Identification and optimization of piperine analogues as neuroprotective agents for the treatment of Parkinson's disease via the activation of Nrf2/keap1 pathway

European Journal of Medicinal Chemistry 199 (2020) 1 12385

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Identiﬁcation and optimization of piperine analogues as

neuroprotective agents for the treatment of Parkinson’s disease via the

activation of Nrf2/keap1 pathway

,

Lun Wang ^{a 1}, Xiaoying Cai ^{a 1}, Mingsong Shi ^{a 1}, Linlin Xue ^a, Shuang Kuang ^a, Ruiling Xu ^a,

Wenyan Qi ^a, Yan Li ^b, Xu Ma ^a, Ruijia Zhang ^a, Feng Hong ^a, Haoyu Ye ^a^**, Lijuan Chen ^a

^aState Key Laboratory of Biotherapy/Collaborative Innovation Center of Biotherapy and Cancer Center, West China Hospital of Sichuan University, Chengdu,

,

, *

610041, China

^bDepartment of Pharmaceutical and Biological Engineering, School of Chemical Engineering, Sichuan University, Chengdu, 610065, 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:

Parkinson’s disease (PD) is a slowly progressive and complex neurodegenerative disorder. Up to date,

there are no approved drugs that could slow or reverse the neurodegenerative process of PD. Here, we

reported the synthesis of series of piperine analogues and the evaluation of their neuroprotective effects

against hydrogen peroxide (H₂O₂) induced damage in the neuron-like PC12 cells. Among these ana-

logues, 3b exhibited the most potent protection effect and its underlying mechanism was further

investigated. Further results indicated that the ROS scavenging and cytoprotection effect of 3b might be

related to the Nrf2 activation and upregulation of related phase II antioxidant enzymes, such as HO-1 and

NQO1. In in vivo study, oral administration (100 mg/kg) of 3b signiﬁcantly attenuated PD-associated

behavioral deﬁcits in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of

PD and protected tyrosine hydroxylase-immunopositive dopaminergic neurons. Our results provided

evidence that 3b might be a promising candidate for Parkinson’s disease treatment.

Received 8 January 2020

Received in revised form

8 April 2020

Accepted 23 April 2020

Available online 5 May 2020

Keywords:

Parkinson’s disease

Piperine analogues

Nrf2 activation

MPTP

1. Introduction

treatment. Among the agents, levodopa remains the cornerstone

for managing PD patients, especially in the early stages [4]. Other

Parkinson’s disease (PD) is one of the most common incurable

neurodegenerative diseases, which affects about 1% of global

elderly over sixty ages and more than 3% over eighty ages, that

brings severe ﬁnancial and society burden [1]. It has been charac-

terized by cardinal motor symptoms, such as resting tremor,

muscular rigidity, akinesia, and postural instability resulting from

the loss of dopamine (DA) neurons in the substantia nigra (SN) and

the depletion of the neurotransmitter DA in the striatum [2].

Currently, dopamine precursor levodopa [3] (dopaminergic agents)

as well as adamantamine hydrochloride and cabergoline (direct or

indirect dopamine agonist) are the most widely used drugs for PD

drugs are generally used in combination with levodopa to control

speciﬁc symptoms or to enhance levodopa activity [3]. However,

those drugs only ameliorate motor symptoms for a short period of

time and even cause side effects, such as L-DOPA-induced dyski-

nesia, when taken for long periods [5,6]. So far, no drugs have been

developed to slow or reverse the neurodegenerative process in PD

[7]. Therefore, it is important to develop new therapies preventing

the death of DA neurons in PD-affected brain areas.

Recently, the role of oxidative stress in neurological abnormal-

ities, including PD, has been particularly addressed. Oxidative stress

is a disturb of the balance of cellular antioxidant defense systems by

over expression of reactive oxygen species (ROS) [8]. Nuclear factor

erythroid 2-related factor 2 (Nrf2) is the master regulator of cellular

defense against oxidative stress [9]. Under normal conditions, Nrf2

is rapidly degraded (half-life ~ 20min) by the Kelch like ECH-

associated protein 1 (Keap1), an adaptor in the Cul3 (cullin3)-

based ubiquitin E3 ligase, mediated ubiquitin proteasome system.

Under stress conditions, oxidants and electrophiles react with

* Corresponding author.

** Corresponding author.

E-mail addresses: yhy1984_hb@163.com (H. Ye), chenlijuan125@163.com

(L. Chen).

1

These authors contributed equally.

https://doi.org/10.1016/j.ejmech.2020.1 12385

2

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

cysteine sensors within Keap1, causing a conformational change of

Keap1 and a detachment of Nrf2 from Keap1, which allows Nrf2 to

translocate to the nucleus where Nrf2 binds to antioxidant

responsive elements (ARE) via hetero-dimerization with small Maf

proteins, activating the expression of its target genes, including

several antioxidant and detoxiﬁcation genes, such as heme

oxygenase-1 (HO-1), superoxide dismutase (SOD), NAD(P)H: qui-

noneoxidoreductase (NQO1), the glutamate-cysteine ligasemodifer

(GCLM) and the catalytic (GCLC) subunit [10,1 1]. Therefore, acti-

vating the Keap1/Nrf2 signaling pathway has been considered to be

able to reduce and prevent neuronal cell death, representing a

promising avenue for PD therapy [12,13].

modulators [9]. The ﬁrst Nrf2-inducer dimethyl fumarate (Tecﬁ-

dera) with the mechanism of reacting with Cys151 of Keap 1 pro-

tein has been approved by FDA for the treatment of remitting-

relapsing multiple sclerosis and psoriasis [14,15]. CDDO, the ﬁrst

phase II clinical drug for the treatment of diabetic nephropathy, and

its methyl ester (bardoxolone methyl or CDDO-Me), phase III for the

treatment of chronic kidney disease and hypertension, pulmonary

arterial are also thought to react with Cys151 for the activation of

Nrf2 through its

a,b-unsaturated scaffold [16]. Some other Nrf2

activators based on different scaffolds are under clinical develop-

ment at various stages, such as cyanoenone triterpenoids, fumaric

acid analogues, curcumin, and isothiocyanate sulforaphane (SFN)

Covalently modifying the thiol-rich Keap1 protein via electro-

philic molecules is the major strategy for Nrf2 activation, which

sparked the interest of the pharmaceutical industry and led to a

substantial improvement in the clinical development of NRF2

[16]. Thus, chemicals with electrophilic scaffold, especially a,b-

unsaturated ketone structure, have the potentials for the treatment

of PD through Nrf2 activation. Piperine (PIP) as a major alkaloid,

isolated from the fruits of Piper longum and Piper nigrum [1 7],

Fig. 1. Neuroprotective effect of 3b against H₂O₂induced PC12 cell damage. (A) The cytoprotection of 3b in H₂O₂induced PC12 cell damage, determined by MTT assays. (B) The

effect on LDH release of 3b in H₂O₂induced PC12 cell damage. (C) The apoptosis inhibition effects of 3b, determined by ﬂow cytometry. (D, E) The ROS level in PC12 cells were

assessed using DCFH-DA staining (D) and ﬂow cytometry (E); The ﬂuorescence images (bottom panel) indicated the elimination of H₂O₂-induced ROS in PC12 cells by 3b. The

corresponding phase contrast images were shown in the top panel. All data represent the means SD of three independent experiments. ***, P < 0.001 vs the vehicle group; ^∧∧∧

P < 0.001 vs the H₂O₂-treated group.

,

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

3

demonstrated various of biological activities [18e24]. Its

a

,

b

-un-

Heterocycles are common fragments of the vast majority of

marketed drugs and play central roles in drug design [28e30]. The

majority nature of heterocycles are served as hydrogen bond do-

nors and receptors [28e30]. Thus, we further used heterocycles to

replace the aromatic group in the scaffold of PIP, such as 2-furan

(3q), 2-thiophene (3r) and N-methyl-2-pyrrole (3u). The results

(Table 2) showed that 3s, 3t and 3v exhibited improved activity

with cell viability of 77%, 77% and 67% respectively, compared with

PIP (3n) (56%) and when piperidine of 3r was substituted with the

diethylamine and ethylamine, the cell viability were obviously

improved with 20%, which was in accordance with 4a.

Since 2-Piperidinone (Chart 1), which contained shorter un-

saturated chain compared with PIP, exhibited good neuroprotective

activity [13]. We hypothesized that shorter unsaturated chain may

be beneﬁcial to improve the neuroprotective activity of PIP. To test

the hypothesis, compound 7a-7n were synthesized (Table 3). For

the length of the unsaturated chain, there was no rules to follow at

least within compounds that we reported. For instance, 7a (73%),

7b (69%), 7f (77%), 7g (72%) and 7h (70%) with an unsaturated

double bond has superior activity than corresponding two unsat-

urated double bonds compounds 3h (65%), 3i (64%), 3n (56%), 3o

(31%) and 3p (22%), while 7c (43%), 7i (81%), 7j (78%), 7k (68%), 7m

(54%) and 7n (57%) has lower activity than 3j (61%), 3b (89%), 3d

(81%), 3e (75%), 3s (77%) and 3t (77%). In sum, the initial SARs study

of PIP provided valuable information for further study of the neu-

roprotective effects of PIP analogues.

The property of blood-brain barrier (BBB) penetration is one of

the primary challenges for neuroprotective agents in vivo. Hence,

ADMET-BBB Level were calculated for all analogues to predict the

BBB penetration ability based on the following parameters: (I) the

calculated cLogP; (II) the calculated cLogD (at pH 7.4); (III) the most

basic center (pKa); (Ⅳ) the molecular weight (MW), (Ⅵ) the to-

pological polar surface area (TPSA) and the number of hydrogen

bond donors (HBD) [31]. Results revealed that all the compounds

showed high BBB penetration level (Tables S2 and S3). We also

performed an in vitro BBB model using bEnd.3 cells to predict the

membrane penetration of 3b (Table S4). The permeability of 3b (Pe:

3033.59 ꢀ 10^ꢁ6cm/s) was found to be better than that of piperine

(Pe: 2551.95 ꢀ 10^ꢁ6cm/s). Considering 3b demonstrated the best

protection effects against H₂O₂-induced PC12 cell damage and good

BBB permeability, its underlying mechanism was further evaluated.

saturated ketone structure makes a perfect lead for the Nrf2 acti-

vator development. Previously, PIP has been reported to exhibit

good neuroprotective effects in both 1-methyl-4-phenyl-1,2,3,6-

tetrahydropyridine hydrochloride (MPTP) or 6-OHDA induced

Parkinson’s animal models through antioxidant, anti-apoptotic and

anti-inﬂammatory mechanism [25,26]. In order to improve the

neuroprotective activity of PIP, we synthesized a series of PIP ana-

logues following the strategy shown in Chart 2 and evaluated their

neuroprotective activities in H₂O₂induced PC12 cell damage

model. Among the forty-one PIP derivatives, 3b exhibited the best

neuroprotective effect. The cellular assay showed that 3b demon-

strated cytoprotection effects via suppression of ROS accumulation

and restoration of mitochondrial membrane potential in PC12 cells,

which might be related to the activation of Nrf2 and the expression

of corresponding antioxidant protein by promoting Nrf2 entry into

the nucleus, thereby activating cellular oxidative stress and pro-

tecting PC12 cells. Moreover, the neuroprotective effect of 3b was

abolished when Nrf2 was knockdown by shRNA transfection, which

further conﬁrmed that Nrf2 was crucial for the neuroprotective

effect of 3b. Molecular docking study with the homology structure

for the all length Keap1 constructed by I-TASSER online service also

showed that 3b might activate Nrf2 through covalently binding

with Cys residues in Keap1 based on the lowest binding energy. In

in vivo experiment, 3b improved motor behavior and rescued

dopaminergic neuronal cell death against MPTP induced PD mice

model.

2. Results and discussion

2.1. Protection of PC12 cells against H₂O₂-induced cell damage by

PIP analogues

Hydrogen peroxide (H₂O₂), an endogenous cellular signaling

molecule could generate exogenous free radicals immediately [27].

Neuronal damage in the nervous system induced by abnormal high

levels of H₂O₂has been implicated in AD, PD and other neurode-

generative diseases [27]. Since H₂O₂at concentration of 650 mM

could induced approximate 50% PC12 cell damage (Fig. S1), it was

then chosen for the further study. Neuroprotective effects of PIP

analogues (12.5

mM) against H₂O₂-induced damage of PC12 cell

were shown in Fig. 1 with TBHQ (tert-tutylhydroquinone) as the

positive control.

2.2. Protection of PC12 cells from H₂O₂-induced damage by 3b

As exhibited in Table 1, different substituted groups were

introduced at the 2-, 3-, and 4-position of phenyl. In this series of

analogues, the 2-methoxy substituted 3b exhibited potency neu-

roprotective effect with cell viability (89%) better than 3c and 3a (2-

OMe > 3-OMe > 4-OMe). However, when the methoxyl was

substituted with hydroxyl group, compound 4c substituted at the

3-position showed better neuroprotective effect than at 2-position,

followed by 4-position substituted 4a (3-OH > 2-OH > 4-OH). Along

with these compounds, we also synthesize several compounds

substituted with phenyl (3f), pyrrolidine (3g), piperazine (3h),

morpholine (3k), piperidine (3l), and chlorine (3m). Unfortunately,

only 3h substituted with piperazine at 4-position of phenyl showed

improved neuroprotective effect compared with piperine (3n) and

most of the 4-substituted compounds showed only marginal or

negative effect due to cytotoxicity (Table S1). To further explore the

contribution of amide moiety to the neuroprotective effect, the

piperidine of 3n, 3b, 3h and 4a was substituted with diethylamine

or ethylamine. Unexpected, these compounds showed different

structure-neuroprotection relationship. The neuroprotective ef-

fects of 3b, 3h and 3n were abolished or decreased when the amide

was substituted with diethylamine or ethylamine, while 4d (67%)

and 4e (69%) showed improved activity compared with 4a.

The viability of PC12 cells treated by 3b with the concentration

ranging from 12.5 to 100

mM was ﬁrst evaluated by MTT assay.

Result showed that 3b signiﬁcantly elevated the cell viability from

72% to 91% at tested concentration compared with H₂O₂treated

group (51%) (Fig. 1A). Lactate dehydrogenase (LDH) was a critical

marker for the membrane integrity. High content of LDH released in

the medium implied cell damage [32]. In our experiment, H₂O₂up-

regulated the content of LDH almost two folds compared with the

control. As we expected, 3b decreased the release of LDH signiﬁ-

cantly in accordance with the cell viability (Fig. 1B). However, it

could also be observed that higher concentration of 3b (50 and

100

mM) demonstrated slight toxicity from the increasing LDH

content.

2.3. Attenuation of apoptosis of PC12 cells induced by H₂O₂

To further evaluate whether apoptosis of PC12 cells induced by

H₂O₂could be attenuated by 3b, cells were stained with Annexin V-

FITC/PI and then analyzed by ﬂow cytometry. As shown in Fig. 1C,

compared with the H₂O₂treated group (47.66%), percentage of

apoptotic cells was signiﬁcantly decreased in a dose-dependent

4

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

Fig. 2. 3b could rescue the mitochondrial membrane potential in PC12 cells and upregulate the phase II antioxidant enzymes. (A) The mitochondrial membrane potential was

measured by JC-1 stain. PC12 cells were pretreated with compound 3b (12.5, 25 and 50 M) for 24 h, induced with H₂O₂for another 24 h and stained with JC-1. Data (red/green

m

ﬂuorescence ratios) were expressed as mean SD of three independent experiments. (B) 3b induced the expression of NQO1 and HO-1. PC12 cells were treated with 3b for 24 h, and

the NQO1 and HO-1 were determined by Western blot. All data represent the means SD of three independent experiments. *, P < 0.05, **, P < 0.01, and ***, P < 0.001 vs the

vehicle group; ^∧, P < 0.05 and ^∧∧, P < 0.01 vs the H₂O₂-treated group. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to the Web version of

this article.)

manner in groups pretreated by 3b (12.5e50

dance with the results of LDH assay, percentage of apoptotic cells

increased again at 100 M due to cytoxicity. Taken together, 3b

m

M). And in accor-

2.5. Protective effect on H₂O₂-induced dissipation of the

mitochondrial membrane potential

m

could protect PC12 cell apoptosis induced by H₂O₂, but exhibited

cytotoxicity at high concentration.

It is generally accepted that the mitochondrial membrane plays

an important role in cell survival and death, especially under the

inﬂuence of oxidative stress [9]. In this study, the membrane-

permeant and sensitive JC-1 dye was used to monitor mitochon-

drial membrane potential (△

(Fig. 2A), which formed red ﬂuorescent J-aggregates in healthy cells

with a normal △ m and retained its original green ﬂuorescence

jm) of H₂O₂-induced PC12 cells

2.4. Prevention of ROS accumulation in PC12 cells

j

under apoptotic condition. Our results suggested that mitochon-

drial membrane potential loss induced by H₂O₂could be sup-

pressed in a dose dependent manner by pretreatment with 3b,

indicating that the neuroprotection of 3b might be related to the

The imbalanced redox states caused by excessive reactive oxy-

gen species (ROS) production is most widely implicated in PD [33].

Further experiments were performed to ascertain whether pro-

tection effects caused by 3b were due to interference with ROS

generation and reduction of the oxidative stress. Intracellular ROS

accumulation can be measured by cell-permeable and nonﬂuo-

rescent DCFH-DA. Once diffusing into cells, it is hydrolyzed by the

cellular esterase activity to DCFH, which then interacts with ROS

forming ﬂuorescent 2⁰,7⁰-dichloroﬂuorescin [34]. Stimulation of

PC12 cells with H₂O₂led to appearance of bright-green ﬂuores-

cence, indicating the burst of ROS in the cells compared with

control group (Fig. 1D and E). Both the results of ﬂuorescence and

ﬂow cytometry indicated that cells pretreated with 3b (12.5, 25 and

△

jm restoration.

2.6. Activation of phase II antioxidant enzymes in PC12 cells

Phase II antioxidant enzymes provide efﬁcient cytoprotection by

regulating the intracellular redox state, including Heme oxygenase-

1 (HO-1) and quinone oxidoreductase 1 (NQO1) and other enzymes

[35]. NQO1 detoxiﬁes benzene-derived quinones and generate

antioxidant forms of ubiquinone and Vitamin E [32], while HO-1

catalyzes the initial and rate-limiting step in the degradation of

heme into carbon monoxide, biliverdin/bilirubin, and ferritin [36].

Therefore, we hypothesized that ROS scavenging activity of 3b

attributed to induction of phase II enzymes. As showed in Fig. 2B,

the HO-1 level pretreated by 3b was remarkably upregulated

50

H₂O_2,while the accumulation of ROS was not reduced properly

when pretreated with 100 M 3b. Consistent with the LDH assay

and ﬂow cytometry results, 3b showed toxicity to cells at 100 M.

mM) signiﬁcantly reduced the accumulation of ROS induced by

m

The result revealed that 3b prevent the accumulation of ROS in

PC12 cells. Further, 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay

was performed to determine the direct free radical scavenging

activity of 3b (Fig. S2). Results indicated that 3b did not act as a

direct free radical scavenger. The above results showed that the

cytoprotection effects of 3b might result from suppression of ROS

accumulation in PC12 cells, but did not directly scavenge free

radical.

compared with control group in

a concentration-dependent

manner, and the NQO1 level was also signiﬁcantly upregulated,

conﬁrming our hypothesis that 3b upregulated the antioxidative

defense system to scavenge ROS.

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

5

Fig. 3. The accumulation of Nrf2 in nucleus. (A) 3b promoted Nrf2 translocation into the nucleus detected by Western blot assay. PC12 cells were incubated with 3b at 25

m

M for 4, 6

and 8 h. Nuclear Nrf2, cytosolic Nrf2, and total Nrf2 were prepared and analyzed by the western blots. (B) Nrf2 accumulation in the nucleus detected by immunoﬂuorescence assays.

PC12 cells were incubated with 3b at 25 M for 4, 6 and 8 h, and then stained with Nrf2 antibody and DAPI. (C) The inductivity of 3b is calculated compared to the blank control. All

data represent the means SD of three independent experiments. *, P < 0.05, **, P < 0.01, and ***, P < 0.001 vs the vehicle group.

m

2.7. Promotion of Nrf2 nuclear accumulation

whether 3b could induce the nucleus translocation of Nrf2 in

PC12 cells by Western blot assay (Fig. 3A). Results showed that

treatment of PC12 cells with 3b (25 M) signiﬁcantly promoted

Nrf2 protein accumulation in the nucleus in a time dependent

manner, which was in accordance with the immunoﬂuorescence

assays results (Fig. 3B), indicating that the protection effect of 3b in

PC12 cells might be due to promotion of Nrf2 accumulation in the

Previous studies have determined that Nrf2 binds to a common

DNA sequence called antioxidant response element (ARE) in nu-

cleus to initiate the transcription of numerous cytoprotective pro-

teins, including phase I and phase II drug-metabolizing enzymes,

drug transporters, and antioxidants [1 1]. Hence, we examined

m

6

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

Fig. 4. Prerequisite of Nrf2 for the cytoprotection of 3b in PC12 cells. (A) Expression level of Nrf2 in PC12-shNrf2-1, 2, 3 cells and PC12-shNT cells were determined by Western blot.

(B) The cytoprotection of 3b in H₂O₂induced PC12-shNT cells and PC12-shNrf2-2 cells damage. Cells were pretreated with 3b (12.5, 25.0, 50.0 and 100 M) for 24 h and induced

m

with H₂O₂for another 24 h, then determined by MTT assays. All data represent the means SD of three independent experiments. **, P < 0.01, and ***, P < 0.001 vs the vehicle

group.

The protecting effect of 3b was almost completely suppressed in the

PC12-shNrf2-2 cells, while a clean promotion of cell viability in

PC12-shNT cells pretreated by 3b was observed, indicating that

Nrf2 played an indispensable role in the cytoprotection of 3b in

PC12 cell.

2.9. Molecular docking study with Keap1

Nrf2 activation was tightly regulated by Keap 1 and many

electrophiles chemicals were reported to modify the cysteine rich

protein Keap1 and thereby mediate interactions with the Cul3/

Rbx1 E3 ubiquitin ligase system and activation of Nrf2.⁹Consid-

ering 3b contained a Michael receptor moiety,

a,b-unsaturated

ketone structure with the potential to assault cysteine residues in

biomolecules, computational molecular docking methods were

then used to predict the potential molecular interaction mecha-

nisms between 3b and Keap1 (Fig. 5 and Fig. S4 and Table S5). There

are 27 Cys in Keap 1 included C77, C151 and C171 in the BTB domain

and C368, C395, C406, C434, C489, C513, C518 and C583 in the

Kelch domain (Fig. S3) with the other Cys residues located on the N-

terminal, IVR domain and C-terminal [37]. Since no full length Keap

1 cristal structure was available, we selected the I-TASSER online

service [38] to construct the homology structure for the full length

Keap1. Upon applying covalent docking software (AutoDock 4.2)

[39], which allowed to integrate Michael addition reactions of 3b

with all Cys residues in Keap1, lower binding energy was used as a

criterion to predict the possible binding site between 3b and Keap 1.

Consistent with the previous report that Cys151, Cys273, and

Cys288 appeared to be the most susceptible cysteines to electro-

phile reaction [9], the binding energy for 3b with the three cyste-

ines in our experiment were estimated with comparably low

binding energy as ꢁ4.86, ꢁ3.99 and ꢁ3.97 kcal/mol, respectively.

Except these common three cysteines, Cys513 and Cys368 were

found as the potential binding site with the lowest binding energy

(ꢁ7.46 kcal/mol and ꢁ6.86 kcal/mol) (Table S5). And the possible

interaction mode of 3b with Cys151 in the BTB domain (4CXI),

Cys249 and 288 in the IVR domain and Cys368, Cys513 in the Kelch

domain (4IFL) were displayed in Fig. 5 and Fig. S4. Further conﬁr-

mation experiment would be done in the near future. The above

results suggesting that 3b might have the potential to covalent

binding with Keap1, alter the conformation of Keap1 leading to

nuclear translocation of Nrf2 and subsequently activate the target

gene expression. To conﬁrm if 3b can covalently bind with Keap1,

Fig. 5. In silico modeling of interaction of 3b with representative Cys in Keap1. (A)

Covalent docking of 3b with Cys 151 in BTB domain (PDB 4CXT). (B) Covalent docking

of 3b with Cys 273 in IVR domain (PDB not available). (C) Covalent docking of 3b with

Cys 513 in Kelch domain (PDB 4IFL).

nucleus. HepG2 cell, transiently transfected with ARE-luciferase,

were used for the evaluation of Nrf2-ARE activation effects of 3b.

3b exhibited ARE inductivity more than three-folds at 12.5

mM

compared with control group, while no normal dose-dependent

manner were observed, conﬁrmed that 3b effectively promotes

Nrf2 nucleus translocation and ARE genes expression.

2.8. Prerequisite of Nrf2 for cytoprotection of 3b

To further demonstrate the role of Nrf2 signaling pathway in the

cellular protection effects of 3b in PC12 cells, Nrf2 knockdown

PC12 cells was constructed by infection of lentiviral carrying

negative control shRNA (PC12-shNT) or Nrf2 shRNA (PC12-shNrf2-

1, 2, 3). As seen in Fig. 4A, PC12-shNrf2-2 successfully silenced the

expression level of Nrf2 detected by Western blot and then PC12-

shNT and PC12-shNrf2-2 were chose for the further test. Silencing

of Nrf2 expression was evaluated in Fig. 4B via Western blot assay.

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

7

Fig. 6. 3b attenuated the deﬁcts of motor behaviors in MPTP-induced PD mice. (A) Experimental protocols for pole and rotarod behavioral tests. (B) The weight of mice during the

experiment. (C) Latency to fall off the rotating rod in rotarod test. (D) Time that mice climb from the top to the bottom of the pole in pole test. All data represent the means SD of

the mean (n ¼ 6 per group). ***, P < 0.001 vs. the vehicle group; ^∧, P < 0.05, ^∧∧, P < 0.01 and ^∧∧∧, P < 0.001 vs. the H₂O₂-treated group.

we performed LC-MS-MS analysis for 3b incubated with recombi-

nant full-length Keap1 (Fig. S5). Although the analysis showed 3b

can covalently bind with Cys288 within Keap1, it only have low

conﬁdence. Whether 3b can covalently bind with Cys288 within

Keap1 needs further experiment to conﬁrm.

neurons [3]. Similar to the previous reports [25], the immunohis-

tochemical staining for TH revealed that MPTP exerted signiﬁcant

neurotoxicity with 30.7% and 74.0% reduction of the TH-positive

cells in the substantia nigra pars compacta (SNpc) and striatum

respectively compared with the saline-treated control while both

prophylactic (50 and 100 mg/kg) and therapeutic administration

(100 mg/kg) of 3b signiﬁcantly increased the number of TH-

positive cells (Fig. 7A), which was indicative of rescue on the

MPTP-induced neurotoxicity, suggesting that 3b might exhibit

neuroprotective effects against dopaminergic neuronal cell death in

PD. Since 3b could active Nrf2 and its activation might protect

neurons from dying in Parkinson’s disease patients [40], the

immunohistochemical staining for Nrf2 was also performed. It

could be observed that Nrf2 was upregulated both in SNpc and

striatum area in prophylactic (100 mg/kg) administration of 3b

(Fig. 7B). Nrf2 and its downstream protein, HO-1 and NQO1 in

mouse brain tissue were further detected by western bolt. Fig. 8

showed that the expression of HO-1 and NQO1 was increased

signiﬁcantly by 3b, indicating it could initiate the antioxidant sys-

tem through Nrf2 activation in vivo, which was inconsistent with

the in vitro results. The upregulation of antioxidative proteins HO-1

and NQO1 as well as the reverse of TH and Nrf2 in MPTP induced

lesion in both striatum and SNpc area conﬁrmed the neuro-

protective and antioxidant effect of 3b.

2.10. 3b improves motor behavior and rescues dopaminergic

neuronal cell death in MPTP induced PD mice

To determine 3b⁰s effects on the behavioral recovery in MPTP-

lesioned mice, motor behavior analysis was conducted through

the rotarod and pole test. In the rotarod test (Fig. 6C), robust motor

deﬁcits were obviously observed in MPTP treated group, which was

determined by decreased sustained rotarod time to 74.0 48.2, a

68.9% decrease compared with the saline-treated control, whereas

both prophylactic (50 and 100 mg/kg) and therapeutic adminis-

tration (100 mg/kg) of 3b signiﬁcantly alleviated the abnormal

movement with increased rotarod time to 253.7

57.2,

226.2 52.5 and 189.5 92.7 s respectively. In the pole test

(Fig. 6D), the MPTP treated group displayed a signiﬁcant prolon-

gation of the total time for climbing down the pole while prophy-

lactic (50 and 100 mg/kg) and therapeutic administration (100 mg/

kg) of 3b reduced the climbing down time by 38.1%, 34.5% and

20.6% compared with MPTP treated group. Taken together, these

behavioral results suggested that 3b elicited a potent protective

effect against MPTP-induced impairments in motor function while

no obvious toxicity were observed (Fig. 6B).

3. Conclusion

Tyrosine hydroxylase (TH) is known to be involved in motor

movement and is usually used as a marker for dopaminergic

PIP as a natural alkaloid, isolated from the fruits of Piper longum

and Piper nigrum [1 7], has been reported that it has the ability to

8

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

Fig. 7. 3b protected dopaminergic neurons and activated the antioxidant system in MPTP-induced PD mice. (A) Representative immunohistochemical staining (brown, TH) in the

striatum and SNpc. Bar graphs summarized TH-immunopositive density to the total area. (B) Representative immunohistochemical staining (brown, Nrf2) in the striatum and SNpc.

Bar graphs summarized Nrf2-immunopositive density to the total area. All data represent the means SD of the mean (n ¼ 6). Magniﬁcation: ꢀ 100. *, P < 0.05, **, P < 0.01, and ***,

P < 0.001 vs. the vehicle group; ^∧, P < 0.05 and ^∧∧, P < 0.01 vs. the MPTP-treated group. (For interpretation of the references to colour in this ﬁgure legend, the reader is referred to

the Web version of this article.)

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

9

series of piperine analogues were designed and synthesized and

their neuroprotective effects were evaluated in H₂O₂induced

PC12 cell damage model. The SAR investigation revealed that 2-

methoxy substituted phenyl derivatives (3b) has the best activity

and also indicated that further exploring the structure of aromatic

group replaced with 2-thiophene and N-methyl-2-pyrrole maybe a

promising strategy. 3b with the best neuroprotective activity was

chosen for the further mechanism research. Flow cytometry and

ﬂuorescence analysis showed that 3b exhibited cytoprotection ef-

fects through suppression of ROS accumulation and restoration of

mitochondrial membrane potential in PC12 cells. As DPPH assays

showed that 3b could not directly scavenge ROS, accompanied with

upregulation of the phase II antioxidant enzymes HO-1 and NQO1

and previous report [41], we hypothesis that these effects may

related to Nrf2-ARE signaling pathway. Western blot and immu-

noﬂuorescence results showed that 3b siniﬁcantly promoted Nrf2

accumulation in the nucleus. Additionally, 3b exhibited signiﬁcant

ARE inductivity in HepG2 cell, transiently transfected with ARE-

luciferase.

Some reported Nrf2 activators can covalently modifying

cysteine within Keap1 protein via Michael receptor, like the ﬁrst

Nrf2-inducer dimethyl fumarate (Tecﬁdera) can react with Cys151

of Keap1 protein to activate Nrf2, identiﬁed by LC-MS-MS analysis

Fig. 8. 3b induced the expression of Nrf2, NQO1 and HO-1 in MPTP-induced PD mice.

[15]. Considering 3b also contains a Michael receptor moiety, a,b-

activate Nrf2 [41]ssss and has in vivo activity in MPTP or 6-OHDA

induced PD mouse model [25,26]. However, the clinical usage

was obstructed partially due to the low activity and undisclosed

exacted mechanism [ref]. In order to improve the activity of PIP, a

unsaturated ketone structure with the potential to assault cysteine

residues in biomolecules, computational molecular docking

methods were applicated for identity the potential covalent bind-

ing target within Keap1 which tightly regulates Nrf2 activation.

Chart 1. Known Keap1ꢁNrf2 pathway electrophilic activator bearing

a,b-unsaturated ketone structure.

Chart 2. Strategy for structure optimization of PIP.

10

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

Table 1

The neuroprotection and ADMET-BBB level of PIP analogues substituted with different groups.

a

b

Compound

R₁

R₂

Cell viability (%) (H₂O₂-induced PC12 cells)

ADMET-BBB Level

H₂O₂

TBHQ

Piperine (3n)

52.81 6.00

70.42 2.01

55.72 1.35

1

3a

3b

3c

4-OCH₃

2-OCH₃

3-OCH₃

26.47 3.91

88.82 7.83

58.12 1.83

3d

3e

2-OCH₃

80.74 4.20

74.62 2.52

1

2

3f

15.15 0.24

37.20 1.95

64.99 4.00

0

1

3g

3h

3i

3j

63.56 1.62

60.79 7.31

1

2

3k

3l

20.18 1.63

18.86 1.06

32.14 4.13

31.41 6.29

22.31 3.23

48.40 1.63

1

0

1

2

1

3m

3o

3p

4a

4-Cl

4-OH

4b

4c

2-OH

3-OH

68.84 4.58

75.67 3.95

1

4d

4e

4-OH

67.31 2.20

69.14 6.37

1

2

a

Cell viability (%) were detected with compounds at the concentration of 12.5

the ADMET-BBB level was calculated by Discovery studio 3.1.

mM; data are expressed as the mean SD of at least three independent experiments.

b

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

11

Table 2

The neuroprotection and ADMET-BBB level of PIP analogues substituted with heterocycles.

b

Compound

Ar

R

Cell viability (%) ^a(H₂O₂-induced PC12 cells)

ADMET-BBB Level

1

H₂O₂

3q

52.81 6.00

33.29 2.66

3r

57.88 1.83

1

3s

3t

77.33 6.66

77.22 9.83

1

3u

3v

40.95 3.05

67.83 3.65

53.81 4.04

1

3w

a

Cell viability (%) were detected with compounds at the concentration of 12.5

the ADMET-BBB level was calculated by Discovery studio 3.1.

mM; data are expressed as the mean SD of at least three independent experiments.

b

Docking study with the homology structure for the all length Keap1

showed that 3b might activates Nrf2 through covalently binding

with Cys residues in Keap1 based on the lowest binding energy.

However, our preliminary LC-MS-MS experiment showed that 3b

covalently binding to Keap1 is a low conﬁdence result, although

some peptides within Keap1 modiﬁed by 3b can be detected.

Future work is required to explore the condition of the LC-MS-MS

experiment or adopt crystall structure or site-directed mutagen-

esis to further conﬁrm the hypothesis. In vivo, 3b improved motor

behavior and rescued dopaminergic neuronal cell death in MPTP

induced PD mice.

1.0 mL/min; UV wavelength, 254e400 nm; temperature, 25 ^ꢂC;

injection volume, 10 L).

m

PIP analogues synthesized with the synthetic routes of the

target compounds (3a-w, 4a-e) depicted in Scheme 1. Brieﬂy,

commercially available (E)-2-crotonyl chloride was reacted with

corresponding amines to form crotonyl amides 2a-c [41], which

was then coupled with substituted benzaldehyde (or aromatic

heterocyclic formaldehyde) using NaOH to achieve the target

molecules 3a-v [41]. Target compounds 4a-e need further reaction

to demethylate in the presence of Boron tribromide (BBr₃) [42].

Synthesis of compound 7a-7n is described in Scheme 2. The

starting material, commercially available substituted benzaldehyde

(or aromatic heterocyclic formaldehyde), reacting with malonic

In a word, this work identiﬁed a promising candidate for the

development of a novel therapeutic for PD, numbered as 3b, which

activated Nrf2-ARE signaling pathway may via covalently binding

with Cys288 within Keap1.

acid to obtain

a,b-unsaturated acids 6a-e through Knoevenagel

reaction [13]. It was then coupled with corresponding amides to

achieve the target molecules 7d-n [13]. And some of the target

molecules needed further reaction to deprotect with triﬂuoroacetic

acid to obtain 7a-c.

4. Experiment part

4.1. Chemistry

4.1.1. General procedure A for the synthesis of 2

To a stirred solution of corresponding lamins (0.58 mol) and

Et₃N (55 g, 0.4175 mol) in DCM (50 mL), crotonyl chloride (1) was

added dropwise at 0 ^ꢂC. After the addition, reaction mixture was

slowly warm to room temperature and stirred for 8 h. Progress of

the reaction was monitored by TLC. After completion, organic layer

was washed with sat. sodium bicarbonate solution, water, dried

over anhyd. Na₂SO₄and concentrated under reduced pressure. A

pale yellow viscous liquid 2 was obtained without further puriﬁed.

All commercially available reagents were used without further

puriﬁcation. All solvents were dried and redistilled prior to use in

the usual manner. Reaction progress was monitored using analyt-

ical thin layer chromatography (TLC) on precoated silica gel GF254

(Yantai, China) plates and the spots were detected under UV light

(254 nm and 360 nm). Column chromatography was performed on

silica gel (200e300 mesh, Yantai, China). ¹H and ¹³C NMR spectra

were on a Bruker Avance 400 spectrometer (Bruker Company,

Germany), using TMS as an internal standard. Chemical shifts are

reported in ppm (d) using the residual solvent line as internal

standard. Splitting patterns are designed as s, singlet; d, doublet; t,

triplet; m, multiplet. Mass spectra were recorded on a Q-TOF Pre-

mier mass spectrometer (Micromass, Manchester, UK). The purity

of all compounds (>95%) was determined on a Waters e2695 series

4.1.2. (E)-1-(piperidin-1-yl)but-2-en-1-one (2a)

Piperidine was reacted with crotonyl chloride (1) following the

general procedure A to give the desired product 2a as a light yellow

oil (yield 98.0%). ¹H NMR (400 MHz, CDCl₃)

d

6.58 (dq, J ¼ 13.9,

6.8 Hz, 1H), 6.06 (d, J ¼ 15.0 Hz,1H), 3.31 (d, J ¼ 34.4 Hz, 4H), 1.64 (d,

LC system (column, Agilent C₁₈, 4.6 mm ꢀ 150 mm, 5

mm; ﬂow rate,

J ¼ 6.8 Hz, 3H), 1.42 (dd, J ¼ 10.7, 5.7 Hz, 2H), 1.33 (d, J ¼ 4.8 Hz, 4H).

12

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

Table 3

The neuroprotection and ADMET-BBB level of PIP analogues.

a

b

Compound

Ar

R

Cell viability (%) (H₂O₂-induced PC12 cells)

ADMET-BBB Level

2

7a

72.61 3.37

7b

7c

69.07 2.03

42.70 1.77

2

7d

7e

7f

30.64 0.51

26.06 1.98

76.95 4.73

1

2

7g

7h

71.74 7.15

69.74 8.74

2

7i

81.26 2.61

1

7j

78.21 2.13

68.45 1.56

1

2

7k

7l

50.42 3.18

54.33 1.90

56.55 4.79

1

2

7m

7n

a

Cell viability (%) were detected with compounds at the concentration of 12.5

the ADMET-BBB level was caculated by Discovery studio 3.1.

m

M; data are expressed as the mean SD of at least three independent experiments.

b

Exact mass calcd for C9H15NO [MþH]^þ: 154.1232; found 154.1235.

4.1.5. General procedure B for the synthesis of 3

To a stirred solution of aromatic aldehyde (1.0 equiv, 2.4 mmol)

in DMSO (2 mL), 1 mL saturate sodium hydroxide solution was

added and stirred for 30 min. Then 2 (1.0 equiv, 2.4 mmol) was

added. Then stirred for another 6e8 h at room temperature. Re-

action was monitored by TLC. After completion, the reaction

mixture was quenched with 5% aq. HCl and extracted with DCM,

washed with water, dried over anhyd. Na₂SO₄and concentrated

under reduced pressure. The obtained crude product was puriﬁed

by column chromatography.

4.1.3. (E)-N,N-diethylbut-2-enamide (2b)

Diethylamine was reacted with crotonyl chloride (1) following

the general procedure A to give the desired product 2b as a light

yellow oil (yield 98.0%). ¹H NMR (400 MHz, CDCl₃)

d 6.91e6.77 (m,

1H), 6.16 (dd, J ¼ 14.9, 1.6 Hz, 1H), 3.41e3.27 (m, 4H), 1.81 (dd,

J ¼ 6.8, 1.6 Hz, 3H), 1.11 (dt, J ¼ 20.7, 6.4 Hz, 6H). Exact mass calcd for

C8H15NO [MþH]^þ: 142.1232; found 142.1234.

4.1.6. (2E,4E)-5-(4-methoxyphenyl)-1-(piperidin-1-yl)penta-2,4-

dien-1-one (3a)

4.1.4. (E)-N-ethylbut-2-enamide (2c)

Ethylamine was reacted with crotonyl chloride (1) following the

general procedure A to give the desired product 2c as a light yellow

2a was reacted with 4-methoxybenzaldehyde following the

general procedure B to give the desired product 3a as a white

oil (yield 98.0%). ¹H NMR (400 MHz, CDCl₃)

d

6.80e6.62 (m, 1H),

powder (yield 88.0%). ¹H NMR (400 MHz, CDCl₃)

d 7.43 (ddd,

5.79 (dd, J ¼ 15.2, 1.6 Hz, 1H), 3.30e3.20 (m, 2H), 1.74 (d, J ¼ 6.9 Hz,

3H), 1.06 (t, J ¼ 7.3 Hz, 3H). Exact mass calcd for C6H11NO [MþH]^þ:

114.0919; found 114.0918.

J ¼ 12.4, 6.3, 1.7 Hz, 1H), 7.40e7.37 (m, 2H), 6.89e6.85 (m, 2H),

6.81e6.77 (m, 2H), 6.43 (d, J ¼ 14.6 Hz,1H), 3.81 (s, 3H), 3.58 (s, 4H),

1.70e1.63 (m, 2H), 1.58 (dt, J ¼ 11.0, 5.5 Hz, 4H). ¹³C NMR (101 MHz,

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

13

Scheme 1. Reagents and conditions: (a) Amines, Et₃N, CH₂Cl₂, 0 ^ꢂC, 8 h; (b) Aromatic aldehydes, sat. NaOH, DMSO, r.t., 6e8 h; (c) 1M BBr₃, dry CH₂Cl₂, -80 ^ꢂC, 6 h.

Scheme 2. Reagents and conditions: (a) Malonic acid, Piperidine, Pyridine, reﬂux, 8 h; (b) Amines, HATU, DMF, r.t., 4 h; (c) TFA, r.t., 2 h.

CDCl₃)

d

165.61, 160.12, 142.87, 138.30, 129.38, 128.40, 125.07,

4.1.7. (2E,4E)-5-(2-methoxyphenyl)-1-(piperidin-1-yl)penta-2,4-

119.72, 114.29, 55.40, 47.04, 43.54, 26.21, 24.76. Exact mass calcd for

dien-1-one (3b)

C17H21NO2 [MþH]^þ: 272.1572; found 272.1576.

2a was reacted with 2-methoxybenzaldehyde following the

general procedure B to give the desired product 3b as a white

14

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

powder (yield 78.0%). ¹H NMR (400 MHz, CDCl₃)

10.3 Hz, 1H), 7.28e7.22 (m, 1H), 7.04 (d, J ¼ 7.7 Hz, 1H), 6.97 (s, 1H),

6.94e6.77 (m, 3H), 6.49 (d, J ¼ 14.7 Hz,1H), 3.82 (s, 3H), 3.59 (s, 4H),

1.74e1.64 (m, 2H), 1.63e1.51 (m, 4H). ¹³C NMR (101 MHz, CDCl₃)

d

7.42 (dd, J ¼ 14.5,

(dd, J ¼ 15.4, 10.5 Hz, 1H), 6.52 (d, J ¼ 8.6 Hz, 2H), 6.35 (d,

J ¼ 14.6 Hz,1H), 3.58 (m, 4H), 3.35e3.28 (m, 4H), 2.03e1.98 (m, 4H),

1.69e1.63 (m, 2H), 1.61e1.53 (m, 4H). ¹³C NMR (101 MHz, CDCl₃)

d

166.01, 148.23, 143.81, 139.70, 128.56, 122.16, 117.61, 111.88, 47.74,

d

165.48, 159.97, 142.33, 138.48, 137.99, 129.80, 127.48, 121.25,

46.96, 43.30, 26.82, 25.56, 24.85. Exact mass calcd for C20H26N2O

119.77, 114.47, 112.13, 55.38, 47.05, 43.39, 26.80, 25.88, 24.76. Exact

[MþNa]^þ: 333.1947; found 333.1947.

mass calcd for C17H21NO2 [MþH]^þ: 272.1572; found 272.1576.

4.1.13. (2E,4E)-5-(4-(piperazin-1-yl)phenyl)-1-(piperidin-1-yl)

penta-2,4-dien-1-one (3h)

4.1.8. (2E,4E)-5-(3-methoxyphenyl)-1-(piperidin-1-yl)penta-2,4-

dien-1-one (3c)

2a was reacted with tert-butyl 4-(4-formylphenyl)piperazine-1-

carboxylate following the general procedure B to give the inter-

mediate p-3h. Then the intermediate p-3h was dissolved in 2 mL

triﬂuoroacetic acid (TFA) and stirred for 1 h. After the reaction was

completed, the reaction was quenched by saturated sodium bicar-

bonate solution and extracted with ethyl acetate (10 mL ꢀ 3), dried

over anhyd. Na₂SO₄and concentrated under reduced pressure to

obtain the desired product 3h as a light yellow powder (yield 68.5%

2a was reacted with 3-methoxybenzaldehyde following the

general procedure B to give the desired product 3c as a white

powder (yield 76.0%). ¹H NMR (400 MHz, CDCl₃)

d

7.48 (dd, J ¼ 7.6,

1.6 Hz,1H), 7.45 (dd, J ¼ 14.8, 11.2 Hz, 1H), 7.28e7.23 (m, 1H), 7.18 (d,

J ¼ 15.7 Hz, 1H), 7.00e6.91 (m, 2H), 6.88 (d, J ¼ 8.3 Hz, 1H), 6.45 (d,

J ¼ 14.8 Hz, 1H), 3.87 (s, 3H), 3.58 (m, 4H), 1.71e1.63 (m, 2H), 1.59

(dt, J ¼ 10.9, 5.4 Hz, 4H). ¹³C NMR (101 MHz, CDCl₃)

d 165.67, 157.45,

143.37, 133.82, 129.74, 127.81, 127.32, 125.61, 120.79, 120.37, 111.18,

55.61, 47.04, 43.35, 26.84, 25.74, 24.79. Exact mass calcd for

C17H21NO2 [MþH]^þ: 272.1572; found 272.1576.

over two steps). ¹H NMR (400 MHz, CDCl₃)

d

7.37 (dd, J ¼ 9.3, 4.5 Hz,

1H), 7.33 (d, J ¼ 8.6 Hz, 2H), 6.82 (d, J ¼ 8.6 Hz, 2H), 6.77e6.73 (m,

2H), 6.39 (d, J ¼ 14.7 Hz, 1H), 3.57 (s, 4H), 3.40 (s, 4H), 3.30 (s, 4H),

1.64 (m, 2H), 1.58 (m, 4H). ¹³C NMR (101 MHz, CDCl₃)

d 166.39,

4.1.9. (2E,4E)-N,N-diethyl-5-(2-methoxyphenyl)penta-2,4-

dienamide (3d)

150.08, 143.77, 138.98, 129.46, 128.51, 125.01, 119.13, 116.71, 46.23,

45.27, 43.88, 43.44, 26.47, 25.87, 24.56. Exact mass calcd for

C20H26N2O [MþNa]^þ: 348.2052; found 348.2056.

2b was reacted with 2-methoxybenzaldehyde following the

general procedure B to give the desired product 3d as a white

powder (yield 78.9%). ¹H NMR (400 MHz, CDCl₃)

d

7.57e7.44 (m,

4.1.14. (2E,4E)-N,N-diethyl-5-(4-(piperazin-1-yl)phenyl)penta-2,4-

dienamide (3i)

2H), 7.30e7.15 (m, 2H), 7.03e6.90 (m, 2H), 6.88 (d, J ¼ 8.3 Hz, 1H),

6.37 (d, J ¼ 14.6 Hz, 1H), 3.86 (s, 3H), 3.53e3.36 (m, 4H), 1.24e1.13

2b was reacted with tert-butyl 4-(4-formylphenyl)piperazine-1-

carboxylate following the general procedure B to give the inter-

mediate p-3i. Then the intermediate p-3i was dissolved in 2 mL

triﬂuoroacetic acid (TFA) and stirred for 1 h. After the reaction was

completed, the reaction was quenched by saturated sodium bicar-

bonate solution and extracted with ethyl acetate (10 mL ꢀ 3), dried

over anhyd. Na₂SO₄and concentrated under reduced pressure to

obtain the desired product 3i as a light yellow powder (yield 68.5%

(m, 6H). ¹³C NMR (101 MHz, CDCl₃)

d 166.05, 157.39, 143.40, 134.03,

129.73, 127.67, 127.26, 125.50, 120.71, 120.40, 111.13, 55.55, 42.27,

41.00, 15.03, 13.29. Exact mass calcd for C16H21NO2 [MþH]^þ:

260.1651; found 260.1649.

4.1.10. (2E,4E)-N-ethyl-5-(2-methoxyphenyl)penta-2,4-dienamide

(3e)

2c was reacted with 2-methoxybenzaldehyde following the

general procedure B to give the desired product 3e as a white

over two steps). ¹H NMR (400 MHz, CDCl₃)

d

7.46 (ddd, J ¼ 14.6, 6.1,

3.8 Hz, 1H), 7.38 (d, J ¼ 8.6 Hz, 2H), 6.87 (d, J ¼ 8.6 Hz, 2H),

6.83e6.75 (m, 2H), 6.34 (d, J ¼ 14.6 Hz, 1H), 3.43 (m, 8H), 3.23 (d,

J ¼ 4.7 Hz, 4H), 1.21 (m, 6H), 0.91e0.79 (m, 1H). ¹³C NMR (101 MHz,

powder (yield 78.9%). ¹H NMR (400 MHz, DMSO‑d₆)

d 8.01 (t,

J ¼ 5.4 Hz, 1H), 7.60 (dd, J ¼ 7.7, 1.4 Hz, 1H), 7.34e7.25 (m, 1H), 7.17

(dd, J ¼ 14.9, 10.7 Hz, 1H), 7.11 (d, J ¼ 15.6 Hz, 1H), 7.04 (d, J ¼ 2.2 Hz,

1H), 7.02e6.97 (m, 1H), 6.95 (t, J ¼ 7.5 Hz, 1H), 6.09 (d, J ¼ 14.9 Hz,

1H), 3.83 (s, 3H), 3.21e3.10 (m, 2H), 1.05 (t, J ¼ 7.2 Hz, 3H). ¹³C NMR

CDCl₃) d 166.17,147.66,143.01,138.55,130.53,128.36,125.00,119.83,

116.45, 47.73, 44.41, 42.38, 41.15. Exact mass calcd for C19H27N3O

[MþNa]^þ: 336.2052; found 336.2005.

(101 MHz, DMSO‑d₆) d 164.89,156.77, 139.42, 132.44,129.85,127.43,

126.98, 125.23, 124.64, 120.61, 111.52, 55.52, 33.44, 14.75. Exact

4.1.15. (2E,4E)-N-ethyl-5-(4-(piperazin-1-yl)phenyl)penta-2,4-

dienamide (3j)

mass calcd for C14H17NO2 [MþH]^þ: 232.1338; found 232.1338.

2d was reacted with tert-butyl 4-(4-formylphenyl)piperazine-1-

carboxylate following the general procedure B to give the inter-

mediate p-3j. Then the intermediate p-3j was dissolved in 2 mL

triﬂuoroacetic acid (TFA) and stirred for 1 h. After the reaction was

completed, the reaction was quenched by saturated sodium bicar-

bonate solution and extracted with ethyl acetate (10 mL ꢀ 3), dried

over anhyd. Na₂SO₄and concentrated under reduced pressure to

obtain the desired product 3j as a light yellow powder (yield 68.5%

4.1.11. (2E,4E)-5-([1,1⁰-biphenyl]-4-yl)-1-(piperidin-1-yl)penta-2,4-

dien-1-one (3f)

2a was reacted with [1,1⁰-biphenyl]-4-carbaldehyde following

the general procedure B to give the desired product 3f as a white

powder (yield 68.5%). ¹H NMR (400 MHz, CDCl₃)

d 7.63e7.56 (m,

4H), 7.52 (d, J ¼ 8.4 Hz, 2H), 7.49e7.40 (m, 3H), 7.36 (dt, J ¼ 9.3,

4.2 Hz, 1H), 6.95 (dd, J ¼ 15.5, 10.1 Hz, 1H), 6.88 (d, J ¼ 15.5 Hz, 1H),

6.50 (d, J ¼ 14.7 Hz, 1H), 3.60 (d, J ¼ 40.1 Hz, 4H), 1.73e1.64 (m, 2H),

over two steps). ¹H NMR (400 MHz, CD₃OD)

d

7.42 (d, J ¼ 8.9 Hz,

1.60 (m, 4H). ¹³C NMR (101 MHz, CDCl₃)

d

165.37, 142.35, 141.28,

2H), 7.31 (ddd, J ¼ 15.0, 8.3, 1.9 Hz, 1H), 6.96 (d, J ¼ 8.9 Hz, 2H),

6.89e6.78 (m, 2H), 6.05 (d, J ¼ 15.0 Hz, 1H), 3.30 (d, J ¼ 7.3, 5.9 Hz,

2H), 3.22 (dd, J ¼ 6.1, 4.0 Hz, 4H), 2.99 (dd, J ¼ 6.1, 4.0 Hz, 4H), 1.19 (t,

140.46, 138.02, 135.53, 128.85, 127.54, 127.41, 127.09, 126.95, 120.94,

46.98, 43.29, 26.77, 25.64, 24.69. Exact mass calcd for C22H23NO

[MþNa]^þ: 340.1677; found 340.1685.

J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz, CD₃OD)

d 161.48, 138.15, 137.55,

131.51, 129.24, 126.18, 124.61, 116.74, 116.64, 50.43, 50.26, 46.46,

46.44, 35.31, 14.80. Exact mass calcd for C17H23N3O [MþNa]^þ:

308.1739; found 308.1740.

4.1.12. (2E,4E)-1-(piperidin-1-yl)-5-(4-(pyrrolidin-1-yl)phenyl)

penta-2,4-dien-1-one (3g)

2a was reacted with 4-(pyrrolidin-1-yl)benzaldehyde following

the general procedure B to give the desired product 3g as a white

4.1.16. (2E,4E)-5-(4-morpholinophenyl)-1-(piperidin-1-yl)penta-

2,4-dien-1-one (3k)

powder (yield 63.2%). ¹H NMR (400 MHz, CDCl₃)

d

7.44 (dd, J ¼ 14.6,

10.5 Hz, 1H), 7.33 (d, J ¼ 8.7 Hz, 2H), 6.78 (d, J ¼ 15.4 Hz, 1H), 6.70

2a was reacted with 4-(4-formylphenyl)morpholine following

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

15

the general procedure B to give the desired product 3k as a light

yellow powder (88.0%). ¹H NMR (400 MHz, CDCl₃)

7.42 (ddd,

1.5 Hz, 1H), 6.97e6.88 (m, 2H), 6.85 (d, J ¼ 15.5 Hz, 1H), 6.07 (d,

J ¼ 15.0 Hz, 1H), 6.05 (s, 2H), 3.21e3.12 (m, 2H), 1.06 (t, J ¼ 7.2 Hz,

d

J ¼ 14.6, 6.0, 4.3 Hz, 1H), 7.37 (d, J ¼ 8.8 Hz, 2H), 6.87 (d, J ¼ 8.6 Hz,

2H), 6.80e6.74 (m, 2H), 6.41 (d, J ¼ 14.6 Hz, 1H), 3.91e3.81 (m, 4H),

3.58 (d, J ¼ 37.6 Hz, 4H), 3.24e3.16 (m, 4H), 1.66 (dd, J ¼ 11.0, 6.1 Hz,

2H), 1.58 (dt, J ¼ 10.9, 5.5 Hz, 4H). ¹³C NMR (101 MHz, CDCl₃)

3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 164.89, 147.90, 147.65, 139.05,

137.68, 130.86, 125.27, 124.67, 122.57, 108.40, 105.60, 101.22, 33.40,

14.75. Exact mass calcd for C14H15NO3 [MþNa]^þ: 268.0950; found

268.0952.

d

165.73, 151.32, 143.09, 138.52, 130.63, 128.26, 124.53, 119.32,

115.35, 66.84, 48.82, 47.03, 43.36, 26.87, 25.80, 24.82. Exact mass

4.1.22. (2E,4E)-5-(furan-2-yl)-1-(piperidin-1-yl)penta-2,4-dien-1-

one (3q)

calcd for C20H26N2O2 [MþNa]^þ: 349.1892; found 349.1890.

2a was reacted with 2-furanaldehyde following the general

procedure B to give the desired product 3q as a light yellow powder

4.1.17. (2E,4E)-1-(piperidin-1-yl)-5-(4-(piperidin-1-yl)phenyl)

penta-2,4-dien-1-one (3l)

(64.5%). ¹H NMR (400 MHz, CDCl₃)

d 7.39 (s, 1H), 7.39e7.31 (m, 1H),

2a was reacted with 4-piperidinobenzaldehyde following the

general procedure B to give the desired product 3l as a light yellow

6.79 (dd, J ¼ 15.4, 11.3 Hz, 1H), 6.60 (d, J ¼ 15.4 Hz, 1H), 6.44 (d,

J ¼ 14.6 Hz, 1H), 6.42e6.36 (m, 2H), 3.56 (d, J ¼ 44.1 Hz, 4H),

1.68e1.61 (m, 2H), 1.57 (d, J ¼ 4.3 Hz, 4H). ¹³C NMR (101 MHz,

powder (78.6%). ¹H NMR (400 MHz, CDCl₃)

d

7.43 (ddd, J ¼ 14.6, 8.5,

1.8 Hz, 1H), 7.33 (d, J ¼ 8.8 Hz, 2H), 6.87 (d, J ¼ 8.5 Hz, 2H),

6.81e6.70 (m, 2H), 6.39 (d, J ¼ 14.6 Hz, 1H), 3.58 (d, J ¼ 36.0 Hz, 4H),

3.28e3.15 (m, 4H), 1.75e1.54 (m, 12H). ¹³C NMR (101 MHz, CDCl₃)

CDCl₃) d 165.45, 152.62, 143.22, 142.03, 125.55, 125.43, 120.85,

112.06, 110.92, 47.02, 43.37, 26.83, 25.72, 24.74. Exact mass calcd for

C14H17NO2 [MþNa]^þ: 254.1157; found 254.1159.

d

165.87, 152.17, 143.41, 138.99, 128.26, 126.92, 123.77, 118.71, 115.69,

49.86, 25.73, 24.86, 24.47. Exact mass calcd for C21H28N2O

4.1.23. (2E,4E)-1-(piperidin-1-yl)-5-(thiophen-2-yl)penta-2,4-

dien-1-one (3r)

2a was reacted with 2-thiophenaldehyde following the general

procedure B to give the desired product 3r as a light yellow powder

[MþNa]^þ: 347.2099; found 347.2101.

4.1.18. (2E,4E)-5-(4-chlorophenyl)-1-(piperidin-1-yl)penta-2,4-

dien-1-one (3m)

(67.8%). ¹H NMR (400 MHz, CDCl₃)

d

7.37 (dd, J ¼ 14.6, 11.1 Hz, 1H),

2a was reacted with 4-chlorobenzaldehyde following the gen-

eral procedure B to give the desired product 3m as a white powder

7.22 (d, J ¼ 5.1 Hz, 1H), 7.06 (d, J ¼ 3.6 Hz, 1H), 6.98 (dd, J ¼ 5.1,

3.6 Hz, 1H), 6.95 (d, J ¼ 15.8 Hz, 1H), 6.69 (dd, J ¼ 15.3, 11.1 Hz, 1H),

6.44 (d, J ¼ 14.6 Hz, 1H), 3.56 (s, 3H), 1.68e1.62 (m, 2H), 1.57 (dt,

(68.2%). ¹H NMR (400 MHz, DMSO‑d₆)

d

7.56 (d, J ¼ 8.5 Hz, 2H), 7.45

(d, J ¼ 8.5 Hz, 2H), 7.25 (dd, J ¼ 14.4, 10.9 Hz, 1H), 7.13 (dd, J ¼ 15.5,

10.9 Hz, 1H), 6.97 (d, J ¼ 15.5 Hz, 1H), 6.80 (d, J ¼ 14.4 Hz, 1H), 3.54

(s, 4H), 1.63 (dt, J ¼ 11.2, 5.6 Hz, 2H), 1.50 (s, 4H). ¹³C NMR (101 MHz,

J ¼ 11.0, 5.5 Hz, 4H). ¹³C NMR (101 MHz, CDCl₃)

d 165.37, 142.02,

142.00, 131.15, 127.94, 127.86, 126.78, 125.97, 120.60, 46.95, 43.56,

26.20, 24.75. Exact mass calcd for C14H17NOS [MþH]^þ: 248.1109;

found 248.1104.

DMSO‑d₆)

d 164.04, 141.12, 136.14, 135.28, 132.79, 128.82, 128.41,

128.23, 122.44, 46.09, 42.46, 26.44, 25.36, 24.11. Exact mass calcd

for C16H18ClNO [MþNa]^þ: 298.0975; found 298.0976.

4.1.24. (2E,4E)-N,N-diethyl-5-(thiophen-2-yl)penta-2,4-dienamide

(3s)

4.1.19. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-1-(piperidin-1-yl)

penta-2,4-dien-1-one (3n)

2b was reacted with 2-thiophenaldehyde following the general

procedure B to give the desired product 3s as a light yellow powder

2a was reacted with piperonyl aldehyde following the general

procedure B to give the desired product 3n as a white powder

(56.3%). ¹H NMR (400 MHz, DMSO‑d₆)

d

7.57 (d, J ¼ 5.1 Hz, 1H), 7.28

(d, J ¼ 3.6 Hz, 1H), 7.20e7.15 (m, 1H), 7.15e7.11 (m, 1H), 7.09 (dd,

J ¼ 5.1, 3.6 Hz,1H), 6.73 (dd, J ¼ 15.4,11.1 Hz,1H), 6.06 (d, J ¼ 15.1 Hz,

1H), 3.55e3.34 (m, 4H),1.24 (t, J ¼ 7.1 Hz, 3H),1.18 (t, J ¼ 6.5 Hz, 3H).

(75.3%). ¹H NMR (400 MHz, DMSO‑d₆)

1H), 7.16 (d, J ¼ 1.2 Hz, 1H), 6.95 (dt, J ¼ 20.7, 8.3 Hz, 3H), 6.86 (d,

J ¼ 15.6 Hz, 1H), 6.67 (d, J ¼ 14.5 Hz, 1H), 6.04 (s, 2H), 3.51 (s, 4H),

1.65e1.55 (m, 2H), 1.47 (s, 4H). ¹³C NMR (101 MHz, DMSO‑d₆)

d

7.21 (dd, J ¼ 14.5, 10.5 Hz,

¹³C NMR (101 MHz, DMSO‑d₆)

d 165.06, 141.05, 138.21, 130.47,

128.19, 127.81, 126.68, 126.09, 125.12, 42.12, 40.89, 15.03, 13.16.

Exact mass calcd for C11H13NOS [MþNa]^þ: 258.0929; found

258.0928.

d

164.20, 147.91, 147.71, 141.71, 137.57, 130.83, 125.66, 122.41, 120.76,

108.48, 105.45, 101.25, 46.06, 42.42, 26.44, 25.41, 24.13. Exact mass

calcd for C17H19NO3 [MþNa]^þ: 308.1263; found 308.1264.

4.1.25. (2E,4E)-N-ethyl-5-(thiophen-2-yl)penta-2,4-dienamide (3t)

2d was reacted with 2-thiophenaldehyde following the general

procedure B to give the desired product 3t as a light yellow powder

4.1.20. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N,N-diethylpenta-2,4-

dienamide (3o)

2b was reacted with piperonyl aldehyde following the general

procedure B to give the desired product 3o as a white powder

(63.3%). ¹H NMR (400 MHz, DMSO‑d₆)

d

8.04 (t, J ¼ 5.3 Hz, 1H), 7.55

(d, J ¼ 5.1 Hz, 1H), 7.26 (d, J ¼ 3.6 Hz, 1H), 7.20e7.15 (m, 1H),

7.15e7.11 (m, 1H), 7.09 (dd, J ¼ 5.1, 3.6 Hz, 1H), 6.73 (dd, J ¼ 15.4,

11.1 Hz, 1H), 6.09 (d, J ¼ 15.1 Hz, 1H), 3.17 (qd, J ¼ 7.2, 5.8 Hz, 2H),

(75.8%). ¹H NMR (400 MHz, CDCl₃)

d

7.44 (ddd, J ¼ 14.6, 7.4, 2.6 Hz,

1H), 6.99 (d, J ¼ 1.5 Hz, 1H), 6.89 (dd, J ¼ 8.0, 1.5 Hz, 1H), 6.77 (d,

J ¼ 8.5 Hz, 2H), 6.75 (d, J ¼ 5.0 Hz, 1H), 6.36 (d, J ¼ 14.6 Hz, 1H), 5.97

(s, 2H), 3.53e3.34 (m, 4H), 1.23 (t, J ¼ 7.1 Hz, 3H), 1.17 (t, J ¼ 6.5 Hz,

1.06 (t, J ¼ 7.2 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 164.76,

141.35, 138.36, 130.66, 128.19, 127.99, 126.79, 126.18, 125.22, 33.42,

14.74. Exact mass calcd for C11H13NOS [MþNa]^þ: 230.0616; found

230.0617.

3H). ¹³C NMR (101 MHz, CDCl₃)

d 165.88, 148.21, 148.14, 142.50,

138.44, 131.05, 125.36, 122.53, 120.27, 108.50, 105.71, 101.28, 42.22,

40.98, 15.03, 13.26. Exact mass calcd for C16H19NO3 [MþNa]^þ:

296.1263; found 296.1265.

4.1.26. (2E,4E)-1-(piperidin-1-yl)-5-(thiophen-3-yl)penta-2,4-

dien-1-one (3u)

4.1.21. (2E,4E)-5-(benzo[d][1,3]dioxol-5-yl)-N-ethylpenta-2,4-

dienamide (3p)

2a was reacted with 3-thiophenaldehyde following the general

procedure B to give the desired product 3u as a light yellow powder

2c was reacted with piperonyl aldehyde following the general

procedure B to give the desired product 3p as a white powder

(65.4%). ¹H NMR (400 MHz, CDCl₃)

d

7.40 (dd, J ¼ 14.6, 10.9 Hz, 1H),

7.32e7.24 (m, 3H), 6.86 (d, J ¼ 15.5 Hz, 1H), 6.73 (dd, J ¼ 15.5,

10.9 Hz,1H), 6.45 (d, J ¼ 14.6 Hz, 1H), 3.58 (m, 4H), 1.68 (m, 2H), 1.59

(78.2%). ¹H NMR (400 MHz, DMSO‑d₆)

(d, J ¼ 1.5 Hz, 1H), 7.15 (dd, J ¼ 15.0, 10.2 Hz, 1H), 7.00 (dd, J ¼ 8.1,

d

8.03 (t, J ¼ 5.4 Hz, 1H), 7.26

(m, 4H). ¹³C NMR (101 MHz, CDCl₃)

d 165.53, 142.65, 139.61, 132.51,

16

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

127.19, 126.57, 125.02, 124.43, 120.50, 26.35, 24.80. Exact mass calcd

4.1.32. (2E,4E)-5-(2-hydroxyphenyl)-1-(piperidin-1-yl)penta-2,4-

dien-1-one (4c)

for C14H17NOS [MþH]^þ: 248.1109; found 248.1080.

3c was demethylated with BBr₃following the general procedure

C to give the desired product 4c as a light brown yellow powder

4.1.27. (2E,4E)-5-(1-methyl-1H-pyrrol-2-yl)-1-(piperidin-1-yl)

penta-2,4-dien-1-one (3v)

(48.2%). ¹H NMR (400 MHz, CD₃OD)

d

7.33 (dd, J ¼ 14.7, 10.9 Hz, 1H),

2a was reacted with 1-methyl-2-pyrrolecarboxaldehyde

following the general procedure B to give the desired product 3v

7.16 (t, J ¼ 7.9 Hz, 1H), 7.03e6.91 (m, 3H), 6.83 (d, J ¼ 15.6 Hz, 1H),

6.73 (dd, J ¼ 8.0, 2.0 Hz, 1H), 6.66 (d, J ¼ 14.7 Hz, 1H), 3.67e3.56 (m,

4H), 1.69 (dd, J ¼ 10.5, 5.5 Hz, 2H), 1.59 (s, 4H). ¹³C NMR (101 MHz,

as a white powder (68.3%). ¹H NMR (400 MHz, CDCl₃)

d 7.43 (dd,

J ¼ 14.5, 10.5 Hz, 1H), 6.73 (d, J ¼ 15.3 Hz, 1H), 6.67 (d, J ¼ 10.5 Hz,

1H), 6.65 (d, J ¼ 2.5 Hz, 1H), 6.51 (dd, J ¼ 3.8, 1.5 Hz, 1H), 6.37 (d,

J ¼ 14.5 Hz, 1H), 6.14 (dd, J ¼ 3.6, 2.8 Hz, 1H), 3.65 (s, 1H), 3.61e3.54

(m, 2H), 1.71e1.63 (m, 1H), 1.59 (dt, J ¼ 10.9, 5.4 Hz, 2H). ¹³C NMR

CD₃OD) d 167.62, 158.88, 144.26, 140.43, 139.20, 130.77, 128.02,

121.54, 119.77, 116.93, 114.43, 48.14, 44.54, 27.86, 26.87, 25.53. Exact

mass calcd for C16H19NO2 [MþNa]^þ: 280.1313; found 280.1314.

(101 MHz, CDCl₃)

d

165.78, 143.33, 131.39, 126.68, 125.03, 123.92,

4.1.33. (2E,4E)-N,N-diethyl-5-(4-hydroxyphenyl)penta-2,4-

dienamide (4d)

118.44, 108.96, 108.65, 34.21, 24.84, 24.58. Exact mass calcd for

C15H20N2O [MþH]^þ: 245.1654; found 245.1647.

2b was reacted with 4-methoxybenzaldehyde following the

general procedure B to give an intermediate without further puri-

ﬁcation and then demethlyated with BBr₃following the general

procedure C to give the desired product 4d as a light yellow powder

4.1.28. (2E,4E)-5-(5-methoxy-1-methyl-1H-indol-3-yl)-1-

(piperidin-1-yl)penta-2,4-dien-1-one (3w)

2a

was

reacted

with

1-isopropyl-5-methoxy-3-

(69.5% over two steps). ¹H NMR (400 MHz, DMSO‑d₆)

d 8.02 (dd,

indolecarboxaldehyde following the general procedure B to give

J ¼ 15.9, 11.2 Hz, 1H), 7.30 (d, J ¼ 8.5 Hz, 2H), 6.80 (d, J ¼ 8.5 Hz, 2H),

6.64 (d, J ¼ 15.9 Hz, 1H), 6.45 (t, J ¼ 11.2 Hz, 1H), 5.59 (d, J ¼ 11.2 Hz,

1H), 2.75 (q, J ¼ 7.2 Hz, 4H), 1.11 (t, J ¼ 7.2 Hz, 6H). ¹³C NMR

the desired product 3w as a white powder (45.3%). ¹H NMR

(400 MHz, CDCl₃)

d

7.56 (dd, J ¼ 14.6, 11.1 Hz, 1H), 7.33 (d, J ¼ 2.3 Hz,

1H), 7.23e7.20 (m, 2H), 7.06 (d, J ¼ 15.5 Hz, 1H), 6.94 (dd, J ¼ 8.9,

2.4 Hz, 1H), 6.83 (dd, J ¼ 15.5, 11.1 Hz, 1H), 6.36 (d, J ¼ 14.6 Hz, 1H),

3.91 (s, 3H), 3.77 (s, 3H), 3.64e3.59 (m, 4H), 1.71e1.65 (m, 2H), 1.62

(101 MHz, DMSO‑d₆) d 169.84, 158.33, 138.98, 136.85, 128.14, 127.55,

123.84,123.09,115.77, 41.74,12.45. Exact mass calcd for C15H17NO2

[MþNa]^þ: 268.1313; found 268.1316.

(m, 4H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 164.73, 154.53, 143.78,

132.76, 132.21, 131.88, 125.96, 121.92, 117.00, 112.22, 111.70, 111.10,

102.41, 55.64, 46.03, 42.38, 32.81, 26.53, 25.46, 24.22. Exact mass

calcd for C20H24N2O2 [MþNa]^þ: 347.1735; found 347.1736.

4.1.34. (2E,4E)-N-ethyl-5-(4-hydroxyphenyl)penta-2,4-dienamide

(4e)

2c was reacted with 4-methoxybenzaldehyde following the

general procedure B to give an intermediate without further puri-

ﬁcation and then demethlyated with BBr₃following the general

procedure C to give the desired product 4e as a light yellow powder

4.1.29. General procedure C for the synthesis of 4

1 M boron tribromide (BBr₃) in DCM (2.66 mL, 2.66 mmol) was

added to a solution of compound 3a (0.2 g, 0.50 mmol) in dry DCM

(20 mL) at ꢁ78 ^ꢂC and stirred for 2 h. The reaction mixture was

allowed to raise to room temperature slowly and stirred for another

4 h, as indicated by TLC. The reaction was carefully mixed with

saturated aqueous NaHCO₃(30 mL) at 0 ^ꢂC and stirred for 30 min.

This mixture was extracted with ethyl acetate twice (20 mL ꢀ 2),

and the organic portion was combined, further washed with brine,

and dried with MgSO₄. The residue was ﬁltered, concentrated, and

puriﬁed by silica gel column chromatography to yield 4.

(65.3% over two steps). ¹H NMR (400 MHz, DMSO‑d₆)

d 8.11 (dd,

J ¼ 15.9, 11.1 Hz, 1H), 7.29 (d, J ¼ 8.4 Hz, 2H), 6.84 (d, J ¼ 8.4 Hz, 2H),

6.58 (d, J ¼ 15.9 Hz, 1H), 6.35 (t, J ¼ 11.1 Hz, 1H), 5.59 (d, J ¼ 11.1 Hz,

1H), 2.79 (q, J ¼ 7.2 Hz, 2H), 1.13 (t, J ¼ 7.2 Hz, 3H). ¹³C NMR

(101 MHz, DMSO‑d₆) d 170.69, 158.54, 137.07, 135.54, 127.90, 127.55,

126.30, 123.50, 115.82, 34.16, 13.63. Exact mass calcd for

C13H15NO2 [MþNa]^þ: 240.1000; found 240.1001.

4.1.35. General procedure D for the synthesis of 6

Malonic acid (0.3 g, 2.86 mmol) was added to the stirred solu-

tion of the corresponding aldehyde 5 (1.3 mmol) in pyridine

(15 mL) and piperidine (1.5 mL). The mixture was heated to reﬂux

for 8 h. The reaction mixture was neutralized with hydrochloric

acid in an ice bath. The precipitates were ﬁltered and washed with

cold water. The crude product was then recrystallized from ethanol

to afford the corresponding acids 6.

4.1.30. (2E,4E)-5-(4-hydroxyphenyl)-1-(piperidin-1-yl)penta-2,4-

dien-1-one (4a)

3a was demethylated with BBr₃following the general procedure

C to give the desired product 4a as a light brown yellow powder

(98.0%). ¹H NMR (400 MHz, CDCl₃)

d 7.46e7.37 (m, 1H), 7.30 (d,

J ¼ 8.4 Hz, 2H), 6.86 (d, J ¼ 8.4 Hz, 2H), 6.74 (m, 2H), 6.40 (d,

J ¼ 14.4 Hz,1H), 3.60 (s, 4H),1.72e1.63 (m, 2H),1.60 (s, 4H). ¹³C NMR

(101 MHz, CDCl₃)

d

166.12, 157.12, 143.73, 139.18, 128.76, 128.63,

4.1.36. (E)-3-(4-(4-(tert-butoxycarbonyl)piperazin-1-yl)phenyl)

acrylic acid (6a)

124.35, 118.75, 115.93, 100.00, 27.10, 26.35, 24.58. Exact mass calcd

for C16H19NO2 [MþNa]^þ: 280.1313; found 280.1314.

Malonic acid was reacted with tert-butyl 4-(4-formylphenyl)

tetrahydro-1(2H)-pyrazinecarboxylate following the general pro-

cedure D to give the desired product 6a as a light yellow powder

4.1.31. (2E,4E)-5-(3-hydroxyphenyl)-1-(piperidin-1-yl)penta-2,4-

dien-1-one (4b)

(67.1%). ¹H NMR (400 MHz, DMSO‑d₆)

d 12.06 (s, 1H), 7.53 (d,

3b was demethylated with BBr₃following the general procedure

C to give the desired product 4b as a light brown yellow powder

J ¼ 8.8 Hz, 2H), 7.48 (d, J ¼ 15.9 Hz, 1H), 6.95 (d, J ¼ 8.8 Hz, 2H), 6.29

(d, J ¼ 15.9 Hz, 1H), 3.50e3.38 (m, 4H), 3.28e3.13 (m, 4H), 1.42 (s,

9H). Exact mass calcd for C18H24N2O4 [M-H]^-: 331.1663; found

331.1664.

(23.1%). ¹H NMR (400 MHz, DMSO‑d₆)

d 9.88 (s, 1H), 7.44 (d,

J ¼ 6.7 Hz,1H), 7.22 (ddd, J ¼ 14.4, 8.4, 2.1 Hz,1H), 7.17e7.03 (m, 3H),

6.86 (d, J ¼ 7.8 Hz, 1H), 6.80 (t, J ¼ 7.4 Hz, 1H), 6.70 (d, J ¼ 14.4 Hz,

1H), 3.52 (s, 4H), 1.65e1.55 (m, 2H), 1.48 (s, 4H). ¹³C NMR (101 MHz,

4.1.37. (E)-3-(4-(pyrrolidin-1-yl)phenyl)acrylic acid (6b)

DMSO‑d₆)

d

164.31, 155.49, 142.52, 133.51, 129.53, 127.26, 127.13,

Malonic acid was reacted with 1-(4-formylphenyl)pyrrolidine

following the general procedure D to give the desired product 6b as

a light yellow powder (77.1%). ¹H NMR (400 MHz, DMSO‑d₆)

123.04, 120.71, 119.30, 115.94, 46.04, 42.43, 26.48, 25.40, 24.16.

Exact mass calcd for C16H19NO2 [MþNa]^þ: 280.1313; found

280.1314.

d

7.52e7.39 (m, 3H), 6.53 (d, J ¼ 8.7 Hz, 2H), 6.17 (d, J ¼ 15.9 Hz, 1H),

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

17

3.28 (t, J ¼ 6.5 Hz, 4H), 2.00e1.90 (m, 4H). Exact mass calcd for

solution of triﬂuoroacetic acid (2 mL) at 0 ^ꢂC. The reaction mixture

was then allowed to warm to room temperature and stirred for 1 h

until completed, as indicated by TLC. Then triﬂuoroacetic acid was

evaporated under reduced pressure. The residue was dissolved

with ethyl acetate (10 mL), and washed with saturated aqueous

NaHCO₃(10 mL ꢀ 2), dried over anhyd. Na₂SO₄and concentrated

under reduced pressure. The obtained crude product was puriﬁed

by column chromatography to get pure product 7b as yellow

powder with a yield of 79.8% over two steps. ¹H NMR (400 MHz,

C13H15NO2 [M-H]^-: 216.1030; found 216.1031.

4.1.38. (E)-3-(benzo[d][1,3]dioxol-5-yl)acrylic acid (6c)

Malonic acid was reacted with piperonyl aldehyde following the

general procedure D to give the desired product 6c as a light yellow

powder (69.5%). ¹H NMR (400 MHz, DMSO‑d₆)

1H), 7.36 (d, J ¼ 1.5 Hz, 1H), 7.15 (dd, J ¼ 8.1, 1.5 Hz, 1H), 6.94 (d,

J ¼ 8.1 Hz, 1H), 6.39 (d, J ¼ 15.9 Hz, 1H), 6.07 (s, 2H). Exact mass

calcd for C10H8O4 [M-H]^-: 191.0350; found 191.0351.

d

7.50 (d, J ¼ 15.9 Hz,

CDCl₃)

d

7.64 (d, J ¼ 15.3 Hz, 1H), 7.44 (d, J ¼ 8.7 Hz, 2H), 6.88 (d,

J ¼ 8.7 Hz, 2H), 6.67 (d, J ¼ 15.3 Hz, 1H), 3.48 (d, J ¼ 2.6 Hz, 4H),

4.1.39. (E)-3-(2-methoxyphenyl)acrylic acid (6d)

3.35e3.19 (m, 4H), 3.08 (dd, J ¼ 6.0, 4.0 Hz, 4H), 1.26 (s, 6H). ¹³C

Malonic acid was reacted with 2-methoxybenzaldehyde

following the general procedure D to give the desired product 6d

NMR (101 MHz, CDCl₃) d 166.21, 152.17, 142.17, 129.08, 126.67,

115.38, 114.36, 48.94, 45.56, 42.26, 41.09, 29.70, 15.06. Exact mass

as a light yellow powder (75.6%). ¹H NMR (400 MHz, CDCl₃)

d

8.10

calcd for C17H25N3O [MþH]^þ: 288.2076; found 288.2077.

(d, J ¼ 16.1 Hz, 1H), 7.54 (dd, J ¼ 7.7, 1.4 Hz, 1H), 7.41e7.34 (m, 1H),

6.98 (t, J ¼ 7.5 Hz, 1H), 6.93 (d, J ¼ 8.3 Hz, 1H), 6.56 (d, J ¼ 16.1 Hz,

1H), 3.90 (s, 3H). Exact mass calcd for C10H10O3 [MþH]^þ:

179.0708; found 179.0702.

4.1.44. (E)-N-ethyl-3-(4-(piperazin-1-yl)phenyl)acrylamide (7c)

6a was reacted with ethylamine following the general proced-

ure E to give an intermediate, which was then added to a solution of

triﬂuoroacetic acid (2 mL) at 0 ^ꢂC. The reaction mixture was then

allowed to warm to room temperature and stirred for 1 h until

completed, as indicated by TLC. Then triﬂuoroacetic acid was

evaporated under reduced pressure. The residue was dissolved

with ethyl acetate (10 mL), and washed with saturated aqueous

NaHCO₃(10 mL ꢀ 2), dried over anhyd. Na₂SO₄and concentrated

under reduced pressure. The obtained crude product was puriﬁed

by column chromatography to get pure product 7c as yellow

powder with a yield of 65.4% over two steps. ¹H NMR (400 MHz,

4.1.40. (E)-3-(thiophen-2-yl)acrylic acid (6e)

Malonic acid was reacted with 2-thenaldehyde following the

general procedure D to give the desired product 6e as a light yellow

powder (65.5%). ¹H NMR (400 MHz, DMSO‑d₆)

d 12.35 (s, 1H), 7.74

(d, J ¼ 15.8 Hz, 1H), 7.70 (d, J ¼ 5.0 Hz, 1H), 7.50 (d, J ¼ 3.4 Hz, 1H),

7.14 (dd, J ¼ 5.0, 3.7 Hz, 1H), 6.17 (d, J ¼ 15.8 Hz, 1H). Exact mass

calcd for C7H6O2S [M-H]^-: 153.0016; found 153.0016.

4.1.41. General procedure E for the synthesis of 7

CDCl₃)

d

7.54 (d, J ¼ 15.5 Hz, 1H), 7.40 (d, J ¼ 8.8 Hz, 2H), 6.86 (d,

6 (1 mmol) was dissolved into 2 mL DMF. O-(7-aza-1H-benzo-

triazole-1-yl)-N,N,N⁰,N⁰-tetramethyluronium hexaﬂuorophosphate

(HATU) (1.5 mmol) was added to a solution, followed by corre-

sponding lamins (1.2 mmol), triethylamine (3.0 mmol). The

mixture was reacted at room temperature for 4 h. After the reaction

was completed, 8 mL water was added to quench the reaction. Then

exacted with ethyl acetate 2 mL (three times). The organic layer was

dried over anhyd. Na₂SO₄and concentrated under reduced pres-

sure. The obtained crude product was puriﬁed by silico column

chromatography to get pure product 7.

J ¼ 8.8 Hz, 2H), 6.21 (d, J ¼ 15.5 Hz, 1H), 5.55 (s, 1H), 3.46e3.35 (m,

2H), 3.27e3.16 (m, 4H), 3.07e2.97 (m, 4H), 1.20 (t, J ¼ 7.3 Hz, 3H).

¹³C NMR (101 MHz, CDCl₃)

d 166.54, 152.62, 140.82, 129.20, 125.80,

117.32, 115.29, 49.43, 46.05, 34.68, 15.11. Exact mass calcd for

C15H21N3O [MþNa]^þ: 260.1763; found 260.1762.

4.1.45. (E)-1-(piperidin-1-yl)-3-(4-(pyrrolidin-1-yl)phenyl)prop-2-

en-1-one (7d)

6b was reacted with piperidine following the general procedure

E to give the desired product 7d as a light yellow powder (75.3%). ¹H

NMR (400 MHz, CDCl₃)

d

7.61 (d, J ¼ 15.2 Hz, 1H), 7.41 (d, J ¼ 8.7 Hz,

4.1.42. (E)-3-(4-(piperazin-1-yl)phenyl)-1-(piperidin-1-yl)prop-2-

en-1-one (7a)

2H), 6.67 (d, J ¼ 15.2 Hz, 1H), 6.52 (d, J ¼ 8.7 Hz, 2H), 3.62 (s, 4H),

3.32 (t, J ¼ 6.6 Hz, 4H), 2.04e1.97 (m, 4H), 1.71e1.63 (m, 2H), 1.60 (d,

6a was reacted with piperidine following the general procedure

E to give an intermediate, which was then added to a solution of

triﬂuoroacetic acid (2 mL) at 0 ^ꢂC. The reaction mixture was then

allowed to warm to room temperature and stirred for 1 h until

completed, as indicated by TLC. Then triﬂuoroacetic acid was

evaporated under reduced pressure. The residue was dissolved

with ethyl acetate (10 mL), and washed with saturated aqueous

NaHCO₃(10 mL ꢀ 2), dried over anhyd. Na₂SO₄and concentrated

under reduced pressure. The obtained crude product was puriﬁed

by column chromatography to get pure product 7a as yellow

powder with a yield of 76.4% over two steps. ¹H NMR (400 MHz,

J ¼ 4.6 Hz, 4H). ¹³C NMR (101 MHz, CDCl₃)

d 166.30, 148.76, 143.11,

129.37,122.73,111.55,111.49, 47.55, 46.85, 43.37, 26.72, 25.46, 24.78.

Exact mass calcd for C18H24N2O [MþNa]^þ: 307.1786; found

307.1784.

4.1.46. (E)-N,N-diethyl-3-(4-(pyrrolidin-1-yl)phenyl)acrylamide

(7e)

6b was reacted with diethylamine following the general pro-

cedure E to give the desired product 7e as a light yellow powder

(68.9%). ¹H NMR (400 MHz, CDCl₃)

d

7.64 (d, J ¼ 15.2 Hz,1H), 7.40 (d,

J ¼ 8.7 Hz, 2H), 6.58 (d, J ¼ 15.2 Hz, 1H), 6.51 (d, J ¼ 8.7 Hz, 2H),

CDCl₃)

d

7.56 (d, J ¼ 15.4 Hz, 1H), 7.44 (d, J ¼ 8.7 Hz, 2H), 6.86 (d,

3.55e3.39 (m, 4H), 3.30 (t, J ¼ 6.6 Hz, 4H), 2.10e1.92 (m, 4H),

J ¼ 8.7 Hz, 2H), 6.77 (d, J ¼ 15.4 Hz, 1H), 3.61 (d, J ¼ 19.9 Hz, 4H),

1.32e1.10 (m, 6H). ¹³C NMR (101 MHz, CDCl₃)

d 166.73, 148.88,

3.49e3.36 (m, 4H), 3.34e3.16 (m, 4H), 1.72e1.64 (m, 2H), 1.60 (d,

143.15, 129.51, 122.77, 111.77, 111.64, 47.63, 42.31, 41.11, 25.54, 15.13,

13.43. Exact mass calcd for C17H24N2O [MþNa]^þ: 295.1786; found

295.1792.

J ¼ 4.1 Hz, 4H). ¹³C NMR (101 MHz, CDCl₃)

d 165.71, 150.99, 141.74,

129.11, 127.96, 116.23,115.19, 47.03, 46.82, 43.77, 43.38, 26.76, 25.64,

24.66. Exact mass calcd for C18H25N3O [MþH]^þ: 300.2076; found

300.2076.

4.1.47. (E)-3-(benzo[d][1,3]dioxol-5-yl)-1-(piperidin-1-yl)prop-2-

en-1-one (7f)

4.1.43. (E)-N,N-diethyl-3-(4-(piperazin-1-yl)phenyl)acrylamide

(7b)

6c was reacted with piperidine following the general procedure

E to give the desired product 7f as a light yellow powder (52.9%). ¹H

6a was reacted with diethylamine following the general pro-

cedure E to give an intermediate, which was then added to a

NMR (400 MHz, CDCl₃)

1H), 6.99 (dd, J ¼ 8.0, 1.4 Hz, 1H), 6.79 (d, J ¼ 8.0 Hz, 1H), 6.73 (d,

d

7.56 (d, J ¼ 15.3 Hz, 1H), 7.03 (d, J ¼ 1.4 Hz,

18

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

J ¼ 15.3 Hz, 1H), 5.98 (s, 2H), 3.61 (s, 4H), 1.71e1.64 (m, 2H),

1.64e1.56 (m, 4H). ¹³C NMR (101 MHz, CDCl₃)

165.62, 148.98,

148.34, 142.13, 130.10, 123.74, 115.86, 108.63, 106.50, 101.53, 26.22,

24.81. Exact mass calcd for C15H17NO3 [MþNa]^þ: 282.1106; found

282.1105.

NMR (400 MHz, CDCl₃)

d

7.78 (d, J ¼ 14.9 Hz, 1H), 7.30 (d, J ¼ 5.0 Hz,

d

1H), 7.20 (d, J ¼ 3.5 Hz, 1H), 7.03 (dd, J ¼ 5.0, 3.5 Hz, 1H), 6.72 (d,

J ¼ 14.9 Hz, 1H), 3.62 (s, 4H), 1.68 (d, J ¼ 4.2 Hz, 2H), 1.62 (s, 4H). ¹³

C

NMR (101 MHz, CDCl₃)

d 164.73, 138.77, 134.73, 130.07, 128.88,

127.32, 116.45, 46.97, 43.58, 26.21, 24.76. Exact mass calcd for

C12H15NOS [MþNa]^þ: 244.0772; found 244.0775.

4.1.48. (E)-3-(benzo[d][1,3]dioxol-5-yl)-N,N-diethylacrylamide (7g)

6c was reacted with diethylamine following the general pro-

cedure E to give the desired product 7g as a light yellow powder

4.1.54. (E)-N,N-diethyl-3-(thiophen-2-yl)acrylamide (7m)

6e was reacted with diethylamine following the general pro-

cedure E to give the desired product 7m as a light yellow powder

(82.7%). ¹H NMR (400 MHz, CDCl₃)

d

7.62 (d, J ¼ 15.3 Hz,1H), 7.03 (d,

J ¼ 1.6 Hz, 1H), 7.00 (dd, J ¼ 8.0, 1.6 Hz, 1H), 6.80 (d, J ¼ 8.0 Hz, 1H),

6.65 (d, J ¼ 15.3 Hz, 1H), 5.99 (s, 2H), 3.47 (s, 4H), 1.25 (t, J ¼ 6.5 Hz,

(41.5%). ¹H NMR (400 MHz, CDCl₃)

d

7.81 (d, J ¼ 15.1 Hz, 1H), 7.29 (d,

J ¼ 5.1 Hz, 1H), 7.20 (d, J ¼ 3.5 Hz, 1H), 7.02 (dd, J ¼ 5.1, 3.5 Hz, 1H),

3H), 1.18 (t, J ¼ 6.5 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)

d 166.01,

6.62 (d, J ¼ 15.1 Hz, 1H), 3.55e3.37 (m, 4H), 1.21 (s, 6H). ¹³C NMR

149.00, 148.32, 142.18, 130.08, 123.83, 115.97, 108.65, 106.51, 101.53,

42.41, 41.22, 15.22, 13.39. Exact mass calcd for C14H17NO3

[MþNa]^þ: 270.1106; found 270.1107.

(101 MHz, CDCl₃) d 205.56, 165.53, 140.77, 135.17, 130.10, 128.08,

127.02, 116.75, 42.41, 41.23, 15.21, 13.35. Exact mass calcd for

C11H15NOS [MþNa]^þ: 232.0772; found 232.0774.

4.1.49. (E)-3-(benzo[d][1,3]dioxol-5-yl)-N-ethylacrylamide (7h)

6c was reacted with ethylamine following the general procedure

E to give the desired product 7h as a light yellow powder (73.7%). ¹H

4.1.55. (E)-N-ethyl-3-(thiophen-2-yl)acrylamide (7n)

6e was reacted with ethylamine following the general proced-

ure E to give the desired product 7n as a light yellow powder

NMR (400 MHz, CDCl₃)

d

7.52 (d, J ¼ 15.5 Hz, 1H), 6.98 (d, J ¼ 1.5 Hz,

(27.4%). ¹H NMR (400 MHz, CDCl₃)

d

7.73 (d, J ¼ 15.3 Hz, 1H), 7.29 (d,

1H), 6.96 (dd, J ¼ 8.0, 1.5 Hz, 1H), 6.78 (d, J ¼ 8.0 Hz, 1H), 6.21 (d,

J ¼ 15.5 Hz, 1H), 5.70 (s, 1H), 3.41 (qd, J ¼ 7.3, 5.9 Hz, 2H), 1.20 (t,

J ¼ 5.1 Hz, 1H), 7.19 (d, J ¼ 3.4 Hz, 1H), 7.02 (dd, J ¼ 5.1, 3.7 Hz, 1H),

6.19 (d, J ¼ 15.3 Hz, 1H), 5.67 (s, 1H), 3.48e3.36 (m, 2H), 1.20 (t,

J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)

d

166.08, 149.11, 148.34,

J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)

d 165.68, 140.18, 133.67,

140.65, 129.45, 123.88, 118.99, 108.65, 106.45, 101.54, 34.73, 15.05.

Exact mass calcd for C12H13NO3 [MþNa]^þ: 242.0793; found

242.0787.

130.24, 128.10, 127.23, 119.88, 34.75, 15.02. Exact mass calcd for

C9H11NOS [MþNa]^þ: 204.0459; found 204.0453.

4.2. Biological study

4.1.50. (E)-3-(2-methoxyphenyl)-1-(piperidin-1-yl)prop-2-en-1-

one (7i)

4.2.1. MTT assay

6d was reacted with piperidine following the general procedure

PC12 cells (1 ꢀ 10⁴cells/well) were seeded in 96-well plates for

E to give the desired product 7i as a light yellow powder (84.5%). ¹H

24 h followed by pretreation with PIP analogues for another 24 h,

NMR (400 MHz, CDCl₃)

d

7.89 (d, J ¼ 15.6 Hz, 1H), 7.48 (dd, J ¼ 7.6,

and then replaced with fresh medium containing 650 mM H₂O₂.

1.5 Hz, 1H), 7.33e7.27 (m, 1H), 6.99 (d, J ¼ 15.6 Hz, 1H), 6.94 (t,

J ¼ 7.8 Hz, 1H), 6.90 (d, J ¼ 8.3 Hz, 1H), 3.87 (s, 3H), 3.61 (s, 4H),

1.72e1.64 (m, 2H), 1.63e1.55 (m, 4H). ¹³C NMR (101 MHz, CDCl₃)

After 24 h exposing to H₂O₂, medium was replaced with fresh

medium again. Cell viability was determined by MTT assay

described as previously described [43].

d

166.15, 158.22, 137.69, 130.55, 128.90, 124.77, 120.72, 118.90,

111.25, 55.62, 47.14, 43.43, 26.87, 25.83, 24.84. Exact mass calcd for

4.2.2. Lactate dehydrogenase (LDH) release assay

C15H19NO2 [MþH]^þ: 246.1494; found 246.1495.

PC12 cells (1 ꢀ 10⁴cells/well) were seeded in 96-well plates for

24 h followed by pretreated with different concentration PIP ana-

4.1.51. (E)-N,N-diethyl-3-(2-methoxyphenyl)acrylamide (7j)

6d was reacted with piperidine following the general procedure

E to give the desired product 7j as a light yellow powder (83.6%). ¹H

logues (12.5, 25, 50 and 100

fresh medium containing 650

medium was measured using an LDH diagnostic kit (KeyGEN

BioTECH, NanJing, Jiang Su, China) according to the instruction.

m

M) for 24 h, and then replaced with

mM H₂O₂. The content of LDH in the

NMR (400 MHz, CDCl₃)

d

7.94 (d, J ¼ 15.6 Hz, 1H), 7.48 (dd, J ¼ 7.6,

1.5 Hz, 1H), 7.33e7.27 (m, 1H), 6.98e6.88 (m, 3H), 3.86 (s, 3H), 3.47

(q, J ¼ 7.1 Hz, 4H), 1.21 (s, 6H). ¹³C NMR (101 MHz, CDCl₃)

d

166.47,

4.2.3. Apoptosis assay

158.30, 137.96, 130.58, 129.09, 124.75, 120.71, 118.97, 111.24, 55.58,

42.31, 41.30,15.02,13.49. Exact mass calcd for C14H19NO2 [MþH]^þ:

234.1494; found 234.1500.

PC12 cells (2 ꢀ 10⁵cells/well) were seeded into 6-well plates for

24 h followed by pretreation with different concentration PIP an-

alogues (12.5, 25, 50 and 100

m

M) for 24 h, and then replaced with

fresh medium containing 650

mM H₂O₂. Apoptosis was detected

4.1.52. (E)-N-ethyl-3-(2-methoxyphenyl)acrylamide

using the Annexin V-FITC/PI Apoptosis Detection Kit (KeyGEN

BioTECH, NanJing, Jiang Su, China) according to the manufacturer’s

instructions. Speciﬁcally, PC12 cells were harvested and washed

with binding buffer, followed by Annexin V-FITC and Propidium

iodide (PI) stained for 5 min and detected by ﬂow cytometry

(Beckman Coulter, Miami, FL, USA). Analysis was performed using

Expo32 software (Beckman Coulter). Each sample was repeated

three times.

6d was reacted with piperidine following the general procedure

E to give the desired product 7k as a light yellow powder (83.6%). ¹H

NMR (400 MHz, CDCl₃)

d

7.85 (d, J ¼ 15.8 Hz, 1H), 7.44 (dd, J ¼ 7.6,

1.5 Hz, 1H), 7.32e7.26 (m, 1H), 6.90 (dd, J ¼ 14.5, 7.8 Hz, 2H), 6.51 (d,

J ¼ 15.8 Hz, 1H), 5.85 (s, 1H), 3.85 (s, 3H), 3.48e3.36 (m, 2H), 1.19 (t,

J ¼ 7.3 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)

d 166.60, 158.30, 136.16,

130.72, 129.02, 124.06, 121.97, 120.73, 111.20, 55.52, 34.66, 15.02.

Exact mass calcd for C12H15NO2 [MþH]^þ: 206.1181; found

206.1183.

4.2.4. Determination of intracellular ROS

ROS levels were monitored using the ﬂuorescent probe 2⁰,7⁰-

dichlorodihydroﬂuorescein diacetate (DCFH-DA). PC12 cells

(1 ꢀ 10⁴cells/well) were seeded into 96-well plates. After pre-

4.1.53. (E)-1-(piperidin-1-yl)-3-(thiophen-2-yl)prop-2-en-1-one

(7l)

6e was reacted with piperidine following the general procedure

treatment with 3b (12.5, 25, 50 and 100 mM) for 24 h, the cells were

E to give the desired product 7l as a light yellow powder (33.5%). ¹H

induced with 300 mM of H₂O₂for 24 h. At the end of induction,

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

19

PC12 cells were rinsed with serum-free medium and treated with

DCFH-DA working solution (DCFH-DA: serum-free

knockdown experiments, and scrambled sequence (shNT) was used

as a control. Exponentially growing cells were transfected with

different shRNAs using GeneTran III transfection reagent according

to the manufacture’s instruction. After 48 h of transfection, the cells

were cultured in DMEM containing 10% FBS, 2 mM glutamine, 100

units/mL penicillin/streptomycin at 37 ^ꢂC in a humidiﬁed atmo-

sphere of 5% CO₂and selected by supplementation with 0.5 mg/mL

of G418. Knockdown of the Nrf2 expression in the cells was

analyzed by Western blotting.

medium ¼ 1:1000) for 15 min at room temperature in a dark

environment. At the end of the incubation, extracellular DCFH-DA

molecules were washed away using serum-free medium. Finally,

ﬂuorescence was detected using an inverted ﬂuorescence micro-

scope (Zeiss Axiovert 200, Shanghai, China) and Inﬁnite M200 PRO

€

Multimode Microplate (Tecan Group Ltd, Mannedorf) (excitation

525 nm, emission 590 nm).

4.2.5. Measurement of mitochondrial membrane potential

4.2.10. Molecular docking

JC-1 dye (KeyGEN BioTECH, NanJing, Jiang Su, China) was used to

detect mitochondrial membrane potential of H₂O₂-induced

PC12 cells. 4 ꢀ 10⁵cells/well were seeded into 24-well plates. After

The Keap1 of homo sapiens has 624 residues. There are two main

domains for the Keap1, one for BTB domain between 67 and 179

residue, the other one for Kelch domain with the 327e611 residue.

There are many crystal structures for BTB or Kelch and no full

length keap1 structure was available [9]. Hence, we selected the I-

TASSER online service as the method to construct the homology

structure for the full length Keap1. The 1U6D [45], 2DYH [46], 3HVE

[47], 3I3N (Murray, unpublished), 3ZGD [48], 4AP2 [49] and 4IFJ

(Pan, unpublished) were used as the temple models. The overall

quality of the ﬁve homology models was veriﬁed by Verify 3D [50].

We selected the best one as our covalent docking homology model.

The covalent docking was done with the AutoDock 4.2 using the

two-point attractor and ﬂexible side chain methods [51]. The side

chain of Cys and the ligand molecule were ﬂexibly. The binding grid

box center was the C_aatom of Cys and the number points in X, Yand

Z were 40 ꢀ 40 ꢀ 40 with 0.375 Å as the spacing. The ten confor-

mation were identiﬁed by using a Lamarkian type of genetic algo-

rithm for every covalent docking and selected the lowest estimated

binding energy by the scoring function in the AutoDock 4.2.

pretreatment with 3b (12.5, 25 and 50 mM) for 24 h, cells were

induced with 650 mM H₂O₂for 24 h. PC12 cells were then treated

with pre-warmed JC-1 working solution (1:1000) for 20 min. After

incubation, the cells were washed three times with Incubation

Buffer (1:10). For signal quantiﬁcation, the intensity of red ﬂuo-

rescence (excitation 525 nm, emission 590 nm) and green ﬂuo-

rescence (excitation 488 nm, emission 525 nm) was detected by

Inﬁnite M200 PRO Multimode Microplate (Tecan Group Ltd,

€

Mannedorf) and the depolarization of mitochondrial membrane

potential was measured by the relative ratio of red ﬂuorescence to

green ﬂuorescence signal. The ﬂuorescent signal in the cells was

also observed and recorded with an inverted ﬂuorescent micro-

scope (Zeiss Axiovert 200, Shanghai, China).

4.2.6. Western blot analysis

Western blotting was performed as previously described [25].

Cytoplasmic and nuclear proteins were prepared from PC12 cells

using a nuclear and cytoplasmic protein extraction kit (KeyGEN

BioTECH, NanJing, Jiang Su, China) according to the manufacturer’s

instructions. Equal amounts of protein samples were separated by

denaturing SDS-PAGE, then transferred to PVDF membrane, and

probed with antibody respectively. Band intensity was analyzed

using Image Lab software (version 5.2.1, BIORAD, Hercules, USA).

4.2.11. Experiment animal and treatment

All animal experiments were performed according to the insti-

tutional guide for the care and use of laboratory animals, and all

mouse protocols were approved by the Animal Care and Use

Committee of Sichuan University Chengdu, Sichuan, China). Male

10-week-old C57BL/6 mice (weight, 20e23 g) were purchased from

Dossy Experimental Animals Co. Ltd. (Chengdu, China). Food and

water were given ad libitum, and mice were housed on a standard

12 h light/12 h dark cycle. Vehicle was treated with saline. In the

prophylactic administration group, saline or 3b (50 or 100 mg/kg)

dissolved in the saline was orally administered for two weeks; on

the third week, MPTP-HCl (25 mg/kg) or saline was intraperitone-

ally injected to create the MPTP-induced PD mouse models (n ¼ 6

per group). In the therapeutic administration group, MPTP-HCl

(25 mg/kg) or saline was intraperitoneally injected for one week

to create the MPTP-induced PD mouse models and followed by

saline or 3b (100 mg/kg) dissolved in the saline was orally

administered for two weeks (Fig. 7A). All mice were trained for pole

and rotarod test in the last three days. Twenty-one days after

treatment, the mice were sacriﬁced for further study.

4.2.7. Immunoﬂuorescence assay

Immunofuorescence assay was used to evaluate the levels of

Nrf2. Brieﬂy, PC12 cells were seeds into slides after incubation with

3b at a concentration of 25 mM for 0 h, 4 h, 6 h and 8 h. After

treatment, the medium was aspirated and the cells were washed

with PBS, ﬁxed with cold methanol at ꢁ20 ^ꢂC for 20 min, rinsed

with PBST for 5 min. Fixed cells were incubated with anti-Nrf2

diluted 1:200 in 0.5% BSA (diluted by PBST) overnight. Fixed cells

were incubated with anti-Nrf2 diluted 1:1000 in PBS overnight and

washed and treated with Dylight 594-conjugated secondary anti-

body diluted 1:500 in 0.5% BSA for 1 h, and then nuclei were

counterstained with 4⁰,6⁰-diamidino-2-phenylindole (DAPI). Sub-

sequently, the cells were observed with a fuorescence microscope

(Zeiss Axiovert 200, Shanghai, China).

4.2.12. Behavior tests

4.2.8. ARE-luciferase activity assay

Behavior tests was performed to detected the motor ability. Pole

and rotarod test were carried out as previously described [52].

Human hepatoma HepG2 cells in 24-well plates were trans-

fected with pGL3-ARE plasmid (gifted by professor Ling Wang,

West China School of Pharmacy, Sichuan University) using Lip-

ofectamine 2000 reagent at a ratio of 1:6 DNA/Lipofectamine and

incubated in opti-MEM medium for 30 min at 37 ^ꢂC, then DNA-lipid

complex were added. After 6 h, cells were cultured in fresh me-

dium. The cells were treated with different concentrations of 3b

after 48 h following the procedure described previously [44].

4.2.13. Immunohistochemistry

Immunohistochemistry assays were performed to detect the

expression of TH and Nrf2 in striatum and SNpc. These experiments

were carried as previously described [53].

4.2.14. Statistical analysis

Data are reported as the mean SD. The signiﬁcant difference

between multiple treatments and the control was analyzed using a

one-way ANOVA with Dunnett’s post-test. P values less than 0.05

4.2.9. Knockdown of Nrf2 expression by shRNA transfection

The shRNAs targeting rat Nrf2 gene was used for Nrf2

20

L. Wang et al. / European Journal of Medicinal Chemistry 199 (2020) 112385

Planta Med. 65 (1999) 600e603.

were considered statistically signiﬁcant.

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a

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The authors declare that they have no known competing

ﬁnancial interests or personal relationships that could have

appeared to inﬂuence the work reported in this paper.

Acknowledgement

The authors appreciate the ﬁnancial support from the National

Natural Science Foundation of China (81527806 and 81874297) and

the 1.3.5 project for disciplines of excellence, West China Hospital,

Sichuan University (ZY2017202).

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Appendix A. Supplementary data

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Identification and optimization of piperine analogues as neuroprotective agents for the treatment of Parkinson's disease via the activation of Nrf2/keap1 pathway

DOI: 10.1016/j.ejmech.2020.112385

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