhibitors were proved failed and weeded out in
the phase II or III clinical trial. The current situation of AD treatment, the complexity of AD and
the involvement of different enzymes in its progression make the modulation of a single protein
might not be sufficient to produce the desired efficacy[12].
Since 1996, the concept of so-called ‘‘non cholinergic action of AChE” was demonstrated by
researchers[13,14], who hold a view that the interaction of Aβ at the peripheral anionic site
(PAS) of AChE greatly accelerates the aggregation of this toxic peptide. Interactions at the PAS
of AChE catalyze some conformational changes in Aβ fibrils to form the β-sheet with increased
aggregating potential[15]. Thus, inhibitors targeting the PAS of the enzyme will decrease the
aggregation rate of Aβ keeping it in solution, and therefore facilitating its clearance. According
to the view stated above, both enzymes, AChE and BACE-1, are connected by the production
aggregation process. Therefore, development of agents targeting at both enzymes should be a
good approach to design effective pharmaceutical agents.
After the single X-ray crystals of AChE of different species, including human, Electrophorus
electricus, Torpedo californica, and mouse, have been confirmed in the past decade[16,17], the
inhibitor binding pocket was more specific and visualized for researchers. These structures
showed that there are two major subsites in the inhibitor-binding gorge-like pocket, as shown in
Fig. 1a, including the catalytic active site (CAS) and the peripheral active site (PAS)[18,19].
Recently, a narrow channel between the CAS and PAS was also identified as an additional
binding site. In addition, the gorge-like pocket is very rich in aromatic amino acid residues,
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especially in the PAS (including Trp286, Phe297, Phe295, Phe338, Tyr124 and Tyr 72) and CAS
(including Trp86, Tyr337 and Trp439), and Tyr341 in the narrow linking channel. Moreover, the
three dimensional structure of enzyme huBACE-1 (PDB code: 1M4H) with polypeptide
inhibitors has been confirmed by single crystal diffraction method, as shown in Fig. 1b. The
residues of Asp32 and Asp228 play a key role in catalyzing hydrolysis of amide bonds of α-APP
synergistically, which are confirmed as catalytic sites of BACE-1 on the basis of former
research[11].
Figure 1. (a) The inhibitor-binding gorge-like channel of AChE (PDB code 4EY7) and (b) The
binding pocket of BACE-1 (PDB code 1M4H).
While discussing the metal ion hypothesis, which was inspired by decades of research[20-22],
the dyshomeostasis of metal ions may correlate with the development of AD. It is also reported
that iron levels of Fe2+,Cu2+, and Zn2+ are higher in AD neuropils compared with normal
neuropils, and iron is concentrated in amyloid plaques abnormally[23]. Giving to this
pathological character, the strategy of designing dual-enzyme inhibitors with metal chelation
properties would be more efficient for the treatment of AD.
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Researches on plants from the genus Corydalis (Papaveraceae) which are used for the
treatment of memory dysfunction in folk medicine, reported the presence of benzylisoquinoline
alkaloids with anti-AChE activity[24]. Our research group has been involved for many years in
the development of N-containing heterocycle AChE inhibitors as potential drugs for AD based
on corydaline. We had optimized the corydaline and obtained a series of ring-opening
derivatives with different carbon linkages and different substituent groups. Based on these virtual
screening results, compound I, as shown in Fig. 2, was synthesized and a pharmacological study
showed that its inhibition activity was three times as high as galantamine[1]. Moreover, we had
made plenty studies about visual screening of novel tetrahydroisoquinoline derivates[25,26]. In
addition to that, we found that 1,2,3,4-tetrahydroisoquinoline moiety may stack with aromatic
amino acid residue in the channel of AChE, according to which we further optimized the
1,2,3,4-tetrahydroisoquinoline scaffold by the strategy of bioisosterism to the skeleton we used
in the present study. On the basis of previous works[27-30], we use the thought that the potential
lead compound should be composed of three aromatic structural units: an aromatic section as the
head to interact with the PAS region for blocking the inhibitor-binding pocket, another aromatic
ring as the tail to bind with the CAS for preventing the hydrolysis of ACh, and an aromatic
moiety to interact with the residues of channel, such as Tyr341. Recently, a novel series of
isatin-based inhibitors of BACE-1 have been identified using a virtual high-throughput screening
approach, which shows synthetic compound with the amide bonds, or with the properties of
mimetic peptides based on the structure of II, may have the potential of inhibition to
BACE-1[11]. Accordingly, in the design of the new molecule, the 1,2,3,4-tetrahydroisoquinoline
moiety of compound I was modified to the structure of 7-amino-1,4-dihydro-2H-isoquilin-3-one
(AQO) ring, which is acted as the central aromatic ring to interact with aromatic residues in
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gorge-like channel and capable of binding to multiple receptors in the central nervous system
with high affinity for bearing N-containing heterocylcle[31]. Another two aromatic rings are
substituent benzene to interact with aromatic residues in CAS and PAS. We introduce amide
bond to connect three aromatic parts, after realizing that amide bonds, including the amide bond
of AQO ring, can provide hydrogen bond donors and acceptors to interact with the catalytic site
of BACE-1, for the goal of enhance inhibitory activity on BACE-1. Additionally, considering
that biometal (Fe, Cu, and Zn) ions play a very important role in many critical aspects of
AD[32], we hoped the introduce of amide bonds may exert metal chelation properties to a certain
degree.
The current study describes the preparation and in vitro activities of peptidomimetic
derivatives of AQO as multifunctional inhibitors of AChE and BACE-1 with metal chelating
abilities. The goal was to identify compounds that were potential in inhibiting AChE and
BACE-1, with metal chelation properties simultaneously, which could be developed for the
treatment of AD and other neurodegenerative diseases.
Figure 2. The chemical structure of corydaline, compound I and isatin derivates II.
2. RESULTS AND DISCUSSION
2.1 Chemistry
The syntheses of the various AQO derivatives are described in Scheme 1, and the synthetic
intermediates and final products are listed in Table 1. The starting material was the
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commercially available indene. Initial oxidation reaction of olefinic bond with formic acid and
hydrogen peroxide gave the corresponding epoxy compound, which was subsequently converted
into monoformate of 1,2-indanediol via ring-opening reaction. Hydrolysis of the monoformate
gave compound 2. Nitration reaction of 2-indanone gave compound 3. After oximation of
5-nitro-2-indanone in the presence of hydroxylamine, the 5-nitro-2-indanone oxime was obtained
and subsequently converted into 7-nitro-1,2,3,4-tetrahydroisoquinolin-2-one via classic Beckman
rearrangement reaction. Reduction of nitro group gave the key intermediate 6. In view of the
amino group is easily oxidized when exposed to air, we benzoylated the amino group with a
series of derivates of benzoyl chloride immediately. With the consideration of comparing the
activities of N-benzoyl compounds to their analogues, some of the compounds were
N-3’-chlorobenzoylated with 3-chlorobenzoyl chloride or 3-methylbenzoylated with
3-methylbenzoyl chloride to give compounds 7b and 7c belonging to category 7. In order to
acquire compounds with three parts of aromatic moieties, we benzoylated the N of lactam in the
compounds of category 7 with electron-rich or electron-deficient benzoyl chloride, including
3-chlorobenzoyl chloride, 3-methyl benzoyl chloride and 2-methyloxyl benzoyl chloride, and
finally gained compounds 10a-n of category 10.
To determine their potential interest as dual-binding AChEIs for the treatment of AD, all
newly synthetic compounds were assayed for their human AChE inhibitory activity. The
inhibitory potency is expressed as IC50, which represents the concentration of inhibitor required
to decrease the enzyme activity by 50%. The inhibit ratio against AChE was calculated
according to the absorbance of the product in the AChE-catalyzed reaction and the results were
shown in Table 1, and as displayed in Table 1, donepezil shows good potency against AChE
with an IC50 value of (3.46±0.051)×103 pM, which is used as positive control. And compounds
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10a-n with three parts of aromatic units showed high efficacy of AChE inhibitory ability.
Furthermore, the inhibition was found to be influenced markedly by o-methoxybenzoyl group at
N-2 position. Among them, compound 10h, an AQO derivative contained 3-chlorobenzoyl and
o-methoxybenzoyl group at N-7 and N-2 position, respectively, was found to have the most
potent inhibitory activity with IC50 value of 10.95±0.80 pM, which was 315-fold more inhibitory
effect compared with donepezil, which might attribute to the three parts aromatic moieties of it.
Scheme 1. General Synthetic Scheme: AQO and their synthetic intermediates a
a Reagents and conditions: (a) (1) 88%HCOOH/30%H2O2,rt,6 h;(2) 7%H2SO4, 100 °C,4 h; (b) 90%HNO3/chloroform, -20 °C, 20
min; (c) HA/ethanol/H2O/pyridine, rt,3 h; (d) (1) PCl5/anhydrous chloroform, -20 °C,20 min; (2) rt, 2 h; (e) Fe/ CH3COOH/
H2O/ethanol, 65 °C,3 h; (f) SOCl2, 70 °C,4 h; (g) 9a-d,K2CO3/chloroform,0°C,4 h; (h) 9a-d,triethylamine/dioxane,100°C,2 h
2.2 Human AChE inhibition and molecular modeling
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Table 1. The inhibitory activities of each derivate of AQO
AChE inhibition BACE-1 inhibition
IC50±SD (pM) at 20 µM,%
Compd.
R1
R2
R3
R4
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(3.46±0.051)×103 79.86±5.51
(1.36±0.053)×106 5.37±8.32
(1.32±0.041)×106 42.35±7.73
(9.14±0.552)×105 10.22±9.06
(7.91±0.008)×105 67.91±7.08
(9.26±0.092) ×105 29.31±8.85
(5.10±0.112) ×102 15.46±7.98
Donepezil
6
-H
-H
-H
-H
7a
-Cl
7b
-CH3
7c
-OCH3 -H
7d
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
10a
10b
10c
10d
10e
10f
-H
-Cl
28.36±6.12
28.17±5.43
20.08±6.73
97.68±8.01
20.74±7.36
82.66±6.63
74.92±7.91
13.22±5.28
16.72±8.79
10.84±7.12
88.54±6.57
22.39±9.13
5.42±9.04
-H
-CH3 26.21±4.86
-H
-OCH3 -H
18.93±1.02
13.23±0.46
14.15±1.04
14.41±0.03
10.95±0.80
20.94±0.99
13.52±0.61
29.38±1.78
16.72±0.79
25.92±1.37
21.58±1.00
-Cl
-Cl
-Cl
-Cl
-H
-H
-H
-H
-Cl
-CH3
10g
10h
10i
-OCH3 -H
-CH3 -Cl
-H
-H
-H
-H
-H
-CH3 -CH3
10j
-OCH3 -H
-OCH3 -H
-OCH3 -H
-OCH3 -H
-H
10k
10l
-Cl
-CH3
10m
10n
-OCH3 -H
11.76±8.47
To elucidate further the binding mechanism of these derivates with AChE, Molecular
Operating Environment (MOE, CCGI, Canada, 2008.10) was used to conduct molecular
modeling (docking) procedure. Default parameters were used unless otherwise indicated. The
crystal structure of human AChE (hAChE) was obtained from the Protein Data Bank (PDB code
4EY7). It is well known that two distinct binding sites exist in the active pocket of AChE
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peripheral binding site (PAS) and catalytic active site (CAS), located at the entrance and the
bottom of the active-site gorge, respectively. Heteroatoms and water molecules in the proteins
were removed, and hydrogen atoms were subsequently added. As shown in Fig. 3a and 3b, the
modeling result of compound 10a (IC50=(5.10±0.112) ×102 pM) demonstrated that phenyl
groups of two ends formed π-π stacking interaction with Trp286 (4.32 Å) in PAS and Trp86
(3.91 Å) in CAS, respectively, while the middle part of aromatic moiety formed π-π stacking
interaction (3.16 Å) with Tyr341 in the narrow linking channel. In addition to that, the potency
of compound 10a was further improved due to the formation of a hydrogen bond between N-7
amide hydrogen and Asp74 (2.46 Å), and a hydrogen bond (2.71 Å) between amide oxygen of
the lactam and the backbone of Phe295.
Compared to that (as shown in Fig. 3c), compound 10h was obtained by replacing the phenyl
group of each end with 3-chlorobenzoyl group in N-7 and o-methoxybenzoyl in N-2 position.
The potency of compound 10h (IC50=10.95±0.08 pM) was improved significantly, which is
owing to blocking of gorge-like pocket by the formation of hydrogen bond between Tyr124 and
amide oxygen of 3-chlorobenzoyl group (2.84 Å), and a π-π stacking interaction between Trp286
and phenyl of 3-chlorobenzoyl group (5.41 Å) in the PAS. Beyond that, electron-rich
o-methoxybenzoyl group of compound 10h formed π-π stacking interaction with Trp86 (5.35 Å)
in the CAS. These results indicated that electron-donating effect of the o-methoxy group may
increase the electronic density of the benzoyl, and consequently improve the π-π stacking
interaction between the phenyl and the CAS of AChE. As shown in Fig.3d, the structure of
compound 10d (IC50=18.93±1.02 pM) is similar to that of compound 10h, with an N-7 benzoyl
group instead. The molecular modeling of 10d with AChE showed that two oxygen of
o-methoxybenzoyl group formed two hydrogen bonds with Tyr124 in the PAS with a distance of
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2.98 Å and 2.89 Å, respectively. Furthermore, the phenyl ring staked with Tyr341 with a
ring-to-ring distance of 3.73 Å in the narrow channel of pocket, and oxygen of N-7 amide bond
formed hydrogen bond with Tyr72 residue (2.62 Å). Besides, the phenyl ring of AQO template
staked a π-π interaction with Trp286 (3.76 Å). Despite that compound 10d did not interact with
CAS of AChE directly, it can block the gorge-like binding pocket and occupy the CAS position,
and eventually prevent the ACh from entering and being hydrolyzed.
Figure 3. Molecule docking of compound 10a with AChE generated with MOE: (a) the 3D
picture of binding and (b) the 2D picture of binding; The 2D picture of docking models of
compound 10h (c) and 10d (d) with AChE.
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It should be noted that all the compounds with N-2 electron-rich aromatic group
(3-methylbenzoyl and o-methoxybenzoyl) showed more effective inhibitory than those
substituted by electron-withdrawing group. This result was in agreement with the results of in
vitro assays, except compound 10l (IC50=16.72±0.79 pM), which showed more potency than
compound 10n (IC50=21.58±1.00 pM). In this regard, an N-2 o-methoxybenzoyl substituent
could be considered as a suitable CAS binding pharmacophore in the development of novel
AQO based small molecules.
According to the results of human AChE inhibitory activity assays, we found that all the
newly synthesized compounds with three parts aromatic moieties show more potential inhibitory
ability than donepezil. The SAR data obtained from this AQO chemical library indicates that
steric and electronic properties at N-2 and N-7 position exert a significant effect on the AChE
inhibitory profiles.
2.3 Human BACE-1 inhibition and molecular modeling
Due to its involvement in the proteolytic cleavage of amyloid precursor protein (APP) to Aβ,
β-secretase has encouraged great interest as a therapeutic target for disease-modifying agents in
AD. Thus, the capability of newly synthesized compounds was firstly evaluated at a single
concentration (20 µM) against β-secretase by fluorometric assay, followed by molecular
modeling of promising candidates. Donepezil was assayed as positive control, with an inhibition
rate of 79.86±5.51% at 20 µM (IC50 to BACE-1=0.169± 0.012 µM[33]). As displayed in Table
1, compounds 10f, 10g, and 10k showed modest potency against β-secretase, whereas compound
10d revealed good potency against β-secretase with an inhibition rate of 97.68±8.01% at 20 µM.
Molecular modeling procedure was conducted by MOE to analyze the interaction between
compound 10d with human BACE-1. The crystal structure of human BACE-1 was obtained from
the Protein Data Bank (PDB code 1M4H), followed by removing heteroatoms and water
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molecules, and hydrogen atoms were subsequently added. As shown in Figs.4a and 4b, a
hydrogen bond was formed between catalytic residue Asp32 with amide oxygen of the lactam
(2.72 Å), which may improve the potency of BACE-1 inhibitory activity. Additionally, oxygen
of o-methoxybenzoyl group formed two hydrogen bonds with Thr231 and Thr72 (2.99 Å and 3.1
Å), and a hydrogen bond was formed between backbone of residue Phe109 with N-7 amide
bond, which may contribute to improving the interaction between small ligand with BACE-1,
and eventually exert good inhibitory potency against BACE-1. In this regard, we found that the
introduce of amide group in the derivates of AQO can enhance the BACE-1 inhibitory activity.
This result is in consistent with what we proposed before the design of novel small non-peptide
molecular as BACE-1 inhibitors.
Figure 4. Molecule docking of compound 10d with BACE-1 generated with MOE: (a) the 3D
picture of binding and (b) the 2D picture of binding.
2.4 Metal-chelating property
In addition, compound 10d was also taken as an example to test for its metal-chelating effect
using difference UV-visible spectra recorded by UV-visible spectrophotometer (Shimadzu
UV-2450) in methanol at 298 K with wavelength ranging from 200 to 500 nm[34]. In Fig. 5a,
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UV-visible spectra of 10d at increasing concentrations of Cu2+ were shown. The increase in
absorbance, which could be better estimated by an inspection of the differential spectra (Fig. 5b),
indicated that there was an interaction between Cu2+ and 10d. These observations indicated that
the compounds could effectively chelate Cu2+, and thereby could serve as metal chelators in
treating AD.
Figure 5. UV-vis (200-500 nm) absorption spectra of compound 10d (35 µM) in methanol after
addition of increasing amounts of CuCl2 (5-50 µM) (a) and differential spectra due to 10d-Cu2+
complex formation obtained by numerical subtraction from the above spectra of those of CuCl2
and 10d at the corresponding concentrations (b).
3. CONCLUSIONS
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In conclusion, AQO was totally synthesized for the first time from indene by the oxidation
with hydrogen peroxide, nitration reaction, oximation in the presence of hydroxylamine,
Beckman rearrangement with phosphorus pentachloride, and reduction of nitro group. The
peptidomimetic derivates of AQO were designed, synthesized, and subjected to biological
evaluated as the dual-binding AChE and BACE-1 inhibitors. All derivatives exhibited excellent
AChE inhibitory activity compared with donepezil in the preliminary bioassay, which implies
that three parts aromatic moieties seemed necessary to inhibit AChE. Among them, compound
10h revealed the most potent inhibitory against AChE. The molecular modeling study further
indicated that this compound can bind both with the CAS and PAS of AChE. Beyond that,
compound 10d exerted excellent AChE (IC50=18.93±1.02 pM, 181-fold more inhibitory effect
compared with donepezil) and BACE-1 inhibitory activity (97.68±8.01% at 20 µM). In addition
to that, compounds 10f, 10g and 10k showed modest potency against β-secretase. Furthermore,
such compounds, with three moieties of aromatic rings and sufficient amide bonds, also exerted
excellent metal chelating properties. Together, these results imply that the AQO is the suitable
ring template to design anti-AD small molecule therapeutics as disease-modifying agents.
4. EXPERIMENTAL PROTOCOLS
4.1 Chemistry
4.1.1 Materials and methods
Unless otherwise noted, all chemical reagents were commercially available and treated with
standard methods before use. Solvents were dried in a routine way and redistilled before use. 1H
NMR spectra were recorded on a BRUKER 500 MHz or 400 MHz spectrometer in CDCl3 with
TMS as the internal reference.13C NMR spectra were recorded in CDCl3 at 100 MHz or 125
MHz on a BRUKER spectrometer. Melting points were taken on an X4 melting point apparatus
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(Beijing Tech Instrument CO., LTD., China) and uncorrected. The purity of the synthesized
compounds was recorded on HPLC system (HP 1100 series, Agilent Technologies, Palo Alto,
USA) by two different gradient methods using an YMC ODS column (50×4.6 mm, 5 µm particle
size), and the purity of the target compounds was above 95%. Reactions were followed by
thin-layer chromatography (TLC) on Merck (0.25 mm) glass-packed precoated silica gel plates
(60 F254), and then visualized with a UV lamp. All chemical yields are unoptimized and
generally represent the result of a single experiment.
4.1.2 General procedure for 2-Indanone (2)
According to the reported methodology[35], 2-indanone was known that it can be obtained by
oxidation of indene with formic acid and hydrogen peroxide. In a typical preparation process, 13
mL indene was added slowly in the mixture of 104 mL formic acid (88%) and 25 mL hydrogen
peroxide (30%) at 35-40 °C to give monoformate of 1,2-indanediol via ring-opening reaction
after stirred for 6 hours. Subsequently, hydrolysis of the monoformate gave 2-indanone by steam
distilled in 150 mL7% (by volume) sulfuric acid. The cold distillate was filtered. The white
crystals were dried further in a vacuum at room temperature for about 12 hours to give
2-indanone 7.53 g (56.2% yield). m.p.: 57–59 °C (lit[35] 57–58 °C).
4.1.3 General synthesis of 5-nitro-2-indanone (3)
It has been reported that 2-indanone is able to convert to 5-nitro-2-indanone by fuming nitric
acid with vigorous stirring at -20 °C according to modified published methodology[36]. To a
cooled solution of 5.6 g 2-indanone in 40 mL chloroform was dropped slowly with 13 mL
fuming nitric acid with vigorous stirring in 45 min at -20 °C. After the addition was completed,
the mixture was then stirred further for 20 min, poured into a mixture of sodium hydroxide (6.5
17

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g) in water and ice (ca. 50 mL), and then extracted twice with ethyl acetate. The combined
extracts were washed with water until neutral, dried over magnesium sulfate, and evaporated in
vacuum to yield yellow solid. Chloroform was used as the eluent in column chromatography.
Finally, the crude product was recrystallized from ethyl acetate/hexane to give 3.7 g of product
(49.5% yield). m.p.:142 -144 °C (lit[36] 141-143 °C).
4.1.4 General synthesis of 5-nitro-2-indanone oxime (4)
To a stirring solution of 4 g 5-nitro-2-indanone in 20 mL pyridine was added with the solution
of 2.2 g hydroxylamine in 5 mL ethanol and 2 mL water at room temperature in 5 min, with
further stirring for 3 hours. 100 mL water was added to the flask and stirred for half an hour. The
mixture was then filtered to give yellow crystal. The crude product was recrystallized from
ethanol / water (60%) to obtain 3.7 g product (72.7% yield). m.p.: 173 -175 °C.
4.1.5 General synthesis of 7-nitro-1,4-dihydro-2H-isoquilin-3-one (5)
To the cooled solution of 2.4 g 5-nitro-2-indanone oxime in 80 mL substantial anhydrous
chloroform was added 2.5 g phosphorus pentachloride at -20 °C in 40 min with vigorous stirring.
The reaction mixture was stirred at -20 °C for further 20 min and then at 25 °C for 2 additional
hours. 80 mL icy water at 0 °C -5 °C was added and then the pH was adjusted to ~7 by addition
of icy solution of 10% sodium hydroxide. The organic layer was separated and the aqueous layer
was extracted by chloroform (2×50mL). The combined organic layer was washed by saturated
salt water (2×50 mL), dried over anhydrous magnesium sulfate, decolorized by activated
charcoal, and evaporated in vacuum to yield yellow solid. Ethyl acetate was used as the eluent in
column chromatography to give 800 mg product (31.8% yield). m.p.: 159-161 °C; 1H NMR (400
18

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MHz, CDCl3) δ 8.21 - 8.07 (m, 2H), 7.39 (d, J = 8.4 Hz, 1H), 6.69 (s, 1H), 4.64 (s, 2H), 3.73 (s,
2H).
4.1.6 General synthesis of compound AQO(6)
In a three-necked flask fitted with stirrer, reflux condenser and thermometer was placed 16 mL
water, 0.9 mL glacial acetic acid and 5 g reduced iron. The mixture was heated to boiling over 10
minutes and then cooled to room temperature. 400 mg 7-nitro-1,4-dihydro-2H-isoquilin-3-one in
10 mL ethanol was added and stirred at 65 °C for 3 hours. 10% sodium carbonate was added to
terminate the reaction. The obtained suspension was filtered to give brown solution, and then
evaporated in vacuum with mixed zinc powder to give crude product. Ethyl acetate/glacial acetic
acid (150:2) was used as the eluent in column chromatography to give 300 mg product (88.9%
yield). m.p.: 156-158 °C; 1H NMR (500 MHz, DMSO) δ 7.87 (d, J = 19.9 Hz, 1H), 6.85 (dd, J =
29.7, 8.0 Hz, 1H), 6.47- 6.36 (m, 2H), 4.98 (d, J = 11.3 Hz, 2H), 4.16 (s, 2H), 3.23 (d, J = 15.8
Hz, 2H).
4.1.7 General synthesis of a series of benzoyl chloride (9a-d)
In a round flask was placed 5 g benzoyl acid and 10 mL thionyl chloride, and the mixture was
refluxed at 70 °C for 4 hours. The solution was then evaporated in vacuum to remove excess
thionyl chloride, and yellow oil residue was yielded as crude product. The residue was then
distilled at reduced pressure to give pure benzoyl chloride. The reduced boiled point for 9b, 9c
and 9d was 125-126 °C/40 mmHg, 122-123 °C/40 mmHg and 163-164°C/40 mmHg
respectively.
4.1.8 General synthesis of compound 7a-d
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The newly synthetic AQO was then solved with equivalent amount of anhydrous potassium
carbonate in substantial anhydrous chloroform at 0 °C, and then added the solution of equivalent
amount of various benzoyl chlorides in 10 mL anhydrous chloroform slowly to the amino
compound solution with stirring for 4 hours. The reaction products were transferred to separating
funnel. 20 mL 28% ammonium hydroxide was added to remove the excess benzoyl chloride. The
organic phase was separated and the upper aqueous phase was extracted by 20 mL chloroform.
The combined organic layer was then washed by saturated salt water to pH~7, dried over
anhydrous magnesium sulfate, and evaporated in vacuum to yield crude product. Ethyl acetate/
glacial acetic acid (50:1) were used as the eluent in column chromatography to give pure
product.
4.1.8.1 N-(3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)benzamide (7a)
100 mg newly synthetic AQO was reacting with 100 µL compound benzoyl chloride to give
1
60 mg solid compound 7a (36.6% yield). m.p.: 115-117 °C; H NMR (500 MHz, DMSO) δ
10.26 (s, 1H), 8.01 (s, 1H), 7.96 (d, J = 7.3 Hz, 2H), 7.87 - 7.38 (m, 5H), 7.21 (dd, J = 31.4, 8.2
Hz, 1H), 4.33 (d, J = 10.1 Hz, 2H), 3.42 (d, J = 13.4 Hz, 2H).
4.1.8.2 3-chloro-N-(3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)benzamide (7b)
100 mg compound AQO was reacting with 100 µL 3-chlorobenzoyl chloride to give 40 mg
1
yellowish viscous oily 7b (21.6% yield). H NMR (500 MHz, DMSO) δ 10.36 (s, 1H), 8.01 (s,
2H), 7.92 (d, J = 7.8 Hz, 1H), 7.72 (s, 1H), 7.68 (d, J = 7.9 Hz, 1H), 7.59 (dd, J = 17.6, 9.8 Hz,
2H), 7.19 (d, J = 7.9 Hz, 1H), 4.33 (d, J = 10.5 Hz, 2H), 3.43 (d, J = 13.8 Hz, 2H).
4.1.8.3 3-methyl-N-(3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)benzamide (7c)
20

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100 mg compound AQO reacting with 100 µL 3-methylbenzoyl chloride to give 80 mg
1
yellowish-brown solid 7c (46.3% yield). m.p.: 98-100 °C; H NMR (500 MHz, DMSO) δ 10.21
(s, 1H), 8.02 (s, 1H), 7.75 (d, J = 19.3 Hz, 2H), 7.60 (dd, J = 21.7, 10.6 Hz, 2H), 7.41 (s, 2H),
7.21 (dd, J = 31.6, 8.0 Hz,1H), 4.32 (d, J = 9.4 Hz, 2H), 3.42 (d, J = 13.1 Hz, 2H), 2.35 (s, 3H).
4.1.8.4 2-methoxy-N-(3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)benzamide (7d)
100 mg compound AQO reacting with 100 µL o-methoxybenzoyl chloride to give 100 mg
1
brown solid compound 7d (54.9% yield). m.p.: 58-60 °C; H NMR (500 MHz, DMSO) δ 10.11
(s, 1H), 8.00 (s, 1H), 7.71 (s, 1H), 7.64 (dd, J = 7.5, 1.5 Hz, 1H), 7.52 (m, 2H), 7.25 - 7.13 (m,
2H), 7.07 (m, 1H), 4.32 (d, J = 9.7 Hz, 2H), 3.90 (s, 3H), 3.41 (d, J = 12.7 Hz, 2H).
4.1.9 General synthesis of compounds 10a-n
To the solution of compound 7a-d and equivalent amount of triethylamine in substantial
dioxane was added with the solution of approximately 1.2 equivalent amount of various benzoyl
chlorides in 5 mL dioxane at room temperature. The reactants were refluxed at 100 °C for 2
hours. When the reaction was finished, the dioxane was evaporated in vacuum and the residue
was solved in 10 mL chloroform. The organic solution then washed by 10 mL 28% ammonium
hydroxide to remove the excess benzoyl chloride to obtain crude product. The ethyl acetate /
hexane (1:1) were used as the eluent in column chromatography to give pure product.
4.1.9.1 N-(2-benzoyl-3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)benzamide(10a)
100 mg compound 7a reacting with 100 µL benzoyl chloride to give 80 mg yellowish solid
1
product 10a (57.6% yield). m.p.: 183-185 °C; HNMR (400 MHz, CDCl3) δ 7.91 (d, J = 8.1,
13
2H), 7.81 (s, 1H), 7.69 -7.26 (m, 11H), 4.98 (d, J = 9.6 Hz, 2H), 3.79 (d, J = 13.5 Hz, 2H); C
21

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NMR (100 MHz, CDCl3) δ 173.5, 171.4, 165.9, 138.0, 137.2, 135.5, 134.6, 133.2, 132.1, 132.0,
129.7, 128.9, 128.4, 128.1, 127.7, 127.1, 126.7, 120.1, 119.0, 118.9, 118.0 48.0, 39.7.
4.1.9.2 N-(2-(3-chlorobenzoyl)-3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)benzamide (10b)
100 mg compound 7a reacting with 100 µL 3-chlorobenzoyl chloride to give 40 mg yellowish
viscous oily 10b (25.8% yield). 1HNMR (500 MHz, CDCl3) δ 7.93 (d, J = 7.3 Hz, 3H), 7.83 (s,
1H), 7.63 -7.24 (m, 9H ), 4.97 (d, J = 10.3 Hz, 2H), 3.79 (d, J = 14.5 Hz, 2H).
4.1.9.3 N-(2-(3-methylbenzoyl)-3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)benzamide (10c)
100 mg compound 7a reacting with 100 µL 3-methylbenzoyl chloride to give 40 mg
yellowish-brown oily 10c (27.7% yield). 1HNMR (500 MHz, CDCl3) δ 7.95 -7.84 (m, 2H), 7.79
(s, 1H), 7.61 - 7.49 (m, 4H),7.45- 7.29(m, 4H), 7.16 (d, J = 7.5 Hz, 2H), 4.96 (d, J = 8.7 Hz, 2H),
3.78 (d, J = 14.1 Hz, 2H), 2.42 (s, 3H).
4.1.9.4 N-(2-(2-methoxybenzoyl)-3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)benzamide (10d)
100 mg compound 7a reacting with 100 µL o-methoxybenzoyl chloride to give 80 mg
yellowish-brown oily 10d (73.3% yield). 1HNMR(500 MHz, CDCl3) δ 7.91 (d, J = 7.2 Hz, 2H),
7.71 (s, 1H), 7.66 -7.48 (m, 4H), 7.45 -7.32 (m, 3H), 7.06 - 6.96 (m, 2H), 6.85 (d, J = 8.3 Hz,
1H), 5.02 (d, J = 3.8 Hz, 2H), 3.76 (d, J = 5.0 Hz, 2H), 3.66 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 176.8, 171.0, 170.0, 155.9, 145.1, 139.8, 137.2, 134.7, 133.8, 133.1, 132.6, 132.0,
131.7, 128.8, 128.6, 127.4, 127.1, 120.8, 120.0, 118.0, 110.8, 55.5, 46.8, 39.8.
4.1.9.5 N-(2-benzoyl-3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)-3-chlorobenzamide (10e)
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100 mg compound 7b reacting with 100 µL benzoyl chloride to give 80 mg yellowish crystine
1
10e (59.4% yield). m.p.: 179-180 °C; HNMR (400 MHz, CDCl3) δ 7.85(s, 1H), 7.62-7.34 (m,
12H), 4.98 (d, J = 4.9 Hz, 2H), 3.66 (d, J = 5.2 Hz, 2H).
4.1.9.6
3-chloro-N-(2-(3-chlorobenzoyl)-3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)benzamide
(10f)
100 mg compound 7b reacting with 100 µL 3-chlorobenzoyl chloride to give 50 mg
1
yellowish-brown oily 10f (34.2% yield). H NMR (500 MHz, CDCl3) δ 8.09 (s, 1H), 7.82 (s,
1H), 7.59 - 7.51 (m, 4H), 7.49 - 7.44 (m, 2H), 7.41 (dd, J = 7.7, 1.4 Hz, 2H), 7.38 -7.30 (m, 2H),
4.96 (d, J = 2.6 Hz, 2H), 3.78 (d, J = 7.7 Hz, 2H).
4.1.9.7
3-chloro-N-(2-(3-methylbenzoyl)-3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)benzamide
(10g)
100 mg compound 7b reacting with 100 µL 3-methylbenzoyl chloride to give 60 mg yellowish
powder 10g (43.1% yield). m.p.: 166-168°C; 1H NMR (500 MHz, CDCl3) δ 7.95 (s, 1H), 7.89 (s,
1H), 7.78 (m, 2H), 7.56 (m, 2H), 7.51 -7.43 (m, 2H), 7.40 (s, 1H), 7.39 - 7.28 (m, 3H), 4.96 (d, J
= 6.5 Hz, 2H), 3.78 (d, J = 12.1 Hz, 2H), 2.38 (s, 3H).
4.1.9.8 3-chloro-N-(2-(2-methoxybenzoyl)-3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)benzamide
(10h)
100 mg compound 7b reacting with 100 µL o-methoxybenzoyl chloride to give 50 mg
1
lamellar crystal 10h (34.6% yield). m.p.: 89-90°C; H NMR (500 MHz, CDCl3) δ 7.98 (d, J =
11.7 Hz, 1H), 7.89( s, 1H), 7.80 -7.67 (m, 2H),7.61 (d, J = 2.0 Hz, 1H) 7.59-7.52 (m, 1H), 7.49-
7.33 (m, 4H), 7.01 (t, J = 7.5 Hz, 1H), 6.85 (d, J = 8.3 Hz, 1H), 5.07 (d, J = 3.8 Hz, 2H), 3.74 (d,
23

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J = 12.3 Hz, 2H), 3.65 (s, 3H);13C NMR (125 MHz, CDCl3) δ 170.8, 169.9, 164.6, 155.8, 137.0,
136.5, 135.0, 133.1, 132.0, 131.7, 130.1, 128.6, 127.5, 126.5, 125.2, 120.8, 120.1, 119.1, 118.8,
118.0, 110.8, 55.5, 46.8, 39.9.
4.1.9.9
N-(2-(3-chlorobenzoyl)-3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)-3-methylbenzamide
(10i).
100 mg compound 7c reacting with 100 µL 3-chlorobenzoyl chloride to give 20 mg light
1
yellow oily 10i (13.4% yield). H NMR (400 MHz, CDCl3) δ 8.04 -7.76 (m, 2H), 7.71 (s, 1H),
7.63 - 7.51 (m, 3H), 7.47 (dd, J = 9.9, 5.2 Hz, 2H), 7.45 -7.38 (m, 2H), 7.38 - 7.31 (m, 2H), 4.97
(d, J = 6.6 Hz, 2H), 3.78 (d, J = 10.4 Hz, 2H), 2.46 (s, 3H).
4.1.9.10 3-methyl-N-(2-(3-methylbenzoyl)-3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)benzamide
(10j)
100 mg compound 7c reacting with 100 µL 3-methylbenzoyl chloride to give 20 mg yellowish
1
oily 10j (14.1% yield). H NMR (400 MHz, CDCl3) δ 8.01 -7.84 (m, 2H), 7.79 (s, 1H), 7.76
-7.64 (m, 3H), 7.57 (dd, J = 8.2, 2.0 Hz, 1H), 7.52- 7.38 (m, 3H), 7.39 - 7.29 (m, 2H), 4.96 (d, J
= 6.6 Hz, 2H), 3.78 (d, J = 11.3 Hz, 2H), 2.47 (s, 3H), 2.39 (s, 3H); 13C NMR (125 MHz, CDCl3)
δ 173.6, 171.3, 164.5, 138.0, 136.9, 135.4, 135.1, 133.3, 132.9, 132.1, 130.2, 128.8,128.1, 128.0,
127.8, 127.4, 126.5, 120.2, 119.2, 119.0, 118.1, 48.0, 39.7, 21.3, 21.2.
4.1.9.11 N-(2-benzoyl-3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)-2-methoxybenzamide (10k)
100 mg compound 7d reacting with 100 µL benzoyl chloride to give 30 mg yellowish oily 10k
1
(22.2% yield). H NMR (400 MHz, CDCl3) δ 9.92 (s, 1H), 8.33 (dd, J = 7.8, 1.7 Hz, 1H), 7.89
24

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-7.77 (m, 1H), 7.67 -7.46 (m, 6H), 7.42 (d, J = 7.8 Hz, 2H), 7.18 (dd, J = 11.2, 4.0 Hz, 1H), 7.07
(d, J = 7.8 Hz, 1H), 4.98 (d, J = 12.6 Hz, 2H), 4.12 (s, 3H), 3.79 (d, J = 15.8 Hz, 2H).
4.1.9.12
N-(2-(3-chlorobenzoyl)-3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)-2-methoxybenzamide(10l)
100 mg compound 7d reacting with 100 µL 3-chlorobenzoyl chloride to give 40 mg yellowish
viscous 10l (27.2% yield). 1H NMR (500 MHz, CDCl3) δ 9.93 (s, 1H ), 8.35 - 8.28 (m, 1H), 7.84
(dd, J = 7.5, 1.7 Hz, 1H), 7.63 - 7.44 (m, 4H), 7.44 - 7.38 (m, 2H), 7.36 - 7.30 (m, 1H), 7.18
(ddd, J = 9.5, 2.3, 1.4 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 4.97 (d, J = 15.7 Hz, 2H), 4.13 (s, 3H),
3.82 (d, J = 14.7 Hz, 2H).
4.1.9.13 2-methoxy-N-(2-(3-methylbenzoyl)-3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)benzamide
(10m)
100 mg compound 7d reacting with 100 µL 3-methylbenzoyl chloride to give 21 mg yellowish
crystaline 10m (15.1% yield). m.p.: 152-154°C; 1H NMR (400 MHz, CDCl3) δ 9.92 (s, 1H), 8.32
(dd, J = 7.8, 1.7 Hz, 1H), 7.92 - 7.77 (m, 2H), 7.66 - 7.49 (m, 2H), 7.40 (d, J = 7.6 Hz, 1H), 7.38
- 7.29 (m, 3H), 7.24 - 7.14 (m, 1H), 7.08 (d, J = 8.3 Hz, 1H), 4.97 (d, J = 12.6 Hz, 2H), 4.13 (s,
3H), 3.78 (d, J = 15.6 Hz, 2H), 2.38 (s, 3H).
4.1.9.14
2-methoxy-N-(2-(2-methoxybenzoyl)-3-oxo-1,2,3,4-tetrahydroisoquinolin-7-yl)
benzamide (10n)
100 mg compound 7d reacting with 100 µL o-methoxybenzoyl chloride to give 20 mg
1
yellowish viscous 10n (13.8% yield). H NMR (400 MHz, CDCl3) δ 9.92 (s, 1H), 8.33 (d, J =
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7.8 Hz, 1H), 7.51 - 7.30 (m, 5H), 7.07 - 6.90 (m, 5H), 5.08 (d, J = 10.9 Hz, 2H), 4.11 (s, 3H),
3.84 (s, 3H), 3.72 (d, J = 4.2 Hz, 2H).
4.2 Bioactivity study
4.2.1 AChE activity assay
The assays on the in vitro inhibition of AChE were measured by a modification of Ellman
methods[37]. Human recombinant AChE was obtained from Sigma Chemical and donepezil was
purchased from Dalian Meilun Biotech (China). Acetylthiocholine (ATC) and
5’,5’-dithio-bis(2-nitrobenzoic acid) (DTNB) purchased from Sigma Chemical were prepared in
water as stock solutions of 0.01 M and frozen at -20 °C. Enzymatic hydrolysis of ATC was
monitored by Shimadzu UV-2450 UV-visible spectrophotometer at 412 nm in the presence or
absence of various concentrations of inhibitor at 30 °C, in 0.1 M potassium phosphate, pH 7.0.
Each experiment was repeated at least three times and the values were averaged.
The inhibition curve was obtained by plotting the inhibition percentage versus the logarithm of
the inhibitor concentration in the reaction system. The standard deviation was calculated from
three independent assays. The linear regression parameters were determined and the IC50 was
extrapolated with the statistical software Statistical Product and Service Solutions (SPSS)
version 10.0.
4.2.2 BACE-1 inhibition assay
BACE-1 in vitro inhibition assay was performed as described in the following procedure by
employing the β-secretase Inhibitor Screening Assay Kit (Shanghai Haling biological
technological CO., LTD., China). Active recombinant human BACE-1 was obtained from
26

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BioVision Incorporated (USA) with specific activity of ≥120 units/µg. The weakly fluorescent
substrate becomes highly fluorescent once the enzymatic cleavage occurs and the increase in
fluorescence is linearly correlated with the rate of proteolysis[38]. The detailed procedures are as
follows: known concentrations of test compound (2.5 µL) or DMSO (control), and appropriate
amounts of enzyme (15 µL) were preincubated in 10 mM Tris-HCl (pH 8.0) at 25 °C. The
substrate (15 µL 400 µM H-RE(EDANS) EVNLDAEFK (Dabcyl)R-OH) was then added to start
the reaction and incubated for 60 min at the same temperature. The fluorescence signal was
recorded at λem=490 nm (λexc=340 nm) by microplate reader (Infinite M200 PRO, Tecan) using
black microwell (96 wells). The inhibition percentages corresponding to the presence of different
concentration of test compound was calculated by the following equation: 100-[AFi/AF0]×100%,
where AFi and AF0 are the fluorescence intensities obtained for BACE-1 in the presence and
absence of an inhibitor, respectively. The standard deviation was calculated from three
independent assays.
ACKNOWLEDGMENT
The financial support from the National Natural Science Foundation of China (No. 20876180)
is gratefully acknowledged. The authors also would like to thank School of Pharmaceutical
Sciences of Central South University for support of MOE and microplate reader for this research
project.
ABBREVIATIONS
AChE, acetylcholinesterase; AChEIs, acetylcholinesterase inhibitors; Aβ, β-amyloid; AD,
Alzheimer's disease; PAS, peripheral anionic sites; BACE-1, β-secretase; MTDLs,
multi-target-directed ligands; PDB, Protein Data Bank; TLC, thin layer chromatography.
REFERENCES
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