Design, synthesis, biological evaluation, and docking study of 4-isochromanone hybrids bearing N-benzyl pyridinium moiety as dual binding site acetylcholinesterase inhibitors (part II)

Article abstract of DOI:10.1111/cbdd.13136

A series of novel 4-isochromanone compounds bearing N-benzyl pyridinium moiety were designed and synthesized as acetylcholinesterase (AChE) inhibitors. The biological evaluation showed that most of the target compounds exhibited potent inhibitory activities against AChE. Among them, compound 1q possessed the strongest anti-AChE activity with an IC₅₀ value of 0.15?nm and high AChE/BuChE selectivity (SI?>?5,000). Moreover, compound 1q had low toxicity in normal nerve cells and was relatively stable in rat plasma. Together, the current finding may provide a new approach for the discovery of novel anti-Alzheimer's disease agents.

Full text of DOI:10.1111/cbdd.13136

DR JIN-YI XU (Orcid ID : 0000-0002-1961-0402)
Article type
: Research Article
Design, synthesis, biological evaluation and docking study of
4-isochromanone hybrids bearing N-benzyl pyridinium moiety as dual
binding site acetylcholinesterase inhibitors (partⅡ)
Jia Wanga, Chaolei Wanga, Zheng Wua, Xinnan Lia, Shengtao Xua,∗, Jie Liub,∗, Qinying
Lanc, Zheying Zhud and Jinyi Xua,∗
aState Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, PR China
bDepartment of Organic Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing,
210009, PR China
cLife Science and Technique Base Department of Life Science, Nanjing Agricultural University, Wei
Gang, Nanjing 210018, China
∗Corresponding authors. Tel.: +86 025 83271299; Fax. +86 025 83302827 (J.X.);
E-mail: jinyixu@china.com (J. Xu); cpuxst@163.com (S. Xu); cpu-jill@163.com (J. Liu).
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process,
which may lead to differences between this version and the Version of Record. Please
cite this article as doi: 10.1111/cbdd.13136
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dDivision of Molecular Therapeutics & Formulation, School of Pharmacy, the University of
Nottingham, University Park Campus, Nottingham NG7 2RD, U.K.
Abstract
A series of novel 4-isochromanone compounds bearing N-benzyl pyridinium
moiety were designed and synthesized as acetylcholinesterase (AChE) inhibitors. The
biological evaluation showed that most of the target compounds exhibited potent
inhibitory activities against AChE. Among them, compound 1q possessed the
strongest anti-AChE activity with an IC50 value of 0.15 nM and high AChE/BuChE
selectivity (SI >5000). Moreover, compound 1q had low toxicity in normal nerve
cells and was relatively stable in rat plasma. Together, the current finding may
provide a new approach for the discovery of novel anti-Alzheimer’s disease agents.
Key words
Alzheimer’s disease, acetylcholinesterase inhibitors, 4-isochromanone skeleton,
benzyl pyridine
Introduction
Alzheimer’s disease (AD) is a progressive and neurodegenerative disease
characterized by dementia, memory loss and other cognitive impairments, which
affects millions of elder people around the world1,2. Although many factors contribute
to this disease, its etiology and pathogenesis remain unclear until now. Studies have
shown that several factors, including obvious reduction in cholinergic neurons3,
accumulation and aggregation of β-amyloid peptide4 (Aβ) and oxidative stress5 play
significant roles in the development of AD6. These physiological changes in turn lead
to the current strategies for the treatment and prevention of AD. Among them,
increasing levels of acetylcholine (Ach) through inhibition of cholinesterase (ChEs)
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has been used most frequently to treat AD7. Up to now, several acetylcholinesterase
inhibitors such as donepezil, rivastigmine and galantamine have been approved and
widely used for the treatment of AD8. There is evidence that two binding sites exist in
AChE according to the crystal structure of the AChE of the eel, including the catalytic
active site (CAS) and the peripheral anionic site (PAS)9. Therefore, it is meaningful to
design dual-site inhibitors, which can interact with both the CAS and PAS to prevent
AD.
It has been reported that N-benzyl pyridinium moiety was introduced into a series
of aromatic scaffolds and emerged as an essential structure for some inhibitors of
AchE10 (Figure 1). In our previous studies, using donepezil as a template11, a series of
dual-site AChE inhibitors were synthesized by introducing N-benzyl pyridinium
moiety into the natural product XJP (Figure 1). Among these compounds, the most
potent compound 1 showed good AChE inhibitory activity (IC50 value : 8.9 nM) and
excellent AChE/BuChE selectivity (SI >230)11. The structure-activity relationships
(SARs) studies demonstrated that benzyl pyridine, 4-isochromanone skeleton and
nitrogen ion were essential for the activity against AChE. Moreover, the activity was
maintained or increased by introducing the fluorine or chlorine to the benzyl group.
Other groups introduced to the para-position of the benzyl group diminished the
inhibitory activity remarkably. However, the effect of methoxy group at
4-isochromanone skeleton on the AChE inhibitory activity was undefined. Besides,
the preferred position of fluorine or chlorine on benzyl group and the importance of
α,β-unsaturated ketone to the AChE inhibitory activity should be explored logically.
Inspired by our previous promising study and preliminary SARs, we embarked on
the second-round design to accomplish the SARs and find more potent AChE
inhibitors. Herein, a series of novel AChE inhibitors bearing 4-isochromanone moiety
were further designed and synthesized using compound 1 as the lead compound. The
ChEs inhibitory activities and selectivity of target compounds were also evaluated.
Moreover, the neurotoxicity and plasma stability studies of potent compounds were
disclosed in this research.
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The synthetic route of target compounds was shown in Schemes 1-2. Based on our
previously developed method for the construction of 4-isochromanone with Weinreb
amide12, we successfully completed the synthesis of 4-isochromanone skeleton to get
the compound 7, which reacted with pyridine formaldehyde to yield the intermediate
9 in the presence of K2CO3. Target compounds 1a-1r were obtained after the
nucleophilic substitution of compound 9.
The intermediate 9 was hydrogenated with Pd-C as a catalyst at the pressure of 0.7
MPa to afford compound 11, which was oxidized with Dess-Martin oxidizing reagent
to yield compound 12. Compounds 1s-1v were obtained by the reaction of compound
12 with various substituted benzyl bromide.
MATERIALS AND METHODS
General procedure for preparation of compounds
Compound 9 or 12 was dissolved in acetonitrile, then substituted bromobenzene
was added. The mixture was refluxed at 80 oC for 1 h. The yellow solid precipitated
and the target compound were obtained by filtration.
(Z)-4-((6,7-dimethoxy-4-oxoisochroman-3-ylidene)methyl)-1-(4-fluorobenzyl)pyri
dinium bromide (1q)
Yellow solid 1H NMR (300 MHz, DMSO-d6) δ: 9.11 (d, J = 6.81 Hz, 2H), 8.42
(d, J = 6.27 Hz, 2H), 7.67 (m, 2H), 7.41 (s, 1H), 7.32 (t, J = 8.88 Hz, 2H), 7.15 (s,
1H), 6.97 (s, 1H), 5.83 (s, 2H), 5.56 (s, 2H),3.91 (s, 3H), 3.86 (s, 3H); 13C NMR (75
MHz, DMSO-d6) δ: 176.37, 164.09, 160.83, 156.25, 154.95, 150.13, 149.13, 143.88,
134.66, 131.35, 131.23, 130.78, 126.66, 120.66, 116.21, 115.93, 107.29, 107.04,
104.55, 67.12, 61.29, 56.26, 55.68; MS-ESI: (ESI, pos.ion): 485.1; HRMS-ESI:
[M-Br-]+ calcd. For C24H20FNO4: 406.1449, found: 406.1452.
(Z)-4-((6,7-dimethoxy-4-oxoisochroman-3-ylidene)methyl)-1-(2-fluorobenzyl)pyri
dinium bromide (1r)
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Yellow solid; 1H NMR (300 MHz, DMSO-d6) δ: 9.06 (s, 2H), 8.45 (s, 2H),
7.67-6.97 (m, 7H), 5.97 (s, 2H), 5.57 (s, 2H), 3.91 (s, 3H), 3.86 (s, 3H); 13C NMR (75
MHz, DMSO-d6) δ: 176.27, 162.07, 158.79, 156.33, 154.93, 150.33, 149.09, 144.16,
134.66, 131.99, 131.89, 131.34, 126.58, 125.23, 121.59, 121.41, 120.59, 116.08,
115.81, 107.24, 107.07, 104.49, 67.14, 56.79, 56.29, 55.66; MS-ESI: (ESI,
pos.ion):485.1; HRMS-ESI: [M-Br-]+ calcd. For C24H20FNO4: 406.1449, found:
406.1456.
The information of other compounds is available in the supporting information.
AChE and BuChE inhibitory activity test assay
The compound was dissolved in DMSO, diluted with buffer A to the desired
concentration, and the DMSO content in the solution was controlled to be less than
1%. To the 96-well plates were added: 160 μL of 1.5 mM DTNB, 50 μL of AChE
(0.22 U/mL, prepared with Buffer B) and 10 μL of different concentrations of
inhibitor, incubated at 37 °C for 10 min, and then 30 μL of iodoacetylcholine (15
mM) was added rapidly. The absorbance changes of 0, 60, 120 and 180 s were
measured at 405 nM wavelengths. BuchE was tested in an assay similar to AChE,
which needed to replace AChE with BuChE (0.12 U/mL, prepared with Buffer B) and
replace substrate iodoacetylcholine with thioiodobenzoyl chloride (15 mM), other
conditions remain unchanged.
Cell viability
SH-SY5Y cells were planted in a 25 mL culture flask containing 1: 1 mixture of
Eagle's minimum essential medium (EMEM) and ham's F-12 medium supplemented
with 10% fetal bovine serum, 100 U/mL penicillin and 100 μg/mL streptomycin,
placed in a 5% carbon dioxide incubator at 37 °C. The cells were then grown at a
density of 10,000 per well in 96-well plates. When the cells were fused, the cells were
placed in serum-free medium and cultured with different concentrations of compound
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1q, 1r (1, 5, 10, 25, 50 μM) for 24 h. After completion of the incubation, 20 μL of
MTT was added at 37 °C for 4 h, and 200 μL of DMSO was added to dissolve
methanine crystals at the end, and the absorbance was measured at 570 nM.
Plasma stability test
A certain amount of compound 1q was dissolved in DMSO, which was adjusted to
the equivalent of free base 1.67 mg/mL of the mother liquor according to the purity
and salinity, and then using 50% MeOH diluted it to 100 μg/mL of the INT solution.
2 μL of INT solution was took and mixed with 998 μL plasma (n = 2, the ratio of
organic phase in the system does not exceed 0.5%), and the concentration of
compound in plasma is 200 mg/mL, it was placed at 37 °C water bath incubation. The
sampling point was set at 0 min, 10 min, 30 min, 1 h, 2 h. each sampling point took
100 μL of sample and was added 300 μL acetonitrile to precipitate, then
centrifuged at 12,000 rpm for 5 min, supernatant 100 μL was added 100 μL 0.1%
formic acid and took to LC-MS/MS analysis.
Determination of pharmacokinetic parameters
Three male Sprague-Dawley (SD) rats (200-220 g) were dosed with 1q
intravenously (i.v.) at dose of 1 mg/kg following Institutional Animal Care and Use
Committee guidelines. The compound was dissolved in a vehicle of 5% DMA, 10%
Solutol HS-15 and 85% saline. Blood samples (0.3 mL) were obtained via orbital
sinus puncture at 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 24 h time
points and collected into heparinized tubes. Blood samples were centrifuged for cell
removal, and the plasma supernatant was then transferred to a clean vial and
subsequently stored in 4 oC prior to analysis. Test sample concentrations were
determined by LC-MS/MS (Agela Technologies Venusil XBP C8(L) 2.1*5mm, 5µm,
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150A) using propranolol as an internal standard. Pharmacokinetic parameters were
calculated using winnonlin 5.2. software.
Results and discussion
To determine the inhibitory activities of these target compounds against AChE and
BuChE13, IC50 of all compounds were obtained. Lead compound 1 and donepezil were
used as the reference compounds, and the results were shown in Table 1. It was found
that the inhibitory activities of most compounds reached the nanomolar level against
AChE and the micromolar level against BuChE. Among them, compound 1q showed
the most potent activity against AChE with IC50 of 0.15 nM, which was 250 and 160
times higher compared to the positive drug donepezil and the lead compound 1,
respectively. Furthermore, its selectivity index of AChE/BuChE reached up to 5903.
The SARs studies showed that when keeping fluorine group at the para-position of
the benzyl group, the anti-AChE activity was enhanced or maintained by introducing
halogens to the ortho-position. On the contrast, introducing halogens to the
meta-position decreased the activity dramatically. As to compounds 1o-1r, the
number and position of the methoxy group on 4-isochromanone skeleton had a
significant influence on the anticholinesterase activity. The compounds bearing
methoxy group at both R1 and R2 positions exhibited more potent anticholinesterase
activity than those with methoxy group at both R2 and R3 positions. Besides, those
compounds with reduced double bond also have good anticholinesterase activity
(1s-1v).
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Molecular docking study
In order to investigate the interaction mode of compounds 1 and 1q with AChE, a
molecular modeling study14 was carried out by using Molecular Operating
Environment (MOE) software and the PDB structure of 1EVE was taken from the
RCSB Protein Data Bank for the docking procedure. Previous studies showed that
two methoxy groups of donepezil interacted with Trp286 of acetylcholinesterase
through hydrogen bond15,16. In the present study, the methoxy group at the C7 position
of compound 1 interacted with Trp286 to form a hydrogen bond, while the methoxy
group at the C8 position could not form a hydrogen bond with Trp286 due to steric
hindrance (Figure 2b). However, both methoxy groups of compound 1q could
interacted with Trp286 through hydrogen bond, which was similar to that of
donepezil, and at the same time, according to previous study, phenyl pyridine
interacted with the CAS of AChE and the 4-isochromanone moiety interacted with the
PAS of the AChE simultaneously. This interaction mode might explain why the
activity of compound 1q was significantly increased.
Cell viability test of compounds 1q and 1r
To measure the toxicity of target compounds against nerve cells17, the viability of
SH-SY5Y cells treated with representative compounds 1q and 1r was determined and
the result was shown in Table 2. When SH-SY5Y cells were exposed to compounds
1q and 1r at concentrations of 5 and 50 μM for 24 h, the survival rates of nerve cells
did not decrease obviously. Even at the concentration of 100 μM, the survival rate just
decreased slightly (cell viability >90%) and the morphology of the cells was not
affected, indicating the low toxicity of these compounds.
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Plasma stability test and pharmacokinetic data of compound 1q
Compound 1q has a good inhibitory activity on enzyme, in order to provide
information for pharmacokinetic evaluation, representative compound 1q was
selected for preliminary plasma stability test. The results showed the concentration of
compound 1q in plasma decreased with time, but changed slightly in the period of 0
to 2 h, indicating that compound 1q was relatively stable in rat plasma (Table 3).
The further pharmacokinetic evaluation showed that compound 1q had undesirable
pharmacokinetic properties and its average half-life was only 1.94 hours (Table 4).
Therefore, compound 1q might be considered as a promising lead for the next round
structural modification.
Conclusions
In conclusion, a series of novel AChE inhibitors derived from natural product XJP
were further designed and synthesized based on our previous studies. The biological
evaluation showed that these compounds had potent activities against AChE and good
selectivity against BuChE. Among them, compound 1q showed the strongest
anti-AChE activity (IC50 = 0.15 nM), high AChE/BuChE selectivity (SI >5903) and
low neurotoxicity. Additionally, compound 1q possessed high plasma stability. As a
result, compound 1q might be considered as a promising lead compound for further
research to develop new anti-AD agents.
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Acknowledgments
This study was financially supported by the National Natural Science Foundation
of China (No. 81302635), the Project Program of the State Key Laboratory of Natural
Medicines, China Pharmaceutical University (No.SKLNMKF201710), China
Postdoctoral Science Foundation (No. 2015M581903).
The authors declare no competing financial interest.
Support information
The photographs of the docking experiment and information of compounds
synthesized are available in the supporting information.
Figure legends
Figure 1. Discovery of potent AchE inhibitor 1 and the design of its derivatives
Scheme 1 Reagents and conditions: (a) K2CO3, H2O/toluene, r.t., 30 min; (b)
NaOMe, MeOH, r.t. then H2O, 95% yield over two steps; (c) Oxalyl chloride, then
N, O-Dimethyl hydroxylamine hydrochloride, 80% yield; (d) t-BuLi, THF, -78 °C,
1-5 min, 70% yield; (e) K2CO3, MeOH, r.t., 4 h, 60% yield; (f) CH3CN, reflux, 1 h,
60%-80% yields.
Scheme 2 Reagents and conditions: (a) H2/Pd-C, 0.7 Mpa, r.t., overnight, 70% yield;
(b) Dess-Martin reagent, r.t. 85% yield; (c) CH3CN, reflux, 1 h, 60%-80% yields.
Figure 2. (a) 3D docking graph of compound 1; (b) 2D docking graph of compound
1; (c) 3D docking graph of compound 1q; (d) 2D docking graph of compound 1q.
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Table 1. Inhibition of ChEs by target compounds, and their selectivity index
compound
R1
R2
R3
R4
R5
R6
IC50 (nM)
Selectivity
Index
AChE
BuChE
1
8.93 0.4
2.07 0.06
232
9.8
1a
1b
1c
1d
H
H
H
H
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
=
=
=
=
F
F
F
F
F
H
Cl
H
H
F
91.80 3.92 902.13 37.92
25.63 1.09 438.92 20.14
17.1
57.4
31.1
H
Cl
97.27 4.82
5580 21
23.44 1.12 728.61 35.43
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1e
1f
H
H
H
H
H
H
H
H
H
H
OCH3 OCH3
OCH3 OCH3
=
=
=
=
=
=
=
=
=
=
=
=
=
=
-
H
H
F
CF3
H
F
H
CF3
H
F
48.69 2.31
4280 12
87.9
8.5
70.03 3.19 592.36 28.18
1g
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
1023 45
5.42 0.25
96.82 4.32
5.09 0.23
31.92 1.52
30.57 1.32
28.22 1.28
33.32 1.48
59120 2580
950 4.5
57.8
1h
F
H
Cl
H
H
H
F
175.3
39.9
1i
F
H
Cl
H
H
H
F
3860 130
1210 48
2020 90
1130 43
2540 90
2410 80
1j
F
237.7
63.3
1k
H
F
1l
H
36.9
1m
1n
H
H
H
H
H
F
90.0
H
H
H
F
72.3
1o
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
H
H
H
F
0.29 0.013 968.68 48.21
0.34 0.015 329.04 15.12
0.15 0.007 885.53 43.17
0.44 0.021 798.35 37.29
3340.3
967.8
5903.5
1814.4
158.7
30.6
1p
1q
H
H
H
H
F
1r
H
H
F
1s
H
H
H
H
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
OCH3 OCH3
H
H
H
F
27.13 1.29
161.28 8.00
88.95 4.23
44.38 2.33
37.54 1.92
4306 25
4920 18
3210 15
4560 18
3410 150
1t
-
1u
-
H
H
36.1
1v
-
H
102.7
90.8
donepezil
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Table 2. Cell viability of compounds 1q and 1r on nerve cells SH-SY5Y
Compound Concentrations (μM)
Cell viability (%)
98.3 12.1
97.8 15.2
93.4 10.2
97.8 11.5
97.6 15.2
91.4 13.1
5
50
100
5
1q
1r
50
100
Table 3. Plasma stability test of compound 1q
Analyte Peak Area
(counts)
IS Peak Area
Time
Area Ratio
(counts)
0 min
10min
30min
1 h
3.46E+05
3.12E+05
2.79E+05
2.76E+05
2.58E+05
2.55E+05
1.11E+00
1.22E+00
1.21E+00
1.18E+00
1.11E+00
3.41E+05
3.34E+05
3.04E+05
2 h
2.84E+05
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Table 4. Pharmacokinetic parameters (mean S.D) of 1q 1.0 mg/kg, i.v. in rats
(n=3)
HL.Lambda_z(h)
Tmax
(h)
Cmax
AUClast
AUCinf
Vz_obs
(ml/kg)
Cl_obs
(ml/h/kg)
407 31
MRTlast
(h)
(ng/ml)
(h*ng/ml) (h*ng/ml)
1.94 0.05
0.083 0.0 3947 311 2460 182 2465 182 1139 116
0.70 0.03
Note: The pharmacokinetic parameters were calculated using winnonlin 5.2.
HL: half life of the drug
Tmax: the time to reach the peak concentration of drug
Cmax: peak concentration of drug
AUC: area under the curve, it is an important indicator of the degree of drug absorption
Vz: apparent distribution volume
Cl: drug internal clearance rate
MRT: average dwell time
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