Novel cannabidiol?carbamate hybrids as selective BuChE inhibitors: Docking-based fragment reassembly for the development of potential therapeutic agents against Alzheimer's disease

European Journal of Medicinal Chemistry 223 (2021) 1 13735

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

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

Novel cannabidiolꢀcarbamate hybrids as selective BuChE inhibitors:

Docking-based fragment reassembly for the development of potential

therapeutic agents against Alzheimer's disease

,

Xia Jiang ^{a 1}, Ziwen Zhang ^{a 1}, Jiawei Zuo ^a, Chengyao Wu ^a, Liang Zha ^a, Yingying Xu ^a,

Sheng Wang ^b, Jingbo Shi ^a, Xin-Hua Liu ^a, Jing Zhang ^{c **}, Wenjian Tang ^a

,

, *

^aSchool of Pharmacy, Inﬂammation and Immune Mediated Diseases Laboratory of Anhui Province, Anhui Medical University, Hefei, 230032, China

^bCenter for Scientiﬁc Research, Anhui Medical University, Hefei, 230032, China

^cAnhui Prevention and Treatment Center for Occupational Disease, Anhui No. 2 Provincial People's Hospital, Hefei, 230041, China

a r t i c l e i n f o

a b s t r a c t

Article history:

Cannabidiol (CBD) and rivastigmine have been launched as drugs for treating dementia and cholines-

terases (ChEs) are ideal drug targets. This study focused on developing novel ChE inhibitors as drug leads

against dementia through molecular modeling and fragment reassembly approaches. A potent carbamate

fragment binding to active site gorge of BuChE was found via a docking-based structural splicing

approach, thus, 17 novel compounds were designed by structural reassembly. Compound C16 was

identiﬁed as a highly selective potent BuChE inhibitor (IC₅₀¼ 5.3 nM, SI > 4000), superior to CBD

Received 15 June 2021

Received in revised form

8 July 2021

Accepted 28 July 2021

Available online 2 August 2021

(IC₅₀¼ 0.67

mM). C16 possessed BBB penetrating ability, benign safety, neuroprotection, antioxidant and

Keywords:

Cannabidiol

Carbamate

Fragment reassembly

Acetylcholinesterase

Butyrylcholinesterase

Pseudo-irreversible

Alzheimer's disease

pseudo-irreversible BuChE inhibition (K_d¼ 13 nM, k₂¼ 0.26 min^ꢀ1), showing good drug-like properties.

In vivo studies conﬁrmed that C16 signiﬁcantly ameliorated the scopolamine-induced cognition

impairment, almost entirely recovered the Ab_1ꢀ42(icv)-impaired cognitive function to the normal level,

showed better behavioral performance than donepezil and good anti-amyloidogenic effect. Hence, the

potential BuChE inhibitor C16 can be developed as a promising disease-modifying treatment of AD.

1. Introduction

including MF-8622 and cymserine indicated the potential thera-

peutic beneﬁt of inhibiting BuChE in AD and related dementias [5 ‒

Alzheimer's disease (AD) is an irreversible and severe progres-

sive neurodegenerative disorder [1,2]. In the last few decades, the

8]. BuChE activity progressively increases as the severity of de-

mentia advances, while AChE activity declines, therefore, inhibition

of BuChE may provide additional beneﬁts. Hence, BuChE was

considered as an important target for novel drug development to

treat AD, and the development of BuChE-speciﬁc inhibitors could

lead to improved clinical outcomes in the future [9 ‒20]. Moreover,

oxidative stress caused by free radicals has been proved to be the

main factor leading to the development of AD. Therefore, BuChE

inhibitors with the antioxidant potential may have a special ther-

apeutic effect on AD [21 ‒23].

Etiological research implicated cannabis use as a risk factor for

psychotic illness in general and schizophrenia in particular [24 ‒27].

Delta-9-tetrahydrocannabinol (THC) is thought to be the main

psychotropic agent of the cannabis in the Cannabis sativa plant [28].

THC is dose dependently associated to psychiatric symptoms such

as psychotic-like experiences in several studies [29]. In contrast,

approved drugs for treating AD include the N-methyl- -aspartate

D

receptor antagonist memantine and the cholinesterase inhibitors

donepezil, rivastigmine and galantamine, however, they cannot

effectively cure this disease, but they do provide short-term

symptomatic relief [3]. Two main cholinesterases (ChEs), acetyl-

cholinesterase (AChE) and butyrylcholinesterase (BuChE), are

involved in the hydrolysis and regulation of ACh in vertebrates [4].

Various cholinergic drugs, especially BuChE inhibitors also function

as AChE inhibitors, while the use of high selective BuChE inhibitors

* Corresponding author.

** Corresponding author.

E-mail addresses: hfzj2552@163.com (J. Zhang), ahmupharm@126.com

cannabidiol (CBD),

a main non-psychoactive component of

(W. Tang).

1

X. Jiang and Z.W. Zhang contributed equally to this work.

https://doi.org/10.1016/j.ejmech.2021.113735

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

cannabis, was ﬁrstly reported to interfere with the psychomimetic

actions of THC in 1974, indicating that CBD may have potential as an

antipsychotic agent [30]. CBD can not only antagonize the mental

activity caused by THC, but also has wide biological activities,

including anxiety, schizophrenia, addiction, neurodegenerative

diseases [31]. CBD exhibited neuroprotective effect on iron-induced

apoptosis and therapeutic effect in transgenic AD model mouse

[32,33], anti-inﬂammatory and antioxidant effects by inhibiting

BACH1 and weakly activating the transcription factor Nrf2 to ach-

ieve neuroprotective effects [34]. CBD had the potential to resist the

cytotoxicity of Ab _{(31̶ 35)}and Ab _{(25̶ 35)}peptides, indicating that CBD

can improve AD symptoms by reducing the accumulation of amy-

loid and the production of amyloid plaques [35]. As shown in Fig. 1,

CBD can enter the synaptic cleft and display wide pharmacological

effect on some receptors [36,37]. In the synaptic cleft, can CBD bind

into ChEs and improve AD by inhibiting ChEs to increase the level of

ACh?

Clinical drugs containing amide (eCONHe) and/or carbamate

(eOCONHe) motifs can provide privileged structural fragments

[38 ‒40]. These motifs are able to interact with a wide range of re-

ceptors/enzymes to induce biological responses [41,42]. As a result,

there is a growing interest in amide- and carbamate-based scaffolds

for medicinal chemists [43,44]. Based on the cholinergic hypothe-

sis, carbamate-like drugs can be regarded as one of the mainstays

for the contemporary pharmacotherapy for AD by inhibiting ChEs

[45]. Rivastigmine is a pseudo-irreversible inhibitor that forms

covalent carbamate adduct with AChE [46]. The molecular docking

showed that the carbonyl of the carbamate group tends to form

hydrogen bonding interactions, and the N atom is essential for

interaction with the ChE binding site.

Fig. 2. The general design strategy in this study.

were designed by fragment reassembly followed by docking to

predict the bioactivity of the reassembled molecules, followed by

in vitro and in vivo study to identify potent ChE activity (Fig. 2).

2. Results and discussion

CBD has sparked wide attention due to its therapeutic potential,

and there is considerable interest in synthesizing novel CBD de-

rivatives with improved drug-like properties [47,48]. In this study,

using molecular modeling approaches, a series of CBD carbamates

2.1. Docking-based structural splicing design

To better explore the capacity of CBD targeting BuChE, we

investigated the binding modes by the CDOCKER molecular

Fig. 1. The relevant pharmacology of CBD in synaptic cells. As a FAAH inhibitor, CBD increases the AEA and 2-AG concentrations to activate retrograde signals. It acts as an agonist at

TRPV1 to facilitate cation transport and at the somatic 5HT1A receptor to inhibit adenylate cyclase. It also inhibits AEA through binding to the orphan receptor GPR55 [34,37].

However, can CBD act to ChEs?.

2

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

docking module in Discovery Studio 2018. CBD could nicely insert

into the binding groove of BuChE, forming multiple interactions via

p p interaction interactions between the benzene ring and

ꢀ

Trp332, hydrophobic interactions between the cyclohexene ring

and Trp82 and His438 and two hydrogen bonds between the hy-

droxyl and Asp70 and Trp332. The molecular modeling results

showed that although both eOH of CBD have modiﬁable space, only

one has a larger space that is suitable to be modiﬁed or combined

with Thr120 (Fig. 3). Therefore, under the guide of a docking-based

structural fragment reassembly approach and the structure char-

acteristics of CBD and rivastigmine, compounds C1eC6 containing

single- or double-substituted CBD carbamates were synthesized

(Scheme 1).

2.2. Inhibition of equine BuChE and electrophorus electricus AChE

Initial screening of CBD carbamates was performed with equine

BuChE (eqBuChE), electrophorus electricus AChE (eeAChE), recom-

binant human AChE (hAChE) and BuChE from human serum

(hBuChE) using modiﬁed Ellman's method [49,50].

As shown in Table 1, ChEs inhibition was consistent with the

molecular modeling results that one of two hydroxyl groups of CBD

is suitable for being modiﬁed into a larger space at the active site of

hBuChE. The eqBuChE inhibitory activity of the single-substituted

CBD was signiﬁcantly higher than that of the double-substituted

(IC₅₀values for eqBuChE, C1 > C4; C2 > C5; C3 > C6; C7 > C8).

Compounds with aliphatic amine exhibited better BuChE inhibition

than those with cyclic amine (IC₅₀values for eqBuChE, C1, C9,

C10 > C2, C3; C4 > C5, C6), amongst them, compound C1 with

methylethylamine showed similar ChE inhibitory activity to riva-

Scheme 1. Synthesis of CBD carbamates C1eC17. Reagents and conditions: (i) tri-

phosgene, NaHCO₃, CH₂Cl₂, -10e0 ^ꢁC, 6e8 h; (ii) R₁R₂NCOCl (C1eC17), 4-DMAP, K₂CO₃,

CH₃CN, 60e65 ^ꢁC.

compounds C16 and C17 exhibited nanomolar inhibition on eqBu-

ChE (IC₅₀¼ 5.3 and 7.3 nM, respectively) and >4000 selectivity

index for eqBuChE over eeAChE. Substituted group at benzene ring

of tert-amine affected eqBuChE inhibitory activity, for example, 4-

substituted halogen showed better activity than the correspond-

ing 3-substituted (C15 > C12; C16 > C13; C17 > C14). Further, the

order of substituted halogen on eqBuChE inhibition is Cl z Br > F

(C16 z C17 > C15 for 4-halogen; C13 z C14 > C12 for 3-halogen).

The structure-activity relationship (SAR) of CBD carbamates was

illustrated in Fig. 5.

stigmine (IC₅₀values for eeAChE and eqBuChE, 14.95 and 0.077

for C1; 16.35 and 0.058 M for rivastigmine).

mM

m

2.3. Optimization and SARs of CBD carbamates

In our recent work, based on the hydrophobic interactions of

donepezil at the peripheral anionic site (PAS) of ChE, the intro-

duction of a tert-benzylamine signiﬁcantly improved the BuChE

2.4. Inhibition of hAChE and hBuChE

inhibitory activity of the d-sultone-fused pyrazole scaffold [14,19].

In this work, the tert-benzylamine would be introduced into CBD

carbamate by the fragment reassembly. As shown in Fig. 4A and B,

CBD containing tert-benzylamine was well docked into the active

To determine the potency and selectivity of CBD carbamates for

the human enzymes, we tested them for hBuChE and hAChE

inhibitory potential in vitro. As shown in Table 2, promising com-

pounds C1, C16 and C17 showed better hBuChE inhibition than

site, the benzene ring fell into the hydrophobic pocket through pep

interactions between the benzene ring and Trp82 and Try332,

hydrogen bond interaction between the carbonyl of carbamate and

Thr120. Under the guide of the molecular modeling, a series of CBD

carbamates were designed and synthesized. As shown in Table 1,

rivastigmine (IC₅₀values ¼ 1.51, 1.77 and 2.15

m

M for hBuChE,

M.

respectively), while they hardly inhibited hAChE even at 20

m

Hence, compounds C1, C16 and C17 were found to be highly se-

lective inhibitors of hBuChE.

Fig. 3. Binding modes of CBD with hBuChE (PDB code: 5LKR). CBD is shown in pink, important residues of the receptor are presented in green. Hydrophobic interactions are shown

in pink, hydrogen bonds are shown in green and

p-

p

interactions are shown in dark pink.

3

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

Table 1

Chemical Structures of CBD and C1eC17 and Their Inhibitory Activities against

eeAChE and eqBuChE^a.

d

Compound

IC₅₀

20

,

m

M)

M (or inhibition rate % at

RP of DPPH assay

c

eeAChE ^b

eqBuChE

CBD

C1

C2

C3

C4

C5

C6

C7

C8

C9

C10

C11

C12

C13

C14

C15

17.07 2.43

0.67 0.06

0.077 0.005

0.39 0.04

1.89 0.30

0.29 0.02

0.53 0.08

3.36 0.23

0.046 0.008

0.38 0.08

0.20 0.01

0.37 0.03

0.53 0.08

4.87 0.15

2.82 0.26

2.40 0.10

0.069 0.006

0.0053 0.0012

0.0073 0.0012

9.24 1.18

0.058 0.013

66.7%

31.1%

4.9%

21.1%

0.1%

14.95 1.02

na ^e

na

17.8%

2.0%

21.1%

41.8%

27.1%

40.4%

46.8%

na

27.0%

27.1%

41.8%

1.3%

Fig. 5. SARs of the BuChE inhibitor based on compound C16.

36.1 1.8%

28.4 0.8%

30.5 5.1%

na

Table 2

Inhibitory activity on hAChE and hBuChE ^a

.

Compound

IC₅₀

,

m

M (or inhibition rate % at 20

mM)

na

b

c

hAChE

hBuChE

C16

C17

Donepezil

rivastigmine

ascorbic acid

21.4 2.8%

na

0.026 0.003

16.35 1.54

Donepezil

Rivastigmine

C1

C16

C17

0.018 0.002

12.2 1.3%

13.2 2.9%

26.3 3.5%

na ^d

11.86 3.51

2.71 0.73

1.51 0.48

1.77 0.78

2.15 0.77

2.5%

86.2%

a

Each IC₅₀value is the mean SEM from at least three independent experiments.

eeAChE from electric eel.

eqBuChE from horse serum.

b

c

a

Each IC₅₀value is the mean SEM from at least three independent experiments.

hAChE.

hBuChE.

na, no activity.

b

c

d

RP of 1,1-Diphenyl-2-picrylhydrazyl (DPPH) (%) ¼ reduction percentage of

DPPH, compounds at a concentration of 100

m

M (n ¼ 2).

d

e

na, no activity.

oxidative damage to the cells and tissues caused by the trace of free

radicals.

2.5. DPPH radical scavenging activity assay

1,1-Diphenyl-2-picrylhydrazyl (DPPH) is an extremely effective

free radical scavenger that can be used to monitor a chemical re-

action that involves free radicals. The DPPH assay was performed

with ascorbic acid and donepezil as reference antioxidants to

evaluate the ability of the synthesized compounds to scavenge

activated oxygen species. As shown in Table 1, almost all com-

pounds exhibited mild free radical scavenging activity with the

2.6. Kinetic study on the mode of eqBuChE inhibition

Rivastigmine, a carbamate drug, was selected as a reference to

explore pseudo-irreversible inhibition of CBD carbamates on

eqBuChE. The mechanism of pseudo-irreversible enzyme inhibition

by carbamates can be described by three pivotal steps as shown in

Fig. 6 [51,52].

Firstly, the enzyme E forms a reversible enzyme‒inhibitor

complex (ECX) with the carbamate-based inhibitor CX comparable

to reversible competitive inhibitors. This reversible inhibition is

quantiﬁed by K_dwhich describes the apparent afﬁnity between the

enzyme and the inhibitor in an equilibrium state (Fig. S2). In the

second step, the carbamate moiety itself is transferred onto the

DPPH RP of 0e46.8% at a concentration of 100

is 66.7%. CBD exhibited close antioxidant activity to vitamin C at a

concentration of 100 M, when OH of CBD was substituted, the

mM, and that of CBD

m

activity decreased. The antioxidant activity of CBD carbamates was

proportionate to the number of OH. Mono-carbamate of CBD

retained almost half antioxidant activity, which may attenuate the

Fig. 4. (A) Alignment of docking mode of C7 in the receptor. The pocket surface is presented in blue; C7 is shown in yellow. (B) 3D mode of the interaction of C7 with receptor

hBuChE. Hydrophobic interactions are shown in pink, hydrogen bonds are shown in green and

p-p interactions are shown in dark pink.

4

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

Compound C16 was docked into the hBuChE pocket and then

subjected to MD simulation to examine the structural stability of

simulation system. MD simulation of complexes was performed for

100 ns period. In this study, the kinetic energy of compound C16

started to increase by temperature and time until a steady state was

reached. As shown in Fig. 9A, before 60,000 ps, the entire system of

the complex hBuChE‒C16 was in a stable state. After 60,000 ps, an

obvious upward trend was emerged, especially at 70,000 ps, after

which the overall system was relatively stable, and the wave range

of RMSD was within 1 Å. Subsequently, calculated by the MM-

PB(GB)SA method (Fig. 9B), the total binding free energy

was ꢀ41.09 kcal/mol for the complex of hBuChE‒C16, and the van

der Waals energy was ꢀ56.14 kcal/mol. Fig. 9C was the contribu-

tions of hot residues in the binding pocket of hBuChE. Under normal

Fig. 6. Pseudo-irreversible inhibition of an enzyme E by carbamates CX.

enzyme with release of the carrier scaffold X, resulting in the car-

bamoylated enzyme EC. The constant k₂represents the carbamo-

lyation rate of the inhibitor from the reversible complex (ECX) to

the carbamoylated enzyme EC. K_I(k₂/K_d) represents the overall

carbamylation rate. The last step is the recovery of the enzyme

through slow hydrolysis of EC and release of the carbamate group C

with k₃as the decarbamoylation rate constant. Normally, k₃is

signiﬁcantly lower than k₂due to the fact that carbamoylation oc-

curs much faster than decarbamoylation because of the high sta-

bility of EC toward hydrolysis [52,53].

As shown in Figs. 7 and S1 and Table 3, compounds C2 (Fig. 7B)

and C16 (Fig. 7C) inherited the pseudo-irreversible inhibition of

BuChE by rivastigmine (Fig. 7A). The kinetics of ChE inhibition by

carbamates is strongly inﬂuenced by the substituent at the N-car-

bamoyl group, for example, short-chain alkyl carbamates gave

relatively rapid carbamoylation and decarbamoylation, while long-

chain alkyl carbamates generally provided a more stable carba-

moylated enzyme which is formed slower but also undergoes a

slower hydrolysis toward a reactivated enzyme [51]. The carba-

mylation rate of compound C2 with short-chain alkyl was signiﬁ-

cantly higher than that of compound C16 with long-chain alkyl. The

kinetics of enzyme inhibition and reactivation inﬂuences the

duration of action of a carbamate drug. Therefore, CBD carbamates

with high afﬁnity can exert a longer-lasting effect in the body

[53,54].

circumstances,

than ꢀ1 kcal/mol was considered to be essential for ligand recog-

nition and combination. As disclosed in docking studies, in-

a

residue with lower interaction energy

p‒p

teractions between C16 and Trp82 (ꢀ1.71 kcal/mol) and Tyr332

(ꢀ2.35 kcal/mol), hydrogen bonding of C16 with Thr120

(ꢀ1.35 kcal/mol), and

p‒alkyl interactions of C16 with Met437

(ꢀ3.98 kcal/mol) and His438 (ꢀ1.14 kcal/mol) were signiﬁcant for

binding to hBuChE.

2.9. In vitro cytotoxicity evaluation

To examine the cytotoxicity of the CBD and its carbamates, the

effects of CBD and C16 on cell viability were determined at 6.25,

12.5, 25 and 50 mM in parallel with donepezil in human hepato-

blastoma HepG2 cells and human normal liver cells L02 [57]. As

shown in Fig. 10A, exposing HepG2 cells for 24 h to C16 and

donepezil at either 12.5

cell viability. However, at 50

affected viability, and the cytotoxic effect was almost same for C16

and donepezil (88.3% vs 91.1%); while CBD at 25 M signiﬁcantly

mM or 25

mM did not signiﬁcantly reduce

mM, both C16 and donepezil slightly

m

affected viability (p < 0.01), and the cell viability decreased to 20.1%.

Fig. 10B showed that after exposing L02 cells for 24 h to CBD, C16

and donepezil the similar trend was observed. Up to 50

C16 and donepezil slightly affected viability, and the cytotoxic ef-

fect was 96.0% and 92.6%; while CBD at 50 M signiﬁcantly affected

viability (p < 0.01), and the cell viability decreased to 44.9%. The

results showed that C16 is less toxic to HepG2 and L02 cells than

CBD. All in all, the cell toxicity proﬁle of C16 roughly matched that

of donepezil, much lower than that of CBD. CBD may be

cytotoxicity-related antioxidant activity, while C16 reduced the

activity by half, therefore, C16 could be the best candidate for

further investigation.

mM, both

2.7. Further molecular simulation

m

Halogen-containing phenyl moiety is beneﬁcial for lip-

osolubility and membrane permeability, which can inﬂuence the

rate of absorption and transport of drugs. As shown in Fig. 8, the

molecular docking of compounds C16 and C17 showed that the

introduction of Cl and Br atoms increases hydrophobic interactions

through the pep interactions with Met437 and Trp430 to further

improve BuChE inhibitory activity.

2.8. Molecular dynamics simulation

2.10. Oil/water partition coefﬁcient

MD simulation mainly relies on Newtonian mechanics to

simulate the motion of a molecular system. This approach can not

only obtain the trajectory of atoms but also enables the observation

of various microscopic details during atomic motion [55,56].

The log P (o/w) (octanol-water partition coefﬁcient) can reﬂect

the absorption of a drug in an organism and the optimum value was

around 2.0 0.7 for the central nervous system penetration [58,59].

Table 3

Kinetic values for carbamoylation and decarbamoylation on eqBuChE for selected compounds.

k₂(min^ꢀ1

b

)

k₃^d(min^ꢀ1) *10³

a

c

K_I(m )

M^ꢀ1min^ꢀ1

Compound

K_d

(

m

M)

C2

C16

rivastigmine

0.35 0.04^e

0.013 0.001

1.15 0.09

0.33 0.04

0.26 0.01

0.30 0.03

0.94 0.03

20.26 2.75

0.26 0.04

5.13 0.57

5.20 0.56

4.97 0.59

a

The apparent afﬁnity between the enzyme and the inhibitor in an equilibrium state.

The carbamolyation rate.

The overall carbamylation rate.

The decarbamoylation rate.

Each value is the mean SEM from at least three independent experiments.

b

c

d

e

5

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

Fig. 7. Time‒and concentration‒dependent carbamoylation of eqBuChE by rivastigmine (A), C2 (B) and C16 (C). Residual activity indicates the percentage of remaining enzyme

activity that has not been carbamylated (n ¼ 3).

Fig. 8. 3D mode of interactions of C16 (A) and C17 (B) with receptor hBuChE. Cl and Br atoms are presented by bright green and dark brown respectively; p-p interactions are shown

in dark pink; important residues are presented in purple.

Fig. 9. (A) The RMSD plots of complexes vs time; (B) Total binding free energy and its component; (C) Residue contribution for receptor-ligand combination.

Fig. 10. Cytotoxicity of CBD, C16 and donepezil was tested at concentrations in the range 0e50 mM in HepG2 (A) cells and L02 (B) cells for 24 h. Untreated cells were used as control.

Results are expressed as percentage of cell survival vs untreated cell (control) and shown as mean SD (n ¼ 3).

6

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

Therefore, log P values of active compounds were usually used to

predict the ability to cross blood-brain barrier (BBB). As shown in

Table 4, log P values of active compounds ranged from 1.6 to 2.6,

which implied that the modiﬁed CBDs have proper lipophilicity to

cross BBB.

observed at all time points between vehicle and C16 groups.

Parafﬁn sections were observed 36 h after administration of the

vehicle (control group) or C16 (30 mg/kg). Neither pericentral ne-

crosis nor distinct fatty degeneration of the hepatocytes of the

surrounding intermediate and periportal zones was discovered in

immunohistochemical staining images (Fig. 13D). Therefore,

obvious acute hepatoxicity triggered by treatment of C16 was

observed. Taken together, C16 exhibited high in vivo safety,

ensuring its further pharmacology development.

2.11. BBB permeability study

The central nervous system (CNS) is the ultimate therapeutic

target of the anti-AD agent, it is essential to evaluate the BBB

penetration capacity of the synthesized compounds in the drug

discovery process [18,58]. Hence, BBB penetrating ability of CBD, C1

and C16 was estimated using a parallel artiﬁcial membrane

permeation assay (PAMPA) BBB model. To validate the experi-

mental procedure, six commercial drugs with reported values were

chosen. A plot of experimental versus bibliographic data presented

a good linear correlation, P_e(exp.) ¼ 0.9219 P_e(bibl.) e 0.4558

(R²¼ 0.9696) (Fig. 1 1B). CBD, C1 and C16 were tested following this

procedure, and the results in Fig. 1 1A showed that permeability

values of tested compounds are above 4 ꢂ 10^ꢀ6cm/s. CBD, C1 and

C16 were indicated to be capable of BBB penetration.

2.14. In vivo behavioral studies

Cognition-improving potency is of utmost importance for anti-

AD agents. Scopolamine is a nonselective muscarinic ACh recep-

tor antagonist, and it produces cognitive deﬁcits in test subjects

that are comparable to those caused by the cholinergic deﬁcit

characteristic of AD. ChE inhibitors alleviate this cognitive deﬁcits

by increasing the levels of synaptic ACh (notably in the brain),

which results in displacement of scopolamine from muscarinic ACh

receptors. The Morris water maze (MWM) task is a hippocampal-

dependent memory and spatial learning task that is mainly used

to study the effect on long-term memory by reducing the time to

reach the escape platform (ie, the escape time latency). In vitro ChE

inhibitory activity must translate into behavioral effects and

improve the cognition in vivo [18,60,61]. Scopolamine was used to

induce cognitive deﬁcits in test animals (1 mg/kg; intaperitoneal

(ip) injection prior to the acquisition trials in each test). MWM test

was used to study the ameliorating potential of C16 against

scopolamine-induced cognition impairment in mice. The results

demonstrated impairment in learning and memory by a model

group of animals with a signiﬁcant prolongation of ELT. The

administration of C16 and donepezil exhibited a remarkable

reduction in ELT (Fig. 14A) and signiﬁcantly improved the overall

target quadrant preference (the number to cross the platform and

the swimming time in the target quadrant) compared to model

group (Fig. 14B and C). The results signiﬁed that C16 had effect in

ameliorating cognitive dysfunction, which may be attributed to

superior activity.

2.12. Neuroprotective study

The protective effects of CBD and C16 against free radicals

damage were assessed by measuring their ability against H₂O₂-

induced injury according to the reported protocol [18,19]. After

exposing PC12 cells to 100

mM H₂O₂, cell viability obviously

decreased to 38.7% (p < 0.01 vs control), manifesting high sensi-

tivity to H₂O₂-induced injury. As shown in Fig. 12, C16 had weak

protective effect in a dose-dependent manner against H₂O₂-

induced PC12 cell injury. At a concentration of 25 mM, C16 exhibited

signiﬁcant neuroprotective effect with cell viability of 41.9% and

was stronger than CBD with that of 17.9%, which clariﬁed that C16

could efﬁciently capture the hydroxyl radical generated by H₂O₂.

2.13. In vivo acute toxicity evaluation

In order to give more detailed information about the anti-AD

proﬁle of C16, we conducted an Ab_1-42(intracerebroventricular

(icv) injection)-induced cognition-impaired model [18,62,63]. AD-

like cognitive dysfunction was caused by icv injection of the oli-

In vivo safety was carried out by a single-dose acute toxicity

evaluation in ICR mice. After intragastric administration of C16 at a

dose of 1.0 g/kg, no death and abnormal behaviors were observed.

Moreover, the body weights of all mice in vehicle and test groups

were recorded and shown in Fig. 13. Within the 14 days after a

single-dose administration, the body weights gradually enhanced

(Fig. 13A), and the mean body weight of the mice in test group

presented no signiﬁcant differences as those in vehicle group.

Hence, C16 could be well tolerated in vivo at a high dose of 1.0 g/kg.

Additionally, the in vivo hepatotoxicity of C16 was evaluated by

levels of aspartate aminotransferase (AST) and alanine amino-

transferase (ALT) (Fig. 13B) and hematoxylin-eosin (HE) staining

images (Fig. 13C). Heparinized serum was collected 8, 22, and 36 h

after the intragastric administration of the vehicle and C16 (30 mg/

kg), and no signiﬁcant differences in two biomarkers were

gomerized Ab_1-42peptide (10 mg per mouse) on day 1 (Fig. 15A).

Meanwhile, mice icv injected with saline (same volume) were set

as a sham-operation group. Donepezil (positive control) and C16

were administered (po) from days 3e14. Behavioral studies were

carried out by MWM. The water maze study included 5 days of

learning and memory training (on days 10e14) and a probe trial on

the sixth day (on day 15). As shown in Fig. 15B‒E, icv saline did not

affect the cognitive and learning capacity, with no differences from

the blank control group in regard to undifferentiated alternation

performance, latency to target, and trajectory confusion. Treatment

with donepezil (15 mg/kg) could signiﬁcantly shorten the latency,

simpliﬁed the trajectories to the platform, and improved the

alternation performance. Moreover, administration with C16 (5 mg/

kg) or donepezil (15 mg/kg) could obviously prevent the impact of

Table 4

Log P values of active compounds.

A

b_1-42peptide icv injection and almost entirely recover the

Compound

Log P ^a

cognitive function to the normal level. Furthermore, C16 exhibited

better efﬁcacy than the donepezil in the MWM test, reduced target

latency (Time: C16 < donepezil < model group), and much

simpliﬁed trajectory to the target. As shown in Fig .1 5F, compared to

the control or sham-operation groups, the total level of the Ab_1-42

peptide in the icv Ab_1-42group was signiﬁcantly enhanced, indi-

cating a successful modeling. Furthermore, mice treated with

donepezil or C16 presented obvious reduction of the Ab_1-42peptide

CBD

C1

C4

C7

C16

C17

1.67

1.92

2.57

2.20

2.38

2.37

a

Octanolewater partition coefﬁcients were

measured by the shake ﬂask method.

7

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

Fig. 11. PAMPA-BBB penetration study of CBD, C1 and C16. (A) Results of the PAMPA-BBB assay for six commercial drugs used in the experimental procedure validation and CBD, C1

a

b

c

and C16. Bibl. values are reported data from the reference; All tests were obtained from three independent experiments; “CNS þ” (high BBB permeation): P_e( ꢂ 10^ꢀ6cm/

s) > 4.0; “CNS þ/ꢀ” (uncertain BBB permeation): P_e( ꢂ 10^ꢀ6cm/s) from 2.0 to 4.0; “CNS ‒” (low BBB permeation): P_e( ꢂ 10^ꢀ6cm/s) < 2.0. (B) Linear correlation presenting

experimental versus bibliographic data of commercial drugs. P_e(exp.) ¼ 0.9219 P_e(bibl.) e 0.4558 (R²¼ 0.9696).

numerous scattered shrinking neurons and nuclear pyknosis in

hippocampal CA1 and CA3 regions. The quantitative analysis

showed that the increase of neuronal density was prominent at

hippocampal and especially at the CA1 region in C16-treatment

group, compared to the control group (Fig. 16B and C).

3. Conclusions

This study demonstrated the effectiveness of using a docking-

based structural splicing and reassembly strategy to discover

potent and selective BuChE inhibitors. Based on the structure

characteristics of marketed drugs CBD and rivastigmine, a series of

CBD carbamates were synthesized. Subsequently, in vitro ChEs

assay revealed that BuChE inhibition of single-substituted CBD was

superior to that of the double-substituted. In view of the structure

of donepezil, the introduction of a tert-benzylamine signiﬁcantly

increased the activity. All synthesized compounds showed selective

BuChE inhibitory activity, amongst them, compounds C16 and C17

exhibited high inhibition on BuChE (eeAChE IC₅₀> 20

mM, eqBuChE

IC₅₀¼ 5.3 and 7.3 nM, respectively; hAChE IC₅₀> 20

m

M, hBuChE

Fig. 12. Neuroprotective effect of CBD and C16 (0e25

mM) against H₂O₂-induced

IC₅₀¼ 1.77 and 2.15

mM, respectively) and >4000-fold selectivity for

(100 M) PC12 neurons injury for 24 h (cells are given the CBD or C16 and incubated

m

for 3 h and then given H₂O₂peroxide to model). Results represent mean SEM (n ¼ 3).

BuChE over AChE. The enzymic kinetics study revealed that C16

was a pseudo-irreversible BuChE inhibitor (K_d¼ 13 nM, k₂¼ 0.26

min^ꢀ1). Compared to CBD, C16 almost decreased the antioxidant

capacity by half. The molecular docking and dynamics proved that

C16 was well fallen into the active pocket of BuChE and formed

diverse interactions with the key residues around the pocket. C16

also exhibited benign BBB penetrating ability. The results of in vitro

and in vivo safety showed that C16 possessed good neural and

hepatic safety and was tolerated up to a dose of 1.0 g/kg. In the

subsequent in vivo behavioral study, C16 remarkably reduced the

scopolamine-induced cognitive impairment in the mice model. In

the Ab_1ꢀ42-induced cognition-impairment study, treatment with

C16 signiﬁcantly prevented the impact of Ab_1ꢀ42toxicity and

almost entirely recovered the cognitive function to the normal

(19.0% or 31.6%, respectively). Meanwhile, staining of the hippo-

campus in Fig. 15G showed that b-amyloid plaque was obvious in

the hippocampus of model group compared to control group. The

result is consistent with the behavioral test conclusion, supporting

that C16 could effectively improve the cognitive function of AD

mice by decreasing the BuChE level and its further neuroprotective

effects against Ab toxicity. Additionally, none of the treatments (icv

saline or peptide or po compound) inﬂuenced the mice body

weight during therapy period, showing a good safety of C16

(Fig. 15H).

2.15. Histopathology in hippocampal CA1 and CA3 regions

level. Moreover, the evaluation of the

Ab_1ꢀ42total amount

conﬁrmed its anti-amyloidogenic proﬁle. However, as a selective

BuChE inhibitor, C16 can produce remarksble cognitive improving

and anti-amyloidogenic effects. Hence, C16 had signiﬁcant poten-

tiality to be further developed as promising therapeutics for AD

treatment.

Hippocampus is one of the most important parts for spatial

learning and memory, especial for CA3 to CA1 loop [64 ‒67]. To

further study the effect of active compound C16 against the Ab_1-42

-

induced cognitive impairment, the intact brains of the test mice

were collected from the behavioral experiment on the last day to

observe the histopathology of hippocampus CA1 and CA3 regions.

The morphology of pyramidal neurons was assessed by H&E

staining. The number of normal cells in hippocampal CA1 and CA3

regions were counted. Each pyramidal neuron was identiﬁed by its

shape, size, position and visible nucleus. As shown in Fig. 16A,

neuronal density was reduced in Ab_1-42-induced model mice with

4. Experimental section

4.1. General methods of chemistry

All chemicals, reagents and solvents were purchased from

8

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

Fig. 13. Results of in vivo acute toxicity evaluation. (A) Body weight of ICR mice (g)-time points of measurement (day). (B) ALT and AST activities at 8, 22, and 36 h after the

administration of the vehicle only (control) or C16. (C) Histomorphological appearance of mice livers after treatment with control or C16. Results represent mean SEM (n ¼ 10), HE

staining's view is 50 mm.

Fig. 14. Effects of C16, CBD and donepezil on scopolamine-induced memory impairment in the MWM task. (A) Learning curves of the escape latencies for Control (vehicle-treated

mice, red), Model (scopolamine-treated control mice, green), CBD (blue), C16 (purple) and donepezil (black) (scopolamine-induced memory-impaired mice treated with 20 mg/kg

CBD, 20 mg/kg C16 and 10 mg/kg donepezil, respectively) during the acquisition phase; (B) Time to swim in the target quadrant the acquisition phase in Control, Model, CBD,

and C16 or donepezil; (C) The number to cross the platform during the acquisition phase in Control, Model, CBD, and C16 or donepezil. Data are means SEM (n ¼ 8). Statistical

analysis: repeated-measures ANOVA, followed by Bonferroni posthoc comparisons. ^##p < 0.01, ^###p < 0.001 (vs control group), *p < 0.05, ***p < 0.001 (vs scopolamine þ vehicle-

treated mice).

9

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

Fig. 15. Effects of C16 and donepezil on the oligomerized Ab_1-42-induced impairment experiment in the MWM task. (A) Protocol followed for the in vivo experiment. Abbreviations:

icv, intracerebroventricular injection; po, orally administration; MWM, morris water maze; HE, hematoxylin-esin. (B) Learning curves of the escape latencies for vehicle-treated

mice, sham-operation (icv. saline) mice, model (icv. Ab_1-4210 mg) mice and mice treated with 5 mg/kg C16 or 15 mg/kg donepezil during the acquisition phase. (C) Average

tracks of mice in the MWM on the last day of study. (D) Time to swim in the target quadrant the acquisition phase in control (vehicle-treated mice), sham-operation, model, C16 or

donepezil. (E) The number to cross the platform during the acquisition phase in control, sham-operation, model, C16 or donepezil. (F) Ab_1-42total amount in mice brains of different

groups. The Ab_1-42total amount was quantiﬁed by using a mouse Ab_1-42ELISA kit. (G) Histomorphological appearance of mice brains after treatment with the vehicle (control) or

model (icv. Ab_1-4210

m

g). HE staining's view is 100

m

m. (H) Daily weight of mice in different groups during the therapy period. Data are presented as means SEM (n ¼ 8; ^#p < 0.05,

^###p < 0.001 vs control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs Ab_1-42peptide model group).

commercial sources and used without further puriﬁcation. Re-

actions were checked by thin-layer chromatography (TLC) on pre-

coated silica gel plates (Qingdao Marine Chemical Factory, GF254);

spots were visualized by UV at 254 nm. Melting points are deter-

mined on a XT4MP apparatus (Taike Corp., Beijing, China) and are

not corrected. The purity (relative content) of active compounds

d (doublet), t (triplet), or m (multiplet). High resolution mass

spectra (HRMS) were obtained on an Agilent 1260e6221 TOF mass

spectrometry.

4.2. General synthetic information of target compounds

was determined by HPLC through area normalization method. ¹

H

4.2.1. General synthetic information of carbamyl chlorides (B)

A solution of the secondary amine (A) (30.0 mmol) in CH₂Cl₂

(5 mL) was added dropwise, over half of an hour, to a stirred slurry

of sodium bicarbonate (5.04 g, 60 mmol) and triphosgene (5.94 g,

20 mmol) in dichloromethane (30 mL) at ꢀ10 ^ꢁC. The reaction

mixture was left to warm to room temperature and stirred for

NMR and ¹³C NMR spectra were recorded on Bruker AV-400 or AV-

600 MHz instruments using CDCl₃and C₆D₅N as solvent. Chemical

shifts are reported in parts per million (d) downﬁeld from the signal

of tetramethylsilane (TMS) as internal standards. Coupling con-

stants are reported in Hz. The multiplicity is deﬁned by s (singlet),

10

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

Fig. 16. (A) Morphology in hippocampal CA1 and CA3 regions using hematoxylin-eosin-staining. Scale bar in Hippocampus positioning ¼ 500

mm, scale bar in CA1 and CA3

regions ¼ 50

m

m; (B) Neuron counts in CA1 region; (C) Neuron counts in CA3 region. Data were expressed as the mean S.E.M. (N ¼ 3). ^##p < 0.01 vs control group; *p < 0.05 vs Ab_1-

peptide model group.

42

6e8 h. The mixture was then ﬁltered and the ﬁltrate was evapo-

rated under reduced pressure to give the carbamyl chloride crude

product. The crude product was distilled under reduced pressure or

puriﬁed by chromatography on silica, with petroleum

ethereCH₂Cl₂as mobile phase, to give the desired product (B).

CDCl₃)

d

6.54 (s, 1H), 6.49 (d, J ¼ 1.6 Hz, 1H), 6.05 (s, 1H), 5.62 (s, 1H),

4.56 (d, J ¼ 51.8 Hz, 2H), 3.61 (d, J ¼ 36.4 Hz, 1H), 3.60e3.41 (m, 4H),

2.54e2.48 (m, 2H), 2.13 (dd, J ¼ 37.3, 16.7 Hz, 2H), 1.98e1.89 (m,

4H),1.85e1.69 (m, 5H),1.64e1.57 (m, 4H),1.37e1.24 (m, 6H), 0.89 (t,

J ¼ 6.9 Hz, 3H). ¹³C NMR (101 MHz, C₆D₅N)

d 158.1, 153.9, 152.4,

149.7, 142.1, 131.7, 127.4, 122.9, 116.3, 113.5, 111.6, 47.3 (2C), 46.9,

36.1, 32.1, 31.6, 31.4, 30.3, 26.6, 25.6 (2C), 24.0, 23.2, 20.0, 14.6.

HRMS (ESI) m/z [M þ H]^þ: 412.2846, calcd for C₂₆H₃₈NO₃: 412.2846.

4.2.2. General synthetic information of CBD carbamate derivatives

(C1eC17)

Carbamyl chloride B (1 equiv. or 1.5 equiv.) was added to a

mixture of anhydrous K₂CO₃(2 equiv.), 4-DMAP (0.2 equiv.), and

CBD (0.50 mmol) in 5 mL MeCN. The reaction mixture was warmed

to 60e65 ^ꢁC and stirred for 5e10 h under an argon atmosphere. The

reaction was monitored by TLC. On completion of the reaction the

solvent was evaporated under reduced pressure. Water (20 mL)

was added to the residue and the mixture was extracted with

CH₂Cl₂(2 ꢂ 30 mL). The combined organic phases were washed

sequentially with 5% NaOH solution (20 mL), 5% HCl solution

(20 mL), and saturated aqueous sodium chloride (25 mL), dried

over anhydrous Na₂SO₄, and ﬁltered. The solvent was evaporated

under reduced pressure and the residue was puriﬁed by silica gel

ﬂash column chromatography (mobile phase 2:1 CH₂Cl₂eEtOAc) to

give compounds C1eC17.

4.2.5. 6-Hydroxy-5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-

1⁰,2⁰,3⁰,4⁰-tetrahydro-[1,1⁰-biphenyl]-2-yl morpholine-4-carboxylate

(C3)

Light yellow oil, yield 36%; purity, 97.5%. ¹H NMR (400 MHz,

CDCl₃)

d 6.61e6.38 (m, 2H), 6.08 (s, 1H), 5.57 (s, 1H), 4.54 (d,

J ¼ 58.5 Hz, 2H), 3.77e3.46 (m, 8H), 2.61e2.38 (m, 3H), 2.27e1.96

(m, 2H), 1.92e1.48 (m, 11H), 1.35e1.26 (m, 4H), 0.87 (t, J ¼ 6.9 Hz,

3H). ¹³C NMR (101 MHz, C₆D₅N)

d 158.1, 154.6, 152.3, 149.5, 142.2,

131.9, 127.3, 122.8, 115.9, 113.6, 111.7, 67.3 (2C), 45.8, 44.9 (2C), 36.1,

32.1, 31.5, 31.4, 30.3, 24.1, 23.2, 20.0, 14.6. HRMS (ESI) m/z [M þ

Na]^þ: 450.2611, calcd for C₂₆H₃₇NNaO₄: 450.2614.

4.2.6. 5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-1⁰,2⁰,3⁰,4⁰-

tetrahydro-[1,1⁰-biphenyl]-2,6-diyl bis(ethyl(methyl)carbamate)

(C4)

4.2.3. 6-Hydroxy-5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-

1⁰,2⁰,3⁰,4⁰-tetrahydro-[1,1⁰-biphenyl]-2-yl ethyl(methyl)carbamate

(C1)

Light white oil, yield 39%; purity, 95.6%. ¹H NMR (400 MHz,

CDCl₃)

d

6.76 (s, 1H), 6.71 (d, J ¼ 9.5 Hz, 1H), 5.38 (d, J ¼ 8.6 Hz, 1H),

Light yellow oil, yield 36%; purity, 96.0%. ¹H NMR (400 MHz,

4.54 (d, J ¼ 18.2 Hz, 2H), 3.66e3.37 (m, 6H), 2.99 (d, J ¼ 21.8 Hz, 3H),

2.66e2.48 (m, 3H), 2.11e1.71 (m, 8H), 1.66e1.45 (m, 8H), 1.37e1.10

CDCl₃)

d

6.52 (s, 1H), 6.43 (d, J ¼ 11.7 Hz, 1H), 5.59 (s, 1H), 4.55 (d,

J ¼ 56.4 Hz, 2H), 3.57 (s, 1H), 3.04e2.95 (m, 3H), 2.47 (dd, J ¼ 15.0,

7.1 Hz, 3H), 1.81e1.54 (m, 12H), 1.38e1.12 (m, 10H), 0.87 (t,

(m, 8H), 0.87 (t, J ¼ 6.2 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)

d 155.0

(2C), 150.4 (2C), 148.1, 141.9, 137.4 (2C), 137.3, 128.9 (2C), 128.8 (2C),

128.3 (4C), 127.8 (2C), 127.5, 126.7, 125.4 (2C), 111.1, 53.0, 52.8, 46.0,

38.8, 35.5, 34.9, 34.0, 31.9, 30.8, 30.7, 29.2, 23.5, 22.7, 20.3, 14.3.

HRMS (ESI) m/z [M þ Na]^þ: 507.3185, calcd for C₂₉H₄₄N₂NaO₄:

507.3193.

J ¼ 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)

d 155.5, 154.6, 154.3,

149.9, 142.6, 139.9, 123.7, 119.7, 114.5, 114.1, 110.9, 44.0, 43.8, 38.0,

37.9, 35.4, 34.1, 33.7, 31.6, 30.6, 23.6, 22.5, 20.4, 14.1, 12.9. HRMS

(ESI) m/z [M þ Na]^þ: 422.2661, calcd for C₂₅H₃₇NNaO₃: 422.2665.

4.2.4. 6-Hydroxy-5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-

1⁰,2⁰,3⁰,4⁰-tetrahydro-[1,1⁰-biphenyl]-2-yl pyrrolidine-1-carboxylate

(C2)

4.2.7. 5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-1⁰,2⁰,3⁰,4⁰-

tetrahydro-[1,1⁰-biphenyl]-2,6-diyl bis(pyrrolidine-1-carboxylate)

(C5)

Light yellow oil, yield 43%; purity, 98.4%. ¹H NMR (400 MHz,

White oil, yield 44%; purity, 99.0%. ¹H NMR (400 MHz, CDCl₃)

11

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

d

6.76 (s, 2H), 5.39 (s, 1H), 4.53 (d, J ¼ 20.4 Hz, 2H), 3.65 (d,

J ¼ 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl3)

d 155.6, 154.1, 149.8,

J ¼ 12.4 Hz, 1H), 3.45 (m, 8H), 2.58 (m, 3H), 2.04 (s, 2H), 1.93 (dt,

J ¼ 13.3, 5.9 Hz, 8H),1.82e1.69 (m, 2H),1.65e1.45 (m, 8H),1.36e1.23

147.4, 142.6, 140.0, 123.8, 119.7, 114.5, 114.0, 110.9, 45.3, 41.9, 41.5,

38.1, 35.5, 31.6, 30.6, 30.2, 27.8, 23.6, 22.5, 20.5, 14.4, 14.1, 13.4.

HRMS (ESI) m/z [M þ H]^þ: 414.2999, calcd for C₃₀H₄₀NO₃: 414.3002.

(m, 4H), 0.87 (t, J ¼ 6.9 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)

d 152.9

(2C), 150.0 (2C), 147.9, 141.5, 131.6, 126.4 (2C), 125.4 (2C), 110.8, 46.4

(2C), 46.2 (2C), 46.0, 38.4, 35.3, 31.6, 30.8, 30.5, 28.9, 25.9 (2C), 24.9

(2C), 23.2, 22.5, 19.9, 14.1. HRMS (ESI) m/z [M þ Na]^þ: 531.3186,

calcd for C₃₁H₄₄N₂NaO₄: 531.3193.

4.2.13. 6-Hydroxy-5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-

1⁰,2⁰,3⁰,4⁰-tetrahydro-[1,1⁰-biphenyl]-2-yl piperidine-1-carboxylate

(C11)

Light yellow oil, yield 30%; purity, 98.9%. ¹H NMR (400 MHz,

4.2.8. 5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-1⁰,2⁰,3⁰,4⁰-

tetrahydro-[1,1⁰-biphenyl]-2,6-diyl bis(morpholine-4-carboxylate)

(C6)

CDCl₃) d 6.60e6.36 (m, 2H), 6.03 (s, 1H), 5.59 (s, 1H), 4.68e4.41 (m,

2H), 3.77e3.27 (m, 5H), 2.61e2.40 (m, 3H), 2.29e2.00 (m, 2H),

1.90e1.47 (m, 16H), 1.36e1.23 (m, 4H), 0.87 (t, J ¼ 6.9 Hz, 3H). ¹³

C

Light white oil, yield 35%; purity, 98.9%. ¹H NMR (400 MHz,

NMR (101 MHz, CDCl₃) d 155.5, 153.7, 150.0, 149.9, 142.6, 140.2,

CDCl₃)

d

6.74 (s, 2H), 5.34 (s, 1H), 4.60e4.53 (m, 1H), 4.48 (s, 1H),

123.8, 119.7, 114.4, 114.1, 111.1, 45.5, 45.1 (2C), 37.9, 35.4, 31.6, 30.6,

30.1, 26.0, 25.5, 24.3 (2C), 23.6, 22.5, 20.2, 14.1. HRMS (ESI) m/z [M þ

Na]^þ: 448.2818, calcd for C₂₇H₃₉NNaO₃: 448.2822.

3.80e3.38 (m, 16H), 2.59e2.50 (m, 2H), 2.50e2.41 (m, 1H), 2.05 (s,

3H), 1.77 (ddd, J ¼ 19.5, 7.4, 5.1 Hz, 2H), 1.65 (s, 3H), 1.63e1.54 (m,

2H), 1.53 (s, 3H), 1.35e1.27 (m, 4H), 0.88 (t, J ¼ 6.9 Hz, 3H). ¹³C NMR

(101 MHz, CDCl₃)

d

153.3 (2C), 149.9 (2C), 147.6, 141.8, 131.8, 126.6,

4.2.14. 6-Hydroxy-5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-

1⁰,2⁰,3⁰,4⁰-tetrahydro-[1,1⁰-biphenyl]-2-yl (3-ﬂuorobenzyl) (methyl)

carbamate (C12)

124.9, 120.4, 110.9 (2C), 66.6 (2C), 66.5 (2C), 46.1, 44.8 (2C), 44.1

(2C), 38.6, 35.2, 31.5, 30.7, 30.4, 28.8, 23.3, 22.4, 20.2, 14.0. HRMS

(ESI) m/z [M þ Na]^þ: 563.3087, calcd for C₃₁H₄₄N₂NaO₆: 563.3091.

Light yellow oil, yield 33%; purity, 97.7%. ¹H NMR (400 MHz,

C₆D₅N)

d

7.32 (m, 3H), 7.13 (d, J ¼ 7.9 Hz, 1H), 6.96e6.78 (m, 2H),

4.2.9. 6-Hydroxy-5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-

1⁰,2⁰,3⁰,4⁰-tetrahydro-[1,1⁰-biphenyl]-2-yl benzyl(methyl)carbamate

(C7)

5.72 (s, 1H), 5.06 (m, 2H), 4.82e4.60 (m, 2H), 4.42 (d, J ¼ 14.7 Hz,

1H), 3.06 (m, 4H), 2.44 (s, 2H), 2.08e1.79 (m, 7H), 1.66 (m, 3H), 1.48

(s, 2H), 1.14 (s, 5H), 0.74 (s, 3H). ¹³C NMR (101 MHz, C₆D₅N)

d 163.9,

Light yellow oil, yield 41%; purity, 96.6%. ¹H NMR (400 MHz,

158.1, 155.9, 155.5, 152.4, 149.6, 142.3, 141.7, 131.5, 131.3, 127.3, 123.5,

122.7, 115.4, 115.2, 114.9, 111.7, 52.9, 52.7, 36.1, 35.4, 34.7, 32.1, 31.4,

30.5, 30.4, 24.1, 23.1, 20.1, 14.6. HRMS (ESI) m/z [M þ Na]^þ: 502.2717,

calcd for C₃₀H₃₈FNNaO₃: 502.2718.

CDCl₃)

d 7.42e7.30 (m, 5H), 6.87e6.37 (m, 2H), 6.01-5.99 (m, 1H),

5.60 (s, 1H), 4.80e4.32 (m, 4H), 3.59 (s, 1H), 2.97 (s, 3H), 2.61e2.42

(m, 3H), 2.29e2.01 (m, 2H), 1.85e1.44 (m, 10H), 1.35e1.27 (m, 4H),

0.89 (t, J ¼ 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)

d 155.2, 154.9,

153.4, 149.9, 147.4, 137.2, 137.1, 128.8, 128.6, 128.1, 127.6, 127.3, 123.7,

119.7, 114.3, 111.0, 100.0, 52.9, 52.7, 38.3, 38.1, 35.5, 31.6, 30.6, 30.5,

23.6, 22.6, 22.5, 20.4,14.1. HRMS (ESI) m/z [M þ H]^þ: 462.3001, calcd

for C₃₀H₄₀NO₃: 462.3002.

4.2.15. 6-Hydroxy-5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-

1⁰,2⁰,3⁰,4⁰-tetrahydro-[1,1⁰-biphenyl]-2-yl (3-chlorobenzyl) (methyl)

carbamate (C13)

Light yellow oil, yield 30%; purity, 99.6%. ¹H NMR (600 MHz,

CDCl₃)

d

7.37e7.28 (m, 1H), 7.26e7.19 (m, 1H), 6.90 (d, J ¼ 18.0 Hz,

4.2.10. 5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-1⁰,2⁰,3⁰,4⁰-

tetrahydro-[1,1⁰-biphenyl]-2,6-diyl bis(benzyl(methyl)carbamate)

(C8)

1H), 6.81 (t, J ¼ 39.4 Hz, 1H), 6.64e6.34 (m, 1H), 6.04 (s, 1H), 5.46 (d,

J ¼ 133.2 Hz, 1H), 4.83e4.36 (m, 4H), 3.92e3.44 (m, 1H), 3.01e2.90

(m, 2H), 2.65e2.47 (m, 3H), 2.30e1.95 (m, 3H), 1.80e1.58 (m, 10H),

1.38e1.28 (m, 5H), 0.88 (t, J ¼ 5.8 Hz, 3H). ¹³C NMR (151 MHz,

White oil, yield 38%; purity, 99.6%. ¹H NMR (400 MHz, CDCl₃)

d

7.53e7.26 (m, 10H), 6.76 (d, J ¼ 24.5 Hz, 2H), 5.41 (s, 1H),

CDCl₃) d 154.8, 154.2, 151.2, 149.8, 147.9, 141.9, 139.2, 134.6, 130.0,

4.87e4.23 (m, 6H), 3.59e3.57 (m, 1H), 2.97 (s, 6H), 2.53 (m, 3H),

129.9, 128.0, 127.8, 127.2, 126.2, 125.3, 122.9, 111.0, 52.5, 45.9, 38.4,

38.2, 35.3, 31.4, 31.3, 30.3, 29.0, 23.8, 22.4, 19.9, 13.9. HRMS (ESI) m/z

[M þ Na]^þ: 518.2433, calcd for C₃₀H₃₈ClNNaO₃: 518.2432.

2.13e1.89 (m, 2H), 1.84e1.29 (m, 14H), 0.88 (t, J ¼ 6.1 Hz, 3H). ¹³

C

NMR (101 MHz, CDCl₃)

d 155.1 (2C), 150.4 (2C), 148.1, 141.9, 137.4

(2C), 137.3, 128.9 (4C), 128.8 (4C), 128.3 (2C), 127.8, 127.5, 126.7,

125.4, 111.1, 53.0, 52.8, 46.0, 38.8, 35.5, 34.9, 34.0, 31.9, 30.8, 30.7,

29.2, 23.5, 22.7, 20.3, 14.3. HRMS (ESI) m/z [M þ Na]^þ: 631.3502,

calcd for C₃₉H₄₈N₂NaO₄: 631.3506.

4.2.16. 6-Hydroxy-5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-

1⁰,2⁰,3⁰,4⁰-tetrahydro-[1,1⁰-biphenyl]-2-yl (3-bromobenzyl) (methyl)

carbamate (C14)

Light yellow oil, yield 29%; purity, 99.2%. ¹H NMR (400 MHz,

4.2.11. 6-Hydroxy-5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-

1⁰,2⁰,3⁰,4⁰-tetrahydro-[1,1⁰-biphenyl]-2-yl dimethylcarbamate (C9)

Light yellow oil, yield 31%; purity, 98.2%. ¹H NMR (400 MHz,

C₆D₅N) d 7.74 (s, 1H), 7.59 (s, 1H), 7.43 (s, 1H), 7.35e7.24 (m, 1H),

6.88 (d, J ¼ 42.2 Hz, 2H), 5.72 (s, 1H), 5.06 (m, 2H), 4.69 (m, 2H), 4.37

(d, J ¼ 14.0 Hz, 1H), 3.08 (m, 4H), 2.45 (s, 2H), 1.95 (m, 6H), 1.68 (m,

3H), 1.48 (s, 3H), 1.15 (s, 5H), 0.75 (d, J ¼ 4.2 Hz, 3H). ¹³C NMR

CDCl₃)

d 6.79e6.36 (m, 2H), 6.01 (s, 1H), 5.58 (s, 1H), 4.57 (M, 2H),

3.89e3.52 (m, 1H), 3.02 (dd, J ¼ 27.4, 4.5 Hz, 6H), 2.57e2.43 (m, 3H),

(101 MHz, C₆D₅N) d 158.2, 156.1, 152.3, 149.4, 142.4, 142.3, 141.5,

2.27e2.02 (m, 2H), 1.86e1.59 (m, 10H), 1.39e1.28 (m, 4H), 0.87 (t,

141.4, 131.6, 131.3, 127.5, 127.3, 123.6, 122.7, 115.9, 113.7, 111.7, 52.8,

52.7, 36.1, 35.4, 34.6, 32.1, 31.5, 31.4, 30.5, 30.4, 24.1, 23.2, 20.2, 14.6.

HRMS (ESI) m/z [M þ H]^þ: 540.2109, calcd for C₃₀H₃₉BrNO₃:

540.2108.

J ¼ 6.8 Hz, 3H). ¹³C NMR (101 MHz, CDCl₃)

d 155.6, 154.9, 150.3,

149.9, 142.7, 141.9, 123.8, 123.7, 114.5, 114.3, 111.1 (2C), 37.9, 36.8,

36.4, 35.4, 35.3, 31.6, 31.4, 30.5, 23.6, 22.5, 22.5, 20.3, 14.0. HRMS

(ESI) m/z [M þ Na]^þ: 408.2506, calcd for C₂₄H₃₅NNa O₃: 408.2509.

4.2.17. 6-Hydroxy-5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-

1⁰,2⁰,3⁰,4⁰-tetrahydro-[1,1⁰-biphenyl]-2-yl (4-ﬂuorobenzyl) (methyl)

carbamate (C15)

4.2.12. 6-Hydroxy-5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-

1⁰,2⁰,3⁰,4⁰-tetrahydro-[1,1⁰-biphenyl]-2-yl diethylcarbamate (C10)

Light yellow oil, yield 39%; purity, 98.8%. ¹H NMR (400 MHz,

Light yellow oil, yield 38%; purity, 95.7%. ¹H NMR (400 MHz,

CDCl₃)

d

6.62e6.33 (m, 2H), 6.01 (s, 1H), 5.59 (s, 1H), 4.55 (d,

C₆D₅N) d 7.49 (s, 2H), 7.17 (s, 2H), 6.97e6.76 (m, 2H), 5.72 (d,

J ¼ 51.9 Hz, 2H), 3.60 (s, 1H), 3.52e3.29 (m, 4H), 2.60e2.44 (m, 3H),

J ¼ 10.1 Hz, 1H), 4.94 (s, 2H), 4.68 (m, 2H), 4.38 (m, 1H), 3.21e2.88

2.29e2.01 (m, 2H), 1.86e1.51 (m, 10H), 1.33e1.14 (m, 10H), 0.87 (t,

(m, 4H), 2.44 (s, 2H), 1.94 (m, 6H), 1.66 (m, 3H), 1.48e1.10 (m, 8H),

12

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

0.73 (s, 3H). ¹³C NMR (101 MHz, C₆D₅N)

d

163.0, 158.2, 156.0, 155.5,

0.1 M pH 8 phosphoric acid buffer. The test compounds were dis-

solved in DMSO to produce stock solutions with a concentration of

2 ꢂ 10^ꢀ1M. For each compound, a dilution series of ﬁve concen-

152.4, 142.3 (2C), 134.8, 130.7 (2C), 127.3, 123.5, 122.7, 116.5, 116.3,

116.1, 111.7, 52.6, 52.5, 36.1, 35.2, 34.5, 32.1, 31.4, 30.4, 24.1, 23.1, 20.1,

14.6. HRMS (ESI) m/z [M þ Na]^þ: 502.2716, calcd for C₃₀H₃₈FNNaO₃:

502.2718.

trations (200, 100, 10, 1 and 0.1

mM, diluted with ethanol) was

performed. The measurement was conducted using a 96-well plate.

Buffer (40 L), test compounds with a series of concentrations

(10 L), AChE or BuChE (10 L), and DTNB (20 L) were successively

added into the wells. The mixture was then incubated at 37 ^ꢁC for

m

4.2.18. 6-Hydroxy-5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-

1⁰,2⁰,3⁰,4⁰-tetrahydro-[1,1⁰-biphenyl]-2-yl (4-chlorobenzyl) (methyl)

carbamate (C16)

m

5 min. Thereafter, 20 mL of ACh or BuCh was added, after which the

Light yellow oil, yield 35%; purity, 97.0%. ¹H NMR (400 MHz,

reaction was initiated. After incubation at 37 ^ꢁC for 5 min, the ab-

sorption was determined at 412 nm. For the blank value, addition of

CDCl₃)

d 7.40e7.27 (m, 2H), 7.27e7.19 (m, 2H), 6.94e6.71 (m, 1H),

6.66e6.37 (m, 1H), 6.06 (s, 1H), 5.47 (d, J ¼ 86.3 Hz, 1H), 4.78e4.29

(m, 4H), 3.94e3.42 (m, 1H), 2.97 (t, J ¼ 4.1 Hz, 3H), 2.66e2.34 (m,

3H), 2.07 (d, J ¼ 16.7 Hz, 1H), 1.83e1.55 (m, 10H), 1.33e1.15 (m, 5H),

10 mL of water replaced the enzyme solution. A plot of percentage of

enzyme activity (100% for the reference) versus logarithm of

compound concentration was obtained to express the inhibitory

activity. The IC₅₀values were calculated by SPSS 17.0.

0.88 (t, J ¼ 6.8 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃)

d 154.7, 149.8,

147.9, 141.9, 135.6, 133.5, 130.8, 129.5 (2C), 128.9 (2C), 128.8, 128.6,

123.4, 114.3, 113.4, 111.0, 52.3, 45.9, 38.2, 37.5, 35.3, 31.4, 30.4, 30.3,

23.6, 23.4, 22.4, 19.8, 13.9. HRMS (ESI) m/z [M þ Na]^þ: 518.2432,

calcd for C₃₀H₃₈ClNNaO₃: 518.2432.

4.5. Radical scavenging activity (DPPH assay)

The antioxidant capacity of the test compounds were evaluated

by DPPH method in which DPPH free radical chould be scavenged

4.2.19. 6-Hydroxy-5⁰-methyl-4-pentyl-2'-(prop-1-en-2-yl)-

1⁰,2⁰,3⁰,4⁰-tetrahydro-[1,1⁰-biphenyl]-2-yl (4-bromobenzyl) (methyl)

carbamate (C17)

by antioxidant. Brieﬂy, 150

mL of the compound (200 mM) with

150 L of DPPH (140 M) was mixed and incubated in a 96-well

m

plate for 2 h in the dark at 37 ^ꢁC. The relevant absorbance of the

reaction mixture was measured at 520 nm using a microplate

reader (PerkinElmer VICTOR Nivo). The reducing percentage (RP) of

DPPH was determined by the formula: RP ¼ (1 ‒ A_C/A₀) ꢂ 100%,

where A_C/A₀are DPPH absorbance in the presence and absence of

inhibitors, respectively. Ascorbic acid was used as a standard for

DPPH determination.

Light yellow oil, yield 31%; purity, 98.2%. ¹H NMR (400 MHz,

CDCl₃)

d 7.55e7.45 (m, 2H), 7.23e7.13 (m, 2H), 6.54 (s, 2H), 6.43 (d,

J ¼ 29.5 Hz, 0H), 5.95 (s, 1H), 5.58 (d, J ¼ 10.0 Hz, 1H), 4.70e4.36 (m,

4H), 3.50 (s, 1H), 2.97 (s, 3H), 2.59e2.41 (m, 3H), 2.13e2.04 (m, 1H),

1.78 (s, 4H), 1.64e1.48 (m, 6H), 1.39e1.23 (m, 5H), 0.88 (t, J ¼ 6.9 Hz,

3H). ¹³C NMR (101 MHz, CDCl₃)

d 155.5, 149.8, 149.7, 142.9, 136.3,

135.2, 131.8 (2C), 129.9 (2C), 129.0, 124.9, 123.5, 121.5, 114.5, 114.3,

110.9, 52.4, 45.3, 38.2, 35.4, 32.2, 31.5, 30.6, 29.8, 26.5, 23.5, 22.6,

4.6. Kinetic studies of eqBuChE inhibition

20.5, 14.1. HRMS (ESI) m/z [M

₃₀H₃₉BrNO₃: 540.2108.

þ

H]^þ: 540.2108, calcd for

C

Carbamoylation kinetics was measured following the enzyme

inhibition protocol. For this purpose, the enzyme was preincubated

at 1, 2, 4, 8, 15, 25, 40 and 60 min with ﬁve different inhibitor

concentrations before the addition of substrate. Plotting the

enzyme activity in percentage as a function of time for each in-

hibitor concentration gave nonlinear time dependent inhibition

curves from which k_obswas determined by eq (1) (nonlinear

regression / growth curves / exponential plateau).

4.3. EeAChE and eqBuChE inhibition assay

Assays were performed on AChE from electric eel

(C3389e500UN; Sigma) and BuChE from equine serum

(C4290e1KU; Sigma), according to the Ellman's method. The

experiment was performed in 48-well plates in a ﬁnal volume of

500 mL. Each well contained 0.036 U/mL of eeAChE or eqBuChE, and

0.1 M pH 8 phosphate buffer. They were preincubated for 20 min at

different compound concentrations at 37 ^ꢁC. Then 0.35 mM ace-

tylthiocholine iodide (ACh; A5751-1G; Sigma) or 0.5 mM butyr-

ylthiocholine iodide (BuCh; 20820-1G; Sigma) and 0.35 mM 5,5⁰-

dithiobis(2-nitrobenzoic acid) (DTNB; D8130-1G; Sigma) were

added. The DTNB produces the yellow anion 5-thio-2-nitrobenzoic

acid along with the enzymatic degradation of ACh or BuCh. Changes

in absorbance were measured at 410 nm after 20 min in a Perki-

nElmer VICTOR Nivo reader. The IC₅₀values were calculated by

SPSS 17.0. A control experiment was performed under the same

conditions without inhibitor and the blank contained buffer, DMSO,

DTNB, and substrate.

A ¼ A₀$ exp ðꢀk_obs$ tÞ þ A∞

in which A is the activity of the enzyme at a speciﬁc time t, A₀

the activity at t ¼ 0, A_∞the activity at t ¼ ∞ and k_obsrepresents the

apparent ﬁrst order rate constant.

Double reciprocal plot of 1/k_obsagainst 1/[I] (inhibitor) gave a

linear plot from which k₂and K_dwere determined by eq (2).

1

k_d

1

k₂

¼

þ

(2)

k_obsk₂½Iꢃ

Determination of the decarbamylation rate constants (k₃), by

the analysis of the area under. Follow as:

4.4. hAChE and hBuChE inhibition assay

_CE_ðt2Þꢀ _CE

AUC_CE½t1;t2ꢃ

AU_CE½t1;t2ꢃk₃AUC_CE½t1;t2ꢃ

1

K_obs

k₃

¼

,

^ðt1Þþ

(3)

Assays were performed on. AChE from human (C1682; Sigma),

BuChE from human (B4186; Sigma), 5,5⁰-dithiobis(2-nitrobenzoic

acid) (DTNB; D8130-1G; Sigma), acetylthiocholine iodide (ACh;

A5751-1G; Sigma), and butyrylthiocholine iodide (BuCh; 20820-

1G; Sigma). Buffer is 0.1 M pH 8 phosphoric acid buffer, Enzyme

stock solution was prepared in gelatin (1% in deionized water) and

then diluted with water to give 0.125 units per mL. ACh and BuCh

iodide solutions were prepared in deionized water to give a ﬁnal

According to formula eq (1), the percentage of enzyme

remaining activity (A) is calculated, here we use (E) to indicate the

percentage concentration of enzyme activity (E ¼ A), then the

percentage concentration of enzyme that loses activity after being

bound by small carbamate molecules (cE) can be expressed as

(cE ¼ 1 ‒ E), ﬁt (E) and (cE) with time (t) respectively (Fig. S1), and

then use the formula “area under the curve” in the graph-pad to

calculate the active enzyme concentration -The area under the time

concentration of 3.75 mM. DTNB solution (5 mM) was prepared with

13

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

curve (AUC_E) and the concentration of enzyme that loses activity

after being bound-the area under the time curve (AUC_cE). In order to

reduce the inﬂuence of experimental error, we use the most suit-

able ﬁrst-order exponential decay formula (eq (1)) to ﬁt the curve,

and collect the data of the enzyme and inhibitor incubation time

from 15 min to 60 min. A set of minutes to calculate (E) (cE) (AUC_E)

and (AUC_cE).

Since (t₁) starts from 0 min, cE (t₁) is also 0. It can be found that

eq (3) conforms to the characteristics of a linear equation of two

variables, and then a straight line can be ﬁtted (Fig. S1) to obtain

The X-axis intercept (k_obs) and Y-axis intercept (k_obs/k₃) of the line.

Fit the X-axis intercept (k_obs) and Y-axis intercept (k_obs/k₃) to a

straight line, and obtain (k₃) from the slope (Fig. S1). All kinetic

values were determined using the software GraphPad Prism 8.

electrostatics were treated using the Particle Mesh Ewald (PME)

algorithm. The SHAKE algorithm was applied to constrain bonds

involving hydrogen atoms. To removed improper atom contacts,

the system was ﬁrst minimized by (1) the 2500 steps of steepest

descent and the 2500 steps of the conjugate gradient, under a

harmonic constraint of 10.0 kcal/(mol,Å²) on heavy atoms; (2)

relaxing the entire system by steepest descent and conjugate

gradient each in 10000 steps. And then the system was gradually

heated up to 300 K by a 20 ps NVT simulation. The equilibration

phase was carried out in two steps: (1) a 200 ps NPT simulation

with constraints on heavy atoms followed by (2) a 500 ps NVT

simulation without restraint. The temperature and pressure were

maintained at 300 K and 1 atm using the Berendsen thermostat and

Monte Carlo barostat, with coupling constant and relaxation time

of 1 ps. Finally, the system was subjected to a 10 ns NPT simulation

with a time step of 2 fs. The root-mean-square deviation (RMSD),

root-mean-square ﬂuctuation (RMSF), and hydrogen bonds were

analyzed by the Cpptraj module.

4.7. Docking-based structural splicing design

On the basis of the structural novelty and the preliminary

inhibitory activity, we considered a “fragment-linking” strategy

consisting in the chemical linking of these fragments to give higher

afﬁnity-binding compounds by additive effect. X-ray crystallo-

graphic structures and computational docking were used to guide

the fragment linking leading to novel series of BuChE inhibitors. A

structure based in silico procedure was applied to discover the

binding modes of the active compounds to BuChE enzyme active

site. The CDOCKER of Discovery Studio Client v18.1.0 (DS) was

conducted to explain SAR of series compounds and further guide

the design of more effective and concrete BuChE inhibitors. The

ligand binding to the crystal structure of hBuChE with PDB ID: 5LKR

was selected as template. All redundant atoms except chain A were

deleted and then the protein structure carefully treated in several

steps including residue repairing, protonation, and partial charges

assignment in CHARMm force ﬁeld. The target enzyme was pre-

pared with Prepare Protein of DS to ensure the integrity of target.

The ligand was processed by Full Minimization of the Small Mo-

lecular in DS. The binding pocket was deﬁned by the crystal ligand.

Then title compounds were docked into the active site of protein

using CDOCKER. The view results of molecular docking were

extracted after the program running end, each docking result was

analyzed for interaction and their different pose. Those poses with

the lowest -CDOCKER_INTERACTION_ENERGY values were regar-

ded as the most stable and picked to analysis binding interactions

with target enzyme visualized.

4.7.3. MM/PB(GB)SA calculations

The binding free energies were calculated using the Molecular

Mechanics PoissoneBoltzmann Surface Area (MM/PBSA) method

implemented in AmberTools 20 for 200 snapshots from the MD

trajectory. For each snapshot, the free energy was calculated for

protein hBuChE, compound C16, and the complex hBuChE‒C16

using a single trajectory approach. The total binding free energy

was calculated according to the following equation:

D

G_bind

E_MM

G_solv

¼

D

G_complexꢀ ð

D

G_receptor

G_solvꢀ T SÞ

E_ele

G_SA

E_MMdenotes the gas-phase interaction energy between

ꢀ

D

G_ligand

Þ

¼

DE_MM

þ

D

¼

D

E_vdw

D

¼

D

G_PB

þ

D

where

D

the receptor and the ligand (including van der Waals energy

contribution ( E_vdw) and electrostatic energy contribution ( E_ele);

G_PBand G_SAare the polar and nonpolar components of the de-

solvation free energy, respectively; T S represents the conforma-

tional entropy contribution at temperature T. Here, G_PBwas

determined by the Poisson-Boltzmann approximation model, while

G_SAwas estimated based on the solvent accessible surface area

model by the method: G_SA, where the values of

ꢂ SASA þ

the constants and

were 0.00542 kcal Å^ꢀ2and 0.92 kcal mol^ꢀ1

D

4.7.1. System preparations

D

The initial structure of hBuChE was retrieved from the RCSB

Protein Data Bank (PDB code: 5LKR). Receptor was prepared and

missing atoms of residues were ﬁxed by the advanced PDB-

Preparation tool in Yinfo Cloud Computing Platformusing

PDBFixer and the tLEaP module in AmberTools 20. The RESP partial

charges of compound C16 were derived by the Amber antechamber

program based on the electrostatic potentials calculated by

Gaussian 09 at the B3LYP/6-311G (d,p) level.

D

¼

g

b

g

b

,

respectively. The solvent probe radius and ionic strength were set

to be 1.4 Å and 0.15 mM, respectively. The interior and exterior

dielectric constant of MM/PBSA calculation systems was 1.0 and

80.0.

4.7.2. Molecular dynamics simulations

4.8. Oil/water partition coefﬁcient assay

The molecular dynamics (MD) simulations were performed in

Yinfo Cloud Computing Platform [68] using AmberTools 20 package

with AMBER ff19SB and GAFF force ﬁelds for hBuChE and com-

pound C16, respectively [69,70]. The system was solvated by a

truncated octahedron (or cubic) water box using OPC water model

with a margin of 10 Å, the net charge was neutralized by (0.15 M of

NaCl) sodium ions. Periodic boundary condition (PBC) was used and

the net charge neutralized by Na^þ(or Cl^ꢀ) ions (or 0.15 M of NaCl).

Nonbonded van der Waals interactions were calculated using the

Lennard-Jones 12-6 potentials with a 10 Å cutoff, while long-range

Using the classical shake ﬂask method, the oil/water partition

coefﬁcient of the compounds was tested. The same amount of oleic

phase (noctanol) and aqueous phase (PBS pH 7.4) were mixed. And

the mixture was shaken by ultrasonic (400 W, 40 kHz) and allowed

to stand for 24 h to obtain a saturated solution of noctanol. An

appropriate amount of the compounds was added. After sealing,

the test compounds were shaken at 37 ^ꢁC for 48 h to make it fully

equilibrated in the two phases. Then the mixture was measured

with a high performance liquid chromatography.

14

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

4.9. PAMPA-BBB penetration assay

4.12. Animal studies

The ability of the test compound penetrating into the brain was

conducted by a parallel artiﬁcial membrane permeation assay

(PAMPA) for the blood-brain barrier (BBB) based on previous work

of Di et al. [58]. Six commercial drugs were used to validate the

protocol and purchased from Aladdin Reagents. DMSO and

dodecane were obtained from Energy Chemical. Porcine brain lipid

(PBL) was purchased from Avanti Polar Lipids. The donor 96-well

All experiments were performed in accordance with the Na-

tional Institutes of Health Guide for the Care and Use of Laboratory

Animals. The procedures for maintenance and treatment of labo-

ratory animals were approved by the Animal Care and Use Com-

mittee of the Anhui Medical University (LLSC20190274). C57BL/6J

mice from the Animal Breeding Farm in Anhui Changlinhe Phar-

maceutical Technology Co. LTD and male ICR mice from Animal

Center of Anhui Medical University (Hefei, China) were used in the

MWM task. Six-week-old to eight-week-old male mice (18e24 g)

were housed in groups of 10 mice per cage at a room temperature of

22 2 ^ꢁC under a light/dark (12: 12 h) cycle. The animals had free

access to food and water before the tests. The ambient temperature

of the room and relative humidity (50%) were kept consistent

throughout all of the tests. For the behavioral experiments, the

mice were selected at random. Each experimental group consisted

of 8e10 mice/dose. The experiments were performed between

08:00 h and 14:00 h. Immediately after the in vivo assays, the mice

were euthanized by cervical dislocation. As for scopolamine-

induced impairment experiment, CBD and C16 were tested at a

dose 20 mg/kg intraperitoneally in the MWM task. It was sus-

pended in 2% Tween 80 (BioFroxx, Guangzhou) and 0.5% sodium

carboxymethyl cellulose (SCR, Shanghai) and was administered

60 min before the behavioral tests. Donepezil HCl (Sigma-Aldrich,

Poland) was dissolved in distilled water. In the MWM task, it was

tested at 10 mg/kg intraperitoneally. Control mice were given the

appropriate amount of vehicle (2% Tween 80 and 0.5% sodium

carboxymethyl cellulose). (ꢀ)-Scopolamine hydrobromide (Ada-

mas-beta; Shanghai) was used to induce memory impairment in

the mice. It was dissolved in distilled water and was administered

intraperitoneally at 1 mg/kg, 30 min before the acquisition phase of

the MWM tasks. As for the oligomerized Ab_1-42-induced impair-

ment experiment, 40 male ICR mice were separated into ﬁve groups

as follows: (i) vehicle (blank control) group (po), (ii) normal saline

(icv) as the sham-operation group, (iii) oligomerized Ab_1-42peptide

ﬁlter microplate with a PVDF membrane (pore size 0.45 mm) and

acceptor indented 96-well microplate were purchased from Milli-

pore. The 96-well UV plate (COSTAR) was obtained from Corning

Inc. Commercial drugs and test compounds were initially dissolved

in DMSO at a concentration of 20 mg/mL. Subsequently, they were

diluted 200-fold with a solution of PBS (pH 7.4 0.1)/EtOH (70/30,

v/v) to give a ﬁnal concentration of 100

of the donor microplate was coated with 4

(20 mg/mL). Then, 200 L of diluted compound solution was added

into the donor wells and 300 L of PBS/EtOH (70/30, v/v). The donor

m

g/mL. The ﬁlter membrane

mL of PBL in dodecane

m

ﬁlter plate was carefully placed on the top of the acceptor plate to

form a “sandwich” assembly to make the membrane contact with

buffer solution. The sandwich was put undisturbed at 25 ^ꢁC. After

incubation for 18 h, the donor plate was carefully removed; the

concentrations of test compounds in the donor and acceptor wells

were measured with a UV plate spectroscopy reader (PerkinElmer

VICTOR Nivo, Finland). Each sample was analyzed at three wave-

lengths, in at least three independent experiments, in four wells.

4.10. In vitro cytotoxicity assay

Human hepatoblastoma cells HepG2 and human normal liver

cells L02 were maintained at 37 ^ꢁC in a humidiﬁed incubator con-

taining 5% CO₂in DMEM containing 10% fetal bovine serum, 100 U/

mL penicillin, and 100 mg/mL streptomycin. Cell cytotoxicity was

evaluated by methyl thiazolyl tetrazolium (MTT) assay. HepG2 cells

and L02 cells were inoculated at 1 ꢂ 10⁴cells per well in 96-well

plate. After cultured for 24 h, the cells were treated with different

(10

tide (10

positive control group, and (v) oligomerized Ab_1-42peptide (10

per mouse, icv) þ C16 (5 mg/kg, po) as the positive control group.

Ab_1-42peptide was dissolved in DMSO as a 5 mM stock solution and

mg per mouse, icv) model group, (iv) oligomerized Ab_1-42pep-

m

g per mouse, icv) þ donepezil (15 mg/kg, po) as the

compounds which were diluted in DMEM for 24 h. Then 20

5.0 mg/mL MTT reagent was added into the cells and incubated for

4 h. After 4 h, cell culture was removed and then 150 L DMSO was

mL of

mg

m

added to dissolve the formazan. The optical density was measured

at 492 nm (OD₄₉₂). Cell viability was calculated from three inde-

pendent experiments. The density of formazan formed in blank

group was set as 100% of viability. Cell viability (%) ¼ compound

(OD₄₉₂/blank (OD₄₉₂) ꢂ 100%.

incubated for 24 h at 37 ^ꢁC in normal saline at a ﬁnal concentration

of 2 mg/mL to induce Ab_1-42aggregation. The positive and test

compounds were suspended in a mixture of DMSO and normal

saline (1/99, V/V) before the experiments. The behavioral study is

MWM on days 10e15.

Blank: cultured with fresh medium only.

Compound: treated with compounds or donepezil.

4.12.1. In vivo acute toxicity

Compound C16 was suspended in a mixed solution of DMSO,

PEG 400, and saline (10/50/40, v/v/v). Twenty male female mice

(20e25 g) were divided into two groups (n ¼ 10): control groups

and test groups. All mice were fasted overnight and then intra-

gastricaly administered with the vehicle or a dosage of 1.0 g/kg of

test compound C16 on the ﬁrst day. Thereafter, the death, daily

behavior, and body weights of mice were monitored during the

subsequent 7 days. Body masses of mice were compared between

control and test groups and summarized using GraphPad Prism 5.0

software.

4.11. Neuroprotection assay

PC12 cells were dispensed into 96-well microtiter plates at a

density of 1 ꢂ 10⁴cells/well. Following incubation overnight, cells

were treated with a range of CBD and compound C16 concentra-

tions (0e25

media were replaced by fresh media still containing the drug plus

the cytotoxic stimulus represented by 100 M H₂O₂that was left for

an additional 24 h period. Cell viability was measured after 24 h by

using the MTT assay. Brieﬂy, 20 L of 5.0 mg/mL MTT reagent was

added into the cells and incubated for 4 h. After 4 h, cell culture was

removed and then 150 L DMSO was added to dissolve the for-

mM) at time zero and maintained for 3 h. Then, the

m

4.12.2. In vivo hepatotoxicity studies

In vivo hepatotoxicity was evaluated according to the method

previously described in male ICR mice (20e25 g). Compound C16

was suspended in a mixed solution of DMSO, PEG 400, and saline

(10/50/40, v/v/v). A dosage of 30 mg/kg body weight of compound

and the same volume of the vehicle were administered

m

mazan. The optical density was measured at 492 nm (OD₄₉₂) on the

Biotek Synergy HTX Multi-Mode reader. Results were adjusted

considering OD measured in the blank.

15

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

intragastrically (ig). 8, 22, and 36 h after the administration, hep-

arinized serum was collected from the retrobulbar plexus for

hepatotoxicity evaluation. The activities of alanine aminotrans-

ferase (ALT) and aspartate aminotransferase (AST), two indicators

of liver damage, were determined using corresponding assay kits

(EF551 and EF550 for ALT, EH027 and EH548 for AST, Wako, Japan).

The data were processed using a biochemical analyzer (Hitachi

7020, Japan). After the last collection of retrobulbar blood, mice

were sacriﬁced and livers were taken for morphological studies by

the immunohistochemical method. We used an ultrathin semi-

automatic microtome (Leica RM2245, Germany) to isolate two

3 mm sections of each liver extending from the hilus to the margin

of the left lateral lobe, which was immediately placed in 10%

buffered formaldehyde, ﬁxed for 2 days, and embedded together in

a parafﬁn block using a parafﬁn embedding station (Leica EG1150H,

4.13.2. Oligomerized Ab_1-42-induced impairment experiment

As for the oligomerized Ab_1-42-induced impairment experiment,

after the behavioral study, all mice were sacriﬁced and brains were

taken to evaluate the amount of total Ab_1-42using the mouse ELISA

kit (WuHan HuaMei Biotech Co. Ltd., China). Each brain sample was

fully homogenized using a grinder in ten times the amount of PBS

(pH ¼ 7.4 0.1) and then centrifuged at a speed of 5000ꢂg for

5 min. The supernatant was separated for use. The assay procedure

was in accordance with the instructions, and the standard curve is

presented in the Supporting Information (Fig. S3). The content of

brain Ab_1-42was calculated according to the linear regression

equation. All values were expressed as means

GraphPad Prism 5.0 software.

SEM using

4.13.3. Histology examination in hippocampus

Germany). Subsequently, 5

m

m sections were prepared from those

For each dose group, nine entire brains separated from three

mice were collected to observe CA1 and CA3 regions histopathol-

ogy in hippocampus. Fresh collected brain was ﬁxed in 4% para-

formaldehyde and embedded in parafﬁn. Parafﬁn-embedded brains

parafﬁn blocks, deparafﬁnated, and stained with hematoxylin and

eosin or by the method of the periodic acid-Schiff procedure for

glycogen.

were sectioned on serial coronal sections of 5 mm thickness. He-

matoxylin and eosin (H&E) stained brain sections were analyzed

for normal pyramidal neurons hippocampus CA1 and CA3 regions

quantiﬁcation. We performed an image analysis using the Case-

Viewer. In each sample, we observe mainly about two ﬁelds of each

head and the tail and three ﬁelds in the middle of the hippocampal

CA1 and CA3 regions respectively. Finally, the number of normal

pyramidal neuron in all ﬁelds-of-view was recorded at

magniﬁcation ꢂ 400. Each normal pyramidal neuron was identiﬁed

by its shape, size, position and visible nucleus. The reported results

in the present study represent the average results for nine ﬁelds of

CA1 and CA3 regions from three mice in each group.

4.13. Behavioral testing

4.13.1. Scopolamine-induced impairment experiment

MWM Task. The MWM is a circular, plastic, gray-painted pool

(diameter, 1.20 m; height, 0.60 m) that is ﬁlled with water (up to

about 48 cm below the edge, to prevent animals from jumping out)

maintained at 23

1

^ꢁC. The pool was divided into four equal

quadrants (compass locations: NE, NW, SE, SW) using a comput-

erized video tracking system (SMART, version 3.0; Panlab, Spain).

An escape platform made of transparent Plexiglas (diameter, 11 cm;

height, 47 cm) was positioned at a ﬁxed location (the center of the

NW quadrant; i.e., the target quadrant), with this invisible to the

swimming animal. It was immersed 1 cm under the surface of the

water. During the experiments, the maze was lit with a light in-

tensity of 45 lx. In the spatial acquisition trial (6 consecutive days),

the mice were assigned to training sessions (four training sessions

per day, sessions held 4 h apart) in which the mice were trained to

escape from the water by reaching the hidden platform, the loca-

tion of which can be identiﬁed using distal extra-maze cues

attached to the room walls (i.e., A4-sized sheets of black laminated

paper with colored geometric symbols), which constitute the

navigation points. These visual cues have different colors and di-

mensions and were kept constant during the whole experiment.

The whole experiment was conducted by an experimenter who

always remained stationary in a constant location as an additional

distal cue for the swimming mice. For each trial, the mouse was

placed in the water starting from a different randomly chosen

quadrant that did not contain the platform, whereas the platform

was always positioned in the same place. If an animal did not ﬁnd

the hidden platform within 60 s, it was gently placed on the plat-

form for 15 s. The time taken to reach the hidden platform (i.e.,

escape latency time), the distance traveled to reach the hidden

platform, the distance in the target (NW) zone, and mean speed

were recorded. On day 7 (24 h after the last training session), the

platform was removed from the pool and a probe trial was per-

formed (drug-off trial). Each mouse was allowed to swim for 60 s. If

a mouse did not ﬁnd the previous platform position within 60 s, it

was given a latency score of 60 s. The latency time to the ﬁrst

crossing of the former platform location (i.e., the target zone), the

number of crossings of the target zone, the time spent in the target

NW quadrant, the total distance, the distance spent in the NW

quadrant, the entries into the NW quadrant, and the mean speed

were measured and compared across the experimental groups.

Declaration of competing interest

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.

Acknowledgments

Financial support was provided by Natural Science Fundation of

Anhui provincial Department of Education (KJ2019ZD21), and

Anhui Provincial Natural Science Foundation (2008085MH261,

2008085MH272). Thanks are given to Dr. Yong-An YANG at Elion

Nature Biological Technology Co., Ltd, Nanjing 210038, China for

providing the material cannabidiol and its structural identiﬁcation.

Appendix A. Supplementary data

Supplementary data to this article can be found online at

https://doi.org/10.1016/j.ejmech.2021.113735.

Abbreviation

A

b

b-amyloid

ACh

AChE

AD

AEA

AST

ALT

2-AG

BBB

acetylcholine;

acetylcholinesterase

Alzheimer's disease

anandamide;

aspartate aminotransferase

alanine aminotransferase

2-acyl-glycerol

blood-brain barrier

butyrylcholinesterase

cannabidiol

BuChE

CBD

16

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

potent and selective aryl-1,2,3-triazolyl benzylpiperidine inhibitors toward

butyrylcholinesterase in Alzheimer's disease, Bioorg. Med. Chem. 27 (2019)

931e943.

CB1 receptor cannabinoid receptor 1

ChE

cholinesterase

CNS

central nervous system

[14] L. Jing, G. Wu, D. Kang, Z. Zhou, Y. Song, X. Liu, P. Zhan, Contemporary

medicinal-chemistry strategies for the discovery of selective butyr-

ylcholinesterase inhibitors, Drug Discov. Today 24 (2) (2019) 629e635.

[15] J. Marikova, A. Ritomska, J. Korabecny, R. Perinova, A. Al Mamun, T. Kucera,

E. Kohelova, D. Hulcova, T. Kobrlova, J. Kunes, L. Novakova, L. Cahlikova, Ar-

omatic esters of the crinane amaryllidaceae alkaloid ambelline as selective

inhibitors of butyrylcholinesterase, J. Nat. Prod. 83 (2020) 1359e1367.

[16] A. Meden, D. Knez, N. Malikowska-Racia, X. Brazzolotto, F. Nachon, J. Svete,

K. Salat, U. Groselj, S. Gobec, Structure-activity relationship study of

tryptophan-based butyrylcholinesterase inhibitors, Eur. J. Med. Chem. 208

(2020), 112766.

[17] J.A. Miles, J.S. Kapure, G.S. Deora, C. Courageux, A. Igert, J. Dias, R.P. McGeary,

X. Brazzolotto, B.P. Ross, Rapid discovery of a selective butyrylcholinesterase

inhibitor using structure-based virtual screening, Bioorg. Med. Chem. Lett 30

(2020), 127609.

[18] Q. Li, S. Xing, Y. Chen, Q. Liao, B. Xiong, S. He, W. Lu, Y. Liu, H. Yang, Q. Li,

F. Feng, W. Liu, Y. Chen, H. Sun, Discovery and biological evaluation of a novel

highly potent selective butyrylcholinsterase inhibitor, J. Med. Chem. 63 (2020)

10030e10044.

DPPH

DTNB

DS

1,1-diphenyl-2-picrylhydrazyl

5,5⁰-dithiobis(2-nitro-benzoic acid)

Discovery Studio Client v18.1.0

Electrophorus electricus AChE

EeAChE

eqBuChE equine BuChE

FAAH

GPR55

HE

HPLC

HIA

fatty acid amide hydrolase

G protein-coupled receptor 55

hematoxylinꢀeosin

high-perform-ance liquid chromatography

human intestinal absorption

5HT1A

MD

5-hydroxytryptamine receptor 1A

molecular dynamics

MTT

PAS

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

peripheral anionic site;

[19] Z. Zhang, J. Min, M. Chen, X. Jiang, Y. Xu, H. Qin, W. Tang, The structure-based

optimization of delta-sultone-fused pyrazoles as selective BuChE inhibitors,

Eur. J. Med. Chem. 201 (2020), 112273.

PAMPA-BBB parallel artiﬁcial membrane permeation assay of BBB

THC

Delta-9-tetrahydrocannabinol

[20] J.A. Miles, B.P. Ross, Recent advances in virtual screening for cholinesterase

inhibitors, ACS Chem. Neurosci. 12 (2021) 30e41.

TRPV1

transient receptor potential vanilloid-1

[21] J.M. Roldan-Pena, V. Romero-Real, J. Hicke, I. Maya, A. Franconetti, I. Lagunes,

J.M. Padron, S. Petralla, E. Poeta, M. Naldi, M. Bartolini, B. Monti,

M.L. Bolognesi, O. Lopez, J.G. Fernandez-Bolanos, Tacrine-O-protected phe-

nolics heterodimers as multitarget-directed ligands against Alzheimer's dis-

ease: selective subnanomolar BuChE inhibitors, Eur. J. Med. Chem. 181 (2019),

111550.

References

[1] M. Price, A. Comas-Herrera, M. Knapp, M. Guerchet, M. Karagiannidou, The

World Alzheimer Report 2016, Alzheimer ’s Disease International (ADI), Lon-

don, 2016, pp. 1e131.

[22] M.S. Sadegh, A. Azadi, Z. Izadi, M. Kurd, T. Dara, M. Dibaei, Z.M. Sharif,

J.H. Akbari, M. Hamidi, Brain delivery of curcumin using solid lipid nano-

particles and anostructured lipid carriers: preparation, optimization, and

pharmacokinetic evaluation, ACS Chem. Neurosci. 10 (2019) 728e739.

[23] E.N. Agbo, S. Gildenhuys, Y.S. Choong, M.J. Mphahlele, G.K. More, Synthesis of

furocoumarin-stilbene hybrids as potential multifunctional drugs against

multiple biochemical targets associated with Alzheimer's disease, Bioorg.

Chem. 101 (2020), 103997.

[24] E.B. Russo, A. Burnett, B. Hall, K.K. Parker, Agonistic properties of cannabidiol

at 5-ht1a receptors, Neurochem. Res. 30 (2005) 1037e1043.

[25] E. Manrique-Garcia, S. Zammit, C. Dalman, T. Hemmingsson, S. Andreasson,

P. Allebeck, Cannabis, schizophrenia and other non-affective psychoses: 35

years of follow-up of a population-based cohort, Psychol. Med. 42 (2012)

1321e1328.

[26] R. Cooray, V. Gupta, C. Suphioglu, Current aspects of the endocannabinoid

system and targeted THC and CBD phytocannabinoids as potential thera-

peutics for Parkinson's and alzheimer's diseases: a review, Mol. Neurobiol. 57

(2020) 4878e4890.

[27] L. Cristino, T. Bisogno, V. Di Marzo, Cannabinoids and the expanded endo-

cannabinoid system in neurological disorders, Nat. Rev. Neurol. 16 (2020)

9e29.

[2] M. Zhou, H. Wang, X. Zeng, P. Yin, J. Zhu, W. Chen, X. Li, L. Wang, L. Wang,

Y. Liu, J. Liu, M. Zhang, J. Qi, S. Yu, A. Afshin, E. Gakidou, S. Glenn, V.S. Krish,

M.K. Miller-Petrie, W.C. Mountjoy-Venning, E.C. Mullany, S.B. Redford, H. Liu,

M. Naghavi, S.I. Hay, L. Wang, C.J.L. Murray, X. Liang, Mortality, morbidity, and

risk factors in China and its provinces, 1990-2017: a systematic analysis for

the Global Burden of Disease Study 2017, Lancet 394 (10204) (2019)

1145e1158.

[3] S. Mostafavi, C. Gaiteri, S.E. Sullivan, C.C. White, S. Tasaki, J. Xu, M. Taga,

H.U. Klein, E. Patrick, V. Komashko, C. McCabe, R. Smith, E.M. Bradshaw,

D.E. Root, A. Regev, L. Yu, L.B. Chibnik, J.A. Schneider, T.L. Young-Pearse,

D.A. Bennett, P.L. De Jager, A molecular network of the aging human brain

provides insights into the pathology and cognitive decline of Alzheimer's

disease, Nat. Neurosci. 21 (2018) 811e819.

[4] R. Tundis, M. Bonesi, F. Menichini, M.R. Loizzo, Recent knowledge on medic-

inal plants as source of cholinesterase inhibitors for the treatment of de-

mentia, Mini Rev. Med. Chem. 16 (2016) 605e618.

[5] M.A. Kamal, P. Klein, W. Luo, Y. Li, H.W. Holloway, D. Tweedie, N.H. Greig,

Kinetics of human serum butyrylcholinesterase inhibition by a novel experi-

mental Alzheimer therapeutic, dihydrobenzodioxepine cymserine, Neuro-

chem. Res. 33 (2008) 745e753.

[6] H. Hampel, M.M. Mesulam, A.C. Cuello, M.R. Farlow, E. Giacobini,

G.T. Grossberg, A.S. Khachaturian, A. Vergallo, E. Cavedo, P.J. Snyder,

Z.S. Khachaturian, G. Cholinergic Syst Working, The cholinergic system in the

pathophysiology and treatment of Alzheimer's disease, Brain 141 (2018)

1917e1933.

[28] Z.X. Xiong, X.F. Muldoon Wang, G.H. Bi, M.I. Damaj, A.H. Lichtman,

R.G. Pertwee, E.L. Gardner,

D 8 -Tetrahydrocannabivarin has potent anti-

nicotine effects in several rodent models of nicotine dependence, Br. J.

Pharmacol. 176 (2019) 4773e4784.

[29] C.D. Schubart, W.A. van Gastel, E.J. Breetvelt, B.S.L. eetz, R.A. Ophoff,

I.E. Sommer, R.S. Kahn, M.P. Boks, Cannabis use at a young age is associated

with psychotic experiences, Psychol. Med. 41 (2010) 1301e1310.

[7] A. Majdi, S. Sadigh-Eteghad, S.R. Aghsan, F. Farajdokht, V.S.M. atandoust,

A. Namvaran, J. Mahmoudi, Amyloid-beta, tau, and the cholinergic system in

Alzheimer's disease: seeking direction in a tangle of clues, Rev. Neurosci. 31

(4) (2020) 391e413.

[8] S. Xing, Q. Li, B. Xiong, Y. Chen, F. Feng, W. Liu, H. Sun, Structure and thera-

peutic uses of butyrylcholinesterase: application in detoxiﬁcation, Alzheimer's

disease, and fat metabolism, Med. Res. Rev. (2020), https://doi.org/10.1002/

med.21745.

[9] N.H. Greig, T. Utsuki, D.K. Ingram, Y. Wang, G. Pepeu, C. Scali, Q.S. Yu,

J. Mamczarz, H.W. Holloway, T. Giordano, D. Chen, K. Furukawa,

K. Sambamurti, A. Brossi, D.K. Lahiri, Selective butyrylcholinesterase inhibition

elevates brain acetylcholine, augments learning and lowers Alzheimer beta-

amyloid peptide in rodent, Proc. Natl. Acad. Sci. Unit. States Am. 102 (2005)

17213e17218.

[30] S. Bhattacharyya, P.D. Morrison, P. Fusar-Poli, R. Martin-Santos, S. Borgwardt,

T.C. Winton-Brown, Nosarti, C.M. O' Carroll, M. Seal, P. Allen, M.A. Mehta,

J.M. Stone, N. Tunstall, V. Giampietro, S. Kapur, R.M. Murray, A.W. Zuardi,

J.A. Crippa, Z. Atakan, P.K. McGuire, Opposite effects of delta-9-

tetrahydrocannabinol and cannabidiol on human brain function and psy-

chopathology, Neuropsychopharmacology 35 (2010) 764e774.

^

[31] da Silva Vanessa Kappel, de Freitas Betania Souza, Lacerda Garcia Rebeca

Carvalho, Tavares Monteiro Ricardo, Eduardo Hallak Jaime, Waldo

^

ꢀ

€

Zuardi Antonio, S. Jose Alexandre, Nadja Schroder Crippa, Antiapoptotic ef-

fects of cannabidiol in an experimental model of cognitive decline induced by

brain iron overload, transl, Psychiatry 8 (2018) 176e183.

[32] C.D. heng, J.K. Low, W. Logge, B. Garner, T. Karl, Chronic cannabidiol treatment

improves social and object recognition in double transgenic appswe/ps1e9

mice, Psychopharmacology (Berlin) 231 (15) (2014) 3009e3017.

[10] U. Kosak, B. Brus, D. Knez, S. Zakelj, J. Trontelj, A. Pislar, R. Sink, M. Jukic,

M. Zivin, A. Podkowa, F. Nachon, X. Brazzolotto, J. Stojan, J. Kos, N. Coquelle,

K. Salat, C.J.-P. olletier, S. Gobec, The magic of crystal structure-based inhibitor

optimization: development of a butyrylcholinesterase inhibitor with pico-

molar af ﬁ nity and in vivo activity, J. Med. Chem. 61 (2018) 119e139.

[11] Y. Dundar, O. Kuyrukcu, G. Eren, F.S.S. Deniz, T. Onkol, I.E. Orhan, Novel pyr-

idazinone derivatives as butyrylcholinesterase inhibitors, Bioorg. Chem. 92

(2019), 103304.

[12] S.N. Dighe, M. Tippana, S. van Akker, T.A. Collet, Structure-based scaffold

repurposing toward the discovery of novel cholinesterase inhibitors, ACS

Omega 5 (48) (2020) 30971e30979.

[13] P. de Andrade, S.P. Mantoani, P.S. Goncalves Nunes, C.R. Magadan, C. Perez,

D.J. Xavier, E.T. Sakamoto Hojo, N.E. Campillo, A. Martinez, I. Carvalho, Highly

[33] G. Watt, K. Shang, J. Zieba, J. Olaya, H. Li, B. Garner, T. Karl, Chronic treatment

with 50 mg/kg cannabidiol improves cognition and moderately reduces A

b

40

levels in 12-month-old male A b PPswe/PS1 D E9 transgenic mice, J. Alzheimers

Dis. 74 (2020) 937e950.

[34] A. Gururajan D.T. Malone, Does cannabidiol have a role in the treatment of

schizophrenia? Schizophr. Res. 176 (2016) 281e290.

[35] C. Wojciech, P.D. Wojciech, B. Fredrik, A.R. lexander, S. Jan, E. Inna, Component

of cannabis, cannabidiol, as a possible drug against the cytotoxicity of a

35) and a (25-35) peptides: an investigation by molecular dynamics and

well-tempered metadynamics simulations, ACS Chem. Neurosci. 12 (4) (2021)

b (31-

b

17

X. Jiang, Z. Zhang, J. Zuo et al.

European Journal of Medicinal Chemistry 223 (2021) 113735

660e674.

carbamate-based butyrylcholinesterase inhibitors by rational investigation

into their inhibition mode, J. Med. Chem. 59 (2016) 2067e2082.

[36] L. Casares, J.D. Unciti-Broceta, M.E. Prados, C.D. aprioglio, D. Mattoteia,

~

M. Higgins, G. Apendino, Din A.T. kova-Kostova, E. Munoz, L. de la Vega,

Isomeric O-methyl cannabidiolquinones with dual BACH1/NRF2 activity,

Redox Biol 37 (2020), 101689.

[54] S. Montanari, L. Scalvini, M. Bartolini, F. Belluti, S. Gobbi, V. Andrisano,

A. Ligresti, V. Di Marzo, S. Rivara, M. Mor, A. Bisi, A. Rampa, Fatty acid amide

hydrolase (FAAH), acetylcholinesterase (AChE), and butyrylcholinesterase

(BuChE): networked targets for the development of carbamates as potential

anti-alzheimer's disease agents, J. Med. Chem. 59 (2016) 6387e6406.

[55] M. Hoffmann, C. Stiller, E. Endres, M. Scheiner, S. Gunesch, C. Sotriffer,

T. Maurice, M. Decker, Highly selective butyrylcholinesterase inhibitors with

tunable duration of action by chemical modi ﬁ cation of transferable carbamate

units exhibit pronounced neuroprotective effect in an alzheimer's disease

mouse model, J. Med. Chem. 62 (2019) 9116e9140.

[37] H. Li, Y. Liu, Ti D. an, L. Tian, C. Liang, Overview of cannabidiol (cbd) and its

analogues: structures, biological activities, and neuroprotective mechanisms

in epilepsy and alzheimer's disease, Eur. J. Med. Chem. 192 (2020), 112163.

[38] A.K. Ghosh, M. Brindisi, Organic carbamates in drug design and medicinal

chemistry, J. Med. Chem. 58 (2015) 2895e2940.

[39] A.C. Galasiti Kankanamalage, Y. Kim, A.D. Rathnayake, K.R. Alliston,

M.M. Butler, S.C. Cardinale, T.L. Bowlin, W.C. Groutas, K.O. Chang, Design,

synthesis, and evaluation of novel prodrugs of transition state inhibitors of

norovirus 3CL protease, J. Med. Chem. 60 (2017) 6239e6248.

[56] B.D. Freudenthal, W.A. Beard, L. Perera, D.D. Shock, T.

m Ki, T. Schlick,

S.H. Wilson, Uncovering the polymerase-induced cytotoxicity of an oxidized

nucleotide, Nature 517 (2015) 635e639.

[57] Y. Xu, Z. Zhang, X. Jiang, X. Chen, Z. Wang, H. Alsulami, H.L. Qin, W.J. Tang,

[40] B. Li, S. Cai, Y.A. Yang, S.C. Chen, R. Chen, J.B. Shi, X.H. Liu, W.J. Tang, Novel

unsaturated glycyrrhetic acids derivatives: design, synthesis and anti-

in ﬂ ammatory activity, Eur. J. Med. Chem. 139 (2017) 337e348.

Discovery of

d -sultone-fused pyrazoles for treating Alzheimer's disease:

[41] J. Jampilek, K. Brychtova, Azone analogues: classi ﬁ cation, design, and trans-

dermal penetration principles, Med. Res. Rev. 32 (2012) 907e947.

[42] M. Vettorazzi, E. Angelina, S. Lima, T. Gonec, J. Otevrel, P. Marvanova,

T. Padrtova, P. Mokry, B.P. obal, L.M. Acosta, A. Palma, J. Cobo, J. Bobalova,

J. Csollei, I. Malik, S. Alvarez, S. Spiegel, J. Jampilek, R.D. Enriz, An integrative

study to identify novel scaffolds for sphingosine kinase 1 inhibitors, Eur. J.

Med. Chem. 139 (2017) 461e481.

[43] A. Imramovsky, M. Pesko, J. Monreal-Ferriz, K. Kralova, J. Vinsova, J. Jampilek,

Photosynthesis-inhibiting ef ﬁ ciency of 4-chloro-2-(chlorophenylcarbamoyl)

phenyl alkylcarbamates, Bioorg. Med. Chem. Lett 21 (2011) 4564e4567.

[44] Jiri Kos, Nevin Eoghan, Soral Michal, Kushkevych Ivan, Gonec Tomas,

Bobal Pavel, Kollar peter, coffey aidan, O'mahony jim, liptaj, tibor, kralova

katarina, jampilek josef, synthesis and antimycobacterial properties of ring-

substituted 6-hydroxynaphthalene-2-carboxanilides, Bioorg. Med. Chem. 23

(2015) 2035e2043.

design, synthesis, biological evaluation and SAR studies, Eur. J. Med. Chem.

181 (2019), 111598.

[58] L. Di, E.H. Kerns, I.F. Bezar, S.L. Petusky, Y. Huang, Comparison of blood-brain

barrier permeability assays: in situ brain perfusion, MDR1-MDCKII and

PAMPA-BBB, J. Pharm. Sci. 98 (2009) 1980e1991.

[59] W.J. Tang, J. Wang, X. Tong, J.B. Shi, X.H. Liu, J. Li, Design and synthesis of

celastrol derivatives as anticancer agents, Eur. J. Med. Chem. 95 (2015)

166e173.

[60] S.N. Dighe, G.S. Deora, De E. la Mora, F. Nachon, S. Chan, M.O. Parat,

X. Brazzolotto, B.P. Ross, Discovery and structure-activity relationships of a

highly selective butyrylcholinesterase inhibitor by structure-based virtual

screening, J. Med. Chem. 59 (2016) 7683e7689.

[61] M. Wu, J. Ma, L. Ji, M. Wang, Z. Li, Design, synthesis, and biological evaluation

of rutacecarpine derivatives as multitarget-directed ligands for the treatment

of alzheimer's disease, Eur. J. Med. Chem. 177 (2019) 198e211.

[62] M.J. ehla, S.H. Deibel, F.J. araji, T. Saito, T.C. Saido, M.H. Mohajerani,

R.J. McDonald, Looking beyond the standard version of the Morris water task

in the assessment of mouse models of cognitive de ﬁ cits, Hippocampus 29

(2019) 3e14.

[45] S. Pospisilova, J. Kos, H. Michnova, I. Kapustikova, S.T. trharsky, M. Oravec,

A.M. Moricz, J. Bakonyi, T. Kauerova, P. Kollar, A. Cizek, J. Jampilek, Synthesis

and spectrum of biological activities of novel N-arylcinnamamides, Int. J. Mol.

Sci. 19 (2018), 2318.

[46] P. Bar-On, C.B. Millard, M. Harel, H. Dvir, A. Enz, S.J.L. ussman, I. Silman, Kinetic

and structural studies on the interaction of cholinesterases with the anti-

Alzheimer drug rivastigmine, Biochemistry 41 (2002) 3555e3564.

[47] Pizova Hana, Havelkova Marketa, Bobal Pavel, Stepankova Sarka,

Kauerova Tereza, Andrzej Bak, Kollar Peter, Kozik Violetta, Oravec Michal,

Imramovsky Ales, Jampilek Josef, Proline-based carbamates as cholinesterase

inhibitors, Molecules 22 (2017) 1969.

[63] L.E. Berkowitz, R.E. Harvey, E. Drake, S.M. Thompson, B.J. Clark, Progressive

impairment of directional and spatially precise trajectories by TgF344-

Alzheimer's disease rats in the Morris Water Task, Sci. Rep. 8 (2018), 16153.

[64] T.E. Evans, H.H.H. Adams, S. Licher, F.J. Wolters, A. van der Lugt, M.K. Ikram,

M.J. O'Sullivan, M.W. Vernooij, M.A. Ikram, Subregional volumes of the hip-

pocampus in relation to cognitive function and risk of dementia, Neuroimage

178 (2018) 129e135.

[48] F. Pollastro, D. Caprioglio, P. Marotta, A.S. Moriello, L. De Petrocellis,

O. Taglialatela-Scafati, G. Appendino, Iodine-promoted aromatization of p-

menthane-type phytocannabinoids, J. Nat. Prod. 81 (2018) 630e633.

[49] S.N. Dighe, G.S. Deora, E. De la Mora, F. Nachon, S. Chan, M.O. Parat,

X. Brazzolotto, B.P. Ross, Discovery and structure-activity relationships of a

highly selective butyrylcholinesterase inhibitor by structure-based virtual

screening, J. Med. Chem. 59 (2016) 7683e7689.

[50] G.L. Qiu, S.S. He, S.C. Chen, B. Li, H.H. Wu, J. Zhang, W.J. Tang, Design, synthesis

and biological evaluation of tricyclic pyrazolo 1,5-c 1,3 benzoxazin-5(5H)-one

scaffolds as selective BuChE inhibitors, J. Enzym. Inhib. Med. Chem. 33 (2018)

1506e1515.

[65] J.F.O. da Cruz, A. Busquets-Garcia, Z.Z. hao, M. Varilh, G. Lavanco, L. Bellocchio,

L. Robin, A. Cannich, F. Julio-Kalajzic, T. Leste-Lasserre, M. Maitre, F. Drago,

G. Marsicano, E. Soria-Gomez, Speci ﬁ c hippocampal interneurons shape

consolidation of recognition memory, Cell Rep. 32 (2020), 108046.

[66] P.T. Nelson, C.D. Smith, E.L. Abner, B.J. Wilfred, W.-X. Wang, J.H. Neltner,

B.M. aker, D.W. Fardo, R.J. Kryscio, S.W. Scheff, G.A. Jicha, K.A. Jellinger, Van

L.J. Eldik, F.A. Schmitt, Hippocampal sclerosis of aging, a prevalent and high-

morbidity brain disease, Acta Neuropathol. 126 (2013) 161e177.

[67] H. Zhang, H. Kuang, Y. Luo, S. Liu, L. Meng, P.Q. ang, R. Fan, Low-dose

bisphenol A exposure impairs learning and memory ability with alterations of

neuromorphology and neurotransmitters in rats, Sci. Total Environ. 697

(2019), 134036.

[51] R. Dash, M. Junaid, S. Mitra, A.M. rifuzzaman, S.M.Z. Hosen, Structure-based

identi ﬁ cation of potent VEGFR-2 inhibitors from in vivo metabolites of a

herbal ingredient, J. Mol. Model. 25 (2019) 98.

[68] A Friendly and Versatile Cloud Computing Platform for Biomedicinal, Material,

and Statistical Researches. https://cloud.yinfotek.com/.

[52] M. Marta, F. Gatta, M. Pomponi, Physostigmine analogs anticholinesterases:

effects of the lengthening of the N-carbamic chain on the inhibition kinetics,

Biochim. Biophys. Acta Protein Struct. Mol. Enzymol. 1120 (1992) 262e266.

[53] E. Sawatzky, S. Wehle, B. Kling, J. Wendrich, G. Bringmann, C.A. Sotriffer,

J. Heilmann, M. Decker, Discovery of highly selective and nanomolar

[69] J. Wang, R.M. Wolf, J.W. Caldwell, P.A. Kollman, D.A. Case, Development and

testing of

a general AMBER force ﬁ eld, J. Comput. Chem. 25 (2004)

1157e1174.

[70] T. Darden, D. York, L. Pedersen, Particle mesh Ewald: an N,log(N) method for

Ewald sums in large systems, J. Chem. Phys. 98 (1993) 10089e10092.

18

Article Doi

Novel cannabidiol?carbamate hybrids as selective BuChE inhibitors: Docking-based fragment reassembly for the development of potential therapeutic agents against Alzheimer's disease

DOI: 10.1016/j.ejmech.2021.113735

Source and publish data:

Authors:

Article abstract of DOI:10.1016/j.ejmech.2021.113735

Full text of DOI:10.1016/j.ejmech.2021.113735

Products guided by the article

R&D Labs maybe for 55245-71-1

Relevant to this article

Hot Product