Discovery of Novel Tacrine-Pyrimidone Hybrids as Potent Dual AChE/GSK-3 Inhibitors for the Treatment of Alzheimer's Disease

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Discovery of Novel Tacrine−Pyrimidone Hybrids as Potent Dual

AChE/GSK‑3 Inhibitors for the Treatment of Alzheimer’s Disease

Hong Yao,^#Giuseppe Uras,^#Pengfei Zhang,^#Shengtao Xu, Ying Yin, Jie Liu, Shuai Qin, Xinuo Li,

Stephanie Allen, Renren Bai, Qi Gong, Haiyan Zhang,* Zheying Zhu,* and Jinyi Xu *

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ABSTRACT: Based on a multitarget strategy, a series of novel

tacrine−pyrimidone hybrids were identiﬁed for the potential

treatment of Alzheimer’s disease (AD). Biological evaluation

results demonstrated that these hybrids exhibited signiﬁcant

inhibitory activities toward acetylcholinesterase (AChE) and

glycogen synthase kinase 3 (GSK-3). The optimal compound

27g possessed excellent dual AChE/GSK-3 inhibition both in

terms of potency and equilibrium (AChE: IC₅₀= 51.1 nM; GSK-

3β: IC₅₀= 89.3 nM) and displayed signiﬁcant amelioration on

cognitive deﬁcits in scopolamine-induced amnesia mice and

eﬃcient reduction against phosphorylation of tau protein on Ser-

199 and Ser-396 sites in glyceraldehyde (GA)-stimulated diﬀerentiated SH-SY5Y cells. Furthermore, compound 27g exhibited

eligible pharmacokinetic properties, good kinase selectivity, and moderate neuroprotection against GA-induced reduction in cell

viability and neurite damage in SH-SY5Y-derived neurons. The multifunctional proﬁles of compound 27g suggest that it deserves

further investigation as a promising lead for the prospective treatment of AD.

becoming a hot research topic for the potentially curable

treatment of AD.¹⁶

INTRODUCTION

■

AD is a progressive neurodegenerative disorder with a

currently incurable dementia syndrome;¹it aﬄicts millions of

individuals globally in a chronic and fatal way.²The anatomical

features of AD are extracellular senile plaques (SPs) and

intracellular neuroﬁbrillary tangles (NFTs) in the brain,³while

the pathogenesis of AD is still inconclusive and undeﬁned since

it was ﬁrst reported by Alois Alzheimer in 1907.⁴Accumulated

research data have conﬁrmed that the progression of AD is

mainly evident in cholinergic deﬁcits,⁵tau protein hyper-

phosphorylation,⁶β-amyloid (Aβ) aggregation,⁷oxidative

damage,⁸unbalanced homeostasis of biometals,⁹glutamate-

related excitotoxicity,¹⁰serotonergic dysfunction,¹¹histaminer-

gic dysfunction,¹²and reduced signal transduction mediated by

second messengers.¹³The present anti-AD therapies rationally

regulate key targets in related signaling pathways, which could

be classiﬁed as single-target strategies and multitarget

strategies.¹⁴Approved drugs based on a single target (Figure

1A) show obvious improvements in cognition and memory in

clinical applications,¹⁵but the progression of AD cannot be

stopped or reversed by these drugs because of the

compensatory eﬀects of other morbid signaling pathways. In

consideration of the complicated interrelation among multiple

pathways, a multitarget strategy possesses a more eﬀective and

promising capacity to manage the special disease network of

AD with synergistic adjustment toward more pivotal targets,

The cholinergic hypothesis, a classical theory in AD

pathology proposed in 1976,¹⁷states that the neurotransmitter

acetylcholine (ACh) in the hippocampus and the neocortex

regions plays a key role in learning and memory,¹⁸and the

remarkable decline in the level of ACh is primarily responsible

for the dementia of AD patients. Under physiological

conditions, acetylcholinesterase (AChE) can hydrolyze ACh

to choline and acetate with a high eﬃciency.¹⁹Thus, inhibition

against AChE is an advisable way to enhance the concentration

of ACh in the synaptic cleft, making it a crucial target in the

cholinergic system. Drugs approved by the FDA for the

treatment of AD have demonstrated the therapeutic value of

AChE, among which donepezil (1), galantamine (2),

rivastigmine (3), tacrine (4), and huperzine A (5, approved

by CFDA) are all AChE inhibitors (AChEIs) (Figure 1A).²⁰In

addition, appropriate AChEIs could inhibit the oligomerization

and ﬁbrillization of Aβ induced by AChE,^21,22indicating the

Received: January 28, 2021

Published: May 24, 2021

https://doi.org/10.1021/acs.jmedchem.1c00160

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Figure 1. (A) Drugs approved by the FDA or CFDA for the treatment of AD. (B) Representative hybrids bearing a tacrine unit as anti-AD agents

through multiple regulatory mechanisms. (C) Structures of several typical GSK-3β inhibitors.

multifunctional proﬁles of AChEIs in potential anti-AD

therapy.

highly upregulated expression of AChE, which would

consequently lead to suppression of the cholinergic system,

and initially, increased levels of AChE around NFTs would

result in disturbing tau phosphorylation.²⁹Thus, simultaneous

modulation targeting AChE and GSK-3β has mutually

beneﬁcial eﬀects for the amelioration of both cholinergic and

tau protein signaling pathways, and new chemical entities

(NCEs) with dual AChE/GSK-3β inhibition have promising

potential to improve AD conditions through multiple

regulatory mechanisms, such as ACh enhancement, NFT

reduction, and Aβ suppression. However, related anti-AD

studies based on AChE/GSK-3β targets are rare.³⁰⁻³²To

broaden research on multifunctional anti-AD drugs, the

discovery of novel and promising lead compounds with

multiple anti-AD properties is extremely urgent.

Tacrine (4) (Figure 1A) was the ﬁrst AChEI drug approved

in 1993, but it was regretfully withdrawn by the FDA due to

liver toxicity.¹Evidence has shown that the structure of 4 is still

popular in anti-AD studies, especially in multitarget-related

studies.³³For example (Figure 1B), a tacrine−hydroxyquino-

line hybrid³⁴exhibited both AChE inhibitory activity and

metal chelating ability, a tacrine−benzofuran hybrid³⁵was

proven to be a dual inhibitor targeting AChE and β-site

amyloid precursor protein cleaving enzyme (BACE-1), a

tacrine−chromane hybrid³⁶was found to exert multiple

AChE inhibition and antioxidative eﬀects, and a tacrine−

pyridinthiazole hybrid was recently found to possess AChE and

GSK3β dual inhibition activities.³²Therefore, the unique

Meanwhile, the tau protein hypothesis is another signiﬁcant

inference regarding AD pathology, stating that microtubule

depolymerization and NFT generation, which are mostly

caused by tau protein hyperphosphorylation, accelerate the

degeneration and necrosis of neuronal cells, resulting in the

progressive deterioration of disease conditions.⁶Disordered

phosphorylation of tau protein catalyzed by glycogen synthase

kinase 3 beta (GSK-3β), primarily at Ser-396, Ser-199, and Ser-

404 sites,²³increases the number of additional 2−7 phosphate

groups and weakens its adhesion with microtubules. Sub-

sequently, hyperphosphorylated tau protein separates from

microtubules and aggregates into insoluble NFTs, leading to

instability of the microtubule structure and, ultimately, cell

death.²⁴Therefore, GSK-3β upstream of the tau protein

signaling pathway has become a vital target for a new anti-AD

drug design,²⁵and its inhibition could directly beneﬁt the

microtubule system and reduce intracellular NFTs, as well as

radiate to Aβ signaling pathways by reducing the formation of

Aβ.²⁶

The cholinergic signaling pathway has a close connection

with the tau protein signaling pathway in AD pathogenesis.

Evidence has shown that the neuroprotective mechanism of

AChEI donepezil (1) is related to the inactivation of GSK-3β²⁷

and further induces a corresponding decrease in the

insolubility and hyperphosphorylation of tau protein.²⁸On

the other hand, phosphorylated tau protein (p-Tau) can trigger

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Figure 2. Design strategy of tacrine−pyrimidone hybrids with dual AChE/GSK-3β inhibition.

three-ring pharmacophore of 4 was used in our design of new

anti-AD hybrids.

RESULTS AND DISCUSSION

■

Design and Synthesis. First, the tacrine moiety of the

Compound 7 is a typical GSK-3β inhibitor, and further

modiﬁcation on its pyrimidone moiety oﬀered derivative 8

bearing more potent inhibitory activity toward GSK-3β (Figure

1C).³⁷Subsequent optimization on substituent groups of the

pyrimidone scaﬀold oﬀered compound 9,³⁸which showed

similar high aﬃnity for GSK-3β and better pharmacokinetic

proﬁles than compound 8 (Figure 1C). Indeed, the

pyrimidone unit is the key pharmacophore of the above

compounds exerting GSK-3β inhibition. Given the signiﬁcance

of pyrimidone for druggability³⁹and the possible GSK-3β

inhibitory activity of its derivatives, the structure of pyrimidone

was also used as a pivotal moiety in our design of NCEs for

anti-AD.

As shown in Figure 2, the tacrine moiety of hybrids was

principally designed to retain AChE inhibition, the structure of

aromatic ring-substituted pyrimidone was applied to maintain

potential GSK-3β inhibition, and the alkylamine linker was

selected as an appropriate bridge to realize the incorporation of

the two pharmacophores above. Furthermore, the hybrids

underwent ﬁne-tuning of structures to obtain a better target

binding eﬀect. For example, in the case of AChE inhibition, the

substitution patterns of the tacrine unit, leading to optimal

cholinesterase inhibition, are very well known, with higher

potencies observed when halogen atoms or methyl groups are

introduced into the position at C6.³⁶Therefore, the

substitution patterns of the tacrine moiety are worth

investigating. In addition, the introduction of the linker and

pyrimidone moiety into the tacrine might form a new

interaction with AChE, so the linker and pyrimidone moiety

should be slightly modiﬁed to maintain or enhance AChE

inhibition. Herein, we have identiﬁed a novel series of dual

AChE/GSK-3β inhibitors by fusing the key skeletons of tacrine

(4) and pyrimidone (8) based on the multitarget anti-AD

strategy, aiming to alleviate cholinergic deﬁcits and synergisti-

cally eliminate intracellular NFTs. These hybrids underwent

two iterative cycles of optimization, and abundant pharmaco-

logical experiments were performed to investigate their drug-

like properties, which provided optimistic feedback. Through

eﬀective regulation of both cholinergic and tau protein

signaling pathways, we envisaged that such promising

tacrine−pyrimidone hybrids could exhibit favorable eﬀects

under real disease conditions of AD, oﬀering valuable

information for the development of anti-AD therapies.

novel scaﬀold of hybrids was investigated mainly for potential

AChE inhibition. Since higher AChE inhibition potencies are

observed when halogen atoms or methyl groups are introduced

to the phenyl ring of tacrine,³⁶the substituent of the phenyl

moiety was investigated. As depicted in Scheme 1 and Scheme

S1, the starting material 11a was treated with N,N′-

carbonyldiimidazole (CDI) and ethyl potassium malonate to

produce 12a. Cyclization of 12a with N-methylthiourea and

successive chlorination by POCl₃aﬀorded the key intermediate

14a.³⁷Esters with diﬀerent substituents (15), another kind of

starting material, were treated with NaOH to provide

carboxylic acid (16), which underwent cyclization with

cyclohexanone to achieve three-ring structures (17). Sub-

sequently, the corresponding diamines were attached to 17

under the catalysis of NaI to form key intermediates 18a−

18m,³⁰which were coupled with 14a under alkaline conditions

to yield hybrids 21a−21m with diverse substituents (series 1).

Second, the pyrimidone moiety of the novel scaﬀold of

hybrids was investigated mainly for potential GSK-3β

inhibition. It was found that the introduction of a ﬂuorine

atom or the change of pyridine by a pyrimidine ring linked to

the pyrimidone moiety would be beneﬁcial for binding with

GSK-3β;^37,38,40therefore, compounds 14a−14c were synthe-

sized as described in Scheme 1. Compared with 14a, key

intermediates 14b and 14c were synthesized from starting

materials 11b and 11c similarly.^38,40Then, 14b and 14c were

treated with corresponding intermediates from 18a−18m in

the presence of K₂CO₃to yield target compounds 22a−22j

(series 2).

Third, the linker of the novel scaﬀold was investigated

mainly for balanced dual inhibition against AChE/GSK-3β, as

depicted in Scheme 1. Key intermediates 19a−19e were

prepared by amination of compound 17a with diverse

diamines in the presence of NaI, and the subsequent

condensation reaction with 14a in the tetrahydrofuran

(THF) solvent produced hybrids 23a−23e (series 3).

The ﬁrst round of evaluation of the dual AChE/GSK-3β

inhibitory potency of compounds in series 1−3 conﬁrmed our

design strategy and provided explicit guidance for further

optimization of hybrids (Table 1). Thus, in the fourth

modiﬁcation, more promising compounds were designed and

synthesized by assembling the positive building blocks

conﬁrmed by series 1−3, as described in Scheme 2. Key

intermediates 24a−24e or 25a−25e were obtained from the

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Scheme 1. Synthesis of Key Intermediates 14a−14c, 18a−18m, and 19a−19e and Final Hybrids 21a−21m, 22a−22j, and 23a−

a

23e

a

Reagents and conditions: (a) (i) N,N′-carbonyldiimidazole, dry THF, reﬂux, 1−2 h; (ii) ethyl potassium malonate, MgCl₂, dry THF, reﬂux,

overnight; (b) N-methylthiourea, DBU, EtOH, reﬂux, overnight; (c) POCl₃, DMF, 1,2-dichloroethane, 65 °C, 1 h; (d) (i) 2 N NaOH, r.t.,

overnight; (ii) conc. HCl, r.t., 10 min; (e) cyclohexanone, POCl₃, 115 °C, 3−5 h; (f) corresponding diamine, NaI, n-pentanol, reﬂux, 12−24 h; (g)

K₂CO₃, THF, reﬂux, overnight.

amination of relative compound 17 using 1,6-heptanediamine

or 1,7-hexanediamine agents in the presence of NaI. Then, key

intermediates 14a and 14c were adopted to react with 19d and

19e, 24a−24e, or 25a−25e, giving corresponding target

compounds 26a−26k (series 4) and 27a−27k (series 5),

which underwent the second round of evaluation of dual

AChE/GSK-3β inhibitory activities soon after (Table 2).

In total, 50 derivatives in ﬁve series (i.e., 21a−21m, 22a−

22j, 23a−23e, 26a−26k, and 27a−27k) were designed and

synthesized. Related details of the synthetic procedures and

structural characterization of key intermediates and target

compounds are described in the Experimental Section and

Supporting Information.

In Vitro AChE and GSK-3β Inhibitory Activities. In the

ﬁrst round of biological evaluation, the inhibitory potency of

compounds 21a−21m (series 1), 22a−22j (series 2), and

23a−23e (series 3) toward AChE/GSK-3β was assessed in

vitro. The commercially available compounds donepezil (1),

tacrine (4), and AR-A014418⁴¹were used as positive controls

for AChE and GSK-3β, respectively. Due to the high homology

between the murine and human AChE enzymes (with

identities of 89%) and the consistent IC₅₀data obtained with

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Table 1. Structures and Inhibitory Activities toward AChE and GSK-3β of Compounds 21a−21m, 22a−22j, and 23a−23e

a

b

Data are expressed as the mean of three independent determinations. The zymogen of AChE was from the cerebral cortex of a mouse.

c

d

Recombinant human GSK-3β was used. The inhibitory activities of compounds were not favorable at primary screening (inhibitory eﬀect less

e

than 50% at 1 μM), of which the IC₅₀values were not further tested. A typical GSK-3β inhibitor used as the positive control.

reference compounds (huperzine A,⁴²huperzine B,⁴³and

donepezil (Table S1)) across the two species in our previous

studies, a murine AChE-based screening was applied in the

current study. The inhibition against two targets of all

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a

Scheme 2. Synthesis of Key Intermediates 24a−24e and 25a−25e and Final Hybrids 26a−26k and 27a−27k

a

Reagents and conditions: (a) 1,7-heptanediamine or 1,6-hexanediamine, NaI, n-pentanol, reﬂux, 12−24 h; (b) K₂CO₃, THF, reﬂux, overnight.

compounds at 1.0 μM was tested in primary screening, and

compounds with favorable results were further measured to

obtain their IC₅₀values, which are presented in Table S2 and

Table 1.

compounds toward AChE increased. For the target GSK-3β,

compound 22f (n = 1) was the best inhibitor with an IC₅₀

value of 1.18 μM in series 1 and 2, and novel hybrid 23e (n =

6) had more favorable potency with an IC₅₀value of 0.33 μM.

Therefore, the GSK-3β inhibition of hybrids increased in a

major way along with the increase in the carbon chain,

suggesting that the short carbon chain of compounds in series

1 and 2 (21a−21m and 22a−22j) limited the capacity of

pyrimidone moiety binding to enzyme GSK-3β. In addition to

the enhanced potency, a favorable balance of inhibition against

two targets was also achieved when the alkyl chain had six or

seven carbon atoms. Considering that 23d and 23e were the

most promising dual AChE/GSK-3β inhibitors among

compound series 1−3, 23d (IC₅₀= 0.12 μM for AChE; IC₅₀

= 0.52 μM for GSK-3β) had better AChE inhibition and weak

GSK-3β inhibition than 23e (IC₅₀= 0.13 μM for AChE; IC₅₀=

0.33 μM for GSK-3β), and alkylamine linkers with six or seven

carbon atoms were both adopted for further exploration.

Additionally, hybrids holding alkyl alcohol linkers were

designed, synthesized (Scheme S2), and evaluated in our

work with positive feedback, and the alkyl alcohol linker was

abandoned in our further research because its integral results

on dual inhibition had a slightly inferior eﬀect than the

alkylamine linker (Table S3, Supporting Information).

In short, the ﬁrst round of evaluation of compounds from

series 1−3 (21a−21m, 22a−22j, and 23a−23e) showed that

halogen groups (Br-/Cl-/F-) as substituent R¹, ﬂuorine as

substituent R², a longer alkyl linker (n = 5 or 6), and pyridine-

substituted (X = C−F or C−H) pyrimidone were more

beneﬁcial for dual AChE/GSK-3β inhibition of compounds.

Therefore, hybrids based on the above building blocks were

further designed and synthesized as series 4 (26a−26k) and

series 5 (27a−27k), and their dual AChE/GSK-3β inhibitory

activities were assessed in vitro (Table 2). In the same way, the

Compounds 21a−21m and 22a−22j had poor to good

inhibitory activity against AChE (0.16 to 23 μM, Table 1),

suggesting that substituent groups in both the tacrine moiety

and pyrimidine moiety were important for the inhibition of

AChE (see the Supporting Information for more details of the

discussion of inhibitory activity against AChE). It was found

that substituent groups in the R¹site had better inhibitory

activity than the same group in the R²site (see 21g vs 21k, 21c

vs 21j, 21h vs 21i, 21b vs 21d, and 21m vs 21l in Table 1), and

compounds with halogen groups (Br-/Cl-/F-) in the R¹site

and ﬂuorine in the R²site were beneﬁcial for anti-AChE

activity. However, none of them had suﬃcient inhibitory

potency against GSK-3β (Table 1). Although the biological

data were not suﬃcient in series 1 and 2, pyrimidone with an

F-substituted pyridine group (X = C−F) demonstrated its

ability to inhibit GSK-3β (Table 1), which was adopted in

posterior design. Meanwhile, although pyridine-substituted

pyrimidone (X = C−H) had similar potency with imidazole-

substituted pyrimidone (X = N), the structure containing X =

C−H was chosen as a representative core for further study

along with the core containing X = C−F, since the potency of

21a was slightly better than 22a (Table 1).

The alkylamine linker between the tacrine unit and the

pyrimidone unit was studied in compound series 3 (23a−23e)

with the length of the carbon chain changed from three (n = 2)

to seven (n = 6). Among the novel compounds in series 3,

hybrid 23d (n = 5) had the best IC₅₀value of 0.12 μM against

AChE. Compound 22h (n = 1), the best AChE inhibitor in

series 1 and 2, had an IC₅₀value of 0.16 μM. Thus, along with

the increase in carbon chains, the inhibitory potency of

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Table 2. Structures and Dual AChE/GSK-3β Inhibitory Activities of Compounds 26a−26k and 27a−27k

a

b

Data are expressed as the mean of three independent determinations. The zymogen of AChE was from the cerebral cortex of a mouse.

c

d

Recombinant human GSK-3β was used. The inhibitory activities of compounds were relatively not potent enough at primary screening

e

(inhibitory eﬀect less than 60% at 1 μM). Under comprehensive consideration, the exact IC₅₀values of them were not detected. A typical GSK-3β

inhibitor used as a positive control.

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Figure 3. Proposed binding models of 23e (A/C) or 27g (B/D) with AChE (PDB code: 4EY7). For secondary bonds, the hydrogen bond was

shown as a black dotted line, the halogen bond was shown as a pink dotted line, and π−π stacking contacts were shown as yellow dotted lines. (A)

Relative position of compound 23e (shown in brown) in the active pocket of AChE. (B) Relative position of 27g (shown in brown) in the active

pocket of AChE. (C) Interactions between 23e (shown in brown) and the amino acid residue of AChE. (D) Interactions between 27g (shown in

brown) and the amino acid residue of AChE. (E) 2D binding mode of compound 23e with AChE; the π−π stacking contact was shown in purple.

(F) 2D binding mode of compound 27g with AChE; the π−π stacking contacts were shown in purple, the hydrogen bond was shown in green, and

the halogen bond was shown in cyan.

typical AChEI donepezil (1) and GSK-3β inhibitor AR-

A014418 were used as corresponding positive controls in the

second round of biological evaluation. In particular, only the

IC₅₀values of compounds that exhibited potent inhibition at

1.0 μM in primary screening were measured for the GSK-3β

target.

From the perspective of AChE inhibition, the positive results

of compound series 4 and 5 (26a−26k and 27a−27k) could

possibly be ascribed to tacrine units bearing diverse

substituents. Compared to substituent R², the ﬂuoro group

of R¹showed superiority without controversy in hybrids

bearing longer alkyl tethers (26c vs 26d, 26h vs 26i, 27c vs

27d, and 27h vs 27i). Halogen groups Br- and Cl- were more

powerful for enhancing AChE inhibition, with IC₅₀values of

26a and 26b, 26f and 26g, 27a and 27b, and 27f and 27g

lower than 100 nM, constituting the ﬁrst level of inhibitor

echelon. Furthermore, 27a, 27b, 27f and 27g, bearing seven

carbon atoms in the linker, possessed more potent activities

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Figure 4. Proposed binding models of 23e (A/C) or 27g (B/D) with GSK-3β (PDB code: 3F88). For secondary bonds, hydrogen bonds were

shown as black dotted lines, and π−cation interactions were shown as yellow dotted lines. (A) Relative position of compound 23e (shown in

brown) in the active pocket of GSK-3β. (B) Relative position of 27g (shown in brown) in the active pocket of GSK-3β. (C) Interactions between

23e (shown in brown) and the amino acid residue of GSK-3β. (D) Interactions between 27g (shown in brown) and the amino acid residue of

GSK-3β. (E) 2D binding mode of compound 23e with GSK-3β; the hydrogen bond was shown in green, and the π−cation bond was shown in

orange. (F) 2D binding mode of compound 27g with GSK-3β; the hydrogen bond was shown in green, and the π−cation bond was shown in

orange.

than 26a, 26b, 26f, and 26g, respectively, of which 27b was the

most eﬀective AChE inhibitor with an IC₅₀value of 37.5 nM. It

is interesting to note that in compound series 4 and 5 (26a−

26k and 27a−27k), only the introduction of Br and Cl groups

to the tacrine unit (26a and 26b, 26f and 26g, 27a and 27b,

and 27f and 27g) could further enhance the inhibitory activity

of new compounds compared to 23d, which had the best

AChE inhibition among series 1−3 in the ﬁrst round of

biological evaluation.

From the perspective of GSK-3β inhibition, the objective

results of series 5 indicated that the pyrimidone unit bearing

ﬂuorine-substituted pyridine (X = C−F) was the pivotal

pharmacophore to obtain stronger aﬃnity with the enzyme,

and the pyridine-substituted pyrimidone moiety (X = C−H)

was relatively weak. Changing groups in the tacrine units of

hybrids had a minor inﬂuence on the inhibitory activities

against GSK-3β, while alkylamine linkers with more carbon

atoms could increase GSK-3β inhibition (27f−27k) compared

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to compounds 26f−26k (series 4). In series 5, hybrids 27f,

27g, 27i, and 27j belonged to the ﬁrst level of the inhibitor

group with IC₅₀values lower than 100 nM, of which 27j was

the most potent GSK-3β inhibitor (IC₅₀= 67.2 nM).

were consistent with the structure−activity relationships

(SARs) of hybrids.

As shown in Figure 4, both pyrimidone fragments of 23e

and 27g stretched into the interior part of the binding groove

of GSK-3β, and tacrine units adhered to the exterior section of

the active pocket (Figure 4A,B). The binding positions of

compounds with GSK-3β still had notable diﬀerences in

molecular orientation mainly because of the pyrimidone

moiety bearing a ﬂuorine-substituted pyridine group, generat-

ing optimal steric conﬁgurations for each other. Hybrid 23e

formed a hydrogen bond (2.0 Å) with the interior residue Val-

135 and a π−cation interaction (3.0 Å) with the exterior

residue Lys-183 (Figure 4C). Hybrid 27g had two hydrogen

bonds with Val-135 (2.6 Å) and Lys-85 (2.7 Å) and a π−cation

interaction (4.5 Å) with residue Arg-141 located on the

opposite side of the peripheral groove (Figure 4D). Even

though the ﬂuorine group did not participate in the formation

of secondary bonds directly, its introduction had a favorable

eﬀect on forming more hydrogen bonds with GSK-3β by

adjusting the steric conﬁguration of 27g to a more suitable

binding position. Similarly, the docking results of GSK-3β were

consistent with the SARs of hybrids.

To investigate whether the linker length of 27g (hepta-

methylene) was extended to octa- or nona-methylene, the

relevant compounds were docked with AChE and GSK-3β,

respectively. As shown in Figure S3a −f, octa- or nona-

methylene-linked hybrids formed fewer secondary bonds

with either AChE or GSK-3β than 27g, which suggested that

a linear chain of seven carbons was more suitable for the

balance of dual-target potencies.

Hence, compound 27g could ﬁt the binding pockets of

AChE and GSK-3β and exhibited good aﬃnity with the

interactions of several secondary bonds through the cooper-

ation of the tacrine unit, alkylamine linker, and pyrimidone

moiety, making it an excellent dual AChE/GSK-3β inhibitor.

The introduction of a chlorine group on the tacrine unit and a

ﬂuorine group on the pyrimidone moiety is a feasible

modiﬁcation strategy, as demonstrated by SARs and molecular

docking.

Inhibition against the Tau Protein Phosphorylation.

The ability of 27g to regulate the tau protein signaling pathway

was further investigated using SH-SY5Y neuronal cultures⁴⁵

derived from the human neuroblastoma cell line SH-SY5Y

under continuous treatment with retinoic acid (RA) (for more

details, see the Experimental Section).

The exposure of SH-SY5Y neurons to glyceraldehyde (GA),

an inhibitor of glycolysis, results in glycolytic inhibition, cell

apoptosis, and an increase in tau protein phosphorylation.⁴⁶

For GA-induced SH-SY5Y-derived neurons, the p-Tau levels

on serine-199 (S199) and serine-396 (S396) sites, the primary

phosphorylation sites of tau protein catalyzed by GSK-3β,⁴⁷

were measured to evaluate the inhibitory capacity of 27g for

tau protein phosphorylation, with tideglusib (10) as a positive

control, which is a typical selective GSK-3β inhibitor (Figure

1C).⁴⁸

Figure 5 shows dramatically induced tau protein phosphor-

ylation in SH-SY5Y neurons compared with the blank neuronal

group. In contrast with the model group, compound 27g

presented a signiﬁcant reduction in p-Tau levels at both the

S199 and S396 sites in a dose-dependent manner. In detail,

27g exerted an obvious inhibitory eﬀect on the p-Tau level at a

relatively low concentration (10 nM) and a favorable

ameliorative eﬀect close to that of the blank group at superior

The results from the second round were successful and

valuable, conﬁrming the speculation from the ﬁrst round and

further clarifying the value of alternative building blocks, such

as the ﬂuorine group in R¹or R², the type of X in the

pyrimidone unit, and the length of the alkyl chain for the

linker. Remarkably, the seven-carbon alkyl linker made an

outstanding contribution to the hybrids to achieve a favorable

inhibitory balance toward AChE/GSK-3β, which is an

important factor in administration dosage and in vivo toxicity

aspects. Thus, hybrids 23d, 23e, 26h, 26k, 27f, 27g, 27h, and

27k possessed excellent inhibitory equilibrium and activities

and could be considered as promising dual AChE/GSK-3β

inhibitors for further study to ﬁnd new potential treatments for

AD; of these, 23e (IC₅₀= 0.13 μM for AChE; IC₅₀= 0.33 μM

for GSK-3β) is the representative hybrid among series 1−3,

and 27g (IC₅₀= 51.1 nM for AChE; IC₅₀= 89.3 nM for GSK-

3β) is the optimal compound among series 1−5.

In summary, for the novel scaﬀold fused from tacrine and

pyrimidone, the introduction of halogen groups Br- and Cl- as

substituents of R¹of the tacrine unit is very favorable for AChE

inhibition, pyrimidone units bearing ﬂuorine-substituted

pyridine groups are the best pharmacophores toward the

GSK-3β target, and alkylamine linkers with a linear chain of

seven carbons are the most beneﬁcial moiety for inhibitory

balance.

Molecular Modeling. As the most promising compound

after two rounds of design, 27g (IC₅₀= 51.1 nM for AChE;

IC₅₀= 89.3 nM for GSK-3β) exhibited a notable increase in

inhibitory potency against dual targets compared with the

representative hybrid 23e (IC₅₀= 0.13 μM for AChE; IC₅₀

=

0.33 μM for GSK-3β) from the ﬁrst round of design. To gain

insights into the binding patterns with the AChE and GSK-3β

enzymes responsible for the diﬀerence in activities, molecular

docking studies were performed using Schrodinger Glide

(Figure S1). The docking results are shown in Figures 3 and 4

and Figures S2 and S3 after preparation by PyMOL.

As shown in Figure 3, both compounds 23e and 27g could

occupy the catalytic anionic site (CAS) and the peripheral

anionic site (PAS) of AChE, of which the tacrine units were

located in the interior of the AChE gorge, and pyrimidone

fragments were located in the exterior of the AChE gorge,

forming the foundation of AChE inhibition (Figure 3A,B). The

speciﬁc orientation of 23e and 27g in the binding pocket was

evident, resulting in diﬀerent binding patterns and correspond-

ing inhibitory potency (Figure 3C,D). There were only two

π−π stacking contacts between 23e and Trp-86, while there

were ﬁve secondary bonds between 27g and the binding gorge

mainly due to the introduction of Cl- groups on the tacrine

unit. Compared with 23e, the hydrogen bond (3.5 Å) between

the chlorine of 27g and oxygen of Tyr-72 propels the reversal

of the tacrine unit, losing one π−π stacking contact with Trp-

86, inducing two more π−π stacking contacts with Trp-286

and one more hydrogen bond with Ser-293 (2.2 Å) for the

pyrimidone unit through the relay of the alkyl linker. Halogen

bonds frequently occur in the presence of chlorine or bromine,

which are large enough to contain σ-holes,⁴⁴meaning that it is

diﬃcult for tacrine units bearing ﬂuorine groups to form

halogen bonds with Tyr-72. Thus, the docking results of AChE

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Figure 6. Cell viability of diﬀerentiated SH-SY5Y neurons following

the treatment of GA, compound 27g, or tideglusib at diﬀerent

concentrations. Ordinary one-way ANOVA followed by Tukey’s post-

hoc test was used to compare diﬀerences between diﬀerent groups.

Data are presented as the mean SEM (at least n = 9 independent

experiments were performed; *p < 0.05, ***p < 0.001, ****p <

0.0001).

detrimental eﬀect of GA by inhibiting GSK-3β and improving

the tau protein signaling pathway.

Figure 5. Quantiﬁcation of phosphorylation ratio of p-Tau/total tau

of GA-treated diﬀerentiated SH-SY5Y neurons after 27g or tideglusib

treatment at diﬀerent concentrations. Kruskal−Wallis test followed by

Dunn’s post-hoc test was used to compare the diﬀerences between

diﬀerent groups. Data are expressed as mean SEM (at least n = 3

independent experiments were performed; *p < 0.05, **p < 0.01,

***p < 0.001, ****p < 0.0001. (A) Phosphorylation results of p-Tau/

total tau on the S199 site of tau protein. (B) Phosphorylation results

of p-Tau/total tau on the S396 site of tau protein.

Alleviation against GA-Induced Morphological Dam-

age in Diﬀerentiated SH-SY5Y Cells. Cytoskeletal

architecture plays a crucial role in maintaining neural

morphology and synaptic plasticity,⁵¹forming a correct

network between multiple neurons. During the progression

of AD, this architecture is jeopardized due to the formation of

NFTs, which leads to neuronal destruction and apoptosis.

Incubation of SH-SY5Y cells with RA results in typical neural

networking, with a signiﬁcant increase in axon length

compared to SH-SY5Y cells.⁴⁵Treatment with 0.7 mM GA

results in a dramatic increase in tau protein phosphorylation

and, in turn, results in neurite dystrophy and loss of network

between SH-SY5Y-derived neurons.

Next, morphology studies were conducted to determine

whether compound 27g can maintain the axon length of

neurons in the presence of GA. As shown in Figure 7A,

compound 27g (0.5 and 5 μM) signiﬁcantly prevented neurite

shortening in a dose-dependent manner and exhibited more

potent improvement eﬀects than tideglusib at the same

concentrations. Confocal images of the corresponding neuro-

nal groups are displayed in Figure 7B to show the destruction

of GA (Figure 7B3) and the reversible ability of 27g (Figure

7B4/5) and tideglusib (Figure 7B6/7) in a more visual way.

These similar results with 27g and tideglusib suggested that the

adverse eﬀect of axon length induced by GA was implicated in

the disorder of the tau protein signaling pathway, indicating

once again that compound 27g could exert neuroprotection

against SH-SY5Y-derived neurons as a GSK-3β inhibitor.

Kinase Selectivity Proﬁling of 27g. To further

investigate kinase selectivity, a panel of 205 kinase selectivity

proﬁles of compound 27g was measured at a concentration of

1000 nM. As shown in Figure 8, the results indicated that

compound 27g displayed weak inhibitory activities against

almost all of the tested kinases except GSK-3α and GSK-3β.

This result suggested that compound 27g was a pan-GSK-3

inhibitor and possessed good kinase selectivity proﬁles.

concentrations (0.5 and 5 μM). The results of SH-SY5Y-

derived neurons treated with tideglusib conﬁrmed again that

the key enzyme GSK-3β was responsible for tau protein

phosphorylation in the GA-induced neuronal model. There-

fore, compound 27g with potent GSK-3β inhibition was

proven to have the ability to regulate the tau protein pathway

in SH-SY5Y-derived neurons.

Alleviation against GA-Induced Cytotoxicity in Diﬀer-

entiated SH-SY5Y Cells. Neuronal damage caused by NFTs

and SP is one of the major issues in AD progression,

aggravating the disease condition and threatening the lives of

patients.⁴⁹As neurons cannot actively replicate,⁵⁰preventing

neuronal loss is a crucial goal for any potential therapy. To

assess whether compound 27g was able to protect neuronal

cells from cytotoxic GA, cell viability experiments were

conducted using SH-SY5Y-derived neurons.

As depicted in Figure 6, incubation of neurons with 0.7 mM

GA caused serious toxicity.⁴⁶In contrast with the model group,

the presence of compound 27g or the positive control

tideglusib at diﬀerent concentrations (0.5 and 5 μM) resulted

in a signiﬁcant increase in cell viability, indicating that these

two compounds had neuroprotection toward SH-SY5Y

neurons. The holistic results suggested the central role of tau

protein phosphorylation, which played a pivotal function in

GA-induced cytotoxicity, and 27g could reverse the

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Figure 7. Morphology studies of SH-SY5Y-derived neurons. Ordinary one-way ANOVA followed by Tukey’s post-hoc test was used to compare

diﬀerences between diﬀerent groups. Data are presented as mean SEM (at least n = 3 independent experiments were performed; *p < 0.05, **p <

0.01, ***p < 0.001, ****p < 0.0001). (A) Axon length quantiﬁcation of 0.7 mM GA-treated diﬀerentiated SH-SY5Y neurons after 27g or tideglusib

treatment at diﬀerent concentrations. (B) Confocal images of 0.7 mM GA-treated diﬀerentiated SH-SY5Y neurons after 27g or tideglusib treatment

at diﬀerent concentrations.

Pharmacokinetic Property Investigation. The pharma-

cokinetic proﬁles of a promising compound can provide

important information with regard to its absorption,

distribution, metabolism, and excretion (ADME) properties,

which become crucial references to predict its manifestation in

the human body.⁵²Therefore, an in vivo pharmacokinetic

investigation of compound 27g in ICR mice was conducted to

validate its ADME characteristics, and the results are shown in

Table 3 and Tables S4 −S6.

(0.033 h) and higher C_max(2807 ng/mL), similar values for the

area under curve (AUC) and apparent volume of distribution

(V_d), and a longer half-life (t_1/2= 3.65 h), longer mean

residence time (MRT = 3.05 h), and smaller clearance rate

(CL = 3.08 L/h/kg) in terms of excretion when compared

with i.p. administration. Notably, the data derived from i.p.

injection of 27g were still valid and positive under

comprehensive estimation. Moreover, the pharmacokinetic

data of i.p. administration were acceptable for moving

compound 27g to in vivo study as a tool to validate the

potential of the series with a half-life of 1.15 h and

bioavailability (F) of 35.3%.

Obviously, the intravenous (i.v.) administration (5 mg/kg)

showed better performance for the use of 27g than i.p.

injection (15 mg/kg). The i.v. injection had a shorter t_max

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Figure 8. TREEspot maps for 27g against 205 kinases (ICE Bioscience Inc.). A concentration of 1000 nM was used for the measurements, and the

aﬃnity was deﬁned as a percent of the DMSO control (% control).

a

Table 3. Pharmacokinetic Parameters of 27g after Its Administration

administration

t_1/2(h)

t_max(h)

C_max(ng/mL)

AUC_last(h ng/mL)

V_d(L/kg)

CL_{F_obs}(L/h/kg)

MRT_last(h)

F (%)

i.v. 5 mg/kg

i.p. 15 mg/kg

3.65 1.05

1.15 1.18

0.033 0.000

0.139 0.096

2807 1113

1868 392

1564 93

1658 222

16.42 5.94

14.14 13.14

3.08 0.24

9.12 1.30

3.05 2.09

0.78 0.33

35.3

a

Values are expressed as the mean SD (n = 3).

The eligible performance of compound 27g demonstrated

the brain after their administration.⁵³Thus, an in vivo assay of

27g in ICR mice was performed to evaluate its permeability to

the BBB and its qualiﬁcation as a truly available AChE/GSK-3

inhibitor.

From the results presented in Table 4 and Table S7, hybrid

27g was clearly detected in both the plasma and brain after

intraperitoneal (i.p.) administration at the dosage of 15 mg/kg.

Along with the change in time (0.5−4 h), the concentration of

27g in plasma decreased in a relatively rapid way (866.5 to 3.5

ng/mL), while the downtrend of 27g in the brain was torpid,

its potential as a lead compound and suggested that further

modiﬁcation based on its ADME properties is needed to

acquire more extensive application approaches and more

potent adjustments toward the disease network of AD.

Blood−Brain Barrier Permeability Assay. AD pathology

is fundamentally implicated in disordered enzymes and morbid

regions inside the brain, and drugs aiming to regulate AD-

related targets need the ability to penetrate the blood−brain

barrier (BBB) and form gradual enrichment at the interior of

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buﬀered saline (PBS) with 10% dimethyl sulfoxide (DMSO)

and 20% (2-hydroxypropyl)-β-cyclodextrin, and the dosages of

agents for other groups were uniﬁed as 15 mg/kg in the blank

solvent. The entire experimental period was 25 days, and the

blank control group was treated with blank solvent each day.

Other groups were treated with scopolamine (i.p.) for the

initial 10 consecutive days.⁵⁶Tacrine, 23e, and 27g for the

respective group were intraperitoneally injected into the ICR

mice 30 min after the administration of scopolamine in the

following 15 consecutive days. Days 21−24 were the training

period for mice in all groups to incrementally learn the location

of the ﬁxed and hidden platform in the fourth quadrant of the

maze, and a spatial probe trial was conducted and recorded on

days 23−25. The mean values presented in Figure 9 and Table

S8 show the latencies and distances for mice to reach the ﬁrst

escape entry on days 23−24. An additional test item on day 25

was used to assess the memory retention of mice by removing

the hidden platform from the maze, and entry times for mice

arriving at the virtual platform were recorded, as shown in

Figure 9 and Table S8.

Excellent behavior performance for mice required faster

escape latency (Figure 9A), shorter searching distance (Figure

9B), and more arrival times (Figure 9C). The cognitive

competence of the blank control group exhibited the optimal

performance (9.40 s, 2.07 m, n = 6.79, respectively), and the

model group treated with scopolamine had relatively poor

results (23.81 s, 5.00 m, n = 3.63, respectively), indicating that

the scopolamine-induced cognition-impaired animal model

worked well. As the positive control, tacrine eﬃciently

improved the overall memory and cognition of mice (10.76

s, 2.42 m, n = 6.56, respectively). Moreover, sample groups

treated with 23e and 27g displayed remarkable behavior

enhancement based on the data of the model group, and 27g

(11.86 s, 2.46 m, n = 6.44, respectively) showed slightly more

Table 4. Concentration of 27g in Plasma and Brain after Its

i.p. Administration (15 mg/kg)

a

concentration of 27g

T (h)

plasma (ng/mL)

brain (ng/g)

ratio of brain/plasma

0.5

1

2

866.5 205.8

76.75 14.41

26.75 12.74

3.469 1.433

23.63 4.78

11.13 1.18

12.23 2.06

5.994 1.043

0.028 0.007

0.148 0.025

0.522 0.189

1.924 0.759

4

a

Values are expressed as the mean SD (n = 4).

and there was even an increase in the period of 1−2 h (11.2 to

12.2 ng/g). The value of blood/plasma steadily increased from

the initial 0.028 to 1.924, and the amount of 27g in the brain

surpassed its quantities in plasma after 4 h of metabolism. Data

from this assay demonstrated that 27g could partially penetrate

the brain and accumulate in this tissue, being removed slower

than that from the plasma. Therefore, further modiﬁcation is

still needed to acquire molecules with better BBB permeability.

Behavioral Studies in the AD-Related Mouse Model.

The paramount goal of outstanding anti-AD drugs, such as

donepezil (1) and memantine (6), is to strongly improve the

cognition and memory conditions of patients. The most

common approach to investigate the ability of promising anti-

AD agents to improve the behavior of mice is the classical

Morris water maze (MWM) test in preclinical research.⁵⁴

Thus, scopolamine-induced cognition-impaired ICR mice were

adopted to evaluate the overall amelioration eﬀects of

compounds 23e and 27g, two representative hybrids in our

work, using a modiﬁed MWM test.⁵⁵

Forty female ICR adult mice (8−10 weeks) were randomly

distributed into ﬁve groups (n = 8 for each group): blank

control, model (scopolamine), tacrine, 23e, and 27g. The

blank solvent for the blank control group was phosphate-

Figure 9. Eﬀects of i.p. administration of 27g (15 mg/kg), 23e (15 mg/kg), and tacrine (4, 15 mg/kg) on scopolamine-induced cognitive

impairment in ICR mice evaluated by the Morris water maze test. Data are presented as the mean SEM (n = 8; ^#p ≤ 0.05, ^##p ≤ 0.01 vs blank

control group; *p ≤ 0.05, **p ≤ 0.01 vs scopolamine group). (A) Latency for mice to reach the ﬁrst escape entry. (B) Distance for mice to reach

the ﬁrst escape entry. (C) Number of entering times for mice to the virtual platform (the original platform location). (D) Representative tracks of

mice in MWM during the spatial probe trial period.

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potent amelioration of cognition and memory than 23e (14.08

s, 2.50 m, n = 6.10, respectively), indicating that the in vitro

advantage of 27g in AChE/GSK-3β inhibition could reﬂect an

authentic in vivo AD-related animal model. Given the same

dosage of agents (15 mg/kg), the smaller molecular weight of

tacrine (M.W. = 237.72) led to its higher concentration, while

mice treated with compound 27g (M.W. = 549.09) still

exhibited similar performance on the MWM test, approaching

the therapeutic eﬀects of tacrine. Furthermore, the i.p.

injection of 23e and 27g caused no abnormal events (emesis

or diarrhea) or swimming motility impairment in mice

throughout the entire experimental period, which demon-

strated their safety for long-term administration at the dosage

of 15 mg/kg for each day.

pharmacokinetic parameters of 27g were conﬁrmed by in

vivo assays in mice with the auxiliary detection of UPLC-MS/

MS. In the preliminary hepatotoxicity study, oral admin-

istration of compound 27g to mice showed only weak

hepatotoxicity, which would not disrupt the physiological

function of the liver (Table S9). In particular, the ability of 27g

to improve cholinergic deﬁcits and disorder of the tau protein

pathway was investigated. Scopolamine-induced ICR mice

treated with 27g (15 mg/kg/day) showed signiﬁcant

amelioration of memory performance and spatial behavior in

the MWM test, indicating that the cholinergic signaling

pathway could be enhanced by 27g as an AChE inhibitor.

In conclusion, this work validates the multitarget anti-AD

strategy by fusing tacrine and pyrimidone for the ﬁrst time,

identifying a series of novel hybrids with excellent dual AChE/

GSK-3 inhibition. All of the pharmacological results highlight

that the most promising compound, 27g, possesses multiple

anti-AD proﬁles with benign safety and drug-like properties.

Therefore, 27g can be considered a potential prototype to be

further developed for the better treatment of AD.

Taken together, the overall behavioral performance in the

MWM assay observed in the 23e and 27g groups supports

their capacities to signiﬁcantly ameliorate the impairment

caused by scopolamine, possessing an analogous eﬀect to

tacrine at the same administered dosage.

CONCLUSIONS

■

In summary, it is not easy to ﬁnd the potentially curable

treatment of AD, whereas regulating cholinergic and tau

protein signaling pathways by a single molecule in AD-related

studies will play an essential role in achieving a better

treatment eﬀect of AD. Based on the multitarget strategy, a

series of dual AChE/GSK-3 inhibitors were innovatively

designed and easily synthesized by using tacrine (4) and

pyrimidone structures as parent building blocks. Optimization

has resulted in 50 tacrine−pyrimidone hybrids, which

exhibited integrated and detailed SARs with IC₅₀levels

enhanced from the micromole to nanomole level against

AChE and GSK-3β in vitro. Furthermore, several promising

compounds were discovered to possess excellent character-

istics, of which compound 27b displayed the best AChE

inhibition, compound 27j showed the most potent GSK-3β

inhibition, and compound 27g simultaneously possessed

favorable inhibitory potency and inhibitory equilibrium against

two targets, becoming the optimal hybrid among the series of

new compounds. Furthermore, molecular docking studies

rationally predicted the binding patterns between hybrids (23e

and 27g) and enzymes and clearly demonstrated the binding

diﬀerence inﬂuenced by key substituted groups, which is in

agreement with the actual assay results of compounds against

dual targets.

EXPERIMENTAL SECTION

■

Chemistry. General Methods. Unless otherwise stated, all

reagents were purchased from Energy Chemical (China) or

Bidepharm (China). AR-A014418 was purchased from Selleck

(catalog no.S7435). All commercially available reagents (reagent

grade) were used without further puriﬁcation. THF was dried through

routine protocols using metal sodium. Flash column chromatography

was performed on 200−300 mesh silica gel (Qingdao Haiyang

Chemical, China). Reactions were monitored by thin-layer

chromatography (TLC) on 0.25 mm silica gel GF₂₅₄plates and

visualized under UV light (λ = 254 nm). The ¹H NMR and ¹³C NMR

spectra were recorded by a Bruker AV-300 spectrometer (Bruker

Company, Germany) in the indicated solvents (CDCl₃, CD₃OD, or

DMSO-d₆) and referenced to the internal standard tetramethylsilane.

Chemical shifts were reported in ppm (δ) values and the coupling

constants (J) in Hz. Proton coupling patterns were described as

singlet (s), broad singlet (brs), doublet (d), doublet of doublets (dd),

triplet (t), quartet (q), and multiplet (m). The high-resolution mass

spectrometry (HRMS) was measured on a Finnigan MAT 95

spectrometer (Finnigan, Germany). The analytical samples of target

compounds possessed satisfactory purity (>95%), according to their

respective NMR spectra and high-performance liquid chromatography

(HPLC) and HRMS measurements (for more details, see the

Supporting Information).

For key intermediates shown in Schemes 1 and 2, compounds 14a,

14b, and 14c were successfully synthesized according to Uehara et

al.’s work (compound 28)³⁷and Fukunaga et al.’s work (compound

6³⁸and compound 26⁴⁰), respectively. Also, analogues 18a−18m,

19a−19e, 24a−24e, and 25a−25e, which are diﬀerent tacrine

derivatives bearing respective alkylamine, were smoothly synthesized

according to Sun et al.’s work using the similar method.³⁰The

synthesis of representative intermediates is described in the

Supporting Information.

General Procedures for the Synthesis of Target Compounds

21a−21m, 22a−22j, 23a−23e, 26a−26k, and 27a−27k. To a

solution of key intermediate 14 (0.45 mmol, 1 equiv) in THF (8 mL)

was added a speciﬁc tacrine derivative (0.45 mmol, 1 equiv) from 18,

19, 24, or 25. Subsequently, base K₂CO₃(1.35 mmol, 3 equiv) was

added to the solution, and the reaction mixture was stirred overnight

at 66 °C. Then, the mixture was extracted with EtOAc (3 × 20 mL)

after the addition of water, and the combined organic layers were

washed with saturated brine and dried over anhydrous Na₂SO₄.

Removal of the solvent in vacuo produced a residue, which was

puriﬁed by column chromatography, using a gradient eluent from

petroleum ether/EtOAc (1:3) to dichloromethane/methanol (10:1),

to aﬀord the corresponding target compound in good yield.

The GA-induced phosphorylation of tau protein on S199

and S396 sites could be dose-dependently suppressed with

treatment by 27g in SY-SY5Y-derived neurons, indicating the

beneﬁcial regulation of 27g on the tau protein signaling

pathway as a GSK-3β inhibitor. Thus, evidence has shown that

the inhibitory activities of compound 27g at the enzyme level

could present macroscopic improvement at the nervous tissue

and animal levels, suggesting that 27g has the potential to

synergistically adjust the two pathways, which is an out-

standing therapeutic option for the prospective treatment of

AD. For adverse eﬀects induced by GA, the evident

neuroprotective eﬀect of 27g on SH-SY5Y-derived neurons

was veriﬁed through cell viability assays and morphology

surveys. Kinase selectivity proﬁling study showed that

compound 27g is a pan-GSK-3 inhibitor and possessed good

kinase selectivity proﬁles.

Furthermore, the capacity of 27g to successfully permeate

the BBB and locate in the brain and the eligible

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3-Methyl-6-(pyridin-4-yl)-2-((2-((1,2,3,4-tetrahydroacridin-9-yl)-

2-((2-((6-Bromo-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)ethyl)amino)pyrimidin-4(3H)-one (21a). White solid, 62%

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (21g). White

1

yield; R_f= 0.4 (10% methanol in dichloromethane). H NMR (300

solid, 61% yield; R_f= 0.5 (10% methanol in dichloromethane). H

MHz, CD₃OD) δ 8.54 (d, J = 5.7 Hz, 2H), 8.38 (d, J = 8.7 Hz, 1H),

7.75 (dd, J = 13.0, 6.5 Hz, 3H), 7.58 (d, J = 8.5 Hz, 1H), 7.43 (t, J =

7.5 Hz, 1H), 6.29 (s, 1H), 4.33 (s, 2H), 4.06 (s, 2H), 3.31 (s, 3H),

2.78 (d, J = 5.9 Hz, 2H), 2.44 (s, 2H), 1.84−1.59 (m, 4H). ¹³C NMR

(75 MHz, CD₃OD) δ 167.0, 161.3, 160.8, 159.0, 154.4, 154.0, 154.0,

148.6, 142.1, 136.6, 129.3, 129.0, 124.8, 124.8, 123.4, 120.2, 115.9,

102.3, 45.5, 32.2, 31.4, 31.4, 28.4, 25.7, 24.5. HRMS (ESI) m/z: calcd

for C₂₅H₂₇N₆O [M + H]⁺, 427.2241; found, 427.2236.

NMR (400 MHz, CD₃OD) δ 8.58 (dd, J = 4.6, 1.6 Hz, 2H), 8.28 (d, J

= 9.1 Hz, 1H), 7.76 (dd, J = 4.6, 1.6 Hz, 2H), 7.72 (d, J = 1.9 Hz,

1H), 7.48 (dd, J = 9.2, 1.9 Hz, 1H), 6.35 (s, 1H), 4.36−4.27 (m, 2H),

4.12−4.00 (m, 2H), 3.33 (s, 3H), 2.82 (t, J = 6.2 Hz, 2H), 2.44 (s,

2H), 1.76 (m, 4H). ¹³C NMR (75 MHz, CD₃OD): Given the

property of the 300 MHz spectrometer and the low solubility of the

compound in deuterated solvents, the ¹³C NMR spectrum of 21g was

not successfully acquired. However, the ¹H NMR spectrum and

HRMS data of 21g aﬀorded here could be the valid evidence for the

structure of 21g. HRMS (ESI) m/z: calcd for C₂₅H₂₆BrN₆O [M +

H]⁺, 505.1346; found, 505.1349.

2-((2-((6-Methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (21b). White

1

solid, 53% yield; R_f= 0.5 (9% methanol in dichloromethane). H

NMR (400 MHz, CD₃OD) δ 8.56 (dd, J = 4.6, 1.6 Hz, 2H), 8.29 (d, J

= 9.5 Hz, 1H), 7.77 (dd, J = 4.6, 1.6 Hz, 2H), 7.01 (dd, J = 9.5, 2.6

Hz, 1H), 6.88 (d, J = 2.6 Hz, 1H), 6.35 (s, 1H), 4.36−4.19 (m, 2H),

4.10−4.02 (m, 2H), 3.96 (s, 3H), 3.32 (s, 3H), 2.76 (t, J = 6.1 Hz,

2H), 2.37 (t, J = 5.8 Hz, 2H), 1.81−1.61 (m, 4H). ¹³C NMR (75

MHz, CD₃OD) δ 167.8, 166.7, 161.8, 160.2, 159.2, 153.5, 153.5,

152.9, 149.2, 144.8, 130.7, 125.1, 125.1, 120.8, 114.5, 114.1, 103.1,

102.5, 59.6, 45.0, 31.8, 31.6, 31.6, 27.6, 25.4, 24.4. HRMS (ESI) m/z:

calcd for C₂₆H₂₉N₆O₂[M + H]⁺, 457.2347; found, 457.2342.

2-((2-((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (21c). White

3-Methyl-2-((2-((6-methyl-1,2,3,4-tetrahydroacridin-9-yl)amino)-

ethyl)amino)-6-(pyridin-4-yl)pyrimidin-4(3H)-one (21h). White

1

solid, 65% yield; R_f= 0.5 (10% methanol in dichloromethane). H

NMR (400 MHz, CD₃OD) δ 8.56 (dd, J = 4.7, 1.4 Hz, 2H), 8.27 (t, J

= 8.9 Hz, 1H), 7.76 (dd, J = 4.7, 1.5 Hz, 2H), 7.34 (s, 1H), 7.25 (d, J

= 8.9 Hz, 1H), 6.34 (s, 1H), 4.37−4.28 (m, 2H), 4.10−4.01 (m, 2H),

3.30 (s, 3H), 2.79 (t, J = 6.1 Hz, 2H), 2.51 (s, 3H), 2.43 (s, 2H),

1.83−1.65 (m, 4H). ¹³C NMR (75 MHz, CD₃OD) δ 167.8, 161.8,

160.7, 159.0, 153.4, 153.3, 153.3, 149.1, 148.1, 142.4, 131.1, 128.7,

125.1, 125.1, 121.9, 117.9, 115.2, 102.6, 45.2, 31.8, 31.3, 31.3, 27.8,

25.4, 24.8, 24.3. HRMS (ESI) m/z: calcd for C₂₆H₂₉N₆O [M + H]⁺,

441.2397; found, 441.2396.

1

solid, 58% yield; R_f= 0.5 (10% methanol in dichloromethane). H

NMR (400 MHz, CD₃OD) δ 8.57 (dd, J = 4.6, 1.6 Hz, 2H), 8.36 (d, J

= 9.2 Hz, 1H), 7.76 (dd, J = 4.6, 1.6 Hz, 2H), 7.56 (t, J = 4.3 Hz, 1H),

7.36 (dd, J = 9.2, 2.1 Hz, 1H), 6.35 (s, 1H), 4.36−4.26 (m, 2H),

4.10−4.02 (m, 2H), 3.66 (d, J = 3.2 Hz, 1H), 3.33 (s, 3H), 2.82 (t, J =

6.2 Hz, 2H), 2.44 (s, 2H), 1.84−1.65 (m, 4H). ¹³C NMR (75 MHz,

CD₃OD) δ 167.8, 161.8, 160.1, 159.2, 154.8, 153.5, 153.5, 149.2,

143.2, 142.7, 130.7, 129.7, 125.1, 125.1, 122.4, 118.2, 116.1, 103.3,

45.0, 32.2, 31.7, 31.7, 28.0, 25.3, 24.3. HRMS (ESI) m/z: calcd for

C₂₅H₂₆ClN₆O [M + H]⁺, 461.1852; found, 461.1850.

3-Methyl-2-((2-((7-methyl-1,2,3,4-tetrahydroacridin-9-yl)amino)-

ethyl)amino)-6-(pyridin-4-yl)pyrimidin-4(3H)-one (21i). White solid,

1

63% yield; R_f= 0.5 (10% methanol in dichloromethane). H NMR

(400 MHz, CD₃OD) δ 8.58 (dd, J = 4.6, 1.6 Hz, 2H), 8.04 (s, 1H),

7.76 (dd, J = 4.6, 1.6 Hz, 2H), 7.58 (dd, J = 8.6, 1.3 Hz, 1H), 7.47 (d,

J = 8.6 Hz, 1H), 6.26 (s, 1H), 4.37−4.27 (m, 2H), 4.08−3.97 (m,

2H), 3.28 (s, 3H), 2.81 (t, J = 6.2 Hz, 2H), 2.58 (s, 2H), 2.37 (s, 3H),

1.88−1.65 (m, 4H). ¹³C NMR (75 MHz, CD₃OD) δ 167.7, 161.5,

160.4, 158.8, 153.3, 153.3, 149.0, 140.2, 139.6, 138.5, 127.2, 124.9,

124.9, 122.7, 120.2, 115.6, 102.7, 97.2, 45.2, 31.8, 31.5, 31.5, 28.2,

25.4, 25.0, 24.3. HRMS (ESI) m/z: calcd for C₂₆H₂₉N₆O [M + H]⁺,

441.2398; found, 441.2396.

2-((2-((7-Methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (21d). White

1

solid, 56% yield; R_f= 0.5 (10% methanol in dichloromethane). H

NMR (400 MHz, CD₃OD) δ 8.57 (dd, J = 4.6, 1.6 Hz, 2H), 7.71 (dd,

J = 4.6, 1.6 Hz, 2H), 7.48 (dd, J = 7.4, 5.9 Hz, 2H), 7.37 (dd, J = 9.2,

2.5 Hz, 1H), 6.21 (s, 1H), 4.33−4.23 (m, 2H), 4.05−3.95 (m, 2H),

3.80 (s, 3H), 3.27 (s, 3H), 2.82 (t, J = 6.2 Hz, 2H), 2.66 (t, J = 5.8 Hz,

2H), 1.89−1.71 (m, 4H). ¹³C NMR (75 MHz, CD₃OD) δ 167.8,

161.5, 161.1, 160.0, 158.7, 153.3, 153.3, 152.9, 149.1, 136.9, 127.9,

125.0, 125.0, 124.5, 121.9, 115.9, 107.5, 102.5, 59.8, 45.6, 32.0, 31.5,

31.5, 28.8, 25.7, 24.4. HRMS (ESI) m/z: calcd for C₂₆H₂₉N₆O₂[M +

H]⁺, 457.2347; found, 457.2343.

2-((2-((7-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (21j). White

1

solid, 60% yield; R_f= 0.5 (10% methanol in dichloromethane). H

NMR (400 MHz, CD₃OD) δ 8.59 (dd, J = 4.6, 1.6 Hz, 2H), 8.28 (d, J

= 1.8 Hz, 1H), 7.79 (dd, J = 4.6, 1.6 Hz, 2H), 7.70 (dd, J = 9.0, 2.0

Hz, 1H), 7.56 (d, J = 9.0 Hz, 1H), 6.31 (s, 1H), 4.35−4.22 (m, 2H),

4.09−3.97 (m, 2H), 3.30 (s, 3H), 2.85 (t, J = 6.3 Hz, 2H), 2.60 (s,

2H), 1.80 (m, 4H). ¹³C NMR (75 MHz, CD₃OD): Given the

property of the 300 MHz spectrometer and the low solubility of the

compound in deuterated solvents, the ¹³C NMR spectrum of 21j was

not successfully acquired. However, the ¹H NMR spectrum and

HRMS data of 21j aﬀorded here could be the valid evidence for the

structure of 21j. HRMS (ESI) m/z: calcd for C₂₅H₂₆ClN₆O [M +

H]⁺, 461.1852; found, 461.1855.

2-((2-((6-Fluoro-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (21e). White

1

solid, 59% yield; R_f= 0.4 (9% methanol in dichloromethane). H

NMR (400 MHz, CD₃OD) δ 8.58 (dd, J = 4.6, 1.6 Hz, 2H), 8.49 (dd,

J = 8.8, 5.4 Hz, 1H), 7.79 (dd, J = 4.6, 1.6 Hz, 2H), 7.24 (dd, J = 14.4,

5.6 Hz, 2H), 6.37 (s, 1H), 4.38−4.27 (m, 2H), 4.16−4.03 (m, 2H),

3.35 (s, 3H), 2.80 (t, J = 6.2 Hz, 2H), 2.41 (s, 2H), 1.73 (m, 3H). ¹³C

NMR (75 MHz, CD₃OD) δ 170.0, 167.8, 166.6, 161.9, 160.6, 159.2,

154.4, 153.5, 153.5, 149.3, 132.4, 125.1, 125.1, 118.7, 116.7, 115.5,

108.0, 103.1, 45.0, 31.9, 31.6, 31.6, 27.9, 25.3, 24.3. HRMS (ESI) m/

z: calcd for C₂₅H₂₆FN₆O [M + H]⁺, 445.2147; found, 445.2146.

2-((2-((7-Fluoro-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (21f). White

2-((2-((7-Bromo-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (21k). White

1

solid, 59% yield; R_f= 0.5 (10% methanol in dichloromethane). H

NMR (400 MHz, CD₃OD) δ 8.59 (dd, J = 4.6, 1.6 Hz, 2H), 8.43 (d, J

= 1.7 Hz, 1H), 7.80 (ddd, J = 6.8, 6.2, 1.8 Hz, 3H), 7.48 (d, J = 9.0

Hz, 1H), 6.30 (s, 1H), 4.34−4.20 (m, 2H), 4.09−3.98 (m, 2H), 3.31

(s, 3H), 2.84 (t, J = 6.3 Hz, 2H), 2.61 (s, 2H), 1.88−1.69 (m, 4H).

¹³C NMR (75 MHz, CD₃OD): Given the property of the 300 MHz

spectrometer and the low solubility of the compound in deuterated

solvents, the ¹³C NMR spectrum of 21k was not successfully acquired.

1

solid, 60% yield; R_f= 0.4 (9% methanol in dichloromethane). H

NMR (400 MHz, CD₃OD) δ 8.58 (dd, J = 4.6, 1.6 Hz, 2H), 8.04 (dd,

J = 10.6, 2.3 Hz, 1H), 7.77 (dd, J = 4.6, 1.6 Hz, 2H), 7.67−7.56 (m,

2H), 6.31 (s, 1H), 4.34−4.22 (m, 2H), 4.11−3.99 (m, 2H), 3.30 (s,

3H), 2.84 (t, J = 6.3 Hz, 2H), 2.55 (t, J = 5.7 Hz, 2H), 1.85−1.67 (m,

4H). ¹³C NMR (75 MHz, CD₃OD) δ 167.8, 164.7, 161.7, 161.4,

158.9, 154.7, 153.3, 153.3, 149.1, 139.1, 126.4, 125.9, 125.8, 125.0,

125.0, 121.0, 116.2, 102.6, 45.3, 32.1, 31.2, 31.2, 28.4, 25.4, 24.3.

HRMS (ESI) m/z: calcd for C₂₅H₂₆FN₆O [M + H]⁺, 445.2147;

found, 445.2141.

1

However, the H NMR spectrum and HRMS data of 21k aﬀorded

here could be the valid evidence for the structure of 21 k. HRMS

(ESI) m/z: calcd for C₂₅H₂₆BrN₆O [M + H]⁺, 505.1346; found,

505.1346.

3-Methyl-6-(pyridin-4-yl)-2-((2-((7-(triﬂuoromethyl)-1,2,3,4-tetra-

hydroacridin-9-yl)amino)ethyl)amino)pyrimidin-4(3H)-one (21l).

Brown solid, 64% yield; R_f= 0.4 (10% methanol in dichloromethane).

¹H NMR (300 MHz, CD₃OD) δ 8.52 (d, J = 18.9 Hz, 3H), 7.88 (d, J

7498

https://doi.org/10.1021/acs.jmedchem.1c00160

J. Med. Chem. 2021, 64, 7483−7506

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

= 8.9 Hz, 1H), 7.80−7.68 (m, 3H), 6.30 (s, 1H), 4.25 (s, 2H), 4.01

(s, 2H), 3.33 (s, 3H), 2.87 (d, J = 5.7 Hz, 2H), 2.60 (s, 2H), 1.78 (m,

4H). ¹³C NMR (75 MHz, CD₃OD) δ 167.9, 161.6, 160.0, 158.8,

157.9, 153.3, 153.3, 149.0, 145.6, 131.2, 130.4, 130.0, 129.8, 126.4,

124.9, 124.9, 120.1, 118.0, 102.5, 45.5, 33.3, 31.1, 31.1, 28.5, 25.5,

24.6. HRMS (ESI) m/z: calcd for C₂₆H₂₆F₃N₆O [M + H]⁺, 495.2115;

found, 495.2114.

3-Methyl-6-(pyridin-4-yl)-2-((2-((6-(triﬂuoromethyl)-1,2,3,4-tetra-

hydroacridin-9-yl)amino)ethyl)amino)pyrimidin-4(3H)-one (21m).

Brown solid, 55% yield; R_f= 0.4 (10% methanol in dichloromethane).

¹H NMR (300 MHz, CD₃OD) δ 8.56 (d, J = 5.9 Hz, 2H), 8.23 (d, J =

8.9 Hz, 1H), 7.90 (s, 1H), 7.76 (d, J = 6.0 Hz, 2H), 7.32 (d, J = 8.7

Hz, 1H), 6.28 (s, 1H), 4.03 (s, 2H), 3.85 (s, 2H), 3.15 (s, 3H), 2.87

(d, J = 6.2 Hz, 2H), 2.65 (s, 2H), 1.77 (m, 4H). ¹³C NMR (75 MHz,

CD₃OD) δ 167.9, 162.9, 161.9, 158.7, 155.3, 153.2, 153.2, 149.2,

149.0, 133.9, 129.5, 128.4, 128.0, 125.0, 125.0, 124.7, 122.5, 120.6,

102.6, 46.0, 36.6, 30.9, 30.9, 28.9, 26.2, 25.8. HRMS (ESI) m/z: calcd

for C₂₆H₂₆F₃N₆O [M + H]⁺, 495.2115; found, 495.2116.

CD₃OD): Given the property of the 300 MHz spectrometer and the

low solubility of the compound in deuterated solvents, the ¹³C NMR

spectrum of 22e was not successfully acquired. However, the ¹H

NMR spectrum and HRMS data of 22e aﬀorded here could be the

valid evidence for the structure of 22e. HRMS (ESI) m/z: calcd for

C₂₄H₂₅ClN₇O [M + H]⁺, 462.1804; found, 462.1809.

6-(3-Fluoropyridin-4-yl)-3-methyl-2-((2-((1,2,3,4-tetrahydroacri-

din-9-yl)amino)ethyl)amino)pyrimidin-4(3H)-one (22f). White

1

solid, 65% yield; R_f= 0.4 (10% methanol in dichloromethane). H

NMR (300 MHz, CD₃OD) δ 8.56 (d, J = 3.4 Hz, 1H), 8.34 (dd, J =

9.3, 7.0 Hz, 2H), 7.82−7.70 (m, 2H), 7.59 (d, J = 7.8 Hz, 1H), 7.43

(t, J = 7.8 Hz, 1H), 6.32 (d, J = 1.2 Hz, 1H), 4.37−4.25 (m, 2H),

4.10−3.98 (m, 2H), 3.33 (s, 3H), 2.86 (t, J = 5.9 Hz, 2H), 2.51 (t, J =

5.7 Hz, 2H), 1.81 (m, 4H). ¹³C NMR (75 MHz, CD₃OD) δ 163.6,

156.8, 154.9, 153.6, 150.2, 145.5, 139.0, 138.6, 138.3, 132.8, 132.7,

125.2, 124.8, 123.6, 119.1, 116.0, 111.8, 103.6, 41.3, 28.0, 27.5, 27.5,

24.1, 21.5, 20.5. HRMS (ESI) m/z: calcd for C₂₅H₂₆FN₆O [M + H]⁺,

445.2147; found, 445.2146.

2-((2-((6-Fluoro-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)-6-(3-ﬂuoropyridin-4-yl)-3-methylpyrimidin-4(3H)-one

(22g). White solid, 70% yield; R_f= 0.4 (10% methanol in

dichloromethane). ¹H NMR (300 MHz, CD₃OD) δ 8.58 (d, J =

3.1 Hz, 1H), 8.43−8.29 (m, 2H), 7.76 (t, J = 5.9 Hz, 1H), 7.31−7.09

(m, 2H), 6.34 (s, 1H), 4.19 (d, J = 5.1 Hz, 2H), 3.99 (t, J = 5.1 Hz,

2H), 3.32 (s, 3H), 2.86 (t, J = 5.8 Hz, 2H), 2.54 (d, J = 5.8 Hz, 2H),

1.81 (m, 4H). ¹³C NMR (75 MHz, CD₃OD) δ 165.3, 163.6, 161.9,

154.9, 153.6, 153.3, 145.6, 142.5, 139.0, 138.6, 132.7, 127.3, 123.5,

114.4, 114.1, 112.6, 106.1, 103.7, 41.4, 29.6, 27.5, 27.5, 24.1, 21.8,

20.9. HRMS (ESI) m/z: calcd for C₂₅H₂₅F₂N₆O [M + H]⁺, 463.2053;

found, 463.2051.

2-((2-((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)-6-(3-ﬂuoropyridin-4-yl)-3-methylpyrimidin-4(3H)-one

(22h). White solid, 68% yield; R_f= 0.5 (10% methanol in

dichloromethane). ¹H NMR (300 MHz, CD₃OD) δ 8.58 (d, J =

3.0 Hz, 1H), 8.36 (d, J = 5.0 Hz, 1H), 8.25 (d, J = 9.2 Hz, 1H), 7.72

(t, J = 5.8 Hz, 1H), 7.55 (s, 1H), 7.29 (d, J = 9.0 Hz, 1H), 6.33 (s,

1H), 4.20 (d, J = 4.9 Hz, 2H), 3.99 (d, J = 5.3 Hz, 2H), 3.32 (s, 3H),

2.85 (d, J = 5.7 Hz, 2H), 2.53 (s, 2H), 1.81 (m, 4H). ¹³C NMR (75

MHz, CD₃OD) δ 163.6, 158.9, 155.0, 154.8, 153.7, 153.0, 145.5,

141.3, 139.0, 138.6, 137.5, 126.1, 125.2, 123.6, 120.4, 115.3, 113.1,

103.6, 41.4, 29.5, 27.5, 27.5, 24.2, 21.7, 20.9. HRMS (ESI) m/z: calcd

for C₂₅H₂₅FClN₆O [M + H]⁺, 479.1759; found, 479.1760.

1-Methyl-2-((2-((1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)-[4,4′-bipyrimidin]-6(1H)-one (22a). White solid, 65% yield;

1

R_f= 0.4 (10% methanol in dichloromethane). H NMR (300 MHz,

CD₃OD) δ 9.19 (s, 1H), 8.76 (d, J = 5.1 Hz, 1H), 8.24 (d, J = 8.8 Hz,

1H), 7.83 (d, J = 5.0 Hz, 1H), 7.62 (d, J = 7.0 Hz, 2H), 7.35 (d, J =

6.7 Hz, 1H), 6.76 (s, 1H), 4.19 (s, 2H), 3.95 (s, 2H), 3.23 (s, 3H),

2.83 (s, 2H), 2.55 (s, 2H), 1.75 (m, 4H). ¹³C NMR (75 MHz,

DMSO-d₆) δ 162.7, 161.2, 159.0, 158.8, 156.3, 155.1, 155.0, 153.5,

143.1, 130.4, 124.5, 124.4, 118.9, 117.8, 114.9, 114.6, 98.6, 47.3, 31.4,

27.9, 27.9, 25.4, 22.6, 21.9. HRMS (ESI) m/z: calcd for C₂₄H₂₆N₇O

[M + H]⁺, 428.2194; found, 428.2201.

2-((2-((6-Fluoro-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)-1-methyl-[4,4′-bipyrimidin]-6(1H)-one (22b). White solid,

1

64% yield; R_f= 0.4 (10% methanol in dichloromethane). H NMR

(300 MHz, CD₃OD) δ 9.20 (s, 1H), 8.79 (d, J = 5.0 Hz, 1H), 8.46−

8.29 (m, 1H), 7.89 (d, J = 5.0 Hz, 1H), 7.20 (dd, J = 20.5, 9.3 Hz,

2H), 6.76 (s, 1H), 4.26 (s, 2H), 4.00 (s, 2H), 3.29 (s, 3H), 2.80 (d, J

= 5.3 Hz, 2H), 2.48 (s, 2H), 1.76 (d, J = 5.6 Hz, 4H). ¹³C NMR (75

MHz, DMSO-d₆) δ 162.9, 161.7, 161.1, 160.8, 158.4, 158.3, 156.3,

155.2, 154.8, 117.6, 114.4, 114.1, 112.9, 112.6, 105.7, 103.5, 98.8,

29.7, 27.5, 27.5, 24.6, 21.9, 21.0, 20.0. HRMS (ESI) m/z: calcd for

C₂₄H₂₅FN₇O [M + H]⁺, 446.2100; found, 446.2105.

2-((2-((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)-1-methyl-[4,4′-bipyrimidin]-6(1H)-one (22c). White solid,

6-(3-Fluoropyridin-4-yl)-2-((2-((6-methoxy-1,2,3,4-tetrahydroa-

cridin-9-yl)amino)ethyl)amino)-3-methylpyrimidin-4(3H)-one

(22i). White solid, 67% yield; R_f= 0.5 (10% methanol in

1

57% yield; R_f= 0.5 (10% methanol in dichloromethane). H NMR

(300 MHz, CD₃OD) δ 9.23 (s, 1H), 8.79 (d, J = 5.3 Hz, 1H), 8.29 (d,

J = 9.2 Hz, 1H), 7.89 (d, J = 5.2 Hz, 1H), 7.57 (d, J = 2.1 Hz, 1H),

7.33 (d, J = 9.0 Hz, 1H), 6.80 (s, 1H), 4.27 (s, 2H), 4.01 (s, 2H), 3.29

(s, 3H), 2.83 (d, J = 6.1 Hz, 2H), 2.51 (s, 2H), 1.77 (m, 4H). ¹³C

NMR (75 MHz, DMSO-d₆) δ 162.8, 161.2, 158.8, 158.6, 156.4,

154.8, 151.9, 146.4, 141.1, 133.7, 125.9, 125.6, 124.0, 118.4, 117.7,

116.0, 98.6, 42.3, 33.0, 27.5, 27.5, 25.4, 22.7, 22.3. HRMS (ESI) m/z:

calcd for C₂₄H₂₅ClN₇O [M + H]⁺, 462.1804; found, 462.1812.

2-((2-((6-Methoxy-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)-1-methyl-[4,4′-bipyrimidin]-6(1H)-one (22d). White solid,

1

dichloromethane). H NMR (300 MHz, CD₃OD) δ 8.57 (s, 1H),

8.37 (d, J = 5.0 Hz, 1H), 8.06 (d, J = 9.3 Hz, 1H), 7.74 (t, J = 5.8 Hz,

1H), 7.00−6.80 (m, 2H), 6.34 (s, 1H), 4.17−4.00 (m, 2H), 3.96−

3.82 (m, 5H), 3.26 (s, 3H), 2.94−2.67 (m, 2H), 2.54 (s, 2H), 1.81

(m, 4H). ¹³C NMR (75 MHz, CD₃OD) δ 163.7, 161.0, 159.0, 154.6,

154.1, 153.7, 153.4, 145.5, 145.2, 138.9, 138.5, 132.7, 125.1, 123.6,

116.3, 112.7, 112.5, 102.4, 55.2, 41.8, 30.7, 29.5, 27.3, 24.2, 22.2, 21.5.

HRMS (ESI) m/z: calcd for C₂₆H₂₈FN₆O₂[M + H]⁺, 474.2253;

found, 475.2247.

2-((2-((7-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)-6-(3-ﬂuoropyridin-4-yl)-3-methylpyrimidin-4(3H)-one (22j).

White solid, 62% yield; R_f= 0.5 (10% methanol in dichloromethane).

¹H NMR (300 MHz, CD₃OD) δ 8.56 (d, J = 3.1 Hz, 1H), 8.42 (d, J =

4.9 Hz, 1H), 8.07 (s, 1H), 7.81 (t, J = 5.9 Hz, 1H), 7.61 (d, J = 9.0

Hz, 1H), 7.47 (d, J = 8.9 Hz, 1H), 6.32 (s, 1H), 4.01 (d, J = 5.2 Hz,

2H), 3.85 (d, J = 5.4 Hz, 2H), 3.21 (s, 3H), 2.96−2.80 (m, 2H), 2.73

(d, J = 5.6 Hz, 2H), 1.84 (m, 4H). ¹³C NMR (75 MHz, CD₃OD) δ

163.8, 156.9, 154.5, 153.7, 151.6, 145.4, 143.1, 138.9, 138.6, 132.7,

130.0, 129.6, 127.2, 123.6, 122.1, 119.9, 116.1, 103.6, 42.1, 31.9, 27.2,

27.2, 25.0, 22.3, 21.9. HRMS (ESI) m/z: calcd for C₂₅H₂₅FClN₆O [M

+ H]⁺, 479.1757; found, 479.1763.

1

61% yield; R_f= 0.5 (12.5% methanol in dichloromethane). H NMR

(300 MHz, CD₃OD) δ 9.23 (s, 1H), 8.77 (d, J = 5.1 Hz, 1H), 8.27 (d,

J = 9.9 Hz, 1H), 7.88 (s, 1H), 7.01 (d, J = 9.6 Hz, 1H), 6.87 (s, 1H),

6.79 (s, 1H), 4.31 (s, 2H), 4.04 (s, 2H), 3.96 (s, 3H), 3.66 (s, 3H),

2.77 (s, 2H), 2.42 (s, 2H), 1.74 (m, 4H). ¹³C NMR (75 MHz,

CD₃OD): Given the property of the 300 MHz spectrometer and the

low solubility of the compound in deuterated solvents, the ¹³C NMR

spectrum of 22d was not successfully acquired. However, the ¹H

NMR spectrum and HRMS data of 22d aﬀorded here could be the

valid evidence for the structure of 22d. HRMS (ESI) m/z: calcd for

C₂₅H₂₈N₇O₂[M + H]⁺, 458.2300; found, 458.2303.

2-((2-((7-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)ethyl)-

amino)-1-methyl-[4,4′-bipyrimidin]-6(1H)-one (22e). White solid,

3-Methyl-6-(pyridin-4-yl)-2-((3-((1,2,3,4-tetrahydroacridin-9-yl)-

1

60% yield; R_f= 0.4 (12.5% methanol in dichloromethane). H NMR

amino)propyl)amino)pyrimidin-4(3H)-one (23a). Yellow solid, 59%

1

(300 MHz, CD₃OD) δ 9.19 (s, 1H), 8.81 (s, 1H), 8.11 (s, 1H), 7.92

(s, 1H), 7.51 (s, 2H), 6.72 (s, 1H), 4.12 (s, 2H), 3.89 (s, 2H), 3.20 (s,

3H), 2.86 (s, 2H), 2.67 (s, 2H), 1.81 (m, 4H). ¹³C NMR (75 MHz,

yield; R_f= 0.4 (10% methanol in dichloromethane). H NMR (300

MHz, CD₃OD) δ 8.52 (d, J = 4.3 Hz, 2H), 8.22 (d, J = 8.3 Hz, 1H),

7.79 (d, J = 4.6 Hz, 2H), 7.68−7.57 (m, 1H), 7.52 (d, J = 8.1 Hz,

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Article

1H), 7.35 (t, J = 7.4 Hz, 1H), 6.28 (s, 1H), 4.10 (s, 2H), 3.74 (s, 2H),

3.31 (s, 3H), 2.77 (s, 2H), 2.60 (s, 2H), 2.23 (s, 2H), 1.79 (m, 4H).

¹³C NMR (75 MHz, CD₃OD) δ 164.2, 157.8, 155.5, 154.4, 151.3,

149.3, 149.3, 145.3, 139.7, 131.9, 124.7, 124.4, 121.1, 121.1, 120.3,

116.3, 111.9, 97.9, 39.4, 29.9, 28.8, 27.4, 27.4, 24.1, 21.8, 20.8. HRMS

(ESI) m/z: calcd for C₂₆H₂₉N₆O [M + H]⁺, 441.2398; found,

441.2407.

NMR (300 MHz, CD₃OD) δ 8.59 (d, J = 5.3 Hz, 2H), 8.16 (d, J = 9.2

Hz, 1H), 7.95 (d, J = 5.1 Hz, 2H), 7.71 (s, 1H), 7.36 (d, J = 9.1 Hz,

1H), 6.40 (s, 1H), 3.70 (t, J = 7.2 Hz, 2H), 3.58 (t, J = 6.9 Hz, 2H),

3.41 (s, 3H), 2.95 (s, 2H), 2.66 (s, 2H), 1.91 (s, 4H), 1.76 (d, J = 6.1

Hz, 4H), 1.50 (m, 4H). ¹³C NMR (75 MHz, CD₃OD) δ 164.6, 158.3,

155.5, 154.4, 153.5, 149.2, 149.2, 145.8, 144.0, 136.2, 125.8, 124.6,

122.6, 121.2, 121.2, 116.4, 114.0, 97.6, 48.4, 41.8, 31.0, 30.8, 28.9,

27.0, 26.5, 26.4, 24.1, 22.2, 21.5. HRMS (ESI) m/z: calcd for

C₂₉H₃₄ClN₆O [M + H]⁺, 517.2478; found, 517.2479.

3-Methyl-6-(pyridin-4-yl)-2-((4-((1,2,3,4-tetrahydroacridin-9-yl)-

amino)butyl)amino)pyrimidin-4(3H)-one (23b). Yellow solid, 63%

1

yield; R_f= 0.4 (10% methanol in dichloromethane). H NMR (300

2-((6-((6-Fluoro-1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-

MHz, CD₃OD) δ 8.56 (d, J = 4.6 Hz, 2H), 8.28 (d, J = 8.5 Hz, 1H),

7.90 (d, J = 4.4 Hz, 2H), 7.72 (q, J = 8.4 Hz, 2H), 7.43 (t, J = 7.5 Hz,

1H), 6.34 (s, 1H), 3.96 (d, J = 6.4 Hz, 2H), 3.61 (s, 2H), 3.25 (s,

3H), 2.87 (s, 2H), 2.58 (s, 2H), 2.02−1.71 (m, 8H). ¹³C NMR (75

MHz, CD₃OD) δ 164.4, 157.9, 155.9, 154.5, 151.5, 149.5, 149.5,

145.6, 139.9, 132.2, 125.0, 124.8, 121.3, 121.3, 120.4, 116.5, 112.3,

97.5, 41.6, 28.9, 27.7, 26.8, 26.8, 25.6, 23.9, 21.9, 20.9. HRMS (ESI)

m/z: calcd for C₂₇H₃₁N₆O [M + H]⁺, 455.2554; found, 455.2562.

3-Methyl-6-(pyridin-4-yl)-2-((5-((1,2,3,4-tetrahydroacridin-9-yl)-

amino)pentyl)amino)pyrimidin-4(3H)-one (23c). Yellow solid, 57%

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (26c). Yellow

1

solid, 67% yield; R_f= 0.4 (10% methanol in dichloromethane). H

NMR (300 MHz, CD₃OD) δ 8.60 (d, J = 5.9 Hz, 2H), 8.29 (dd, J =

9.3, 5.8 Hz, 1H), 7.96 (d, J = 5.9 Hz, 2H), 7.38 (d, J = 9.8 Hz, 1H),

7.25 (t, J = 8.7 Hz, 1H), 6.40 (s, 1H), 3.75 (t, J = 7.1 Hz, 2H), 3.59 (t,

J = 7.0 Hz, 2H), 3.41 (s, 3H), 2.96 (s, 2H), 2.66 (s, 2H), 1.93 (d, J =

11.5 Hz, 4H), 1.87−1.65 (m, 4H), 1.48 (m, 4H). ¹³C NMR (75 MHz,

CD₃OD) δ 164.6, 162.3, 158.3, 154.6, 154.4, 154.1, 149.2, 149.2,

145.8, 144.1, 127.2, 121.3, 121.3, 114.6, 113.9, 113.0, 107.5, 97.6,

48.4, 41.8, 30.7, 30.4, 28.9, 27.1, 26.5, 26.4, 23.9, 22.2, 21.4. HRMS

(ESI) m/z: calcd for C₂₉H₃₄FN₆O [M + H]⁺, 501.2773; found,

501.2780.

1

yield; R_f= 0.5 (10% methanol in dichloromethane). H NMR (300

MHz, CD₃OD) δ 8.51 (d, J = 4.7 Hz, 2H), 8.21 (d, J = 8.5 Hz, 1H),

7.84 (d, J = 4.7 Hz, 2H), 7.70−7.59 (m, 2H), 7.39 (t, J = 7.5 Hz, 1H),

6.31 (s, 1H), 3.83 (t, J = 6.7 Hz, 2H), 3.59 (t, J = 6.5 Hz, 2H), 3.33

(d, J = 6.3 Hz, 3H), 2.90 (s, 2H), 2.62 (s, 2H), 1.87 (m, 6H), 1.79−

1.68 (m, 2H), 1.55 (d, J = 6.5 Hz, 2H). ¹³C NMR (75 MHz,

CD₃OD) δ 164.5, 158.0, 155.3, 154.7, 152.3, 149.4, 149.4, 145.7,

140.6, 131.8, 124.7, 124.7, 121.2, 121.2, 121.0, 116.8, 112.5, 97.4,

41.5, 30.2, 29.4, 28.6, 26.9, 26.9, 24.2, 23.7, 22.1, 21.2. HRMS (ESI)

m/z: calcd for C₂₈H₃₃N₆O [M + H]⁺, 469.2711; found, 469.2704.

3-Methyl-6-(pyridin-4-yl)-2-((6-((1,2,3,4-tetrahydroacridin-9-yl)-

amino)hexyl)amino)pyrimidin-4(3H)-one (23d). Yellow solid, 53%

2-((6-((7-Fluoro-1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (26d). Brown

1

solid, 64% yield; R_f= 0.4 (10% methanol in dichloromethane). H

NMR (300 MHz, CD₃OD) δ 8.59 (d, J = 4.9 Hz, 2H), 7.95 (d, J = 4.9

Hz, 2H), 7.80 (d, J = 9.4 Hz, 2H), 7.43 (t, J = 8.4 Hz, 1H), 6.39 (s,

1H), 3.56 (s, 4H), 3.40 (s, 3H), 2.97 (s, 2H), 2.72 (s, 2H), 1.90 (s,

4H), 1.72 (s, 4H), 1.48 (s, 4H). ¹³C NMR (75 MHz, CD₃OD) δ

164.6, 160.1, 158.3, 156.3, 154.4, 152.0, 149.2, 149.2, 145.8, 142.1,

128.0, 121.2, 121.2, 119.9, 119.2, 115.7, 107.0, 97.6, 48.3, 41.8, 32.0,

31.0, 29.0, 27.0, 26.5, 26.5, 24.6, 22.5, 22.0. HRMS (ESI) m/z: calcd

for C₂₉H₃₄FN₆O [M + H]⁺, 501.2773; found, 501.2780.

1

yield; R_f= 0.5 (10% methanol in dichloromethane). H NMR (300

MHz, CD₃OD) δ 8.57 (d, J = 4.3 Hz, 2H), 8.35 (d, J = 8.6 Hz, 1H),

7.94 (d, J = 4.4 Hz, 2H), 7.87−7.71 (m, 2H), 7.54 (t, J = 7.6 Hz, 1H),

6.38 (s, 1H), 3.94 (t, J = 7.0 Hz, 2H), 3.60 (t, J = 6.6 Hz, 2H), 3.40 (s,

3H), 2.99 (s, 2H), 2.66 (s, 2H), 1.91 (m, 6H), 1.77 (s, 2H), 1.54 (m,

4H). ¹³C NMR (75 MHz, CD₃OD) δ 164.6, 158.3, 156.7, 154.7,

150.4, 149.4, 149.4, 145.9, 138.6, 132.9, 125.4, 125.2, 125.2, 121.4,

119.0, 115.8, 111.6, 97.5, 41.88, 30.3, 29.0, 28.2, 27.0, 27.0, 26.4, 26.4,

23.8, 21.8, 20.7. HRMS (ESI) m/z: calcd for C₂₉H₃₅N₆O [M + H]⁺,

483.2867; found, 483.2862.

3-Methyl-2-((6-((6-methyl-1,2,3,4-tetrahydroacridin-9-yl)amino)-

hexyl)amino)-6-(pyridin-4-yl)pyrimidin-4(3H)-one (26e). Yellow

1

solid, 62% yield; R_f= 0.5 (10% methanol in dichloromethane). H

NMR (300 MHz, CD₃OD) δ 8.59 (d, J = 5.8 Hz, 2H), 8.24 (d, J = 8.8

Hz, 1H), 7.95 (d, J = 5.8 Hz, 2H), 7.51 (s, 1H), 7.38 (d, J = 8.9 Hz,

1H), 6.39 (s, 1H), 3.94 (t, J = 7.1 Hz, 2H), 3.62 (t, J = 6.8 Hz, 2H),

3.42 (s, 3H), 2.98 (s, 2H), 2.63 (s, 2H), 2.56 (s, 3H), 1.92 (m, 6H),

1.81 (d, J = 6.4 Hz, 2H), 1.57 (m, 4H). ¹³C NMR (75 MHz,

CD₃OD) δ 164.6, 158.3, 156.2, 154.5, 149.5, 149.2, 149.2, 145.8,

144.3, 138.8, 127.1, 124.9, 121.2, 121.2, 118.1, 113.7, 111.0, 97.6,

47.8, 41.8, 30.3, 28.9, 28.0, 27.1, 26.4, 26.3, 23.4, 21.8, 21.0, 20.6.

HRMS (ESI) m/z: calcd for C₃₀H₃₇N₆O [M + H]⁺, 497.3024; found,

497.3027.

2-((6-((6-Bromo-1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-

amino)-6-(3-ﬂuoropyridin-4-yl)-3-methylpyrimidin-4(3H)-one

(26f). Yellow solid, 68% yield; R_f= 0.5 (10% methanol in

dichloromethane). ¹H NMR (300 MHz, CD₃OD) δ 8.52 (d, J =

3.2 Hz, 1H), 8.42 (d, J = 4.9 Hz, 1H), 8.02 (t, J = 7.4 Hz, 2H), 7.89

(s, 1H), 7.42 (d, J = 9.1 Hz, 1H), 6.42 (s, 1H), 3.55 (dt, J = 20.2, 7.1

Hz, 4H), 3.40 (s, 3H), 2.95 (s, 2H), 2.68 (s, 2H), 1.89 (s, 4H), 1.70

(s, 4H), 1.42 (m, 4H). ¹³C NMR (75 MHz, CD₃OD) δ 164.3, 158.8,

158.1, 156.2, 154.2, 152.1, 146.7, 145.3, 138.8, 133.2, 128.3, 126.7,

125.1, 123.9, 123.0, 117.9, 115.1, 102.7, 48.8, 41.9, 32.5, 31.2, 29.0,

27.1, 26.6, 26.5, 24.5, 22.5, 22.1. HRMS (ESI) m/z: calcd for

C₂₉H₃₃BrFN₆O [M + H]⁺, 579.1878; found, 579.1884.

2-((6-((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-

amino)-6-(3-ﬂuoropyridin-4-yl)-3-methylpyrimidin-4(3H)-one

(26g). Yellow solid, 63% yield; R_f= 0.5 (10% methanol in

dichloromethane). ¹H NMR (300 MHz, CD₃OD) δ 8.52 (d, J =

3.3 Hz, 1H), 8.42 (d, J = 5.0 Hz, 1H), 8.14 (d, J = 9.1 Hz, 1H), 8.03

(t, J = 5.9 Hz, 1H), 7.71 (s, 1H), 7.34 (d, J = 9.1 Hz, 1H), 6.41 (s,

1H), 3.67 (t, J = 7.1 Hz, 2H), 3.54 (t, J = 7.0 Hz, 2H), 3.41 (s, 3H),

2.96 (s, 2H), 2.68 (s, 2H), 1.93 (d, J = 12.1 Hz, 4H), 1.74 (d, J = 5.8

Hz, 4H), 1.49 (m, 4H). ¹³C NMR (75 MHz, CD₃OD) δ 164.3, 158.8,

156.4, 154.2, 153.0, 145.3, 144.8, 138.8, 138.5, 135.8, 133.2, 125.5,

124.6, 123.9, 123.5, 116.8, 114.3, 102.8, 48.6, 41.9, 31.5, 31.0, 28.9,

3-Methyl-6-(pyridin-4-yl)-2-((7-((1,2,3,4-tetrahydroacridin-9-yl)-

amino)heptyl)amino)pyrimidin-4(3H)-one (23e). Yellow solid, 63%

1

yield; R_f= 0.6 (9% methanol in dichloromethane). H NMR (300

MHz, CD₃OD) δ 8.55 (d, J = 5.0 Hz, 2H), 8.31 (d, J = 8.8 Hz, 1H),

7.92 (d, J = 4.9 Hz, 2H), 7.80 (t, J = 7.6 Hz, 1H), 7.71 (d, J = 8.3 Hz,

1H), 7.51 (t, J = 7.8 Hz, 1H), 6.35 (s, 1H), 3.89 (t, J = 6.9 Hz, 2H),

3.57 (t, J = 6.9 Hz, 2H), 3.39 (s, 3H), 2.98 (s, 2H), 2.63 (s, 2H), 1.93

(m, 4H), 1.82 (s, 2H), 1.72 (s, 2H), 1.46 (m, 6H). ¹³C NMR (75

MHz, CD₃OD) δ 164.7, 158.4, 154.5, 154.3, 153.7, 149.3, 149.3,

145.9, 142.3, 130.9, 124.4, 124.2, 122.7, 121.3, 121.3, 117.7, 113.3,

97.6, 41.9, 30.8, 30.4, 29.5, 29.0, 28.9, 27.1, 26.7, 26.7, 24.1, 22.3,

21.5. HRMS (ESI) m/z: calcd for C₃₀H₃₇N₆O [M + H]⁺, 497.3024;

found, 497.3017.

2-((6-((6-Bromo-1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (26a). Yellow

1

solid, 60% yield; R_f= 0.5 (10% methanol in dichloromethane). H

NMR (300 MHz, CD₃OD) δ 8.59 (d, J = 5.4 Hz, 2H), 8.00 (d, J = 9.1

Hz, 1H), 7.94 (d, J = 5.3 Hz, 2H), 7.89 (s, 1H), 7.41 (d, J = 9.2 Hz,

1H), 6.39 (s, 1H), 3.65−3.49 (m, 4H), 3.40 (s, 3H), 2.94 (s, 2H),

2.66 (s, 2H), 1.88 (s, 4H), 1.71 (s, 4H), 1.46 (m, 4H). ¹³C NMR (75

MHz, CD₃OD) δ 164.6, 158.3, 157.8, 154.4, 152.3, 149.2, 149.2,

146.5, 145.8, 128.1, 126.7, 125.2, 123.1, 121.2, 121.2, 117.8, 115.0,

97.6, 48.7, 41.9, 32.4, 31.1, 29.0, 27.0, 26.5, 26.5, 24.5, 22.5, 22.0.

HRMS (ESI) m/z: calcd for C₂₉H₃₄BrN₆O [M + H]⁺, 561.1972;

found, 561.1984.

2-((6-((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (26b). Yellow

1

solid, 68% yield; R_f= 0.5 (10% methanol in dichloromethane). H

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27.1, 26.5, 26.5, 24.2, 22.3, 21.8. HRMS (ESI) m/z: calcd for

C₂₉H₃₃ClFN₆O [M + H]⁺, 535.2383; found, 535.2390.

1H), 6.41 (s, 1H), 3.69 (t, J = 6.8 Hz, 2H), 3.58 (t, J = 6.9 Hz, 2H),

3.44 (s, 3H), 2.97 (s, 2H), 2.69 (s, 2H), 1.93 (s, 4H), 1.73 (s, 4H),

1.46 (m, 6H). ¹³C NMR (75 MHz, CD₃OD) δ 164.6, 158.3, 155.3,

154.4, 153.6, 149.2, 149.2, 145.8, 143.8, 136.3, 125.8, 124.6, 122.4,

121.3, 121.3, 116.3, 113.8, 97.6, 48.4, 41.9, 30.9, 30.8, 28.9, 28.9, 27.0,

26.7, 26.6, 24.1, 22.2, 21.5. HRMS (ESI) m/z: calcd for C₃₀H₃₆ClN₆O

[M + H]⁺, 531.2634; found, 531.2642.

2-((6-((6-Fluoro-1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-

amino)-6-(3-ﬂuoropyridin-4-yl)-3-methylpyrimidin-4(3H)-one

(26h). Yellow solid, 68% yield; R_f= 0.4 (10% methanol in

1

dichloromethane). H NMR (300 MHz, CD₃OD) δ 8.53 (d, J =

3.3 Hz, 1H), 8.43 (d, J = 4.8 Hz, 1H), 8.35 (dd, J = 9.4, 5.6 Hz, 1H),

8.05 (t, J = 5.9 Hz, 1H), 7.40 (d, J = 9.6 Hz, 1H), 7.30 (t, J = 8.8 Hz,

1H), 6.41 (s, 1H), 3.82 (t, J = 7.1 Hz, 2H), 3.57 (t, J = 7.0 Hz, 2H),

3.42 (s, 3H), 2.98 (s, 2H), 2.66 (s, 2H), 1.94 (s, 4H), 1.80 (dd, J =

16.4, 7.0 Hz, 4H), 1.53 (m, 4H). ¹³C NMR (75 MHz, CD₃OD) δ

165.1, 164.3, 162.6, 158.8, 156.2, 155.0, 154.2, 153.2, 145.3, 142.8,

138.7, 133.2, 127.8, 123.8, 114.1, 112.4, 105.7, 102.4, 48.3, 41.8, 30.5,

29.6, 28.8, 27.1, 26.4, 26.4, 23.7, 22.0, 21.0. HRMS (ESI) m/z: calcd

for C₂₉H₃₃F₂N₆O [M + H]⁺, 519.2679; found, 519.2684.

2-((6-((7-Fluoro-1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-

amino)-6-(3-ﬂuoropyridin-4-yl)-3-methylpyrimidin-4(3H)-one (26i).

Brown solid, 60% yield; R_f= 0.4 (10% methanol in dichloromethane).

¹H NMR (300 MHz, CD₃OD) δ 8.52 (s, 1H), 8.43 (d, J = 4.8 Hz,

2-((7-((6-Fluoro-1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)-

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (27c). Yellow

1

solid, 63% yield; R_f= 0.5 (10% methanol in dichloromethane). H

NMR (300 MHz, CD₃OD) δ 8.61 (d, J = 4.8 Hz, 2H), 8.25−8.15 (m,

1H), 7.97 (d, J = 4.9 Hz, 2H), 7.37 (d, J = 10.4 Hz, 1H), 7.21 (t, J =

8.3 Hz, 1H), 6.42 (s, 1H), 3.61 (dt, J = 19.0, 7.0 Hz, 4H), 3.43 (s,

3H), 2.97 (s, 2H), 2.69 (s, 2H), 1.92 (s, 4H), 1.72 (s, 4H), 1.42 (m,

6H). ¹³C NMR (75 MHz, CD₃OD) δ 164.6, 164.3, 161.8, 158.3,

156.8, 154.4, 153.0, 149.2, 149.2, 145.8, 126.5, 121.3, 121.3, 115.6,

113.8, 113.4, 108.6, 97.6, 48.7, 41.9, 31.7, 30.9, 28.9, 28.9, 27.0, 26.7,

26.6, 24.2, 22.4, 21.8. HRMS (ESI) m/z: calcd for C₃₀H₃₆FN₆O [M +

H]⁺, 515.2930; found, 515.2939.

1H), 8.05 (d, J = 5.4 Hz, 1H), 7.81 (dd, J = 18.7, 8.9 Hz, 2H), 7.48 (t,

J = 8.2 Hz, 1H), 6.41 (s, 1H), 3.62 (t, J = 7.1 Hz, 2H), 3.55 (t, J = 6.8

Hz, 2H), 3.41 (s, 3H), 2.99 (s, 2H), 2.73 (s, 2H), 1.92 (s, 4H), 1.74

(d, J = 5.7 Hz, 4H), 1.44 (d, J = 27.9 Hz, 4H). ¹³C NMR (75 MHz,

CD₃OD) δ 164.3, 160.1, 158.8, 157.7, 156.2, 155.2, 154.2, 152.7,

145.3, 140.8, 138.7, 133.1, 126.9, 123.8, 119.5, 114.9, 107.4, 102.6,

48.2, 41.8, 31.3, 30.9, 28.9, 27.0, 26.5, 26.4, 24.5, 22.4, 21.7. HRMS

(ESI) m/z: calcd for C₂₉H₃₃F₂N₆O [M + H]⁺, 519.2679; found,

519.2680.

2-((7-((7-Fluoro-1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)-

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (27d). Yellow

1

solid, 60% yield; R_f= 0.5 (10% methanol in dichloromethane). H

NMR (300 MHz, CD₃OD) δ 8.61 (d, J = 5.8 Hz, 2H), 7.96 (d, J = 5.8

Hz, 2H), 7.80 (d, J = 9.7 Hz, 1H), 7.45 (t, J = 8.6 Hz, 1H), 6.40 (s,

1H), 3.57 (s, 4H), 3.43 (s, 3H), 2.99 (s, 2H), 2.74 (s, 2H), 1.86 (d, J

= 36.9 Hz, 4H), 1.71 (d, J = 5.7 Hz, 4H), 1.42 (m, 6H). ¹³C NMR

(75 MHz, CD₃OD) δ 164.6, 160.1, 158.4, 157.7, 156.0, 154.4, 152.1,

149.2, 149.2, 145.8, 141.9, 127.8, 121.3, 121.3, 119.0, 115.5, 107.0,

97.6, 48.2, 41.9, 31.9, 31.0, 28.9, 28.9, 26.9, 26.7, 26.6, 24.6, 22.5,

22.0. HRMS (ESI) m/z: calcd for C₃₀H₃₆N₆O [M + H]⁺, 515.2930;

found, 515.2924.

6-(3-Fluoropyridin-4-yl)-3-methyl-2-((6-((1,2,3,4-tetrahydroacri-

din-9-yl)amino)hexyl)amino)pyrimidin-4(3H)-one (26j). Yellow

1

solid, 63% yield; R_f= 0.4 (10% methanol in dichloromethane). H

NMR (300 MHz, CD₃OD) δ 8.53 (s, 1H), 8.43 (d, J = 4.7 Hz, 1H),

8.31 (d, J = 8.6 Hz, 1H), 8.05 (t, J = 5.8 Hz, 1H), 7.77 (s, 2H), 7.52

(d, J = 6.3 Hz, 1H), 6.41 (s, 1H), 3.87 (t, J = 7.1 Hz, 2H), 3.57 (t, J =

6.9 Hz, 2H), 3.42 (s, 3H), 3.01 (s, 2H), 2.69 (s, 2H), 1.96 (s, 4H),

1.81 (d, J = 28.3 Hz, 4H), 1.53 (m, 4H). ¹³C NMR (75 MHz,

CD₃OD) δ 164.3, 158.8, 156.2, 155.5, 154.2, 151.6, 145.3, 140.1,

138.7, 133.1, 131.9, 124.8, 124.6, 123.8, 120.6, 116.5, 112.2, 102.6,

48.1, 41.8, 30.5, 29.0, 28.8, 27.1, 26.4, 26.3, 23.8, 21.9, 20.9. HRMS

(ESI) m/z: calcd for C₂₉H₃₄FN₆O [M + H]⁺, 501.2773; found,

501.2779.

6-(3-Fluoropyridin-4-yl)-3-methyl-2-((6-((6-methyl-1,2,3,4-tetra-

hydroacridin-9-yl)amino)hexyl)amino)pyrimidin-4(3H)-one (26k).

Yellow solid, 62% yield; R_f= 0.5 (10% methanol in dichloromethane).

¹H NMR (300 MHz, CD₃OD) δ 8.51 (d, J = 3.3 Hz, 1H), 8.41 (d, J =

4.9 Hz, 1H), 8.04 (dt, J = 16.3, 9.3 Hz, 2H), 7.52 (s, 1H), 7.27 (d, J =

8.7 Hz, 1H), 6.36 (d, J = 19.6 Hz, 1H), 3.72 (t, J = 7.1 Hz, 2H), 3.54

(t, J = 7.0 Hz, 2H), 3.40 (s, 3H), 2.96 (s, 2H), 2.66 (s, 2H), 2.51 (s,

3H), 1.88 (d, J = 22.5 Hz, 4H), 1.84−1.65 (m, 4H), 1.50 (m, 4H).

¹³C NMR (75 MHz, CD₃OD) δ 164.3, 158.8, 156.2, 154.2, 154.0,

3-Methyl-2-((7-((6-methyl-1,2,3,4-tetrahydroacridin-9-yl)amino)-

heptyl)amino)-6-(pyridin-4-yl)pyrimidin-4(3H)-one (27e). Yellow

1

solid, 66% yield; R_f= 0.6 (10% methanol in dichloromethane). H

NMR (300 MHz, CD₃OD) δ 8.58 (d, J = 5.8 Hz, 2H), 8.21 (d, J = 8.9

Hz, 1H), 7.94 (d, J = 5.9 Hz, 2H), 7.51 (s, 1H), 7.38 (d, J = 9.0 Hz,

1H), 6.38 (s, 1H), 3.90 (t, J = 7.2 Hz, 2H), 3.61 (t, J = 7.0 Hz, 2H),

3.43 (s, 3H), 2.98 (s, 2H), 2.61 (d, J = 15.3 Hz, 2H), 2.55 (s, 3H),

1.96 (s, 4H), 1.81 (d, J = 24.7 Hz, 4H), 1.51 (m, 6H). ¹³C NMR (75

MHz, CD₃OD) δ 164.6, 158.3, 156.2, 154.5, 149.5, 149.2, 149.2,

145.8, 144.3, 138.8, 127.0, 124.9, 121.2, 121.2, 118.1, 113.6, 110.9,

97.5, 47.9, 41.9, 30.3, 28.9, 28.8, 28.0, 27.1, 26.6, 26.5, 23.4, 21.8,

21.0, 20.6. HRMS (ESI) m/z: calcd for C₃₁H₃₉N₆O [M + H]⁺,

511.3180; found, 511.3179.

2-((7-((6-Bromo-1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)-

amino)-6-(3-ﬂuoropyridin-4-yl)-3-methylpyrimidin-4(3H)-one

(27f). Yellow solid, 65% yield; R_f= 0.6 (10% methanol in

1

dichloromethane). H NMR (300 MHz, CD₃OD) δ 8.53 (s, 1H),

8.45 (d, J = 4.9 Hz, 1H), 8.32−8.20 (m, 1H), 8.05 (t, J = 5.8 Hz, 1H),

7.38 (d, J = 10.0 Hz, 1H), 7.25 (t, J = 8.5 Hz, 1H), 6.43 (s, 1H), 3.71

(t, J = 7.1 Hz, 2H), 3.55 (t, J = 7.1 Hz, 2H), 3.43 (s, 3H), 2.98 (s,

2H), 2.69 (s, 2H), 1.94 (s, 4H), 1.74 (s, 4H), 1.42 (m, 6H). ¹³C

NMR (75 MHz, CD₃OD) δ 164.3, 158.8, 156.7, 156.2, 154.2, 152.3,

145.3, 145.3, 138.7, 138.4, 133.2, 126.9, 125.4, 123.9, 123.7, 117.2,

114.5, 102.6, 48.6, 41.9, 31.7, 30.9, 28.9, 28.9, 27.0, 26.7, 26.6, 24.3,

22.4, 21.8. HRMS (ESI) m/z: calcd for C₃₀H₃₅BrFN₆O [M + H]⁺,

593.2035; found, 593.2043.

153.4, 145.3, 142.7, 141.6, 138.7, 138.5, 133.1, 126.4, 123.8, 121.9,

115.7, 112.7, 102.6, 48.3, 41.8, 30.8, 30.4, 28.9, 27.1, 26.5, 26.4, 24.0,

22.3, 21.5, 20.9. HRMS (ESI) m/z: calcd for C₃₀H₃₆FN₆O [M + H]⁺,

515.2930; found, 515.2934.

2-((7-((6-Bromo-1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)-

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (27a). Yellow

1

solid, 61% yield; R_f= 0.6 (10% methanol in dichloromethane). H

NMR (300 MHz, CD₃OD) δ 8.60 (s, 2H), 8.02 (d, J = 8.8 Hz, 1H),

7.93 (d, J = 15.4 Hz, 3H), 7.44 (d, J = 8.7 Hz, 1H), 6.42 (s, 1H),

3.69−3.48 (m, 4H), 3.43 (s, 3H), 2.96 (s, 2H), 2.68 (s, 2H), 1.92 (s,

4H), 1.71 (s, 4H), 1.38 (m, 6H). ¹³C NMR (75 MHz, CD₃OD) δ

164.6, 158.4, 157.4, 154.4, 152.5, 149.3, 149.3, 146.1, 145.8, 127.7,

126.8, 125.3, 123.4, 121.3, 121.3, 117.6, 114.8, 97.7, 48.7, 42.0, 32.2,

31.1, 29.0, 28.9, 27.0, 26.7, 26.7, 24.4, 22.5, 22.0. HRMS (ESI) m/z:

calcd for C₃₀H₃₆BrN₆O [M + H]⁺, 575.2129; found, 575.2134.

2-((7-((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)-

amino)-3-methyl-6-(pyridin-4-yl)pyrimidin-4(3H)-one (27b). Yellow

2-((7-((6-Chloro-1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)-

amino)-6-(3-ﬂuoropyridin-4-yl)-3-methylpyrimidin-4(3H)-one

(27g). Yellow solid, 69% yield; R_f= 0.6 (10% methanol in

1

dichloromethane). H NMR (300 MHz, CD₃OD) δ 8.53 (s, 1H),

8.45 (d, J = 4.9 Hz, 1H), 8.32−8.22 (m, 1H), 8.05 (t, J = 5.8 Hz, 1H),

7.38 (d, J = 10.0 Hz, 1H), 7.25 (t, J = 8.5 Hz, 1H), 6.43 (s, 1H), 3.71

(t, J = 7.1 Hz, 2H), 3.55 (t, J = 7.1 Hz, 2H), 3.43 (s, 3H), 2.98 (s,

2H), 2.69 (s, 2H), 1.94 (s, 4H), 1.74 (s, 4H), 1.45 (m, 6H). ¹³C

NMR (75 MHz, CD₃OD) δ 164.3, 156.5, 154.2, 153.0, 145.3, 144.9,

138.7, 138.5, 135.7, 133.2, 125.5, 124.4, 123.9, 123.5, 116.8, 114.3,

102.6, 102.5, 48.6, 41.9, 31.6, 31.0, 28.9, 28.9, 27.1, 26.7, 26.6, 24.2,

22.3, 21.8. HRMS (ESI) m/z: calcd for C₃₀H₃₅ClFN₆O [M + H]⁺,

549.2540; found, 549.2542.

1

solid, 62% yield; R_f= 0.6 (10% methanol in dichloromethane). H

NMR (300 MHz, CD₃OD) δ 8.61 (d, J = 4.4 Hz, 2H), 8.16 (d, J = 9.3

Hz, 1H), 7.97 (d, J = 4.5 Hz, 2H), 7.73 (s, 1H), 7.38 (d, J = 9.2 Hz,

7501

https://doi.org/10.1021/acs.jmedchem.1c00160

J. Med. Chem. 2021, 64, 7483−7506

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

2-((7-((6-Fluoro-1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)-

amino)-6-(3-ﬂuoropyridin-4-yl)-3-methylpyrimidin-4(3H)-one

(27h). Yellow solid, 69% yield; R_f= 0.5 (10% methanol in

GSK-3β Activity Assay In Vitro. The in vitro inhibitory eﬀects of

compounds on GSK-3β activity were evaluated with human

recombinant GSK-3β (gifted from Prof. Yechun Xu from Shanghai

Institute of Materia Medica, Chinese Academy of Sciences) by using

the Kinase-Glo reagent kit (Promega, Madison, WI, USA) according

to a previous method⁵⁸with modiﬁcation. Test compounds were

dissolved and diluted with DMSO. The primary screening and IC₅₀

evaluation were carried out in a 96-well black plate. The reaction was

initiated by a mixture of 1 μL of test compounds at diﬀerent

concentrations (10 μM ﬁnal concentration for the ﬁrst-round

compounds and 1 μM ﬁnal concentration for the second-round

compounds at primary screening), 10 μL (20 ng) of GSK-3β enzyme,

and 89 μL of assay buﬀer containing a substrate (25 μM ﬁnal

concentration) and adenosine triphosphate (ATP, 5 μM ﬁnal

concentration). After a 30 min incubation at 30 °C, the enzymatic

reaction was terminated by adding 100 μL of Kinase-Glo reagent.

Luminescence was recorded after 10 min using a microplate reader.

The inhibitory rates of tested compounds were normalized by GSK-

3β activity of the control group, and the IC₅₀was deﬁned as the

concentration of the compound that reduced 50% of the enzymatic

activity without an inhibitor.

Molecular Modeling. In our study, the protein structure of AChE

was derived from the complex of recombinant human AChE and

donepezil (1),⁵⁹downloaded from the Protein Data Bank (PDB code:

4EY7). Also, the protein structure of GSK-3β was derived from the

complex of GSK-3β and an oxadiazole derivative,⁶⁰downloaded from

the Protein Data Bank (PDB code: 3F88). The preprocessing of two

proteins was conducted using Protein Preparation Wizard, and

compounds 23e and 27g were prepared using LigPrep, both in

Schrodinger software. The molecular docking between enzymes and

compounds was performed by employing the Schrodinger Glide

procedure, respectively. Subsequently, the docking results were

disposed as ﬁgures using PyMOL software.

BBB Assay. A UPLC-MS/MS system was employed to detect the

concentration of compound 27g in the brain and plasma. The

experimental protocols were evaluated and approved by the Ethics

Committee of the China Pharmaceutical University (IACUC no.

2020-06-004). Adult female ICR mice were purchased from the

Qinglongshan Animal Breeding Ground (Nanjing, China). Com-

pound 27g was dissolved in PBS containing 10% DMSO and 20% (2-

hydroxypropyl)-β-cyclodextrin. The instrumentation system included

an ACQUITY UPLC I-Class Plus chromatograph, a Xevo TQ-XS

mass spectrometer, and MassLynx V4.2 operational software. Animals

were divided into four groups (n = 4 in each group) and treated with

27g at a dosage of 15 mg/kg (i.p.). After the administration for 0.5, 1,

2, and 4 h, mice were executed by drawing blood from femoral artery

(about 300 μL). Brain tissues were immediately taken out and washed

with normal saline. All samples after centrifugation for 5 min (8000

rpm) were detected for the concentration of 27g using the UPLC-

MS/MS system.

Pharmacokinetic Property Investigation. A UPLC-MS/MS

system was also employed to detect the concentration of compound

27g in plasma. The experimental protocols were evaluated and

approved by the Ethics Committee of the China Pharmaceutical

University (IACUC no. 2020-06-004). Adult female ICR mice were

purchased from the Qinglongshan Animal Breeding Ground

(Nanjing, China). Compound 27g was dissolved in PBS containing

10% DMSO and 20% (2-hydroxypropyl)-β-cyclodextrin, and the

mixed solution of DMSO/PEG300/normal saline (5:45:50, v/v/v)

was used for i.v. injection. The instrumentation system included an

ACQUITY UPLC I-Class Plus chromatograph, a Xevo TQ-XS mass

spectrometer, and MassLynx V4.2 operational software. Animals were

divided into two groups (n = 3 in each group) and administered with

compound 27g by i.p. (15 mg/kg) or i.v. (5 mg/kg) injection. After

the administration for 2 min, 5 min, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, 12

h, and 24 h, heparinized blood (about 50 μL for each mouse) was

collected from orbital venous plexus to anticoagulant tubes.

Subsequently, the centrifugation of the above blood aﬀorded serum

samples (8000 rpm for 5 min), which were tested for the

concentration of 27 g using the UPLC-MS/MS system. Also, related

1

dichloromethane). H NMR (300 MHz, CD₃OD) δ 8.53 (s, 1H),

8.45 (d, J = 4.9 Hz, 1H), 8.30−8.20 (m, 1H), 8.05 (t, J = 5.8 Hz, 1H),

7.38 (d, J = 10.0 Hz, 1H), 7.25 (t, J = 8.5 Hz, 1H), 6.43 (s, 1H), 3.71

(t, J = 7.1 Hz, 2H), 3.55 (t, J = 7.1 Hz, 2H), 3.43 (s, 3H), 2.98 (s,

2H), 2.69 (s, 2H), 1.94 (s, 4H), 1.74 (s, 4H), 1.45 (m, 6H). ¹³C

NMR (75 MHz, CD₃OD) δ 164.3, 162.2, 158.8, 156.2, 155.2, 154.2,

153.8, 145.3, 138.7, 133.2, 127.0, 123.9, 114.9, 114.0, 113.7, 113.2,

107.5, 102.6, 48.5, 41.9, 30.8, 30.8, 28.9, 28.9, 27.0, 26.7, 26.6, 24.0,

22.2, 21.5. HRMS (ESI) m/z: calcd for C₃₀H₃₅F₂N₆O [M + H]⁺,

533.2835; found, 533.2841.

2-((7-((7-Fluoro-1,2,3,4-tetrahydroacridin-9-yl)amino)heptyl)-

amino)-6-(3-ﬂuoropyridin-4-yl)-3-methylpyrimidin-4(3H)-one (27i).

Brown solid, 61% yield; R_f= 0.5 (10% methanol in dichloromethane).

¹H NMR (300 MHz, CD₃OD) δ 8.52 (d, J = 3.1 Hz, 1H), 8.44 (d, J =

4.9 Hz, 1H), 8.04 (t, J = 5.9 Hz, 1H), 7.85−7.73 (m, 2H), 7.44 (t, J =

8.6 Hz, 1H), 6.42 (s, 1H), 3.55 (dd, J = 16.5, 7.7 Hz, 4H), 3.42 (s,

3H), 2.98 (s, 2H), 2.74 (s, 2H), 1.92 (s, 4H), 1.69 (s, 4H), 1.42 (m,

6H). ¹³C NMR (75 MHz, CD₃OD) δ 164.3, 160.1, 158.8, 157.6,

156.2, 155.9, 154.2, 152.2, 145.3, 141.7, 138.7, 133.2, 127.7, 123.9,

119.3, 115.3, 107.4, 102.6, 48.3, 41.9, 31.8, 31.0, 28.9, 28.9, 27.0, 26.7,

26.6, 24.6, 22.5, 21.9. HRMS (ESI) m/z: calcd for C₃₀H₃₅F₂N₆O [M

+ H]⁺, 533.2835; found, 533.2843.

6-(3-Fluoropyridin-4-yl)-3-methyl-2-((7-((1,2,3,4-tetrahydroacri-

din-9-yl)amino)heptyl)amino)pyrimidin-4(3H)-one (27j). Yellow

1

solid, 61% yield; R_f= 0.5 (10% methanol in dichloromethane). H

NMR (300 MHz, CD₃OD) δ 8.52 (d, J = 3.1 Hz, 1H), 8.45 (d, J = 4.8

Hz, 1H), 8.25 (d, J = 8.7 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 7.80−7.67

(m, 2H), 7.49 (d, J = 5.9 Hz, 1H), 6.42 (s, 1H), 3.78 (t, J = 7.0 Hz,

2H), 3.56 (t, J = 7.0 Hz, 2H), 3.43 (s, 3H), 3.01 (s, 2H), 2.71 (s, 2H),

1.95 (s, 4H), 1.84−1.63 (m, 4H), 1.46 (m, 6H). ¹³C NMR (75 MHz,

CD₃OD) δ 164.3, 158.8, 156.2, 154.7, 154.2, 152.9, 145.3, 141.5,

138.7, 133.2, 131.2, 124.5, 124.3, 123.8, 121.9, 117.2, 112.9, 102.5,

48.3, 41.9, 30.7, 29.9, 28.9, 28.8, 27.1, 26.6, 26.6, 24.0, 22.1, 21.2.

HRMS (ESI) m/z: calcd for C₃₀H₃₆FN₆O [M + H]⁺, 515.2930;

found, 515.2937.

6-(3-Fluoropyridin-4-yl)-3-methyl-2-((7-((6-methyl-1,2,3,4-tetra-

hydroacridin-9-yl)amino)heptyl)amino)pyrimidin-4(3H)-one (27k).

Yellow solid, 61% yield; R_f= 0.6 (10% methanol in dichloromethane).

¹H NMR (300 MHz, CD₃OD) δ 8.50 (d, J = 3.2 Hz, 1H), 8.43 (d, J =

4.8 Hz, 1H), 8.21 (d, J = 8.9 Hz, 1H), 8.05 (t, J = 5.9 Hz, 1H), 7.52

(s, 1H), 7.38 (d, J = 8.9 Hz, 1H), 6.39 (s, 1H), 3.90 (t, J = 7.3 Hz,

2H), 3.58 (t, J = 7.0 Hz, 2H), 3.44 (s, 3H), 3.00 (s, 2H), 2.62 (d, J =

16.0 Hz, 2H), 2.56 (s, 3H), 1.97 (s, 4H), 1.80 (d, J = 26.0 Hz, 4H),

1.50 (m, 6H). ¹³C NMR (75 MHz, CD₃OD) δ 164.3, 158.8, 156.2,

154.2, 149.6, 145.3, 144.2, 138.9, 138.7, 138.4, 133.1, 127.1, 124.8,

123.9, 118.2, 113.7, 111.0, 102.5, 47.9, 41.9, 30.4, 28.9, 28.8, 28.1,

27.2, 26.6, 26.5, 23.4, 21.8, 21.0, 20.6. HRMS (ESI) m/z: calcd for

C₃₁H₃₈FN₆O [M + H]⁺, 529.3086; found, 529.3096.

Pharmacology. AChE Activity Assay In Vitro. The in vitro

inhibitory assay against AChE was performed using Ellman’s method

with modiﬁcation.⁵⁷The cerebral cortex of a mouse was homogenized

in cold 75 mM sodium phosphate buﬀer (pH = 7.4) with 0.4 mM

tetraisopropylpyrophosphoramide as the AChE source. The reaction

was initiated by a mixture of 1 μL of test compounds (40 μM ﬁnal

concentration for primary screening) or solvent, 50 μL of 0.1 M

phosphate buﬀer, 50 μL of 0.2% dithionitrobenzoic acid (Sigma), 109

μL of deionized water, 10 μL of mouse cortex homogenate, and 30 μL

of 2 mM acetylthiocholine iodide (Sigma) as the substrate of the

AChE enzymatic reaction and then incubated for 20 min at room

temperature. The reaction was terminated by adding 50 μL of 3%

sodium-dodecyl sulfate (SDS). The production of the yellow anion of

5-thio-2-nitrobenzoic acid was measured with a microplate reader at

450 nm. The inhibitory rates of tested compounds were normalized

by AChE activity of the control group, and the IC₅₀was deﬁned as the

concentration of the compound that reduced 50% of the enzymatic

activity without an inhibitor.

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pharmacokinetic parameters of 27g were calculated using WinNonlin

version 6.4 software.

Table 5. Speciﬁc Ingredients of Media Used in This Study

basic growth

medium

diﬀerentiation

medium #1

diﬀerentiation

medium #2

diﬀerentiation

medium #3

Behavioral Studies. The experimental protocols were evaluated

and approved by the Ethics Committee of the China Pharmaceutical

University (IACUC no. 20201111135). Behavioral studies were

performed with adult female ICR mice (8−10 weeks old; weight, 25−

30 g), which were purchased from the Zhejiang Academy of Medical

Sciences (Hangzhou, China). Scopolamine hydrobromide and tacrine

were supplied by Aladdin Reagents (Shanghai, China). The test

agents were prepared in a blank solution, which was PBS containing

10% DMSO and 20% (2-hydroxypropyl)-β-cyclodextrin.

EMEM

neurobasal

15% hiFBS

2.5% hiFBS

1% hiFBS

1 × Pen/Strep

20 mM KCl

1 × Pen/Strep

1 × Pen/Strep 1 × Pen/Strep

2 mM

glutamine

2 mM glutamine 2 mM glutamine 2 mM glutamine

10 μM RA

50 ng/mL BDNF

The mice were divided into ﬁve groups and compounds were given

to each group as follows: (i) vehicle, (ii) scopolamine, (iii) tacrine

plus scopolamine, (iv) compound 23e plus scopolamine, and (v)

compound 27g plus scopolamine. In the ﬁrst period (days 1−10),

scopolamine (15 mg/kg) was administered to groups (iii), (iv), and

(v). In the second period (days 11−25), after the i.p. injection of

scopolamine (15 mg/kg) for 30 min, tacrine (15 mg/kg), 23e (15

mg/kg), and 27g (15 mg/kg) were intraperitoneally injected to mice

in groups (iii), (iv), and (v), respectively. In the entire period (days

1−25), the blank solution was treated to group (i) each day and

scopolamine (15 mg/kg) was administered to group (ii) each day.

Cognitive function was evaluated by the ANY-maze Video

Tracking System. MWM was placed in a well-lit room and the

opaque circular pool (120 cm in diameter and 60 cm in height) was

ﬁlled with water (40 cm in depth) and sustained at 25 °C. The maze

was divided into four equal quadrants, and an escape platform (10 cm

in diameter) was located in the center of the fourth quadrant of the

maze, labeled by a small ﬂag (5 cm tall). The behavioral studies of

each mouse included 4 consecutive days of learning and memory

training (days 21−24) and 3 days of cognitive evaluation (days 23−

25). The animal starting positions, facing the pool wall, were

pseudorandomized for each trial. During days 21−24, mice were

trained once to ﬁnd the platform in each quadrant for one trial (four

times in total for each mouse). If an animal failed to ﬁnd the platform

within 120 s, then the test ended and the animal was gently navigated

to the platform by hand. Whether a mouse found or failed to ﬁnd the

platform within 120 s, it was kept on the platform for 10 s. For the

cognitive evaluation, each mouse was individually evaluated on both

visible-platform (days 23−24) and hidden-platform (day 25) versions

of the water maze. On days 23−24, the latency and distance taken for

mouse to reach the platform (a successful escape) from the second

quadrant were recorded. On day 25, the platform was removed and

the animals were given a probe trial in which mice had 120 s to search

for the platform. The time taken to reach the missing platform and the

number of times that animals crossed the missing platform location

were recorded.

Data for the escape latency (latency to the ﬁrst entry), the distances

traveled (distance to the ﬁrst entry), and the number of platform

location crossings (entering times) were recorded by the ANY-maze

Video Tracking System and processed by GraphPad Prism 8.

Diﬀerentiation of SH-SY5Y Cells. SH-SY5Y neuroblastoma cells

were diﬀerentiated into corresponding neurons using the method

described by Shipley and co-workers⁴⁵with some modiﬁcations.

Brieﬂy, SH-SY5Y cells were plated with basic growth medium and

allowed to reach 70% conﬂuency. Subsequently, the medium was

changed to diﬀerentiation medium #1 and replaced every 48 h for the

following 7 days. Cells were then split 1:1 and moved into fresh ﬂasks

and diﬀerentiation medium #2 was added. Then, diﬀerentiation

medium #2 was replaced every 48 h for the following 4 days.

Subsequently, the medium was changed with diﬀerentiation medium

#3 and replaced every 48 h for the following 7 day. After this period,

SH-SY5Y-derived neurons could be acquired and used for subsequent

assays and analyses. The speciﬁc ingredients of each medium were

shown as follows (Table 5; the full name of the corresponding

ingredient could be found in the Abbreviations section).

Takeuchi’s group.⁴⁶Compounds to be tested were co-incubated with

GA for 24 h at appropriate concentrations. Levels of total tau and p-

Tau were measured as follows. A total of 3 × 10⁵cells were lysed

using 100 μL of Cell Extraction Buﬀer (Thermo Fisher), followed by

incubation for 20 min on ice. Cell lysates were subsequently

centrifuged at the force of 14,000g for 15 min, and supernatants

were used for subsequent analysis. Phosphorylation levels of tau

protein on S199 and S396 were quantiﬁed using the methodology of

enzyme-linked immunosorbent assay (ELISA). ELISA kit KHB7041

(Thermo Fisher) was used to quantify p-Tau on the S199 site

following the manufacturer’s recommended protocol. ELISA kit

KHB7031 (Thermo Fisher) was used to quantify p-Tau on the S396

site following the manufacturer’s recommended protocol. ELISA kit

KHB0041 (Thermo Fisher) was used to quantify total tau protein

following the manufacturer’s recommended protocol. Phosphoryla-

tion percentage was obtained for the analyzed residues by normal-

ization against total Tau.

Cell Viability Assay of SH-SY5Y-Derived Neurons. A total of 10⁵

cells per well were seeded into a 96-well plate. After SH-SY5Y

diﬀerentiation by the continuous treatment of RA,⁴⁶cells were treated

with 0.7 mM GA. Along with GA treatment, an appropriate

concentration of test compounds was added. After 24 h, 25 μL of 5

mg/mL Thiazolyl blue (MTT) (Sigma) was added into each well

without removing cell culture media and incubated at 37 °C with 5%

CO₂for 2 h. Subsequently, 100 μL of lysing buﬀer (50% SDS

solution, 25% DMF, and 25% demineralized water) was added. After

overnight incubation (20 h) at 37 °C, the optical densities at 490 nm

were measured using a microplate reader. The medium/MTT/lysing

buﬀer incubated under the same conditions was used as the control.

Morphology Studies in the Neuronal System. Cells were ﬁxed in

4% paraformaldehyde (PFA) for 20 min at room temperature.

Samples were then washed three times using 1% PBS in Tween-20

(PBS-T) for 2 min and subsequently placed in 5% normal goat serum

(NGS) in PBS-T (NGS-T) for 2 h at room temperature. After

removing 5% NGS-T, the mouse anti-β tubulin primary antibody was

added at a ﬁnal concentration of 1:1000 and left to incubate overnight

at 4 °C. The primary antibody was then washed three times with 1%

PBS-T, and subsequently, the rabbit anti-mouse secondary antibody

was added at a ﬁnal 1:1200 concentration. The sample was allowed to

incubate for 1 h at room temperature and subsequently mounted for

confocal imaging. Cells images were acquired using a Zeiss880

confocal microscope. The laser power was set at 4%, gained 650, and

was digitally oﬀset at 350. The acquisition speed was set at 1.3 m,

averaging two times per image. Morphological analysis of SH-SY5Y-

derived neuron confocal images was performed using ImageJ software

with the NeuronJ plug-in.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00160.

■

sı

Experimental methods for biological assays and ﬁgures,

tables, NMR spectra, HPLC spectra, and MS spectra

(PDF)

Measurements of p-Tau and Total Tau Protein Levels. SH-SY5Y

cells were cultured on diﬀerent types of media and diﬀerentiated to

SH-SY5Y neurons as described above. Then, SH-SY5Y-derived

neurons were treated with 0.7 mM GA for 24 h as described by

Molecular formula strings and some data (CSV)

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AUTHOR INFORMATION

ACKNOWLEDGMENTS

■

Corresponding Authors

This study was supported from the National Natural Science

Foundation of China (nos. 81874289 and 81903446), The

Open Project of State Key Laboratory of Natural Medicines,

China Pharmaceutical University (SKLNMKF 201710), the

Natural Science Foundation of Jiangsu Province

(BK20190564), the China Postdoctoral Science Foundation

grant 2019M652037, and “Double First-Class” University

Project (nos. CPU2018GY04 and CPU2018GY35), China

Pharmaceutical University. This study was also funded by

School of Pharmacy, The University of Nottingham (RJ/

T5433).

Haiyan Zhang − CAS Key Laboratory of Receptor Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China; orcid.org/0000-0002-

2877-2040; Email: hzhang@simm.ac.cn

Zheying Zhu − Division of Molecular Therapeutics &

Formulation, School of Pharmacy, The University of

Nottingham, Nottingham NG7 2RD, U.K.;

Email: Zheying.Zhu@nottingham.ac.uk

Jinyi Xu − State Key Laboratory of Natural Medicines and

Department of Medicinal Chemistry, China Pharmaceutical

University, Nanjing 210009, China; orcid.org/0000-

0002-1961-0402; Email: jinyixu@china.com

ABBREVIATIONS

■

Authors

Aβ, β-amyloid; ACh, acetylcholine; AChE, acetylcholinester-

ase; AChEIs, AChE inhibitors; AD, Alzheimer’s disease; ATP,

adenosine triphosphate; AUC, area under curve; BACE-1, β-

site amyloid precursor protein cleaving enzyme; BBB, blood−

brain barrier; BDNF, brain-derived neurotrophic factor; CAS,

catalytic anionic site; CDI, N,N′-carbonyldiimidazole; CL,

clearance rate; CMC-Na, sodium carboxymethyl cellulose;

DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immuno-

sorbent assay; EMEM, Eagle’s minimum essential medium;

GA, glyceraldehyde; GSK-3β, glycogen synthase kinase 3 beta;

HFIP, hexaﬂuoroisopropanol; hiFBS, heat-inactivated fetal

bovine serum; HPLC, high-performance liquid chromatog-

raphy; HRMS, high-resolution mass spectrometry; i.p., intra-

peritoneal; i.v., intravenous; MRT, mean residence time; MTT,

Thiazolyl blue; MWM, Morris water maze; NCEs, new

chemical entities; NFTs, neuroﬁbrillary tangles; NGS, normal

goat serum; NGS-T, NGS in PBS-T; NMDA, N-methyl-^D-

aspartate; PAS, peripheral anionic site; PBS, phosphate buﬀer

saline; PBS-T, PBS in Tween-20; Pen/Strep, penicillin−

streptomycin solution; PFA, paraformaldehyde; p.o., oral

administration; p-Tau, phosphorylated tau protein; RA,

retinoic acid; S199, serine-199; S396, serine-396; SARs,

structure−activity relationships; SDS, sodium-dodecyl sulfate;

SPs, senile plaques; THF, tetrahydrofuran; ThT, Thioﬂavine-

T; TLC, thin-layer chromatography

Hong Yao − State Key Laboratory of Natural Medicines and

Department of Medicinal Chemistry, China Pharmaceutical

University, Nanjing 210009, China

Giuseppe Uras − Division of Molecular Therapeutics &

Formulation, School of Pharmacy, The University of

Nottingham, Nottingham NG7 2RD, U.K.

Pengfei Zhang − State Key Laboratory of Natural Medicines

and Department of Medicinal Chemistry, China

Pharmaceutical University, Nanjing 210009, China

Shengtao Xu − State Key Laboratory of Natural Medicines

and Department of Medicinal Chemistry, China

Pharmaceutical University, Nanjing 210009, China;

orcid.org/0000-0002-5705-6377

Ying Yin − State Key Laboratory of Natural Medicines and

Department of Medicinal Chemistry, China Pharmaceutical

University, Nanjing 210009, China; CAS Key Laboratory of

Receptor Research, Shanghai Institute of Materia Medica,

Chinese Academy of Sciences, Shanghai 201203, China

Jie Liu − State Key Laboratory of Natural Medicines and

Department of Medicinal Chemistry, China Pharmaceutical

University, Nanjing 210009, China

Shuai Qin − State Key Laboratory of Natural Medicines and

Department of Medicinal Chemistry, China Pharmaceutical

University, Nanjing 210009, China

Xinuo Li − Division of Molecular Therapeutics &

Formulation, School of Pharmacy, The University of

Nottingham, Nottingham NG7 2RD, U.K.

Stephanie Allen − Division of Molecular Therapeutics &

Formulation, School of Pharmacy, The University of

Nottingham, Nottingham NG7 2RD, U.K.

Renren Bai − College of Pharmacy, School of Medicine,

Hangzhou Normal University, Hangzhou 311121, China;

orcid.org/0000-0002-3511-5794

Qi Gong − CAS Key Laboratory of Receptor Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China

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Discovery of Novel Tacrine-Pyrimidone Hybrids as Potent Dual AChE/GSK-3 Inhibitors for the Treatment of Alzheimer's Disease

DOI: 10.1021/acs.jmedchem.1c00160

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