Synthesis and evaluation of multi-target-directed ligands for the treatment of Alzheimer's disease based on the fusion of donepezil and melatonin

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Synthesis and evaluation of multi-target-directed ligands for the

treatment of Alzheimer’s disease based on the fusion of donepezil

and melatonin

⇑

Jin Wang, Zhi-Min Wang, Xue-Mei Li, Fan Li, Jia-Jia Wu, Ling-Yi Kong , Xiao-Bing Wang

State Key Laboratory of Natural Medicines, Department of Natural Medicinal Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, People’s Republic

of China

a r t i c l e i n f o

a b s t r a c t

Article history:

A novel series of compounds obtained by fusing the acetylcholinesterase (AChE) inhibitor donepezil and

the antioxidant melatonin were designed as multi-target-directed ligands for the treatment of

Alzheimer’s disease (AD). In vitro assay indicated that most of the target compounds exhibited a signif-

icant ability to inhibit acetylcholinesterase (eeAChE and hAChE), butyrylcholinesterase (eqBuChE and

hBuChE), and b-amyloid (Ab) aggregation, and to act as potential antioxidants and biometal chelators.

Especially, 4u displayed a good inhibition of AChE (IC₅₀value of 193 nM for eeAChE and 273 nM for

hAChE), strong inhibition of BuChE (IC₅₀value of 73 nM for eqBuChE and 56 nM for hBuChE), moderate

Received 20 April 2016

Revised 26 June 2016

Accepted 13 July 2016

Available online xxxx

Keywords:

Alzheimer’s disease

Donepezil

inhibition of Ab aggregation (56.3% at 20 lM) and good antioxidant activity (3.28 trolox equivalent by

ORAC assay). Molecular modeling studies in combination with kinetic analysis revealed that 4u was a

mixed-type inhibitor, binding simultaneously to catalytic anionic site (CAS) and the peripheral anionic

site (PAS) of AChE. In addition, 4u could chelate metal ions, reduce PC12 cells death induced by oxidative

stress and penetrate the blood–brain barrier (BBB). Taken together, these results strongly indicated the

hybridization approach is an efﬁcient strategy to identify novel scaffolds with desired bioactivities, and

further optimization of 4u may be helpful to develop more potent lead compound for AD treatment.

Melatonin

Antioxidant activity

Cholinesterase

Biometal chelators

1. Introduction

Alzheimer’s disease, and the primary therapeutic options currently

approved by the U.S. Food and Drug Administration for the treat-

Alzheimer’s disease (AD), the most common form of dementia

among the elderly, is a dreadful neurological illness characterized

by loss of brain function, affecting memory, cognition, and behav-

ior.¹Although the etiology of AD remains elusive, several condi-

tions, such as b-amyloid (Ab) aggregates, soluble Ab oligomers

(AbOs), oxidative stress, dyshomeostasis of biometals, and levels

of acetylcholine, and neuroinﬂammation, likely play signiﬁcant

roles in the pathogenesis of AD.^2,3Many different approaches to

the treatment of AD have been developed over the past several

decades.^4–9At present, there is no drug that can deﬁnitively cure

ment of AD are acetylcholinesterase inhibitors (AChEIs), namely,

tacrine, donepezil, rivastigmine, and galantamine.^10–12Among

them, donepezil is the most effective pharmacological agent for

AD treatment. However, it is effective in reversing the symptoms

for only a short period of time.

Current clinical therapy for AD patients is mainly based on the

cholinergic hypothesis. This hypothesis asserts that the decline of

acetylcholine (ACh) level lead to cognitive and memory deﬁcits,

and sustaining or recovering the cholinergic function is supposed

to be clinically beneﬁcial.¹³ACh can be degraded by two types of

cholinesterases in the central nervous system (CNS), namely

acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE).

The crystallographic structure of AChE reveals that it includes

two separate ligand binding sites: a peripheral cationic site (PAS)

at the entrance and a catalytic active site (CAS) at the bottom.

Hence, inhibitors that bind to either site could inhibit the AChE.¹⁴

In addition, the assumption that AChE promotes amyloid ﬁbril for-

mation by interaction through the PAS of AChE has led to the

Abbreviations: AchE, Acetylcholinesterase; BuChE, Butyrylcholinesterase; AD,

Alzheimer’s disease; Ab, b-Amyloid; CAS, Catalytic anionic site; PAS, Peripheral

anionic site; BBB, Blood–brain barrier; AbOs, Soluble Ab oligomers; CNS, Central

nervous system; NFTS, Neuroﬁbrillary tangles; ChEs, Cholinesterases; ChEIs,

Cholinesterase inhibitors; CDI, 1,1⁰-Carbonyldiimidazole; ORAC, Oxygen radical

absorbance capacity; PAMPA, Parallel artiﬁcial membrane permeation assay.

⇑

Corresponding authors.

E-mail addresses: cpu_lykong@126.com (L.-Y. Kong), xbwang@cpu.edu.cn

(X.-B. Wang).

http://dx.doi.org/10.1016/j.bmc.2016.07.025

Please cite this article in press as: Wang, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.025

2

J. Wang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

development of dual binding site inhibitors of both CAS and PAS.¹⁵

For this reason, the dual binding inhibitors may be promising ther-

apeutic strategy.^16,17Furthermore, in healthy brains, AChE is more

active than BuChE and can hydrolyze about 80% of ACh.^18–20As AD

progresses, the ability of BuChE signiﬁcantly increases, and that of

AChE diminishes in the hippocampus and temporal cortex. As a

result, both AChE and BuChE are important targets in the therapy

of AD.²¹

During aging, the endogenous antioxidant protection system

progressively decays and may be further diminished in AD.^22,23

In fact, recent research had demonstrated that oxidative damage

in cellular structures is an event that precedes the appearance of

other pathological hallmarks of AD, namely, senile plaques and

neuroﬁbrillary tangles.^24,25Many evidences proved that antioxi-

dants could attenuate the syndrome of AD, and prevent the pro-

gression of the disease.²⁶Thus, drugs with speciﬁcally

antioxidants activity could be useful for either the prevention or

the treatment of AD.²⁷

Continuing with our interest in the design, synthesis, and bio-

logical evaluation of multifunctional molecules, our work is cur-

rently focused on taking advantage of the potential neurogenic

proﬁle of melatonin-based hybrids, which are endowed with addi-

tional anticholinergic properties. Thus, in this work, we have

designed new melatonin–donepezil hybrids (4a–u) by binding

two fragments with interesting and complementary properties.

The melatonin framework, in addition to its above-mentioned neu-

rogenic proﬁle, could demonstrate antioxidant and neuroprotec-

tive features and could also interact with the AChE-PAS via

p–p

aromatic stacking for its aromatic character, as we observed in

the tacrine–melatonin series. The second selected fragment, the

protonable N-benzyl piperidine which is present in the well-

known AChE inhibitor donepezil can interact with AChE-CAS

through cation–p interaction. The last feature of the designed com-

pounds is the linker between the groups interact with PAS and CAS

site (Fig. 1).

In this study, a series of new multifunction compounds by fus-

ing donepezil with melatonin were designed and synthesized as

multifunctional anti-AD agents. The pharmacological evaluation

of these new compounds included AChE and BuChE inhibition,

the kinetics of enzyme inhibition, self-induced Ab aggregation,

metal chelation, antioxidant, and neuroprotection.

Converging lines of evidences suggest that accumulation of Ab

peptide aggregation in the brain play an important role in the

pathogenesis and development of AD, as its accumulation may

result in senile plaque, neuroﬁbrillary tangles, neuronal cell death,

and ultimately dementia.^28,29The Ab_1–40and Ab_1–42are the major

components of amyloid plaques found in the brain of AD patients.

2. Results and discussion

2.1. Chemistry

However, Ab_1–42is more prone to self-assembly into ﬁbrils and

7,30

more neuronal toxicity compared to relatively soluble Ab_1–40

.

Therefore, the prevention of Ab_1–42aggregation currently attracts

much attention.

The synthetic pathways of the target and intermediate com-

pounds were summarized in Scheme 1. The synthesis of target

and intermediate compounds applied readily available starting

material and was completed using well-established methods.

Reﬂuxing 4-piperidyl esters with benzyl bromide derivations

(1a–j) in the presences of MeCN, K₂CO₃under nitrogen for 8 h at

85 °C gave the respective intermediates 2a–j.^10,51Intermediates

2a–j were then hydrolyzed to provide the acids 3a–j.

Recent studies indicated that another hypothesis, called metal

hypothesis, also contribute to AD pathology.³¹It is observed that

abnormally high levels of metal ion (Fe, Zn, Cu) in the brain is clo-

sely to associated with the formation of Ab aggregates and neu-

roﬁbrillary tangles (NFTs), which the promoted inﬂammation of

Ab and activate neurotoxic pathways, leading to dysfunction and

the death of brain cell.^31,32,26,33Therefore, lowering of concentra-

tion of brain metals by chelating metals has been proposed as a

rational therapeutic approach for halting AD pathogenesis.

Donepezil (1-benzyl-4-[5,6-dimethoxy-(1-indanone)-2-yl]methyl

piperidine, 1) also known as E2020, was found to be highly selec-

tive AChE inhibitor. Donepezil was approved by FDA in 1996 for

the treatment of mild to moderate AD.^34,35Donepezil is recognized

by AChE active site by interactions with benzyl moiety (CAS of

AChE), the atom of the piperidine (mid-gorge) and dimethoxyin-

danone moiety (PAS of AChE).³⁶Melatonin (N-acetyl-5-methoxyl-

tryptamine, 2) is an endogenous neurohormone whose level

decreases during aging, especially in AD patients.^{6,7,18,20,37,38}It

had been reported to possess strong antioxidant property and is

able to directly scavenge a variety of reactive oxygen species.³⁹

Moreover, it has been demonstrated to be a moderate inhibitor

of Ab aggregation by disrupting Asp^ꢀ-His⁺salt bridges of Ab or

affecting the synthesis and maturation of amyloid precursor

protein (APP).⁴⁰

Due to the pathological complexity found in AD, multifunc-

tional molecules with two or more complementary biological

activities may represent an important advance for the treatment

of this disease.^41–44In recent years, many interesting MTLs have

been developed, such as tacrine–melatonin hybrid, melatonin–N,

N-dibenzyl(N-methyl)amine hybrid, and curcumin-melatonin

hybrid, among others.^45–47Tacrine–melatonin hybrid is an AChE

inhibitor with additional antioxidant and Ab anti-aggregating

properties.^48–50Melatonin–N,N-dibenzyl(N-methyl)amine hybrid

is able to reduce amyloid burden and behavioral deﬁcits in a mouse

model of AD.¹⁰Curcumin–melatonin hybrid show signiﬁcant neu-

roprotection in MC65 cells. Our group has also reported several

families of MTLs that combined neuroprotective, cholinergic, and

antioxidant properties.

Finally, activation of 3a–j with 1,1⁰-carbonyldiimidazole (CDI)

and subsequent coupling with tryptamine analogues afforded tar-

gets compounds 4a–s. On the other hand, activation of 3-

Indolepropionic acid (3) with 1,1⁰-carbonyldiimidazole (CDI) and

subsequent coupling to 3t–u provided target compound 4t–u.²⁷

2.2. Cholinesterase inhibitory activity

The inhibitory activities of hybrids 4a–u against AChE (from

electric eel) and BuChE (from equine serum) were measured

according to the spectrophotometric method of Ellman et al.⁵²

For comparison purpose, tacrine and donepezil were used as refer-

ence compounds. The IC₅₀values of all tested compounds were

summarized in Table 1. As illustrated in Table 1, it could be seen

that all new target compounds showed good inhibitory activities

to both ChEs with IC₅₀values ranging from sub-micromolar to

nanomolar. All new target compounds displayed little ﬂuctuation

after the introduction of different substituents in the benzene or

indole ring. Most of compounds exhibited higher inhibitory activ-

ity for BuChE than for AChE, indicating that these compounds were

selective inhibitors for BuChE. According to the above results, sub-

stituents with varying electronic properties were introduced to the

benzene ring. Compared with 4a, 4h, 4j and 4l bearing a chlorine

group exhibited a decrease in AChE inhibition. However, the com-

pounds 4b, 4d and 4f with a methyl group was a slight increase in

AChE inhibition. The compound 4b bearing a methyl group at the

2-position of the phenyl group is more active than the compound

4d with a methyl group at the 3-position of the phenyl group

and the compound 4f with a methyl group at the 4-position of

the phenyl group. These results implied that electron-donating

Please cite this article in press as: Wang, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.025

J. Wang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

3

Figure 1. Structure of melatonin, donepezil and donepezil–melatonin hybrids.

groups at the 2-position of the phenyl group seemed beneﬁcial to

AChE inhibitory activity.

BuChE (h-BuChE) inhibition were listed in Table 2. From the

results, it can be seen that all compounds are also potent inhibitors

of human ChEs. For BuChE inhibition, most of these compounds

showed improved inhibitory activity for hBuChE compared to

eqBuChE. Compound 4u displayed the highest inhibitory activity

for hBuChE with the IC₅₀value of 56.3 nM. However, no signiﬁcant

change between hAChE and eeAChE was observed. Although com-

pound 4u exhibited the inhibitory activity for hAChE was slightly

lower than for eeAChE (193 nM vs 273 nM), it was still the most

potent inhibitor of hAChE. Such an activity change made 4u more

balanced for both ChE inhibition (IC₅₀= 273 nM for hAChE;

IC₅₀= 56 nM for hBuChE), which would be more beneﬁcial for AD

treatment. Based on the above assay results, we then selected

the most potent AChE and BuChE inhibitor, 4u, for kinetic analysis

to investigate the type of inhibitor.

To extend the series of the hybrids, compounds 4n–s with N-

benzyl piperidine–indole moiety and 4t–u were synthesized. As

the trend with the compounds 4b–c, 4h–i for AChE inhibition,

the compounds 4p–s were also sensitive to substituents at 2-posi-

tion of phenyl ring. Interestingly, compared with the compounds

4n–o, the compounds 4p–q with the methyl group at the 2-posi-

tion of the phenyl group increased remarkably in AChE inhibitory

activities. However, compared with the compounds 4n–o, the

compounds 4r–s with the nitro group at the 2-position of the phe-

nyl group decreased strongly in AChE inhibitory activities. This

suggested the electron-donating group at the 2-position of the

phenyl group seemed favorable for AChE inhibitory activity. How-

ever, these results implied that electron-drawing groups at the 2-

position of the phenyl group seemed unbeneﬁcial to AChE inhibi-

tory activity.

2.3. Kinetic study of ChE inhibition

The designed compounds are composed of two fragments: a

CAS binding motif (N-benzyl piperidine) and a PAS binding motif

(indole) which are joined either via a carboxamide (compounds

4n, 4p, 4q) or ethyl acid carboxamide (4a, 4b, 4c) linkers. It was

observed that the compounds 4a, 4b and 4c are more active than

their counterparts 4n, 4p and 4q respectively. In hybrid 4t–4u,

n = 0, 2 is taken. Compound 4u with a two-carbon spacer exhibited

the most potent AChE inhibitory activity, which was stronger than

the compound 4t with a zero-carbon spacer. It is possible to con-

clude that the length linker is effect to AChE inhibitory activity.

In general, modiﬁcations indole ring introducing methoxy group

showed a higher inhibitory potency than compound with indole

ring without substituted group for AChE. The effect of linker length

and substituted group on BuChE inhibitory activity revealed a

trend similar from those observed for the AChE inhibitory activity.

Subsequently, a selection of the compounds (4a–g, 4p–q and

4t–u) were then evaluated as inhibitors of human ChEs (h-ChEs).

The IC₅₀values of test compounds for human AChE (h-AChE) and

To gain further insight into the ChE inhibitory mechanism for

this class of melatonin–donepezil hybrids, the most potent com-

pound 4u was selected for kinetic study. Graphical analysis

revealed both increasing slopes (decreased V_max) and increasing

intercepts (increased K_max) at increasing concentration of com-

pound 4u (Fig. 2). This pattern indicated 4u was a mixed-type inhi-

bitor to AChE and therefore revealed that compound 4u might be

able to bind to the CAS and the PAS of AChE. In contrast, a different

plot for BuChE was obtained, showing different K_mand constant

V_maxin different inhibitor concentrations (Fig. 3). This suggested

a competitive inhibition, revealing that these compounds compete

for the same binding site (CAS) as the substrate acetylcholine.

2.4. Metal-chelating properties of compound 4u

The ability of 4u to chelate biometals such as Cu(II), Fe(II), Fe(III),

and Zn(II) was studied by UV–vis spectroscopy (Fig. 4).^53–56,20

Please cite this article in press as: Wang, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.025

4

J. Wang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

Scheme 1. Synthesis of donepezil–melatonin hybrids. Reagents and conditions: (a) MeCN, K₂CO₃, N₂, reﬂux, overnight; (b) NaOH, 65 °C, 3 h; (c) 1,1⁰-carbonyldiimidazone

(CDI), rt, overnight.

Upon the addition of CuSO₄the maximum absorption at 212 nm

suffered a bathochromic shift at 219 nm, pointing out the formation

of complex 4u–Cu(II). To establish the time required for the

complexation process, the spectrum of the mixture 4u plus CuSO₄

was recorded at different times, observing that the curve did not

change after the ﬁrst minute and the absorbance dropped dramat-

ically, indicating the formation of a 4u–Cu(II) complex (Fig. 4A).

When the same experiment was performed using compound 4u

and FeSO₄, FeCl₃, or ZnCl₂, the maximum absorption at 212 nm

shifted to 216, 216, or 214 nm, respectively, suggesting that 4u

assay.^6,7,54,57Curcumin and resveratrol, a known active natural

product for the inhibition of Ab_1–42self-aggregation, were used

as reference compounds and the results were gathered in Table 1

and showed in Figure 5. It was necessary to mention that most

hybrids exhibited moderate-to-good potencies (32.8–65.1% at

20

l

M) compared to that of curcumin (51.5% at 20

lM) and resver-

atrol (54.2% at 20

lM). The result indicated that 4j was the most

potent inhibitor of Ab_1–42among the series of compounds. From

the inhibition values of compounds 4t–u, it appeared that the

linker length did not play a role in determining the inhibition of

Ab_1–42self-aggregation, since no signiﬁcant change of the percent-

ages of inhibition was observed along with the lengthening the

spacer length. In contrast, compounds 4c–g, 4p–q showed the per-

centages of inhibition ranging from 32.8% to 42.1%. These results

indicated that an electron-donating group at benzene ring might

not be favorable for inhibition of Ab_1–42self-aggregation. Com-

pounds featuring chloride group on the benzene ring, exhibited

good inhibitory activities (4h–m, 4r, 4s, from 52.8% to 65.1%).

These results implied that an electron-drawing group at the ben-

zene ring seemed to be beneﬁcial for Ab_1–42self-aggregation inhi-

bitory activity.

coordinate Fe²⁺, Fe³⁺, and Zn²⁺

.

To determine the stoichiometry of the complex 4u–Cu(II), the

technique of continuous variations (also called Job’s method) was

used by preparing solutions of compound 4u and CuSO₄so that

the sum of concentrations of both species was constant in all

samples, but the proportions of both components varied between

0% and 100%.⁵⁴The UV spectra were recorded and the absorbance

change at 219 nm was plotted versus the mole fraction of com-

pound 4u, giving two straight lines whose equations were calcu-

lated. The equations gave a solution at a mole fraction of 0.50 for

compound 4u, revealing a 1:1 stoichiometry for complex 4u–Cu

(II) (Fig. 4B).

2.6. In vitro antioxidant activity assays

2.5. Inhibition of Ab_1–42self-induced aggregation

Some compounds were tested for their antioxidant activities by

using the oxygen radical absorbance capacity (ORAC) method

(Table 1) following a well-established protocol.⁵⁸Trolox, the

All compounds were tested for their ability to inhibit Ab_1–42

self-induced aggregation by using a thioﬂavin-T based ﬂuorometric

Please cite this article in press as: Wang, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.025

J. Wang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

5

Table 1

Inhibition of eeAChE, eqBuChE, self-induced Ab_1–42aggregation activities and oxygen radical absorbance capacity by ﬂuorescence (ORAC-FL) of hybrids 4a–u

a

Compounds

IC₅₀

(

lM)

Selectivity index^b

Ab_(1–42)aggregation

ORAC-FL^d

AChE

BuChE

Inhibition^c(%)

4a

4b

4c

4d

4e

4f

4g

4h

4i

4j

4k

4l

4m

4n

4o

4p

2.10 0.10

0.94 0.05

2.20 0.12

1.63 0.01

9.50 0.52

1.94 0.13

5.26 0.32

28.2 1.23

16.8 1.06

1.63 0.01

9.50 0.52

22.4 1.35

20.6 1.63

5.55 0.30

9.27 0.62

2.25 0.10

4.42 0.32

31.5 1.32

54.2 1.43

5.75 0.23

0.193 0.02

0.04 0.003

0.23 0.05

n.t.^e

1.10 0.06

1.49 0.08

1.28 0.14

0.95 0.04

4.40 0.34

1.29 0.08

1.48 0.10

1.49 0.08

1.28 0.14

0.95 0.04

4.40 0.34

5.29 0.58

6.48 0.60

3.30 0.20

8.21 0.40

1.50 0.20

3.21 0.20

10.8 1.24

28.5 1.72

0.37 0.02

0.073 0.01

3.36 0.05

0.05 0.003

n.t.

0.52

1.59

0.58

0.46

0.66

0.28

0.05

0.08

0.58

0.46

0.24

0.31

0.6

0.89

0.67

0.73

0.34

0.53

0.06

0.37

84

55.2 1.19

42.1 1.23

40.5 1.13

38.1 1.20

40.2 1.15

42.1 1.53

40.5 1.13

62.1 1.53

60.5 1.28

65.1 1.62

60.5 1.28

63.5 1.53

60.5 1.20

50.6 1.22

46.8 1.16

36.6 1.02

32.8 1.06

58.6 1.52

52.8 1.26

56.2 1.21

56.3 1.32

n.t.

2.36 0.02

3.32 0.01

3.38 0.02

3.06 0.03

3.10 0.02

3.12 0.04

3.28 0.02

1.35 0.04

1.30 0.02

1.35 0.04

1.30 0.02

1.25 0.01

1.30 0.02

2.22 0.01

2.30 0.03

2.30 0.01

3.94 0.04

0.82 0.01

0.90 + 0.03

3.24 0.02

3.28 0.01

n.t.

4q

4r

4s

4t

4u

Donepezil

Tacrine

Melatonin

Resveratrol

Curcumin

0.22

n.t.

45.2 1.10

43.5 1.18

n.t.

2.30 0.01

n.t.

a

b

c

IC₅₀: 50% inhibitory concentration (means SEM of three experiments).

Selectivity index = IC₅₀(eqBuChE)/IC₅₀(eeAChE).

Inhibition of Ab_1–42self-induced aggregation, the thioﬂavin-T ﬂuorescence method was used, the mean SD of at least three independent experiments and the

M compounds.

mol of tested compound.

measurements were carried out in the presence of 20

l

d

Data are expressed as

n.t. = not tested.

lmol of trolox equivalents/l

e

Table 2

Inhibitory activities of hAChE, hBuChE

3.28 trolox equivalents respectively. All of these selected com-

pounds demonstrated good antioxidant activities ranging from

0.82- to 3.38-fold of the trolox value. The best activity was

observed for compounds bearing methyl group at benzene ring

and methoxy group at indole ring.

Compounds

IC₅₀(l

M)

Selectivity index^d

a

hAChE^b

hBuChE^c

4a

3.80 0.12

0.95 0.06

1.42 0.08

1.03 0.14

0.85 0.04

1.40 0.34

0.29 0.08

1.06 0.10

2.00 0.20

2.21 0.40

0.40 0.02

3.01 0.08

0.12 0.01

0.056 0.01

4.76 0.05

0.03 0.003

0.25

0.92

0.29

0.18

0.1

0.06

0.11

0.25

0.18

0.11

0.41

0.01

0.21

39.7

0.07

4b

4c

4d

4e

4f

4g

4n

4o

4p

4q

4t

4u

Donepezil

Tacrine

1.54 0.08

3.60 0.10

4.63 0.07

13.50 0.72

4.94 0.15

9.26 0.32

7.85 0.20

12.22 0.52

3.56 0.20

7.42 0.25

8.32 0.53

0.273 0.05

0.12 0.0004

0.45 0.05

2.7. Cell toxicity and effect on H₂O₂-induced oxidative cell

damage in PC12 cells

To gain insight into the therapeutic potential of hybrids, cell

viability was assayed using the neuroblastoma cell line PC12.⁵⁹

a

b

c

IC₅₀: 50% inhibitory concentration (means SEM of three experiments).

AChE (EC 3.1.1.7) from human erythrocytes.

BuChE (EC 3.1.1.8) from human serum.

d

Selectivity index = IC₅₀(hBuChE)/IC₅₀(hAChE).

aromatic part of vitamin E and the part that is responsible for rad-

ical capturing, was used as an internal standard to which the unit

value is arbitrarily given, ORAC (trolox) = 1. Thus, results were

expressed as trolox equivalents (micromoles of trolox/micromoles

of tested compound) on a comparative scale that shows if the prod-

ucts are better (ORAC > 1) or worse than trolox (ORAC < 1). Mela-

tonin was used as a positive standard for comparative purposes.

As expected, the ORAC of these compounds is mainly located in

the melatonin-like fragment. Compounds 4b–j had the ability to

scavenge the ORAC radical with 3.32, 3.38, 3.06, 3.10, 3.12,

Figure 2. Kinetic study on the mechanism of AChE inhibition by compound 4u

Overlaid Line weaver–Burk reciprocal plots of AChE initial velocity at increasing

substrate concentration (0.05–0.50 mM) in the absence of inhibitor and in the

presence of 4u are shown. Lines were derived from a weighted least-squares

analysis of the data points.

Please cite this article in press as: Wang, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.025

6

J. Wang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

2.8. In vitro blood–brain barrier permeation study

Because one of the major requirements of anti-AD drugs is to

reach their therapeutic targets, screening of the blood–brain bar-

rier (BBB) penetration is of great importance. BBB permeability

properties of CNS drug candidates should be determined as early

as possible in the drug discovery process. To explore whether the

selected donepezil–melatonin hybrids would be able to penetrate

into the brain, we used a parallel artiﬁcial membrane permeation

assay for blood–brain barrier (PAMPA–BBB). This simple and rapid

model, described by Di et al., has the advantage of predicting pas-

sive BBB permeation with high success.⁶⁰Assay validation was

made by comparing experimental permeabilities of nine commer-

cial drugs with reported values (Table 3). A plot of experimental

data versus bibliographic values gave a good linear correlation,

Pe (exp.) = 0.8437 Pe (Bibl.) ꢀ 0.2587 (R²= 0.9618) (Fig. 8). From

this equation and taking into account the limit established by Di

et al. for blood–brain barrier permeation, we classiﬁed compounds

as follows:

Figure 3. Kinetic study on the mechanism of BuChE inhibition by compound 4u

Overlaid Line weaver–Burk reciprocal plots of BuChE initial velocity at increasing

substrate concentration (0.05–0.50 mM) in the absence of inhibitor and in the

presence of 4u are shown. Lines were derived from a weighted least-squares

analysis of the data points.

(a) ‘CNS+’ (high BBB permeation predicted): Pe (10^ꢀ6cm s^ꢀ1

)

Compounds 4b, 4c, 4p, 4q, 4t and 4u were selected as representa-

tive compounds of different types. First, the colorimetric MTT [3-

(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide]

assay was performed to examine the potential cytotoxic effects

of compounds 4b, 4c, 4p, 4q, 4t and 4u. As indicated in Figure 6,

compounds 4b, 4c, 4p, 4q, 4t and 4u did not show signiﬁcant effect

> 3.54;

(b) ‘CNS’ (low BBB permeation predicted): Pe (10^ꢀ6cm s^ꢀ1

)

< 1.51;

(c) ‘CNS ’ (BBB permeation uncertain): Pe (10^ꢀ6cm s^ꢀ1) from

1.51 to 3.54;

on cell viability at 1–50 lM after incubation for 24 h. This sug-

gested that compounds 4b, 4c, 4p, 4q and 4u were nontoxic to neu-

roblastoma PC12 cells.

Compounds 4b, 4c, 4p, 4q and 4u were tested for their capacity

to protect the rat pheochromocytoma cell line PC12 cells against

oxidative stress-associated death induced by H₂O₂. Trolox and

melatonin were also used as the reference compounds. In this

Finally, six compounds (4a, 4b, 4c, 4p, 4q and 4u) exhibited

good activities against ChE and Ab_1–42aggregation were chosen

as the test compounds. The results summarized in Table 3 indicate

that all of the selected compounds could penetrate the BBB.

2.9. Molecular modeling studies

assay, addition of 200 lM H₂O₂to the growth medium reduced cell

To further study the interaction mode of 4u for AChE, molecular

docking study was performed using software package MOE.⁶¹For

the docking studies, X-ray crystallographic structure of hAChE in

complex with donepezil (PDB ID: 4EY7) was utilized. As shown

in Figure 9A and C, the aromatic ring of indole moiety of 4u binding

viability to 48.7% compared to control. The tested compounds were

added to the media at different concentrations immediately prior

to the H₂O₂insult. As can be seen in Figure 7, all of the compounds

exhibited neuroprotective effects at concentrations ranging from

1.25 to 10

protective capability almost the same as trolox at the concentra-

tion of 10 M and much better than melatonin. Compounds 4p,

4q showed moderate protective capabilities similar to melatonin

at the concentration of 10 M. These observations further indi-

cated that these new derivatives had the potential to be efﬁcient

multifunctional agents, including antioxidant activity, for the

treatment of AD.

lM. Compounds 4b, 4c and 4u had showed the highest

to the PAS site interacts with the indole ring of Trp286 via the

stacking with distance of 3.42 Å, Moreover, the charged nitrogen of

p–p

l

benzyl piperidine group bound to the PAS was via a cation–

action with Tyr337 and Trp 86.

p inter-

l

At the bottom of the gorge, the N-benzyl piperidine moiety of

4u was bound to the CAS of AChE via aromatic stacking inter-

p–p

actions with the indole ring from Trp86 with the ring-to-ring dis-

tance of 3.16 Å. All these results clearly indicated that compound

Figure 4. (A) UV spectrum of compound 4u (2.5

lM) alone and in the presence of CuSO₄(5.0 lM), FeSO₄(5.0 lM), FeCl₃(5.0 lM), or ZnCl₂(5.0 lM) in 20% (v/v) ethanol/

buffer (20 mM HEPES, 150 mM NaCl, pH 7.4). (B) Determination of the stoichiometry of complex 4uꢀCu (II) by Job’s method.

Please cite this article in press as: Wang, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.025

J. Wang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

7

Figure 5. Inhibition of Ab_1–42self-induced aggregation by compounds (4a–u) comparing with those of curcumin and resveratrol. The thioﬂavin-T ﬂuorescence method was

used and the measurements were carried out in the presence of 20

lM test compound. The mean SD values from three independent experiments were shown.

produced by the fusion of donepezil and melatonin. These com-

pounds were evaluated as potential multivalent inhibitors of ChEs,

antioxidant, anti-Ab aggregation, metal-chelating, and neuropro-

tective properties. Among the synthesized compounds, compound

4u showed the potent ChEs inhibitory activities. Interestingly,

molecular modeling studies, necessary to validate the results

derived from the kinetic assay, revealed that 4u binds to the

enzyme in reverse fashion as compared to other dual-binding-site

inhibitors of AChE. As one of the building blocks in the design

strategy of the target compounds was an antioxidant compound,

melatonin. The ORAC assay conﬁrmed that these hybrids could

exert ROS scavenging ability as well. In addition, the promising

AChE inhibitor 4u exhibited moderate inhibition of Ab_1–42

self-aggregation by using a thioﬂavin-T based ﬂuorometric assay,

and good biometal-chelating ability was studied by UV–vis

spectroscopy. Besides, to gain insight into the therapeutic potential

of hybrids, cell viability was assayed using the PC12 cell and com-

pounds were tested for their capacity to protect the PC12 cells

against death induced by H₂O₂. Compound 4u could penetrate the

BBB by using a PAMPA of BBB. These results showed that compound

4u was a potential multifunctional agent for the treatment of AD.

Figure 6. Effects of compounds on cell viability in PC12 cells. The cell viability was

determined by the MTT assay after 24 h of incubation with various concentrations

of 4a, 4b, 4p, 4q, 4t and 4u. The results were expressed as a percentage of control

cells. Values are reported as the mean SD of three independent experiments.

4. Experimental section

4.1. Chemistry

4u could simultaneously bind to the CAS and PAS of AChE, which

demonstrated the rationality of our molecular design.

In order to explore the interaction modes of the potential com-

pound 4u for BuChE, docking studies was employed using the

Molecular Operating Environment (MOE) software package. The

protonation level of the compound at physiological pH was calcu-

lated using the Marvin software package (Version 14.12.8 2014).

Crystallographic structures of hBuChE (PDB code 1P0I) were used

to dock the derivatives under study. As shown in Figure 9B and D,

Reagents and solvents were purchased from common commer-

cial suppliers and were used without further puriﬁcation. Reaction

progress was monitored using analytical thin layer chromatography

(TLC) on precoated silica gel GF254 (Qingdao Haiyang Chemical

Plant, Qingdao, China) plates and the spots were detected under

UV light (254 nm). IR (KBr-disc) spectra were recorded by Bruker

Tensor 27 spectrometer. Melting points (uncorrected) were deter-

mined with an XT-4 micromelting point instrument. ¹H and ¹³C

NMR spectra were recorded in CDCl3 or DMSO solutions using a

Bruker ACF-500 spectrometer at 25 °C. Chemical shifts are reported

in d (ppm) relative to internal Me4Si. J values are given in hertz, and

spin multiplicities are expressed as s (singlet), d (doublet), t (triplet),

q (quartet), or m (multiplet). The purity of all compounds were con-

ﬁrmed to be higher than 95% through analytical HPLC performed

the indole moiety of 4u stacked against the Trp82 through a

p–p

interaction with the distance of 2.86 Å at the CAS.

3. Conclusion

In conclusion, our study involved the synthesis of a new series of

multi-target-directed ligands for the treatment of AD that were

Please cite this article in press as: Wang, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.025

8

J. Wang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

Figure 7. Neuroprotection against H₂O₂toxicity. Compounds 4b, 4c, 4p, 4q and 4u were tested for neuroprotective activity against H₂O₂toxicity in PC12 cell cultures. Trolox

and melatonin (10 lM) was used as the reference compound. Results are expressed as percent viability compared to cells not treated with H₂O₂. Data represent the mean SD

of three observations. ^*p <0.05, ^**p <0.01.

Table 3

Permeability (Pe, 10^ꢀ6cm s^ꢀ1) in the PAMPA–BBB assay for 9 commercial drugs (Used

in the Experiment Validation) and selected donepezil–melatonin hybrids^a

was performed on silica gel (90–150

Inc.).

lm; Qingdao Marine Chemical

Compounds

Bibl.^bPe (10^ꢀ6cm s^ꢀ1

)

Compounds Pe (10^ꢀ6cm s^ꢀ1

)

4.1.1. General procedures for the preparation of intermediate

A mix of methyl piperidine-4-carboxylate or ethyl 2-(piperidin-

4-yl)acetate (10 mmol) in acetonitrile (20 ml), 1a–j (11 mmol),

anhydrous K₂CO₃(12 mmol) was reﬂuxed under nitrogen for 8 h

at 85 °C. The mixture was then cooled and ﬁltered and the solvent

removed in vacuum. The residue was dissolved in ethyl acetate

(50 ml) and washed with water (15 ml). Drying and removal of

the solvent followed by chromatography (ethyl acetate/petroleum

ether = 1:4) afforded desired product 2a–j.

Testosterone

Clonidine

Hydrocortisone 1.9

Corticosterone

Verapamil

b-Estradiol

Progesterone

Piroxicam

17

5.3

15.6

4.7

4a

4b

4c

4p

4q

4u

7.4

6.91

10.9

8.9

9.37

6.98

1.57

4.92

13.2

5.1

16

12

8.7

6.5

2.3

0.2

5.62

1.71

0.12

Dopamine

a

Experimental data are the mean SD of 3 independent experiments, using PBS/

EtOH (70:30) as solvent.

b

4.1.1.1. Ethyl 2-(1-benzylpiperidin-4-yl)acetate (2a).

Ethyl

Data from Ref. 45.

2-(piperidin-4-yl)acetate was treat with benzyl bromide according

to the general procedure to give the desired 2a as yellow oil, 90%

yield; ESI-MS m/z: 262.0 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) d

7.36 (s, 1H), 7.35 (s, 1H), 7.32–7.29 (s, 3H), 4.17 (q, J = 7.1 Hz,

2H), 3.54 (s, 2H), 2.91 (d, J = 11.6 Hz, 2H), 2.27 (d, J = 7.1 Hz, 2H),

2.04 (td, J = 11.8, 1.8 Hz, 2H), 1.83 (m, J = 11.3, 7.4, 4.0 Hz, 1H),

1.73 (d, J = 12.9 Hz, 2H), 1.38 (d, J = 12.5, 3.7 Hz, 2H), 1.29 (t,

J = 7.1 Hz, 2H).

with Agilent 1200 HPLC System (Supporting information, Table S1).

Mass spectra were obtained on

a MS Agilent 1100 Series

LC/MSD Trap mass spectrometer (ESI-MS) and a Mariner ESI-TOF

spectrometer (HRESIMS), respectively. Column chromatography

4.1.1.2. Ethyl 2-(1-(2-methylbenzyl)piperidin-4-yl)acetate (2b).

Ethyl 2-(piperidin-4-yl)acetate was treated with 1-(bro-

momethyl)-2-methylbenzene according to the general procedure

to give the desired product 2b as yellow oil, 88% yield. ESI-MS m/

z: 276.0 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) d 7.31 (s, 1H), 7.18

(d, J = 5.5 Hz, 3H), 4.17 (q, J = 7.1 Hz, 2H), 3.47 (s, 2H), 2.89 (d,

J = 11.5 Hz, 2H), 2.40 (s, 3H), 2.26 (d, J = 7.1 Hz, 2H), 2.05 (dd,

J = 16.5, 6.6 Hz, 2H), 1.84 (m, J = 11.1, 7.1, 3.4 Hz, 1H), 1.72 (d,

J = 12.8 Hz, 2H), 1.40–1.32 (m, 2H), 1.30 (t, J = 7.1 Hz, 3H).

4.1.1.3. Ethyl 2-(1-(3-methylbenzyl)piperidin-4-yl)acetate (2c).

Ethyl 2-(piperidin-4-yl)acetate was treated with 1-(bro-

momethyl)-3-methylbenzene according to the general procedure

to give the desired product 2c as yellow oil, 89% yield. ESI-MS m/

z: 276.0 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) d 7.24 (t, J = 7.5 Hz,

1H), 7.18 (s, 1H), 7.15 (d, J = 7.5 Hz, 1H), 7.10 (d, J = 7.4 Hz, 1H),

4.17 (q, J = 7.1 Hz, 2H), 3.51 (s, 2H), 2.92 (d, J = 11.6 Hz, 2H), 2.39

(s, 3H), 2.27 (d, J = 7.1 Hz, 2H), 2.03 (dd, J = 11.5, 10.2 Hz, 2H),

1.83 (m, J = 11.1, 7.2, 3.9 Hz, 1H), 1.73 (d, J = 12.9 Hz, 2H), 1.38

(qd, J = 12.4, 3.5 Hz, 2H), 1.29 (t, J = 7.1 Hz, 3H).

Figure 8. Linear correlation between experimental and reported permeability of

commercial drugs using the PAMPA-BBB assay.

Please cite this article in press as: Wang, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.025

J. Wang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

9

Figure 9. (A) 3D docking model of compound 4u with hAChE. Atom colors: yellow—carbon atoms of 4u, gray—carbon atoms of residues of hAChE, dark blue—nitrogen atoms,

red—oxygen atoms. The dashed lines represent the interactions between the protein and the ligand. (B) 3D docking model of compound 4u with hBuChE. (C) 2D schematic

diagram of docking model of com pound 4u with hAChE. (D) 2D schematic diagram of docking model of compound 4u with hBuChE. The ﬁgure was prepared using the ligand

interactions application in MOE.

4.1.1.4. Ethyl 2-(1-(3-methylbenzyl)piperidin-4-yl)acetate (2d).

Ethyl 2-(piperidin-4-yl)acetate was treated with 1-(bro-

momethyl)-4-methylbenzene according to the general procedure

to give the desired product 2d as yellow oil, 88% yield. ESI-MS m/

z: 276.0 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) d 7.23 (d, J = 7.9 Hz,

2H), 7.15 (d, J = 7.8 Hz, 2H), 4.16 (q, J = 7.1 Hz, 2H), 3.49 (s, 3H),

2.89 (d, J = 11.6 Hz, 2H), 2.37 (s, 3H), 2.26 (d, J = 7.1 Hz, 2H), 2.01

(td, J = 11.7, 1.8 Hz, 2H), 1.82 (m, J = 3.9 Hz, 1H), 1.72 (d,

J = 12.9 Hz, 2H), 1.46–1.31 (m, 2H), 1.29 (t, J = 7.1 Hz, 3H).

to give the desired product 2f as yellow oil, 87% yield. ESI-MS m/

z: 295.0 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) d 7.36 (s, 1H), 7.31

(s, 1H), 7.26 (d, J = 6.3 Hz, 2H), 7.22 (d, J = 4.6 Hz, 1H), 4.17 (d,

J = 7.1 Hz, 2H), 3.49 (s, 2H), 2.87 (d, J = 11.6 Hz, 2H), 2.27 (d,

J = 7.1 Hz, 2H), 2.03 (td, J = 11.7, 1.9 Hz, 3H), 1.95–1.79 (m, 1H),

1.73 (d, J = 12.9 Hz, 2H), 1.37 (td, J = 12.2, 3.4 Hz, 2H), 1.29 (t,

J = 7.1 Hz, 3H).

4.1.1.7. Ethyl 2-(1-(4-chorobenzyl)piperidin-4-yl)acetate (2g).

Ethyl 2-(piperidin-4-yl)acetate was treated with 1-(bro-

momethyl)-4-chlorobenzene according to the general procedure

to give the desired product 2g as yellow oil, 85% yield. ESI-MS m/

z: 295.0 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) d 7.30 (s, 4H), 4.15

(d, J = 7.1 Hz, 2H), 3.51 (s, 2H), 2.88 (d, J = 11.5 Hz, 2H), 2.25 (d,

J = 7.1 Hz, 2H), 2.05 (t, J = 11.3 Hz, 2H), 1.82 (m, 1H), 1.72 (d,

J = 12.9 Hz, 2H), 1.48–1.32 (m, 1H), 1.29–1.25 (t, 7.1 Hz, 3H).

4.1.1.5. Ethyl 2-(1-(2-chlorobenzyl)piperidin-4-yl)acetate (2e).

Ethyl 2-(piperidin-4-yl)acetate was treated with 1-(bro-

momethyl)-2-chlorobenzene according to the general procedure

to give the desired product 2e as yellow oil, 86% yield. ESI-MS m/

z: 295.0 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) d 7.55 (d, J = 7.5 Hz,

1H), 7.38 (d, J = 7.1 Hz, 1H), 7.27 (t, J = 7.0 Hz, 1H), 7.22 (dd,

J = 10.8, 4.3 Hz, 1H), 4.17 (q, J = 7.1 Hz, 2H), 3.67 (s, 2H), 2.95 (d,

J = 11.3 Hz, 2H), 2.28 (d, J = 7.1 Hz, 2H), 2.18 (t, J = 11.3 Hz, 2H),

1.86 (ddt, J = 15.1, 7.8, 3.7 Hz, 1H), 1.75 (d, J = 12.9 Hz, 2H), 1.51

(d, J = 7.3 Hz, 2H), 1.41 (dd, J = 21.7, 11.6 Hz, 2H), 1.29 (t,

J = 7.1 Hz, 3H).

4.1.1.8. Methyl 1-benzylpiperidine-4-carboxylate (2h).

Methyl piperidine-4-carboxylate was treated with (bromomethyl)

benzene according to the general procedure to give the desired

product 2h as yellow oil, 78% yield. ESI-MS m/z: 233.0 [M+H]⁺;

¹H NMR (500 MHz, CDCl₃) d 7.36 (d, J = 4.4 Hz, 4H), 7.31 (s, 1H),

3.72 (s, 3H), 3.55 (s, 2H), 2.90 (d, J = 11.5 Hz, 2H), 2.35 (dd,

J = 14.9, 10.9, 3.8 Hz, 1H), 2.09 (t, J = 11.1 Hz, 2H), 2.01–1.89 (m,

2H), 1.82 (td, J = 14.2, 3.4 Hz, 2H).

4.1.1.6. Ethyl 2-(1-(3-chlorobenzyl)piperidin-4-yl)acetate (2f).

Ethyl 2-(piperidin-4-yl)acetate was treated with 1-(bro-

momethyl)-3-chlorobenzene according to the general procedure

Please cite this article in press as: Wang, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.025

10

J. Wang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

4.1.1.9. methyl 1-(2-methylbenzyl)piperidine-4-carboxylate

(2i). Methyl piperidine-4-carboxylate was treated with 1-

J = 7.9 Hz, 1H), 7.24 (d, J = 5.9 Hz, 1H), 7.16 (s, 2H), 7.14 (s, 2H),

7.07 (t, J = 7.5 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H), 3.42 (s, 2H), 3.34 (t,

J = 7.1 Hz, 2H), 2.83 (t, J = 7.1 Hz, 2H), 2.77 (d, J = 8.1 Hz, 2H), 2.32

(s, 3H), 2.00 (t, J = 6.9 Hz, 2H), 1.92 (t, J = 6.9 Hz, 2H), 1.69 (m,

1H), 1.58 (d, J = 12.7 Hz, 2H), 1.21–1.09 (m, 2H). ¹³C NMR

(bromomethyl)-2-methylbenzene according to the general proce-

dure to give the desired product 2i as yellow oil, 88% yield. ESI-

MS m/z: 247.0 [M+H]; ¹H NMR (500 MHz, CDCl₃) d 7.29–7.25 (m,

1H), 7.20–7.12 (m, 4H), 3.69 (s, 3H), 3.45 (s, 2H), 2.85 (d,

J = 11.6 Hz, 2H), 2.37 (s, 3H), 2.32 (tt, J = 11.2, 4.1 Hz, 1H), 2.05

(dd, J = 16.2, 6.4 Hz, 2H), 1.94–1.82 (m, 2H), 1.82–1.69 (m, 2H).

(125 MHz, DMSO)

d 171.43, 137.49, 136.72, 130.51, 130.03,

127.73, 125.84, 123.04, 121.33, 118.70, 118.64, 112.36, 111.79,

60.76, 53.69, 42.97, 41.86, 33.35, 32.11, 25.75, 19.29. HRMS: calcu-

lated for C₂₅H₃₂N₃O [M+H]⁺390.2537, found 390.2540.

4.1.1.10. Methyl 1-(3-methylbenzyl)piperidine-4-carboxylate

(2j). Methyl piperidine-4-carboxylate was treated with 1-(bro-

momethyl)-3-methylbenzene according to the general procedure

to give the desired product 2j as yellow oil, 85% yield. ESI-MS m/

z: 247.0 [M+H]⁺; ¹H NMR (500 MHz, CDCl₃) d 7.24 (t, J = 7.5 Hz,

1H), 7.18 (s, 1H), 7.15 (d, J = 7.5 Hz, 1H), 7.10 (d, J = 7.4 Hz, 1H),

3.70 (s, 3H), 3.42 (s, 2H), 2.85 (d, J = 11.6 Hz, 2H), 2.37 (s, 3H),

2.35 (ddd, J = 14.9, 10.9, 3.8 Hz, 1H), 2.09 (t, J = 11.1 Hz, 2H),

2.01–1.89 (m, 2H), 1.82 (td, J = 14.2, 3.4 Hz, 2H).

4.1.3.3. N-(2-(1H-Indol-3-yl)ethyl)-2-(1-(2-methylbenzyl)piperi-

din-4-yl)acetamide (4c).

tryptamine according to the general procedure to give the desired

product 4c as yellow oil in 35.2% yield. IR (KBr) 3414, 2924, 1731,

Intermediate 3b was treated with

m

1642, 1545, 1097, 974, 843, 741 cm^ꢀ1; ESI/MS m/z: 420.2 [M+H]⁺;

¹H NMR (500 MHz, DMSO) d 10.62 (s, 1H), 7.86 (s, 1H), 7.24 (s, 1H),

7.22 (s, 1H), 7.15 (d, J = 3.1 Hz, 1H), 7.15 (s, 1H), 7.14 (s, 1H), 7.10 (s,

1H), 7.03 (s, 1H), 6.72 (dd, J = 8.7, 2.3 Hz, 1H), 3.77 (s, 3H), 3.43 (s,

2H) 3.33 (t, J = 7.3 Hz, 2H), 2.78 (t, J = 7.3 Hz, 2H), 2.72 (d,

J = 10.0 Hz, 2H), 2.32 (s, 3H), 1.99 (d, J = 7.1 Hz, 2H), 1.93 (t,

J = 10.8 Hz, 2H), 1.77–1.59 (m, 1H), 1.61–1.49 (d, 2H), 1.14 (d,

J = 11.6 Hz, 2H). ¹³C NMR (125 MHz, DMSO) d 171.42, 153.46,

137.39, 137.25, 130.45, 129.87, 128.07, 127.19, 125.78, 123.75,

112.39, 112.15, 111.42, 100.82, 60.97, 55.91, 53.78, 43.07, 33.49,

32.34, 25.77, 19.25. HRMS: calculated for C₂₆H₃₄N₃O [M+H]⁺

420.2645, found 420.2646.

4.1.2. General procedure for the preparation of methyl

1-benzylpiperidine-4-carboxylic acids (3a–j)

A mixture of the corresponding b-carboline-3-acid methyl ester

(1 mmol), NaOH (4 mmol), C₂H₅OH (5 ml) and H₂O (10 ml) was

reﬂuxed for 3 h, and the ethanol was removed on the rotary evap-

orator. The mixture was neutralized (pH = 5) with 5 N HCl and

cooled. The precipitate was collected, washed well with H₂O and

dried in vacuum. The material was used without further puriﬁca-

tion for the following steps.

4.1.3.4. N-(2-(1H-Indol-3-yl)ethyl)-2-(1-(3-methylbenzyl)piperi-

din-4-yl)acetamide (4d).

5-methoxytryptamine according to the general procedure to give

the desired product 4d as yellow oil in 48.2% yield. IR (KBr)

Intermediate 3c was treated with

4.1.3. General procedure for the preparation of compounds 4a–s

A solution of methyl 1-benzylpiperidine-4-carboxylic acids

(1 mmol) and 1,1⁰-carbonyldiimidazole (1.1 mmol) in 12 mL of

anhydrous THF was stirred at ambient temperature for 1 h. The

tryptamine analogue (1 mmol) was added to the solution, and stir-

ring was continued overnight. The reaction mixture was diluted

with H₂O and extracted with EtOAc. The organic extracts were

combined, washed with brine, and dried with anhydrous Na₂SO₄,

and the solvent was evaporated in vacuum to give the crude pro-

duct, which was puriﬁed by chromatography (CH₂Cl₂–

MeOH = 50:1) on silica gel.

m

3399, 2925, 1727, 1647, 1553, 1095, 844, 744 cm^ꢀ1; ESI/MS m/

z:390.2 [M+H]⁺; ¹H NMR (500 MHz, DMSO) d 10.86 (s, 1H), 7.96

(t, J = 5.5 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.36 (t, J = 7.2 Hz, 1H),

7.22 (d, J = 7.5 Hz, 1H), 7.15 (s, 1H), 7.13 (d, J = 13.7 Hz, 2H), 7.09

(d, J = 6.4 Hz, 1H), 7.06 (d, J = 7.6 Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H),

3.50 (s, 2H), 3.35 (t, 6.9 Hz, 2H), 3.05 (t, 6.9 Hz, 2H), 2.85 (d, 2H,

J = 13.2 Hz), 2.73 (m, 2H), 2.47 (s, 3H), 2.02 (t, J = 9.6 Hz, 2H),

1.84–1.64 (m, 1H), 1.59 (d, J = 12.2 Hz, 2H), 1.24 (d, J = 13.2 Hz,

2H). ¹³C NMR (125 MHz, DMSO) d 171.42, 137.71, 136.74, 130.22,

128.54, 128.27, 127.74, 126.70, 123.07, 121.32, 118.71, 118.64,

112.35, 111.82, 62.42, 53.31, 42.85, 41.75, 31.63, 25.75, 21.47,

11.43. HRMS: calculated for C₂₅H₃₂N₃O [M+H]⁺390.2541, found

390.2540.

4.1.3.1.

dine-4-carboxamide (4a).

tryptamine according to the general procedure to give the desired

product 4a as a white power in 48.5% yield. IR (KBr) 3343, 2985,

N-(2-(1H-Indol-3-yl)ethyl)-1-(2-methylbenzyl)piperi-

Intermediate 3a was treated with

m

2815, 1640, 1531, 1450, 864, 738 cm^ꢀ1; mp 132–136 °C; ESI/MS m/

z: 376.2 [M+H]⁺; ¹H NMR (500 MHz, DMSO) d 10.82 (s, 1H), 7.91 (t,

J = 5.5 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 3.9 Hz, 1H), 7.34

(d, J = 7.4 Hz, 1H), 7.32 (s, 1H), 7.31 (s, 1H), 7.29 (s, 1H), 7.26 (t,

J = 6.8 Hz, 1H), 7.14 (d, J = 1.7 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.99

(t, J = 7.4 Hz, 1H), 3.45 (s, 2H), 3.35 (t, J = 7.0 Hz, 2H), 2.83 (t,

J = 7.0 Hz, 2H), 2.77 (m, 2H), 2.01 (d, J = 7.1 Hz, 2H), 1.91 (d,

J = 9.7 Hz, 2H), 1.73–1.60 (m, 1H), 1.57 (td, J = 12.3 Hz, 7.1 Hz,

2H), 1.18 (td, J = 17.1, 9.7 Hz, 2H). ¹³C NMR (125 MHz, DMSO) d

171.41, 136.74, 129.22, 128.56, 127.75, 127.27, 123.05, 121.32,

118.71, 118.63, 112.38, 111.80, 62.90, 53.59, 43.03, 42.43, 33.38,

32.18, 25.77. HRMS: calculated for C₂₄H₃₀N₃O [M+H]⁺376.2384,

found 376.2383.

4.1.3.5. N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-2-(1-(3-methyl-

benzyl)piperidin-4-yl)acetamide (4e).

Intermediate 3c was

treated with 5-methoxytryptamine according to the general proce-

dure to give the desired product 4e as yellow oil in 56.42% yield. IR

(KBr)

m

3417, 2941, 1641, 1452, 1069, 797 cm^ꢀ1; ESI/MS m/z:

420.3 [M+H]⁺; ¹H NMR (500 MHz, DMSO) d 10.64 (s, 1H), 7.89 (d,

J = 5.3 Hz, 1H), 7.23 (d, J = 4.2 Hz, 1H), 7.22 (d, J = 3.0 Hz, 1H), 7.11

(s, 1H), 7.10 (s, 1H), 7.08 (s, 1H), 7.06 (s, 1H), 7.03 (s, 1H), 6.72 (dd,

J = 8.6, 1.8 Hz, 1H), 3.77 (s, 3H), 3.42 (s, 2H), 3.33 (t, J = 13.2, 2H),

2.78 (t, J = 13.3 Hz, 2H), 2.56 (t, J = 13.3 Hz, 2H), 2.31 (s, 3H), 2.00

(d, J = 7.0 Hz, 2H), 1.92 (s, 2H), 1.66 (m, 1H), 1.57 (d, J = 12.2 Hz,

2H), 1.22–0.95 (m, 2H). ¹³C NMR (125 MHz, DMSO) d 171.40,

153.45, 137.61, 131.89, 129.92, 128.47, 128.06, 128.00, 126.41,

123.75, 112.40, 112.13, 111.42, 100.79, 62.81, 55.90, 50.23, 49.08,

48.99, 43.02, 35.76, 33.32, 32.54, 32.06, 25.77, 21.48. HRMS: calcu-

lated for C₂₆H₃₄N₃O₂[M+H]⁺420.2644, found 420.2646.

4.1.3.2. N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-2-(1-(2-methyl-

benzyl)piperidin-4-yl)acetamide (4b).

Intermediate 3b was

treated with 5-methoxytryptamine according to the general proce-

dure to give the desired product 4b as yellow oil in 51.2% yield. IR

4.1.3.6. N-(2-(1H-Indol-3-yl)ethyl)-2-(1-(4-methylbenzyl)piperi-

(KBr)

m

3410, 2925, 1641, 1550, 1485, 1215, 1070, 922, 796,

din-4-yl)acetamide (4f).

methoxytryptamine according to the general procedure to give the

desired product 4f as yellow oil in 35.2% yield. IR (KBr) 3418,

Intermediate 3d was treated with 5-

745 cm^ꢀ1; ESI/MS m/z: 390.3 [M+H]⁺; ¹H NMR (500 MHz, DMSO)

d 10.79 (s, 1H), 7.88 (s, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.34 (d,

m

Please cite this article in press as: Wang, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.025

J. Wang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

11

2924, 2854, 1654, 1551, 1456, 743 cm^ꢀ1; ESI/MS m/z: 390.2 [M

+H]⁺; ¹H NMR (500 MHz, CDCl₃) d 8.51 (s, 1H), 8.02 (s, 1H), 7.62

(t, J = 7.0 Hz, 1H), 7.49 (s, 1H), 7.31 (s, 1H), 7.28 (s, 1H), 7.26 (s,

1H), 7.22 (dd, J = 10.1, 4.5 Hz, 1H), 7.21–7.18 (s, 1H), 7.17 (d,

J = 9.9 Hz, 1H), 7.14 (d, J = 4.1 Hz, 1H), 3.74 (t, 2H), 3.60 (s, 2H),

3.02 (dd, J = 12.1, 5.0 Hz, 2H), 2.96 (d, J = 12.9 Hz, 2H), 2.45 (s,

3H), 2.10 (d, J = 12.8, 2H), 2.09–1.95 (m, 2H), 1.76 (m, 1H), 1.65

(d, J = 12.7 Hz, 2H). 1.22–0.95 (m, 2H). ¹³C NMR (125 MHz, CDCl₃)

d 171.69, 136.53, 129.72, 129.11, 127.37, 122.20, 122.15, 119.46,

118.69, 118.02, 112.85, 111.37, 62.59, 53.18, 43.52, 39.64, 31.76,

31.25, 29.71, 25.38, 21.13. HRMS: calculated for C₂₅H₃₂N₃O [M

+H]⁺390.2541, found 390.2540.

desired product 4j as yellow solid in 45.2% yield. IR (KBr)

m

3342,

2917, 1734, 1640, 1075, 841, 743 cm^ꢀ1; mp 92–95 °C; ESI/MS m/z:

410.2 [M+H]⁺; ¹H NMR (500 MHz, DMSO) d 10.79 (s, 1H), 7.88 (s,

1H), 7.54 (d, J = 7.9 Hz, 1H), 7.38 (s, 1H), 7.36 (s, 1H), 7.35 (s, 1H),

7.34–7.29 (m, 1H), 7.27 (s, 1H), 7.14 (s, 1H), 7.07 (t, J = 7.5 Hz, 1H),

6.98 (t, J = 7.4 Hz, 1H), 3.45 (s, 2H), 3.04 (m, 2H), 2.82 (t, J = 7.4 Hz,

2H), 2.79–2.69 (d, J = 14.2 Hz, 2H), 2.07–1.97 (m, 2H), 1.91 (dd,

J = 25.7, 14.2 Hz, 2H), 1.73–1.63 (m, 1H), 1.57 (d, J = 12.5 Hz, 2H),

1.22–1.08 (m, 2H). ¹³C NMR (125 MHz, DMSO) d 171.47, 138.03,

133.39, 130.46, 128.76, 127.77, 127.74, 127.25, 123.06, 121.34,

118.72, 118.65, 62.04, 53.55, 43.01, 33.32, 32.17, 25.74. HRMS: cal-

culated for C₂₅H₂₉ClN₃O [M+H]⁺410.1991, found410.1994.

4.1.3.7. N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-2-(1-(4-methyl-

4.1.3.11. 2-(1-(3-Chlorobenzyl)piperidin-4-yl)-N-(2-(5-methoxy-

benzyl)piperidin-4-yl)acetamide (4g).

Intermediate 3d was

1H-indol-3-yl)ethyl)acetamide (4k).

Intermediate 3f was

treated with 5-methoxytryptamine according to the general proce-

dure to give the desired product 4g as yellow oil in 46.2% yield. IR

dure to give the desired product 4k as yellow solid in 39.8.2% yield.

(KBr)

m

3406, 2924, 2852, 1644, 1441, 1261, 1171, 1071, 802 cm^ꢀ1

;

IR (KBr) m

3422, 2927, 1732, 1484, 1215, 845 cm^ꢀ1; mp 82–85 °C;

ESI/MS m/z: 420.2 [M+H]⁺; ¹H NMR (500 MHz, DMSO) d 10.65 (s,

1H), 7.90 (s, 1H), 7.62 (s, 1H), 7.23 (d, J = 8.7 Hz, 1H), 7.20 (s, 1H),

7.19 (s, 1H), 7.14 (d, J = 7.7 Hz, 1H), 7.13 (s, 1H), 7.10 (s, 1H), 7.03

(s, 1H), 3.77 (s, 3H), 3.45 (s, 2H), 3.24 (m, 2H), 2.91–2.74 (m, 2H),

2.30 (s, 3H), 2.08–1.92 (m, 4H), 1.66 (s, 1H), 1.58 (d, J = 12.1 Hz,

2H), 1.29 (d, J = 11.3 Hz, 2H). ¹³C NMR (125 MHz, DMSO) d

171.39, 153.46, 131.91, 129.40, 129.18, 128.06, 123.75, 112.41,

112.27, 112.13, 111.42, 100.80, 62.36, 55.90, 53.36, 42.96, 33.22,

31.89, 25.77, 21.17. HRMS: calculated for C₂₆H₃₄N₃O₂[M+H]⁺

420.2644, found 420.2646.

ESI/MS m/z: 440.2 [M+H]⁺; ¹H NMR (500 MHz, DMSO) d 10.62 (s,

1H), 7.87 (t, J = 5.2 Hz, 1H), 7.37 (s, 1H), 7.36 (s, 1H), 7.34 (s, 1H),

7.28 (d, J = 3.1 Hz, 1H), 7.23 (d, J = 8.7 Hz, 1H), 7.10 (d, J = 1.6 Hz,

1H), 7.03 (d, J = 2.0 Hz, 1H), 6.74–6.64 (S, 1H), 3.77 (s, 3H), 3.60

(d, J = 3.8 Hz, 2H), 2.83–2.68 (m, 6H), 2.01 (d, J = 7.0 Hz, 2H),

1.95–1.80 (m, 2H), 1.67 (s, 1H), 1.59 (d, J = 11.6 Hz, 2H), 1.18 (d,

J = 9.5 Hz, 2H). ¹³C NMR (125 MHz, DMSO) d 171.35, 153.46,

133.41, 131.90, 130.50, 128.07, 123.76, 112.39, 112.13, 111.41,

100.82, 55.91, 53.45, 44.86, 42.93, 32.05, 25.77. HRMS: calculated

for C₂₅H₃₁ClN₃O₂[M+H]⁺440.2097, found 440.2099.

4.1.3.8. N-(2-(1H-Indol-3-yl)ethyl)-2-(1-(2-chlorobenzyl)piperi-

4.1.3.12. N-(2-(1H-Indol-3-yl)ethyl)-2-(1-(4-chlorobenzyl)piper-

din-4-yl)acetamide (4h).

tryptamine according to the general procedure to give the desired

product 4h as yellow oil in 51.2% yield. IR (KBr) 3413, 2976, 2819,

Intermediate 3e was treated with

idin-4-yl)acetamide (4i).

tryptamine according to the general procedure to give the desired

product 4l as yellow solid in 45.2% yield. IR (KBr) 3339, 2924,

Intermediate 3g was treated with

m

2487, 1637, 1456, 942, 751 cm^ꢀ1; ESI/MS m/z: 410.2 [M+H]⁺; ¹H

NMR (500 MHz, CDCl₃) d 8.57 (s, 1H), 7.61 (t, J = 6.2 Hz, 1H), 7.60

(d, J = 5.0 Hz, 1H), 7.48 (s, 1H), 7.31 (s, 1H), 7.30 (s, 1H), 7.27 (s,

1H), 7.26 (s, 1H), 7.20 (d, J = 7.9 Hz, 1H), 7.13 (d, J = 7.2 Hz, 1H),

7.05 (s, 1H), 3.76 (s, 2H), 3.61 (t, J = 12.7, 2H), 3.04 (t, J = 12.7 Hz,

2H), 2.98 (d, J = 6.9 Hz, 2H), 2.24 (t, J = 11.5 Hz, 2H), 2.03 (t,

J = 11.1 Hz, 2H), 1.93–1.74 (m, 1H), 1.66 (d, J = 12.6 Hz, 2H), 1.47

(t, J = 7.3 Hz, 2H). ¹³C NMR (125 MHz, CDCl₃) d 171.74, 136.50,

134.65, 129.58, 128.93, 127.40, 126.93, 122.25, 122.11, 119.42,

118.68, 112.84, 111.37, 58.78, 53.32, 43.43, 42.37, 39.68, 32.70,

1737, 1640, 1445, 803, 741 cm^ꢀ1; mp 132–135 °C; ESI/MS m/z:

410.2 [M+H]⁺; ¹H NMR (500 MHz, DMSO) d 10.79 (s, 1H), 7.88 (t,

J = 5.3 Hz, 1H), 7.65 (s, 1H), 7.54 (d, J = 7.7 Hz, 1H), 7.40 (s, 1H),

7.38 (d, J = 4.4 Hz, 1H), 7.35 (s, 1H), 7.33 (s, 1H), 7.14 (d,

J = 1.9 Hz, 1H), 7.07 (t, J = 7.3 Hz, 1H), 6.98 (t, J = 7.2 Hz, 1H), 3.60

(s, 2H), 3.44 (d, J = 17.0 Hz, 2H), 2.82 (t, J = 17.4 Hz, 2H), 2.25 (d,

J = 6.9 Hz, 2H), 2.00 (d, J = 7.0 Hz, 2H), 1.98–1.74 (m, 2H), 1.64 (s,

1H), 1.58 (d, J = 12.2 Hz, 2H), 1.16 (d, J = 11.7 Hz, 2H). ¹³C NMR

(125 MHz, DMSO)

d 171.37, 136.72, 131.05, 129.69, 129.33,

128.58, 127.73, 123.05, 121.32, 118.70, 118.64, 112.36, 111.79,

61.80, 53.44, 51.61, 42.94, 31.74, 25.75. HRMS: calculated for

31.21, 25.35. HRMS: calculated for

410.1990, found 410.1994.

C

₂₄H₂₉ClN₃O [M+H]⁺

C

₂₄H₂₉ClN₃O [M+H]⁺410.1995, found 410.1994.

4.1.3.9. 2-(1-(2-Chlorobenzyl)piperidin-4-yl)-N-(2-(5-methoxy-

1H-indol-3-yl)ethyl)acetamide (4i). Intermediate 3e was

4.1.3.13. 2-(1-(4-Chlorobenzyl)piperidin-4-yl)-N-(2-(5-methoxy-

1H-indol-3-yl)ethyl)acetamide (4m). Intermediate 3g as

treated with 5-methoxytryptamine according to the general proce-

reacted with 5-methoxytryptamine according to the general proce-

dure to give the desired product 4i as yellow oil in 35.2% yield. IR

dure to give the desired product 4m as yellow oil in 45.9% yield. R

(KBr)

m

3427, 2923, 1693, 1441, 1209, 1140, 844, 804 cm^ꢀ1; ESI/MS

(KBr) m

3431, 2926, 1732, 1641, 1286, 1016, 845 cm^ꢀ1; ESI/MS m/z:

m/z: 440.2 [M+H]^{+; 1}H NMR (500 MHz, DMSO) d 10.62 (s, 1H), 7.87

(t, J = 5.2 Hz, 1H), 7.37 (s, 1H), 7.35 (d, J = 9.6 Hz, 1H), 7.34 (s, 1H),

7.28 (d, J = 3.1 Hz, 1H), 7.23 (d, J = 8.7 Hz, 1H), 7.10 (d, J = 1.6 Hz,

1H), 7.03 (d, J = 2.0 Hz, 1H), 6.72 (dd, J = 8.7, 2.3 Hz, 1H), 3.77 (s,

3H), 3.56–3.45 (m, 2H), 3.36 (s, 2H), 2.89–2.77 (m, 2H), 2.77 (m,

2H), 2.01 (d, J = 7.0 Hz, 2H), 1.92 (d, J = 11.6 Hz, 2H), 1.67 (m, 1H),

1.59 (d, J = 11.6 Hz, 2H), 1.18 (d, J = 9.5 Hz, 2H). ¹³C NMR

440.2 [M+H]⁺; ¹H NMR (500 MHz, DMSO) d 10.62 (s, 1H), 7.89 (s,

1H), 7.65 (s, 1H), 7.42 (s, 2H), 7.36 (s, H), 7.23 (d, J = 8.7 Hz, 1H),

7.09 (d, J = 1.8 Hz, 1H), 7.02 (d, J = 2.1 Hz, 1H), 6.72 (dd, J = 8.7,

2.3 Hz, 1H), 3.77 (s, 3H), 3.60 (s, 2H), 3.19 (d, J = 4.5 Hz, 2H), 2.78

(t, J = 7.3 Hz, 2H), 2.26 (d, J = 6.7 Hz, 2H), 2.01 (d, J = 6.8 Hz, 2H),

1.65 (dd, J = 12.5, 6.0 Hz, 2H), 1.46–1.33 (m, 1H), 1.28 (d,

J = 23.7 Hz, 2H), 0.90–0.79 (m, 2H). ¹³C NMR (125 MHz, DMSO) d

153.45, 131.89, 130.08, 129.12, 128.73, 128.06, 123.76, 112.40,

112.11, 111.41, 100.81, 67.93, 64.67, 55.90, 51.63, 49.08, 38.72,

(125 MHz, DMSO)

128.58, 127.73, 123.05, 121.32, 118.70, 118.64, 112.36, 111.79,

61.80, 53.44, 51.61, 42.94, 31.74, 25.75. HRMS: calculated for C₂₅

₃₁ClN₃O₂[M+H]⁺440.2100, found 440.2099.

d 171.37, 136.72, 131.05, 129.69, 129.33,

-

28.85, 25.76. HRMS: calculated for

440.2097, found440.2099.

C

₂₅H₃₁ClN₃O₂[M+H]⁺

H

4.1.3.10. N-(2-(1H-Indol-3-yl)ethyl)-2-(1-(3-chlorobenzyl)piper-

idin-4-yl)acetamide (4j). Intermediate 3f was treated with 5-

methoxytryptamine according to the general procedure to give the

4.1.3.14. N-(2-(1H-Indol-3-yl)ethyl)-1-benzylpiperidine-4-car-

boxamide (4n). Intermediate 3h was treated with tryptamine

according to the general procedure to give the desired product 4n

Please cite this article in press as: Wang, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.025

12

J. Wang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

as a white powder in 55.5% yield. IR (KBr)

m

3312, 2936, 2621,

¹H NMR (500 MHz, DMSO) d 10.77 (s, 2H), 7.87 (d, J = 8.0 Hz, 1H),

1634, 1535, 1055, 742 cm^ꢀ1; mp 158–162 °C; ESI/MS m/z: 362.3

[M+H]⁺; ¹H NMR (500 MHz, DMSO) d 10.79 (s, 1H), 7.83 (t,

J = 5.2 Hz, 1H), 7.55 (d, J = 7.8 Hz, 1H), 7.36 (s, 1H), 7.34 (s, 2H),

7.32 (s, 1H), 7.31 (s, 1H), 7.30 (s, 1H), 7.14 (s, 1H), 7.08 (t,

J = 7.5 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H), 3.45 (s, 2H), 3.33 (d,

J = 5.1 Hz, 4H), 2.98–2.76 (m, 4H), 2.18–2.01 (m, 1H), 1.98–1.77

(m, 2H), 1.63 (s, 2H). ¹³C NMR (125 MHz, DMSO) d 174.75,

139.04, 136.72, 129.19, 128.58, 127.76, 127.27, 123.05, 121.33,

7.82 (t, J = 5.5 Hz, 1H), 7.67 (t, J = 7.4 Hz, 1H), 7.63 (d, J = 7.0 Hz,

1H), 7.55 (s, 1H), 7.53 (s, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.12 (d,

J = 10.1 Hz, 1H), 7.07 (d, J = 7.3 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H),

3.70 (s, 2H), 3.20 (d, J = 14.7 Hz, 2H), 2.82 (t, J = 14.4 Hz, 2H),

2.76–2.59 (m, 2H), 2.15–2.02 (m, 1H), 2.01–1.80 (m, 2H), 1.57

(dd, J = 20.2, 7.0 Hz, 2H), 1.60–1.43 (m, 2H). ¹³C NMR (125 MHz,

DMSO) d 174.66, 150.14, 136.73, 133.58, 133.03, 131.51, 128.88,

127.77, 124.54, 123.05, 121.34, 118.73, 118.64, 112.39, 111.81,

58.93, 53.28, 49.09, 42.32, 29.03, 25.67. HRMS: calculated for

118.73, 118.63, 112.38, 111.80, 62.88, 53.21, 42.59, 29.10, 25.70.

+

HRMS: calculated for

362.2227.

C₂₃H₂₈N₃O [M+H]

362.2229, found

C

₂₃H₂₇N₄O₃[M+H]⁺407.2075, found 407.2078.

4.1.3.19. N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-1-(2-nitroben-

zyl)piperidine-4-carboxamide (4s). Intermediate 3n as trea-

ted with 5-methoxytryptamine according to the general procedure

4.1.3.15. 1-Benzyl-N-(2-(5-methoxy-1H-indol-3-yl)ethyl)piperi-

dine-4-carboxamide (4o). Intermediate 3h was treated with

5-methoxytryptamine according to the general procedure to give

the desired product 4o as yellow oil in 45.2% yield. IR (KBr)

to give the desired product 4s as yellow solid in 45.9% yield. IR

m

(KBr) m

3418, 2953, 2896, 1652, 1362, 1025, 796 cm^ꢀ1; mp 132–

3297, 2937, 1644, 1269, 921, 793 cm^ꢀ1; ESI/MS m/z: 390.2 [M

+H]⁺; ¹H NMR (500 MHz, DMSO) d 10.63 (s, 1H), 7.82 (s, 1H),

7.33 (s, 1H), 7.32 (s, 1H), 7.31 (s, 2H), 7.30 (s, 1H), 7.23 (s, 1H),

7.10 (d, J = 1.9 Hz, 1H), 7.04 (d, J = 2.2 Hz, 1H), 6.73 (dd, J = 8.7,

2.3 Hz, 1H), 3.78 (s, 3H), 3.44 (s, 2H), 3.35 (m, 4H), 2.80 (dd,

J = 16.4, 9.2 Hz, 4H), 2.09 (tt, J = 10.1, 5.0 Hz, 1H), 1.91 (td,

J = 11.2, 3.6 Hz, 2H), 1.20 (t, J = 7.1 Hz, 2H). ¹³C NMR (125 MHz,

DMSO) d 174.78, 153.49, 139.06, 131.93, 129.18, 128.57, 128.11,

127.25, 123.76, 112.42, 112.19, 111.46, 100.85, 62.89, 55.91,

53.22, 42.65, 29.13, 25.74. HRMS: calculated for C₂₄H₃₀N₃O₂[M

+H]⁺392.2332, found 390.2333.

140 °C; ESI/MS m/z: 437.2 [M+H]⁺; ¹H NMR (500 MHz, DMSO) d

10.61 (s, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.81 (t, J = 5.5 Hz, 1H), 7.67

(t, J = 7.4 Hz, 1H), 7.63 (d, J = 6.8 Hz, 1H), 7.53 (dd, J = 11.0, 4.1 Hz,

1H), 7.23 (d, J = 8.7 Hz, 1H), 7.09 (d, J = 1.6 Hz, 1H), 7.03 (d,

J = 2.0 Hz, 1H), 6.73 (dd, J = 8.7, 2.2 Hz, 1H), 3.78 (s, 3H), 3.70 (s,

2H), 2.78 (t, J = 7.3 Hz, 2H), 2.69 (d, J = 11.3 Hz, 2H), 2.30 (d,

J = 9.6 Hz, 2H), 2.12–2.02 (m, 1H), 1.98 (dd, J = 11.4, 9.6 Hz, 2H),

1.59 (d, J = 10.6 Hz, 2H), 1.52 (dd, J = 12.1, 2.9 Hz, 2H). ¹³C NMR

(125 MHz, DMSO)

d 174.65, 153.48, 150.13, 133.58, 133.03,

131.91, 131.51, 128.88, 128.08, 124.54, 123.76, 112.42, 112.17,

111.47, 100.82, 58.93, 55.92, 53.28, 42.35, 29.04, 25.70. HRMS: cal-

culated for C₂₄H₂₉N₄O₄[M+H]⁺437.2186, found 437.2183.

4.1.3.16. N-(2-(1H-Indol-3-yl)ethyl)-1-(2-methylbenzyl)piperi-

dine-4-carboxamide (4p). Intermediate 3k was treated with

tryptamine according to the general procedure to give the desired

4.1.4. General procedure for the preparation of compounds 4t–u

A solution of 3-(1H-indol-3-yl)propanoic acid (1 mmol) and 1,1⁰-

carbonyldiimidazole (1.1 mmol) in 12 mL of anhydrous THF was

stirred at ambient temperature for 1 h. The 1-benzylpiperidin-4-

amine (1 mmol) was added to the solution, and stirring was contin-

ued overnight. The reaction mixture was diluted with H₂O and

extracted with EtOAc. The organic extracts were combined, washed

with brine, and dried with anhydrous Na₂SO₄, and the solvent was

evaporated in vacuo to give the crude product, which was puriﬁed

by chromatography (CH₂Cl₂–MeOH = 50:1) on silica gel.

product 4p as yellow oil in 44.8% yield. IR (KBr)

m 3425, 3268, 2922,

2855, 1630, 1269, 921, 739 cm^ꢀ1; ESI/MS m/z: 376.2 [M+H]⁺; ¹H

NMR (500 MHz, DMSO) d 10.79 (s, 1H), 7.85 (s, 1H), 7.54 (d,

J = 7.8 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.25 (s, 1H), 7.17 (s, 2H),

7.14 (s, 2H), 7.08 (t, J = 7.5 Hz, 1H), 6.99 (t, J = 7.4 Hz, 1H), 3.43 (s,

2H), 2.82 (t, J = 7.4 Hz, 4H), 2.34 (s, 3H), 2.12 (s, 2H), 2.07–1.91

(m, 1H), 1.64 (s, 2H), 1.61 (m, 2H), 1.20 (t, J = 7.3 Hz, 2H). ¹³C

NMR (125 MHz, DMSO) d 174.62, 136.74, 130.63, 127.76, 125.93,

123.06, 121.35, 118.73, 118.65, 112.37, 111.82, 53.13, 49.09,

46.19, 33.79, 28.82, 25.67, 19.34. HRMS: calculated for C₂₄H₃₀N₃O

[M+H]⁺376.2382, found 376.2383.

4.1.4.1. N-(1-Benzylpiperidin-4-yl)-3-(1H-indol-3-yl)propana-

mide (4t). 3-(1H-indol-3-yl)propanoic acid was treated with

1-benzylpiperidin-4-amine according to the general procedure to

give the desired product 4t as white power in 45.2% yield. IR

4.1.3.17.

N-(2-(5-Methoxy-1H-indol-3-yl)ethyl)-1-(2-methyl-

benzyl)piperidine-4-carboxamide (4q). Intermediate 3k as

treated with 5-methoxytryptamine according to the general proce-

dure to give the desired product 4q as white solid in 45.9% yield. IR

(KBr) m

3310, 2933, 2816, 1644, 1553, 1220, 808, 743 cm^ꢀ1; mp

164–168 °C; ESI/MS m/z: 362.3 [M+H]⁺; ¹H NMR (500 MHz, DMSO)

d 10.74 (s, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.35

(s, 1H), 7.33 (s, 1H), 7.32 (s, 1H), 7.30 (s, 1H), 7.29 (s, 1H), 7.26 (t,

J = 7.0 Hz, 1H), 7.09 (d, J = 4.9 Hz, 1H), 7.07 (t, J = 7.6 Hz, 1H), 6.98

(t, J = 7.4 Hz, 1H), 3.69–3.49 (m, 1H), 3.45 (s, 2H), 2.93 (t,

J = 7.7 Hz, 2H), 2.74 (d, J = 11.5 Hz, 2H), 2.43 (t, J = 7.7 Hz, 2H),

2.00 (dd, J = 12.7, 8.8 Hz, 2H), 1.70 (d, J = 10.3 Hz, 2H), 1.38 (dd,

J = 11.5, 2.6 Hz, 2H). ¹³C NMR (125 MHz, DMSO) d 171.58, 139.17,

136.71, 129.16, 128.60, 128.43, 127.57, 127.50, 127.28, 122.54,

121.31, 118.83, 118.54, 114.41, 111.73, 62.64, 52.86, 52.46, 52.08,

46.38, 36.88, 33.20, 32.14, 31.09, 21.53. HRMS: calculated for

(KBr)

m 3453, 3319, 2939, 2845, 1640, 1585, 1265, 1212, 808,

741 cm^ꢀ1; mp 125–128 °C; ESI/MS m/z: 406.3 [M+H]⁺; ¹H NMR

(500 MHz, DMSO) d 10.61 (s, 1H), 7.81 (t, J = 4.6 Hz, 1H), 7.24 (d,

J = 2.0 Hz, 1H), 7.23 (d, J = 2.3 Hz, 1H), 7.17 (s, 1H), 7.15 (d,

J = 8.5 Hz, 1H), 7.14 (s, 1H), 7.09 (s, 1H), 7.03 (s, 1H), 6.73 (dd,

J = 8.7, 2.2 Hz, 1H), 3.78 (s, 3H), 3.40 (s, 2H), 3.33 (d, J = 6.4 Hz,

4H), 2.80 (d, J = 4.6 Hz, 4H), 2.33 (s, 3H), 2.10 (t, J = 10.9 Hz, 1H),

1.94 (t, J = 10.6 Hz, 2H), 1.69–1.47 (m, 2H). ¹³C NMR (125 MHz,

DMSO) d 174.79, 153.48, 137.48, 137.11, 131.91, 130.51, 129.94,

128.09, 127.28, 125.81, 123.77, 112.42, 112.18, 111.46, 100.83,

60.96, 55.92, 53.40, 42.72, 29.20, 25.71, 19.25. HRMS: calculated

for C₂₅H₃₂N₃O₂[M+H]⁺406.2486, found 406.2489.

C

₂₃H₂₈N₃O [M+H]⁺362.2230, found 362.2227.

4.1.4.2. N-(2-(1-Benzylpiperidin-4-yl)ethyl)-3-(1H-indol-3-yl)

propanamide (4u). 3-(1H-Indol-3-yl)propanoic acid was treated

with 2-(1-benzylpiperidin-4-yl)ethanamine according to the gen-

eral procedure to give the desired product 4u as white solid in

4.1.3.18.

N-(2-(1H-Indol-3-yl)ethyl)-1-(2-nitrobenzyl)piperi-

dine-4-carboxamide (4r). Intermediate 3n was treated with

tryptamine according to the general procedure to give the desired

45.2% yield. IR (KBr) m 3305, 2924, 2852, 1631, 1557, 1452, 1104,

product 4r as yellow oil in 51.2% yield. IR (KBr)

m

3412, 2964, 1647,

844, 771 cm^ꢀ1; mp 105–110 °C; ESI/MS m/z: 390.2 [M+H]⁺; ¹H

NMR (500 MHz, DMSO) d 10.75 (s, 1H), 7.76 (t, J = 5.3 Hz, 1H), 7.53

1263, 1026, 804 cm^ꢀ1; mp 125–128 °C; ESI/MS m/z: 407.2 [M+H]⁺;

Please cite this article in press as: Wang, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.025

J. Wang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

13

(d, J = 7.8 Hz, 1H), 7.34 (d, J = 8.2 Hz, 1H), 7.33 (s, 1H), 7.32 (s, 1H),

7.30 (s, 1H), 7.29 (s, 1H), 7.25 (t, J = 6.9 Hz, 1H), 7.09 (d, J = 2.1 Hz,

1H), 7.07 (d, J = 7.2 Hz, 1H), 6.98 (t, J = 7.4 Hz, 1H), 3.45 (s, 2H),

3.08 (d, J = 6.3 Hz, 2H), 2.92 (t, J = 7.5 Hz, 2H), 2.77 (d, J = 11.3 Hz,

2H), 2.43 (t, J = 7.7 Hz, 2H), 1.88 (t, J = 11.0 Hz, 2H), 1.60 (d,

J = 12.1 Hz, 2H), 1.43–1.29 (m, 2H), 1.20 (s, 1H), 1.12 (d, J = 9.2 Hz,

2H). ¹³C NMR (125 MHz, DMSO) d 172.13, 136.73, 129.36, 128.60,

127.55, 127.37, 122.53, 121.33, 118.81, 118.57, 114.38, 111.77,

62.80, 53.60, 36.82, 36.53, 36.34, 33.11, 32.10, 21.56. HRMS: calcu-

lated for C₂₅H₃₂N₃O [M+H]⁺390.2539, found 390.2540.

proportions of both components varied between 0% and 100%.

The UV spectra were normalized to the respective blanks. The

absorbance differences at 219 nm were plotted versus the mole

fraction, showing a maximum at 0.5 that revealed a stoichiometry

of 1:1 for complex 4u–Cu(II).

4.5. Inhibition of Ab

self-induced aggregation⁴²

1-42

Inhibition of Ab_1–42aggregation was measured using a Thioﬂa-

vinT (ThT)-binding assay. Ab_1–42was dissolved in HFIP to a concen-

tration of 1 mg/mL, sonicated in a water bath for 10 min, aliquoted

into microcentrifuge tubes, dried under vacuum, and stored in

ꢀ20 °C. For the inhibition of self-induced Ab_1–42aggregation exper-

iment, the Ab was diluted with 50 mM phosphate buffer (pH = 7.4)

4.2. In vitro inhibition experiments of ChEs³⁷

Acetylcholinesterase (AChE, E.C. 3.1.1.7) from electric eel and

human erythrocytes, butyrylcholinesterase (BuChE, E.C. 3.1.1.8)

from equine serum and human serum, S-butyrylthiocholine iodide

(BTCI), acetylthiocholine iodide (ATCI), 5,5⁰-dithiobis-(2-nitroben-

zoic acid) (Ellman’s reagent, DTNB) were purchased from

Sigma–Aldrich (St. Louis, MO, USA). The inhibitory activities of test

compounds 4a–u ware evaluated by Ellman’s method. The proto-

types tacrine and donepezil were used as reference compounds.

The compounds were dissolved in DMSO and diluted with the

buffer solution (50 mM Tris–HCl, pH = 8.0, 0.1 M NaCl, 0.02 M

MgCl₂ꢁ6H₂O) to yield corresponding test concentrations (DMSO

to 50

lM before use. Each tested compound was ﬁrstly prepared in

DMSO at a concentration of 10 mM and 1

lL of each was added to

the well of black, opaque Corning 96-well platessuch that the ﬁnal

solvent concentration was 10%. The ﬁnal concentration of each

compound was 20

lM and was prepared in independent tripli-

cates. The solvent control was also included. Then, 9

lL of 25 lM

Ab_1–42sample was added to each well and the samples mixed by

gentle trapping. Plates were covered to minimize evaporation

and incubated in dark at room temperature for 46–48 h with no

agitation After incubation, the samples were diluted to a ﬁnal vol-

less than 0.01%). In each well of the plate, 160

50 L of AChE (0.22 U/mL eeAChE or 0.05 U/mL hAChE) or 50

BuChE (0.12 U/mL eqBuChE or 0.024 U/mL hBuChE) were incubated

with 10 L of different concentrations of test compounds (0.001–

100 M) at 37 °C for 6 min. After this period, acetylthiocholine

iodide (15 mM) or S-butyrylthiocholine iodide (15 mM) as the sub-

strate (30 L) was added and the absorbance was measured with a

lL of 1.5 mM DTNB,

ume of 200 L with 50 mM glycine NaOH buffer (pH = 8.0) contain-

l

lL of

ing ThT (5 lM). Fluorescence was measured on a SpectraMax M5

(Molecular Devices, Sunnyvale, CA, USA) multi-mode plate reader

with excitation and emission wavelengths at 446 nm and

490 nm, respectively. The ﬂuorescence intensities were compared

and the percent inhibition due to the presence of the inhibitor

was calculated by the following formula: 100 ꢀ (IF_i/IF_oꢂ 100)

where IF_iand IF_oare the ﬂuorescence intensities obtained for

Ab_1–42in the presence and in the absence of inhibitor, respectively.

l

wave length of 405 nm at different time intervals (0, 60, 120, and

180 s). The concentration of compound producing 50% of enzyme

activity inhibition (IC₅₀) was calculated by nonlinear regression

analysis of the response–concentration (log) curve, using the

Graph-Pad Prism program package (Graph Pad Software; San

Diego, CA). Results are expressed as the mean SEM of at least

three different experiments performed in triplicate.

4.6. Oxygen radical absorbance capacity assay⁴³

The ORAC method was followed. 2,2⁰-Azobis(amidinopropane)

dihydrochloride (AAPH), ( )-6-hydroxy-2,5,7,8-tetramethylchro-

mane-2-carboxylic acid (trolox), and ﬂuorescein (FL) were pur-

chased from Sigma–Aldrich. All the assays were conducted with

75 mM phosphate buffer (pH = 7.4), and the ﬁnal reaction mixture

4.3. Kinetic analysis of ChE inhibition³⁷

To obtain of the mechanism of action 4u, reciprocal plots of 1/

velocity versus 1/substrate were constructed at different concen-

trations of the substrate thiocholine iodide (0.05–0.5 mM) by using

Ellman’s method. Three concentrations of 4u were selected for the

studies: 400.0, 200.0 and 100.0 nM for the kinetic analysis of AChE

inhibition; 80.0, 40.0 and 20.0 nM for the kinetic analysis of BuChE

inhibition. The plots were assessed by a weighted least-squares

analysis that assumed the variance of velocity (v) to be a constant

percentage of v for the entire data set. Data analysis was performed

with Graph Pad Prism 4.03 software (San Diego, CA, USA).

was 200 lL. Antioxidant (20 lL) and ﬂuorescein (120 lL, 70 nM

ﬁnal concentration) were placed in the wells of a black 96-well

plate. The mixture was pre-incubated for 15 min at 37 °C, and then

AAPH solution (60 lL, 12 mM ﬁnal concentration) was added

rapidly using an autosampler. The plate was immediately placed

in a Varioskan Flash Multimode Reader (Thermo Scientiﬁc) and

the ﬂuorescence recorded every minute for 120 min with excita-

tion at 485 nm and emission at 538 nm. The plate was automati-

cally shaken prior to each reading. Trolox was used as standard

(1–10 lM, ﬁnal concentration). A blank (FL + AAPH) using phos-

phate buffer instead of antioxidant and trolox calibration were car-

ried out in each assay. The samples were measured at different

4.4. Metal-chelating study⁴⁰

concentration (1–10 lM). All the reaction mixture was prepared

The chelating studies were performed with a UV–vis spec-

trophotometer. The absorption spectra of each compound

in duplicate, and at least three independent assays were performed

for each sample. Raw data were exported from Fluostar Galaxy

Software to an Excel sheet for further calculations. Antioxidant

curves (ﬂuorescence vs time) were ﬁrst normalized to the curve

of the blank corresponding to the same assay, and the area under

the ﬂuorescence decay curve (AUC) was calculated. The net

AUC corresponding to a sample was calculated by subtracting the

AUC corresponding to the blank. Regression equations between

net AUC and antioxidant concentration were calculated for all sam-

ples. ORACFL values were expressed as trolox equivalents by using

the standard curve calculated for each assay, where the ORAC-FL

value of trolox was taken to be 1.0.

(2.5

lM, ﬁnal concentration) alone or in the presence of CuSO₄,

FeSO₄, FeCl₃or ZnCl₂(5

l

M, ﬁnal concentration) for 30 min in20%

(v/v) ethanol/buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) were

recorded at room temperature in a 1 cm quartz cell. The stoichiom-

etry of the compoundꢀCu²⁺complex was determined by employ-

ing Job’s method. Separate solutions of compound and CuSO₄

were used to prepare solutions in which the sum of the concentra-

tions of both species (2.5 ꢂ 10^ꢀ6M) was constant in all samples, 21

solutions were obtained with the condition that the sum of con-

centrations of both species was a constant in all samples but the

Please cite this article in press as: Wang, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.025

14

J. Wang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

4.7. Cell culture and measurement of cell viability⁴⁴

The X-ray crystallographic complex of AChE with donepezil (PDB

code 4EY7) and hBuChE (PDB code 1P0I) were obtained from the

Protein Data Bank. All water molecules in PDB ﬁles were removed

and hydrogen atoms were subsequently added to the protein. The

compound 4u was built using the builder interface of the MOE pro-

gram and energy minimized using MMFF94x force ﬁeld. Then the

4u was docked into the active site of the protein by the ‘Triangle

Matcher’ method, which generated poses by aligning the ligand tri-

plet of atoms with the triplet of alpha spheres in cavities of tight

atomic packing. The Dock scoring in MOE software was done using

ASE scoring function and Force ﬁeld was selected as the reﬁnement

method. The best 10 poses of molecules were retained and scored.

After docking, the geometry of resulting complex was studied

using the MOE’s pose viewer utility.

The toxicity effect of the tested compounds on the rat

pheochromocytoma (PC12) cells was examined according to previ-

ously reported methods. The PC12 cells routinely grown at 37 °C in

a humidiﬁed incubator with 5% CO₂in Dulbecco’s modiﬁed Eagle’s

medium (DMEM) supplemented with 10% bovine calf serum,

100 Units per mL penicillin, and 100 Units per mL of streptomycin.

Cells were sub-cultured in 96-well plates at a seeding density of

10,000 cells per well and allowed to adhere and grow. When cells

reached the required conﬂuence, they were placed into serum-free

medium and treated with compounds 4b, 4c, 4p, 4q, 4t, 4u.

Twenty-four hours later the survival of cells was determined by

MTT assay. Brieﬂy, after incubation with 20

for 4 h, living cells containing MTT formazan crystals were solubi-

lized in 200 L DMSO. The absorbance of each well was measured

lL of MTT at 37 °C

l

Acknowledgements

using a microculture plate reader with a test wavelength of 570 nm

and a reference wavelength of 630 nm. Results are expressed as the

mean SD of three independent experiments.

PC 12 cells was obtained from the Cell Bank of the Chinese

Academy of Sciences (Shanghai, China) and grown in RPMI-1640

medium containing 10% (v/v) foetal bovine serum, 100 U peni-

cillin/mL and 100 mg streptomycin/mL under 5% CO₂at 37 °C. The

culture media were changed every other day. After pretreatment

with different concentrations of test compound (1.25, 2.5, 5.0,

We gratefully acknowledge the ﬁnancial support of Program for

Changjiang Scholars and Innovative Research Team in University

(IRT_15R63), Project Funded by the Priority Academic Program

Development of Jiangsu Higher Education Institutions (PAPD) and

the National Natural Science Foundation of China (81573313).

Supplementary data

10 lM) for 2 h, PC 12 cells were incubated with 100 lM of H₂O₂

for 24 h. The cell viability was evaluated using MTT assay. Brieﬂy,

the cells were treated with 10 ml MTT (5 mg/ml in PBS) for 4 h at

Supplementary data associated with this article can be found, in

the online version, at http://dx.doi.org/10.1016/j.bmc.2016.07.025.

37 °C. Then, 200 lL of dimethylsulfoxide (DMSO) was added to dis-

References and notes

solve the dark blue formazan crystals formed in intact cells, and the

absorbance at 570 nm was detected by a microplate reader. PC12

cells were cultured without test compound or H₂O₂as control

group and the results were expressed by percentage of control.

1. Finder, V. H. J. Alzheimers Dis. 2010 , 22 , 5.

2. Scarpini, E.; Scheltens, P.; Feldman, H. Lancet Neurol. 2003 , 2 , 539.

3. Folch, J.; Petrov, D.; Ettcheto, M.; Abad, S.; Sánchez-López, E.; García, M. L.;

Olloquequi, J.; Beas-Zarate, C.; Auladell, C.; Camins, A. Neural Plast. 2016 , 2016 , 1.

4. Sang, Z.; Qiang, X.; Li, Y.; Yuan, W.; Liu, Q.; Shi, Y.; Ang, W.; Luo, Y.; Tan, Z.;

Deng, Y. Eur. J. Med. Chem. 2015 , 94 , 348.

5. Li, S. Y.; Wang, X. B.; Kong, L. Y. Eur. J. Med. Chem. 2014 , 71 , 36.

6. Nepovimova, E.; Uliassi, E.; Korabecny, J.; Pena-Altamira, L. E.; Samez, S.;

Pesaresi, A.; Garcia, G. E.; Bartolini, M.; Andrisano, V.; Bergamini, C.; Fato, R.;

Lamba, D.; Roberti, M.; Kuca, K.; Monti, B.; Bolognesi, M. L. J. Med. Chem. 2014 ,

57 , 8576.

7. Huang, L.; Lu, C.; Sun, Y.; Mao, F.; Luo, Z.; Su, T.; Jiang, H.; Shan, W.; Li, X. J. Med.

Chem. 2012 , 55 , 8483.

8. Castro, A.; Conde, S.; Rodriguez-Franco, M. I.; Martinez, A. Mini-Rev. Med. Chem.

2002 , 2 , 37.

9. Kuca, K.; Soukup, O.; Maresova, P.; Korabecny, J.; Nepovimova, E.; Klimova, B.;

Honegr, J.; Ramalhob, T. C.; Françab, T. C. J. Braz. Chem. Soc. 2016 ,

27 , 641.

10. Lopez-Iglesias, B.; Perez, C.; Morales-Garcia, J. A.; Alonso-Gil, S.; Perez-Castillo,

A.; Romero, A.; Lopez, M. G.; Villarroya, M.; Conde, S.; Rodriguez-Franco, M. I. J.

Med. Chem. 2014 , 57 , 3773.

4.8. In vitro blood–brain barrier permeation assay⁴⁵

The ability of test compounds that penetrate into brain was

evaluated using a parallel artiﬁcial membrane permeation assay

(PAMPA) for blood–brain barrier according to the method estab-

lished by Di et al. Commercial drugs were purchased from Sigma

and Alfa Aesar. The porcine brain lipid (PBL) was obtained from

Avanti Polar Lipids. The donor microplate (PVDF membrane, pore

size is 0.45 mm) and the acceptor microplate were both from Mil-

lipore. The 96-well UV plate (COSTAR@) was from Corning Incorpo-

rated. The compound was ﬁrstly dissolved in DMSO at

a

concentration of 5 mg/mL. Then, it was diluted 100-fold with a

mixture of PBS/EtOH (70:30) to give a ﬁnal concentration of

11. Nepovimova, E.; Korabecny, J.; Dolezal, R.; Babkova, K.; Ondrejicek, A.; Jun, D.;

Sepsova, V.; Horova, A.; Hrabinova, M.; Soukup, O.; Bukum, N.; Jost, P.;

Muckova, L.; Kassa, J.; Malinak, D.; Andrs, M.; Kuca, K. J. Med. Chem. 2015 , 58 ,

8985.

12. Chen, Y.; Sun, J.; Fang, L.; Liu, M.; Peng, S.; Liao, H.; Lehmann, J.; Zhang, Y. J.

Med. Chem. 2012 , 55 , 4309.

50

with PBL dissolved in dodecane (4

200 L of diluted solution and 300 L of PBS/EtOH (70:30) were

lg/mL. The ﬁlter membrane in donor microplate was coated

l

L, 20 mg/mL). After that,

l

added to the donor wells and the acceptor wells, respectively.

The acceptor ﬁlter plate was carefully placed on the donor plate

to form a sandwich, which was left undisturbed for 16 h at 25 °C.

After incubation, the donor plate was carefully removed and the

concentration of compound in the acceptor wells was determined

using a UV plate reader (Flexstation@ 3). Every sample was ana-

lyzed at ﬁve wave lengths, in four wells, in at least three indepen-

dent runs, and the results are given as the mean standard

deviation. In each experiment, 9 quality control standards of

known BBB permeability were included to validate the analysis set.

13. Xie, S. S.; Lan, J. S.; Wang, X. B.; Jiang, N.; Dong, G.; Li, Z. R.; Wang, K. D.; Guo, P.

P.; Kong, L. Y. Eur. J. Med. Chem. 2015 , 93 , 42.

14. Viayna, E.; Sabate, R.; Munoz-Torrero, D. Curr. Top. Med. Chem. 2013 , 13 , 1820.

15. Jiang, N.; Li, S. Y.; Xie, S. S.; Li, Z. R.; Wang, K. D.; Wang, X. B.; Kong, L. Y. Eur. J.

Med. Chem. 2014 , 87 , 540.

16. Rees, T.; Hammond, P. I.; Soreq, H.; Younkin, S.; Brimijoin, S. Neurobiol. Aging

2003 , 24 , 777.

17. Inestrosa, N. C.; Dinamarca, M. C.; Alvarez, A. FEBS J. 2008 , 275 , 625.

18. Li, S. Y.; Wang, X. B.; Xie, S. S.; Jiang, N.; Wang, K. D.; Yao, H. Q.; Sun, H. B.; Kong,

L. Y. Eur. J. Med. Chem. 2013 , 69 , 632.

19. Wang, Y.; Guan, X. L.; Wu, P. F.; Wang, C. M.; Cao, H.; Li, L.; Guo, X. J.; Wang, F.;

Xie, N.; Jiang, F. C.; Chen, J. G. J. Med. Chem. 2012 , 55 , 3588.

20. Sang, Z.; Li, Y.; Qiang, X.; Xiao, G.; Liu, Q.; Tan, Z.; Deng, Y. Bioorg. Med. Chem.

2015 , 23 , 668.

21. Fernández-Bachiller, M. I.; Pérez, C.; Monjas, L.; Rademann, J.; Rodríguez-

Franco, M. I. J. Med. Chem. 2012 , 55 , 1303.

4.9. Molecular modeling studies³⁸

22. Reddy, V. P.; Zhu, X.; Perry, G.; Smith, M. A. J. Alzheimers Dis. 2009 , 16 , 763.

23. Ansari, M. A.; Scheff, S. W. J. Neuropath. Exp. Neur. 2010 , 69 , 155.

24. Moreira, P. I.; Santos, M. S.; Oliveira, C. R.; Shenk, J. C.; Nunomura, A.; Smith, M.

A.; Zhu, X.; Perry, G. CNS Neurol. Disord-Dr. 2008 , 7 , 3.

Molecular modeling calculations and docking studies were per-

formed using Molecular Operating Environment (MOE) software

version 2008.10 (Chemical Computing Group, Montreal, Canada).

Please cite this article in press as: Wang, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.025

J. Wang et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx

15

25. Gu, F.; Zhu, M.; Shi, J.; Hu, Y.; Zhao, Z. Neurosci. Lett. 2008 , 440 , 44.

26. Jiang, C. S.; Fu, Y.; Zhang, L.; Gong, J. X.; Wang, Z. Z.; Xiao, W.; Zhang, H. Y.; Guo,

Y. W. Bioorg. Med. Chem. Lett. 2015 , 25 , 216.

27. Lan, J. S.; Xie, S. S.; Li, S. Y.; Pan, L. F.; Wang, X. B.; Kong, L. Y. Bioorg. Med. Chem.

2014 , 22 , 6089.

28. Digiacomo, M.; Chen, Z.; Wang, S.; Lapucci, A.; Macchia, M.; Yang, X.; Chu, J.;

Han, Y.; Pi, R.; Rapposelli, S. Bioorg. Med. Chem. Lett. 2015 , 25 , 807.

29. Yang, S.; Liu, W.; Lu, S.; Tian, Y.-Z.; Wang, W.-Y.; Ling, T.-J.; Liu, R.-T. ACS Chem.

Neurosci. 2016.

30. Beniyama, Y.; Matsuno, K.; Miyachi, H. Bioorg. Med. Chem. Lett. 2013 , 23 , 1662.

31. Fernandez-Bachiller, M. I.; Perez, C.; Gonzalez-Munoz, G. C.; Conde, S.; Lopez,

M. G.; Villarroya, M.; Garcia, A. G.; Rodriguez-Franco, M. I. J. Med. Chem. 2010 ,

53 , 4927.

32. Xie, S.; Chen, J.; Li, X.; Su, T.; Wang, Y.; Wang, Z.; Huang, L.; Li, X. Bioorg. Med.

Chem. 2015 , 23 , 3722.

33. Liang, S. H.; Southon, A. G.; Fraser, B. H.; Krause-Heuer, A. M.; Zhang, B.; Shoup,

T. M.; Lewis, R.; Volitakis, I.; Han, Y.; Greguric, I.; Bush, A. I.; Vasdev, N. ACS

Med. Chem. Lett. 2015 , 6 , 1025.

34. Costanzo, P.; Cariati, L.; Desiderio, D.; Sgammato, R.; Lamberti, A.; Arcone, R.;

Salerno, R.; Nardi, M.; Masullo, M.; Oliverio, M. ACS Med. Chem. Lett. 2016.

35. Luo, Z.; Sheng, J.; Sun, Y.; Lu, C.; Yan, J.; Liu, A.; Luo, H. B.; Huang, L.; Li, X. J. Med.

Chem. 2013 , 56 , 9089.

43. Zha, G.-F.; Zhang, C.-P.; Qin, H.-L.; Jantan, I.; Sher, M.; Amjad, M. W.; Hussain,

M. A.; Hussain, Z.; Bukhari, S. N. A. Bioorg. Med. Chem. 2016 , 24 , 2352.

44. Zemek, F.; Drtinova, L.; Nepovimova, E.; Sepsova, V.; Korabecny, J.; Klimes, J.;

Kuca, K. Expert Opin. Drug Saf. 2014 , 13 , 759.

45. Cheng, S.; Zheng, W.; Gong, P.; Zhou, Q.; Xie, Q.; Yu, L.; Zhang, P.; Chen, L.; Li, J.;

Chen, J.; Chen, H.; Chen, H. Bioorg. Med. Chem. 2015 , 23 , 3110.

46. Buendia, I.; Navarro, E.; Michalska, P.; Gameiro, I.; Egea, J.; Abril, S.; López, A.;

González-Lafuente, L.; López, M. G.; León, R. Future Med. Chem. 2015 , 7 , 1961.

47. Spuch, C.; Antequera, D.; Isabel Fernandez-Bachiller, M.; Isabel Rodriguez-

Franco, M.; Carro, E. Neurotox. Res. 2010 , 17 , 421.

48. Zawadzka, A.; Lozinska, I.; Moleda, Z.; Panasiewicz, M.; Czarnocki, Z. J. Pineal

Res. 2013 , 54 , 435.

49. Spuch, C.; Antequera, D.; Fernandez-Bachiller, M. I.; Rodríguez-Franco, M. I.;

Carro, E. Neurotox. Res. 2010 , 17 , 421.

50. Fernandez-Bachiller, M. I.; Perez, C.; Campillo, N. E.; Paez, J. A.; Gonzalez-

Munoz, G. C.; Usan, P.; Garcia-Palomero, E.; Lopez, M. G.; Villarroya, M.; Garcia,

A. G.; Martinez, A.; Rodriguez-Franco, M. I. ChemMedChem 2009 , 4 , 828.

51. Pourshojaei, Y.; Gouranourimi, A.; Hekmat, S.; Asadipour, A.; Rahmani-Nezhad,

S.; Moradi, A.; Nadri, H.; Moghadam, F. H.; Emami, S.; Foroumadi, A.; Shaﬁee, A.

Eur. J. Med. Chem. 2015 , 97 , 181.

52. Ellman, G. L.; Courtney, K. D.; Andres, V.; Featherstone, R. M. Biochem.

Pharmacol. 1961 , 7 , 88.

36. Korabecny, J.; Dolezal, R.; Cabelova, P.; Horova, A.; Hruba, E.; Ricny, J.; Sedlacek,

L.; Nepovimova, E.; Spilovska, K.; Andrs, M.; Musilek, K.; Opletalova, V.;

Sepsova, V.; Ripova, D.; Kuca, K. Eur. J. Med. Chem. 2014 , 82 , 426.

37. Yanovsky, I.; Finkin-Groner, E.; Zaikin, A.; Lerman, L.; Shalom, H.; Zeeli, S.;

Weill, T.; Ginsburg, I.; Nudelman, A.; Weinstock, M. J. Med. Chem. 2012 , 55 ,

10700.

53. Geng, J.; Li, M.; Wu, L.; Ren, J.; Qu, X. J. Med. Chem. 2012 , 55 , 9146.

54. Sharma, A. K.; Pavlova, S. T.; Kim, J.; Finkelstein, D.; Hawco, N. J.; Rath, N. P.;

Kim, J.; Mirica, L. M. J. Am. Chem. Soc. 2012 , 134 , 6625.

55. Baum, L.; Ng, A. J. Alzheimers Dis. 2004 , 6 , 367.

56. Prati, F.; Bergamini, C.; Fato, R.; Soukup, O.; Korabecny, J.; Andrisano, V.;

Bartolini, M.; Bolognesi, M. L. ChemMedChem 2016.

38. Pevet, P. Therapie 1998 , 53 , 411.

57. Jameson, L. P.; Dzyuba, S. V. Bioorg. Med. Chem. Lett. 2013 , 23 , 1732.

58. Davalos, A.; Gomez-Cordoves, C.; Bartolome, B. J. Agric. Food Chem. 2004 , 52 , 48.

59. Magliaro, B. C.; Saldanha, C. J. Brain Res. 2009 , 1283 , 14.

60. Di, L.; Kerns, E. H.; Fan, K.; McConnell, O. J.; Carter, G. T. Eur. J. Med. Chem. 2003 ,

38 , 223.

39. Chojnacki, J. E.; Liu, K.; Yan, X.; Toldo, S.; Selden, T.; Estrada, M.; Rodriguez-

Franco, M. I.; Halquist, M. S.; Ye, D.; Zhang, S. ACS Chem. Neurosci. 2014 , 5 , 690.

40. Stevens, C. B.; Hanna, J. M., Jr.; Lammi, R. K. Bioorg. Med. Chem. Lett. 2013 , 23 ,

1703.

41. Bukhari, S. N. A.; Jantan, I.; Masand, V. H.; Mahajan, D. T.; Sher, M.; Naeem-ul-

Hassan, M.; Amjad, M. W. Eur. J. Med. Chem. 2014 , 83 , 355.

42. Nasir Abbas Bukhari, S.; Jantan, I. Mini-Rev. Med. Chem. 2015 , 15 , 1110.

61. Muzammil, S.; Armstrong, A.; Kang, L.; Jakalian, A.; Bonneau, P.; Schmelmer, V.;

Amzel, L.; Freire, E. J. Virol. 2007 , 81 , 5144.

Please cite this article in press as: Wang, J.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.07.025

Article Doi

Synthesis and evaluation of multi-target-directed ligands for the treatment of Alzheimer's disease based on the fusion of donepezil and melatonin

DOI: 10.1016/j.bmc.2016.07.025

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.bmc.2016.07.025

Full text of DOI:10.1016/j.bmc.2016.07.025

Products guided by the article

R&D Labs maybe for 897094-25-6

Relevant to this article

Hot Product