Development of Bisindole-Substituted Aminopyrazoles as Novel GSK-3β Inhibitors with Suppressive Effects against Microglial Inflammation and Oxidative Neurotoxicity

In ﬂ ammation and Oxidative Neurotoxicity

Research Article

Development of Bisindole-Substituted Aminopyrazoles as Novel

GSK-3β Inhibitors with Suppressive Eﬀects against Microglial

Jian-Guo Liu, Danfeng Zhao, Qi Gong, Fengxia Bao, Wen-Wen Chen,* Haiyan Zhang,*

and Ming-Hua Xu*

Cite This: https://dx.doi.org/10.1021/acschemneuro.0c00520

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sı Supporting Information

ABSTRACT: Development of glycogen synthase kinase-3β

GSK-3β) inactivation-centric agents with polypharmacological

(

proﬁles is increasingly recognized as a promising therapeutic

strategy against the multifactorial etiopathology of Alzheimer’s

disease (AD). In this respect, a series of disubstituted amino-

pyrazole derivatives were designed and synthesized as a new class

of GSK-3β inhibitors. Most of these derivatives possess GSK-3β

inhibitory activities with IC₅₀values in the micromolar ranges,

among which bisindole-substituted aminopyrazole derivative 6h

displayed moderate GSK-3β inhibition (IC₅₀= 1.76 ± 0.19 μM),

and alleviative eﬀects against lipopolysaccharide (LPS)-induced

glial inﬂammation in BV-2 cells and glutamate-induced oxidative

neurotoxicity in HT-22 cells. Further in vivo studies indicated that compound 6h had potent anti-inﬂammatory eﬀect, by showing

markedly reduced microglial activation and astrocyte proliferation in the brain of LPS-injected mice. Overall, the simultaneous

modulation of 6h on multiple dysfunctions of disease network highlights this structural distinctively bisindole-substituted

aminopyrazole could be a useful prototype for the discovery of novel therapeutic agents to tackle AD and other GSK-3β associated

complex neurological syndromes.

KEYWORDS: Glycogen synthase kinase-3, bisindole-substituted aminopyrazoles, Alzheimer’s disease, oxidative neurotoxicity,

microglial inﬂammation

INTRODUCTION

Glycogen synthase kinase 3β (GSK-3β) is a ubiquitously

expressed serine/threonine kinase that plays critical roles in

multiple cellular functions, encompassing growth signaling, cell

■

Alzheimer’s disease (AD) is an irreversible and progressive

neurodegenerative disorder characterized by memory and

cognitive dysfunction. It is increasingly recognized that the

pathogenesis of AD is an intricately linked network,

characterized by multiple pathological alterations including

abnormal accumulation of β-amyloid peptide (Aβ), hyper-

phosphorylation of tau protein, oxidative stress, glial

fate and metabolism, etc. Notably, GSK-3β is also widely

expressed in the central nervous system and considered a key

player in AD pathophysiology, since dysregulation of this

enzyme inﬂuences most of the key features of the disease,

including tau phosphorylation, Aβ accumulation, inﬂamma-

tion, neurogenesis impairment, reduced synaptic plasticity,

−11

inﬂammation, etc. As most anti-AD drugs exhibit poor

disease modifying eﬀects clinically, the Aβ cascade hypothesis

has been believed as the key dogma in the development of

novel anti-AD drugs in the past decades. Nevertheless, this

hypothesis has been recently challenged due to the continued

etc.

Therefore, GSK-3β is believed to be a promising

therapeutic target for the treatment of AD. In fact,

considerable eﬀorts have attempted to develop novel GSK-

3−21

3β inhibitors.

Representative ones include natural

products bisindolyl alkaloid staurosporine, indirubin,

−5

failures of anti-Aβ approaches in clinical studies.

These

dilemmas together with the fact of the complex nature of AD

etiopathology drive researchers to embrace an alternative

approach to simultaneously hamper the multiple components

of the disease network, which is expected to have synergistic

eﬀects and would consequently lead to be more eﬀective

modiﬁcation in the disease progression.

Received: August 10, 2020

Accepted: September 22, 2020

Published: September 22, 2020

XXXX American Chemical Society

ACS Chemical Neuroscience

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Figure 1. Representative chemical structures of GSK-3 β inhibitors.

Research Article

Figure 2. Design strategy of aminopyrazoles as GSK-3β inhibitors.

kenpaullone, hymenialdisine, and various chemical classes

On these premises, great eﬀorts have been continually put

into reﬁning and evaluating multifunctional GSK3β inhibitors,

such as CHIR98014, AR-A014418, and SB-216763

Figure 1). Currently, two potent GSK3β inhibitors, lithium

carbonate and tideglusib, have been investigated in clinical

trials in AD and exhibited statistically insigniﬁcant drug eﬃcacy

but with some positive trends,

including agents possessing additional regulative eﬀects on

other key therapeutic targets besides GSK-3β inhibitory

(

5,26

activity, such as BACE-1,

AChE),

acetylcholinesterase

27,28

(

and p38 MAPK; however, all these multifunc-

22−24

indicating that single

tional ligands have not yet been approved for the treatment of

AD. In another aspect, a great number of studies suggest that

two key pathological phenomena merit more attentionglial

intervention of GSK-3β could be important but not suﬃcient

for halting the disease progression.

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Research Article

Scheme 1. Synthesis of Monoindole-Substituted Aminopyrazole Derivatives 4a−d and 5a−d

Reagents and conditions: (a) NaH, MeI, DMF, rt, 3 h; (b) NaH, CDI, DMF, rt, 20 h; (c) N H ·H O, AcOH, EtOH, reﬂux, 3 h; (d) LHMDS,

THF, 0 °C → rt, 3 h.

Scheme 2. Synthesis of Bisindole-Substituted Aminopyrazole Derivatives 6a−m

Reagents and conditions: (a) Cl CCOCl, pyridine, THF, rt, 5 h; (b) MeOH, MeONa, rt, 0.5 h; (c) LHMDS, THF, 0 °C → rt, 3 h; (d) N H ·

H O, AcOH, EtOH, reﬂux, 3 h; (e) TFA, DCM, rt, 1 h.

inﬂammation and oxidative stress, both are present in the

preliminary and secondary phases of the disease, and

contribute signiﬁcantly to the pathogenesis and progression

disubstituted aminopyrazole derivatives (4−7) (Figure 2)

through structure elaboration of staurosporine, a classical GSK-

β inhibitor, by breaking its planar molecular conformation

0−33

of AD.

Cumulative evidence has also revealed that a large

and replacing the γ-lactam core with other heterocycles while

maintaining the required hydrogen-bonding capability, follow-

ing the principles of bioisosterism and association, as well as

taking into consideration the antioxidative or anti-inﬂamma-

window of opportunity exists to successfully block these two

pathological changes, which might eﬀectively arrest the

initiation and progress of AD and consequently block the

32,33

late manifestation of the disease.

Taken together, discovery

38,39

of GSK-3β inactivation-centric agents, especially those exhibit

potent eﬀects against both oxidative neuronal damage and

microglial inﬂammation, would bring new opportunities for

disease-modifying AD therapy.

tory activities of indole

or aminopyrazole skeletons. In

this paper, we delineate the general structure−activity

relationships of aminopyrazole derivatives against GSK-3β,

and outline the multipotent pharmacological proﬁles of the

most promising bisindole-substituted aminopyrazole deriva-

tives.

To fulﬁll this objective, on the basis of the promising GSK-

21,34−36

β inhibitory activities of maleimide

and pyrazolone

compounds such as 1 and 2, we constructed a new series of

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Research Article

Scheme 3. Synthesis of Bisindole-Substituted Aminopyrazole Derivatives 7a−c

Reagents and conditions: (a) pyridine-BH , HOAc, rt, 8 h; (b) HCHO, TFA, EtOH, rt, 10 h; (c) piperidine-1-carbonyl chloride, TEA, DCM, rt,

0 h; (d) (Boc) O, THF, rt; (e) for 22a, Pd/C, cyclohexene, xylene, 140 °C, 8 h; for 22b, DDQ, THF, rt, 1h; (f) Cl CCOCl, pyridine, THF, rt, 5

h; (g) MeOH, MeONa, rt, 0.5 h; (h) 9, LHMDS, THF, 0 °C → rt, 3 h; (i) N H ·H O, AcOH, EtOH, reﬂux, 3 h; (j) TFA, DCM, rt, 1 h.

RESULTS AND DISCUSSION

the presence of TFA led to the cyclized N-free intermediate

1c. Subsequent N-protection, followed by indoline oxidation,

provided the disired indole precursor 22. Following the

■

Chemistry. The synthetic routes of the designed target

aminopyrazole compounds are outlined in Schemes 1−3. As

illustrated in Scheme 1, the aminopyrazoles (4a−d) having an

indole-substitution at the 4-position and a phenyl group at the

previously mentioned method, new aminopyrazole compounds

a−c were readily prepared in a couple steps from 22.

In Vitro GSK-3β Inhibitory Activity. The synthesized

-position were synthesized through α-acylation of N-

aminopyrazole derivatives (4−7) were then examined for their

inhibitory activities against GSK-3β and the results are

summarized in Table 1. 4-Indolyl-based aminopyrazoles with

the indolyl group orientated to the amino group (4a−d) were

not active against GSK-3β at a maximum concentration of 10

μM, while the 5-indolyl-based aminopyrazole (5a) with

opposite orientation of the indoyl group displayed micromolar

^p^o^t^eⁿ^c^y^a^g^aⁱⁿ^s^t^G^S^K^-³^β⁽^I^C50 = 7.46 ± 1.07 μM). However,

variation of the substituents on the phenyl or indole ring of 5a

led to decreased GSK-3β inhibitory activities (5b−d).

Gratifyingly, the bisindole-substituted aminopyrazole (6a)

exhibited good GSK-3β inhibitory activity in the in vitro

^a^s^s^a^y⁽^I^C50 = 4.20 ± 0.87 μM). Moreover, introduction of a

halogen or a methoxy group at the 5-position of the indole ring

methylated indolyl-acetonitrile 9 upon treatment with NaH/

CDI in DMF followed by cyclization with hydrazine hydrate

under acidic conditions. The aminopyrazoles 5a−d with

indolyl substitution at the 5-position were also prepared

using the same protocol with β-keto cyanide compound 14 as

the key intermediate. It is noteworthy that the use of LHMDS

instead of NaH as the base can dramatically improve the

reaction yield of α-acylation (up to 99%).

The synthesis of bisindole-substituted aminopyrazole

compounds (6) commenced with Friedel−Crafts acylation of

indole derivative 15 with trichloroacetyl chloride to furnish

intermediate 16 (Scheme 2). The latter compound was then

converted to the corresponding methyl ester 17 under the

conditions of MeOH/NaOMe at room temperature. α-

Acylation of commercially available 2-(1-methyl-1H-indol-3-

yl)acetonitrile 9 with the methyl ester 17 in the presence of

LHMDS provided bisindole-substituted 3-oxopropanenitrile

enhanced the inhibitory activities against GSK-3β (IC = 1.28

0.18 μM for 6c; IC = 1.93 ± 0.22 μM for 6e; IC = 1.76 ±

50 50

.19 μM for 6h). Replacement of the ﬂuorine (6d) by a

bromine atom (6f) at the 6-position of the indole ring resulted

in a signiﬁcant loss in the potency (IC = 2.26 ± 0.35 μM for

18, which was then subjected to cyclization with hydrazine

hydrate to aﬀord the desired aminopyrazole compound 6. By

d). Substituents located at the 4- or 7-position of the indole

introducing diﬀerent R and R groups to the starting indole

material, a series of structurally interesting bisindole-containing

aminopyrazole compounds 6a−m were readily obtained.

By taking a similar approach, aminopyrazole compounds

ring led to an unfavorable inhibition on GSK-3β with

inhibitory rate less than 50% at a maximum concentration of

10 μM (6b, 6g, 6i). An aminoethyl substitution at the indole

nitrogen (6k) diminished the GSK-3β inhibition by approx-

imately 2-fold in comparison with compound 6a (IC50 = 8.81

± 0.90 μM for 6k), whereas the piperidinyl substitution (6m)

maintained the same activity as compound 6a (IC50 = 3.92 ±

0.09 μM for 6m). In contrast, the presence of N-Boc

substituents of aminoethyl or piperidinyl (6j and 6l) caused

a signiﬁcant decrease in GSK-3β inhibition. The introduction

of diazepinoindole scaﬀold, which was previously used by Eli

a−c bearing both an indole unit and a 1,2,3,4-tetrahydro-

[1,4]diazepino[6,7,1-hi]indole unit were also synthesized. As

illustrated in Scheme 3, the synthesis began with the

preparation of 1,2,3,4-tetrahydro-[1,4]diazepino[6,7,1-hi]-

indole 22 from previously obtained N-aminoethyl-indole 19.

Under acidic conditions, the indole ring was reduced with

pyridine borane at room temperature to give the correspond-

ing indoline 20, which upon treatment with formaldehyde in

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glutamate exposure. As shown in Figure 4 , stimulation of

Research Article

Table 1. In Vitro GSK-3β Inhibitory Activities of

Aminopyrazole Derivatives 4−7

Figure 3. Eﬀect of compounds 6c, 6e, 6h, 7a, and 7c on NO release

in LPS-stimulated BV-2 cells. Data are presented as percentage of the

LPS group, shown as the mean ± SEM (n = 3). p < 0.001 vs the

control group (Con), *p < 0.05, **p < 0.01, ***p < 0.001 vs the LPS

group (Veh). S: staurosporine.

and 7a at 20 μM exhibited most potent anti-inﬂammatory

eﬀects, by showing an approximately 50% reduction in NO

production, as compared to the LPS alone group.

Protective Eﬀects of Compounds 6c, 6e, 6h, 7a, and

c against Glutamate-Induced Oxidative Cytotoxicity in

HT-22 Cells. A large amount of studies depict that oxidative

30,32

neurotoxicity is closely associated with AD pathogenesis.

Glutamate-stimulated HT-22 cell death has been extensively

employed to mimic endogenous oxidative neuronal damage

44−46

and discover associated active agents.

In order to evaluate

the protective eﬀects of compounds 6c, 6e, 6h, 7a, and 7c

against glutamate-induced oxidative neuronal damage, HT-22

cells were treated with these compounds for 2 h before

Results are expressed as mean ± SEM of at least three independent

experiments. Inhibitory activities of compounds were not favorable

at primary screening (inhibitory rate less than 50% at 10 μM), of

which IC₅₀values were not further tested.

Lilly & Co. for glycogen synthase kinase-3 inhibitors,

resulted in a ∼3-fold increase in potency compared with

compound 6a (IC = 1.48 ± 0.30 μM for 7c). The attachment

of an amide moiety to the diazepinoindole nitrogen atom

(

compound 7a) resulted in a similar GSK3β inhibitory potency

IC₅₀= 1.46 ± 0.04 μM for 7a), as compared to 7c. Taken

Figure 4. Compounds 6c, 6e, 6h, 7a, and 7c protected HT-22 cells

together, compounds 6c, 6e, 6h, 7a, and 7c are found to be the

most potent GSK3β inhibitors and thus chosen for further

biological studies.

against glutamate-induced cytotoxicity. Data are presented as

###

percentage of control group, shown as mean ± SEM (n = 3).

0.001 vs the control group (Con), *p < 0.05, **p < 0.01, ***p <

.001 vs the glutamate group (Veh). Glu: glutamate; S: staurosporine.

Inhibitory Eﬀects of Compounds 6c, 6e, 6h, 7a, and

c against LPS-Induced NO Overproduction in BV-2

Microglial Cells. Accumulating evidence revealed that

microglial inﬂammation plays a critical role in the pathogenesis

of AD, while therapeutic interventions aimed at modulating

glutamate alone on HT-22 cells signiﬁcantly reduced the cell

survival, while pretreatment with 1 and 10 μM of compounds

6c, 6e, and 6h all exhibited potent protective eﬀects against

glutamate-induced oxidative neurotoxicity in HT-22 cells.

Compounds 7a and 7b at 10 μM also markedly alleviated the

oxidative neuronal damage in HT-22 cells; however, no

detectable protection was observed at a lower concentration (1

μM). The cytotoxic eﬀects of compounds 6c, 6e, 6h, 7a, and

7c were further measured on both HT-22 cells and BV-2 cells.

As shown in Figure S1 in the Supporting Information, all of the

tested compounds showed no noticeable inﬂuence on the cell

viabilities of both BV-2 and HT-22 at their pharmacological

31,33

inﬂammatory activity is favorable to treat AD.

To evaluate

the potential ploypharmacological eﬀects of aforementioned

promising GSK3β inhibitors, in vitro anti-inﬂammatory eﬀects

were ﬁrst assessed through measuring NO release in LPS-

stimulated BV-2 cells, a well-known microglial inﬂammatory

2,43

cellular model.

As shown in Figure 3, 100 ng/mL LPS

exposure for 24 h signiﬁcantly induced NO production in BV-2

cells, while preincubation with compounds 6c, 6e, 6h, 7a, and

c all markedly suppressed LPS-induced NO production in a

dose-dependent manner. Among these tested compounds, 6h

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Research Article

Figure 5. Pretreatment with 6h signiﬁcantly reduced glial activation in the hippocampus of LPS-injected mice. (A) Experimental scheme. (B)

Microglia activation and astrocyte proliferation were measured by immunoﬂuorescence staining. Scale bar = 100 μm. (C) Quantiﬁcation of IBA1

positive cells. (D) Quantiﬁcation of GFAP positive cells. The protein levels of CD11b (E) and GFAP (F) were determined by Western blot

analysis. GAPDH was used as loading control. Data are presented as percentage of the control group (Con), shown as the mean ± SEM (n = 4−5).

###

p < 0.05, p < 0.01, p < 0.001 vs the control group (Con), *p < 0.05, **p < 0.01, ***p < 0.001 vs the LPS alone group (Vehicle).

eﬀective concentrations (1 or 10 μM). Compound 6h at 40

μM and compound 7c at both 40 μM and 80 μM showed no

signiﬁcant changes on the cell survival of HT-22 cells,

suggesting these two compounds are less cytotoxic. Taken

with the less potent eﬀect of compound 7c on glutamate-

induced oxidative damage in HT-22 cells, compound 6h is

expected as the most promising lead compound with good

biological activity and safety window (in vitro).

In Vivo Eﬀect of 6h against Glial Inﬂammation in the

Brain of LPS-Injected Mice. Together with the results of

above in vitro studies, compound 6h was then chosen for

further in vivo study. To assess whether compound 6h has an

anti-inﬂammatory eﬀect in animals, microglial activation and

astrocyte proliferation in the brain of LPS-injected mice were

measured by IBA-1 and GFAP immunoﬂuorescence staining,

respectively. The LPS-injected mice showed an increased

number of IBA-1 and GFAP positive cells in the hippocampus

(

Figure 5B−D), indicating microglial activation and astrocyte

proliferation in the brain. In contrast, compound 6h (200 mg/

kg) signiﬁcantly reduced the number of activated microglia

(

Figure 5B,C) and astrocytes (Figure 5B,D) induced by LPS.

To further conﬁrm the anti-inﬂammatory eﬀect of 6h, we

measured the CD11b and GFAP protein levels in the

hippocampus of LPS-injected mice by Western blot analysis.

Similar to the results of immunoﬂuorescence staining, CD11b

and GFAP levels were markedly increased in the hippocampus

of the LPS-injected mice, while 6h treatment signiﬁcantly

reduced the levels of CD11b (Figure 5E) and GFAP (Figure

5F) in the hippocampus of LPS-exposed mice, suggesting 6h

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Research Article

may play a role in mitigating inﬂammatory responses in the

brain of mice.

chromatography (hexane−EtOAc, 2:1) to give the substituted

carboxylates.

General Procedure D for the Condensation to Give 3-

Oxopropanenitriles. To a suspension of indolyl-3-carboxylate (1

equiv) and 2-(1-methyl-1H-indol-3-yl) acetonitrile (1.1 equiv) in dry

THF (10 mL/mmol) at 0−5 °C was added a 1.6 M solution of

LHDMS in THF (3 equiv) in a dropwise manner. The reaction

mixture was stirred at room temperature overnight. Upon completion

of the reaction as indicated by TLC, the reaction mixture was

CONCLUSION

■

In summary, we have designed and synthesized a series of 4,5-

disubstituted aminopyrazole derivatives as a novel class of

multifunctional GSK-3β inhibitors. Five bisindolyl-substituted

aminopyrazoles (6c, 6e, 6h, 7a, and 7c) possess potent GSK-

quenched with saturated aqueous NH Cl and extracted with EtOAc

β inhibitory activities with suppressive eﬀects against glial

(

3 × 20 mL). The combined organic phases were washed with water

inﬂammation and oxidative neurotoxicity. In addition,

compound 6h signiﬁcantly reduces the microglial activation

and astrocyte proliferation in the brain of LPS-injected mice,

showing potent in vivo anti-inﬂammatory eﬀect. This new

multipotent GSK-3β inhibitor could be a useful prototype for

the discovery of therapeutic agents to tackle AD and other

GSK-3β associated complex neurological syndromes.

50 mL) then brine (50 mL), dried over Na SO , and evaporated in

vacuo. The residue was puriﬁed by ﬂash chromatography (hexane−

EtOAc, 1:1) to give the cyclization precursors3-oxopropanenitriles.

General Procedure E for the Condensation to Give Target

Aminopyrazoles 4a−d, 5a−d, 6a−m, and 7a−c. To a solution of

the substituted 3-oxopropanenitriles (1 equiv) in ethanol (5 mL/

mmol) was added N H ·H O (10 equiv) and HOAc (10 equiv) at

ambient temperature. The reaction mixture was stirred at 80 °C oil

bath for 3 h. Upon completion of the reaction as ascertained by TLC,

the reaction mixture was cooled to room temperature, quenched with

METHODS

■

saturated aqueous NaHCO , and extracted with CH Cl (3 × 20 mL).

Chemistry. General Method. Starting materials, reagents, and

solvents were purchased from commercial suppliers and used without

further puriﬁcation unless otherwise stated. Anhydrous THF and

CH Cl were obtained by distillation over sodium wire or CaH ,

The combined organic phases were washed with water (50 mL) then

brine (50 mL), dried over Na SO , and evaporated in vacuo. The

residue was puriﬁed by ﬂash chromatography (CH Cl −MeOH,

0:1) to give the desired aminopyrazoles.

respectively. The progress of reactions was monitored by TLC on

-(1-Methyl-1H-indol-3-yl)-5-phenyl-1H-pyrazol-3-amine (4a).

SiO . For ﬂash chromatography, 100−200 mesh particle size SiO was

Brown solid, 88% yield. H NMR (300 MHz, DMSO) δ 11.94 (br,

H), 7.46−7.36 (m, 3H), 7.33 (s, 1H), 7.26−7.14 (m, 3H), 7.10 (t, J

7.6 Hz, 1H), 6.96 (d, J = 7.8 Hz, 1H), 6.84 (t, J = 7.4 Hz, 1H), 4.37

used. H NMR and C NMR spectra were recorded on a Bruker

spectrometer at 300 or 400 MHz, and were referenced to the residual

peaks of CDCl at 7.26 ppm or DMSO-d at 2.50 ppm ( H NMR)

(s, 2H), 3.81 (s, 3H). C NMR (125 MHz, DMSO) δ 136.79,

and CDCl at 77.2 ppm or DMSO-d at 39.51 ppm ( C NMR).

28.59, 128.17, 127.03, 126.14, 121.04, 119.66, 118.52, 109.67,

Chemical shifts were reported in parts per million (ppm) downﬁeld of

tetramethylsilane (TMS) and the following abbreviations used to

denote coupling patterns: s = singlet; d = doublet, t = triplet, q =

quartet, br = broad. HRMS experiments were performed on a Q-TOF

mass spectrometer with ESI resource.

General Procedure A for the Indole N-Alkylation. To a solution of

substituted indole (1 equiv) in dry DMF (3 mL/mmol) cooled with

an ice bath was added NaH (60% suspension in mineral oil, 1.5

equiv). After stirring at 0 to 5 °C for 30 min, an alkyl chloride,

bromide, or iodomethane (1.2 equiv) was added in one portion. The

reaction mixture was stirred overnight at ambient temperature. Upon

completion of the reaction as indicated by TLC, the reaction mixture

was poured into ice−water and extracted with EtOAc (3 × 25 mL).

The combined organic phases were washed with water (50 mL) then

brine (50 mL), dried over anhydrous Na SO , and concentrated in

105.81, 32.43. HRMS (ESI) for C H N [M + H] : calcd 289.1448,

18 17 4

found 289.1445.

-(1-Methyl-1H-indol-3-yl)-5-(p-tolyl)-1H-pyrazol-3-amine (4b).

Brown solid, 96% yield. H NMR (300 MHz, DMSO) δ 11.87 (br,

1H), 7.42 (d, J = 8.3 Hz, 1H), 7.35−7.22 (m, 3H), 7.10 (t, J = 7.4 Hz,

1H), 7.04−6.91 (m, 3H), 6.84 (t, J = 7.4 Hz, 1H), 4.31 (s, 2H), 3.81

(s, 3H), 2.21 (s, 3H). C NMR (125 MHz, DMSO) δ 136.8, 136.3,

128.8, 128.5, 127.0, 126.0, 121.0, 119.7, 118.5, 109.6, 105.9, 32.4,

20.7. HRMS (ESI) for C19

[M + H] : calcd 303.1604, found

303.1601.

4-(1-Methyl-1H-indol-3-yl)-5-(m-tolyl)-1H-pyrazol-3-amine (4c).

Brown solid, 89% yield. H NMR (300 MHz, DMSO) δ 11.90 (br,

1H), 7.42 (d, J = 8.1 Hz, 1H), 7.32 (d, J = 6.3 Hz, 2H), 7.10 (s, 2H),

7.01 (dd, J = 18.2, 7.7 Hz, 3H), 6.85 (t, J = 7.4 Hz, 1H), 4.34 (s, 2H),

vacuo. The crude product was either puriﬁed by ﬂash chromatography

3.81 (s, 3H), 2.16 (s, 3H). C NMR (125 MHz, DMSO) δ 137.1,

(

hexane to hexane−EtOAc, 20:1) or directly used for further reaction

136.7, 128.6, 128.0, 127.7, 127.0, 126.8, 123.5, 121.0, 119.7, 118.5,

without additional puriﬁcation.

109.6, 105.9, 32.4, 21.0. HRMS (ESI) for C H N [M + H] : calcd

General Procedure B for the Indole Trichloroacetylation. To a

solution of the substituted N-alkylindole (1 equiv) and pyridine (2

equiv) in dry THF (5 mL/mmol) cooled with an ice bath was added

trichloroacetyl chloride (1.5 equiv) dropwise. The reaction mixture

was allowed to slowly warm to room temperature overnight. Upon

reaction completion as indicated by TLC, the reaction mixture was

cooled with an ice bath and carefully quenched by slow addition of

303.1604, found 303.1603.

5-(3-Bromophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrazol-3-

amine (4d). Brown solid, 83% yield. H NMR (300 MHz, DMSO) δ

12.02 (br, 1H), 7.66 (s, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.32 (d, J = 8.1

Hz, 3H), 7.18−7.03 (m, 2H), 7.01−6.80 (m, 2H), 4.48 (s, 2H), 3.82

(s, 3H). C NMR (125 MHz, DMSO) δ 136.8, 130.2, 129.5, 128.7,

128.5, 126.9, 124.9, 121.6, 121.1, 119.5, 118.7, 109.7, 105.3, 32.5.

saturated aqueous NH Cl. The reaction mixture was extracted with

HRMS (ESI) for C18

H16BrN

[M + H] : calcd 367.0553, found

CH Cl (3 × 25 mL), and the combined organic phases were washed

367.0551.

with saturated aqueous NaHCO (2 × 50 mL), water (50 mL), and

5-(1-Methyl-1H-indol-3-yl)-4-phenyl-1H-pyrazol-3-amine (5a).

Brown solid, 84% yield. H NMR (300 MHz, DMSO) δ 11.76 (br,

brine (50 mL), dried over anhydrous Na SO , and concentrated in

vacuo. The residue was puriﬁed by ﬂash chromatography (hexane−

EtOAc, 10:1) to give the substituted trichloroacetylates.

1H), 7.41 (d, J = 8.1 Hz, 1H), 7.27 (d, J = 16.7 Hz, 4H), 7.12 (d, J =

6.4 Hz, 3H), 6.86 (t, J = 7.2 Hz, 1H), 4.52 (s, 2H), 3.77 (s, 3H).

General Procedure C for the Indole 3-Carboxylation. To a

solution of the substituted indole 3-trichloroacetylates (1 mmol) in

methanol (5 mL) was added MeONa (1.5 equiv, 5 mol/L) at ambient

temperature. The mixture was stirred for 30 min and quenched by

NMR (125 MHz, DMSO) δ 136.5, 134.3, 128.5, 128.3, 128.2, 125.6,

125.1, 121.4, 120.2, 119.1, 109.8, 32.5. HRMS (ESI) for C H N [M

+ H] : calcd 289.1448, found 289.1444.

5-(5-Methoxy-1-methyl-1H-indol-3-yl)-4-phenyl-1H-pyrazol-3-

slow addition of saturated aqueous NH Cl. The reaction mixture was

amine (5b). Brown solid, 78% yield. H NMR (300 MHz, DMSO) δ

extracted with EA (3 × 25 mL), and the combined organic phases

11.63 (br, 1H), 7.26 (dd, J = 13.9, 6.6 Hz, 6H), 7.13 (d, J = 4.2 Hz,

1H), 6.69 (d, J = 8.9 Hz, 1H), 6.39 (s, 1H), 4.48 (s, 2H), 3.75 (s,

3H), 3.36 (s, 3H). C NMR (125 MHz, DMSO) δ 153.3, 134.5,

were washed with brine (50 mL), dried over anhydrous Na SO , and

concentrated in vacuo. The residue was puriﬁed by ﬂash

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

Research Article

31.7, 128.6, 128.4, 128.3, 125.5 125.1, 111.9, 110.6, 109.5, 101.5,

HRMS (ESI) for C H BrN [M + H] : calcd 420.0818, found

21 19 5

420.0821.

4.7, 32.7. HRMS (ESI) for C H N O [M + H] : calcd 319.1553,

found 319.1552.

5-(7-Bromo-1-methyl-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-yl)-

-(5-Bromo-1-methyl-1H-indol-3-yl)-4-phenyl-1H-pyrazol-3-

1H-pyrazol-3-amine (6g). Brown solid, 85% yield. H NMR (300

amine (5c). Brown solid, 80% yield. H NMR (300 MHz, DMSO) δ

MHz, DMSO) δ 8.93 (s, 1H), 7.62 (d, J = 7.7 Hz, 1H), 7.40 (d, J =

8.3 Hz, 1H), 7.26 (d, J = 6.9 Hz, 2H), 7.15 (s, 1H), 7.09 (dd, J = 7.5,

4.4 Hz, 2H), 6.82 (dt, J = 15.4, 7.6 Hz, 2H), 4.36 (s, 2H), 3.95 (s,

1.78 (br, 1H), 7.40 (d, J = 8.7 Hz, 1H), 7.30 (t, J = 9.3 Hz, 3H),

.27−7.12 (m, 5H), 4.60 (s, 2H), 3.75 (s, 3H). C NMR (125 MHz,

DMSO) δ 135.2, 134.1, 129.3, 128.8, 128.4, 127.2, 125.4, 123.8,

3H), 3.78 (s, 3H), 3.17 (s, 3H). C NMR (125 MHz, DMSO) δ

122.8, 111.9, 111.9, 32.7. HRMS (ESI) for C H 6BrN [M + H] :

168.6, 167.9, 136.6, 132.2, 131.1, 129.4, 128.5, 127.1, 126.3, 120.9,

calcd 367.0553, found 367.0555.

120.6, 120.5, 119.7, 118.4, 109.6, 106.1, 102.9, 36.4, 32.4. HRMS

-(1-Methyl-1H-indol-3-yl)-4-(o-tolyl)-1H-pyrazol-3-amine (5d).

(ESI) for C21

19BrN

[M + H] : calcd 420.0818, found 420.0820.

Brown solid, 87% yield. H NMR (300 MHz, DMSO) δ 11.69 (s,

5-(5-Methoxy-1-methyl-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-

yl)-1H-pyrazol-3-amine (6h). Brown solid, 84% yield. H NMR (300

H), 7.47 (d, J = 7.6 Hz, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.27−7.05

MHz, DMSO) δ 11.59 (br, 1H), 7.40 (d, J = 8.2 Hz, 1H), 7.20 (dd, J

= 10.5, 6.9 Hz, 4H), 7.09 (t, J = 7.6 Hz, 1H), 6.86 (t, J = 7.4 Hz, 1H),

(m, 5H), 6.96−6.84 (m, 2H), 4.27 (s, 2H), 3.65 (s, 3H), 2.06 (s, 3H).

C NMR (125 MHz, DMSO) δ 137.6, 136.3, 133.5, 131.5, 129.9,

.65 (d, J = 8.7 Hz, 2H), 4.25 (s, 2H), 3.74 (s, 3H), 3.64 (s, 3H), 3.25

27.1, 126.8, 125.7, 125.5, 121.4, 120.5, 119.1, 109.6, 32.4, 19.8.

(s, 3H). C NMR (125 MHz, DMSO) δ 153.3, 136.7, 131.5, 128.5,

HRMS (ESI) for C H N [M + H] : calcd 303.1604, found

28.0, 127.5, 125.8, 121.0, 119.7, 118.5, 111.8, 110.3, 109.5, 106.7,

03.1601.

01.7, 54.4, 32.6, 32.3. HRMS (ESI) for C H N O [M + H] : calcd

,5-Bis(1-methyl-1H-indol-3-yl)-1H-pyrazol-3-amine (6a). Brown

22 22

372.1819, found 372.1817.

solid, 84% yield. H NMR (300 MHz, DMSO) δ 11.66 (br, 1H), 7.58

d, J = 7.9 Hz, 1H), 7.38 (dd, J = 13.2, 8.2 Hz, 2H), 7.28 (s, 1H),

-(7-Methoxy-1-methyl-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-

(

yl)-1H-pyrazol-3-amine (6i). Brown solid, 84% yield. H NMR (300

MHz, DMSO) δ 11.59 (br, 1H), 7.39 (d, J = 8.2 Hz, 1H), 7.26 (s,

.15−7.04 (m, 4H), 6.92 (t, J = 7.5 Hz, 1H), 6.84 (t, J = 7.4 Hz, 1H),

.27 (s, 2H), 3.78 (s, 3H), 3.63 (s, 3H). C NMR (125 MHz,

(

H), 7.08 (t, J = 8.0 Hz, 3H), 6.99 (s, 1H), 6.91−6.72 (m, 2H), 6.59

DMSO) δ 136.7, 136.3, 128.6, 127.8, 127.2, 125.8, 121.3, 120.9,

d, J = 7.7 Hz, 1H), 4.26 (s, 2H), 3.83 (s, 6H), 3.77 (s, 3H).

NMR (125 MHz, DMSO) δ 147.2, 136.6, 128.8, 128.5, 128.3, 127.2,

25.7, 120.9, 119.8, 119.7, 118.3, 113.5, 109.5, 106.4, 102.7, 55.5,

20.6, 119.8, 119.1, 118.3, 109.6, 106.4, 32.4, 32.4. HRMS (ESI) for

C H N [M + H] : calcd 342.1713, found 342.1709.

-(4-Fluoro-1-methyl-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-yl)-

36.2, 32.4. HRMS (ESI) for C H N O [M + H] : calcd 372.1819,

22 22 5

H-pyrazol-3-amine (6b). Brown solid, 81% yield. H NMR (300

found 372.1817.

MHz, DMSO) δ 11.50 (br, 1H), 7.34 (d, J = 8.2 Hz, 1H), 7.23 (d, J =

.9 Hz, 2H), 7.19−6.98 (m, 4H), 6.78 (dd, J = 13.2, 5.2 Hz, 2H), 4.19

tert-Butyl (2-(3-(3-amino-4-(1-methyl-1H-indol-3-yl)-1H-pyrazol-

-yl)-1H-indol-1-yl)ethyl)carbamate(6j). Brown solid, 91% yield. H

(s, 2H), 3.74 (s, 3H), 3.66 (s, 4H). C NMR (125 MHz, DMSO) δ

NMR (300 MHz, DMSO-d ) δ 11.63 (br, 1H), 7.65 (s, 1H), 7.40

55.8 (J_C₋_F= 295.5 Hz), 139.0 (J_C₋_F= 11.3 Hz), 136.5, 129.5, 128.2,

27.0, 122.0 (J_C₋_F= 8.8 Hz), 120.8, 119.7, 118.2, 115.0 (J_C₋_F= 18.8

(

dd, J = 8.4, 3.7 Hz, 2H), 7.28 (s, 1H), 7.16−7.02 (m, 4H), 6.94 (t, J

7.5 Hz, 1H), 6.83 (t, J = 7.2 Hz, 2H), 4.31 (s, 1H), 4.03 (s, 2H),

.79 (s, 2H), 3.08 (d, J = 6.5 Hz, 2H), 1.32 (s, 9H).

-(1-(2-Aminoethyl)-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-yl)-

Hz), 109.5, 106.4 (J_C₋_F= 2.5 Hz), 106.2, 104.7 (J

= 18.8 Hz),

C−F

32.8, 32.3. HRMS (ESI) for C H FN [M + H] : calcd 360.1619,

21 19 4

found 360.1617.

H-pyrazol-3-amine (6k). Brown solid, 91% yield. H NMR (300

-(5-Fluoro-1-methyl-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-yl)-

MHz, DMSO-d ) δ 7.71 (br, 1H), 7.52 (d, J = 8.3 Hz, 1H), 7.41 (d, J

H-pyrazol-3-amine (6c). Brown solid, 88% yield. H NMR (300

MHz, DMSO) δ 7.47−7.24 (m, 4H), 7.20−7.05 (m, 3H), 7.00−6.81

m, 2H), 4.34 (s, 2H), 3.80 (s, 3H), 3.62 (s, 3H). ¹³C NMR (125

MHz, DMSO) δ 157.1 (J_C₋_F= 231.3 Hz), 136.7, 133.1, 129.4, 128.6,

27.2, 125.9 (J_C₋_F= 10.0 Hz), 121.0, 119.7, 118.4, 110.7 (J_C₋_F= 10.0

8.1 Hz, 1H), 7.34 (s, 1H), 7.19−6.93 (m, 5H), 6.83 (t, J = 7.4 Hz,

H), 4.36 (br, 1H), 4.23 (d, J = 6.7 Hz, 2H), 3.80 (s, 3H), 2.90 (m,

(

H). HRMS (ESI) for C H N [M + H] : calcd 370.1979, found

70.1986.

tert-Butyl 4-(3-(3-amino-4-(1-methyl-1H-indol-3-yl)-1H-pyrazol-

^H^z⁾^,¹⁰⁹^.⁶^,¹⁰⁹^.⁶^,¹⁰⁹^.³^,¹⁰⁶^.²^,¹⁰⁵^.⁴⁽^JC−F = 25.0 Hz), 32.7, 32.4.

HRMS (ESI) for for C H FN [M + H] : calcd 360.1619, found

5-yl)-1H-indol-1-yl)piperidine-1-carboxylate (6l). Brown solid, 80%

yield. H NMR (300 MHz, DMSO) δ 11.62 (br, 1H), 7.54 (s, 1H),

60.1617.

(

.40 (d, J = 8.4 Hz, 1H), 7.28 (s, 1H), 7.08 (s, 3H), 6.99−6.76 (m,

-(6-Fluoro-1-methyl-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-yl)-

H), 4.62 (s, 2H), 4.31 (s, 1H), 4.09 (s, 2H), 3.78 (s, 3H), 3.77−3.69

H-pyrazol-3-amine (6d). Brown solid, 90% yield. H NMR (300

m, 2H), 3.02 (s, 4H), 1.47 (s, 6H). C NMR (125 MHz, DMSO) δ

MHz, DMSO) δ 11.66 (br, 1H), 7.58 (s, 1H), 7.41 (d, J = 8.3 Hz,

53.7, 136.6, 135.1, 128.5, 126.8, 125.8, 123.6, 121.2, 120.8, 120.1,

H), 7.34−7.19 (m, 2H), 7.16−7.02 (m, 3H), 6.90−6.71 (m, 2H),

19.3, 118.2, 109.7, 109.5, 106.5, 78.8, 51.8, 33.1, 32.3, 31.7, 28.1.

.33 (s, 2H), 3.79 (s, 3H), 3.59 (s, 3H). C NMR (125 MHz,

HRMS (ESI) for C H N O [M + H] : calcd 511.2816, found

DMSO) δ 159.4 (J_C₋_F= 233.8 Hz), 137.1, 136.8 (J_C₋_F= 12.5 Hz),

29.1, 128.7, 127.7, 123.1, 122.4, 121.4, 120.3, 118.9, 110.1, 107.9

^JC−F = 25.0 Hz), 106.7, 96.5 (J = 25.0 Hz), 33.1, 32.9. HRMS

11.2822.

-(1-Methyl-1H-indol-3-yl)-5-(1-(piperidin-4-yl)-1H-indol-3-yl)-

(

C−F

1H-pyrazol-3-amine (6m). Brown solid, 91% yield. H NMR (300

ESI) for C H FN [M + H] : calcd 360.1619, found 360.1625.

MHz, DMSO-d ) δ 7.80 (br, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.52−

-(5-Bromo-1-methyl-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-yl)-

.37 (m, 2H), 7.31 (s, 1H), 7.17−6.88 (m, 5H), 6.86−6.73 (m, 1H),

H-pyrazol-3-amine (6e). Brown solid, 86% yield. H NMR (300

.80 (s, 3H), 3.10−2.90 (m, 4H), 2.64 (m, 2H), 1.71 (m, 2H), 1.54−

MHz, DMSO) δ 11.68 (br, 1H), 7.80 (s, 1H), 7.43 (d, J = 8.1 Hz,

.37 (m, 2H). HRMS (ESI) for C H N [M + H] : calcd 411.2292,

H), 7.34 (d, J = 8.7 Hz, 1H), 7.29 (s, 1H), 7.21 (dd, J = 8.7, 1.8 Hz,

H), 7.10 (t, J = 8.7 Hz, 3H), 6.86 (t, J = 7.5 Hz, 1H), 4.38 (s, 2H),

found 411.2290.

(

7-(3-Amino-4-(1-methyl-1H-indol-3-yl)-1H-pyrazol-5-yl)-3,4-di-

.81 (s, 3H), 3.60 (s, 3H). C NMR (125 MHz, DMSO) δ 136.7,

35.0, 128.9, 128.7, 127.4, 127.3, 123.7, 123.3, 121.0, 119.7, 118.4,

11.9, 111.7, 109.6, 106.1, 32.6, 32.4. HRMS (ESI) for C H BrN

hydro-[1,4]diazepino[6,7,1-hi]indol-2(1H)-yl)(piperidin-1-yl)-

methanone (7a). Brown solid, 66% yield. H NMR (300 MHz,

DMSO) δ 11.62 (br, 1H), 7.54 (s, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.28

(s, 1H), 7.08 (s, 3H), 6.99−6.76 (m, 3H), 4.62 (s, 2H), 4.31 (s, 1H),

4.09 (s, 2H), 3.78 (s, 3H), 3.77−3.69 (m, 2H), 3.02 (s, 4H), 1.47 (s,

[

M + H] : calcd 420.0818, found 420.0823.

-(6-Bromo-1-methyl-1H-indol-3-yl)-4-(1-methyl-1H-indol-3-yl)-

H-pyrazol-3-amine (6f). Brown solid, 87% yield. H NMR (300

6H). C NMR (125 MHz, DMSO) δ 163.2, 136.6, 135.3, 128.7,

MHz, DMSO) δ 11.70 (br, 2H), 7.64 (d, J = 1.5 Hz, 1H), 7.56 (s,

128.6, 127.7, 127.2, 123.9, 120.9, 120.2, 119.8, 119.6, 119.4, 118.3,

109.6, 106.2, 53.2, 49.5, 48.9, 47.7, 32.4, 25.2, 24.1. HRMS (ESI) for

H), 7.41 (d, J = 8.1 Hz, 1H), 7.29 (s, 1H), 7.17−6.98 (m, 4H), 6.84

(

t, J = 7.4 Hz, 1H), 4.42 (s, 2H), 3.79 (s, 3H), 3.62 (s, 3H). C NMR

125 MHz, DMSO) δ 137.2, 136.7, 128.6, 128.5, 127.2, 124.8, 122.5,

C H N O [M + H] : calcd 494.2663, found 494.2672.

tert-Butyl 7-(3-amino-4-(1-methyl-1H-indol-3-yl)-1H-pyrazol-5-

yl)-3,4-dihydro-[1,4]diazepino[6,7,1-hi]indole-2(1H)-carboxylate

121.9, 121.0, 119.8, 118.4, 114.3, 112.5, 109.6, 106.2, 32.6, 32.4.

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

The Supporting Information is available free of charge at

Research Article

(

7b). Brown solid, 83% yield. H NMR (300 MHz, DMSO-d ) δ

treated with 0.5 mg/mL MTT for 3 h. The formed formazan was

dissolved with DMSO (100 μL/well), and the absorbance was

measured at 490 nm with a DTX 800 Multimode Detector (Beckman

Coulter, Fullerton, CA).

Animals. Male C57/B6J mice (6-week-old) were obtained from

Shanghai SLAC Laboratory Animal Co. Ltd. Mice were housed in a

pathogen-free facility and kept under standard housing conditions

with a 12 h light/dark cycle. All the animal experimental procedures

were approved by the Animal Care and Use Committee of Shanghai

Institute of Materia Medica.

1.65 (br, 1H), 7.64−7.47 (m, 1H), 7.40 (d, J = 8.2 Hz, 1H), 7.28 (s,

H), 7.16−7.00 (m, 3H), 6.97−6.77 (m, 3H), 4.75 (d, J = 9.8 Hz,

H), 4.33 (s, 2H), 4.14 (d, J = 12.8 Hz, 2H), 3.80−3.72 (m, 2H),

.78 (s, 3H), 1.32 (d, J = 24.9 Hz, 9H). C NMR (100 MHz,

DMSO) δ 137.1, 135.7, 135.5, 129.0, 128.2, 127.5, 124.5, 124.4,

21.4, 120.6, 120.3, 120.1, 119.75, 118.7, 110.1, 106.7, 79.7, 79.6,

9.9, 49.5, 48.4, 32.9, 28.5. HRMS (ESI) for C H N O [M + H] :

calcd 483.2503, found 483.2506.

-(1-Methyl-1H-indol-3-yl)-5-(1,2,3,4-tetrahydro-[1,4]diazepino-

6,7,1-hi]indol-7-yl)-1H-pyrazol-3-amine (7c). Brown solid, 77%

yield. H NMR (300 MHz, DMSO-d ) δ 7.79 (br, 1H), 7.40 (d, J

[

Experimental Procedure of LPS Injection in Mice. Mice were

randomly divided into ﬁve groups: Control group, LPS (5 mg/kg, i.p.)

group, LPS + compound 6h (50 mg/kg, i.g.) group, LPS + compound

6h (100 mg/kg, i.g.) group, LPS + compound 6h (200 mg/kg, i.g.)

group. LPS (Sigma, St. Louis, MO, USA) was dissolved in saline

(0.9% NaCl), whereas compound 6h was dissolved in 0.4% CMC−

Na. Mice were daily administrated with compound 6h at indicated

doses (50, 100, or 200 mg/kg, i.g.) or vehicle (0.4% CMC-Na, i.g.) as

illustrated in Figure 5A. Forty minutes after the second drug

administration, mice were administrated with LPS (5 mg/kg, i.p.)

with an equal volume of vehicle injected to the control group. The

brains of mice were collected immediately at 24 h after LPS injection.

For immnunoﬂuorescence staining, half of the brain of mice was

ﬁxed in 4% PFA at 4 °C followed by 5 days incubation with 30%

sucrose at 4 °C. Subsequently, the brains were sectioned in a cryostat

(30 μm thick). Free-ﬂoating sections were processed for immuno-

ﬂuorescence staining. In brief, the sections were rinsed in PBS and

blocked in 1% bovine serum albumin (BSA), 5% goat serum, and

0.2% Triton X-100 in PBS for 2 h. Then, the sections were incubated

with rabbit anti-IBA1 (1:100, Wako, Japan) for microglia or rabbit

anti-GFAP (1:200, CST) for astrocytes at 4 °C overnight. Sections

were then incubated with Alexa Fluor 564-conjugated anti-rabbit IgG

(1:200) or ﬂuorescein 488-conjugated anti-mouse IgG (1:200) for 2 h

at room temperature. The sections were subsequently mounted on

lysine-coated glass slides. Images of the stained sections were captured

using a ﬂuorescence microscope (NanoZoomer HT, Hamamatsu,

Japan).

8.3 Hz, 1H), 7.27 (s, 1H), 7.16−7.01 (m, 3H), 6.92−6.77 (m, 3H),

.29 (br, 2H), 4.11 (s, 2H), 4.04−3.88 (m, 2H), 3.78 (s, 3H), 2.89−

.71 (m, 2H). HRMS (ESI) for C H N [M + H] : calcd 383.1979,

found 383.1983.

Assessment of GSK-3β Inhibitory Activity in Vitro. The in

vitro inhibitory eﬀects of compounds on GSK-3β were evaluated with

human recombinant GSK-3β (gifted from Prof. Yechun Xu from

Shanghai Institute of Materia Medica, Chinese Academy of Sciences)

by using Kinase-Glo reagent kit (Promega, Madison, WI, USA)

according to previous report with a slight modiﬁcation. All the test

compounds were dissolved and diluted in dimethyl sulfoxide

(

DMSO). The primary screening was carried out in 384-well black

plates and IC₅₀evaluation was carried out in 96-well black plates. The

reaction of primary screening was initiated by mixture of 1 μL of test

compounds (10 μM ﬁnal concentration), 29 μL of assay buﬀer

containing substrate (25 μM ﬁnal concentration) and ATP (5 μM

ﬁnal concentration), and 10 μL of human recombinant GSK-3β (8

ng). For IC₅₀determination, reaction was initiated by mixture of 1 μL

of compounds (at least 6 gradient concentrations), 89 μL of assay

buﬀer containing substrate (25 μM ﬁnal concentration) and ATP (5

μM ﬁnal concentration), and 10 μL of human recombinant GSK-3β

(

20 ng). After incubation at 30 °C for 30 min, the enzymatic reaction

was terminated by addition of 40 μL (for primary screening) or 100

μL (for IC₅₀determination) of Kinase-Glo reagent, respectively.

Luminescence was recorded after 10 min using a microplate reader

For Western blot analysis, the hippocampus was dissected from the

other half of the brain and lysed with RIPA buﬀer supplemented with

a protease inhibitor cocktail on ice. Protein concentrations were

determined using a BCA kit (Thermo, Rockford, IL, USA). Proteins

were separated by 10% SDS−polyacrylamide gels and transferred to

nitrocellulose blotting membranes (0.45 μm). The membranes were

blocked with 5% skimmed milk in TBST and incubated overnight at 4

(

PerkinElmer, Waltham, MS, USA). The inhibitory rate of tested

compounds were normalized by GSK-3β activity of control group,

and the IC₅₀was deﬁned as the concentration of the compound that

reduced 50% of the enzymatic activity without inhibitor.

LPS Stimulation and Nitrite Determination in BV-2 Cells.

BV-2 microglial cell line was cultured in DMEM (Life Tech, Grand

Island, USA) supplemented with 10% fetal bovine serum (FBS,

C with antibodies to CD11b (1:1000; Abcam, Cambridge, UK),

Gibco, Grand Island, USA). The cells were seeded at a density of 2 ×

GFAP (1:2000; Cell Signaling Technology, Beverly, MA, USA), and

GAPDH (1:20 000; Cell Signaling Technology, Beverly, MA, USA).

The membranes were washed three times with TBST, followed by

incubation with horseradish peroxidase-conjugated secondary anti-

bodies (1:5000, Kangchen Biotechnology, Shanghai, China). Density

measurements of the bands were analyzed using ImageJ.

0 cells/mL in 96-well plates with 100 μL/well and maintained in a

humidiﬁed atmosphere of 5% CO₂and 95% air at 37 °C. To

determine the in vitro anti-inﬂammatory eﬀects of compounds, the

nitrite level from culture media of lipopolysaccharide (LPS, Sigma, St.

Louis, MO, USA) exposed BV-2 cells were measured. Brieﬂy, BV-2

cells were pretreated with 5, 10, or 20 μM of test compounds for 2 h,

followed by 100 ng/mL of LPS exposure for 24 h. For measurement

of nitrite, 50 μL of culture medium from each well was mixed with 50

μL of Greiss buﬀer (Sigma-Aldrich, St. Louis, MO, USA) in 96-well

plates, and then incubated for 15 min at room temperature. The

absorbance of mixture was measured at 540 nm using a microplate

reader (PerkinElmer, Waltham, MS, USA), and the level of nitrite was

calculated from a standard curve of sodium nitrite.

Glutamate Stimulation and Cell Viability Assay in HT-22

Cells. HT-22 cell line was cultured in DMEM (Life Tech, Grand

Island, USA) supplemented with 10% fetal bovine serum (FBS,

Gibco, Grand Island, USA). The cells were seeded at a density of 5 ×

Statistical Analysis. All data are presented as the mean ± SEM.

Student’s t test was used to determine the statistical signiﬁcance

between two groups. One-way ANOVA followed by Turkey’s tests

were used to analyze the diﬀerences of multiple comparisons. P < 0.05

was considered statistically signiﬁcant.

ASSOCIATED CONTENT

Supporting Information

https://pubs.acs.org/doi/10.1021/acschemneuro.0c00520.

■

sı

0 cells/mL into 96-well plates with 100 μL/well and maintained in a

Characterization data of synthetic intermediates and

copies of NMR spectra (PDF)

humidiﬁed atmosphere of 5% CO₂and 95% air at 37 °C. To

determine the protective eﬀects of compounds against glutamate-

induced neurotoxicity, the cell viability of HT-22 was measured by 3-

AUTHOR INFORMATION

Corresponding Authors

■

(

4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,

Sigma, St. Louis, MO, USA) assay. Brieﬂy, HT-22 cells were

pretreated with 1 or 10 μM of test compounds for 2 h, followed by 10

mM of glutamate exposure for 24 h. The HT-22 cells were then

Wen-Wen Chen − State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

ACS Chemical Neuroscience

Sciences, Shanghai 201203, China; orcid.org/0000-0002-

Research Article

Sciences, Shanghai 201203, China; Email: wenwen@

shnu.edu.cn

Haiyan Zhang − CAS Key Laboratory of Receptor Research,

Shanghai Institute of Materia Medica, Chinese Academy of

(5) Panza, F., Lozupone, M., Seripa, D., and Imbimbo, B. P. (2019)

Amyloid-beta immunotherapy for alzheimer disease: Is it now a long

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strategies for the improved treatment of Alzheimer ’s disease. Eur. J.

Med. Chem. 176, 228−247.

877-2040; Email: hzhang@simm.ac.cn

(7) Souder, D. C., and Anderson, R. M. (2019) An expanding GSK3

Ming-Hua Xu − Shenzhen Key Laboratory of Small Molecule

Drug Discovery and Synthesis, Department of Chemistry,

Southern University of Science and Technology, Shenzhen

network: implications for aging research. Geroscience 41, 369 −382.

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synthase kinase-3 signaling in Alzheimer ’s disease. Biochim. Biophys.

Acta, Mol. Cell Res. 1867, 118664.

18055, China; State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

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hyperphosphorylation in drug design for neurodegenerative diseases.

Nat. Rev. Drug Discovery 6, 464−79.

Sciences, Shanghai 201203, China; orcid.org/0000-0002-

692-2718; Email: xumh@sustech.edu.cn

(10) Martin, L., Latypova, X., Wilson, C. M., Magnaudeix, A., Perrin,

Authors

M. L., Yardin, C., and Terro, F. (2013) Tau protein kinases:

involvement in Alzheimer ’s disease. Ageing Res. Rev. 12, 289−309.

Jian-Guo Liu − Shenzhen Key Laboratory of Small Molecule

Drug Discovery and Synthesis, Department of Chemistry,

Southern University of Science and Technology, Shenzhen

(11) Yagishita, S., Murayama, M., Ebihara, T., Maruyama, K., and

Takashima, A. (2015) Glycogen Synthase Kinase 3beta-mediated

Phosphorylation in the Most C-terminal Region of Protein Interacting

with C Kinase 1 (PICK1) Regulates the Binding of PICK1 to

Glutamate Receptor Subunit GluA2. J. Biol. Chem. 290, 29438−48.

18055, China; State Key Laboratory of Drug Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China

(12) Duda, P., Wisniewski, J., Wojtowicz, T., Wojcicka, O.,

Danfeng Zhao − CAS Key Laboratory of Receptor Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China

Jaskiewicz, M., Drulis-Fajdasz, D., Rakus, D., McCubrey, J. A., and

Gizak, A. (2018) Targeting GSK3 signaling as a potential therapy of

neurodegenerative diseases and aging. Expert Opin. Ther. Targets 22,

Qi Gong − CAS Key Laboratory of Receptor Research, Shanghai

Institute of Materia Medica, Chinese Academy of Sciences,

Shanghai 201203, China

Fengxia Bao − CAS Key Laboratory of Receptor Research,

Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai 201203, China

33−848.

(13) Jope, R. S., and Roh, M. S. (2006) Glycogen synthase kinase-3

(GSK3) in psychiatric diseases and therapeutic interventions. Curr.

Drug Targets 7, 1421−34.

Complete contact information is available at:

14) Cohen, P., and Goedert, M. (2004) GSK3 inhibitors:

development and therapeutic potential. Nat. Rev. Drug Discovery 3,

79−87.

15) Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto,

(

https://pubs.acs.org/10.1021/acschemneuro.0c00520

M., and Tomita, F. (1986) Staurosporine, a potent inhibitor of

phospholipid/Ca++dependent protein kinase. Biochem. Biophys. Res.

Commun. 135, 397−402.

Author Contributions

J.-G. Liu and D. Zhao contributed equally to this work.

(

16) Meijer, L., Skaltsounis, A. L., Magiatis, P., Polychronopoulos,

P., Knockaert, M., Leost, M., Ryan, X. P., Vonica, C. A., Brivanlou, A.,

Dajani, R., Crovace, C., Tarricone, C., Musacchio, A., Roe, S. M.,

Pearl, L., and Greengard, P. (2003) GSK-3-selective inhibitors derived

from Tyrian purple indirubins. Chem. Biol. 10, 1255−66.

Notes

The authors declare no competing ﬁnancial interest.

(

17) Leost, M., Schultz, C., Link, A., Wu, Y. Z., Biernat, J.,

ACKNOWLEDGMENTS

■

Mandelkow, E. M., Bibb, J. A., Snyder, G. L., Greengard, P.,

Zaharevitz, D. W., Gussio, R., Senderowicz, A. M., Sausville, E. A.,

Kunick, C., and Meijer, L. (2000) Paullones are potent inhibitors of

glycogen synthase kinase-3beta and cyclin-dependent kinase 5/p25.

Eur. J. Biochem. 267, 5983−94.

Financial support from the National Science & Technology

Major Project “Key New Drug Creation and Manufacturing

Program”, China (2018ZX09711002-006, 2018ZX09711002-

18), the Strategic Priority Research Program of the Chinese

(

18) Meijer, L., Thunnissen, A. M., White, A. W., Garnier, M.,

Academy of Sciences (XDA 12040106), the National Natural

Science Foundation of China (81402798 and 81521005) and

the Shenzhen Science and Technology Innovation Committee

Nikolic, M., Tsai, L. H., Walter, J., Cleverley, K. E., Salinas, P. C., Wu,

Y. Z., Biernat, J., Mandelkow, E. M., Kim, S. H., and Pettit, G. R.

2000) Inhibition of cyclin-dependent kinases, GSK-3beta and CK1

by hymenialdisine, a marine sponge constituent. Chem. Biol. 7, 51−63.

19) Selenica, M. L., Jensen, H. S., Larsen, A. K., Pedersen, M. L.,

(

ZDSYS20190902093215877) are greatly acknowledged.

(

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