Switching reversibility to irreversibility in glycogen synthase kinase 3 inhibitors: Clues for specific design of new compounds

ARTICLE

pubs.acs.org/jmc

Switching Reversibility to Irreversibility in Glycogen Synthase

Kinase 3 Inhibitors: Clues for Specific Design of New Compounds

Daniel I. Perez,^†Valle Palomo,^†Concepciꢀon Pꢀerez,^†Carmen Gil,^†Pablo D. Dans,^‡F. Javier Luque,^§

Santiago Conde,^†and Ana Martínez*^,†

^†Instituto de Química Medica-CSIC, Juan de la Cierva 3, 28006 Madrid (Spain)

^‡Biomolecular Simulations Group, Institut Pasteur de Montevideo, Mataojo 2020, 11400 Montevideo (Uruguay)

^§Departamento de Fisicoquímica e Instituto de Biomedicina (IBUB), Facultad de Farmacia, Universidad de Barcelona,

Avda. Diagonal 643, 08028 Barcelona (Spain)

S Supporting Information

b

ABSTRACT: Development of kinase-targeted therapies for central

nervous system (CNS) diseases is a great challenge. Glycogen

synthase kinase 3 (GSK-3) oﬀers a great potential for severe CNS

unmet diseases, being one of the inhibitors on clinical trials for

diﬀerent tauopathies. Following our hypothesis based on the

enhanced reactivity of residue Cys199 in the binding site of

GSK-3, we examine here the suitability of phenylhalomethylke-

tones as irreversible inhibitors. Our data conﬁrm that the halomethylketone unit is essential for the inhibitory activity. Moreover,

addition of the halomethylketone moiety to reversible inhibitors turned them into irreversible inhibitors with IC₅₀values in the

nanomolar range. Overall, the results point out that these compounds might be useful pharmacological tools to explore physiological

and pathological processes related to signaling pathways regulated by GSK-3 opening new avenues for the discovery of novel GSK-3

inhibitors.

’ INTRODUCTION

disease.¹⁰Recently, the reversion of these pathological features

in vivo has been shown after oral treatment with a GSK-3

inhibitor in a double transgenic animal model of AD.¹¹

Protein kinases are important targets for cancer, inﬂammatory

diseases, diabetes, and neurodegenerative disorders, because

aberrant protein kinase signaling is implicated in many of these

human diseases.¹To date, ten protein kinase (PK) inhibitors are

in clinical use for diﬀerent cancer treatments.²While some of

these agents are active in a subset of patients, most of them

develop resistance within the course of one year. To overcome

such a problem, irreversiblePK inhibitors are gaining importance³

as a new class of therapeutic agents. This knowledge is now

being applied to other therapeutic areas such as inﬂammatory

and neurodegenerative pathologies, where PK inhibitors have not

reached clinical use yet.

Although several candidate therapeutics for PKs in the central

nervous system (CNS) are now in diﬀerent preclinical and

clinical stages,⁴the development of kinase-targeted therapies

for CNS diseases remains a challenge. Speciﬁcally inhibition of

kinases that phosporylate tau protein could be beneﬁcial for

neurodegenerative diseases.⁵Among them, glycogen synthase

kinase 3 (GSK-3) and cyclin-dependent kinase 5 (CDK-5) play

a major hyperphosphorylating role in vivo.⁶GSK-3 inhibitors

oﬀer a valuable approach for a future therapy against Alzheimer’s

disease (AD).^7ꢀ9Thus, abnormally high GSK-3 activity found in

AD patient brains accounts not only for tau hyperphosphoryla-

tion, but also for memory impairment, increased β-amyloid

production and local plaque-associated microglial-mediated in-

ﬂammatory responses, which are characteristic hallmarks of the

Many drug discovery programs are focused on the develop-

ment of selective GSK-3 inhibitors. The discovery of non ATP-

competitive inhibitors with high GSK-3 speciﬁcity is particularly

challenging in order to minimize unfavorable oﬀ-target eﬀects.

In this context, allosteric GSK-3 modulation, as suggested for the

basic alkaloid manzamine,^12,13or GSK-3 substrate competition,¹⁴

as noted for a small peptide in a study for diabetes, have been

valuable strategies to modulate the activity of GSK-3. Other

approaches to target GSK-3 avoiding ATP competition have also

been envisaged. Speciﬁcally, on the basis of previous studies on

GSK-3 inhibitors,^15,16we have recently hypothesized a new way

to selectively target this important kinase based on the existence

of a cysteine (Cys199) located in the ATP binding site.¹⁷Since

Cys199 in GSK-3 is replaced by diﬀerent amino acids in other

structurally related kinases, such as CDK-1, CDK-2, or CDK-5,

this approach would oﬀer the possibility to design speciﬁc drugs

via covalent modiﬁcation of this key residue. Indirect support for

this strategy comes from the inhibitory activity determined for

thienylhalomethylketones and thiadiazolidindiones (TDZDs),¹⁸

which are the ﬁrst reported non ATP-competitive GSK-3 inhibitors.¹⁹

Among TDZDs, tideglusib (formerly known as NP-12) is now

Received: December 22, 2010

Published: April 18, 2011

r

2011 American Chemical Society

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Table 1. GSK-3 Inhibitory Activity of HMKs and Related

Compounds

was reinforced by the inhibitory activity found for a small set of

commercially available 4-substituted chloro and bromoacetyl-

phenyl derivatives (1ꢀ9), as expected from the known bioisos-

terism between thiophene and benzene rings. Although these

compounds showed an interesting range of activities, no clear

relationship could be identified between the chemical features of

the para-substitution and the biological activity (Table 1).

To gain insight into the structureꢀactivity relationships of

phenylhalomethylketones as GSK-3 inhibitors, a chemically

more diverse set of molecules was evaluated using the 4-bro-

mo-substituted derivative 5 as lead compound due to its high

inhibitory activity (IC₅₀= 0.5 μM). The novel derivatives were

designed to explore the inﬂuence played by the nature of the

halide atom and/or the carbonyl group, which were replaced by

diﬀerent heteroatoms or moieties such as iodine (10), oxygen

(16, 17), nitrogen (11, 12), sulfur (13ꢀ15), sulfur monoxide

(20), oxime (18), and alkyloxime (19) (Scheme 1).

The GSK-3 inhibitory activity was measured following a

previously described method that uses [γ-³²P]ATP and the small

peptide GS-1 (based on residues of glycogen synthase phos-

phorylated by GSK-3) as substrate.²²Among the diﬀerent

chemical modiﬁcations examined here, only replacement of the

bromo acetyl moiety in compound 5 by the iodoacetyl one in 10

retained the inhibitory activity (IC₅₀= 3.0 μM). All the other

compounds did not show signiﬁcant inhibitory activity (Table 1).

Therefore, these ﬁndings point out that the halomethylketone

(HMK) moiety is a chemical feature necessary for the inhibition

of GSK-3.

To further examine the inﬂuence of the substituent in the

benzene ring, diﬀerent commercial acetophenones were con-

verted to their bromomethylketo derivatives (21, 23, 25, and 27)

by bromine treatment in chloroform at low temperature

(Scheme 2). In some cases, the dibromo methyl keto compound

(22, 24, 26, and 28) was also isolated. When p-aminoacetophe-

none was used as starting material, bromination not only in the

methylketone group but also in the benzene ring was obtained

(29). Furthermore, the benzene ring was replaced by the

π-deﬁcient 2-pyridine heterocycle, and bromination of 2-acet-

ylpyridine yielded the dibromo derivative 30. In all cases, an

IC₅₀value in the micromolar range was found (Table 2), thus

reinforcing the assumption that the bromoacetyl moiety is the

key structural feature responsible for the inhibitory activity.

Chemical Reactivity of Phenylhalomethylketones versus

Thiol Group. It can be hypothesized that the GSK-3 inhibitory

activity of haloacetylphenyl derivatives stems from the reactivity

of the HMK moiety with the thiol unit of Cys199, which is

located in the ATP binding site. It is therefore necessary to

characterize the potential reactivity of haloacetylphenyl com-

pounds in front of thiol groups. To this end, we have analyzed by

HPLCꢀMS the reaction of compound 3 or 4 with 2-mercap-

toethanol at room temperature (Table 3). The reaction afforded

11% and 20% of the S-alkylated product for 3 and 4, respectively,

in a 1:1 mix ratio after 1 h. When the mixture was stirred for 18 h,

the yield of the S-alkylation was 74% and 50%, respectively.

When the concentration of mercaptoethanol was increased

10-fold, 78% and 100% of the S-alkylated product was formed

after 18 h. On the other hand, upon addition of a tertiary amine,

such as triethylamine, at equimolar concentrations of compound

3 or 4 and mercaptoethanol, the S-alkylated product was formed

in 65% (3) and 66% (4) after 1 h. The mixture was stirred

for 18 h, but a significant increase of the S-alkylated product

was not observed. Overall, the results confirm the susceptibility

no

X

R

Y

IC₅₀(μM)

1^a

2^a

3^a

4^a

5^a

6^a

7^a

8^a

Cl

Br

Cl

Br

I

H

O

50

5.0

H

Cl

2.5

Cl

1.0

Br

0.5

Me

Ph

OMe

NO₂

Br

2.5

1.0

9^a

2.0

10

3.0

11

phthalimide

NH₂

SEt

Br

100

>10

>100

>10

>100

50

12

Br

13

Br

14

SPh

Br

15

SBn

Br

16

OCOMe

OH

Br

17

Br

18

Br

NOH

NOMe

SO

19

Br

20

Cl

Br

>100

^aSee ref ²¹

in phase II clinical trials both for AD and for an orphan tauopathy

called progressive supranuclear palsy (PSP).

Simultaneously with the writing of this manuscript, some

data underlying the importance of cysteine identiﬁcation on

the kinase nucleotide binding site for the design of covalent

speciﬁc protein kinase inhibitors have been reported.²⁰

Our aim here is to examine the suitability of targeting Cys199,

at the entrance of the ATP binding site of GSK-3, as a new drug

discovery strategy to develop compounds useful as selective

pharmacological tools against this important protein kinase. To

this end, we ﬁrst report the synthesis and biological characteriza-

tion of phenylhalomethylketones chosen as bioisosters of the

irreversible inhibitors previously described in our studies.^17,21

Moreover, based on X-ray crystallographic and molecular mod-

eling data of GSK-3 complexes, the suitability of our strategy is

reinforced by switching compounds with reversible inhibitory

properties into irreversible inhibitors. This strategy led us to

design, synthesize, and evaluate chemically diverse GSK-3 in-

hibitors with diﬀerent kinetics regarding this enzyme. Overall,

the results support the implications of Cys199 in the irreversible

inhibition of GSK-3 and discover new clues for the synthesis of

novel inhibitors for the treatment of human neurodegenerative

diseases.

’ RESULTS AND DISCUSSION

Lead Modification and StructureꢀActivity Relationship.

Screening of our compound library led to the discovery of

thienylhalomethylketones as GSK-3 inhibitors.²¹This behavior

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Scheme 1

(i) KI, acetone, Δ; (ii) potassium phthalimide, AcN, Δ; (iii) RSH, K₂CO₃, EtOH; (iv) (AcO)₂O, NaAcO, AcOH, Δ; (v) CAL-B, ^tBuOH:citrate-

phosphate buﬀer (9:1); (vi) NH₂OH HCl or NH₂OMe HCl, EtOH, pyr; (vii) SO₂Cl₂, AgNO₃, AcN.

3

Scheme 2.

Table 2. GSK-3 Inhibitory Activity of Bromoacetyl Aryl

Derivatives

(i) Br₂, CHCl₃, 0 °C.

of phenylhalomethylketones to react with thiol groups and

the enhanced reactivity upon addition of a suitable base, which

might presumably enhance the nucleophilicity of the sulfur

atom.

Turning Reversible Inhibitors into Irreversible Inhibition.

To confirm the implication of Cys199 in the inactivation of

GSK3 by incubation with phenylhalomethylketones, we checked

the possibility of turning reversible inhibitors into irreversible

ones upon addition of the HMK unit in a proper position that

facilitates the reaction with the thiol group of Cys199. To this

end, three structurally distinct compounds were selected taking

advantage of the known binding mode to GSK-3 from the X-ray

crystallographic structures deposited in the Protein Data Bank:

an analogue of ATP (PDB entry 1J1B), and two reversible

inhibitors (PDB entries 3F88 and 1R0E). Accordingly, three classes

of HMK-containing compounds—derivatives of adenine (31,

32), benzimidazole (35, 36), and maleimide (37ꢀ40)—were

considered for chemical modification with Cys199 (Figure 1).

The feasibility of this strategy was ﬁrst explored by means of

docking calculations, which were performed using rDock (see

Experimental Section. Molecular Modeling). The reliability of

rDock was assessed by docking a set of known GSK-3 inhibitors

taking advantage of the X-ray crystallographic structures of their

complexes with the enzyme (PDB entries 1J1B, 3F88, 1R0E,

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ARTICLE

1Q5K, 1UV5, and 2O5K). It is noteworthy that the unrestrained

docking protocol was able to predict satisfactorily the X-ray

orientation of the compounds in ﬁve out of the six cases, as the

experimental binding mode was identiﬁed as the ﬁrst or second

ranked pose with root-mean-square deviations less than 1.8 Å

(see Table S1 in Supporting Information). The X-ray binding

mode of the ATP analogue had a higher rank (PDB entry 1J1B),

as expected from the larger ﬂexibility of the phosphodiester chain

(see Table S1). Inspection of the predicted poses derived for

docking calculations for compounds 32, 36, and 40 revealed the

existence of ligand orientations in the binding site that yielded

the HMK moiety suitably positioned for chemical modiﬁca-

tion by Cys199. This is illustrated for 36 in Figure 2 (see also

Figure S1 for compounds 32 and 40), which shows the second

best pose of the compound, which places the C_Ratom of the

HMK moiety at around 3.6 Å from the sulfur atom in Cys199).

N-Acylation of adenine was achieved by treatment of acetic

anhydride and pyridine in DMSO²³at room temperature over-

night (Scheme 3). The precipitate formed was ﬁltered and

washed with cold pyridine and ether to achieve the desired

product 31 (51% yield). No bromoacetyl derivative of com-

pound 31 was obtained after treatment of this compound with

bromine either at low temperature or reﬂux. In order to

synthesize the haloacetyl derivative 32 of compound 31, reaction

was carried out employing adenine, pyridine, and chloroacetic

anhydride in THF.

The synthesis of benzimidazole compounds is described in

Scheme 4.²⁴1-Bromo-2-nitro-4-acetyl-benzene was treated with

Table 3. Formation of S-Alkylated Products in the Reaction

of Compounds 3 and 4 with Mercaptoethanol

1 h

18 h

reaction conditions

SM^a

FP^b

SM^a

FP^b

3:SHEtOH 1:1

88%

81%

33%

80%

47%

33%

11%

19%

65%

20%

53%

66%

24%

20%

34%

50%

----

74%

78%

65%

50%

100%

75%

3:SHEtOH 1:10

3:SHEtOH:Et₃N 1:1:1

4:SHEtOH 1:1

4:SHEtOH 1:10

Figure 2. Docked pose for compound 36 with the halomethyketone

moitey suitably positioned for chemical modiﬁcation by Cys199 (shown

in spheres; distance from S atom in Cys199 to the C_Ratom around or

less than 4 Å).

4:SHEtOH:Et₃N 1:1:1

^aSM: starting material (compound 3 or 4). ^bFP: Final product

25%

(S-adduct).

Figure 1. (Top) Representation of the binding mode of an ATP analogue (1J1B), and two reversible inhibitors (3F88 and 1R0E; shown in sticks) in the

ATP-binding site of GSK-3. Cys199 is shown in spheres. (Bottom) Structure-based design of novel compounds with potential GSK-3 inhibitory activity.

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4-methoxyaniline and sodium acetate in DMF to obtain com-

pound 33 in good yield (73%) after recrystallization. The nitro

moiety was reduced under catalytic hydrogenation conditions to

yield quantitatively derivative 34. Final cyclization to synthesize

benzimidazole 35 was achieved employing formic acid and HCl 4

N solution. The desired halomethylketone 36 was obtained by

reaction of the benzimidazole 35 with bromine in acetic acid

under reﬂux.

Finally, the direct formation of the maleimide moiety was

chosen for synthesis of maleimide derivatives (Scheme 5).²⁵

Because commercial indole products required to synthesize those

compounds do not contain a ﬂuorine atom in their structure, we

eliminated the ﬂuorine atom from the structure originally pro-

posed. The reaction conditions were carefully optimized to avoid

the formation of the major byproduct indoleglyoxylic acid. Thus,

methyl 2-indoleglyoxylate or 2-(1-methyl-indol)-glyoxylate was

added to a solution of potassium tert-butoxide and acetamide in

tetrahydrofuran at ꢀ60 °C. Under these conditions, compound

37 was obtained with good yield (74%), whereas the methyl

indole compound 38 reached 56% yield. These compounds were

treated with bromine in acetic acid under reﬂux to yield HMK

compounds 39 and 40, respectively.

All the newly prepared acetyl and halomethylketo heterocycles

31, 32, and 35ꢀ40 were evaluated as GSK-3 inhibitors using

a recently well described luminescent technique²⁶as a safer

nonradioactive assay (see Table 4). The four HMK-containing

compounds (32, 36, 39, and 40) are more potent that their cor-

responding acetyl derivatives (31, 35, 37, and 38, respectively),

showing in one case an IC₅₀value in the nanomolar range.²⁷

More importantly, the inhibitory activity determined at diﬀerent

preincubation times conﬁrmed the switch from reversible to

irreversible inhibition (Figure 3). Thus, the inhibition of the

acetyl-containing compounds 37 and 38 remained unaltered at

diﬀerent preincubation times, mimicking the behavior of alster-

paullone, a known reversible inhibitor. In contrast, the enzyme

inhibition increased with preincubation time for the thiophene

irreversible inhibitor HMK-32¹⁷and for the HMK-containing

compounds 36, 39, and 40. Overall, these ﬁndings are in agree-

ment with our working hypothesis, which provides new clues

for the speciﬁc design of irreversible GSK-3 inhibitors.

The covalent binding of compound 40 to GSK-3 was con-

ﬁrmed biophysically by MALDI-TOF analyses.²⁸Compounds

38 and 40 were added in two diﬀerent experiments to the

GSK-3 enzyme under the same conditions as that used for

Scheme 3

Table 4. GSK-3 Inhibition Values and Binding Mode of

Computer Aided-Designed New Inhibitors

no.

IC₅₀(μM)

binding mode no.

IC₅₀(μM)

binding mode

31 >50

----

35 >10

----

32 13.4

irreversible 36

0.58 ( 0.07

0.89 ( 0.19

irreversible

reversible

37

39

4.47 ( 0.35

reversible

38

(i) Acetic anhydride, DMSO, rt; (ii) Chloroacetic anhydride, THF, rt.

0.047 ( 0.007 irreversible 40

0.005 ( 0.001 irreversible

Scheme 4

(i) Sodium acetate, DMF, 100 °C; (ii) H₂, Pd(C), 20 psi THF, rt; (iii) Formic acid, HCl; (iv) Br₂, acetic acid, Δ.

Scheme 5

(i) ^tBuOK, THF, ꢀ60 °C to 20 °C; (ii) Br₂, acetic acid, Δ.

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Figure 3. Time-dependent GSK-3 inhibition of controls (alsterpaullone and HMK-32) and the newly synthetized protein kinase inhibitors.

IC₅₀measurements. Mass analysis revealed that, when the rever-

sible inhibitor 38 was used, only the mass corresponding to GSK-3

(48 794 Da) was detected, whereas the use of an irreversible

inhibitor (compound 40) showed a mass peak of 49 036 Da con-

ﬁrming its covalent irreversible binding to the enzyme (Figure 4).

Cellular Effects of Phenylhalomethylketones on GSK-3

Inhibition. All the preceding results suggest that phenylhalo-

methylketones may be versatile pharmacological tools to explore

GSK-3-mediated cellular processes in physiological or patholo-

gical conditions. As GSK-3 is involved in tau phosphorylation in

transfected cells and in vivo,²⁹we further explored their ability to

interfere with the GSK-3 mediated tau phosphorylation in cells

using primary granule cerebellar neurons (GCNs), which also

allow us to evaluate the cell permeability of the new inhibitors.

The eﬀect on tau phosphorylation was determined by

Western blotting, and detection was carried out using speciﬁc

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Figure 4. Maldi-TOF spectra. (A) Reversible inhibitor 38 (MW = 268) with GSK-3 (MW = 48 794) showed a 48 794 mass peak. (B) Irreversible

inhibitor 40 (MW = 346) with GSK-3 showed a 49 036 mass peak corresponding to the covalent bond SꢀC formed between the GSK-3 enzyme and the

inhibitor 40.

antibodies: Tau-1 for tau inmunoreactivity, and PHF-1 as tau

phosphospeciﬁc Ser396 reagent. This last epitope is speciﬁcally

phosphorylated by GSK-3 on tau protein.³⁰Lithium chloride, the

ﬁrst reported GSK-3 inhibitor, was used as standard reference

and was added as positive control to the medium at 20 mM. The

bands were quantiﬁed by densitometric analysis. Following this

procedure, data showed that the addition of phenylhalomethylk-

etone 6 or 8 reduced (PHF-1) or increased (Tau-1) the tau-

immunoreactivity, thus mimicking the eﬀect observed when

lithium chloride was used as a GSK-3 inhibitor (IC₅₀= 1.5 mM)

(Figure 5).

Kinase and Neurotransmitter Binding Profiles of Phenyl-

halomethyl Ketones. To examine the PK selectivity of phenyl-

halomethylketones for GSK-3, compounds 3, 5, and 6 were

tested as inhibitors of serine/threonine cAMP-dependent protein

kinase (PKA). All of them remained inactive at the highest con-

centration (100 μM) used. Furthermore, the same set of com-

pounds was evaluated against a panel of eight different serine/

threonine and tyrosine kinases (CaM-K II, MAP-K, EGFR-K, IR-K

MeK1-K, Abl-K, PKp56, and Src), which are involved in very

different signaling pathways not only in neurodegeneration

(Cam-KII and MAP-K), but also in other physiological processes

Figure 5. Representative Western blot of soluble extracts from Granule

cerebellar neurons (GCNs), obtained after 16 h in the presence of

lithium chloride (Li) or diﬀerent phenylhalomethylketones (6 and 8). C

represents cell extracts from control GCNs. Identical samples were

incubated with antitau-1, anti PHF-1, or anti-β-actin, which was used as a

and diseases, such as insulin signal pathway (IR-K) or cancer

(Abl-K, EGFR-K, MeK1-K, and Src). The inhibitory assays

(performed using a 10 μM concentration for the different

compounds) showed that, in general, HMKs did not exhibit a

significant inhibitory effect on the whole set of kinases (Table 5).

loading control. In each experiment, PHF-1 was normalized with respect

to the amount of actin present in each cell extract.

Only the proto-oncogenic Src, which is elevated in some human

tumors, is smoothly inhibited by the three selected compounds.

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Table 5. Inhibitory Activity (% Inhibition)^aShowed by Selected Phenylhalomethylketones for Several Kinases

compound (10 μM)

Abl-K

Cam-KII

EGFR-K

IR-K

MAP-K

MEK-1 K

PK p56

Src-K

3

5

6

ꢀ

16

ꢀ

18

10

ꢀ

29

38

19

38

69

15

^aThe symbol ꢀ indicates an inhibitory activity of less than 10%.

Table 6. Inhibitory Activity (% Inhibition)^aExhibited by Selected Phenylhalomethylketones for Neurotransmitter Receptors

compound (10 μM)

R2

hD2

hD3

AMPA

NMDA

hM

N (RBGT insensitive)

N (RBGT sensitive)

5-HT

3

5

6

ꢀ

15

ꢀ

16

ꢀ

10

13

^aThe symbol ꢀ indicates inhibitory activity of less than 10%.

Furthermore, it is also important for pathway analysis in vivo

and in cellular systems to evaluate the potential oﬀ-targeteﬀects of

these new pharmacological tools. This is of particular relevance in

phenylhalomethylketones due to the presence of a potential

reactive group in their chemical structure, which might react with

diﬀerent neurotransmitter receptors, thus masking a neat GSK-3

response. In order to determine the potential oﬀ-target eﬀects

of these compounds, we evaluated the in vitro binding to diﬀerent

neurotransmitter receptors: R-adrenergic (R₂), dopaminergic

(hD2 and hD3), glutamatergic (AMPA and NMDA), muscarinic

(hM), nicotinic (bungarotoxin sensitive and insensitive), and

serotoninergic (5-HT) receptors. Results showed that com-

pounds 3, 5, and 6 (at 10 μM concentration) did not bind to

any of these neurotransmitter receptors (Table 6).

These ﬁndings are especially relevant, as an emerging aspect in

the eﬀectiveness of protein kinases inhibitors in vivo is the ability

to compete with cellular concentrations of ATP (typically about

1ꢀ10 mM). For epidermal growth factor receptor kinase (EGFR-

K), such problems have been overcome with compound HKI-

272, which binds covalently to a cysteine residue.³¹A similar

strategy has been recently reported for the irreversible inhibition

of HCV NS3/4A viral protease by targeting a noncatalytic

cysteine residue.³²In this context, the strategy explored here,

which relies on the key role of Cys 199 in GSK-3 inhibition, might

then represent a promising strategy for the development of novel

GSK-3 inhibitors for the treatment of severe human diseases.

’ EXPERIMENTAL SECTION

’ CONCLUSIONS

Chemistry. General. Substrates were either purchased from com-

mercial sources or used without further purification. Melting points were

determined with a Reichert-Jung Thermovar apparatus and a Mettler

Toledo MP70 apparatus and are uncorrected. Flash column chroma-

tography was carried out at medium pressure using silica gel (E. Merck,

grade 60, particle size 0.040ꢀ0.063 mm, 230ꢀ240 mesh ASTM) with

the indicated solvent as eluent. Compounds were detected with UV light

GSK-3 is a therapeutically valuable kinase that is very promising

for severe unmet diseases such as AD. To the best of our

knowledge, only a small molecule acting as GSK-3 inhibitor, the

TDZD compound called Tideglusib, has reached phase IIb clinical

trials for the treatment of AD. Additionally, lithium is used in the

clinic as the ﬁrst option for treatment of bipolar disorders, and it is

known to inhibit GSK-3 among other mechanisms of action.

The irreversible inhibition of GSK-3 reported for thienylha-

lomethylketones was suggested to involve covalent modiﬁcation

of Cys199,¹⁷which is located in the ATP-binding site. Modiﬁca-

tion of Cys199 might then be a new strategy for the design of

speciﬁc GSK-3 inhibitors. At this point, the series of phenylha-

lomethylketone derivatives examined conﬁrms unequivocally the

key role of the HMK moiety on GSK-3 inhibition. Furthermore,

the implication of Cys199 is supported by the structure-based

design of novel compounds characterized by reversible inhibi-

tion, which turn out to be irreversible inhibitors upon addition of

an HMK unit to their chemical structure.

Overall, chemical modiﬁcation of Cys199 represents a new

mechanism of action that opens new avenues for the develop-

ment of potent, selective GSK-3 inhibitors. In this context, it is

worth highlighting that phenylhalomethylketones (i) act as

cell-permeable inhibitors, (ii) decrease tau phosphorylation in

cell cultures, (iii) are rather selective against a panel of protein

kinases, and (iv) do not bind to diﬀerent neurotransmitter

receptors. Thus, they may be considered useful pharmacological

tools to explore physiological and pathological cellular signaling

pathways in which GSK-3 is involved.

1

(254 nm). H NMR spectra were obtained on a Gemini-200 spectro-

meter working at 200 MHz and on a Bruker AVANCE-300 spectrometer

working at 300 MHz. Typical spectral parameters: spectral width

10 ppm, pulse width 9 μs (57°), data size 32 K. ¹³C NMR experiments

were carried out on the Varian Gemini-200 spectrometer operating at

50 MHz and on the Bruker AVANCE-300 spectrometer operating at

75 MHz. The acquisiton parameters: spectral width 16 kHz, acquisition

time 0.99 s, pulse width 9 μs (57°), data size 32 K. Chemical shifts are

reported in values (ppm) relative to internal Me₄Si and J values are

reported in Hertz. Elemental analyses were performed by the analytical

department at CENQUIOR (CSIC), and the results obtained were

within (0.4% of the theoretical values. The combustion analyses are

provided in the Supporting Information. Mass spectra were obtained

by electronic impact in a Hewlett-Packard 5973 spectrophotometer.

All compounds are >95% pure by HPLC analyses. HPLC analyses for

compounds 10ꢀ30 were performed on a Waters 6000 instrument, with

UV detector (214ꢀ254 nm), using different columns; μ Bondapack

C18, 10 μm, 125 Å, (300 ꢁ 3.9 mm) and Symmetry C18, 5 μm, 100 Å,

(150 ꢁ 3.9 mm). Acetonitrile/H₂O [(0.05% H₃PO₄þ 0.04% Et₃N)]

50/50 was used as mobile phase. For the chemical reactivity of HMKs

versus thiol groups and HPLC analyses for compounds 31ꢀ40, experi-

ments were performed on Alliance Waters 2690 equipment with a UV

detector photodiode array Waters 2996 with MS detector MicromassZQ

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(Waters) positive electrospray, using the Sunfire column C18, 3.5 μm

(50 ꢁ 4.6 mm) and acetonitrile (0.08% formic acid) and MilliQ water

(0.1% formic acid) as mobile phase in a 10 min run from 10% to 100%

acetonitrile gradient at a flow rate of 0.25 mL/min.

MALDI-MS measurements were performed on a Voyager DE-PRO

mass spectrometer (Applied Biosystems, Foster City, CA) equipped

with a pulsed nitrogen laser (λ = 337 nm, 3 ns pulse width, and 3 Hz

frequency) and a delayed extraction ion source. Ions generated by the

laser desorption were introduced into a time-of-ﬂight analyzer (1.3 m

ﬂight path) with an acceleration voltage of 25 kV, a 88% grid voltage,

a 0.3% ion guide wire voltage, and a delay time of 500 ns in the linear

positive ion mode.

Mass spectra were obtained over the m/z range 30 000ꢀ125 000 μ.

External mass calibration was applied using the monoisotopic [MþH]þ

value of Enolase. Sinapinic acid (>99%; Fluka, Buchs, Switzerland)

at 10 mg mL^ꢀ1in 0.3% triﬂuoroacetic acid/acetonitrile, 70:30 (v/v)

was used as matrix. Samples were mixed with the matrix at a ratio of

approximately 1:1, and 1 μL of this solution was spotted onto a ﬂat

stainless steel sample plate and dried in air.

2H), 7.50 (d, J = 8.7 Hz, 2H), 3.65 (s, 2H), 2.46 (q, J= 14.6, 7.3 Hz, 3H),

1.16 (t, J= 7.3 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃): δ 194.3, 134.7,

132.8, 131.2, 129.4, 37.5, 27.2, 14.9. m/z (EI): 260, 258 (M ^þ, 10, 9%),

185, 183 (M-SCH₂CH₂CH₃, 100, 99%). HPLC: Column Symmetry

C18, 5 μm, 100 Å, (150 ꢁ 3.9 mm), Purity 99%, r.t = 5.85 min,

acetonitrile/H₂O (0.05% H₃PO₄þ 0.04% Et₃N) 50/50.

1-(4-Bromophenyl)-2-(phenylthio)ethanone (14).³⁶2-Bromo-1-(4-

bromophenyl) ethanone (5) (0.5 g, 1.80 mmol), potassium carbonate

(0.25 g, 1.80 mmol), benzenethiol (0.20 g, 1.80 mmol). Purified by silica

gel chromatography (hexane:ethyl acetate 4:1), 0.29 g (53%), colorless

solid, mp 60ꢀ61 °C. ¹H NMR (200 MHz, CDCl₃): δ 7.68 (d, J= 8.7 Hz,

2H), 7.49 (d, J= 8.7 Hz, 2H), 7.24ꢀ7.20 (m, 5H), 4.10 (s, 2H). ¹³C

NMR (75 MHz, CDCl₃): δ 192.8, 134.0, 133.7, 130.4, 130.1, 129.9,

128.8, 128.4, 127.0, 40.7. m/z (EI): 308, 306 (M^þ, 55, 52%), 185, 183

(M-SCH₂Ph, 100, 98%). HPLC: Column Symmetry C18, 5 μm, 100 Å,

(150 ꢁ 3.9 mm), Purity 99%, r.t = 8.15 min, acetonitrile/H₂O (0.05%

H₃PO₄þ 0.04% Et₃N) 50/50.

2-(Benzylthio)-1-(4-bromophenyl)ethanone (15).³⁷2-Bromo-1-(4-

bromophenyl) ethanone (5) (0.5 g, 1.80 mmol), potassium carbonate

(0.25 g, 1.80 mmol), benzylthiol (0.22 g, 1.80 mmol). Purified by silica

gel chromatography (hexane:ethyl acetate 4:1), 0.32 g (55%), colorless

solid, mp 158ꢀ159 °C. ¹H NMR (200 MHz, CDCl₃): δ 7.68 (d, J= 8.7

Hz, 2H), 7.49 (d, J= 8.7 Hz, 2H), 7.24ꢀ7.20 (m, 5H), 3.63 (s, 2H), 3.52

(s, 2H). ¹³C NMR (75 MHz, CDCl₃): δ 192.3, 136.1, 133.0, 130.9,

1-(4-Bromophenyl)-2-iodoethanone (10).³³A mixture of 2-bromo-

1-(4-bromophenyl)ethanone (5) (2.0 g, 7.20 mmol) and potassium

iodide (8.40 g, 50 mmol) in acetone (50 mL) was stirred and refluxed for

3 h. The solvent was evaporated, and CH₂Cl₂(100 mL) and H₂O

(100 mL) were added to the reaction mixture. The organic layer was

dried over MgSO₄and the solvent was evaporated. Compound 10 was

purified by silica gel chromatography (hexane/ethyl acetate 6:1), 1.91 g

(82%), colorless solid, mp 95ꢀ96 °C. ¹H NMR (200 MHz, CDCl₃): δ

7.83 (d, J= 8.3 Hz, 2H), 7.60 (d, J= 8.3 Hz, 2H), 4.30 (s, 2H). ¹³C NMR

(75 MHz, CDCl₃): δ 190.7, 131.6, 129.5, 127.8, 123.7, 24.3. m/z (EI):

326, 324, (M ^þ, 12, 13%), 185, 183 (M-CH₂I, 97, 100%). HPLC:

Column Symmetry C18, 5 μm, 100 Å, (150 ꢁ 3.9 mm), Purity 99%, r.t =

14.15 min, acetonitrile/H₂O (0.05% H₃PO₄þ 0.04% Et₃N) 50/50.

129.2, 128.3, 127.6, 126.4, 126.3, 35.1, 34.7. m/z (EI): 322, 320 (M ^þ

,

17, 17%), 200, 198 (M-SCH₂Ph, 85, 83%). HPLC: Column Symmetry

C18, 5 μm, 100 Å, (150 ꢁ 3.9 mm), Purity 99%, r.t = 9.76 min,

acetonitrile/H₂O (0.05% H₃PO₄þ 0.04% Et₃N) 50/50.

2-(4-Bromophenyl)-2-oxoethyl acetate (16).³⁸To a solution of

2-bromo-1-(4-bromophenyl)ethanone (5) (0.5 g, 1.80 mmol) in acetic

acid (50 mL) was added sodium acetate (0.44 g, 5.40 mmol) and acetic

anhydride (0.55 g, 5.40 mmol). The mixture was stirred and refluxed for

5 h. CH₂Cl₂(100 mL) and H₂O (100 mL) were added to the reaction

mixture. The organic layer was washed with H₂O (2 ꢁ 100 mL), a

saturated solution of NaHCO₃(100 mL), and a saturated solution of

NaCl (100 mL). The organic layer was dried over MgSO₄and the

solvent was evaporated. Compound 16 was purified by silica gel

chromatography (hexane:ethyl acetate 7:1), 0.41 g (87%), colorless

solid, mp 84ꢀ85 °C. ¹H NMR (200 MHz, CDCl₃): δ 7.67 (d, J = 8.6 Hz,

2H), 7.35 (d, J = 8.6 Hz, 2H), 5.18 (s, 2H), 2.12 (s, 3H). ¹³C NMR (50

MHz, CDCl₃): δ 190.3, 169.3, 131.8, 131.2, 128.2, 128.1, 64.8, 19.5. m/z

(EI): 260, 258 (M ^þ, 15, 15%), 183, 181, 179 (M-C₃H₅O₂, 25, 89,

100%). HPLC: Column Symmetry C18, 5 μm, 100 Å, (150 ꢁ 3.9 mm),

Purity 100%, r.t = 6.41 min, acetonitrile/H₂O (0.05% H₃PO₄þ 0.04%

Et₃N) 50/50.

2-(2-(4-Bromophenyl)-2-oxoethyl)isoindoline-1,3-dione (11).³⁴

A

mixture of 2-bromo-1-(4-bromophenyl)ethanone (5) (0.3 g, 1.08

mmol) and potassium phthalimide (0.21 g, 1.13 mmol) in acetonitrile

(50 mL) was stirred and refluxed for 2 h. The solvent was evaporated,

and CH₂Cl₂(100 mL) and H₂O (100 mL) were added to the mixture.

The organic layer was washed with a 0.2 N sodium hydroxide solution

(50 mL) and water (50 mL). Then the organic layer was dried over

MgSO₄and the solvent was evaporated. Compound 11 was purified by

silica gel chromatography (hexane:ethyl acetate 3:1), 0.28 g (76%),

colorless solid, mp 223ꢀ225 °C. ¹H NMR (200 MHz, CDCl₃): δ 7.91

(m, 2H), 7.87 (d, J= 8.4 Hz, 2H), 7.76 (m, 2H), 7.67 (d, J= 8.4 Hz, 2H),

5.01 (s, 2H). ¹³C NMR (75 MHz, CDCl₃): δ 190.1, 167.8, 134.2, 132.2,

129.6, 123.5, 133.1, 132.7, 132.1, 129.4, 44.0. m/z (EI): 345, 343 (M ^þ

,

21, 21%), 185, 183 (M-CH₂phthalimide, 98, 100%). HPLC: Column

Symmetry C18, 5 μm, 100 Å, (150 ꢁ 3.9 mm), Purity 99%, r.t = 3.66

min, acetonitrile/H₂O (0.05% H₃PO₄þ 0.04% Et₃N) 50/50.

2-Amino-4⁰-bromoacetophenone hydrochloride (12). Commer-

cially available from Sigma Aldrich.

1-(4-Bromophenyl)-2-hydroxyethanone (17).³⁹Enzymatic deacyla-

tion of 16, was done employing different lipases (PPL (Pancreatic Porcine

Lipase), PSL (Pseudomonas cepacia Lipase), MML (Mucor miehi Lipase),

and CAL-B (Candida Antartica Lipase-B) under the same reaction

conditions (citrate-phosphate 9:1 buffer pH 7, tert-butanol, 45 °C,

24 h). No product was obtained using MML, while partial hydrolysis

was done when lipases PPL and PSL were employed. However, the use

of CAL-B gave exclusively compound 17 with an 85% yield. Accordingly,

a mixture of 2-(4-bromophenyl)-2-oxoethyl acetate (16) (20 mM) and

CAL-B (20 mM) in tert-butanol:citrate phosphate buffer (pH = 7) 9:1

(85 mL) was orbitally stirred and refluxed for 24 h. The reaction was

filtered and ethyl acetate (75 mL) and H₂O (50 mL) were added to the

solution. The organic layer was dried over MgSO₄and the solvent was

evaporated. Compound 17 was purified by silica gel chromatography

(hexane:ethyl acetate 1:1), 0.36 g (85%), colorless solid, mp

General Procedure for the Synthesis of Sulfur Derivatives (Compounds

13ꢀ15). A mixture of 2-bromo-1-(4-bromophenyl)ethanone (5), po-

tassium carbonate, and the corresponding thiol (ethanethiol, thiophe-

nol, and benzylthiol) in ethanol (50 mL) was stirred and refluxed for

2 h. The mixture was filtered, and CH₂Cl₂(75 mL) and water (75 mL)

were added to the solution. The organic layer was washed with water

(2 ꢁ 75 mL). Then the organic layer was dried over MgSO₄and the

solvent was evaporated. The compounds were purified by silica gel

chromatography.

1-(4-Bromophenyl)-2-(ethylthio)ethanone (13).³⁵2-Bromo-1-(4-

bromophenyl) ethanone (5) (0.5 g, 1.80 mmol), potassium carbonate

(0.25 g, 1.80 mmol), ethanethiol (0.11 g, 1.80 mmol). Purified by silica

gel chromatography (hexane:ethyl acetate 4:1), 0.20 g (45%), colorless

solid, mp 64ꢀ65 °C. ¹H NMR (200 MHz, CDCl₃): δ 7.74 (d, J = 8.7 Hz,

1

103ꢀ104 °C. H NMR (200 MHz, CDCl₃): δ 7.80 (d, J = 8.6 Hz,

2H), 7.65 (d, J = 8.6 Hz, 2H), 4.85 (s, 2H). ¹³C NMR (75 MHz, CDCl₃):

δ 197.8, 132.9, 132.4, 130.1, 129.5, 65.7. m/z (EI): 216, 214 (M ^þ, 11,

11%), 185, 183, (M-CH₃O, 100, 100%). HPLC: Column Symmetry

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Journal of Medicinal Chemistry

ARTICLE

C18, 5 μm, 100 Å, (150 ꢁ 3.9 mm), Purity 99%, r.t = 3.81 min,

acetonitrile/H₂O (0.05% H₃PO₄þ 0.04% Et₃N) 50/50.

Bondapack C18, 10 μm, 125 Å, (300 ꢁ 3.9 mm), Purity 97%, r.t = 4.41

min, acetonitrile/H₂O (0.05% H₃PO₄þ 0.04% Et₃N) 50/50. Com-

pound (22). 0.31 g (15%), colorless solid, mp 95ꢀ96 °C. ¹H NMR (200

MHz, CDCl₃): δ 8.23 (d, J = 8.79 Hz, 2H), 7.83 (d, J = 8.79 Hz, 2H),

6.58 (s, 1H). ¹³C NMR (75 MHz, CDCl₃): δ 185.0, 134.4, 132.9, 130.7,

117.9, 117.8, 39.0. m/z (EI): 305, 303, 301 (M ^þ, 4, 12, 4%),

130 (M-COCHBr₂, 100%). HPLC: μ Bondapack C18, 10 μm, 125 Å,

(300 ꢁ 3.9 mm), Purity 98%, r.t = 4.11 min, acetonitrile/H₂O (0.05%

H₃PO₄þ 0.04% Et₃N) 50/50.

General Procedure for Oxime (18) and Methyl Oxime Synthesis

(19). To a mixture of hydroxylamine hydrochloride or methylhydrox-

ylamine hydrochloride and pyridine in ethanol (50 mL) was added

2-bromo-1-(4-bromophenyl)ethanone (5). The mixture was stirred and

refluxed for 4 h. The solvent was evaporated and cold water (25 mL) was

added to the mixture. The mixture is left at 0 °C for 12 h and the

precipitate formed was filtered. The compounds were purified by silica

gel chromatography.

2-Bromo-1-(4-(trifluoromethyl)phenyl)ethanone (23)⁴⁵and 2,2-

Dibromo-1-(4-(trifluoromethyl)phenyl)ethanone (24).⁴⁶1-(4-(Tri-

fluoromethyl)phenyl) ethanone (1.0 g, 5.31 mmol), bromine (1.10 g,

6.90 mmol). Mixture of compounds separated by silica gel chromatog-

raphy (hexane:ethyl acetate 8:1), 0.18 g (6%) (23), colorless solid, mp

55ꢀ56 °C. ¹H NMR (200 MHz, CDCl₃): δ 8.12 (d, J = 8.06 Hz, 2H),

7.79 (d, J = 8.06 Hz, 2H), 4.47 (s, 2H). ¹³C NMR (75 MHz, CDCl₃): δ

(E,Z)-2-Bromo-1-(4-bromophenyl)ethanone Oxime (18).⁴⁰Hydro-

xylamine hydrochloride (0.26 g, 3.81 mmol), pyridine (0.31 g, 3.81

mmol), and 2-bromo-1-(4-bromophenyl)ethanone (5) (1.0 g, 3.59

mmol). Purified silica gel column chromatography (hexane:ethyl acetate

1

6:1), 0.82 g (78%), colorless solid, mp 115ꢀ116 °C. H NMR (200

MHz, CDCl₃): δ 11.8, 11.7 (s, 1H), 7.52ꢀ7.41 (m, 4H), 4.48, 4.35

(s, 2H). ¹³C NMR (75 MHz, CDCl₃): δ 154.0, 153.9, 132.7, 132.3,

128.2, 124.7, 32.1, 27.2. m/z (EI): 295, 293, 291 (M ^þ, 36, 72, 38%), 102

(M-CH₃Br₂O, 100%). HPLC: Column Symmetry C18, 5 μm, 100 Å,

(150 ꢁ 3.9 mm), Purity 99%, r.t = 8.15 min, acetonitrile/H₂O (0.05%

H₃PO₄þ 0.04% Et₃N) 50/50.

190.4, 136.5, 135.0 (q, J_CF= 33.09 Hz), 129.3, 125.9, 123.3. (q, J_CF

=

272.9 Hz), 30.4. m/z (EI): 268, 266 (M ^þ, 6, 6%), 172 (M-COCH₂Br,

100%). HPLC: Column μBondapack C18, 10 μm, 125 Å, (300 ꢁ 3.9 mm),

Purity 97%, r.t = 6.53 min, acetonitrile/H₂O (0.05% H₃PO₄þ 0.04%

Et₃N) 50/50. Compound (24). 0.64 g (45%), colorless solid, mp

41ꢀ42 °C. ¹H NMR (200 MHz, CDCl₃): δ 8.22 (d, J = 8.3 Hz, 2H),

7.81 (d, J = 8.3 Hz, 2H), 6.6 (s, 1H). ¹³C NMR (75 MHz, CDCl₃): δ

184.1, 134.7 (q, J = 272.9 Hz), 132.7, 129.2, 124.8, 119.5 (q, J = 33.09),

38.2. m/z (EI): 348, 346, 344 (M ^þ, 1, 6, 1%), 173 (M-CHBr₂, 100%).

HPLC: Column μ Bondapack C18, 10 μm, 125 Å, (300 ꢁ 3.9 mm),

Purity 97%, r.t = 9.96 min, acetonitrile/H₂O (0.05% H₃PO₄þ 0.04%

Et₃N) 50/50.

(E,Z)-2-Bromo-1-(4-bromophenyl)ethanone O-Methyl Oxime (19).⁴¹

Methyl hydroxylamine hydrochloride (0.30 g, 3.59 mmol), pyridine

(0.28 g, 3.59 mmol), and 2-bromo-1-(4-bromophenyl)ethanone (5)

(1.0 g, 3.59 mmol). Purified by silica gel chromatography (hexane:ethyl

acetate 6:1), 0.56 g (82%), colorless solid, mp 48ꢀ49 °C. ¹H NMR (200

MHz, CDCl₃): δ 7.65ꢀ7.51 (m, 4H), 4.52, 4.35 (s, 2H), 4.15, 4.05

(s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 151.2, 132. 4, 131.9, 127.7, 124.1,

63.0, 62.9, 32.2, 32.1. m/z (EI): 309, 307, 305 (M ^þ, 13, 26, 13%), 102

(M-C₂H₅Br₂O, 100%). HPLC: Column Symmetry C18, 5 μm, 100 Å,

(150 ꢁ 3.9 mm), Purity 99%, r.t = 8.10 min, acetonitrile/H₂O (0.05%

H₃PO₄þ 0.04% Et₃N) 50/50.

2-Bromo-1-(4-morpholinophenyl)ethanone (25).⁴⁷1-(4-Morpho-

linophenyl) ethanone (1.0 g, 4.87 mmol), bromine (1.01 g, 6.33 mmol).

Purified by silica gel chromatography (hexane:ethyl acetate 1:1), 0.56 g

1

(42%) (25), colorless solid, mp 108ꢀ109 °C. H NMR (200 MHz,

1-Bromo-4-(chloromethylsulfinyl)benzene (20).⁴²A solution of

sulfuryl chloride (0.33 g, 2.46 mmol) in acetonitrile (5 mL) was added

dropwise to a mixture of bromothioanisol (0.50 g, 2.46 mmol) and silver

nitrate (0.42 g, 2.46 mmol) in acetonitrile (10 mL) at 0 °C. The mixture

was then stirred 1 h at room temperature. CH₂Cl₂(10 mL) and H₂O

(10 mL) were added to the reaction mixture. The organic layer was

washed with H₂O (2 ꢁ 100 mL), dried over MgSO₄, and the solvent was

evaporated. Compound 20 was purified by silica gel chromatography

(hexane:ethyl acetate 1:1), 0.44 g (71%), colorless solid, mp 91ꢀ92 °C.

¹H NMR (200 MHz, CDCl₃): δ 7.71 (d, J = 8.7 Hz, 2H), 7.58 (d, J = 8.7

Hz, 2H), 4.38 (s, 2H). ¹³C NMR (75 MHz, CDCl₃): δ 142.1, 132.6,

126.4, 93.1, 6.8. m/z (EI): 256, 254, 252 (M ^þ, 4, 16, 12%), 205, 203 (M-

CH₂Cl, 100, 100%). HPLC: Column Symmetry C18, 5 μm, 100 Å,

(150 ꢁ 3.9 mm), Purity 99%, r.t = 4.73 min, acetonitrile/H₂O (0.05%

H₃PO₄þ 0.04% Et₃N) 50/50.

CDCl₃): δ 7.95 (d, J = 4 Hz, 2H), 7.04 (d, J = 4 Hz, 2H), 4.60 (s, 2H),

3.83 (m, 4H), 3.65 (m, 4H). ¹³C NMR (75 MHz, CDCl₃): δ 189.9,

154.9, 130.5, 120.3, 113.5, 67.8, 47.6, 26.7. m/z (EI): 285, 283 (M ^þ, 14,

13%), 190 (M-CH₂Br, 100%). HPLC: Column μ Bondapack C18,

10 μm, 125 Å, (300 ꢁ 3.9 mm), Purity 99%, r.t = 4.56 min, acetonitrile/

H₂O (0.05% H₃PO₄þ 0.04% Et₃N) 50/50.

2,2-Dibromo-1-(4-iodophenyl)ethanone (26).⁴⁶1-(4-Iodophe-

nyl)ethanone (0.40 g, 1.60 mmol), bromine (0.1 mL, 1.70 mmol).

Purified by silica gel chromatography (hexane:ethyl acetate 6:1), 0.31 g

(48%) (26), colorless solid, mp 77ꢀ78 °C. ¹H NMR (200 MHz,

CDCl₃): δ 7.82 (d, J = 4.2 Hz, 2H), 7.73 (d, J = 4.2 Hz, 2H), 6.3

(s, 1H). ¹³C NMR (75 MHz, CDCl₃): δ 185.3, 138.6, 131.3, 130.4,

103.4, 39.6. m/z (EI): 406, 404, 402 (M ^þ, 9, 17, 9%), 231, (M-CHBr₂,

100%). HPLC: Column Symmetry C18, 5 μm, 100 Å, (150 ꢁ 3.9 mm),

Purity 98%, r.t = 4.51 min, acetonitrile/H₂O (0.05% H₃PO₄þ 0.04%

Et₃N) 50/50.

General Procedure for Bromoacetyl Benzene (21, 23, 25, 27, and 29),

Dibromoacetyl Benzene (22, 24, 26, 28), and Pyridine (30) Synthesis.

To a solution of acetophenone derivative or 2-acetyl pyridine dissolved

in CHCl₃(35 mL) was added dropwise a solution of bromine in CHCl₃

(10 mL) at 0 °C. The mixture was stirred 2 h. The solvent was evapo-

rated and to the mixture was added CH₂Cl₂(50 mL) and a saturated

solution of NaHCO₃(50 mL). The organic layer was dried over MgSO₄

and the solvent was evaporated. Compounds were purified/separated by

silica gel chromatography.

3-(2-Bromoacetyl)benzonitrile (27)⁴⁸and 3-(2,2-Dibromoace-

tyl)benzonitrile (28).⁴⁹3-Acetylbenzonitrile (0.5 g, 3.44 mmol), bro-

mine (0.72 g, 4.48 mmol). Mixture of compounds separated by silica gel

chromatography (hexane:ethyl acetate 5:2), 0.10 g (13%) (27), color-

less solid, mp 70ꢀ71 °C. ¹H NMR (200 MHz, CDCl₃): δ 8.21 (t, J = 1.5

Hz, 1H), 8.15 (td, J = 7.9, 1.5 Hz, 1H), 7.95 (td, J = 7.9, 1.5 Hz, 1H), 7.80

(t, J = 7.9 Hz, 1H), 4.32 (s, 2H). ¹³C NMR (75 MHz, CDCl₃): δ 189.8,

137.0, 135.1, 133.2, 133.0, 130.2, 117.9, 113.9, 30.1. m/z (EI): 225, 223

(M ^þ, 2, 2%), 130 (M-CH₂Br, 100%). HPLC: Column μ Bondapack

C18, 10 μm, 125 Å, (300 ꢁ 3.9 mm), Purity 97%, r.t = 3.60 min,

acetonitrile/H₂O (0.05% H₃PO₄þ 0.04% Et₃N) 50/50. Compound

(28). 0.38 g (37%), colorless solid, mp 85ꢀ86 °C. ¹H NMR (200 MHz,

CDCl₃): δ 8.25 (t, J = 1.5 Hz, 1H), 8.15 (td, J = 7.9, 1.5 Hz, 1H),

7.85 (td, J = 7.9, 1.5 Hz, 1H), 7.70 (t, J = 7.9 Hz, 1H), 6.40 (s, 1H, CH).

¹³C NMR (75 MHz, CDCl₃): δ 184.5, 137.4, 134.1, 133.8, 132.1,

130.2, 117.8, 114.0, 38.9. m/z (EI): 305, 303, 301 (M ^þ, 3, 9, 3%), 130

4-(2-Bromoacetyl)benzonitrile (21)⁴³and 4-(2,2-Dibromoacetyl)-

benzonitrile (22).⁴⁴4-Acetylbenzonitrile (1.0 g, 6.89 mmol), bromine

(1.12 g, 7.02 mmol). Mixture of compounds separated by silica gel

chromatography (hexane:ethyl acetate 3:1), 0.69 g (45%) (21), color-

1

less solid, mp 90ꢀ91 °C. H NMR (200 MHz, CDCl₃): δ 8.10 (d,

J = 8.79 Hz, 2H), 7.82 (d, J = 8.79 Hz, 2H), 4.44 (s, 2H). ¹³C NMR (75

MHz, CDCl₃): δ 190.4, 137.3, 133.0, 129.8, 118.0, 117.6, 30.4. m/z (EI):

225, 223 (M ^þ, 4, 4%), 130 (M-COCH₂Br, 100%). HPLC: Column μ

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(M-COCHBr₂, 100%). HPLC: Column μ Bondapack C18, 10 μm,

125 Å, (300 ꢁ 3.9 mm), Purity 98%, r.t = 4.10 min, acetonitrile/H₂O

(0.05% H₃PO₄þ 0.04% Et₃N) 50/50.

without further purification. ¹H NMR (300 MHz, CDCl₃): δ 7.43

(m, 1H), 7.40 (s, 1H), 7.37 (m, 1H), 7.26 (m, 1H), 6.96ꢀ6.86 (m, 2H),

6.86ꢀ6.68 (m, 2H), 5.64 (s, 1H), 3.81 (s, 3H), 2.51 (s, 3H), 1.44 (s,

1H). HPLC: Purity 88%, r.t = 4.15 min. MS (ESIþ): m/z 257 [MþH]^þ.

1-(1-(4-Methoxyphenyl)-1H-benzo[d]imidazol-5-yl)ethanone (35).

A stirred solution of 34 (0.93 g, 3.64 mmol) and formic acid (1.58 mL, 42

mmol) in HCl 4 N (25 mL) was refluxed for 3 h. Then dichloromethane

(25 mL) was added to the mixture. The organic layer was washed with

saturated NaHCO₃solution (3 ꢁ 25 mL) and then dried over MgSO₄.

The solvent was evaporated under reduced pressure to give 35 as a

1-(4-Amino-3,5-dibromophenyl)-2-bromoethanone (29).⁴⁶1-(4-

Aminophenyl) ethanone (0.5 g, 3.70 mmol), bromine (0.20 mL, 4.06

mmol). Purified by silica gel chromatography (hexane:ethyl acetate 2:1),

1

0.80 g (58%) (29), colorless solid, mp 146ꢀ147 °C. H NMR (200

MHz, CDCl₃): δ 8.0 (s, 2H), 5.1 (s, 2H), 4.2 (s, 2H). ¹³C NMR (75

MHz, CDCl₃): δ 187.5, 147.3, 132.7, 123.3, 106.2, 38.3. m/z (EI): 375,

373, 371, 369 (M ^þ, 10, 31, 31, 10%), 281, 279, 277 (M-CHBr₂, 64, 100,

67%). HPLC: Column Symmetry C18, 5 μm, 100 Å, (150 ꢁ 3.9 mm),

Purity 96%, r.t = 9.98 min, acetonitrile/H₂O (0.05% H₃PO₄þ 0.04%

Et₃N) 50/50.

1

brown solid (803 mg, 83%), mp 139ꢀ140 °C. H NMR (300 MHz,

CDCl₃): δ 8.46 (d, J = 1.1 Hz, 1H), 8.13 (s, 1H), 7.99 (dd, J = 8.6, 1.5 Hz,

1H), 7.50ꢀ7.42 (m, 1H), 7.40 (d, J = 9.0 Hz, 2H), 7.08 (d, J = 9.0 Hz,

2H), 3.89 (s, 3H), 2.69 (s, 3H).¹³C NMR (75 MHz, CDCl₃): δ 198.0,

159.9, 144.6, 132.6, 128.7, 126.0, 123.9, 122.4, 115.5, 110.6, 55.9, 26.9.

HPLC: Purity 99%, r.t = 4.18 min. MS (ESIþ): m/z 267 [MþH]^þ.

2-Bromo-1-(1-(4-methoxyphenyl)-1H-benzo[d]imidazol-5-yl)-

ethanone (36). To a solution of 35 (1.14 g, 4.28 mmol) dissolved in

AcOH (25 mL) at reflux was added a solution of bromine (0.23 mL, 4.50

mmol) in AcOH (5 mL). The mixture was refluxed and stirred for 5 h.

Dichloromethane (50 mL) and water (50 mL) were added to the

mixture. The organic layer was extracted and washed with saturated

solutions of NaHCO₃(2 ꢁ 50 mL) and NaCl (50 mL). The organic

layer was dried over MgSO₄and the solvent was evaporated. The

product was purified by flash chromatography (hexane:ethyl acetate

1:1), 162 mg (11%) (36), red solid, mp 247ꢀ248 °C. ¹H NMR (300

MHz, CDCl₃): δ 8.52 (s, J = 1.1 Hz, 1H), 8.18 (s, 1H), 8.03 (dd, J = 8.6,

1.6 Hz, 1H), 7.51 (d, J = 8.6, 1H), 7.41 (d, J = 9.0 Hz, 2H), 7.10 (d, J = 9.0

Hz, 2H), 4.57 (s, 2H), 3.91 (s, 3H).¹³C NMR (75 MHz, CDCl₃): δ

191.2, 160.0, 144.9, 126.0, 124.7, 122.9, 115.5, 111.1, 55.9. HPLC: Purity

95%, r.t = 4.70 min. MS (ESIþ): m/z 345 [MþH]^þ.

2,2-Dibromo-1-(pyridin-2-yl)ethanone (30).⁵⁰1-(Pyridin-2-yl)-

ethanone (1.0 g, 8.25 mmol), bromine (1.45 g, 9.08 mmol). Purified

by silica gel chromatography (hexane:ethyl acetate 6:1), 1.69 g (74%)

(30), colorless solid, mp 196ꢀ197 °C. ¹H NMR (200 MHz, CDCl₃): δ

8.71 (m, 1H), 8.10 (m, 2H), 7.82 (m, 1H), 6.13 (s, 1H). ¹³C NMR (75

MHz, CDCl₃): δ 187.0, 149.5, 148.1, 138.4, 129.0, 124.0, 45.0. m/z (EI):

281, 279, 277 (M ^þ, 9, 19, 9%), 106 (M-CHBr₂, 100%). HPLC: Column

Symmetry C18, 5 μm, 100 Å, (150 ꢁ 3.9 mm), Purity 98%, r.t = 3.66

min, acetonitrile/H₂O (0.05% H₃PO₄þ 0.04% Et₃N) 50/50.

1-(6-Amino-9H-purin-9-yl)ethanone (31).²³To a solution of ade-

nine (1.23 g, 9.14 mmol) in DMSO anhydrous (50 mL) was added a

solution of pyridine anhydrous (20 mL) and acetic anhydride (5 mL).

The mixture was stirred for 24 h at room temperature and a white

precipitate formed. The precipitate was filtered and washed with several

portions of cold pyridine and ether, to afford a white solid (629 mg,

1

51%), mp 363ꢀ364 °C. H NMR (300 MHz, DMSO-d₆): δ 8.61 (s,

1H), 8.28 (s, 1H), 7.52 (s, 2H), 2.88 (s, 3H). ¹³C NMR (75 MHz,

DMSO-d₆): δ 173.97, 162.01, 159.50, 154.36, 144.05, 125.17, 30.41.

HPLC: Purity >99%, r.t = 0.55 min. MS (ESIþ): m/z 178 [MþH]^þ.

1-(6-Amino-9H-purin-9-yl)-2-chloroethanone (32). To a solution

of adenine (1.00 g, 7.4 mmol) in THF anhydrous (50 mL) was added

a mixture of pyridine anhydrous (20 mL) and chloroacetic anhydride

(1.39 g, 8.14 mmol). The mixture was stirred for 24 h at room

temperature and a white precipitate was formed. The precipitate was

filtered and washed with several portions of cold pyridine and cold ether.

This solid was purified by column chromatography (methanol:dichlor-

omethane 1:10) to afford a white solid (312 mg, 20%), mp 199ꢀ200 °C.

¹H NMR (400 MHz, DMSO-d₆): δ 12.28 (s, 1H), 11.48 (s, 2H), 8.65

(s, 1H), 8.45 (s, 1H), 4.55 (s, 2H). ¹³C NMR (101 MHz, DMSO-d₆):

δ 166.32, 151.25, 145.74, 144.20, 118.15, 43.40. HPLC: Purity 95%,

r.t = 2.87 min. MS (ESIþ): m/z 212 [MþH]^þ.

General Procedure for Maleimide Synthesis (37ꢀ38). To a solution

t

of acetoacetamide (1 equiv) and BuOK (1 M solution in THF) (3.5

equiv) dissolved in THF (20 mL) at ꢀ60 °C was added methyl

3-indoleglyoxylate or methyl 2-(1-methyl-1H-indol-3-yl)-2-oxoacetate

(1 equiv). The mixture was stirred until room temperature was reached.

Then a concentrated HCl solution (11.5 mL), water (30 mL), and

dichlorometane (30 mL) were added in the reaction mixture. The

organic layer was washed with a saturated solution of NaHCO₃(2 ꢁ

30 mL), dried over MgSO₄, and evaporated. Products were purified by

recrystallization from ethyl acetate/pentane.

3-Acetyl-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (37). Acetoaceta-

mide (0.372 g, 3.69 mmol), ^tBuOK (1 M solution in THF) (12.9 mL,

12.9 mmol), methyl 3-indoleglyoxylate (0.750 g, 3.69 mmol). 0.70 g

(74%) (37), orange solid, mp 225ꢀ226 °C. ¹H NMR (300 MHz,

DMSO-d₆): δ 12.29 (s, 1H), 11.17 (s, 1H), 8.24 (s, 1H), 7.50 (d, J = 7.01

Hz 1H), 7.27ꢀ7.17 (m, 1H), 7.13 (s, 2H), 2.50ꢀ2.46 (s, 3H). ¹³C NMR

(75 MHz, DMSO-d₆): δ 195.4, 171.7, 139.8, 137.5, 135.5, 125.5, 124.9,

123.5, 122.6, 121.7, 113.3, 106.0, 31.7. HPLC/MS: Purity >99%, r.t =

3.76 min. MS (ESIþ): m/z 255 [MþH]^þ.

1-(4-(4-Methoxyphenylamino)-3-nitrophenyl)ethanone (33).⁵¹To

a solution of 1-(4-bromo-3-nitrophenyl)ethanone (1 g, 4.09 mmol) and

4-methoxyaniline (0.504 g, 4.09 mmol) in anhydrous DMF (23 mL) was

added sodium acetate (0.504 g, 6.14 mmol). The mixture was heated 2 h

at 100 °C and then stirred at room temperature overnight. Ethyl acetate

(100 mL) and H₂O (100 mL) was added to the reaction mixture.

The organic layer was washed with H₂O (2 ꢁ 100 mL), dried over

MgSO₄, and the solvent was evaporated. The residue was recrystallized

from MeOH/H₂O to afford 33 as a red solid (853 mg, 73%), mp

132ꢀ133 °C. ¹H NMR (300 MHz, CDCl₃): δ 9.77 (s, 1H), 8.82

(s, 1H), 7.94 (d, J = 8.9 Hz, 1H), 7.21 (d, J = 8.7 Hz, 2H), 7.10 ꢀ 6.83

(m, 3H), 3.86 (s, 3H), 2.58 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ

194.7, 158.4, 147.6, 134.4, 131.1, 129.8, 128.4, 127.2, 126.1, 115.6, 115.1,

55.4, 25.92. HPLC: Purity >99%, r.t = 5.09 min. MS (ESIþ): m/z 287

[MþH]^þ.

3-Acetyl-4-(1-methyl-indol-3-yl)-1H-pyrrole-2,5-dione (38). Acetoa-

cetamide (0.325 g, 3.22 mmol), ^tBuOK (1 M solution in THF) (11.3 mL,

11.3 mmol), 2-(1-methyl-1H-indol-3-yl)-2-oxoacetate (0.700 g, 3.22

1

mmol). 0.482 g (56%) (38), red solid, mp 224ꢀ225 °C. H NMR

(300 MHz, DMSO-d₆): δ 11.19 (s, 1H), 8.27 (s, 1H), 7.58 (d, J = 8.1 Hz,

1H), 7.35ꢀ7.06 (m, 3H), 3.92 (s, 3H), 2.51 (s, 3H).¹³C NMR (75 MHz,

DMSO-d₆): δ 194.6, 170.9, 138.5, 138.1, 137.3, 125.2, 123.8, 122.8,

122.1, 121.3, 111.0, 104.3, 33.3, 30.9. HPLC/MS: Purity 95%, r.t = 4.06

min. MS (ESIþ): m/z 269 [MþH]^þ.

1-(4-(4-Methoxyphenylamino)-3-aminophenyl)ethanone (34). To

a solution of 33 (0.23 g, 0.8 mmol) dissolved in THF (20 mL) was added

Pd(C) (23 mg). The mixture was stirred at room temperature overnight

at 20 psi. The mixture was filtered and the solvent was evaporated

to afford a brown solid. The title compound was used in the next step

General Procedure for Maleimide Bromation (39 and 40). Malei-

mide derivative 37 or 38 (1 equiv) is dissolved in AcOH. Under reflux

conditions, bromine (1.05 equiv) is slowly added diluted in AcOH. The

mixture is stirred at reflux temperature for 3 h. Then water and AcOEt

are added and the mixture is extracted, and the organic layer washed with

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several portions of a NaHCO₃saturated solution and then dried and

evaporated. The products are purified by recrystallization MeOH/H₂O

(39) or by flash chromathography (40) using a dichloromethane/

methanol 80:1 mixture as eluent.

(Millipore Iberica S.A.U.). Kinase-Glo Luminescent Kinase Assay was

obtained from Promega (Promega Biotech Ibꢀerica, SL). ATP and all

other reagents were from Sigma-Aldrich (St. Louis, MO). Assay buffer

contained 50 mM HEPES (pH 7.5), 1 mM EDTA, 1 mM EGTA, and

15 mM magnesium acetate.

3-(2-Bromoacetyl)-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione (39).

Title compound was prepared using the same procedure described

for the synthesis of compound 36. Maleimide 37 (0.670 g, 2.63 mmol),

bromine (0.135 mL, 2.63 mmol). The residue was recrystallized from

methanol/water to give 39 as a black solid (0.345 g, 39%), mp

260ꢀ261 °C. ¹H NMR (300 MHz, DMSO-d₆): δ 12.54 (s, 1H),

11.34 (s, 1H), 8.39 (m, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.32ꢀ6.98 (m,

3H), 4.75 (s, 2H). ¹³C NMR (126 MHz, acetone-d₆): δ 186.0, 169.6,

169.4, 142.1, 135.9, 135.7, 124.7, 122.8, 121.0, 112.1, 106.4, 35.0.

HPLC/MS: Purity 95%, r.t = 4.35 min. MS (ESIþ): m/z 333 [MþH]^þ.

3-(2-Bromoacetyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-

dione (40). Title compound was prepared using the same procedure

described for the synthesis of compound 36. Maleimide 38 (0.603 g,

2.25 mmol), bromine (0.115 mL, 2.25 mmol). The product was purified

by flash chromatography (dichloromethane:methanol 80:1), 120 mg

(15%) (40), purple solid, mp 252ꢀ253 °C. ¹H NMR (300 MHz,

CDCl₃): δ 8.46 (s, 1H), 7.38 (m, 4H), 5.31 (s, 1H), 4.64 (s, 2H), 3.94

(s, 3H). ¹³C NMR (126 MHz, acetone-d₆): δ 185.80, 169.78, 169.55,

141.80, 139.52, 137.66, 125.37, 122.94, 122.88, 121.34, 119.28, 110.46,

105.53, 35.02, 32.71. HPLC: Purity 99%, r.t = 4.47 min. MS (ESIþ):

m/z 347 [MþH]^þ.

The method of Baki et al.⁵²was followed for the inhibition of GSK-3β.

Kinase-Glo assays were performed in assay buﬀer using black 96-well

plates. In a typical assay, 10 μL (10 μM) of test compound (dissolved in

dimethyl sulfoxide [DMSO] at 1 mM concentration and diluted in

advance in assay buﬀer to the desired concentration) and 10 μL (20 ng)

of enzyme were added to each well followed by 20 μL of assay buﬀer

containing 25 μM substrate and 1 μM ATP. The ﬁnal DMSO concentra-

tionin the reactionmixture did notexceed 1%. Aftera 30 min incubation at

30 °C, the enzymatic reaction was stopped with 40 μL of Kinase-Glo

reagent. Glow-type luminescence was recorded after 10 min using a

FLUOstar Optima (BMG Labtechnologies GmbH, Oﬀenburg, Germany)

multimode reader. The activity is proportional to the diﬀerence of the total

and consumed ATP. The inhibitory activities were calculated on the basis

of maximal activities measured in the absence of inhibitor. The IC₅₀was

deﬁned as the concentration of each compound that reduces a 50% the

enzymatic activity with respect to that without inhibitors.

GSK-3 Reversibility Studies. To study the type of enzymatic inhibition

for the compounds, assays were performed to determine the activity

of the enzyme after several times of incubation of the enzyme with the

inhibitor. A reversible inhibitor does not increase the inhibition of the

enzyme with the time of incubation, while an irreversible inhibitor

increases the inhibition percentage as it increases the time of incubation

with the enzyme.

Antibodies and Western Blot Analysis. The antibodies used in this

study were: anti-β-actin mAb (Sigma); PHF-1, which is an antiphospho

Ser^396/404Tau mAb and Tau monoclonal antibody Tau-1, from Chemi-

con, that recognizes residues Ser 195, 198, 199, 202 non-phosphorylated.

Cell extracts were prepared from cells washed with 1 ꢁ PBS and then

resuspended in a buﬀer containing the following: 20 mM HEPES, pH

7.4; 100 mM NaCl; 100 mM NaF; 1 mM sodium ortho-vanadate; 5 mM

EDTA; Okadaic Acid 1 μM; 1% Triton X-100; and a protease inhibitor

cocktail (Complete, Roche). The soluble fraction was obtained by

centrifugation at 14 000 g for 10 min at 4 °C, and the proteins

(10ꢀ50 μg) were separated by SDS-PAGE before being transferred

to a nitrocellulose ﬁlter. The ﬁlters were blocked with 5% nonfat milk in

PBS and 0.1% Tween-20 (PBS-T) and then incubated with primary

antibodies overnight at 4 °C. Subsequently, the ﬁlters were rinsed three

times in PBST buﬀer before being exposed to the corresponding

peroxidase-conjugated secondary antibody (diluted 1:5000, Promega)

for 1 h at room temperature. Immunoreactivity was visualized using

an enhanced chemiluminescence detection system (Perkin-Elmer Life

Sci.). Each experiment was normalized with respect to the amount of

actin present in each cell extract. The data are expressed as the mean

of three independent experiments and the data from control cells were

considered 100 relative unit (r.u.).

Chemical Reactivity of HMKs. 100 μL of an acetonitrile solution of

HMK 3 or 4 (50 mM) and 100 μL of an acetonitrile solution of

2-mercaptoethanol (50 mM) were added to a mixture of methanol/

water (1/1, 0.8 mL) and stirred for 1 or 18 h. The reaction progress was

followed by HPLC-MS. HMK 3 (MW = 189) showed a retention time

of 4.90 min, while retention time for HMK 4 (MW = 233) was 5.05 min.

On the other hand, the S-alkylated derivative (MW = 214) presented a

retention time of 4.15 min. An additional experiment was carried out

when the 2-mercaptoethanol concentration was increased 10 times.

In this case, 100 μL of an acetonitrile solution of 2-mercaptoethanol

(500 mM) was added. The same procedure described above was

followed when triethylamine was used; 100 μL of an acetonitrile solution

of triethylamine (50 mM) was added to a methanol/water (1/1, 0.7 mL)

solution.

MALDI-TOF Analyses. Human recombinant GSK-3β was purchased

from Millipore (Millipore Iberica S.A.U). A stock solution of 10 μg of

the enzyme in MiliiQ water (100 μL) was prepared. Compound 38

(17.8 mM) and compound 40 (10 μM) were dissolved in MeOH, then 1

μL of each compound solution was added to 10 μL of the enzyme stock

solution reproducing IC₅₀experimental conditions prior to MALDI-

TOF analysis.

Biological Studies. Inhibition of GSK-3 (Radiometric Assay).

GSK-3β enzyme (Sigma) was incubated with 15 μM of ATP, 0.2 μCi

of [γ-³²P]ATP, GS-1 substrate, and different concentrations of test

compound. GSK-3β activity was assayed in 50 mM Tris, pH 7.5, 10 mM

MgCl₂, 1 mM EGTA, and 1 mM EDTA buffer at 37 °C, in the presence

of 15 mM GS-1 (substrate), 15 μM of ATP, 0.2 μCi of [γ-³²P] ATP in a

final volume of 12 μL. After 20 min of incubation at 37 °C, 4 μL aliquots

of the supernatant were spotted onto 2 ꢁ 2 pieces of Whatman P81

phosphocellulose paper, and the filter was washed four times (at least

10 min each time) in 1% phosphoric acid. The dried filters were

transferred into scintillation vials, and the radioactivity was measured

in a liquid scintillation counter. Blank values were subtracted, and the

GSK-3β activity was expressed in percentage of maximal activity. The

IC₅₀is defined as the concentration of each compound that reduces

enzyme activity by 50% with respect to that without inhibitor present.

Inhibition of GSK-3 (Luminescent Assay). Human recombinant

GSK-3β was purchased from Millipore (Millipore Iberica S.A.U.) The

prephosphorylated polypeptide substrate was purchased from Millipore

Inhibition of Protein Kinases. The experimental procedures for the

inhibition of different protein kinases are described in each paragraph:

- Abl kinase:⁵³Mouse recombinant (E.coli), staurosporine was used

as reference compound and poly GT (0.4 μg.mL^ꢀ1) as a substrate.

Fluorescence polarization was used as the method of detection for

the reaction product (phosphopoly GT).

- CAM kinase II:⁵⁴From rat brain, staurosporine was used as

reference compound and [γ³³-P] ATP þ autocamtide-2 (5 μM)

as a substrate. Liquid scintillation was used as method of detection

for the reaction product ([γ³³-P] autocamtide-2).

- EGFR kinase:⁵⁵From A-431 cells, PD 153035 was used as reference

compound and [γ³³-P] ATP þ poly GAT (0.48 mg.mL^ꢀ1) as a

substrate. Liquid scintillation was used as method of detection for

the reaction product ([γ³³-P] poly GAT).

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ARTICLE

- IRK (h):⁵⁶Human recombinant, staurosporine was used as refer-

ence compound and [γ³³-P] ATP þ poly GT (0.03 mg.mL^ꢀ1) as a

substrate. Liquid scintillation was used as method of detection for

the reaction product ([γ³³-P] poly GT).

derivatives of adenine (32) and maleimide (40) containing the

halomethyketone moiety; elemental analyses of compounds.

This material is available free of charge via the Internet at

http://pubs.acs.org.

- MAP kinase (ERK 42):⁵⁷Rat recombinant (E.coli), staurosporine

was used as reference compound and [γ³³-P] ATP þ MBP (0.5 mg.

mL^ꢀ1) as a substrate. Liquid scintillation was used as method of

detection for the reaction product ([γ³³-P] MBP).

’ AUTHOR INFORMATION

Corresponding Author

*Phone: þ34 91 5680010. Fax: þ34 91 5644853. E-mail:

- MEK1 kinase:⁵⁸Rabbit recombinat (E.coli), staurosporine was

used as reference compound and ATP þ unactivated MAP kinase

(0.01 mg.mL^ꢀ1) as a substrate. Liquid scintillation was used as

method of detection for the reaction product ([γ³³-P] MBP).

amartinez@iqm.csic.es.

- Protein kinase p56^{lck 59}

:

From bovine thymus, staurosporine was

’ ACKNOWLEDGMENT

used as reference compound and [γ³³-P] ATP þ poliGT (0.5 mg.

mL^ꢀ1) as a substrate. Liquid scintillation was used as method of

detection for the reaction product ([γ³³-P] poliGT).

Financial support from MICINN and ISCiii (projects nos.

SAF2009-13015-CO2-01, SAF2008-05595, SAF2006-01249

and RD07-0060/0015) and computational facilities from the

CESCA are also acknowledged. D. I. P. acknowledges a post-

doctoral grant to the CSIC (JAEDoc program) and V. P. a

predoctoral grant (JAEPre program).

Neurotransmitter Receptors Binding Assays. The neurotransmitter

binding assays are described in each paragraph:

- R-2 (non selective) adrenergic receptor:⁶⁰From rat cerebral cortex,

yohimbine was used as reference compound and [³H] RX 821002

as a ligand (0.5 nM).

- D2 (h) dopamine receptor:⁶¹Human recombinant (CHO cells),

(þ)-buctaclamol was used as reference compound and [³H]

spiperone as a ligand (0.3 nM).

’ ABBREVIATIONS

GSK-3, glycogen synthase kinase 3; CNS, central nervous sys-

tem; AD, Alzheimer’s disease; HMK, halomethylketone; TDZD,

thiadiazolidindione; CDK-n, cyclin dependent kinase n, where

n is 1, 2 or 5; GS-1, glycogen synthase 1; PDB, protein data

bank; PK, protein kinase

- D3 (h) dopamine receptor:⁶²Human recombinant (CHO cells),

(þ)-buctaclamol was used as reference compound and [³H]

spiperone as a ligand (0.3 nM).

- AMPA glutaminergic receptor:⁶³From rat cerebral cortex, L-glutamate

was used as reference compound and [³H] AMPA as a ligand (8 nM).

- NMDA glutaminergic receptor:⁶⁴From rat cerebral cortex, CGS

19755 was used as reference compound and [³H] CGP 39653 as a

ligand (5 nM).

’ REFERENCES

(1) Cohen, P. Protein kinases--the major drug targets of the twenty-

ﬁrst century? Nat Rev. Drug Discovery 2002, 1, 309–315.

(2) Duckett, D. R.; Cameron, M. D. Metabolism considerations for

kinase inhibitors in cancer treatment. Expert Opin. Drug Metab. Toxicol.

2010, 6, 1175–1193.

(3) Belani, C. P. The role of irreversible EGFR inhibitors in the

treatment of non-small cell lung cancer: overcoming resistance to

reversible EGFR inhibitors. Cancer Invest. 2010, 28, 413–423.

(4) Chico, L. K.; Van Eldik, L. J.; Watterson, D. M. Targeting protein

kinases in central nervous system disorders. Nat. Rev. Drug Discovery

2009, 8, 892–909.

(5) Martinez, A.; Castro, A. Inhibition of tau phosphorylation: A new

therapeutical strategy for the treatment of Alzheimer’s disease and

other neurodegenerative disorders. Expert Opin. Ther. Pat. 2000, 10,

1519–1527.

- M (nonselective) muscarinic receptor:⁶⁵From rat cerebral cortex,

atropine was used as reference compound and [³H] QNB as a

ligand (0.05 nM).

- N (neuronal) (R-BGTX-insensitive) nicotinic receptor:⁶⁶From rat

cerebral cortex, nicotine was used as reference compound and [³H]

cytisine as a ligand (1.5 nM).

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cerebral cortex, R-bungarotoxin was used as reference compound

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S

Supporting Information. Poses for selected GSK-3 in-

b

hibitors upon docking to their targets; docked poses for

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Switching reversibility to irreversibility in glycogen synthase kinase 3 inhibitors: Clues for specific design of new compounds

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