Rational Design and Multibiological Profiling of Novel Donepezil-Trolox Hybrids against Alzheimer's Disease, with Cholinergic, Antioxidant, Neuroprotective, and Cognition Enhancing Properties

Article abstract of DOI:10.1021/acschemneuro.7b00257

A novel series of donepezil-trolox hybrids were designed, synthesized, and evaluated as multifunctional ligands against Alzheimer's disease (AD). Biological assays showed that these derivatives possessed moderate to good inhibitory activities against acetylcholinesterase (AChE) and monoamine oxidase B (MAO-B) as well as remarkable antioxidant effects. The optimal compound 6d exhibited balanced functions with good inhibition against hAChE (IC₅₀ = 0.54 μM) and hMAO-B (IC₅₀ = 4.3 μM), significant antioxidant activity (41.33 μM IC₅₀ by DPPH method, 1.72 and 1.79 trolox equivalent by ABTS and ORAC methods), excellent copper chelation, and Aβ_1-42 aggregation inhibition effect. Furthermore, cellular tests indicated that 6d has very low toxicity and is capable of combating oxidative toxin (H₂O₂, rotenone, and oligomycin-A) induced neurotoxicity. Most importantly, oral administration of 6d demonstrated notable improvements on cognition and spatial memory against scopolamine-induced acute memory deficit as well as d-galactose (d-gal) and AlCl₃ induced chronic oxidative stress in a mouse model without acute toxicity and hepatotoxicity. In summary, both in vitro and in vivo results suggested that 6d is a valuable candidate for the development of a safe and effective anti-Alzheimer's drug.

Full text of DOI:10.1021/acschemneuro.7b00257

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Article
Rational design and multi-biological profiling of novel donepezil-
trolox hybrids against Alzheimer’s disease, with cholinergic,
antioxidant, neuroprotective and cognition enhancing properties
Pei Cai, Si-Qiang Fang, Xue-Lian Yang, Jia-Jia Wu, Qiao-
Hong Liu, Hao Hong, Xiao-Bing Wang, and Lingyi Kong
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00257 • Publication Date (Web): 14 Aug 2017
Downloaded from http://pubs.acs.org on August 15, 2017
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Rational design and multi-biological profiling of novel donepezil-trolox hybrids
against Alzheimer’s disease, with cholinergic, antioxidant,
neuroprotective and cognition enhancing properties
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†
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Pei Cai, SiꢀQiang Fang, XueꢀLian Yang, JiaꢀJia Wu, QiaoꢀHong Liu, Hao
†
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†
Hong, XiaoꢀBing Wang, * and LingꢀYi Kong *
†
Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key
Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tong Jia
Xiang, Nanjing 210009, People’s Republic of China
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*
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Corresponding Authors. Tel/Fax: +86ꢀ25ꢀ83271405; Eꢀmail: cpu_lykong@126.com
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(LingꢀYi Kong); xbwang@cpu.edu.cn (XiaoꢀBing Wang)
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Abstract
A novel series of donepezilꢀtrolox hybrids were designed, synthesized and
evaluated as multifunctional ligands against Alzheimer's disease (AD). Biological
assays showed that these derivatives possessed moderate to good inhibitory activities
against acetylcholinesterase (AChE) and monoamine oxidase B (MAOꢀB) as well as
remarkable antioxidant effects. The optimal compound 6d exhibited balanced
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functions with good inhibition against hAChE (IC50 = 0.54 ꢁM) and hMAOꢀB (IC50
=
4.3 ꢁM), significant antioxidant activity (41.33 ꢁM of IC50 by DPPH method, 1.72 and
1.79 trolox equivalent by ABTS and ORAC methods), excellent copper chelation and
Aβ1−42 aggregation inhibition effect. Furthermore, cellular tests indicated that 6d was
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2 2
very low toxic and capable of combating oxidative toxins (H O , rotenone and
3
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oligomycinꢀA) induced neurotoxicity. Most importantly, oral administration of 6d
demonstrated notable improvements on cognition and spatial memory against
scopolamineꢀinduced acute memory deficit as well as Dꢀgalactose (Dꢀgal) and AlCl3
induced chronic oxidative stress in mice model without acute toxicity and
hepatotoxicity. In summary, both in vitro and in vivo results suggested that 6d is a
valuable candidate for the development of safe and effective antiꢀAlzheimer’s drug.
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Key words: Alzheimer’s disease, Acetylcholinesterase inhibitors, Antioxidant,
βꢀAmyloid aggregation, Neuroprotection, Cognitive improvement.
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1. Introduction
Alzheimer’s disease (AD) is an ageꢀrelated neurodegenerative disease featuring
in a progressive memory loss, language skills decline and other cognitive
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impairments. AD has been demonstrated to possess a highly complex network of
diverse factors and etiological hallmarks, including the accumulation of abnormal
deposits of βꢀamyloid peptide (Aβ), hyperphosphorylated tau protein,
neuroinflammation of the central nervous system (CNS), oxidative stress,
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dyshomeostasis of biometals and low level of acetylcholine (ACh). Thus, an
appropriate strategy to achieve better therapeutic efficacy for AD is proposed by
development of multiꢀtargetꢀdirected ligands (MTDLs) that can simultaneously
modulate different targets or mechanisms involved in the neurodegenerative AD
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cascade.
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Currently, the dominating treatment agent for AD is acetylcholinesterase
inhibitors (AChEIs), which increases the cholinergic neurotransmission in the
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synaptic cleft by inhibiting degradation of ACh. Also, central acetylcholinesterase
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(AChE) would play noncholinergic functions in the development of AD. The
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peripheral anionic site (PAS) of AChE would catalyze Aβ aggregation. The
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aggregation of Aβ, especially Aβ1ꢀ42, may lead to the formation of senile plaques in
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the brain, associated with neurodegeneration. Hence, dual AChEIs which are able to
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interact with both the catalytic anionic site (CAS) and PAS of AChE are expected not
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only to alleviate the symptoms, but also to slow down the progression of AD.
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The hyperꢀproduction of reactive oxygen species (ROS) has been observed in AD
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and antioxidant enzymes have also been found to be increased in specific AD brain
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regions. Recently, researches have proved that oxidative damage in cellular
structures precedes the appearance of other pathological hallmarks of AD, namely,
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senile plaques and neurofibrillary tangles.
In addition, redoxꢀactive metal ions like
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Cu
Fe and Fe is contributed to the production of ROS, which promotes oxidative
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stress thus leading to AD pathogenesis. Therefore, antioxidant and modulation of
such biometals in the brain have been proposed as a promising therapeutic strategy for
the treatment of AD.
Donepezil, first choice drug currently used for AD treatment, is among the most
popular pharmacophore inspirations in the design of novel drug candidates for its
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potent, low toxic and well tolerated AChE inhibitory activities. Trolox, a
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waterꢀsoluble analogue of vitamin E, is a powerful antioxidant widely used in
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biological or biochemical applications to reduce oxidative stress.
Additionally, it
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showed neuroprotective effects through scavenging ROS and attenuating the
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neurotoxicity mediated by Aβ and H
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on hippocampus neurons.
These premise
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of trolox consolidate its neuroprotective capacity and make it an excellent lead
compound for the design of multifunctional drugs for treating AD.
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In recent years, many interesting MTDLs have been developed, such as,
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memoquin, ladostigil, and huprine X, among others. Our group has also reported
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several families of MTDLs that combined neuroprotective, cholinergic, and
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antioxidant properties, including tacrine–trolox hybrid, melatonin–donepezil hybrids
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and others.
Following our previous work and considering both the donepezil and
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trolox behaving a very concomitant biological properties for AD treatment, we fused
the pharmacophores of donepezil and trolox into one molecule (Figure 1).
Compounds with zero and two carbon amido linkage were designed to tether these
two fragments to meet the requirement of simultaneous binding to PAS and CAS of
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AChE. The biological activities with regards to the inhibition of ChEs, MAOs and
antioxidants activities were evaluated. Noticeably, an optimal compound, 6d, was
assessed neuroprotection in PC12 and BVꢀ2 cells and conducted behavioral
performance in mice model of AD.
2. Results and Discussion
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2.1. Chemistry
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1
The synthetic method for these derivatives was shown in Scheme 1.
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Commercial 4ꢀbocꢀaminopiperidine (1) and 4ꢀ(2ꢀbocꢀaminoethyl)piperidine (2) as
starting materials were reacted with different substituted benzyl bromides to give the
key intermediate 1a-m and 2a-m, respectively, then removal of the protecting group
with trifluoroaceticacid (TFA) obtained the corresponding 1ꢀbenzylꢀsubstituted
4ꢀaminopiperidine 3a-m and 1ꢀbenzylꢀsubstituted 4ꢀaminoethylpiperidine 4a-m in
good yields without further purification. Finally, the target compounds 5a-m and
6a-m were prepared by the reaction between trolox and 3a-m and 4a-m using
Nꢀ(3ꢀ(dimethylamino)propyl)ꢀN′ꢀethylcarbodiimide hydrochloride (EDCI) and
1ꢀhydroxybenzotriazole hydrate (HOBt) as catalyst in dichloromethane (DCM).
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Structures of all target compounds were characterized by H NMR, C NMR,
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ESIꢀMS and HRMS.
2.2. Biological Assays.
2.2.1. Inhibitory activities against AChE and BuChE
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Cholinesterases (ChEs) in human body comprise of two types, namely,
acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). Normally, AChE
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hydrolyzes about 80% of acetylcholine while BuChE plays only a secondary role.
Therefore, simultaneous inhibition of both AChE and BuChE would be more
meaningful for AD treatment.
The inhibitory activities of all target compounds against ChEs were evaluated
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following the Ellman’s method, with donepezil as the reference. Initially,
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compounds were tested with enzymes from animal source (AChE from electric eel，
eeAChE, and BuChE from equine serum, eqBuChE), considering of the high degree
of homology and lower cost compared with human enzymes. As shown in Table 1, all
target compounds exhibited moderate to good inhibitory towards ChEs. Compound
6a-m showed more potent inhibitory activity for eeAChE than for eqBuChE.
Conversly, compound 5a-m exhibited more BuChE selective. Obviously, the length of
the alkyl spacer between trolox and donepezil moiety could significantly influence the
selective of ChE inhibitory activity. Compound 6d (eeAChE: IC50 = 0.31 ꢁM,
eqBuChE: IC = 3.91 ꢁM) possessed the most significant bioactivities, revealing 2ꢀF
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5
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group in the benzene was the best choice for AChE inhibition. Specifically, the AChE
inhibition potency of 6a-m was widely higher than that of 5a-m having a lessꢀcarbon
length of alkyl spacer. While the BuChE inhibition was no substantial difference
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between 5a-m and 6a-m. It may be ascribed to the fact that the functional manner of
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AChE is different with that of BuChE. Comparing with different substituents (H,
CH , OCH , F, Cl, Br and NO ), compounds bearing electronꢀwithdrawing group
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were in favor of the inhibitory activities of AChE. Conversely, the length of the alkyl
spacer and substituent effect exhibited no clear trend for BuChE inhibition.
Compounds 6a-m with potent inhibitory activities for eeChEs were then tested on
human ChEs (hChEs) (Table 1). It can be observed that compounds 6a-m were also
potent inhibitors of hChEs. Most of these compounds showed decreased inhibitory
activity for hChEs compared to eeChEs. Compound 6d with the inhibitory activity
being 0.56 ꢁM was still the most potent inhibitor of hAChE.
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Table 1. In vitro inhibition of AChE and BuChE inhibitory activity of the target
compounds.
a)
IC50 (µM)
Compds
n
d)
b)
b)
c)
c)
SI
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eeAChE
eqBuChE
hAChE
hBuChE
e)
5
5
a
12.53 ± 1.22
19.54 ± 0.62
15.78 ± 0.22
9.25 ± 0.35
11.27 ± 0.25
9.51 ± 0.04
9.84 ± 0.04
13.42 ± 0.03
14.88 ± 1.02
17.92 ± 1.05
11.79 ± 0.11
10.23 ± 0.22
11.56 ± 0.24
0.82 ± 0.11
1.63 ± 0.22
1.44 ± 0.21
0.31 ± 0.03
0.59 ± 0.05
0.43 ± 0.12
0.63 ± 0.10
0.48 ± 0.02
0.77 ± 0.01
0.61 ± 0.03
0.53 ± 0.04
0.85 ± 0.10
0.07 ± 0.01
5.38 ± 0.02
5.49 ± 0.06
5.94 ± 1.02
5.88 ± 0.22
5.42 ± 0.32
5.32 ± 0.24
6.01 ± 1.04
5.93 ± 0.37
5.15 ± 0.45
5.83 ± 0.02
5.66 ± 0.43
6.73 ± 0.62
6.34 ± 0.04
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
b
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
5
c
5d
5
e
5
f
5
5
g
0
h
5i
5
j
5k
5
l
5
m
6
a
3.24 ± 1.01 0.97 ± 0.02
3.82 ± 0.90 2.34 ± 0.21
3.61 ± 0.81 2.75 ± 0.22
5.62 ± 0.11
4.91 ± 0.15
5.80 ± 0.30
5.79
2.10
2.11
6
b
6c
6
d
3.91 ± 0.11
0.56 ± 0.04
5.97 ± 0.13 10.66
6e
3.87 ± 0.13 1.73 ± 0.06
4.09 ± 0.10 1.67 ± 0.09
4.54 ± 0.92 1.04 ± 0.01
3.69 ± 0.71 2.46 ± 0.02
3.71 ± 0.21 2.11 ± 0.09
4.58 ± 0.22 1.92 ± 0.05
4.52 ± 0.32 1.58 ± 0.08
4.95 ± 0.55
7.04 ± 1.21
9.27 ± 1.03
5.81 ± 0.29
4.86 ± 1.22
4.29 ± 0.77
3.26 ± 0.44
5.97 ± 0.06
2.86
4.43
8.91
2.26
2.30
2.55
2.06
3.47
66
6
f
2
6
6
g
h
6
i
6
j
6
k
6l
Donepezil
3.94 ± 0.11
1.72 ± 0.32
－
2.48 ± 0.11 0.048 ± 0.003 3.17 ± 0.10
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a) IC50: 50% inhibitory concentration (mean ± SD of three experiments);
b) AChE from electric eel and BuChE from equine serum were used;
c) AChE from human erythrocytes and BuChE from human serum were used;
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d) SI means selectivity index, hBuChE/hAChE.
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e) Not tested
2. 2. 2. Kinetic study of hAChE inhibition
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To assess the hAChE inhibition mechanism of donepezilꢀtrolox hybrids, the
kinetic test of 6d was investigated. As presented in Figure S1 (Supporting
Information), the LineweavereꢀBurk reciprocal plots with increasing slopes and
intercepts at higher inhibition concentrations intersected in the fourthꢀquadrant,
indicating a mixedꢀtype inhibitory pattern for compound 6d to AChE with the
inhibition constant Ki of 0.44 ꢁM. Therefore, compound 6d can simultaneously bind
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to the CAS and PAS of hAChE. The docking results in Figure S2 (Supporting
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Information) also indicated that compound 6d could fit into the activeꢀsite gorge of
the enzyme and simultaneously interact with the PAS and CAS of hAChE in
agreement with the kinetic study, which demonstrated the rationality of our molecular
design.
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2.2.3. Inhibitory activities against MAOꢀA and MAOꢀB
Monoamine oxidases (MAOs), including MAOꢀA and MAOꢀB, catalyze the
deamination of amines and are responsible for the regulation and metabolism of major
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monoamine neurotransmitters. The chemical reaction catalyzed by MAOs resulted
in a number of potentially neurotoxic species, such as hydrogen peroxide and
ammonia. In particular, hydrogen peroxide can trigger the production of ROS and
induce mitochondrial damage and neuronal apoptosis. MAOꢀB inhibitors have been
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considered as rational bases in AD management.
Considering MAOs being highly relevant to oxidative stress, the inhibitory
activities on human MAOs (hMAOs) of all the target compounds were evaluated. The
protocol was carried out with a fluorescenceꢀbased method using kynuramine as a
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nonselective substrate of hMAOꢀA and hMAOꢀB. According to the Table2, most of
5a-m displayed a better inhibition towards hMAOꢀB than hMAOꢀA, while 6a-m
presented unselective action for MAOs. Those suggested the linkage length between
two different moieties plays some effects on its selectivity. Compound 5e was the
most active inhibitors (hMAOꢀA: IC50 = 9.3 ꢁM, hMAOꢀB: IC50 = 1.6 ꢁM).
Comparing the two series of compounds, the MAOꢀB inhibition of 6a-m was feebly
weaker than that of 5a-m. The results indicated that the linker plays no substantial
impacts on the MAOꢀB inhibitory activity. Additionally, most compounds with the
electronꢀwithdrawing groups displayed a increased MAOꢀB potency.
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Table 2. The MAOs inhibition bioactivity and radical scavenging capacity of the
target compounds.
a)
b)
IC50 (µM)
hMAO-B
Trolox equivalent
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Compds
hMAO-A
DPPH assay
ABTS assay
ORAC assay
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a
15.3 ± 0.2
13.1 ± 0.3
12.7 ± 0.3
8.9 ± 0.1
2.5 ± 0.1
3.1 ± 0.2
3.3 ± 0.2
1.7 ± 0.2
44.38 ± 0.33
50.29 ± 1.21
50.88 ± 1.52
54.30 ± 1.30
1.54 ± 0.22
1.51 ± 0.11
1.43 ± 0.14
1.48 ± 0.12
1.44 ± 0.01
1.41 ± 0.02
1.47 ± 0.12
1.28 ± 0.22
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5
d
5
e
9.3 ± 0.2
1.6 ± 0.3
57.30 ± 2.42
1.59 ± 0.11
1.61 ± 0.12
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f
11.4 ± 0.1
11.8 ± 1.2
12.5 ± 1.5
12.7 ± 2.1
13.8 ± 1.5
12.6 ± 0.3
13.2 ± 0.5
11.1 ± 0.4
7.8 ± 0.6
1.8 ± 0.3
1.9 ± 0.2
2.3 ± 0.5
1.7 ± 0.3
3.1 ± 0.1
2.7 ± 0.9
1.9 ± 0.3
3.2 ± 0.3
7.3 ± 0.2
58.87 ± 2.43
59.64 ± 3.12
56.33 ± 2.11
52.31 ± 3.45
51.90 ± 2.11
53.47 ± 3.14
51.35 ± 1.21
40.77 ± 1.03
43.38 ± 1.22
1.21 ± 0.13
1.24 ± 0.11
1.12 ± 0.05
1.18 ± 0.07
1.15 ± 0.05
1.21 ± 0.04
1.08 ± 0.02
1.04 ± 0.03
1.76 ± 0.07
1.29 ± 0.14
1.34 ± 0.13
1.42 ± 0.08
1.28 ± 0.12
1.25 ± 0.08
1.22 ± 0.08
1.28 ± 0.11
1.09 ± 0.13
1.56 ± 0.02
5g
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h
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i
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j
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k
5l
5
m
6a
6
b
8.4 ± 0.1
8.9 ± 0.1
4.4 ± 0.2
5.3 ± 0.5
4.8 ± 0.1
5.7 ± 0.3
8.1 ± 0.2
7.2 ± 0.4
5.8 ± 0.5
6.9 ± 0.1
7.5 ± 0.1
4.3 ± 0.2
4.6 ± 0.2
4.5 ± 0.3
5.8 ± 1.0
5.3 ± 0.7
6.1 ± 0.4
4.8 ±0.3
47.43 ± 1.22
54.90 ± 1.10
43.33 ± 1.32
44.99 ± 0.07
54.20 ± 1.32
49.55 ± 1.89
44.32 ± 2.43
47.12 ± 3.21
48.89 ± 3.41
1.65 ± 0.34
1.71 ± 0.08
1.79 ± 0.21
1.75 ± 0.02
1.68 ± 0.03
1.59 ±0.11
1.61± 0.04
1.54 ± 0.03
1.49 ± 0.01
1.45 ± 0.07
1.79 ± 0.01
1.62 ± 0.03
1.55 ± 0.01
1.48 ± 0.02
1.56 ± 0.02
1.51 ± 0.05
1.54 ± 0.06
1.49 ± 0.10
6
c
6
d
6e
6
f
6g
6
h
6
i
6
j
6
k
6.3±0.2
5.7± 0.2
48.24 ± 2.11
1.55 ± 0.05
1.45 ± 0.02
6
l
5.4 ± 0.2
5.6 ± 0.2
ꢀ
4.9 ± 0.1
5.1 ± 0.2
ꢀ
46.30 ± 2.21
47.30 ± 2.20
45.2 ± 2.30
1.60 ± 0.12
1.49 ± 0.11
1
1.51 ± 0.05
1.57 ± 0.05
1
6
m
Trolox
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a) IC50: 50% inhibitory concentration (means ± SD of three experiments);
b) Data are expressed as (mmol trolox)/(mmol tested compound);
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2.2.4. In vitro free radical scavenging activities
As an ample evidence reported, oxidative stress plays an critical role in the
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development of AD.
Drugs preventing the formation or clearing of the free
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radicals in the brain would be beneficial for AD. Three independent approaches,
namely, DPPH (diphenylꢀ1ꢀpicrylhydrazyl) radical scavenging method, ABTS (2,
2’ꢀazinoꢀbis(3ꢀethylbenzthiazolineꢀ6ꢀsulfonicacid)) radical scavenging method and
ORAC (oxygen radical absorbance capacity) assay were used to coꢀelucidate the
antioxidant activities of this series of analogs in vitro.
2.2.4.1. DPPH radical scavenging assay
DPPH radical can be used in preliminary screening of compounds capable of
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scavenging reactive oxygen species. For comparison, trolox was used as reference.
The results summarized in Table 2 indicated that all compounds retained the
antioxidant activity comparing with trolox. Connecting the trolox with donepezil
moiety and variation of substituents did not significantly affect the antioxidant activity,
indicating that hybridizing these two scaffolds was rational.
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2.2.4.2. ABTS method and ORAC assay
2
These compounds were also tested for their antioxidant activities by using the
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ABTS method and ORAC assay. Their antioxidant activities were provided as
trolox equivalent (mmol of trolox/mmol of tested compound). As shown in Table 2,
all the trolox equivalent of the target compounds is larger than 1. It means that most of
the compounds demonstrated more superior antioxidant activities compared with
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trolox.
Although the antioxidant results from three kinds of free radical scavenging
methods performed a little difference, the same trend of free radical scavenging
activity was observed according to the results. After above biological evaluation,
compound 6d was chosen as the most promising compound for further study based on
its strong and balanced inhibition for both ChEs and antioxidant activity closed to
trolox.
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2.2.5. MetalꢀChelating properties of compound 6d
The chelating selectivity and ability of compound 6d to chelate biometals, such as
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Cu , Zn , Fe and Fe , were studied by UV−vis spectroscopy assay and
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fluorescence spectrometry. The UV result was presented in Figure 2A showed that
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new optical band was detected at 246 nm after the addition of CuSO to the solution
2
of compound 6d, which demonstrated the production of the corresponding complex
via metal chelation. Meanwhile, from the fluorescence spectra in Figure 2B
(excitation wavelength at 291 nm), the specific fluorescence emission peak of 6d can
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be observed at 320 nm with the highest intensity. After Cu was added to the HEPES
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solution of 6d, the fluorescence intensity of emission spectra was dramatically
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3+
decreased, and even disappeared in 320nm. The spectra of 6dꢀZn , Fe and Fe
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were in a moderate decrease of their fluorescence intensity. Overall, the above
mentioned different changes indicated that all test metals may possess chelation effect
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with compound 6d, especially chelating with Cu .
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The chelating effect of 6d for Cu in HEPES buffer was further investigated by
UVꢀvis spectrometry. Following the absorption at 246 nm, a series of UV−vis spectra
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2.2.6. Modulation of selfꢀ and metalꢀinduced Aβ1ꢀ42 aggregation
we further studied 6d’s regulation of selfꢀ and metalꢀinduced Aβ aggregation by
Thioflavin T (ThT) fluorescence assay and transmission electron microscopy
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(TEM).
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The ThT assay results were reported in Figure 3A, and the curcumin was used as
reference. Compound 6d inhibited 56.3 ± 4.1% of the selfꢀinduced Aβ1−42 aggregation
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2+
and 63.9 ± 3.6% of the Cu ꢀinduced Aβ1ꢀ42 aggregation, which were similar with
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curcumin (52.9 ± 7.2% and 66.5 ± 3.2%). These results indicated that compound 6d
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+
could effectively inhibit selfꢀinduced Aβ aggregation and Cu ꢀinduced Aβ1ꢀ42
aggregation. As indicated in Figure 3B, Aβ1−42 alone (Figure 3B-a) can aggregate
into wellꢀdefined Aβ1ꢀ42 fibrils induced by themselves (Figure 3B-b). More complex
2
2+
2
Aβ fibrils were observed in the presence of Cu (Figure 3B-c) than with Aβ1−42 alone.
By contrast, few Aβ1ꢀ42 fibrils were observed in the presence of compounds curcumin
and 6d (Figure 3B-d~g) under the identical conditions. The consistent results of TEM
images and the ThT binding assay suggested that 6d can inhibit Aβ1−42 fibril
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2
formation and Cu ꢀinduced Aβ1ꢀ42 aggregation effectively.
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2.2.7. The cytotoxic effect of the compound 6d on HepG2, PC12 and BVꢀ2 cells
Considering the critical importance of evaluating the possible hepatotoxicity and
the safety index for developing a nervous system drug, we initially screened the
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cytotoxic effect of 6d on HepG2, PC12 and BVꢀ2 cells, respectively.
As presented in Figure 4A, compound 6d displayed negligible hepatotoxicity
profile up to 100 ꢁM after 24 h incubation on HepG2 cells and showed no obvious
cytotoxicity less than 50 ꢁM on PC12 and BVꢀ2 cells. Collectively, the results
suggested 6d was safe and worthy of further investigations.
2.2.8. Neuroprotective effect of 6d against oxidative injuries on PC12 cells
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The over production of ROS and the unbalance in detoxification systems produce
severe oxidative stress conditions in neurons affected by AD. Reduction of oxidative
stress has been claimed as a viable strategy to slow down the progression of the
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disease. Therefore, we tested the ability of selected 6d to protect PC12 cells against
oxidative injuries against three different toxic insults (hydrogen peroxide,
oligomycinꢀA and rotenone).
2
The cytoprotective effect were determined by measuring the cell viability after
2
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incubation with a radical initiator (hydrogen peroxide, H O ) and two mitochondrial
poisons (oligomycinꢀA and rotenone), both capable of arresting respiratory chain and
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energy production. Compound 6d under investigation was incubated at three
concentrations (5, 10 and 20 ꢁM) using the untreated cells as control. As depicted in
Figure 4B, 6d exerted a relatively poor neuroprotective activity against cellular
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damage induced by oligomycinꢀA, whereas it showed a moderate to good
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neuroprotective activity against H O and rotenone at the same concentration. 6d
markedly protected PC12 cells against three different toxic insults in a significant
doseꢀdependent manner. These results showed that 6d was effective against all the
oxidative injuries insults on PC12 cells.
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2.2.9. Neuroprotective effects of 6d against LPSꢀstimulated inflammation on BV2
microglial cells
Neuroinflammation plays a crucial role in causing neuronal death and damage,
which in turn leads to neurodegenerative diseases such as AD, PD, and multiple
sclerosis. The activation of brain microglial cells in the CNS and the subsequent
excess production of inflammatory mediators, such as nitric oxide (NO), may result in
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uncontrolled neuroinflammation in neurodegenerative diseases. Therefore, by
inhibiting the production of inflammatory mediators NO in microglial cells,
antineuroinflammatory therapy could delay or halt the disease progression prior to
irreversible damage and the occurrence of clinical symptoms.
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The latest research unclosed that the close relationship was observed between the
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cholinergic nerves, oxidative stress and inflammation.
So we determined the
antineuroinflammatory properties of 6d by the Griess assay through detecting the
suppression of NO production, using trolox and donepezil as positive control to help
illuminate smoothly. Results in Figure 4C presented that donepezil and trolox showed
lower NO production inhibitory, while 6d exhibited significantly higher inhibition of
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NO production in the LPSꢀstimulated BV2 cells than the leading compounds trolox
and donepezil. The unique structure of 6d resulted in preferred antineuroinflammatory
activities which further stated the rationality of our molecular design.
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2.2.10. 6d reduced LPSꢀinduced intracellular ROS accumulation
Intracellular ROS act as second messengers in regulating LPSꢀstimulated
production of neurotoxic factors in microglial cells. Correspondingly, specifically
inhibiting the production of intracellular ROS is a general way to suppress
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intracellular proinflammatory signals. As shown in Figure 4D, when BVꢀ2 cells
were exposed to 1 ꢁg/ml LPS for 18 h, the intracellular ROS increased obviously by
using the DCFHꢀDA probe. While treated with compound 6d, the level of ROS
reduced in a concentrationꢀindependent manner. This results suggested that LPS could
induce ROS production and 6d effectively decreased the LPSꢀinduced intracellular
ROS accumulation.
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Taken together, these all cellꢀbased results highlighted that 6d was a promising
neuroprotective agent and deserved a deeper exploration in vivo.
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2.2.11. In vitro bloodꢀbrain barrier permeation assay
Since brain penetration is essential for successful antiꢀAD drugs, we evaluated
the bloodꢀbrain barrier (BBB) penetrating potency of these derivatives. Parallel
artificial membrane permeation assay for BBB (PAMPAꢀBBB) was used according to
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a previous report by Di et al. Assay validation was conducted by comparing
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experimental permeability of eight commercial drugs with reported values. A plot of
experimental data versus bibliographic values gave a good linear correlation, Pe (exp.)
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= 1.2014Pe (bibl.) ꢀ 0.3139 (R = 0.9486). From this equation and considering the
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limit established by Di et al. for BBB permeation, we established that compounds
ꢀ6
ꢀ1
with permeability values over 4.5 × 10 cm s should be able to cross the BBB. From
the results presented in Table S3 (Supporting Information), it could be seen that 6d
ꢀ6
ꢀ1
had the permeability value of 6.7 × 10 cm s , and exhibited good BBB permeability.
2.2.12. Acute toxicity test and hepatotoxicity studies
Since acute toxicity and hepatotoxicity are two important criterions in new drugs
development, the corresponding studies of 6d was launched. Twenty ICR male mice
were randomly allocated into 4 groups, and the test compound 6d was given in single
oral doses of 0, 677, 1333, or 2000 mg/kg, respectively. After administration of the 6d,
mice were monitored continuously for the first 4 h for any abnormal behavior and
mortality changes, intermittently for the next 24 h, and occasionally thereafter for 14
days to supervise the onset of any delayed effects. During the experimental period, no
acute toxicity symptoms, such as mortality, or unnormal behavior / changes in water
or food consumption or body weight were observed (Figure 5A). Furthermore, all
mice were sacrificed on the 14th day after drug administration, and no damage to the
internal organs was macroscopically detected.
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Considering that the liver is the main drug metabolic organ, so we further
evaluated whether the preferred 6d had an effect on the liver. After the acute toxicity
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study, the heparinized serum of mice of different groups were collected, and the levels
of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were
determined. As described in Figure 5B, all groups at the experimental doses did not
cause significant hepatotoxicity compared to the control, as indicated by no obvious
increased activities of ALT and AST. To further confirm its nonꢀhepatotoxicty,
morphological study of the highest dosage on liver tissue stained with hematoxylin
and eosin (HE) was performed. Results showed that no significant area of necrosis
and distinct fatty degeneration of the hepatocytes or other substantial lesions were
observed (Figure 5C), which means no signally morphological changes after the
treatment with 14d even in the highest concentration. These results were consistent
with the previous HepG2 cell model evaluation. All the results coꢀilluminate the 6d
had no acute hepatotoxicity and possessed similar safety index to donepezil and trolox
for the treatment of AD. Overall, compound 6d had no acute toxic and well tolerated
at doses up to 2000 mg/kg.
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2.2.13. Cognitive and memory improvement test of scopolamineꢀinduced cognition
impairment
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Cognitionꢀimproving potency is of utmost importance for antiꢀAD agents.
Compound 6d's ability to ameliorate scopolamineꢀinduced cognition impairment in
ICR mice was investigated in a behavioral study using the stepꢀthrough passive
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avoidance test and the Morris water maze test.
Donepezil and 6d were orally
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administered to the ICR mice 30 min before intraperitoneal (i.p.) administration of
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scopolamine (3 mg/kg) or saline solution for 10 consecutive days to adapt the
apparatus.
2.2.13.1 Passive avoidance task
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Firstly, the stepꢀthrough passive avoidance test was used to assess 6d
cognitionꢀimproving potency. As shown in Figure 6A, the stepꢀthrough latency of
mice treated with scopolamine alone (model group) was significantly shorter than that
of vehicleꢀtreated mice (control group), which means the scopolamineꢀinduced
cognition impairment has been established. The treatment with 6d (5mg/kg, 10mg/kg,
20mg/kg) significantly increased the latency time in a doseꢀdependent manner. In
particular, the effect of 6d (20 mg/kg) was comparable with that of donepezil (5
mg/kg). These results indicated that 6d can reverse cognitive deficit against
scopolamineꢀinduced cognition impairment.
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2.2.13.2 Morris water maze test
3
After the preliminary evaluation of compound 6d cognitionꢀimproving effect by
passive avoidance task, we further confirmed by Morris water maze test which is the
most common and recognized widely in assessing the learning and memory ability.
During the training trials, the mean escape latency and searching distance for the mice
in each group declined progressively; however, the model mice normally spent more
time and required farther distance to find the hidden platform. Figure 6B-a showed
the mean values of the escape latencies to the hidden platform at day 1 and day 5.
Controlꢀoperated mice exhibited a reduction of mean escape latency from 47.3 to 19.4
s over the course of five training days. Compared with the control group, the high
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dosage 6dꢀtreated group (20mg/kg) had similar escape latency to donepezil treated
group, displaying a reduction of mean escape latency from 54.9 to 24.3 s, which the
donepezil group showed a reduction from 47.6 to 23.9 s. Meanwhile, the average
swimming speed (Figure 6B-b) for each group of mice was virtually equivalent,
which further demonstrated that the longꢀterm uptake of 6d did not impact the
animals’ normal physiology activity. Furthermore, in the probe trial on day 6, the
administration of 6d improved the overall target quadrant preference (28.88%)
compared with the model group (20.62%) (Figure 6B-c). Also, the Figure 6B-d
indicated that the control group had significantly higher numbers of virtual platform
(the original platform location) crossings (3.6 ± 0.6) compared to the model group
mice (1.5 ± 0.4), which strongly suggested that scopolamine led to a spatial memory
deficiency in the mice. The numbers of virtual platform crossings for the mice that
were administered donepezil (3.0 ± 0.5), 6d (3.5 ± 0.8) were remarkably improved
compared to the model group. The representative tracks of the mice in Morris water
maze during the spatial probe trial period (Figure 6B-e) formed a more clear and
definite spatial preference in the correct quadrant of the platform. These results further
revealed that administration of 6d led to a substantial improvement of the
conventional reference spatial memory and cognitive abilities.
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2.2.14. Cognitive and memory improvement test of Dꢀgal and AlCl ꢀinduced chronic
cognition impairment
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Reports has demonstrated that the combination of Dꢀgalactose (Dꢀgal) and AlCl
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eventually damages learning and memory as well as increased production of ROS.
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Therefore, mice continuously treated with Dꢀgal and AlCl
3
might be a better model
for studying the mechanisms of AD and for drug screening.
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To investigate the effects of 6d on Dꢀgal and AlCl induced cognitive impairment,
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trolox and 6d were orally administered to the ICR mice for 40 consecutive days, and
th
from the 15 day, Dꢀgal (150mg/kg/d) and AlCl
3
(10mg/kg/d) were intraperitoneal
injectioned after 6 h giving the compounds. A spatial memory test was subsequently
performed as previously described by passive avoidance task. The stepꢀthrough
latency of mice treated with Dꢀgal and AlCl alone (model group) was significantly
3
shorter than that of controlꢀtreated mice. As reported in Figure 7A, the treatment with
6d (20mg/kg) significantly increased the latency time compared with model group.
The effect of 6d was slightly superior to trolox (20mg/kg). In particular, it did not
cause any adverse or abnormal events (such as emesisꢀlike or diarrhea behavior) or
affect the survival. The results were also proved by intuitional histopathological
studies for hippocampal neurons as showed in Figure 7C. The control group
demonstrated significant neuronal normalities, while the model’s presented with
nuclear pyknosis, neuronal shrinkage and an irregular shape, which indicated a
necrotic morphology. In the trolox and 6d group, fewer significant neuronal
abnormalities were detected in the hippocampus. These results indicated that 6d can
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reverse and protect cognitive deficit of Dꢀgal and AlCl ꢀinduced chronic cognition
impairment to some extent.
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2.2.15 Estimation of biochemical parameters
To further verify 6d’s effects on this chronic oxidative stress model, we
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determined the biochemical parameters related to oxidative damages.
After the
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behavior assessments, the mice were sacrificed, and the brain were collected rapidly,
rinsed with cold phosphateꢀbuffered saline (PBS). The biochemical parameters in
different groups were shown in Figure 7B. The levels of malondialdehyde (MDA),
the oxidative stress markers, exhibited a significant increase in the model group
compared with the control group. 6d treatment significantly decreased the enhanced
levels of MDA in model mice (Figure 7B-a). The model group had a notable
reduction in the quantity of superoxide dismutase (SOD) (Figure 7B-b), the activities
of glutathione peroxidase (GSHꢀPX) (Figure 7B-c) were significantly inhibited in
model groups compared with the normal groups, which were greatly reversed by
compound 6d.
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The above impressive profile maybe attribute to the facts that 6d increased brain
cholinergic activity by inhibition of AChE and protected the cholinergic nerve owing
to its significant neuroprotective effects.
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3. Conclusion
This study involved the design, synthesis and biological evaluation of a series of
multifunctional agents against AD by fusing the pharmacophore of donepezil and
trolox. Among all the compounds, compound 6d behaved balanced functions with
excellent hAChE inhibition and MAOꢀB inhibition, efficient antioxidant capacity by
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DPPH, ABTS and ORAC assays, significant copper chelating properties and effective
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+
inhibitory activity against selfꢀ and Cu ꢀinduced Aβ1−42 aggregation. Furthermore,
cellular tests indicated that 6d was very low toxic on three cells models (HepG2,
PC12 and BVꢀ2) and capable of combating oxidative toxins (H O , rotenone and
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oligomycinꢀA) induced neurotoxicity on PC12 cells. Besides, 6d presented a
significant effect on protecting neuronal cells against LPSꢀstimulated inflammation on
BVꢀ2 cells. Most importantly, oral administration of 6d demonstrated notable
improvements on cognition and spatial memory against scopolamineꢀinduced acute
memory deficit as well as Dꢀgalactose (Dꢀgal) and AlCl induced chronic oxidative
3
stress models in mice without acute toxicity and hepatotoxicity. A hypothetical
scheme for the pharmacologic mechanisms of 6d in the treatment of Alzheimer’s
disease mice model was summarized in Figure S5 (Supporting Information) in
which 6d played good profile through its good cholinergic and noncholinergic
pathways. Altogether, both the results in vitro and in vivo suggested that 6d was a
valuable candidate for the development of safe and effective antiꢀAlzheimer’s drug.
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4. Methods
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4.1 Chemistry
4.1.1 General methods
4
All common reagents and solvents were obtained from commercial suppliers and
used without further purification. Reaction progress was monitored using analytical
thin layer chromatography (TLC) on precoated silica gel GF254 plates (Qingdao
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Haiyang Chemical Plant, Qingdao, China) and detected under UV light (254 nm).
Column chromatography was performed on silica gel (90–150 mm; Qingdao Marine
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Chemical Inc.). H NMR and C NMR spectra were measured with a Bruker
ACFꢀ500 spectrometer at 25 °C and referenced to TMS. Chemical shifs are reported
in ppm (δ) using the residual solvent line as the internal standard. Mass spectra were
obtained with an Agilent 1100 Series LC/MSD Trap mass spectrometer (ESIꢀMS) and
a Mariner ESIꢀTOF spectrometer (HRESIꢀMS), respectively.
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4.1.2 General procedures for the preparation of 5aꢀm and 6aꢀm
The commercially available starting material 1 and 2 (1.0 mmol) was respectively
suspended in ethyl alcohol (10 mL) containing triethylamine (TEA) (2.0 mmol). The
reaction was treated with appropriately substituted benzyl bromides (1.2 mmol) and
heated under reflux for 8 h to give the key intermediate 1a-m and 2a-m, then the
product was treated with trifluoroaceticacid (TFA) (4eq) to give 1ꢀbenzylꢀsubstituted
4ꢀaminopiperidine 3a-m and 1ꢀbenzylꢀsubstituted 4ꢀaminoethylpiperidine 4a-m in
good yields without further purification. Finally, trolox (1eq) were reacted with 3a-m
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and
4a-m
in
dichloromethane
(DCM)
with
Nꢀ(3ꢀ(dimethylamino)propyl)ꢀN′ꢀethylcarbodiimide hydrochloride (EDCI) (1eq) and
1ꢀhydroxybenzotriazole hydrate (HOBt) (1eq) catalytic system at room temperature
for 12h. The reaction mixture was washed with saturated aqueous solution of sodium
bicarbonate and extracted with DCM. The combined organic layers were dried over
5
5
anhydrous Na
2 4
SO and evaporated under vacuum. Purification of the crude product
5
was achieved by column chromatography. Structures of all targeted compounds were
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characterized by H NMR, C NMR, ESIꢀMS and HRMS.
6ꢀhydroxyꢀNꢀ(1ꢀ(4ꢀmethoxybenzyl)piperidinꢀ4ꢀyl)ꢀ2,5,7,8ꢀtetramethylchromaneꢀ2
ꢀcarboxamide (5a)
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Yield 37%, white oil. H NMR (500 MHz, DMSOꢀd ) δ 7.56 (s, 1H), 7.20 – 7.10
6
(m, 4H), 6.83 (d, J = 7.5 Hz, 1H), 3.72 (s, 3H), 3.65 – 3.62 (m, 1H), 3.28 (dd, J = 11.8,
5.1 Hz, 2H), 2.58 – 2.53 (m, 1H), 2.44 (d, J = 7.0 Hz, 2H), 2.22 (dt, J = 11.8, 8.1 Hz,
2H), 2.13 (s, 3H), 2.12 (s, 3H), 2.01 (s, 3H), 1.78 – 1.65 (m, 2H), 1.47 – 1.42(m, 2H),
13
1.41 (s, 3H), 1.23ꢀ1.20 (m, 3H). C NMR (151 MHz, DMSOꢀd
6
) δ 173.01, 146.49,
144.28, 130.46, 129.90, 127.41, 125.80, 123.16, 121.29, 120.87, 117.72, 77.74, 60.61,
55.60, 31.22, 30.00, 24.62, 20.63, 19.22, 13.38, 12.47, 12.27. ESIꢀMS m/z: 453.3
+
+
527
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30
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32
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537
38
[M+H] ; HRMS (ESI) m/z 453.2750 [M+H] (calcd for 453. 2753, C27
6ꢀhydroxyꢀ2,5,7,8ꢀtetramethylꢀNꢀ(1ꢀ(2ꢀmethylbenzyl)piperidinꢀ4ꢀyl)chromaneꢀ2ꢀc
arboxamide (5b)
37 2 4
H N O ).
5
1
5
Yield 30%, white oil. H NMR (500 MHz, DMSOꢀd ) δ 7.56 (s, 1H), 7.30 – 7.12
6
(m, 4H), 6.80 (d, J = 7.5 Hz, 1H), 3.58 – 3.55 (m, 1H), 3.25 (dd, J = 12.4, 5.1 Hz, 2H),
2.58 – 2.54 (m, 1H), 2.44 (d, J = 7.0 Hz, 2H), 2.30 (s, 3H), 2.22 (dt, J = 11.8, 8.1 Hz,
5
2H), 2.13 (s, 3H), 2.12 (s, 3H), 2.01 (s, 3H), 1.78 – 1.75 (m, 2H), 1.49 – 1.44 (m, 2H),
13
5
1.41 (s, 3H), 1.25 – 1.21 (m, 3H). C NMR (151 MHz, DMSOꢀd
6
) δ 173.01, 146.49,
144.28, 130.46, 129.90, 127.41, 125.80, 123.16, 121.29, 120.87, 117.72, 77.74, 60.61,
+
5
31.22, 30.00, 24.62, 20.63, 19.22, 13.38, 12.47, 12.27. ESIꢀMS m/z: 437.3 [M+H] ;
+
HRMS (ESI) m/z 437.2797 [M+H] (calcd for 437.2799, C27
H
37
N
2
O
3
).
5
6ꢀhydroxyꢀ2,5,7,8ꢀtetramethylꢀNꢀ(1ꢀ(4ꢀmethylbenzyl)piperidinꢀ4ꢀyl)chromaneꢀ2ꢀc
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arboxamide (5c)
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Yield 43%, pale yellow oil. H NMR (500 MHz, DMSOꢀd
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) δ 7.54 (s, 1H), 7.25
– 7.11 (m, 4H), 6.81 (d, J = 7.5 Hz, 1H), 3.65 – 3.62 (m, 1H), 3.26 (dd, J = 12.1, 4.1
Hz, 2H), 2.58 – 2.56 (m, 1H), 2.45 (d, J = 7.0 Hz, 2H), 2.30 (s, 3H), 2.25 – 2.21(m,
2H), 2.13 (s, 3H), 2.12 (s, 3H), 2.01 (s, 3H), 1.76 – 1.75 (m, 2H), 1.45 – 1.40 (m, 2H),
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1.41 (s, 3H), 1.33 – 1.23 (m, 3H). C NMR (151 MHz, DMSOꢀd ) δ 173.01, 146.49,
144.28, 130.46, 129.90, 127.41, 125.80, 123.16, 121.29, 120.87, 117.72, 77.74, 60.61,
+
31.22, 30.00, 24.62, 20.63, 19.22,13.38, 12.47, 12.27. ESIꢀMS m/z: 437.3 [M+H] ;
+
HRMS (ESI) m/z 437.2796 [M+H] (calcd for 437.2799, C H N O ).
27 37
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Nꢀ(1ꢀ(2ꢀfluorobenzyl)piperidinꢀ4ꢀyl)ꢀ6ꢀhydroxyꢀ2,5,7,8ꢀtetramethylchromaneꢀ2ꢀc
arboxamide (5d)
6
Yield 47%, pale yellow oil. H NMR (500 MHz, DMSOꢀd ) δ 7.58 (s, 1H), 7.34 –
549
50
551
52
553
54
555
56
557
58
559
60
1
5
7.29 (m, 3H), 7.23 (d, J = 7.3 Hz, 1H), 6.83 (d, J = 7.0 Hz, 1H), 3.61 – 3.55 (m, 1H),
3.31 (d, J = 14.1 Hz, 1H), 3.22 – 3.20(m, 1H), 2.57 (d, J = 5.9 Hz, 1H), 2.46 – 2.43
(m, 2H), 2.21 (dt, J = 14.1, 7.4 Hz, 2H), 2.13 (s, 3H), 2.12 (s, 3H), 2.01 (s, 3H), 1.78 –
5
13
5
1.76 (m, 2H), 1.49 (dd, J = 10.6, 7.3 Hz, 2H), 1.41 (s, 3H), 1.27 – 1.25 (m, 3H). C
6
NMR (151 MHz, DMSOꢀd ) δ 161.87 – 161.74, 146.45, 144.22, 142.30, 130.51,
5
125.19, 123.28, 121.40, 120.90, 117.73, 115.38, 114.00 – 113.86, 77.74, 61.81, 31.29,
+
29.95, 24.52, 20.50, 13.32, 12.46, 12.26. ESIꢀMS m/z: 441.3 [M+H] ; HRMS (ESI)
+
5
m/z 441.2550 [M+H] (calcd for 441.2548, C26
H34FN
2
O
3
).
Nꢀ(1ꢀ(3ꢀfluorobenzyl)piperidinꢀ4ꢀyl)ꢀ6ꢀhydroxyꢀ2,5,7,8ꢀtetramethylchromaneꢀ2ꢀc
arboxamide (5e)
5
2
7
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Yield 47%, white oil. H NMR (500 MHz, DMSOꢀd ) δ 7.58 (s, 1H), 7.34 – 7.30
(m, 3H), 7.23 (d, J = 7.3 Hz, 1H), 6.83 (d, J = 7.0 Hz, 1H), 3.61 – 3.58 (m, 1H), 3.32
(d, J = 12.1 Hz, 1H), 3.24 – 3.21 (m, 1H), 2.59 (d, J = 7.4 Hz, 1H), 2.45 – 2.43 (m,
2H), 2.20 (dt, J = 11.9, 7.4 Hz, 2H), 2.13 (s, 3H), 2.12 (s, 3H), 2.01 (s, 3H), 1.72 –
0
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1.69 (m, 2H), 1.48 – 1.47 (m, 2H), 1.41 (s, 3H), 1.28 – 1.25 (m, 3H). C NMR (151
MHz, DMSOꢀd ) δ 161.87, 146.45, 144.22, 142.30, 130.51, 125.19, 123.28, 121.40,
120.90, 117.73, 115.38, 114.00 – 113.86, 77.74, 61.81, 31.29, 29.95, 24.52, 20.50,
6
+
+
13.32, 12.46, 12.26. ESIꢀMS m/z: 441.3 [M+H] ; HRMS (ESI) m/z 441.2550 [M+H]
(calcd for 441.2548, C H FN O ).
26 34
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Nꢀ(1ꢀ(4ꢀfluorobenzyl)piperidinꢀ4ꢀyl)ꢀ6ꢀhydroxyꢀ2,5,7,8ꢀtetramethylchromaneꢀ2ꢀc
arboxamide (5f)
Yield 35%, pale yellow oil. H NMR (500 MHz, DMSOꢀd
571
72
573
74
575
76
577
78
579
80
581
1
5
6
) δ 7.58 (s, 1H), 7.34
– 7.28 (m, 4H), 6.83 (d, J = 7.0 Hz, 1H), 3.58 – 3.55 (m, 1H), 3.29 – 3.26 (m, 2H),
2.57 (d, J = 7.9 Hz, 1H), 2.46 – 2.42 (m, 2H), 2.23 (dt, J = 14.2, 7.9 Hz, 2H), 2.13 (s,
5
3H), 2.12 (s, 3H), 2.01 (s, 3H), 1.74 – 1.72 (m, 2H), 1.47 – 1.45 (m, 2H), 1.41 (s, 3H),
13
5
1.26 – 1.25 (m, 3H). C NMR (151 MHz, DMSOꢀd
6
) δ 171.87, 146.45, 144.22,
142.30, 130.51, 125.19, 123.28, 121.40, 120.90, 117.73, 115.38, 113.86, 77.74, 61.81,
+
5
31.29, 29.95, 24.52, 20.50, 13.32, 12.46, 12.26. ESIꢀMS m/z: 441.3 [M+H] ; HRMS
+
(ESI) m/z 441.2545 [M+H] (calcd for 441.2548, C H FN O ).
26
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Nꢀ(1ꢀ(2,4ꢀdifluorobenzyl)piperidinꢀ4ꢀyl)ꢀ6ꢀhydroxyꢀ2,5,7,8ꢀtetramethylchromaneꢀ
2ꢀcarboxamide (5g)
2
8
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Yield 31%, white oil. H NMR (500 MHz, DMSOꢀd ) δ 7.58 (s, 1H), 7.37 – 7.35
(m, 2H), 7.23 (d, J = 7.3 Hz, 1H), 6.83 (d, J = 7.0 Hz, 1H), 3.60 – 3.57 (m, 1H),3.27 –
3.25 (m, 2H), 2.57 (d, J = 5.9 Hz, 1H), 2.46 – 2.36 (m, 2H), 2.24 – 2.22 (m, 2H), 2.13
(s, 3H), 2.12 (s, 3H), 2.01 (s, 3H), 1.95 – 1.93 (m,1H), 1.78 – 1.75 (m, 2H), 1.49 (dd,
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13
J = 13.1, 6.3 Hz, 2H), 1.41 (s, 3H), 1.25 – 1.21 (m, 3H). C NMR (151 MHz,
DMSOꢀd ) δ 171.87, 146.45, 144.22, 142.30, 130.51, 125.19, 123.28, 121.40, 120.90,
117.73, 115.38, 113.86, 77.74, 61.81, 31.29, 29.95, 24.52, 20.50, 13.32, 12.46, 12.26.
6
+
+
ESIꢀMS m/z: 459.3 [M+H] ; HRMS (ESI) m/z 459.2457 [M+H] (calcd for 459.2459,
C H F N O ).
26 33
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Nꢀ(1ꢀ(3,4ꢀdifluorobenzyl)piperidinꢀ4ꢀyl)ꢀ6ꢀhydroxyꢀ2,5,7,8ꢀtetramethylchromaneꢀ
592
93
594
95
596
97
598
99
600
01
602
03
2ꢀcarboxamide (5h)
1
5
Yield 40%, white oil. H NMR (500 MHz, DMSOꢀd
6
) δ 7.58 (s, 1H), 7.36 (m,
2H), 7.23 (d, J = 7.3 Hz, 1H), 6.83 (d, J = 7.2 Hz, 1H), 3.58 – 3.56 (m, 1H), 3.29 –
3.27 (m, 2H), 2.57 (d, J = 5.9 Hz, 1H), 2.46 – 2.36 (m, 2H), 2.21 (dt, J = 11.9, 7.4 Hz,
2H), 2.13 (s, 3H), 2.12 (s, 3H), 2.01 (s, 3H), 1.96 –1.93(m, 1H), 1.78 – 1.75 (m, 2H),
5
13
5
1.49 (dd, J = 10.6, 7.3 Hz, 2H), 1.41 (s, 3H), 1.30 – 1.27 (m, 3H). C NMR (151
MHz, DMSOꢀd
6
) δ 171.87, 146.45, 144.22, 142.30, 130.51, 125.19, 123.28, 121.40,
5
120.90, 117.73, 115.38, 113.86, 77.74, 61.81, 31.29, 29.95, 24.52, 20.50, 13.32, 12.46,
+
+
12.26. ESIꢀMS m/z: 459.3 [M+H] ; HRMS (ESI) m/z 459.2456 [M+H] (calcd for
459.2459, C26 ).
Nꢀ(1ꢀ(2ꢀchlorobenzyl)piperidinꢀ4ꢀyl)ꢀ6ꢀhydroxyꢀ2,5,7,8ꢀtetramethylchromaneꢀ2ꢀc
arboxamide (5i)
6
33 2 2 3
H F N O
6
2
9
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