Identification of glycogen synthase kinase-3 inhibitors with a selective sting for glycogen synthase kinase-α

Article

pubs.acs.org/jmc

Identiﬁcation of Glycogen Synthase Kinase-3 Inhibitors with

a Selective Sting for Glycogen Synthase Kinase-3α

Fabio Lo Monte,*^,†Thomas Kramer,^†Jiamin Gu,^†Upendra Rao Anumala,^†Luciana Marinelli,^‡

Valeria La Pietra,^‡Ettore Novellino,^‡Benedicte Franco,^§David Demedts,^§Fred Van Leuven,^§

́

Ana Fuertes,^∥Juan Manuel Dominguez,^∥Batya Plotkin,^⊥Hagit Eldar-Finkelman,^⊥and Boris Schmidt*^,†

^†Clemens SchopfInstitute of Organic Chemistry and Biochemistry, Technische Universitat Darmstadt, 64287 Darmstadt, Germany

̈

^‡Dipartimento di Chimica Farmaceutica e Tossicologica, Universita

̀

di Napoli “Federico II”, 80131 Napoli, Italy

^§Experimental Genetics Group, Department of Human Genetics, Katholieke Universiteit Leuven, 3000 Leuven, Belgium

^∥Noscira SA, Drug Discovery, Tres Cantos 28760-Madrid, Spain

^⊥Department of Human Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University,

69978 Tel Aviv, Israel

S

* Supporting Information

ABSTRACT: The glycogen synthase kinase-3 (GSK-3) has

been linked to the pathogenesis of colorectal cancer, diabetes,

cardiovascular disease, acute myeloid leukemia (AML), and

Alzheimer’s disease (AD). The debate on the respective

contributions of GSK-3α and GSK-3β to AD pathology and

AML is ongoing. Thus, the identiﬁcation of potent GSK-3α-

selective inhibitors, endowed with favorable pharmacokinetic

properties, may elucidate the eﬀect of GSK-3α inhibition in AD

and AML models. The analysis of all available crystallized GSK-

3 structures provided a simpliﬁed scheme of the relevant hot

spots responsible for ligand binding and potency. This resulted

in the identiﬁcation of novel scorpion shaped GSK-3 inhibitors. It is noteworthy, compounds 14d and 15b showed the highest

GSK-3α selectivity reported so far. In addition, compound 14d did not display signiﬁcant inhibition of 48 out of 50 kinases in the

test panel. The GSK-3 inhibitors were further proﬁled for eﬃcacy and toxicity in the wild-type (wt) zebraﬁsh embryo assay.

been proposed as the major kinase of tau phosphorylation,

suggesting it as a potential, yet risky target for the therapy of

AD.^1,5Dysregulation of GSK-3β has been associated with

diseases such as diabetes, Down’s syndrome, bipolar disorder,

colorectal cancer, and AD.¹¹The inhibition of GSK-3α was sug-

gested for the treatment of AD and other CNS diseases.¹²⁻¹⁴

Furthermore, GSK-3α inhibition was proposed to modulate

β-adrenergic signaling.¹⁵Recently, it was suggested that GSK-

3α is involved in acute myeloid leukemia (AML), supporting a

potential role for GSK-3α directed therapy.¹⁶Yet, the distinct

contributions of both GSK-3 isoforms are still unknown.

Appropriately, a number of pan-GSK-3α/β inhibitors have been

disclosed because of the structure determination of GSK-3β.¹⁷

Lithium chloride is the most thoroughly investigated GSK-3

inhibitor in AD animal models; it results in decreased tau

hyperphosphorylation and decreased Aβ levels.⁶However, it is

limited by a small therapeutic window. GSK-3 inhibitors were

identiﬁed from remarkably diﬀerent classes: organometallic com-

pounds, paullones, indirubins, maleimides, thiadiazolidinones,

L803-mts, ureas, and other small organic molecules.^4,10,18−24

INTRODUCTION

■

Alzheimer’s disease (AD), ﬁrst described by Alois Alzheimer

in 1906, is the most common dementia at old age. AD is

characterized by the presence of two abnormal protein

deposits: amyloid plaques composed of extracellular deposits

of β-amyloid (Aβ) peptides and neuroﬁbrillary tangles (NFTs)

are formed by the accumulation of insoluble and hyper-

phosphorylated tau.¹⁻³The 40−42 amino acid β-amyloid

peptide is the major component of the amyloid deposits. It is

produced from a larger protein, the amyloid precursor protein

(APP), by proteolytic cleavage.⁴Tau is a soluble microtubule-

binding protein which stabilizes the microtubules in axons.²

Hyperphosphorylation of tau protein causes destabilization of

microtubules and subsequent dissociation of tau, which in turn

aggregates to form NFTs.⁵GSK-3 was identiﬁed ∼30 years ago

and is a serine/threonine protein kinase that participates in a

plethora of cellular processes, e.g., cell proliferation, micro-

tubule dynamics, and gene transcription.⁶⁻⁸Several studies

have linked glycogen synthase kinase-3 (GSK-3) to the primary

abnormalities associated with AD, particularly the phosphor-

ylation of tau.^4,9Two closely related isoforms GSK-3α and

GSK-3β are present in mammals.¹⁰They share 97% sequence

similarity within their catalytic kinase domains.⁷GSK-3β has

Received: March 5, 2012

Published: April 25, 2012

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Figure 1. Synthesis strategy based on hot spot analysis of GSK-3 inhibition. The denoted acceptor (A) and donor (D) domains outline the necessary atoms

respectively functional groups in the designated areas (left/right). The scaﬀold I used for the synthesis is marked on the right. X stands for heteroatoms.

a

Scheme 1

a

Reagents and conditions: (a) MeOH, SOCl₂, 0−50 °C, 83−89%; (b) NH₂NH₂·H₂O, EtOH, reﬂux, 67−75%; (c) CS₂, Et₃N, EtOH, reﬂux, 79−

89%; (d) benzyl halides, 1N NaOH, DMF, rt, 41−84%.

All GSK-3 inhibitors, except for the thiadiazolidinones and

L803-mts, are ATP competitive inhibitors and all of them inhibit

the two isoforms, GSK-3α and GSK-3β, with similar potency.⁶

The design of selective ligands remains a challenge despite

several crystallized GSK-3 inhibitor complexes and substantial

diﬀerences revealed by GSK-3α/GSK-3β sequence comparison

as the major part of the ligand binding site is conserved.¹⁷Here

we report the synthesis and optimization of novel GSK-3

inhibitors, along with their α/β selectivity and the evaluation of

their in vivo eﬃcacy in zebraﬁsh embryos, which is an established

model system for the validation of GSK-3 inhibitors. The

oxadiazole moiety (scaﬀold I; Figure 1) was chosen as lead

structure as it provided, if appropriately decorated, high

inhibition of GSK-3β.^5,25,26

occupy three or at most four acceptor/donor domains in the

active site. Our main intention was the engagement with as

many as possible acceptor/donor areas as depicted in Figure 1.

Initially, we investigated the enlargement of I to reach R141.

Subsequently, diﬀerent substituents on the heterocyclic scaﬀold

were explored in order to enhance the interaction with the

enzyme backbone and to improve solubility at the same time.

Most of the resulting compounds were tested for the selective

inhibition of GSK-3α/β.

CHEMISTRY

■

The esteriﬁcation of the carboxylic acids 1A−C aﬀorded the

compounds 2A−C,^27,28which were converted to the

hydrazides 3A−C.^28,29Reaction of the hydrazides 3A−C with

carbon disulﬁde (CS₂) resulted in the oxadiazoles 4A−C.^30,31

The heterocyclic derivatives 5a−c, 6a−c, and 7a−c³²were pre-

pared by benzylation of the mercaptanes 4A−C (Scheme 1).²⁶

The optimization process took advantage of the available

cocrystallized GSK-3β inhibitor complexes and the analysis of

the relevant hot spots (Figure 1). Most GSK-3 inhibitors

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a

Scheme 2

a

Reagents and conditions: (a) 1N LiOH, THF, 60 °C, 83−91%; (b) SOCl₂, toluene, reﬂux; (c) amine, K₂CO₃, acetone, 0 °C to rt, 79−92%; (d)

NaN₃, NH₄Cl, DMF, 100 °C, 67−79%.

a

Scheme 3

a

Reagents and conditions: (a) aryl bromide, toluene, EtOH, Pd(PPh₃)₄, 2-tolylboronic acid, 2N Na₂CO₃(aq), 80 °C; (b) NBS, AIBN, CCl₄, reﬂux;

(c) 4(A−C), 1N NaOH, DMF, rt, 53−75%.

Compound 7d is commercially available. The esters 5c and 6c

were converted to the carboxylic acids 8a and 9a, followed by

treatment with thionyl chloride (SOCl₂) to form the acyl

chlorides 8b and 9b.^33,34Coupling of the acyl chlorides with

primary amines gave the amides 8c−d and 9c−e.³⁴The

tetrazoles 8e and 9f were prepared from the nitriles 5b

and 6b using sodium azide under microwave irradiation

(Scheme 2).³⁵

The biphenylic derivatives 13a−d were prepared in two steps

from the commercially available p-tolylboronic acid and

substituted bromobenzenes. The 4′-(bromomethyl)biphenyl-

2-carbonitrile is commercially available. The biphenylmethyl

halides were coupled to the mercaptothiadiazoles 4A−C to

obtain the thioethers 14a−d, 15a−b, and 16a (Scheme 3).²⁶

3,4-Dihydroxybenzoic acid 17 was esteriﬁed to the methyl ester

18, followed by cyclization with ( )-glycidyl tosylate or

epibromohydrin to aﬀord compound 19a³⁶as a mixture of

enantiomers.³⁷⁻³⁹The hydrazide 21a was prepared by

methylation of 19a, followed by the addition of hydrazine.⁴⁰

The reaction of the hydrazide 21a with CS₂gave the oxadiazole

22a, which was coupled to 4′-(bromomethyl)biphenyl-2-

carbonitrile to aﬀord the thioether 23a. The methyl ether in

23a was cleaved by boron tribromide (BBr₃) to result in the

alcohol 24a (Scheme 4).⁴⁰The compounds 23b−c and 24a−b

were prepared under similar conditions; see Scheme 4. (S)-

(+)-Glycidyl tosylate was used to obtain the R-enantiomer of

compound 19b.³⁷The S-enantiomer of compound 19 was

synthesized using (R)-(−)-glycidyl tosylate.³⁷Mesylation of the

alcohol 24b and subsequent displacement of the mesylate by an

amine aﬀorded the acetal 26 (Scheme 4).⁴⁰

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a

Scheme 4

a

Reagents and conditions: (a) SOCl₂, MeOH, 0−50 °C, 97%; (b) (R/S)-( )-glycidyl tosylate or epibromohydrin, (S)-(+)-glycidyl tosylate or (R)-

(−)-glycidyl tosylate, K₂CO₃, acetone or DMF, rt or 60 °C, 93−95%; (c) NaH, CH₃I, THF, 0 °C to rt, 68−71%; (d) NH₂NH₂·H₂O, EtOH, reﬂux,

78−87%; (e) CS₂, Et₃N, EtOH, reﬂux, 81−91%; (f) biphenyl halide, 1N NaOH, DMF, rt, 84−88%; (g) BBr₃, DCM, −78 °C to rt; 73−79%; (h)

CH₃SO₂Cl, Et₃N, DCM, 0 °C to rt, 98%; (i) amine, THF, Et₃N, 0 °C to reﬂux, 83%.

RESULTS AND DISCUSSION

■

Molecular Modeling. Compound 15a, one of the most

active inhibitors of the series, was docked, through Glide

software, into the GSK-3β active site (PDB code: 3F88) with

the aim to assess the ligand−protein interactions and to

rationalize the SARs.²⁶The docking experiments suggest that

the oxadiazole ring positions itself in between the V70 and

C199 side chains with one of the two nitrogens establishing an

H-bond with the K85 side chain (Figure 2). The biphenyl

branch forms a T-shaped interaction with P67 and hydrophobic

contacts with the Q185 and Y140 carbons.⁴¹Furthermore, as

Figure 2. Molecular docking of compound 15a into the X-ray

structure of GSK-3β (PDB code: 3F88). This ﬁgure was prepared with

Glide software.

shown in Figure 2, the CN substituent forms an H-bond with

the T138 hydroxyl group. The latter interaction seems to

improve the activity of our ligands, in fact, for example, 14c is

more active against GSK-3β than its analogue 14a, which lacks

the cyano group, and its analogue 14d, equipped with the cyano

group at the R2 site. As regards the 15a binding mode, the

dihydrobenzodioxine moiety establishes several hydrophobic

interactions with L132, I62, A83, V110, and L188.

the rest of the ring establishes hydrophobic contacts with the

Y134. The latter interaction seems to be lost by 14c, featuring

the smaller benzodioxolane ring, while 16a, a pyridine

containing compound, forms a weaker H-bond with the same

residue due to the position and the distance of the nitrogen

atom of the pyridine ring from the NH of V135. The substitu-

tions on the dihydrobenzodioxine moiety of 23 and 24,

Moreover, one of the two oxygens of the dihydrodioxine ring

forms an H-bond with the V135 NH in the hinge region, while

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respectively, do not provide any further interaction with the

enzyme, as the groups point out into the solvent. The same

holds true for 26, where the bulky substituent on the dihydro-

benzodioxine ring may negatively aﬀect the horseshoe shape

(scorpion shape). The proposed binding mode also clariﬁes

the undesirable eﬀect of the substitution of the ﬂuorine atom

at the R2 site of the biphenyl branch in 15b, which is

37-fold less potent for GSK-3β in comparison to 15a. In fact,

the electron-withdrawing atom weakens the H-bond between

the cyano substituent and the T138 hydroxyl group; moreover,

it comes in proximity of the negative ring density of Y140,

providing repulsive edgewise interaction. The good selectivity

toward GSK-3α versus GSK-3β which was observed for several

compounds and especially for compound 15b, is far to be trivial

to explain. The superposition of the GSK-3β crystallographic

structure (PDB code: 3F88) with a homology model built with

Prime software (Schrodinger) shows that the diﬀerences

between the two isoforms are all located out of the binding

site and especially in the loop at C-terminus fragment (see the

Supporting Information). Thus, it is conceivable that the

selectivity of our compounds may be due to subtle enzyme

diﬀerences, which may aﬀect the ligand entrance/exit processes.

This process may include an antechamber site, a step known to

play a pivotal role in the inhibitor/enzyme recognition

process.^42,43By analyzing the enzyme surface and the residue

mutations, the antechamber site in the GSK-3α or GSK-3β

could be represented by the loop at C-terminus fragment as

highlighted in Figure S1 in the Supporting Information.

Obviously, the latter is a pure speculative hypothesis that has

to be conﬁrmed by more advanced theoretical work, mutational

analysis, and additional experiments.

Biological Assays and Structure−Activity Relationship

(SAR) Studies. The synthesized compounds were tested for

their inhibitory activity against GSK-3β in an in house in vitro

assay and further proﬁled in a commercial system based on the

Z’-LYTE technology, available from Invitrogen Life Technol-

ogies (Carlsbad, CA, USA), using human recombinant GSK-3α

or GSK-3β as the enzyme source. Most compounds displayed

signiﬁcant inhibitory activity against GSK-3β at 10 μM, and

several compounds exerted more than 50% inhibitory activity

against GSK-3β at the initial concentration (10 μM). The

potent compounds were selected for IC₅₀determination. We

observed diﬀerences in the IC₅₀determination between the in-

house and the commercial assay and decided to use the results

of the commercial system for comparison.

The structure−activity analysis suggested interactions with

the GSK-3 backbone, Y134/D133, and the polar binding

pocket, K85/E97/D200, to be essential for potent inhibition.

These interactions require an acceptor−donor−acceptor motif

on the inhibitor. We generated a simpliﬁed illustration in which

we denoted acceptor (A) and donor (D) domains and drafted

scaﬀold I as lead structure (Figure 1). We examined the eﬀect

of three heterocycles 5−7 as potential hinge binders and

diﬀerent substituents on the S-benzyl group (Scheme 1). The

oxadiazole derivatives of the heterocycles 5 and 6 provided

several GSK-3 inhibitors with an IC₅₀below 100 nM (Table 1)

and conﬁrmed previously reported activity.²⁶The pyridines

7a−c displayed decreased activity in comparison to the

heterocycles 5 and 6; this may be due to the position of the

pyridine moiety in the ATP binding pocket. Thus, they were

not pursued further. The compounds 5a and 6a indicated that

an electron-withdrawing group is required at the 3-position. We

introduced the cyano and ester group at the 4-position in order

to reach out to R141 and the correlated acceptor/donor

domain and thus to engage the ATP binding pocket in its

entirety. Our data indicated that the electron-withdrawing

group at the 3-position was also tolerated at the 4-position. The

oxadiazoles 5b and 6b−c showed comparable activity to the

3-substituted derivatives and indicated space in the ATP binding

pocket. On the basis of these results, we further examined the

para-position of our lead structure. The carboxylic acids 8a and

9a resulted in a 4-fold less inhibitory activity against GSK-3β at

10 μM concentration compared to their esters 5c and 6c

(Scheme 2). In the case of 5c, the percentage of GSK-3β activity

increased from 17% of up to 81% (Table 2). In addition,

compound 8e, bearing a hydrophilic tetrazole at the 4-position

(IC₅₀value of 107 nM for GSK-3α and 172 nM for GSK-3β),

showed decreased inhibitory activity compared to the ester 6c

(Scheme 2). Conversion of the carboxylic acids to the amides

8c−d and 9c−e resulted in an increased activity. Especially,

compound 8c showed good inhibitory property against GSK-3β

with a remaining kinase activity of 9% at 10 μM (Table 2).

These results and the molecular modeling suggested that

compounds containing polar groups at the 4-position were less

active than compounds containing hydrophobic groups. Hence,

we tried to elongate our compounds with a phenyl ring at the

4-position. Compounds bearing a phenyl group in the para-

position showed very good inhibitory activity. The docking

analysis of the biphenylic derivatives suggested several hydro-

phobic contacts, which may be responsible for the enhanced

potency. Especially, compound 15a with an IC₅₀value of <5 nM

for GSK-3α and GSK-3β was found to be a potent inhibitor of

GSK-3 (Table 3). Slightly decreased activity was observed for

compound 14a with an IC₅₀value of 9 nM for GSK-3α and

176 nM for GSK-3β. We observed an IC₅₀of 2 nM for GSK-3α

and 22 nM for GSK-3β for structure 14b, whereas 14c, which

lacks the ﬂuorine substituent, resulted in slightly decreased IC₅₀

values in comparison to 14b.

The selectivity for the GSK-3α isoform was higher when the

substituents were absent in the series 14a−c. The absence of

selectivity for GSK-3α in 15a in comparison with 14c may

be explained by the interaction with Y134 (see Molecular

Modeling), which seems to be lost in 14c. Remarkably,

compound 14d displayed up to 52-fold selectivity in the

inhibition of GSK-3α versus GSK-3β. This selectivity was even

enhanced with compound 15b, which is characterized by

an IC₅₀of 2 nM for GSK-3α and 185 nM for GSK-3β. Thus,

only the interplay respectively of a combination of diﬀerent

substituents was adequate to gain selectivity against one GSK-3

isoform. This observation will be helpful if a discrimination of

one GSK-3 isoform is needed. In addition, the physiological

functions and pathological roles of GSK-3α can be addressed in

vitro and eventually in vivo with these tools. The biphenyl

derivative 16a from the pyridine series showed remarkably

increased activity in comparison to the used reference 7d,

20-fold for GSK-3α, and 19-fold for GSK-3β (Table 1). The

eﬀect of the second phenyl suggested an interaction with the

glycine-rich loop, such an interaction was reported to have

signiﬁcant eﬀects on binding potency and selectivity recently.⁴¹

Furthermore, the SAR and molecular modeling suggested that

an electron-withdrawing group in the ortho position of the

second phenyl ring, such as the cyano-group, contributes to

the inhibitory activity by interaction with the amino acid T138.

We examined diﬀerent linker systems on the dihydrobenzo-

dioxine moiety with the aim to enhance the interaction with the

backbone. Unfortunately, they do not provide any further

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Table 1. Inhibitory Activity against GSK-3α and GSK-3β, IC₅₀(μM)

interaction (see Molecular Modeling). The eﬀect of the

Good selectivity was obtained for all compounds tested.

methoxy group on compound 23a resulted in an IC₅₀value

of 54 nM for GSK-3α and 233 nM for GSK-3β, respectively,

195 nM and 758 nM for compound 23b. In the case of 23c,

good inhibitory activity was observed against both isoforms of

GSK-3, especially for GSK-3α. These results suggest that the

R-enantiomer 23b is the distomer of this compound, whereas

the S-enantiomer 23c was found to be the eutomer. Despite our

expectations that an amine may improve the activity, compound

26 showed markedly reduced potency.

The potent GSK-3 inhibitors 6c, 14a−d, 15a−b, 16a, and

23a−c were selected for selectivity proﬁling and tested against

four structurally related protein kinases (Cdk5/p35, CK1ε,

AurKA, and PKCα).

Particularly, the biphenyl derivatives 14c, 15a, and 16a showed

more than 2000-fold selectivity against these kinases (Table 4).

Compound 14d was not just GSK-3α selective, it was even

more selective over the other kinases than compound 15b.

The broader selectivity of compound 14d was screened at a

concentration of 1 μM against 50 human protein kinases

(Figure 3); 48 out of the 50 kinases in this panel showed an

activity higher than 80%, whereas GSK-3α displayed a residual

activity of 5% only. The only kinase which was also signiﬁcantly

inhibited by this compound was GSK-3β with a remaining

activity of 27.2%. Therefore, it can be concluded that, within

the test panel, compound 14d is a selective inhibitor of GSK-

3α. A bioavailability proﬁle of compound 14d was evaluated,

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Table 2. Inhibitory Activity against GSK-3β at 10 μM

and the results are shown in the Supporting Information. 14d

possesses a log D value of 3.58 and moderate metabolic stability.

Nevertheless, the poor aqueous solubility and permeability

are adverse properties which limit the potential use of the

compound.

dominant factors on the in vivo assay of the α-selective

inhibitors. The inhibition of GSK-3α was proposed to regulate

β-adrenergic signaling in mice, thus we monitored the heart

development of the zebraﬁsh embryo after administration of

compound 14d until day 5 (see the Supporting Information).^15,46

However, no eﬀect was observed until the ﬁfth day of develop-

ment. All compounds displayed no lethality in our concentration

range (<30 μM).

The biphenyl derivatives 14c−d, 15a−b, and 16a were

further tested for their in vivo activity on wild-type zebraﬁsh

embryos. We exposed the embryos to these compounds at

early stages of development. The embryos were collected and

maintained in E2 medium at ∼28 °C. The compounds were

added 5 h post fertilization (hpf), and the phenotypes

compared at 44−48 hpf. Compound 14c causes the eyeless

phenotype at 0.5 μM and a stunted and crooked tail at 1 μM.

Similar phenotypes were obtained for compound 15a at 2.5 μM

and for compound 16a at 20 μM (Figure 4). This correlates

with the observation that Wnt signaling, and thus GSK-3β plays

a crucial role in the development of metazoan and that known

GSK-3 inhibitors like LiCl and the ruthenium complex (R)-7

perturb the zebraﬁsh development.^44,45The zebraﬁsh embryo

assay provides evidence of exposure and cell penetration of the

biphenyl derivatives, especially for compound 14c. Interest-

ingly, compounds 14d and 15b showed no eﬀect on wild-type

zebraﬁsh embryos, suggesting that GSK-3α plays a minor

role in the zebraﬁsh Wnt signaling pathway. The lack of response

in the zebraﬁsh assay by compounds 14d and 15b may be

explained by poor cell permeability. However, the structurally

analogues compounds 14c and 15a, which are characterized by

comparable solubility, did result in a GSK-3β-phenotype. Thus,

the comparison of these compounds 14c/15a and 14d/15b

does not support poor exposure and cell penetration as the

SH-SY5Y neuroblastoma cells stably transfected with

Tau.P301L were incubated with increasing concentrations of

compounds 14c and 15a (0, 30, and 100 μM) for 6 and 24 h.

Cells were analyzed for total protein tau by Western blotting

with antibody Tau5, directed against nonphosphorylated

protein tau. In addition, the same samples were probed with

a selection of phospho-speciﬁc antibodies that recognize typical

GSK-3 dependent epitopes on protein tau. Moreover, the

electrophoretic mobility of protein tau is a reliable index of the

degree of phosphorylation of protein Tau.P301L: the lesser

mobile isoforms carry the most phosphate groups (Figure 5).

Both compounds dose- and time-dependently decreased the

phosphorylation of protein tau, expressed relatively to total tau,

as demonstrated by the decreased immunoreaction with

antibodies pS199, pT231, pS396, and pS404 in Western blotting

(Figure 5). Moreover, the marked increase in electrophoretic

mobility of the various phospho-isoforms of protein tau induced

by treatment of the stably transfected SH-SY5Y cells

corroborates their eﬀectiveness in preventing phosphoryla-

tion on typical GSK-3 dependent epitopes. The comparison of

the biphenyl derivatives indicated that compound 14c is more

eﬀective in preventing phosphorylation than compound 15a.

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Table 3. Inhibitory Activity of the Biphenyls against GSK-3α and GSK-3β, IC₅₀(μM)

This correlates with the observations made in the zebraﬁsh

embryo assay in which 14c showed the best results.

in the zebraﬁsh embryo assay without lethality at 30 μM. In

addition, two inhibitors decreased the phosphorylation of tau

protein in SH-SY5Y cells. The docking analysis of the potent

inhibitors suggested an interaction with the glycine-rich loop,

which was reported to have signiﬁcant eﬀects on the binding

potency and selectivity by Li Feng et al.⁴¹To our knowledge,

the selective inhibition of GSK-3α versus GSK-3β by the

compounds 14d and 15b is the highest reported so far. In

addition, compound 14d did not show any strong inhibition

for 48 out of 50 kinases. The contribution of GSK-3α and

GSK-3β to the pathology of Alzheimer’s disease is still subject

of an ongoing debate.^47,48Thus, these compounds may be

useful tools and starting points for the synthesis of GSK-3α

selective inhibitors with enhanced pharmacokinetic properties.

CONCLUSION

■

On the basis of a simpliﬁed scheme of known and important

interactions of GSK-3 inhibitors with the ATP binding pocket,

we generated hypotheses for improved interaction of with this

site. These hypotheses were challenged by three series of

structurally closely related inhibitors which are all based on a

central oxadiazole moiety. An appropriate decoration resulted

in a more extended occupation of the ATP binding site.

The most potent inhibitors displayed IC₅₀values in the low

nanomolar range and good kinase selectivity versus four closely

related kinases. Several inhibitors showed reported phenotypes

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phase, Zorbax Eclipse XDB-C18, 4.6 mm × 150 mm; 254 nm).

Solvent gradient = 90% A at 0 min, 30% A at 2 min, 10% A at 5 min;

solvent A = 0.1% triﬂuoroacetic acid in water; solvent B = acetonitrile;

ﬂow rate 1.0 mL/min; temperature 35 °C. Flash column

chromatography was carried out using Merck silica gel 60 (40−63

and 15−40 μm) and 60G (5−40 μm). Thin-layer chromatography

(TLC) was carried out using aluminum sheets precoated with silica gel

60 F254 (0.2 mm; E. Merck). All compounds that were evaluated in

biological assays had >95% purity using the HPLC method described

above.

General Procedure A: Coupling of Aromatic Rings by a

Suzuki Reaction (12a−d).⁴⁹To a solution of the aryl bromide 11

(5 mmol) in 15 mL of toluene/EtOH (1/1) was added 0.17 g

(0.14 mmol) of Pd(PPh₃)₄, and the mixture was stirred under argon

atmosphere. Then 2 N aqueous Na₂CO₃(7.5 mL) and 0.80 g

(6 mmol) of 2-tolylboronic acid 10 were added. The mixture was

reﬂuxed at 80 °C for 1−2 days until reaction was completed (TLC).

After cooling to room temperature, the product was diluted with water

and extracted with EtOAc. The organic layers were dried with MgSO₄,

ﬁltered, and concentrated. Puriﬁcation was performed by column

chromatography using a mixture of cyclohexane/EtOAc.

General Procedure B: Bromination at the Benzylic Position

(13a−d).⁴⁹To a stirred solution of the appropriately substituted

toluene in CCl₄(10 mL per mmol) were added 0.95 equiv of NBS and

AIBN (5 mg per mmol). The reaction mixture was reﬂuxed at 80 °C

for about 40 h and then cooled to room temperature. The product

was diluted with water and extracted with EtOAc. The organic layers

were dried with MgSO₄, ﬁltered, and concentrated. Puriﬁcation was

performed by column chromatography using a mixture of cyclo-

hexane/EtOAc.

Methyl Benzo[d][1,3]dioxole-5-carboxylate (2A). To a stirred

solution of benzo[d][1,3]dioxole-5-carboxylic acid (1.66 g, 10 mmol)

in MeOH (20 mL) was added SOCl₂(1.45 mL, 20 mmol) dropwise

over 1 h at 0 °C. The mixture solution was further stirred 12 h at

50 °C. The mixture was cooled to room temperature and diluted

with water (25 mL). MeOH was evaporated and the pH adjusted to

∼6 with aqueous NaHCO₃. The mixture was extracted three times

Table 4. Kinase Selectivity of Several Derivatives

IC₅₀(μM)

compd GSK-3α

GSK-3β

Cdk5/p35

CK1ε

AurKA

PKCα

6c

0.012

0.009

0.003

<0.005

0.006

<0.005

0.002

0.019

0.054

0.195

0.015

0.036

0.176

0.022

0.039

0.316

<0.005

0.185

0.041

0.233

0.758

0.129

>100

20

>100

30

>100

14a

14b

14c

14d

15a

15b

16a

23a

23b

23c

>100

60

>100

30

>100

5

>100

30

>100

EXPERIMENTAL SECTION

■

General Information. All reactions using anhydrous conditions

were carried out under argon atmosphere with dry solvents unless

otherwise noted. All commercial chemicals were used without further

puriﬁcation. The ¹H NMR spectra were recorded on a Bruker AC 300

spectrometer at 300 MHz and Bruker AC 500 spectrometer at

500 MHz. The ¹³C NMR spectra were recorded on a Bruker AC 300

spectrometer at 75 MHz and Bruker AC 500 spectrometer at

125 MHz. Chemical shifts are reported as ppm downﬁeld from Me₄Si.

Abbreviations used to explain the multiplicities: s = singlet, d =

doublet, t = triplet, q = quartet, n = nonett, m = multiplet, br = broad.

Coupling constants (J values) are given in hertz (Hz). Mass

spectrometry was performed on a Bruker−Franzen Esquire LC mass

spectrometer and a MAT 95 double focusing sector ﬁeld MS.

Microwave experiments were carried out using a Biotage Initiator

microwave apparatus. All microwave experiments were carried out in

sealed microwave process vials utilizing the standard absorbance level

(300 W maximum power). High performance liquid chromatographies

(HPLC) were carried out on an Agilent 1100 (column: reversed

Figure 3. Screening of compound 14d against a panel of human protein kinases. Each bar represents the activity of one individual protein kinase.

Compound 14d was tested at a concentration of 1 μM against 50 protein kinases. See Supporting Information for more details.

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Figure 4. Eﬀects on wild-type zebraﬁsh embryos by compounds 14c, 15a, and 16a. The embryos were collected and maintained in E2 medium at

∼28 °C, compounds were added 5 hpf, and the phenotypes were compared at 44−48 hpf. (A,E) Head and tail of control embryos: DMSO (2%).

(B,F) Head and tail of embryos treated with 14c. This compound causes the eyeless phenotype at 0.5 μM and a stunted and crooked tail at 1.0 μM.

(C,G) Head and tail of embryos treated with 15a. A ﬂuﬀy eye pigmentation and a stunted and crooked tail were observed at 2.5 μM. (D,H) Head

and tail of embryos treated with 16a. This compound causes the eyeless phenotype and a crooked tail at 20 μM.

added carbon disulﬁde (397 μL, 6.60 mmol) and NEt₃(469 μL,

3.30 mmol), and the mixture was heated at reﬂux overnight. The

reaction mixture was diluted with EtOAc, and the organic layer was

washed with 0.1 N HCl and brine and dried over Na₂SO₄. The solvent

was evaporated under reduced pressure, and the obtained residue was

recrystallized from cyclohexane/EtOAc to give 4A (521 mg, 79%) as a

with EtOAc and successively washed with brine. The organic layer was

dried over MgSO₄and concentrated under reduced pressure to give

2A (1.6 g, 89%) as a colorless solid. ¹H NMR (DMSO-d₆, 500 MHz):

δ [ppm] = 3.81 (3H, s), 6.14 (2H, s), 7.03 (1H, d, J = 8.1 Hz), 7.38

(1H, d, J = 1.7 Hz), 7.57 (1H, dd, J = 8.1 Hz, J = 1.7 Hz). ¹³C NMR

(DMSO-d₆, 125 MHz): δ [ppm] = 52.0, 102.1, 108.2, 108.5, 123.4,

125.0, 147.6, 151.4, 165.6. EI-MS: m/z = 180 (M⁺).

1

pale-yellow solid. H NMR (DMSO-d₆, 500 MHz): δ [ppm] = 6.14

The following compound 2B was prepared in a similar manner to

that described for 2A.

(2H, s), 7.10 (1H, d, J = 8.1 Hz), 7.33 (1H, d, J = 1.6 Hz), 7.42 (1H,

dd, J = 8.1 Hz, J = 1.6 Hz), SH signal was not observed. ¹³C NMR

(DMSO-d₆, 125 MHz): δ [ppm] = 102.1, 105.6, 109.1, 116.1, 121.5,

148.1, 150.6, 160.3, 177.2. EI-MS: m/z = 222 (M⁺).

The compounds 4B−C were prepared in a similar manner to that

described for 4A.

5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazole-2-

thiol (4B). Yield 89%, light-brown solid. ¹H NMR (DMSO-d₆,

500 MHz): δ [ppm] = 4.32 (2H, m), 4.34 (2H, m), 7.05 (1H, d, J =

8.4 Hz), 7.30 (1H, d, J = 2.0 Hz), 7.36 (1H, dd, J = 8.4 Hz, J = 2.0 Hz),

SH signal was not observed. ¹³C NMR (DMSO-d₆, 125 MHz): δ

[ppm] = 64.4, 64.8, 115.0, 115.7, 118.6, 120.0, 144.2, 147.3, 160.6,

177.6. EI-MS: m/z = 236 (M⁺).

5-(Pyridin-4-yl)-1,3,4-oxadiazole-2-thiol (4C). Yield 83%, yel-

low solid. ¹H NMR (DMSO-d₆, 500 MHz): δ [ppm] = 7.81 (2H, dd,

J = 4.4 Hz, J = 1.6 Hz), 8.81 (2H, dd, J = 4.4 Hz, J = 1.6 Hz), SH signal

was not observed. ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 119.6,

129.7, 150.8, 158.7, 177.8. EI-MS: m/z = 179 (M⁺).

Methyl 2,3-Dihydrobenzo[b][1,4]dioxine-6-carboxylate (2B).

Yield 83%, colorless solid. ¹H NMR (DMSO-d₆, 500 MHz): δ [ppm] =

3.80 (3H, s), 4.19 (2H, m), 4.23 (2H, m), 6.80 (1H, m), 7.47 (2H, m).

¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 50.5, 62.7, 63.2, 115.7,

117.6, 122.0, 141.7, 146.4, 165.2. EI-MS: m/z 194 (M⁺).

Benzo[d][1,3]dioxole-5-carbohydrazide (3A). To a solution of

2A (1.08 g, 6.0 mmol) in EtOH (30 mL) was added hydrazine hydrate

(2.91 mL, 60 mmol), and the mixture was heated at reﬂux for 2 days.

After cooling to room temperature, pure crystals are formed, collected

by ﬁltration, and washed several times with EtOH to give compound

3A (0.72 g, 67%) as a colorless solid. ¹H NMR (DMSO-d₆, 500 MHz):

δ [ppm] = 4.42 (2H, s), 6.07 (2H, s), 6.96 (1H, d, J = 8.1 Hz), 7.35

(1H, d, J = 1.7 Hz), 7.42 (1H, dd, J = 8.1 Hz, J = 1.7 Hz), 9.59 (1H, s).

¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 101.5, 106.9, 107.8,

121.8, 127.2, 147.2, 149.5, 165.2. EI-MS: m/z = 180 (M⁺).

Compound 3B was prepared in a similar manner to that described

for 3A.

3-((5-Benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-ylthio)-

methyl)benzonitrile (5a). To a solution of 4A (55 mg, 0.25 mmol)

and 1 N NaOH (0.25 mL, 0.25 mmol) in DMF (1 mL) was added

1-(bromomethyl)-3-methoxybenzene (75 mg, 0.38 mmol) at room

temperature, and the mixture was stirred for 5 h. The precipitate

formed was collected by ﬁltration and washed once with less DMF

(∼1 mL) and thereafter several times with EtOH to give compound 5a

2,3-Dihydrobenzo[b][1,4]dioxine-6-carbohydrazide (3B).

1

Yield 75%, light-yellow solid. H NMR (methanol-d₄, 500 MHz): δ

[ppm] = 4.28 (2H, m), 4.30 (2H, m), 6.89 (1H, d, J = 8.3 Hz), 7.30 (1H,

dd, J = 8.3 Hz, J = 2.1 Hz), 7.33 (1H, d, J = 2.1 Hz), NH signals were not

observed. ¹³C NMR (methanol-d₄, 125 MHz): δ [ppm] = 65.9, 66.3,

117.9, 118.6, 121.9, 127.5, 145.2, 148.6, 169.6. EI-MS: m/z 194 (M⁺).

1

(55 mg, 64%) as a brown solid. H NMR (DMSO-d₆, 500 MHz):

5-(Benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazole-2-thiol (4A).

To a solution of 3A (535 mg, 3.00 mmol) in EtOH (5 mL) were

δ [ppm] = 4.61 (2H, s), 6.16 (2H, s), 7.10 (1H, d, J = 8.1 Hz), 7.41

(1H, d, J = 1.4 Hz), 7.47 (1H, dd, J = 8.1 Hz, J = 1.5 Hz), 7.57 (1H, t,

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Figure 5. Western blotting for protein Tau.P301L expressed in stably transfected SH-SY5Y neuorblastoma cells, untreated (lanes marked 0) or

treated for 6 h with compounds 14c or 15a (lanes marked 30 and 100 μM). Total protein tau was detected with antibody Tau5 (A). Phospho-

epitopes on protein tau were detected with speciﬁc antibodies (B−E). Experiments were performed in triplicate, and representative blots are shown.

Similar observations were obtained after 24 h of incubation. Note that compound 14c speciﬁcally decreases the total concentration of protein tau,

while levels of the internal marker (actin) remain unchanged.

J = 7.7 Hz), 7.76 (1H, d, J = 7.7 Hz), 7.84 (1H, d, J = 7.8 Hz), 7.96

(1H, s). ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 34.7, 102.1,

106.1, 109.1, 111.4, 116.6, 118.5, 121.7, 129.8, 131.4, 132.6, 133.9,

138.8, 148.1, 150.4, 162.2, 165.2. HPLC: 98%; t_R7.21 min. EI-MS:

m/z = 337 (M⁺).

The compounds 5b−c, 6a−c, and 7a−c were prepared in a similar

manner to that described for 5a. Note: Compounds which did not

precipitate in solution were puriﬁed as follows. The reaction mixture

was diluted with EtOAc and the organic layer was washed with water

and brine, dried over MgSO₄, and concentrated in vacuo. The residue

was puriﬁed by silica gel column chromatography (cyclohexane/

EtOAc).

4-((5-Benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-ylthio)-

methyl)benzonitrile (5b). Yield 67%, brown solid. ¹H NMR

(DMSO-d₆, 500 MHz): δ [ppm] = 4.64 (2H, s), 6.16 (2H, s), 7.11

(1H, d, J = 8.1 Hz), 7.42 (1H, d, J = 1.6 Hz), 7.48 (1H, dd, J = 8.1 Hz,

J = 1.7 Hz) 7.68 (2H, d, J = 8.3 Hz), 7.82 (2H, d, J = 8.3 Hz). ¹³C

NMR (DMSO-d₆, 125 MHz): δ [ppm] = 35.2, 102.1, 106.1, 109.1,

110.4, 116.5, 118.6, 121.7, 130.0, 132.4, 142.8, 148.1, 150.5, 162.2,

165.2. HPLC: 95%; t_R7.09 min. EI-MS: m/z = 337 (M⁺).

Methyl 4-((5-(Benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-

ylthio)methyl Benzoate (5c). Yield 71%, pale-brown solid. ¹H

NMR (DMSO-d₆, 500 MHz): δ [ppm] = 3.82 (3H, s), 4.63 (2H, s),

6.15 (2H, s), 7.10 (1H, d, J = 8.1 Hz), 7.41 (1H, d, J = 1.7 Hz), 7.48

(1H, dd, J = 8.1 Hz, J = 1.7 Hz), 7.62 (2H, d, J = 8.4 Hz), 7.92 (2H, d,

J = 8.4 Hz). ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 35.4, 52.2,

102.2, 106.1, 109.2, 116.6, 121.7, 128.8, 129.4, 142.4, 148.1, 150.4,

162.3, 165.1, 165.9. HPLC: 96%; t_R7.61 min. EI-MS: m/z = 370 (M⁺).

3-((5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-

1

2ylthio)methyl)benzonitrile (6a). Yield 73%, light-brown solid. H

NMR (DMSO-d₆, 500 MHz): δ [ppm] = 4.31 (2H, m), 4.34 (2H, m),

4.61 (2H, s), 7.05 (1H, d, J = 8.4 Hz), 7.39 (1H, d, J = 2.0 Hz), 7.42

(1H, dd, J = 8.4 Hz, J = 2.1 Hz), 7.57 (1H, t, J = 7.8 Hz), 7.77 (1H, dt,

J = 7.8 Hz, J = 1.3 Hz), 7.84 (1H, dt, J = 7.8 Hz, J = 1.1 Hz), 7.96 (1H,

t, J = 1.3 Hz). ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 35.2, 64.5,

64.7, 111.8, 115.5, 116.3, 118.5, 118.9, 120.4, 130.1, 131.8, 133.0,

134.4, 139.3, 144.2, 147.1, 162.7, 165.5. HPLC: 99%; t_R7.53 min. EI-

MS: m/z = 351 (M⁺).

4-((5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-

2-ylthio)methyl)benzonitrile (6b). Yield 56%, brown solid. ¹H

NMR (DMSO-d₆, 500 MHz): δ [ppm] = 4.31 (2H, m), 4.34 (2H, m),

4.63 (2H, s), 7.05 (1H, d, J = 8.4 Hz), 7.37 (1H, d, J = 2.0 Hz), 7.41

(1H, dd, J = 8.4 Hz, J = 2.0 Hz), 7.67 (2H, d, J = 8.3 Hz), 7.81 (2H, d,

J = 8.3 Hz). ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 35.3, 64.0,

64.4, 110.4, 115.0, 115.8, 118.2, 118.6, 119.9, 130.0, 132.4, 142.8,

143.8, 146.7, 162.2, 165.0. HPLC: 95%; t_R7.49 min. EI-MS: m/z =

351 (M⁺).

Methyl 4-((5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-ox-

adiazol-2-ylthio)methyl) Benzoate (6c). Yield 49%, purple solid.

¹H NMR (DMSO-d₆, 500 MHz): δ [ppm] = 3.89 (3H, s), 4.36 (2H,

m), 4.39 (2H, m), 4.67 (2H, s), 7.10 (1H, d, J = 8.4 Hz), 7.42 (1H, d,

J = 2.0 Hz), 7.47 (1H, dd, J = 8.4 Hz, J = 2.0 Hz), 7.67 (2H, d, J =

8.3 Hz), 7.97 (2H, d, J = 8.3 Hz). ¹³C NMR (DMSO-d₆, 125 MHz): δ

[ppm] = 35.4, 52.1, 64.0, 64.4, 115.0, 115.8, 118.1, 120.0, 128.9, 129.4,

142.4, 143.8, 146.7, 162.3, 165.0, 165.8. HPLC: 99%; t_R7.66 min.

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EI-MS: m/z = 384 (M⁺). HRMS (EI): m/z calcd for C₁₉H₁₆N₂O₅S

384.0780, found 384.0809.

J = 6.7 Hz), 4.60 (2H, s), 6.16 (2H, s), 7.10 (1H, d, J = 8.1 Hz), 7.43

(1H, d, J = 1.6 Hz), 7.50 (1H, dd, J = 8.1 Hz, J = 1.7 Hz), 7.54 (2H, d,

J = 8.2 Hz), 7.78 (2H, d, J = 8.3 Hz), 8.42 (1H, t, J = 5.7 Hz). ¹³C

NMR (DMSO-d₆, 125 MHz): δ [ppm] = 20.2, 28.0, 35.4, 46.6, 102.1,

106.1, 109.2, 116.6, 121.8, 127.5, 128.8, 134.1, 139.8, 148.1, 150.5,

162.4, 165.1, 165.8. HPLC: 95%; t_R7.11 min. EI-MS: m/z = 411 (M⁺).

The following compounds 8d and 9c−e were prepared in a similar

manner to that described for 8c.

3-((5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)methyl)-

benzonitrile (7a). Yield 41%, yellow solid. ¹H NMR (DMSO-d₆,

500 MHz): δ [ppm] = 4.66 (2H, s), 7.58 (1H, t, J = 7.8 Hz), 7.78 (1H,

dt, J = 7.7 Hz, J = 1.3 Hz), 7.86 (1H, t, J = 1.2 Hz), 7.88 (2H, dd, J =

4.4 Hz, J = 1.6 Hz), 7.99 (1H, t, J = 1.4 Hz), 8.82 (2H, dd, J = 4.4 Hz,

J = 1.6 Hz). ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 34.8, 111.4,

118.5, 120.0, 129.8, 130.0, 131.5, 132.7, 134.0, 138.6, 150.9, 163.8,

164.4. HPLC: 96%; t_R4.51 min. EI-MS: m/z = 294 (M⁺).

4-((5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-ylthio)methyl)-

benzonitrile (7b). Yield 77%, pale-yellow solid. ¹H NMR (DMSO-d₆,

500 MHz): δ [ppm] = 4.69 (2H, s), 7.71 (2H, d, J = 8.2 Hz), 7.83

(2H, d, J = 8.2 Hz), 7.88 (2H, dd, J = 4.4 Hz, J = 1.6 Hz), 8.82 (2H,

dd, J = 4.5 Hz, J = 1.5 Hz). ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] =

35.2, 110.5, 118.6, 120.0, 130.0, 130.1, 132.4, 142.6, 150.8, 163.9, 164.4.

HPLC: 95%; t_R4.51 min. EI-MS: m/z = 294 (M⁺).

4-((5-(Benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-ylthio)-

methyl)-N-(2,2-dimethoxyethyl)benzamide (8d). Yield 79%,

1

light-yellow solid. H NMR (DMSO-d₆, 500 MHz): δ [ppm] = 3.27

(6H, s), 3.33 (2H, br), 4.48 (1H, t, J = 5.6 Hz), 4.61 (2H, s), 6.16 (2H,

s), 7.11 (1H, d, J = 8.1 Hz), 7.44 (1H, d, J = 1.7 Hz), 7.50 (1H, dd, J =

8.1 Hz, J = 1.7 Hz), 7.55 (2H, d, J = 8.1 Hz), 7.80 (2H, d, J = 8.1 Hz),

8.52 (1H, t, J = 5.7 Hz). ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] =

35.4, 41.1, 53.2, 101.8, 102.2, 106.2, 109.2, 116.6, 121.7, 127.5, 128.9,

133.5, 140.1, 148.1, 150.4, 162.4, 165.1, 166.0. HPLC: 95%; t_R6.07

min. EI-MS: m/z = 443 (M⁺).

2-(Benzylthio)-5-(pyridin-4-yl)-1,3,4-oxadiazole (7c). Yield

1

79%, light-yellow solid. H NMR (DMSO-d₆, 500 MHz): δ [ppm] =

4-((5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-

2-ylthio)methyl)-N-isobutyl Benzamide (9c). Yield 92%, light-

brown solid. ¹H NMR (DMSO-d₆, 500 MHz): δ [ppm] = 0.93 (6H, d,

J = 6.7 Hz), 1.88 (1H, n, J = 6.7 Hz), 3.12 (2H, t, J = 6.6 Hz), 4.37 (2H,

m), 4.39 (2H, m), 4.66 (2H, s), 7.10 (1H, d, J = 8.4 Hz), 7.45 (1H, d,

J = 2.0 Hz), 7.48 (1H, dd, J = 8.4 Hz, J = 2.0 Hz), 7.60 (2H, d, J = 8.2

Hz), 7.85 (2H, d, J = 8.2 Hz), 8.46 (1H, t, J = 5.7 Hz). ¹³C NMR

(DMSO-d₆, 125 MHz): δ [ppm] = 20.2, 28.1, 35.4, 46.6, 64.1, 64.4,

115.0, 115.8, 118.1, 119.9, 127.4, 128.8, 134.1, 139.7, 143.8, 146.7,

162.4, 164.9, 165.8. HPLC: 96%; t_R7.16 min. EI-MS: m/z = 425 (M⁺).

4-((5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-

2-ylthio)methyl)-N-(2,2-dimethoxy ethyl)benzamide (9d). Yield

4.62 (2H, s), 7.30 (1H, m), 7.36 (2H, m), 7.50 (2H, m), 7.90 (2H, dd,

J = 4.4 Hz, J = 1.6 Hz), 8.82 (2H, dd, J = 4.4 Hz, 1.6 Hz). ¹³C NMR

(DMSO-d₆, 125 MHz): δ [ppm] = 35.8, 120.0, 127.8, 128.6, 129.1,

130.0, 136.4, 150.8, 163.6, 164.7. HPLC: 100%; t_R4.89 min. EI-MS:

m/z = 269 (M⁺).

2-(3-Iodobenzylthio)-5-(pyridin-4-yl)-1,3,4-oxadiazole (7d).

7d was used as reference. It is commercially available from Calbiochem

(361541 GSK-3β Inhibitor II; CAS number, 478482-75-6).

4-((5-(Benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-ylthio)-

methyl)benzoic Acid (8a). Methyl 4-((5-(benzo[d][1,3]dioxol-5-

yl)-1,3,4-oxadiazol-2-ylthio)methyl benzoate 5c (300 mg, 0.81 mmol)

was added in 5 mL of a 2 N lithium hydroxide−tetrahydrofuran

solution. The reaction mixture was stirred overnight at 60 °C under an

argon atmosphere. The reaction mixture was diluted with water and

neutralized with 1 N HCl. Afterward, EtOAc was added and the

organic layer was washed with water and brine, dried over MgSO₄, and

1

84%, beige solid. H NMR (DMSO-d₆, 500 MHz): δ [ppm] = 3.28

(6H, s), 3.35 (2H, d, J = 5.7 Hz), 4.31 (2H, m), 4.34 (2H, m), 4.50

(1H, t, J = 5.6 Hz), 4.61 (2H, s), 7.05 (1H, d, J = 8.3 Hz), 7.40 (1H, d,

J = 2.0 Hz), 7.43 (1H, dd, J = 8.3 Hz, J = 2.0 Hz), 7.55 (2H, d, J = 8.2

Hz), 7.81 (2H, d, J = 8.2 Hz), 8.52 (1H, t, J = 5.8 Hz). ¹³C NMR

(DMSO-d₆, 125 MHz): δ [ppm] = 35.4, 41.1, 53.2, 64.0, 64.4, 101.8,

115.0, 105.8, 108.2, 119.9, 127.4, 128.8, 133.5, 140.0, 143.8, 146.7,

162.4, 164.9, 165.9. HPLC: 95%; t_R6.13 min. EI-MS: m/z = 457 (M⁺).

N-Benzyl-4-((5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-

oxadiazol-2-ylthio)methyl)benzamide (9e). Yield 89%, beige

1

concentrated in vacuo to give 8a (239 mg, 83%) as a rose solid. H

NMR (DMSO-d₆, 500 MHz): δ [ppm] = 4.56 (2H, s), 6.08 (2H, s),

7.04 (1H, d, J = 8.1 Hz), 7.35 (1H, d, J = 1.6 Hz), 7.43 (1H, dd, J =

8.1 Hz, J = 1.7 Hz), 7.53 (2H, d, J = 8.2 Hz), 7.84 (2H, d, J = 8.2 Hz),

12.8 (1H, s, br). ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 35.4,

102.1, 106.1, 109.1, 116.6, 121.7, 129.1, 129.5, 130.4, 141.7, 148.1, 150.4,

162.4, 165.1, 166.9. HPLC: 99%; t_R6.15 min. EI-MS: m/z = 356 (M⁺).

Compound 9a was prepared in a similar manner to that described

for 8a.

Methyl 4-((5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-ox-

adiazol-2-ylthio)methyl)benzoic Acid (9a). Yield 91%, colorless

solid. ¹H NMR (DMSO-d₆, 500 MHz): δ [ppm] = 4.31 (2H, m), 4.34

(2H, m), 4.62 (2H, s), 7.05 (1H, d, J = 8.4 Hz), 7.38 (1H, d, J =

2.0 Hz), 7.43 (1H, dd, J = 8.4 Hz, J = 2.1 Hz), 7.59 (2H, d, J = 8.3 Hz),

7.91 (2H, d, J = 8.3 Hz), 12.95 (1H, s). ¹³C NMR (DMSO-d₆, 125

MHz): δ [ppm] = 35.4, 64.1, 64.4, 115.1, 115.7, 118.2, 119.8, 129.3,

129.5, 130.1, 141.9, 143.8, 146.7, 162.4, 165.0, 166.8. HPLC: 96%;

t_R6.22 min. EI-MS: m/z = 370 (M⁺).

1

solid. H NMR (DMSO-d₆, 500 MHz): δ [ppm] = 4.31 (2H, m),

4.34 (2H, m), 4.47 (2H, d, J = 5.9 Hz), 4.62 (2H, s), 7.05 (1H, d, J =

8.4 Hz), 7.23 (1H, m), 7.32 (4H, m), 7.40 (1H, d, J = 2.0 Hz), 7.43

(1H, dd, J = 8.3 Hz, J = 2.0 Hz), 7.56 (2H, d, J = 8.2 Hz), 7.86 (2H, d,

J = 8.2 Hz), 9.01 (1H, t, J = 5.9 Hz). ¹³C NMR (DMSO-d₆,

125 MHz): δ [ppm] = 35.4, 42.6, 64.1, 64.4, 115.1, 115.8, 118.3, 119.8,

126.7, 127.3, 127.5, 128.4, 128.8, 133.7, 139.6, 140.1, 143.8, 146.7,

162.4, 165.0, 165.8. HPLC: 95%; t_R7.66 min. EI-MS: m/z = 459 (M⁺).

2-(4-(1H-Tetrazol-5-yl)benzylthio)-5-(benzo[d][1,3]dioxol-5-

yl)-1,3,4-oxadiazole (8e). 4-((5-Benzo[d][1,3]dioxol-5-yl)-1,3,4-ox-

adiazol-2-ylthio)methyl)benzonitrile 5b (34 mg, 0.10 mmol), NaN₃

(78 mg, 1.20 mmol), and NH₄Cl (64 mg, 1.20 mmol) were added to

1 mL of DMF and stirred for 5 h at 100 °C under microwave

irradiation. After cooling to room temperature, the reaction solution

was added to water (2−3 mL), acidiﬁed with 2 N HCl, and extracted

three times with ethyl acetate. The combined organic layers were dried

over Na₂SO₄, ﬁltered, and the solvent evaporated oﬀ to provide 8e

4-((5-(Benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-ylthio)-

methyl)-N-isobutylbenzamide (8c). A mixture of 4-((5-(benzo-

[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-ylthio)methyl)benzoic acid 8a

(100 mg, 0.28 mmol) and thionyl chloride (30.5 μL, 0.42 mmol)

was reﬂuxed in dry toluene (1 mL) for about 2 h. Excess thionyl

chloride was removed by repeated evaporation in vacuo with fresh dry

toluene (3 × 1 mL). 2-Methylpropan-1-amine (27.8 μL, 0.28 mmol)

and K₂CO₃(38 mg, 0.28 mmol) were added in dry acetone (1 mL)

cooled to 0 °C and stirred for 30 min. The crude acyl chloride was

dissolved in dry acetone (0.5 mL) and added dropwise to the solution.

After the addition was complete, stirring continued for 2 h. The

reaction mixture was then diluted with water, extracted three times

with EtOAc, and successively washed with brine. The organic layer was

dried over MgSO₄and concentrated under reduced pressure. The

obtained residue was recrystallized from EtOH to give 8c (90 mg,

1

(25 mg, 67%) as a beige solid. H NMR (DMSO-d₆, 500 MHz): δ

[ppm] = 4.65 (2H, s), 6.15 (2H, s), 7.11 (1H, d, J = 8.1 Hz), 7.43 (1H,

d, J = 1.6 Hz), 7.50 (1H, dd, J = 8.1 Hz, J = 1.7 Hz), 7.71 (2H, d, J =

8.3 Hz), 8.00 (2H, d, J = 8.3 Hz), NH signal was not observed. ¹³C

NMR (DMSO-d₆, 125 MHz): δ [ppm] = 35.4, 102.1, 105.0, 106.2,

108.9, 109.1, 116.6, 119.7, 121.7, 127.1, 130.0, 148.1, 150.4, 162.4,

165.1. HPLC: 96%; t_R5.92 min. EI-MS: m/z = 380 (M⁺).

Compound 9f was prepared in a similar manner to that described

for 8e.

2-(4-(1H-Tetrazol-5-yl)benzylthio)-5-(2,3-dihydrobenzo[d]-

1

[1,4]dioxin-5-yl)-1,3,4-oxadiazole (9f). Yield 79%, puce solid. H

1

81%) as a beige solid. H NMR (DMSO-d₆, 500 MHz): δ [ppm] =

NMR (DMSO-d₆, 500 MHz): δ [ppm] = 4.35 (2H, m), 4.38 (2H, m),

4.70 (2H, s), 7.10 (1H, d, J = 8.4 Hz), 7.43 (1H, d, J = 2.0 Hz),

0.86 (6H, d, J = 6.7 Hz), 1.81 (1H, n, J = 6.7 Hz), 3.05 (2H, t,

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7.47 (1H, dd, J = 8.4 Hz, J = 2.1 Hz), 7.75 (2H, d, J = 8.3 Hz), 8.05

(2H, d, J = 8.3 Hz), NH signal was not observed. ¹³C NMR (DMSO-

d₆, 125 MHz): δ [ppm] = 35.5, 64.0, 64.4, 115.0, 115.8, 118.2, 119.9,

123.6, 127.1, 130.0, 140.1, 143.8, 146.7, 162.4, 165.1. HPLC: 95%; t_R

6.01 min. EI-MS: m/z = 394 (M⁺).

7.62 (1H, d, J = 7.7 Hz), 7.68 (2H, d, J = 8.1 Hz), 7.78 (1H, t, J = 7.7

Hz), 7.91 (2H, d, J = 4.1 Hz), 7.95 (1H, d, J = 7.7 Hz), 8.85 (2H, s,

br). ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 35.4, 110.1, 118.5,

120.1, 128.2, 128.9, 129.4, 130.0, 130.1, 133.5, 133.7, 137.2, 137.3,

143.9, 150.8, 163.8, 164.8. HPLC: 99%; t_R6.63 min. EI-MS: m/z =

370 (M⁺). HRMS (EI): m/z calcd for C₂₁H₁₄N₄OS 370.0889, found

370.0926.

The following compounds 14a−d, 15a−b, and 16a were prepared

in a similar manner to that described for 5a.

2-(Benzo[d][1,3]dioxol-5-yl)-5-(biphenyl-4-ylmethylthio)-

1,3,4-oxadiazole (14a). Yield 75%, beige solid. ¹H NMR (DMSO-d₆,

500 MHz): δ [ppm] = 4.55 (2H, s), 6.08 (2H, s), 7.04 (1H, d, J =

8.1 Hz), 7.28 (1H, m), 7.38 (3H, m), 7.45 (1H, dd, J = 8.1 Hz, J =

1.7 Hz), 7.48 (2H, d, J = 8.3 Hz), 7.58 (4H, m). ¹³C NMR (DMSO-d₆,

125 MHz): δ [ppm] = 35.6, 102.1, 106.2, 109.1, 116.6, 121.7, 126.6,

126.8, 127.6, 128.9, 129.7, 135.8, 139.6, 148.1, 150.5, 162.6, 165.1.

HPLC: 95%; t_R9.12 min. EI-MS: m/z = 388 (M⁺).

Methyl 3,4-Dihydroxybenzoate (18). To a stirred solution of

3,4-dihydroxybenzoic acid (2.0 g, 13 mmol) in MeOH (25 mL) was

added SOCl₂(1.88 mL, 26 mmol) dropwise over 1 h at 0 °C. The

solution was further stirred 12 h at 50 °C. The mixture was cooled to

room temperature and diluted with water (30 mL). MeOH was

evaporated and the pH adjusted to ∼6 with aqueous NaHCO₃. The

mixture was extracted three times with EtOAc and successively washed

with brine. The organic layer was dried over MgSO₄and concentrated

under reduced pressure to give 18 (2.1 g, 97%) as a colorless solid. ¹H

NMR (DMSO-d₆, 500 MHz): δ [ppm] = 3.75 (3H, s), 6.80 (1H, d, J =

8.2 Hz), 7.31 (1H, dd, J = 8.2 Hz, J = 2.1 Hz), 7.35 (1H, d, J = 2.1 Hz),

9.35 (1H, s), 9.75 (1H, s). ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] =

51.5, 115.3, 116.2, 120.4, 121.7, 145.0, 150.4, 166.1. EI-MS: m/z =

168 (M⁺).

4′-((5-(Benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-ylthio)-

methyl)biphenyl-2-carbonitrile (14b). Yield 71%, brown solid. ¹H

NMR (DMSO-d₆, 500 MHz): δ [ppm] = 4.66 (2H, s), 6.15 (2H, s),

7.11 (1H, d, J = 8.1 Hz), 7.45 (1H, d, J = 1.7 Hz), 7.51 (1H, dd, J = 8.1

Hz, J = 1.7 Hz), 7.57 (2H, d, J = 8.2 Hz), 7.65 (4H, m), 7.96 (1H, dd,

J = 8.5 Hz, J = 2.3 Hz). ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] =

35.5, 102.1, 106.1, 109.1, 111.5, 111.6, 116.6, 117.3, 117.4, 120.3,

120.5, 121.0, 121.2, 121.7, 129.0, 129.4, 132.3, 132.4, 136.2, 137.5,

140.8, 140.9, 148.1, 150.4, 159.9, 161.9, 162.6, 165.1. HPLC: 95%;

t_R8.77 min. EI-MS: m/z = 431 (M⁺). HRMS (EI): m/z calcd for

C₂₃H₁₄N₃O₃FS 431.0740, found 431.0728.

Methyl 3-(Hydroxymethyl)-2,3-dihydrobenzo[b][1,4]-

dioxine-6-carboxylate (19a). Methyl 3,4-dihydroxybenzoate 18

(0.8 g, 4.75 mmol) and K₂CO₃(0.65 g, 4.75 mmol) were taken in

dry acetone (10 mL) and stirred for 15 min at room temperature.

Afterward, the solution was treated with epibromohydrin (0.40 mL,

4.75 mmol) and stirred overnight at 70 °C. The reaction mixture was

diluted with water and extracted with EtOAc. The organic layer was

washed with brine and dried over Na₂SO₄. The solvent was evaporated

under reduced pressure and the crude product puriﬁed by silica gel

column chromatography (DCM/EtOAc, 4:1) to give 19a (1.01 g, 95%)

́

4-((5-(Benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-ylthio)-

methyl)-4-ﬂuorobiphenyl-2-carbonitrile (14c). Yield 69%, gray

solid. ¹H NMR (DMSO-d₆, 500 MHz): δ [ppm] = 4.66 (2H, s), 6.16

(2H, s), 7.11 (1H, d, J = 8.1 Hz), 7.45 (1H, d, J = 1.7 Hz), 7.51 (1H,

dd, J = 8.1 Hz, J = 1.7 Hz), 7.56 (2H, d, J = 8.2 Hz), 7.65 (4H, m),

7.78 (1H, td, J = 7.7 Hz, J = 1.3 Hz), 7.95 (1H, dd, J = 7.7 Hz, J =

0.9 Hz). ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 35.5, 102.1,

106.1, 109.1, 110.1, 116.6, 118.5, 121.7, 128.3, 128.9, 129.4, 130.1,

133.5, 133.8, 137.2, 137.4, 140.0, 148.1, 150.4, 162.6, 165.1. HPLC:

96%; t_R8.36 min. EI-MS: m/z = 413 (M⁺). HRMS (EI): m/z calcd for

C₂₃H₁₅N₃O₃S 413.0835, found 413.0804.

1

as a colorless oil. H NMR (DMSO-d₆, 500 MHz): δ [ppm] = 3.64

(2H, m), 3.80 (3H, s), 4.10 (1H, m), 4.20 (1H, m), 4.41 (1H, dd, J =

11.4 Hz, J = 2.3 Hz), 5.10 (1H, t, J = 5.7 Hz), 6.98 (1H, d, J = 8.4 Hz),

7.41 (1H, d, J = 2.0 Hz), 7.46 (1H, dd, J = 8.4 Hz, J = 2.0 Hz). ¹³C

NMR (DMSO-d₆, 125 MHz): δ [ppm] = 51.9, 59.7, 65.4, 73.6, 117.0,

117.9, 122.6, 122.7, 142.8, 147.4, 165.6. EI-MS: m/z = 224 (M⁺).

Note: Alternatively (R/S)-( )-glycidyl tosylate can be used to

obtain compound 19a. For more details see the synthesis of compound

19b−c.

4′-((5-(Benzo[d][1,3]dioxol-5-yl)-1,3,4-oxadiazol-2-ylthio)-

methyl)biphenyl-4-carbonitrile (14d). Yield 51%, gray−brown

solid. ¹H NMR (DMSO-d₆, 500 MHz): δ [ppm] = 4.63 (2H, s), 6.15

(2H, s), 7.10 (1H, d, J = 8.1 Hz), 7.43 (1H, d, J = 1.7 Hz), 7.51 (1H,

dd, J = 8.1 Hz, J = 1.7 Hz), 7.61 (2H, d, J = 8.3 Hz), 7.73 (2H, d, J =

8.3 Hz), 7.87 (2H, d, J = 8.6 Hz), 7.91 (2H, d, J = 8.6 Hz). ¹³C NMR

(DMSO-d₆, 125 MHz): δ [ppm] = 35.9, 102.6, 106.6, 109.6, 110.6,

117.1, 119.3, 122.2, 127.7, 128.0, 130.3, 133.3, 138.0, 138.1, 144.5,

148.6, 150.9, 163.0, 165.5. HPLC: 97%; t_R8.77 min. EI-MS: m/z =

413 (M⁺). HRMS (EI): m/z calcd for C₂₃H₁₅N₃O₃S 413.0835, found

413.0825.

Methyl 3-(Methoxymethyl)-2,3-dihydrobenzo[b][1,4]-

dioxine-6-carboxylate (20a). To a suspension of NaH (115 mg,

4.81 mmol) in 5 mL of anhydrous THF was added methyl

3-(hydroxymethyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-carboxylate

19a (900 mg, 4.01 mmol) at 0 °C. The mixture was stirred at room

temperature for 30 min, followed by addition of methyl iodide (374 μL,

6.01 mmol). The reaction mixture was stirred at room temperature for

48 h, quenched with 10 mL of water, and extracted with ethyl acetate.

The combined organic layer was dried over MgSO₄, the solvent was

evaporated under reduced pressure, and the crude product puriﬁed by

silica gel column chromatography (DCM/EtOAc, 20:1) to give 20a

́

4-((5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-[1,3,4]oxadiazol-

2-ylthio)methyl)biphenyl-2-carbonitrile (15a). Yield 74%, color-

less solid. ¹H NMR (DMSO-d₆, 500 MHz): δ [ppm] = 4.31 (2H, m),

4.34 (2H, m), 4.65 (2H, s), 7.05 (1H, d, J = 8.4 Hz), 7.41 (1H, d, J =

2.0 Hz), 7.44 (1H, dd, J = 8.4 Hz, J = 2.0 Hz), 7.60 (6H, m), 7.79 (1H,

td, J = 7.7 Hz, J = 1.2 Hz), 7.95 (1H, dd, J = 7.7 Hz, J = 0.9 Hz). ¹³C

NMR (DMSO-d₆, 125 MHz): δ [ppm] = 35.9, 64.4, 64.8, 110.5, 115.4,

116.3, 118.5, 118.8, 120.3, 128.6, 129.3, 129.7, 130.5, 133.8, 134.2,

137.5, 137.8, 144.1, 144.3, 147.1, 163.0, 165.3. HPLC: 100%; t_R8.39

min. EI-MS: m/z 427 (M⁺). HRMS (EI): m/z calcd for C₂₄H₁₇N₃O₃S

427.0991, found 427.0962.

1

(678 mg, 71%) as a colorless oil. H NMR (DMSO-d₆, 500 MHz): δ

[ppm] = 3.33 (3H, s), 3.60 (2H, m), 3.80 (3H, s), 4.09 (1H, m), 4.40

(2H, m), 6.99 (1H, m), 7.41 (1H, m), 7.47 (1H, m). ¹³C NMR

(DMSO-d₆, 125 MHz): δ [ppm] = 51.8, 58.7, 65.3, 70.3, 71.8, 117.0,

118.0, 122.7, 122.8, 142.6, 147.2, 165.5. EI-MS: m/z = 238 (M⁺).

3-(Methoxymethyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-car-

bohydrazide (21a). To a solution of 20a (600 mg, 2.51 mmol) in

EtOH (15 mL) was added hydrazine hydrate (731 μL, 15.06 mmol).

and the mixture was heated at reﬂux for 2 days. After cooling to room

temperature, the reaction mixture was diluted with water and extracted

with EtOAc. The organic layer was washed with brine and dried over

MgSO₄. The solvent was evaporated under reduced pressure and the

crude product puriﬁed by silica gel column chromatography (MeOH/

EtOAc, 1:10) to give 21a (466 mg, 78%) as a colorless solid. ¹H NMR

(DMSO-d₆, 500 MHz): δ [ppm] = 3.32 (3H, s), 3.59 (2H, m), 4.04

(1H, m), 4.36 (2H, m), 4.41 (2H, s), 6.92 (1H, d, J = 8.2 Hz), 7.35

(1H, dd, J = 8.2 Hz, J = 2.0 Hz), 7.37 (1H, d, J = 2.0 Hz), 9.52 (1H, s).

¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 58.7, 65.1, 70.4, 71.8,

4′-((5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-

2-ylthio)methyl)-4-ﬂuorobiphenyl-2-carbonitrile (15b). Yield

1

29%, light-yellow solid. H NMR (DMSO-d₆, 500 MHz): δ [ppm] =

4.32 (2H, m), 4.34 (2H, m) 4.65 (2H, s), 7.05 (1H, d, J = 8.4 Hz), 7.40

(1H, d, J = 2.1 Hz), 7.45 (1H, dd, J = 8.4 Hz, J = 2.1 Hz), 7.56 (2H, d,

J = 8.2 Hz), 7.63 (2H, d, J = 8.2 Hz), 7.68 (2H, m), 7.97 (1H, dd, J =

9.0 Hz, J = 1.9 Hz). HPLC: 95%; t_R8.76 min. EI-MS: m/z = 445 (M⁺).

HRMS (EI): m/z calcd for C₂₄H₁₆N₃O₃FS 445.0897, found 445.0890.

́

4-((5-(Pyridine-4-yl)-1,3,4-oxadiazol-2-ylthio)methyl)-

1

biphenyl-2-carbonitrile (16a). Yield 53%, colorless solid. H NMR

(DMSO-d₆, 500 MHz): δ [ppm] = 4.71 (2H, s), 7.58 (3H, m),

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115.8, 116.6, 120.3, 126.5, 142.3, 145.3, 165.1. EI-MS: m/z = 238

5.10 (1H, s, br), 6.98 (1H, d, J = 8.4 Hz), 7.41 (1H, d, J = 2.0 Hz),

7.46 (1H, dd, J = 8.4 Hz, J = 2.0 Hz). ¹³C NMR (DMSO-d₆,

125 MHz): δ [ppm] = 51.9, 59.7, 65.5, 73.6, 117.0, 117.9, 122.6, 122.7,

142.8, 147.4, 165.6. EI-MS: m/z = 224 (M⁺).

Compounds 20b−c were prepared in a similar manner to that

described for 20a.

(M⁺).

Compound 22a was prepared in a similar manner to that described

for 4A.

5-(3-(Methoxymethyl)-2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-

1

1,3,4-oxadiazole-2-thiol (22a). Yield 81%, orange solid. H NMR

(DMSO-d₆, 500 MHz): δ [ppm] = 3.32 (3H, s), 3.61 (2H, m), 4.10

(1H, m), 4.43 (2H, m), 7.07 (1H, m), 7.32 (1H, m), 7.37 (1H, m),

14.60 (1H, s). ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 58.7, 65.3,

70.3, 72.1, 114.6, 115.6, 118.0, 119.7, 143.4, 146.4, 160.1, 177.4. EI-

MS: m/z = 280 (M⁺).

(R)-Methyl 3-(Methoxymethyl)-2,3-dihydrobenzo[b][1,4]-

dioxine-6-carboxylate (20b). Yield 68%, as a colorless oil. ¹H

NMR (DMSO-d₆, 500 MHz): δ [ppm] = 3.33 (3H, s), 3.60 (2H, m),

3.81 (3H, s), 4.09 (1H, m), 4.40 (2H, m), 6.99 (1H, d, J = 8.4 Hz),

7.42 (1H, d, J = 2.0 Hz), 7.47 (1H, dd, J = 8.4 Hz, J = 2.0 Hz). ¹³C

NMR (DMSO-d₆, 125 MHz): δ [ppm] = 51.9, 58.7, 65.3, 70.3, 71.8,

117.1, 117.9, 122.8, 122.9, 142.6, 147.3, 165.6. EI-MS: m/z = 238 (M⁺).

(S)-Methyl 3-(Methoxymethyl)-2,3-dihydrobenzo[b][1,4]-

dioxine-6-carboxylate (20c). After extraction and evaporation of

the solvent, compound 20c was used without further puriﬁcation.

Compounds 21b−c were prepared in a similar manner to that

described for 21a.

Compound 23a was prepared in a similar manner to that described

for 5a.

4′-((5-(3-(Methoxymethyl)-2,3-dihydrobenzo[b][1,4]dioxin-

6-yl)-1,3,4-oxadiazol-2ylthio)methyl)biphenyl-2-carbonitrile

1

(23a). Yield 88%, colorless solid. H NMR (DMSO-d₆, 500 MHz): δ

[ppm] = 3.33 (3H, s), 3.61 (2H, m), 4.10 (1H, m), 4.44 (2H, m), 4.66

(2H, s), 7.07 (1H, m), 7.45 (2H, m), 7.57 (3H, m), 7.63 (3H, m), 7.78

(1H, td, J = 7.6 Hz, J = 1.2 Hz), 7.95 (1H, dd, J = 7.7 Hz, J = 1.1 Hz).

¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 35.4, 58.7, 65.2, 70.3,

(R)-3-(Methoxymethyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-

1

carbohydrazide (21b). Yield 87%, as a colorless solid. H NMR

72.0, 110.1, 115.1, 116.2, 118.0, 118.5, 119.9, 128.2, 128.8, 129.4,

130.1, 133.5, 133.8, 137.1, 137.4, 143.3, 143.9, 146.2, 162.6, 164.9.

HPLC: 100%; t_R8.59 min. EI-MS: m/z = 471 (M⁺). HRMS (EI): m/z

calcd for C₂₆H₂₁N₃O₄S 471.1253, found 471.1264.

(DMSO-d₆, 500 MHz): δ [ppm] = 3.32 (3H, s), 3.59 (2H, m), 4.04

(1H, m), 4.36 (2H, m), 4.41 (2H, s), 6.92 (1H, d, J = 8.3 Hz), 7.35

(1H, dd, J = 8.3 Hz, J = 2.0 Hz), 7.37 (1H, d, J = 2.0 Hz), 9.57 (1H, s).

¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 58.7, 65.1, 70.4, 71.7,

4′-((5-(3-(Hydroxymethyl)-2,3-dihydrobenzo[b][1,4]dioxin-

6-yl)-1,3,4-oxadiazol-2-ylthio) methyl)biphenyl-2-carbonitrile

(24a). To a solution of 4′-((5-(3-(methoxymethyl)-2,3-dihydrobenzo-

[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-2ylthio)methyl)biphenyl-2-car-

bonitrile 23a (400 mg, 0.85 mmol) in 10 mL of DCM was added 1 N

solution of BBr₃in hexane (850 μL, 0.85 mmol) under argon

atmosphere at −78 °C. The reaction mixture was stirred at the same

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

further stirred for 24 h. After treatment with saturated NaHCO₃

solution, the reaction mixture was extracted with ethyl acetate. The

combined organic layers were dried over MgSO₄and concentrated

under reduced pressure. The crude product was puriﬁed by silica gel

column chromatography (cyclohexane/EtOAc, 1:2) to give compound

24a (283 mg, 73%) as a light-yellow solid. ¹H NMR (DMSO-d₆,

500 MHz): δ [ppm] = 3.59 (1H, s, br), 3.64 (2H, m), 4.10 (1H, m),

4.24 (1H, m), 4.42 (1H, dd, J = 11.5 Hz, J = 2.2 Hz), 4.65 (2H, s) 7.06

(1H, d, J = 8.3 Hz), 7.43 (2H, m), 7.60 (6H, m), 7.78 (1H, td, J =

7.7 Hz, J = 1.2 Hz), 7.94 (1H, dd, J = 7.7 Hz, J = 1.0 Hz). ¹³C NMR

(DMSO-d₆, 125 MHz): δ [ppm] = 35.4, 59.6, 65.4, 73.7, 110.1, 115.1,

116.1, 117.9, 118.5, 119.8, 128.2, 128.8, 129.4, 130.1, 133.4, 133.8,

137.1, 137.4, 143.5, 144.0, 146.2, 162.6, 165.0. HPLC: 96%; t_R7.39

min. EI-MS: m/z = 457(M⁺).

(R)-Methyl 3-(Hydroxymethyl)-2,3-dihydrobenzo[b][1,4]-

dioxine-6-carboxylate (19b). To a round-bottom ﬂask equipped

with magnetic stirring and a nitrogen inlet was added methyl-3,4-

dihydroxybenzoate 18 (1.0 g, 6 mmol), (2S)-(+)-glycidyl tosylate

(1.37 g, 6 mmol), K₂CO₃(0.99 g, 7.2 mmol), and DMF (15 mL). This

mixture was heated to 60 °C for 5 h. The mixture was cooled to room

temperature, diluted with water, and extracted with EtOAc. The

organic layer was washed with brine and dried over Na₂SO₄. The

solvent was evaporated under reduced pressure and the crude product

puriﬁed by silica gel column chromatography (DCM/EtOAc, 4:1) to

give 19b (1.25 g, 93%) as a colorless oil. ¹H NMR (DMSO-d₆,

500 MHz): δ [ppm] = 3.64 (2H, m), 3.80 (3H, s), 4.10 (1H, m), 4.20

(1H, m), 4.41 (1H, dd, J = 11.4 Hz, J = 2.3 Hz), 5.08 (1H, t, J =

5.7 Hz), 6.98 (1H, d, J = 8.4 Hz), 7.41 (1H, d, J = 2.0 Hz), 7.46 (1H,

dd, J = 8.4 Hz, J = 2.0 Hz). ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] =

51.9, 59.7, 65.5, 73.6, 117.0, 117.9, 122.6, 122.7, 142.8, 147.4, 165.6.

HPLC: 96%; t_R2.44 min. EI-MS: m/z = 224 (M⁺).

115.8, 116.6, 120.4, 126.6, 142.3, 145.3, 165.1. EI-MS: m/z = 238 (M⁺).

(S)-3-(Methoxymethyl)-2,3-dihydrobenzo[b][1,4]dioxine-6-

carbohydrazide (21c). Yield 92%, as a colorless solid. ¹H NMR

(DMSO-d₆, 500 MHz): δ [ppm] = 3.32 (3H, s), 3.59 (2H, m), 4.04

(1H, m), 4.36 (2H, m), 4.41 (2H, s), 6.92 (1H, d, J = 8.3 Hz), 7.36

(1H, dd, J = 8.3 Hz, J = 2.0 Hz), 7.37 (1H, d, J = 2.0 Hz), 9.57 (1H, s).

¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 58.7, 65.1, 70.4, 71.8,

115.8, 116.6, 120.4, 126.6, 142.3, 145.3, 165.2. EI-MS: m/z = 238 (M⁺).

Compounds 22b−c were prepared in a similar manner to that

described for 4A.

(R)-5-(3-(Methoxymethyl)-2,3-dihydrobenzo[b][1,4]dioxin-6-

yl)-1,3,4-oxadiazole-2-thiol (22b). Yield 91%, orange solid. ¹H

NMR (DMSO-d₆, 500 MHz): δ [ppm] = 3.33 (3H, s), 3.61 (2H, m),

4.10 (1H, m), 4.42 (2H, m), 7.07 (1H, m), 7.31 (1H, m), 7.37 (1H,

m), 14.62 (1H, s). ¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 58.8,

65.3, 70.2, 72.1, 114.6, 115.6, 118.0, 119.7, 143.3, 146.4, 160.1, 177.2.

EI-MS: m/z = 280 (M⁺).

(S)-5-(3-(Methoxymethyl)-2,3-dihydrobenzo[b][1,4]dioxin-6-

yl)-1,3,4-oxadiazole-2-thiol (22c). Yield 91%, orange solid. ¹H

NMR (DMSO-d₆, 500 MHz): δ [ppm] = 3.33 (3H, s), 3.61 (2H, m),

4.09 (1H, m), 4.39 (2H, m), 7.02 (1H, m), 7.24 (1H, m), 7.30 (1H,

m), SH signal was not observed. ¹³C NMR (DMSO-d₆, 125 MHz): δ

[ppm] = 58.6, 65.3, 70.0, 72.1, 114.6, 115.6, 117.7, 119.7, 143.3, 146.4,

160.1, 177.2. EI-MS: m/z = 280 (M⁺).

Compounds 23b−c were prepared in a similar manner to that

described for 5a.

(R)-4′-((5-(3-(Methoxymethyl)-2,3-dihydrobenzo[b][1,4]-

dioxin-6-yl)-1,3,4-oxadiazol-2-ylthio) methyl)biphenyl-2-car-

bonitrile (23b). Yield 84%, colorless solid. ¹H NMR (DMSO-d₆,

500 MHz): δ [ppm] = 3.38 (3H, s), 3.66 (2H, m), 4.15 (1H, m), 4.47

(2H, m), 4.71 (2H, s), 7.13 (1H, m), 7.50 (2H, m), 7.65 (6H, m), 7.82

(1H, td, J = 7.7 Hz, J = 1.3 Hz), 8.00 (1H, dd, J = 7.7 Hz, J = 0.9 Hz).

¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 35.4, 58.7, 65.2, 70.4,

72.0, 110.1, 115.1, 116.2, 118.0, 118.4, 119.9, 128.2, 128.8, 129.4,

130.1, 133.5, 133.8, 137.2, 137.4, 143.2, 143.9, 146.2, 162.6, 164.9.

HPLC: 99%; t_R8.70 min. EI-MS: m/z = 471 (M⁺).

(S)-4′-((5-(3-(Methoxymethyl)-2,3-dihydrobenzo[b][1,4]-

dioxin-6-yl)-1,3,4-oxadiazol-2-ylthio) methyl)biphenyl-2-car-

bonitrile (23c). Yield 84%, colorless solid. ¹H NMR (DMSO-d₆,

500 MHz): δ [ppm] = 3.33 (3H, s), 3.61 (2H, m), 4.10 (1H, m), 4.42

(2H, m), 4.66 (2H, s), 7.07 (1H, m), 7.44 (2H, m), 7.60 (6H, m), 7.78

(1H, td, J = 7.6 Hz, J = 1.3 Hz), 7.94 (1H, dd, J = 7.7 Hz, J = 0.9 Hz).

¹³C NMR (DMSO-d₆, 125 MHz): δ [ppm] = 35.2, 58.7, 65.2, 70.1,

Compound 19c was prepared in a similar manner to that described

for 19b. (2R)-(−)-glycidyl tosylate was used instead of (2S)-(+)-glycidyl

tosylate to obtain the S-isomer.

(S)-Methyl 3-(Hydroxymethyl)-2,3-dihydrobenzo[b][1,4]-

dioxine-6-carboxylate (19c). Yield 89%, as a colorless oil. ¹H

NMR (DMSO-d₆, 500 MHz): δ [ppm] = 3.64 (2H, m), 3.80 (3H, s),

4.10 (1H, m), 4.20 (1H, m), 4.41 (1H, dd, J = 11.4 Hz, J = 2.3 Hz),

71.9, 110.1, 115.1, 116.2, 117.9, 118.4, 119.9, 128.2, 128.6, 129.4,

130.1, 133.5, 133.8, 137.2, 137.4, 143.2, 143.9, 146.2, 162.6, 164.9.

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Table 5. Small Kinase Panel Values

enzyme conc

(nM)

ATP conc

(μM)

peptide conc

kinase

peptide used

(μM)

buﬀer

GSK-3β

2

12.5

32

Ser/Thr 9 peptide

2

50 mM Hepes pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% (w/v) Brij-

35

GSK-3α

CKIε

0.5

12

Ser/Thr 9 peptide

Ser/Thr 11 peptide

Ser/Thr 12 peptide

Ser/Thr 1 peptide

Ser/Thr 7 peptide

50 mM Hepes pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% (w/v) Brij-

35

50 mM Hepes pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% (w/v) Brij-

35

Cdk5

10

12.5

10

50 mM Hepes pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% (w/v) Brij-

35

AurKA

PKCα

20

50 mM Hepes pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% (w/v) Brij-

35

0.15

10

50 mM Hepes pH 7.5, 10 mM MgCl₂, 1 mM EGTA, 0.01% (w/v) Brij-

35

HPLC: 100%; t_R8.65 min. EI-MS: m/z = 471 (M⁺). HRMS (EI): m/z

calcd for C₂₆H₂₁N₃O₄S 471.1253, found 471.1269.

Compound 24b was prepared in a similar manner to that described

for 24a.

138.4, 138.8, 144.7, 145.4, 147.6, 163.7, 166.3. HPLC: 95%; t_R6.34 min.

EI-MS: m/z = 544 (M⁺).

GSK-3β in Vitro Assay. Puriﬁed GSK-3β (0.5 μg) was incubated

in a reaction mixture of 50 mM Tris pH 7.3, 10 mM MgAc₂, 0.01%

β-mercaptoethanol, ³²P[γ-ATP](100 μM, 0.5 μci/assay), and 100 μM

of peptide substrate, pIRS-1 (RREGGMSRPAS(p)VDG (1). New

molecules were added at various concentrations (1, 10, and 100 μM),

and the reaction mixture was incubated for 15 min at 30 °C. The

reactions were stopped, spotted on p81 paper (Whatman), washed

with 10 mM phosphoric acid, and counted for radioactivity.⁵⁰GSK-3β

activity was calculated as the percentage of GSK-3β activity in the

absence of inhibitor that was designated to 100%.

(R)-4′-((5-(3-(Hydroxymethyl)-2,3-dihydrobenzo[b][1,4]-

dioxin-6-yl)-1,3,4-oxadiazol-2-ylthio) methyl)biphenyl-2-car-

1

bonitrile (24b). Yield 79%, light-yellow solid. H NMR (DMSO-d₆,

500 MHz): δ [ppm] = 3.40 (1H, s, br), 3.59 (2H, m), 4.04 (1H, m),

4.15 (1H, m), 4.34 (1H, dd, J = 11.5 Hz, J = 2.3 Hz), 4.57 (2H, s),

6.98 (1H, d, J = 8.3 Hz), 7.35 (2H, m), 7.53 (6H, m), 7.70 (1H, td, J =

7.7 Hz, J = 1.3 Hz), 7.85 (1H, dd, J = 7.7 Hz, J = 0.9 Hz). ¹³C NMR

(DMSO-d₆, 125 MHz): δ [ppm] = 35.4, 59.6, 65.4, 73.7, 110.1, 115.0,

116.1, 117.9, 118.5, 119.8, 128.2, 128.8, 129.3, 130.1, 133.4, 133.8,

137.1, 137.4, 143.5, 144.0, 146.2, 162.5, 164.9. HPLC: 95%; t_R7.48

min. EI-MS: m/z = 457 (M⁺).

Small Kinase Panel. Compounds were serially diluted 1/3 in neat

DMSO (10 serial dilutions), and these dilutions were further diluted

1/25 with reaction buﬀer. Then 2.5 μL of these solutions were added

to the reaction mixture described below so that ﬁnal compound

concentration in the assay ranges from 100 μM to 5 nM in 1% (v/v)

DMSO. When compounds showed high inhibition at 5 nM and

therefore the data could not be ﬁtted to the corresponding equation,

they were re-evaluated in a new range from 400 nM to 20 pM (Table 5).

The enzymatic activity of the kinases was determined with a

commercial system based on the Z’-LYTE technology, available from

Invitrogen Life Technologies (Carlsbad, CA, USA), using human

recombinant kinases as the enzyme source. This technology utilizes the

ﬂuorescence resonance energy transfer (“FRET”) process between

ﬂuorescein and coumarin. The assay principle is based on the diﬀerential

sensitivity of phosphorylated and nonphosphorylated peptide to

proteolytic cleavage, which precludes the energy transfer process

between the two ﬂuorophores attached to both sides of the cleavage

site. Hence, enzymatic phosphorylation will yield a phosphopeptide,

which cannot be hydrolyzed by a suitable protease and energy transfer

between the two ﬂuorophores will occur. Opposingly, lack of

phosphorylation will cause peptide hydrolysis hence lack of energy

transfer as. The assay was performed in 96-well black plates, in a ﬁnal

volume of 10 μL, with components as detailed in Tables 1, 3, and 4.

In Vivo Activity on Zebraﬁsh Embryos. The wt zebraﬁsh was

used in this study. The embryos were collected and placed into 24-well

plates, 10 embryos per well, and maintained in E2 medium at ∼28 °C.

Compounds were added 5 hpf (50% epiboly) and the embryos

allowed to grow in chemical compound solution up to 2 days. The

phenotypes were compared using the Axio Scope.A1 microscope

system from Carl Zeiss at 44−48 hpf.^36,37,51

(S)-((7-(5-(2′-Cyanobiphenyl-4-yl)methylthio)-1,3,4-oxadia-

zol-2-yl)-2,3-dihydrobenzo[b][1,4] dioxin-2-yl)-

methylmethanesulfonate (25). To a solution of 24b (238 mg,

0.52 mmol) in 10 mL of DCM was added Et₃N (0.72 mL, 5.2 mmol)

followed by addition of methanesulfonyl chloride (402 μL, 5.2 mmol)

at 0 °C. The reaction mixture was stirred at the same temperature for

1 h and further stirred at room temperature for 4 h. After treating

with saturated NaHCO₃solution, the reaction mixture was extracted

with DCM. The combined organic layer was dried over MgSO₄,

concentrated, and puriﬁed by column chromatography (EtOAc/

1

cyclohexane, 1:1) to provide 25 (273 mg, 98%) yellow oil. H NMR

(DMSO-d₆, 500 MHz): δ [ppm] = 3.26 (3H, s), 4.17 (1H, m), 4.45

(1H, m), 4.50 (1H, dd, J = 11.6 Hz, J = 2.4 Hz), 4.55 (1H, dd, J = 11.6

Hz, J = 3.3 Hz), 4.62 (1H, m), 4.67 (2H, s), 7.11 (1H, d, J = 8.1 Hz),

7.48 (2H, m), 7.61 (6H, m), 7.77 (1H, td, J = 7.6 Hz, J = 1.3 Hz), 7.95

(1H, dd, J = 7.7 Hz, J = 0.8 Hz). ¹³C NMR (DMSO-d₆, 125 MHz): δ

[ppm] = 35.4, 36.8, 64.3, 67.8, 70.9, 110.1, 115.2, 116.4, 118.2, 118.5,

120.3, 128.2, 128.8, 129.4, 130.1, 133.5, 133.8, 137.1, 137.4, 142.7,

143.9, 145.9, 162.7, 164.9. HPLC: 96%; t_R8.46 min. EI-MS: m/z =

535 (M⁺).

(R)-4′-((5-(3-((2,2-Dimethoxyethylamino)methyl)-2,3-

dihydrobenzo[b][1,4]dioxin-6-yl)-1,3,4-oxadiazol-2-ylthio)-

methyl)biphenyl-2-carbonitrile (26). To a stirred solution of 25

(69 mg, 0.13 mmol) in 2 mL of THF was added 2,2-dimethoxyethyl-

amine (140 μL, 1.3 mmol) and NEt₃(180 μL, 1.3 mmol) at 0 °C, and

the reaction mixture was stirred at room temperature for 5 days. The

mixture was diluted with water and extracted with EtOAc. The organic

layer was washed with brine and dried over MgSO₄. The solvent was

evaporated under reduced pressure and the crude product puriﬁed by

silica gel column chromatography (MeOH/EtOAc, 1:10) to give 26

(58 mg, 83%) as a dark-yellow oil. ¹H NMR (DMSO-d₆, 500 MHz): δ

[ppm] = 1.91 (1H, m), 2.61 (2H, dd, J = 5.4 Hz, J = 0.7 Hz), 2.80

(2H, m), 3.18 (3H, s), 3.19 (3H, s), 4.00 (1H, m), 4.17 (1H, m), 4.30

(2H, m), 4.54 (2H, s), 6.87 (1H, m), 7.32 (2H, m), 7.44 (1H, dd, J =

7.6 Hz, J = 1.2 Hz), 7.46 (3H, m), 7.52 (2H, m), 7.64 (1H, td, J = 7.8

Hz, J = 1.3 Hz), 7.74 (1H, m). ¹³C NMR (DMSO-d₆, 125 MHz): δ

[ppm] = 36.7, 50.2, 52.1, 53.8, 53.9, 67.7, 74.1, 104.9, 111.8, 116.5,

117.8, 118.7, 119.1, 120.8, 129.0, 130.0, 130.4, 131.1, 134.1, 134.7,

Animal Husbandry. All animal experiment were conducted and

documented according to the federal and local regulation. All embryo

testing was stopped at day 5 of embryonic development.

SH-SY5Y Neuroblastoma Cells. SH-SY5Y neuroblastoma cells

stably transfected with Tau.P301L in the pcDNA3 vector were grown

to conﬂuency in 6-well cluster plates (∼800000 cells/well) in DMEM-

F12 medium supplemented with Glutamax and 15% fetal calf serum.

The medium contained gentamycin (50 μg/mL) as general antibiotic

and Geneticin (250 μg/mL) to maintain selection pressure on

transfected cells. Cells were grown at 37 °C in a humidiﬁed incubator

in an atmosphere of 5.0% CO₂in air. Stock solutions of the

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Article

compounds in DMSO were added to serum-free culture medium to

the speciﬁed concentrations, using DMSO in the same ﬁnal

concentrations as control. Cells were incubated with the compounds

at 37 °C for the indicated periods of time. After incubation, spent

medium was removed and cells washed once with PBS containing Ca²⁺

and Mg²⁺. The cells were rapidly harvested by mechanical scraping

after addition of hot 62.5 mM Tris pH 6.8 buﬀer, containing 1% SDS

(180 μL per well). Protein extracts were collected by aspiration and

reduced and denatured by addition of 1% β-mercaptoethanol and

boiling for 10 min. After separation by SDS-PAGE on 10% Tris-

glycine SDS-PAGE, proteins were analyzed by Western blotting using

the ECL-system.⁵²In brief, after SDS-PAGE, the separated proteins

were transferred to nitrocellulose ﬁlter-sheets, which were treated

against nonspeciﬁc binding by incubation in 5% nonfat milk in Tris

buﬀered saline (TBS: 10 mM Tris.HCl, pH 7.2, 0.9% sodium chloride,

0.1% Tween). Blots were subsequently incubated with primary

antibodies speciﬁcally directed against total protein tau or against its

selected phosphorylated epitopes, as speciﬁed in Results and

Discussion and in the ﬁgure legends. After incubation with suitably

labeled secondary antibodies, the resulting immune reactions were

recorded and analyzed digitally with dedicated apparatus and software

(LAS4000, Image-QuantTL, GE Healthcare, Brussels, Belgium). Data

for tau and phospho-tau were normalized against actin, revealed by

Western blotting of the same samples on the same blots.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by a collaborative project ﬁnanced by

the 7th Framework Program of the European Union

(neuro.GSK3).

ABBREVIATIONS USED

■

ATP, adenosine triphosphate; AD, Alzheimer’s disease; BBB,

blood−brain barrier; Cdk, cyclin-dependent kinase; GSK-3,

glycogen synthase kinase-3; DMF, dimethylformamide; DMSO,

dimethyl sulfoxide; EtOAc, ethyl acetate; EtOH, ethanol; hpf,

hours post fertilization; HPLC, high performance liquid

chromatography; MeOH, methanol; SAR, structure−activity

relationship

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ASSOCIATED CONTENT

■

S

* Supporting Information

Homology modeling, in vitro pharmacology, bioavailability

proﬁle of compound 14d and NMR data of compounds 5b, 5c,

6b, 6c, 14b, 14c, 14d, 15a, 15b, 16a, 23a, 23b, and 23c. This

material is available free of charge via the Internet at http://

pubs.acs.org.

(12) Kaidanovich-Beilin, O.; Lipina, T. V.; Takao, K.; van Eede, M.;

́

Hattori, S.; Laliberte, C.; Khan, M.; Pkamoto, K.; Chambers, J. W.;

Fletcher, P. J.; MacAulay, K.; Doble, B. W.; Henkelman, M.;

Miyakawa, T.; Roder, J.; Woodgett, J. R. Abnormalities in brain

structure and behavior in GSK-3alpha mutant mice. Mol. Brain 2009,

2, 1−23.

AUTHOR INFORMATION

■

(13) Alon, L. T.; Pietrokovski, S.; Barkan, S.; Avrahami, L.;

Kaidanovich-Beilin, O.; Woodgett, J. R.; Barnea, A.; Eldar-

Finkelman, H. Selective loss of glycogen synthase kinase-3α in birds

reveals distinct roles for GSK-3 isozymes in tau phosphorylation. FEBS

Lett. 2011, 585, 1158−1162.

Corresponding Author

*Phone: +496151-164531. Fax: +496151-163278. E-mail:

schmidt_boris@t-online.de (B.S.); Fabio.Lo-Monte@gmx.de

(F.L.).

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Identification of glycogen synthase kinase-3 inhibitors with a selective sting for glycogen synthase kinase-α

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