Novel Tadalafil Derivatives Ameliorates Scopolamine-Induced

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Novel Tadalafil Derivatives Ameliorates ScopolamineInduced Cognitive Impairment in Mice via Inhibition of Acetylcholinesterase (AChE) and Phosphodiesterase 5 (PDE5) Wei Ni, Huan Wang, Xiaokang Li, Xinyu Zheng, Manjiong Wang, Jian Zhang, Qi Gong, Dazheng Ling, Fei Mao, Haiyan Zhang, and Jian Li ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00014 • Publication Date (Web): 04 Apr 2018 Downloaded from http://pubs.acs.org on April 4, 2018

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ACS Chemical Neuroscience

Novel Tadalafil

Derivatives

Ameliorates Scopolamine-Induced

Cognitive

Impairment in Mice via Inhibition of Acetylcholinesterase (AChE) and Phosphodiesterase 5 (PDE5)

Wei Nia,1, Huan Wangb,c,1, Xiaokang Lia,1, Xinyu Zhenga,1, Manjiong Wanga, Jian Zhangb,d, Qi Gongb,d, Dazheng Linga, Fei Mao a,*, Haiyan Zhangb,d,*, Jian Lia,*

a

Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China

University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China b

CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203, China c

University of Chinese Academy of Science, No.19A Yuquan Road, Beijing 100049,

China d

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica,

University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 201203, China 1 *

These authors contributed equally to this work. To

whom

correspondence

should

be

addressed

[email protected], [email protected].

ACS Paragon Plus Environment

[email protected],

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Based on the drugs repositioning and redeveloping strategy, first-generation dual-target inhibitors of acetylcholinesterase (AChE) and phosphodiesterase 5 (PDE5), have been recently reported as a potentially novel therapeutic method for the treatment of Alzheimer’s disease (AD), and the lead compound 2 has proven this method was feasible in AD mouse models. In this study, our work focused on exploring alternative novel tadalafil derivatives (3a-s). Among the nineteen analogues, compound 3c exhibited good selective dual-target AChE/PDE5 inhibition, good blood-brain barrier (BBB) permeability. Moreover, its citrate (3c·Cit) possessed improved water solubility and good effects against scopolamine-induced cognitive impairment with inhibition of cortical AChE activities and enhancement of cAMP response element-binding protein (CREB) phosphorylation ex vivo. Keywords: Multifunctional agents, AChE inhibitors, PDE5 inhibitors, Alzheimer’s disease


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INTRODUCTION Alzheimer’s disease (AD), an age-related neurodegenerative disorder with complex etiology and pathogenesis, is characterized by progressive memory loss and cognitive impairment.1 According to “World Alzheimer Report 2016”, there were 46.8 million people worldwide living with dementia in 2015 and this number will come up to 131.5 million in 2050.2 AD is the most common cause of dementia and accounts for approximate 60 to 80 percent of the case. However, there is no efficacious drug to cure AD so far due to its complex pathogenesis, including low levels of acetylcholine, β-amyloid (Aβ) deposits, Tau protein aggregation, oxidative stress, inflammation, and dyshomeostasis of biometals.3,

4

Therefore, it arouses a

major and growing public concern and there is an urgent need to develop more effective therapies to treat this disease. Phosphodiesterase 5 (PDE5), localized in the hippocampus, cortex and cerebellum of the brain,5 is specially responsible for the hydrolysis of cyclic guanosine monophosphate (cGMP), which is closely associated with cellular functions such as neurotransmitter release, neuroplasticity, neuroprotection, and strengthening of learning and memory ability. 6, 7 What’s more, research has shown that the cGMP level is significantly lower in the cerebrospinal fluid (CSF) of mild AD patients compared with that of healthy control subjects due to an increased degradation, caused by the increased expression of PDE5 in the cortex of AD patients.8 In fact, PDE5 inhibitors, such as sildenafil,9 tadalafil,10 and icariin,11 have demonstrated significant memory-enhancing effects in different mouse models of AD. Therefore, PDE5 is a novel potential therapeutic target for the treatment of AD. Many efforts have devoted to the research and development of effective therapeutic drugs for AD, but the return is very little. The reasons for the difficulty of developing effective anti-AD therapies are multifactorial. Two of the key reasons are ascribed to its complex pathogenesis and the structure of the brain, which is protected by the blood-brain barrier (BBB) and only few specific small-molecule drugs can cross. Thus, developing multi-target-directed ligands (MTDLs) with properties



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simultaneously targeting multiple pathological processes involved in the disease and good BBB penetration capacity, has been validated as a potential therapeutic approach for AD.12-17 In the light of the facts that acetylcholinesterase (AChE) remains the most widely used therapeutic target nowadays (three of the four AD therapeutic drugs currently approved by the United States Food and Drug Administration are AChE inhibitors, including donepezil, rivastigmine and galanthamine), PDE5 is a novel potential therapeutic target, and the effect of AChE and PDE5 inhibitors on object recognition memory in rats is different and dissociable,18 AChE/PDE5 dual inhibitors may supply a new perspective and breakthrough for the treatment of AD. Previously,

based

on

drugs repositioning

and

redeveloping

strategy,

first-generation dual-target inhibitors of AChE and PDE5 have been recently reported as a potentially novel therapeutic method for the treatment of AD,19 and the lead compound 2 has been confirmed to demonstrate significant memory-enhancing effects in AD mouse models, proving this method was feasible. However, lead compound 2 was nearly insoluble in water, which may limit its further development because water solubility is important to drug’s oral bioavailability. Herein, in this study, to obtain more potent dual-target PDE5/AChE inhibitors with improved water solubility, our work focused on the design, synthesis and evaluation of a new series of tadalafil derivatives (3a-s, Figure 1), anticipating finding a good candidate for the cure of AD.

Figure 1. Design strategy of tadalafil derivatives as novel dual-target AChE/PDE5 inhibitors.

RESULTS AND DISCUSSION ACS Paragon Plus Environment

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Chemistry The synthetic routes of target compounds are outlined in Schemes 1-2. As depicted in Scheme 1, the key intermediates 4a-d (two pairs of enantiomers 4a, 4c and 4b, 4d) were synthesized through Pictet-Spengler reaction of commercially available

D-

or

L-

tryptophan

methyl

ester

hydrochloride

with

4-methoxybenzaldehyde in isopropanol or ethanol. Target compounds 3a-s were synthesized as illustrated in Scheme 2. Reaction of 4a-d with 2-chloroacetyl chloride provided intermediates 5a-d, which further proceeded through the cyclization reaction to

afford

the

target

compounds

3a-d

in

the

presence

of

2-(1-benzylpiperidin-4-yl)ethan-1-amine. Debenzylation of compound 3a in the presence of 10% Pd/C and H2 at room temperature and atmospheric pressure gave intermediate 6, which then reacted with the (bromomethyl)cyclohexane, substituted benzyl chloride or benzyl bromide (RCH2Cl or RCH2Br) to generate target compounds 3e-s. The details of the synthetic procedures and structural characterizations of intermediates 4-6 and target compounds 3a-s are described in the experimental section. The purities of all target compounds were determined by HPLC with ≥ 95% purity (Table S1, Supporting Information) and were identified as non-PAINS on the web of http://fafdrugs3.mti.univparis-diderot.fr/, as recommended by the editors from the ACS (American Chemical Society).20 Scheme 1. Synthesis of Intermediate 4a-da



a

Reagents and conditions: (a) 4-methoxybenzaldehyde, EtOH, reflux, 24 h; (b)

4-methoxybenzaldehyde, i-PrOH, reflux, 24 h. Scheme 2. Synthesis of Compounds 3a-sa

a

Reagents and conditions: (a) 2-chloroacetyl chloride, Et3N, DCM, −10 °C to rt, 6h;

(b) 2-(1-benzylpiperidin-4-yl)ethan-1-amine, Et3N, MeOH, reflux, overnight; (c) Pd/C, H2, MeOH, rt, overnight; (d) RCH2Cl or RCH2Br, K2CO3, rt, 12-24h. ChE Inhibitory Activity


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All the synthetic tadalafil derivatives (19 compounds) were evaluated for the AChE inhibitory activities and some derivatives with good AChE inhibition (14 compounds, AChE inhibition IC50 < 1 µM) were also evaluated for the butyrylcholinesterase (BuChE) inhibitory activities in vitro by modified Ellman method.21 Two well-known ChE inhibitors, huperzine A (Hup A) and donepezil were used as positive references. The inhibitory activities were presented as IC50 (µM) or the percentage of inhibition at 40 µM, and the results were summarized in Table 1 Most of these designed compounds in this study exhibited good AChE inhibition with IC50 values ranged from approximate 40 µM to 0.013 µM (Table 1), manifesting they are more potential AChE inhibitors than those first-generation tadalafil derivatives. The absolute configuration remarkably affected the AChE inhibitory activities. Among the four isomers (3a-d), isomers with 6R configuration (3a, IC50 = 0.031 µM; 3c, IC50 = 0.015 µM) exhibited much more potent AChE inhibitory activity than those with 6S (3b, 55.41% inhibition at 40 µM; 3d, IC50 = 3.070 µM), indicating that the stereo-configuration at position 6, but not position 12, of the tadalafil parent nucleus was more important to AChE inhibitory activity. The influence of R substituent group (3e-s) on AChE inhibitory activity was also evaluated. Compound 3e, obtained by reducing phenyl to a six-membered nonaromatic ring, showed equivalent AChE inhibition (IC50 = 0.029 µM) with 3a, which manifested aromaticity was not a crucial element that favors AChE inhibition. Of these compounds with aryl substituents (3f-s), fluorophenyl-substituted (3f-h), pyridyl-substituted (3l-n) and thienyl-substituted (3s) compounds exhibited more potent AChE inhibitory activity than other compounds (3i-k, 3o-r). The AChE inhibition order of these halogen substituted compounds (3f-k) is as follows: 3-fluorophenyl ≈ 2-fluorophenyl ≈ 4-fluorophenyl >3-chlorophenyl > 3-bromophenyl > 3-iodophenyl. In brief, the bulk of R substituents deeply influenced AChE inhibitory activities. Small R substituents were favorable for the activity (3f-h, 3l-n), while large R substituents were adverse (3i-k, 3o-r). Neither the electron-withdrawing (3o, 3r) nor electron-donating group (3p) was beneficial to AChE inhibition.



Compounds with good AChE inhibition (IC50 < 1 µM, 3a-s except 3b, 3d, 3k, 3p, 3q) were chosen for further assessment of BuChE inhibitory activities. The results shown in Table 1 indicated that these compounds had poor BuChE inhibition and good AChE selectivity. All tested compounds’ IC50 values of BuChE inhibition were more than 5 µM, indicating they were high selective AChE inhibitors over BuChE.


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Table 1. In vitro ChE inhibitory activities and their selectivity for AChE of compounds 3a-sa.

IC50 ± SD (µM) Compd

R

Configuration

IC50 ± SD (µM) for AChEb

for BuChEb

SIb

(Inhibition at 40 µM) 3a

Ph

6R,12aR

0.031 ± 0.005

5.267 ± 0.399

405

3b

Ph

6S,12aR

55.41% c

n.t.d

n.t.d

3c

Ph

6R,12aS

0.015 ± 0.003

7.611 ± 0.718

507

3d

Ph

6S,12aS

3.070 ± 0.249

n.t.d

n.t.d

3e

Cy

6R,12aR

0.029 ± 0.001

6.386 ± 0.738

220

3f

2-fluorophenyl

6R,12aR

0.016 ± 0.003

～40 (50.50%)

～2500

3g

3-fluorophenyl

6R,12aR

0.013 ± 0.002

6.016 ± 0.261

463


ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

a

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3h

4-fluorophenyl

6R,12aR

0.016 ± 0.001

19.328 ± 1.655

1208

3i

3-chlorophenyl

6R,12aR

0.080 ± 0.014

> 40 (39.21%)

> 500

3j

3-bromophenyl

6R,12aR

0.228 ± 0.020

> 40 (32.42%)

> 175

3k

3-iodophenyl

6R,12aR

55.26% c

n.t.d

n.t.d.

3l

2-pyridyl

6R,12aR

0.016 ± 0.001

9.909 ± 0.630

619

3m

3-pyridyl

6R,12aR

0.058 ± 0.008

> 40 (39.27%)

> 690

3n

4-pyridyl

6R,12aR

0.022 ± 0.003

> 40 (35.39%)

> 1818

3o

4-cyanophenyl

6R,12aR

0.101 ± 0.011

> 40 (10.60%)

> 396

3p

4-methylphenyl

6R,12aR

1.309 ± 0.105

n.t.d

n.t.d

3q

4-methoxylphenyl

6R,12aR

3.160 ± 0.324

n.t.d

n.t.d

3r

4-nitrophenyl

6R,12aR

0.730 ± 0.070

> 40 (6.31%)

> 55

3s

3-thienyl

6R,12aR

0.022 ± 0.002

9.448 ± 0.445

429

Tadalafil

-

6R,12aR

26.159 ± 1.300

> 40 (3.86%)

> 1.53

Hup A

-

-

0.084 ± 0.017

36.400 ± 1.838

444

Donepezil

-

-

0.013 ± 0.150

7.704 ± 0.552

593

AChE from rat cortex and BuChE from rat serum. bResults are expressed as mean of at least three experiments. cInhibition at 40 µM. dn.t. indicates no test.


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PDE5 Inhibitory Activity Following, an IMAP-FP (immobilized metal ion affinity-based fluorescence polarization) assay22, 23 was conducted to evaluate the PDE5 inhibitory activities of these tadalafil derivatives with good AChE inhibition (IC50 < 1 µM), using tadalafil as a reference, and the results are shown in Table 2. These data showed that most of the tested compounds could retain PDE5 inhibition, with IC50 values of 0.050-3.231µM. Generally, most of compounds with substituents at the phenyl ring of benzylpiperidine exerted almost equal or a little better PDE5A1 inhibition (3f-h, 3o, 3r), indicating that substituent at the phenyl ring of benzylpiperidine was not bad for PDE5A1 inhibition. Interestingly, compounds with R substituents containing N atom such as pyridyl (3l-n), cyanophenyl (3o), possessed better inhibitory activity than those with non-nitrogen atom substituents. Noticeably, 3a (6R,12aR) exhibited 10 times more potent PDE5A1 inhibitory activity than its diastereoisomer 3c (6R,12aS) (3a, IC50 = 0.203 µM; 3c, IC50 = 3.231 µM), demonstrating the stereo-configuration at position 12 of the tadalafil parent nucleus, was vital for exerting PDE5A1 inhibitory activity. Table 2. In vitro PDE5A1 inhibitory activities of compounds 3a, 3c, 3e-j, 3l-o, 3r-sa.

Compd

R

Configuration

IC50 ± SD (µM)a for PDE5A1

3a

Ph

6R,12aR

0.203 ± 0.027

3c

Ph

6R,12aS

3.231 ± 0.327

3e

Cy

6R,12aR

0.323 ± 0.028

3f

2-fluorophenyl

6R,12aR

0.192 ± 0.031

3g

3-fluorophenyl

6R,12aR

0.193 ± 0.016



a

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3h

4-fluorophenyl

6R,12aR

0.211 ± 0.005

3i

3-chlorophenyl

6R,12aR

0.328 ± 0.005

3j

3-bromophenyl

6R,12aR

0.279 ± 0.022

3l

2-pyridyl

6R,12aR

0.093 ± 0.001

3m

3-pyridyl

6R,12aR

0.074 ± 0.003

3n

4-pyridyl

6R,12aR

0.050 ± 0.018

3o

4-cyanophenyl

6R,12aR

0.062 ± 0.004

3r

4-nitrophenyl

6R,12aR

0.122 ± 0.011

3s

3-thienyl

6R,12aR

0.520 ± 0.024

Tadalafil

-

6R,12aR

0.004 ± 0.0001

Donepezil

-

-

> 100

Results are expressed as mean of at least two experiments.

SAR Studies Based on the AChE and PDE5A1 inhibitory activity data shown in Tables 1-2, the noticeable structure-activity relationships (SARs) for compounds 3a-s were revealed as follows: (1) The stereo-configuration at position 6 of the tadalafil parent nucleus was key to AChE inhibitory activity, and the favored configuration was 6R (3a vs 3b, 3c vs 3d); (2) The stereo-configuration at position 12 of the tadalafil parent nucleus had little influence on AChE inhibitory activity, but was vital for exerting PDE5A1 inhibitory activity (3a vs 3c); (3) The bulk of R substituents deeply influenced AChE inhibitory activities. Specifically, small R substituents were favorable for the activity (3f-h and 3l-n), while large R substituents were adverse (3i-k and 3o-r); (4) The AChE inhibition order of these halogen substituted compounds is as follows: 3-fluorophenyl ≈ 2-fluorophenyl ≈ 4-fluorophenyl >3-chlorophenyl > 3-bromophenyl > 3-iodophenyl; (5) Compounds with R substituents containing N atom such as pyridyl (3l-n), cyanophenyl (3o), possessed better inhibitory activity than those with non-nitrogen atom substituents. In brief, the SARs of the 19 compounds were explicit and provided some insights into the structural modification of effective AChE/PDE5 inhibitors.


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In Vitro Blood-Brain Barrier Permeation Assay and Drug-likeness Evaluation One of the important reasons for the failure of developing effective anti-AD therapies is ascribed to the BBB protection of the brain, and good permeability of the BBB is an essential element for anti-AD drugs, so crossing BBB is of great concern for developing anti-AD drugs. To explore whether the designed tadalafil derivatives have good BBB permeability, a parallel artificial membrane permeation assay (PAMPA) was employed as described by Di et al.24 First, the permeability (Pe) values of 13 commercial drugs in vitro were determined and compared with the reported values to validate the assay (Table S2, Supporting Information). A plot of the experimental data versus bibliographic values showed a good linear correlation, Pe (exp.) = 0.9738Pe (bibl.) – 0.8157 (R2 = 0.9651, Figure S1, Supporting Information). According to this equation and the threshold established by Di et al. for BBB permeability, we could conclude that compounds with Pe values over 3.08 × 10−6 cm s−1 could cross the BBB (CNS +, Table S3, Supporting Information). Following, compounds (3a, 3c, 3e-i, 3l-n, 3s) with good dual-target AChE/PDE5 inhibitors (AChE inhibition: IC50 < 0.2 µM) were tested in the PAMPA-BBB assay, and the results are presented in Table 3. All the tested compounds had the abilities to cross BBB (CNS +), except compounds with cyclohexyl (3e) and pyridyl (3l-n) substituents, which showed uncertain BBB permeability (CNS ±). Among them, compounds 3a and 3c exhibited better BBB permeability than 3f-h and 3s. Therefore, in the light of the activity and druggability, compounds 3a and 3c, with potent AChE/PDE5A1 inhibitory activity and good druggability, were selected as the optimal dual-target AChE/PDE5A inhibitors for further study. Table 4. Permeability results (Pe 10-6 cm s-1) from the PAMPA-BBB assay for selected compounds 3a, 3c, 3e-i, 3l-n, 3s and their prediction of BBB penetration.



a

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Compd

R

Pe (10-6 cm s-1)a

Predictionb

3a

Ph

5.86 ± 1.06

CNS +

3c

-

5.71 ± 0.98

CNS +

3e

Cy

3.05 ± 0.79

CNS ±

3f

2-fluorophenyl

3.61 ± 1.00

CNS +

3g

3-fluorophenyl

4.05 ± 0.91

CNS +

3h

4-fluorophenyl

4.97 ± 0.96

CNS +

3i

3-chlorophenyl

3.39 ± 0.12

CNS +

3l

2-pyridyl

3.06 ± 0.72

CNS ±

3m

3-pyridyl

2.92 ± 0.67

CNS ±

3n

4-pyridyl

2.78 ± 0.52

CNS ±

3s

3-thienyl

3.50 ± 0.45

CNS +

Tadalafil

-

2.16 ± 0.48

CNS ±

Donepezil

-

8.78 ± 2.24

CNS +

PBS/EtOH (70:30) was used as solvent. Values are expressed as mean ± SD of at

least three independent experiments. b CNS + : Compounds with Pe > 3.08 × 10−6 cm s−1 could cross the BBB by passive diﬀusion; CNS−: Compounds with Pe < 1.13 × 10−6 cm s−1 could not cross the BBB; CNS±: Compounds with 1.13 × 10−6 cm s−1 < Pe < 3.08 × 10−6 cm s−1 show uncertain BBB permeability. To determine the potential druggability of optimal compound 3a and 3c, their physicochemical properties were evaluated along with the lead compound 2 and tadalafil. As shown in Table 4, compounds 3a, 3c, 2 fulfilled nearly all of Lipinski’s rules and CNS drug rules.25 Compound 3c has fewer hydrogen-bond acceptor atoms (HBA) and lower tPSA value than lead compound 2, demonstrating better CNS drug-likeness. Although compounds 3a, 3c and 2 were almost insoluble in water


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(water solubility less than 12.5 µg/mL), the water solubility of their corresponding citrates were greatly improved. Importantly, the citrate water solubilities of 3a and 3c were 5.60 and 1.68 times better than that of lead compound 2, indicating it is successful to improve water solubility by replacing benzo[d][1,3]dioxol-5-yl with 4-methoxyphenyl at position 6 of tadalafil. Table 4. Physicochemical properties of compounds 3a, 3c and 2a. water water solubility solubility (µg/mL) of citrate (µg/mL)

compd

MW

HBAb

HBDb

RBb

tPSAb

ClogPb

3a

562.71

7

1

7

68.88

5.18

< 12.5

1400

3c

562.71

7

1

7

68.88

5.18

< 12.5

420

2

576.70

8

1

6

78.11

5.01

< 12.5

250

Tadalafil

389.41

7

1

1

74.88

2.36

< 12.5

-

< 500

< 10

100

3c

> 100

> 100

> 100

> 100

> 100

> 100

Trequinsin

1.062 ± 0.079

0.170 ± 0.001b

0.199 ± 0.021

n.t.c

n.t.c

n.t.c

Dipyridamole

n.t.c

n.t.c

n.t.c

0.943 ± 0.056

58.8 ± 3.21

n.t.b

Zaprinast

n.t.c

n.t.c

n.t.c

n.t.c

n.t.c

7.512 ± 0.687

a

Results are expressed as mean of at least two experiments. bunit: nM. cn.t. indicates

no test. Amelioration on Scopolamine-induced Cognitive Impairment in Mice To evaluate the in vivo effect of optimal compound 3a along with its diastereoisomer 3c on cognitive function improvement, the passageway water maze test was performed on a scopolamine (Scop)-induced cognitive impairment mouse model, a classical AD model to evaluate AChE-related candidate drugs,26 with donepezil (Don) as a positive control. As shown in Figure 2, the model group treated with scopolamine (Scop group, 4.5 mg/kg) exhibited longer escape latency and more frequent errors than the control group (Ctrl group,

###

p < 0.001). Compared with the Scop group, compound 3c·Cit

(citrate of 3c, 10 mg/kg or 30 mg/kg) and donepezil (10 mg/kg) could significantly reduce the escape latency (Figure 2A) and number of errors (Figure 2B). To our satisfaction, 3c·Cit exhibited comparable ability of memory and cognitive function improvement in cognitive impairment mice with donepezil at a dosage of 10 mg/kg. These results above showed 3c·Cit was the most potential anti-AD candidate with a developmental perspective.


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Figure 2. Effects of compounds 3a·Cit and 3c·Cit (10 mg/kg, 30 mg/kg) on scopolamine-induced cognitive impairment in mice. Donepezil (Don, 10 mg/kg) as a reference. (A) The escape latency of mice in the passageway water maze. (B) The number of errors of mice in the passageway water maze. Data are shown as mean ± SEM,

###

p < 0.001 vs Ctrl group, *p < 0.05, **p < 0.01 vs Scop group; N = 12. Ctrl

group: solvent + saline; Scop group: solvent + scopolamine. Efficacy of Compounds 3a·Cit and 3c·Cit on Cortical AChE Activity ex vivo Following, the activities of AChE derived from the cortices of the scopolamine-treated mice were evaluated. Results in Figure 3 showed that the activities of AChE were significantly inhibited by compound 3a·Cit at a dosage of 30 mg/kg (percentage of Ctrl: 82.81 ± 5.65%) and 3c·Cit at both dosages of 10 mg/kg and 30 mg/kg (percentage of Ctrl: 89.50 ± 5.00% and 86.44 ± 7.36%, respectively). The above results indicated that 3a·Cit and 3c·Cit could cross the BBB and target the central cholinergic system in vivo.



Figure 3. Effects of compounds 3a·Cit and 3c·Cit (10 mg/kg, 30 mg/kg) on cortical AChE activities of scopolamine-treated mice. Donepezil (Don, 10 mg/kg) as a reference. Data are shown as mean ± SEM, *p < 0.05. **p

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