Identification of novel scaffold of benzothiazepinones as non-ATP competitive glycogen synthase kinase-3β inhibitors through virtual screening

Article abstract of DOI:10.1016/j.bmcl.2012.09.043

Glycogen synthase kinase-3β (GSK-3β) is an important serine/threonine kinase that has been proved as a key target for neurodegenerative diseases and diabetes. Up to date, most of known inhibitors are bound to the ATP-binding pocket of GSK-3β, which might lead widespread effects due to the high homology between kinases. Recently, some of its non-ATP competitive inhibitors had been confirmed having therapeutical effects owing to their high selectivity. This finding opens a new pathway to study hopeful drugs for treatment of these diseases. However, it is still a challenge nowadays on how to efficiently find non-ATP competitors. Here, we successfully discovered a novel scaffold of benzothiazepinones (BTZs) as selective non-ATP competitive GSK-3β inhibitors through virtual screening approach. A 3D receptor model of substrate binding site of GSK-3β was constructed and applied to screen against drug-like Maybridge database through Autodock program. BTZ compounds were top ranked as efficient hits and were then synthesized for further screening. Among them, the representative compound 4j showed activity to GSK-3β (IC₅₀: 25 μM) in non-ATP competitive mechanism, and nearly no inhibitory effect on other 10 related protein kinases. Overall, the results point out that BTZ compounds might be useful in treatment of Alzheimer's disease and diabetes mellitus as novel GSK-3β inhibitors. It also suggests, on the other hand, that virtual screening would provide a valuable tool in combination with in vitro assays for the identification of novel selective and potent inhibitors.

Full text of DOI:10.1016/j.bmcl.2012.09.043

Bioorganic & Medicinal Chemistry Letters 22 (2012) 7232–7236
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification of novel scaffold of benzothiazepinones as non-ATP
competitive glycogen synthase kinase-3b inhibitors through virtual screening
Peng Zhang a, , Hai-Rong Hu b, , Zhao-Hui Huang a, Jia-Yi Lei a, Yong Chu a, , De-Yong Ye a,
⇑
⇑
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Rd., Shanghai 201203, China
b Key Laboratory of State Genetics Engineering, School of Life Sciences, Fudan University, 220 Handan Rd., Shanghai 200433, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 May 2012
Revised 28 August 2012
Accepted 14 September 2012
Available online 23 September 2012
Glycogen synthase kinase-3b (GSK-3b) is an important serine/threonine kinase that has been proved as a
key target for neurodegenerative diseases and diabetes. Up to date, most of known inhibitors are bound
to the ATP-binding pocket of GSK-3b, which might lead widespread effects due to the high homology
between kinases. Recently, some of its non-ATP competitive inhibitors had been confirmed having ther-
apeutical effects owing to their high selectivity. This finding opens a new pathway to study hopeful drugs
for treatment of these diseases. However, it is still a challenge nowadays on how to efficiently find non-
ATP competitors. Here, we successfully discovered a novel scaffold of benzothiazepinones (BTZs) as selec-
tive non-ATP competitive GSK-3b inhibitors through virtual screening approach. A 3D receptor model of
substrate binding site of GSK-3b was constructed and applied to screen against drug-like Maybridge data-
base through Autodock program. BTZ compounds were top ranked as efficient hits and were then synthe-
sized for further screening. Among them, the representative compound 4j showed activity to GSK-3b
Keywords:
GSK-3b
Kinase inhibitor
Virtual screening
Non-ATP competitive
Benzothiazepinones
(IC50: 25 lM) in non-ATP competitive mechanism, and nearly no inhibitory effect on other 10 related pro-
tein kinases. Overall, the results point out that BTZ compounds might be useful in treatment of Alzhei-
mer’s disease and diabetes mellitus as novel GSK-3b inhibitors. It also suggests, on the other hand,
that virtual screening would provide a valuable tool in combination with in vitro assays for the identifi-
cation of novel selective and potent inhibitors.
Ó 2012 Elsevier Ltd. All rights reserved.
Glycogen synthase kinase-3b (GSK-3b) is an important serine/
threonine protein kinase and has been confirmed playing a critical
role in two serious diseases as type 2 diabetes mellitus (T2DM) and
Alzheimer’s disease (AD). In PI3K pathway, GSK-3b controls many
important functions conducted by insulin, such as glycogen syn-
thesis, transport and uptake of glucose, gluconeogenesis and adi-
pogenesis. As two important substrates of GSK-3b, both insulin
receptor substrate-1 (IRS-1) and glycogen synthase (GS) mediate
the main process of glycogen metabolism in insulin signaling act-
ing in upstream and downstream, respectively. Once GSK-3b is
abnormally much active, GS activity will be inhibited directly
and distinctly, and then the normal process of glycogen synthesis
will slow down, which consequently lead to the elevation of the
blood sugar concentration.1 The inhibition of IRS-1 occurs at the
same time and lets the response ability of the receptors to insulin
be markedly reduced.2 On the other hand, GSK-3b is also the key
enzyme in AD cascade by regulating both the total tau level and
its phosphorylation. Highly active GSK-3b not only exceedingly
phosphorylates protein tau but also leads to the overproduction
of b-Amyloid (Ab) by phosphorylation of amyloid precursor protein
(APP) in the meantime.3 Among the whole process, increased pro-
duction of Ab is the most critical point since Ab can in return fur-
ther activate GSK-3b. The higher activation of GSK-3b will then
abnormally speed up the phosphorylation of tau to a large extent.
As the result, excessive phosphorylated protein tau accumulates to
form AD.4 It has been proved that the accumulation of oligomeric
Ab and hyper-phosphorylation of tau is just the major factors to in-
duce the damages of cholinergic neurons and the representative
cognitive impairments in AD patients.5
Nowadays more and more evidences have been provided that
GSK-3b inhibitors are very powerful in treatment of T2DM and
AD. They have some advantages for treating T2DM. GSK-3b inhib-
itors generally have good hypoglycemic effect, and usually produce
many other positive effects, such as promoting insulin release,
stimulating glucose uptake and improving the sensitivities of cells
to insulin.6 Besides, they normally lead to few of the following
events as weight gain, accumulation of fat tissue, and increase of
blood triglyceride and cholesterol content.7 As for treating AD,
GSK-3b inhibitors also show a lot of positive effects. They are very
effective to reduce the production of Ab,8 prevent cells from death
and thus alleviate external behavior damages to some extent.9 And
the most encouraging thing is that the close internal link between
⇑
Corresponding author. Tel./fax: +86 21 51980125.
E-mail addresses: cy110@fudan.edu.cn (Y. Chu), dyye@shmu.edu.cn (D.-Y. Ye).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.09.043

P. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7232–7236
7233
R
N
R
O
O
X
S
N
S
O
R'
N
N
H
HMKs
IC50=1-5µM
TDZDs
IC50=2-10µM
OH
N
HO
N
O
O
O
Manzamine A
IC50=10.2µM
Palinurin
IC50=4.5µM
O
HO P
HO
O
O
N
O
O
N
O
O
H
O
N
O
H
N
H
N
N
N
N
O
N
N
H
N
N
H
O
H
H
O
O
O
O
OH
O
O
NH2
L803-mts
NH2
IC50=40µM
Figure 1. Structures of reported non-ATP competitive GSK-3b inhibitors.
S
S
S
S
S
N
S
N
S
S
N
H
N
O
O
O
O
score rank
2
3
4
16
Figure 2. Hits of benzothiazepinones analogues by virtual screening.
Figure 4. Amino acid residues with solvent-accessible surface involved in substrate
binding pocket within 6 Å are shown. The compound 4h was in ball-and-stick
model, and the amino acid residues were in line form. The H-bond interactions
between titled compound and the receptor were shown in green dashed line.
Figure 3. The docked 4h and BIO in the binding pocket. The molecule in green stick
is the ATP-competitive ligand BIO; that colored in atom type is the compound 4h.

7234
P. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7232–7236
R1
O
R1
S
S
a
b
c
R1
R1 CHO
COOH
N
N
H
R2
O
1
2
3
4
Scheme 1. Reagents and conditions: (a) CH2(COOH)2, pyridine, piperidine, 2 h, reflux, 69–91%; (b) 2-aminobenzenethiol, 4 Å molecular sieve, 6 h, 180 °C, 25–66%; (c) R2X,
X = Cl, Br, I, NaH, DMF, 0.5 h, À10 °C, 11–86%.
the pathogeneses of T2DM and AD is being revealed and becoming
Table 1
more and more clear in recent years.10 That may partly explain
GSK-3b inhibitory activities of BTZs
why AD has even been referred to type 3 diabetes.11 So, if it is final-
R1
ly identified that GSK-3b inhibitors truly have a kind of unusual
collaborative effects on these two diseases, it is really an exciting
progress just like killing two birds with one stone.
S
N
O
R2
However, though GSK-3b inhibitors have so many good activi-
ties as mentioned above, the risk for side effects really exists and
cannot be ignored. As many kinase inhibitors are, the most known
GSK-3b inhibitors act to the enzyme’s ATP-binding site,12 which is
highly conserved in more than 500 kinases. As a result, these ATP
competitive inhibitors sometimes should offer adverse effects in
a potential chronic treatment.13 So it is quite an encouraging
advance that non-ATP competitive GSK-3b inhibitors be developed,
which paves a new way for getting more promising drugs to T2DM
and AD7a. During the past ten years, five families of non-ATP com-
petitive GSK-3b inhibitors have been reported (Fig. 1). Among
them, thiadiazolidinones (TDZD) is the first one reported by Marti-
nez et al. at 200214 and following confirmed to have both good
selectivity and excellent therapeutical effects on AD.15 Tideglusib
(NP-12), a member of TDZD, is undergoing Phase II b clinical trials
both on AD and orphan tauopathy in Europe now and it is also the
only one as new GSK-3b inhibitor under clinical trials so far in the
world. Another family is halomethylketones (HMK), but its future
of being developed into a real drug has been denied since their
inhibitory activity is very strongly irreversible to enzyme.16 In
addition, the small peptide L803-mts, a kind of substrate compet-
itive inhibitor, has proven to be effective in vivo on T2DM and neu-
rological diseases.2 Lately two marine natural products of alkaloid
manzamine A and sesquiterpene palinurin are reported to well
decrease tau phosphorylation as cell permeable non-ATP competi-
tive inhibitors,17 but the further studies for them were greatly
limited by their chemical structures.
So, it is still urgent to explore more GSK-3b inhibitors acting as
non-ATP competitive with high selectivity. There are at least two
key problems to be concerned about for such type of inhibitors.
The first one is the application of appropriate methods for finding
such type of leads. The widely used high throughput screening
(HTS) is yet not an appropriate tool for finding non-ATP competi-
tive compounds because the affinity of binding to the ATP outside
area is mostly weaker than in ATP-binding site, and the activities of
such type of inhibitors are generally as low as micromolar level,
thus the potential hits are very easily to be abandoned off in
screening.18 Actually, no one of the non-ATP competitive inhibitors
mentioned above is found through HTS. The other problem is about
how to keep the action mode in chemical modifications, which is
due to the fact that the action mode of non-ATP type would often
shift to ATP competitive in structure modification. All the TDZD
analogues modified by Kang’s group had been verified to be ATP
competitive inhibitors rather than expected non-ATP competitive
ones in his case.19
Compound
R1
R2
IC50
(l
M)a
4a
4b
4c
4d
4e
4f
4g
4h
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
2-Furyl
2-Furyl
2-Furyl
2-Furyl
Ph
H
Me
830
480
>100
>100
47.5
>100
>100
77.2
27.0
25.0
1.4
Et
iPr
Bn
Me
iPr
Bn
3-Cl-Bn
2-NO2-Bn
4i
4j
TDZD-8b
a
IC50, the mean value of at least two separate determinations, each determina-
tion were means of triplicate experiments.
TDZD-8, the first reported non-ATP competitive GSK-3b inhibitor, was used as
reference compound in this study.
b
found by this approach. However, to our best knowledge, there is
not yet any report on discovering GSK-3b inhibitors with non-
ATP competitive action mode through VS up to now.
Here, we successfully discovered a novel scaffold of benzothiaz-
epinones (BTZs) as selective non-ATP competitive GSK-3b inhibi-
tors through virtual screening approach. First, a 3D receptor
model of substrate binding pocket of GSK-3b was particularly
constructed and applied to screen against drug-like compounds
library. As the result several BTZ compounds were top ranked as
the effective hits, and some their analogues were then synthesized.
Among them, the representative compound 4j acted in a non-ATP
competitive manner to GSK-3b with IC50 value of 25 lM, which is
better than L803-mts. Moreover, it nearly did not show any inhibi-
tion on other 10 related protein kinases involved in our tests.
The computational search was performed using receptor
structure-based screening by docking with Autodock 3.0.5.21 Of
its three different search algorithms, the Lamarckian Genetic algo-
rithm (LGA) was finally used to do the docking since the other two
(simulated annealing and genetic algorithm) showed less efficient
in preliminary experiments. LGA is an optimization algorithm for
docking flexible ligands into protein binding sites to explore the
full range of ligand conformational flexibility with the rigid
protein,20 utilizing Lamarckian notation that an adaptations of an
individual to its environment can be inherited by its offspring.
The grid maps representing the ligand were calculated with Auto
grid. The dimensions of the grid were 60 Â 60 Â 60 grid points
with a spacing of 0.375 Å between the grid points and centered
on the ligand. The X-ray crystal structure of GSK-3b kinase (PDB
code: 1UV5) was selected as the model of the receptor and the
supposed binding pocket of TDZD composed of Arg 96, Lys 205
and Tyr 216, as screening receptor model since there are still no
As a computational counterpart to HTS, virtual screening (VS) is
developed lately to make up for the deficiencies. VS approach has
some obvious advantages in hits finding such as fast and good
cost-effective.20 A number of new inhibitors have already been

P. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7232–7236
7235
Figure 5. Double-reciprocal plot of kinetic data about assays of GSK-3b protein kinase activity at different concentrations of SB 216763, a known ATP competitive GSK-3b
inhibitor as reference inhibitor (a), and BTZ inhibitors 4h, 4i and 4j (b, c and d, respectively). ATP concentrations in the final reaction mixture varied from 0.5 to 8 M. The
l
concentration of GS-2 was kept constant in all of experiments at 6.25
lM. Compound concentrations were depicted in the plot.
Table 2
any reports on the crystal complex of non-ATP competitive inhib-
itors to GSK-3b, and TDZDs are still the best ones so far. Maybridge
database was prefiltered before screening according to the rules of
lead-likeness.
These jobs were run on a Thinkpad 64-bit 32-processor LINUX
cluster and totally 150,000 drug-like molecules were submitted
to be screened. The average docking cost about 25 min. For all doc-
kings, one hundred independent runs with step sizes of 0.2 Å for
translations, and 5 Å for orientations and torsions, an initial popu-
lation of random individuals with a population size of 150 individ-
Inhibitory activity (% inhibition) of compound 4j (100 lM) against several protein
kinases
Protein kinases
% Inhibitory
Protein kinases
% Inhibitory
Flt-1a
21.8
9.9
À133.3
55.2
0
ErbB2b
ErbB4b
EPH-A2c
Ablc
0
0
0
74.4
0
KDRa
PDGFR-ba
RETa
EGFRb
RONd
a
Su11248 as reference inhibitor for Flt-1 (87.1% inhibitory), KDR (89.7% inhibi-
tion) and PDGFR-b (82.1% inhibition).
b
uals,
a
maximum number of 1.5 Â 106 energy evaluations,
BIBW2992 as reference inhibitor for EGFR (86.9% inhibition), ErbB2 (79.4%
maximum number of generations of 37,000, an elitism value of 1,
and a number of active torsion of 9 were used. All other run param-
eters were kept at their default settings. Final docked conforma-
tions were clustered using a tolerance of 2 Å root mean square
deviations (RMSD). After carefully visual inspection of their poses,
the top 20 compounds greatly matched were identified as reason-
able hits from 100 top dockings ranked with the lowest binding
free energy.
inhibition) and ErbB4 (82.8% inhibition).
c
Dasatinib as reference inhibitor for EPH-A2 (83.1% inhibition), Abl (90.0%
inhibition).
d
PD173074 as reference inhibitor for RON (93.4% inhibition).
study, the predicted active cavity was composed of four amino acid
residues as Phe67, Phe93, Arg 96, Lys205, which is slightly different
to the VS model (Fig. 4).
Finally, ten derivatives of 2,3-dihydro-1,5-benzothiazepin-
4(5H)-one were successfully synthesized through three steps as
Knoevenagel reaction, cyclization and N-alkylation (Scheme 1).23
Kinase-Glo™ luminescent kinase assays were performed to
investigate inhibitory potency of these synthesized BTZs.24 IC50
values of each compound were summarized in Table 1. It could
be seen from these data that the bulk aryl moieties at 5-position
of BTZ might be very important for remaining the inhibitory activ-
ity. Introduction of benzyl moiety to that site makes the activity in-
creased (4e, 4h, 4i and 4j), while compounds with hydrogen atom
or alkyl substituents at 5-position almost show no activity (4a, 4b,
4c and 4d).
To delineate the inhibitory mechanism of compounds with most
potent inhibitory activity (4h, 4i and 4j), kinetic experiments were
carried out under different concentrations of ATP and tested
compounds. Double-reciprocal plot of kinetic profiles are shown
in Figure 5. All of three compounds are demonstrated to be a typical
non-competitive inhibition against phosphorylation substrate ATP
Among them, BTZ analogues aroused our great interest due to
their high-rankings and novel structure as GSK-3b inhibitor
(Fig. 2). It was noteworthy that if there was no methyl group at-
tached at the nitrogen atom, the ranking of hit sharply dropped
to 16th. This result suggested that the substituents at nitrogen
atom might have great impact on the activity or selectivity of the
molecule. In order to verify this assumption, various alkyl and aryl
groups as methyl, isopropyl and benzyl were introduced to the
nitrogen atom of BTZ, respectively. Besides, the furyl group was
also replaced by a thienyl group as its bioisostere.
Before making the synthesis, the binding affinities of these
designed compounds were checked using the VS model. One
known ATP-competitive compound BIO was also docked to this
model as
a
reference.22 In the result, these designed BTZ
compounds could indeed bind to the substrate area while BIO
bound to the ATP site (Fig. 3). In a typical sample, the binding affin-
ity of compound 4h was predicted to be about À7.82 kcal molÀ1
,
and its inhibitory activity to be about 1.86 lM. In this docking

7236
P. Zhang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 7232–7236
4. Martin, L.; Magnaudeix, A.; Esclaire, F.; Yardin, C.; Terro, F. Brain Res. 2009,
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5. Takashima, A. J. Alzheimers Dis. 2006, 9, 309.
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M. Nat. Rev. Drug Disc. 2004, 3, 479; (c) Wagman, A. S.; Johnson, K. W.; Bussiere,
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according to these data, indicating that BTZ derivatives do not share
binding pocket of enzyme with ATP. With increasing concentrations
of ATP, the compounds display intersection of same Km and differ-
ent Vmax values. Of them, the representative compound 4j was fur-
ther assayed against a panel of other 10 related kinases and showed
high selectivity to GSK-3b over these enzymes (Table 2).
8. (a) Leclerc, S.; Garnier, M.; Hoessel, R.; Marko, D.; Bibb, J. A.; Snyder, G. L.;
Greengard, P.; Biernat, J.; Wu, Y. Z.; Mandelkow, E. M. J. Biol. Chem. 2001, 276,
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Chen, P.; Ubeda, O. J. Neurobiol. Dis. 2009, 33, 193.
10. Han, W.; Li, C. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 6557.
11. Steen, Eric; Terry, Benjamin M.; Rivera, Enrique J.; Cannon, Jennifer L.; Neely,
Thomas R.; Tavares, Rose; Julia Xu, X.; Wands, Jack R.; de la Monte, Suzanne M.
J. Alzheimers Dis. 2005, 7, 63.
12. (a) Eglen, R. M.; Reisine, T. Assay Drug Dev. Technol. 2009, 7, 22; (b) Cohen, P.;
Goedert, M. Nat. Rev. Drug Disc. 2004, 3, 479.
As summarized, we built a supposed non-ATP binding pocket as
screening receptor model and employed it to virtually screen out
potential ligands against GSK-3b. In this approach, Autodock pro-
gram was used to perform the screening from a drug-like chemi-
cals library, and the hits were expected to be capable of acting as
non-ATP-competitive mechanism. Successfully a novel scaffold of
BTZ was synthesized and confirmed by kinetic analysis to be in-
deed a kind of non-ATP competitive inhibitor of GSK-3b. The repre-
sentative compound 4j shows inhibitory activity against GSK-3b
with an IC50 value of 25 lM, and little inhibition on other 10 re-
13. Martinez, A.; Castro, A.; Dorronsoro, I.; Alonso, M. Med. Res. Rev. 2002, 22, 373.
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lated protein kinases. These results suggest BTZ compounds might
have expectative selectivity over enzymes. Since GSK-3b inhibitors
with that kind of structure have never been reported before, these
BTZ compounds are much promising candidates worthy of further
studies due to the advantages of non-ATP mechanism for activity
and selectivity. On the other hand, our work has also showed that
VS approach would be a useful tool in primarily search for novel
scaffolding structures especially when HTS is not suitable to find-
ing specific ligands.
15. (a) Kaidanovich-Beilin, O.; Eldar-Finkelman, H. J. Pharmacol. Exp. Ther. 2006,
316, 17; (b) Medina, M.; Castro, A. Curr. Opin. Drug Disc. 2008, 11, 533.
16. (a) Conde, S.; Pérez, D. I.; Martínez, A.; Perez, C.; Moreno, F. J. J. Med. Chem.
2003, 46, 4631; (b) Perez, D. I.; Conde, S.; Pérez, C.; Gil, C.; Simon, D.;
Wandosell, F.; Moreno, F. J.; Gelpí, J. L.; Luque, F. J.; Martínez, A. Bioorg. Med.
Chem. 2009, 17, 6914; Perez, D. I.; Palomo, V.; Prez, C.; Gil, C.; Dans, P. D.;
Luque, F. J.; Conde, S.; Martinez, A. J. Med. Chem. 2011, 54, 4042.
17. (a) Hamann, M.; Alonso, D.; Martín-Aparicio, E.; Fuertes, A.; Pérez-Puerto, M. J.;
Castro, A.; Morales, S.; Navarro, M. L.; del Monte-Millán, M.; Medina, M. J. Nat.
Prod. 2007, 70, 1397; (b) Alonso, D.; Martinez, A. Glycogen Synthase Kinase 3
(GSK-3) and Its Inhibitors; John Wiley & Sons: Hoboken, NJ, USA, 2006. pp 307–
331.
Acknowledgments
18. Eldar-Finkelman, H.; Licht-Murava, A.; Pietrokovski, S.; Eisenstein, M. Biochim.
Biophys. Acta 2010, 1804, 598.
We gratefully acknowledge the financial support by Specialized
Research Fund for the Doctoral Program of Higher Education of
China (Grant No. 20070246089), the Shanghai Committee of
Science and Technology of China (Grant No.10ZR1401800), and
National Drug Innovative Program (Grant No. 2009ZX09301-011).
Thanks are due to Professor Jianzhong Lu (Department of Pharma-
ceutical Analysis, School of Pharmacy, Fudan University) and to
Professor Meiyu Geng (Division of Antitumor Pharmacology, State
Key Laboratory of Drug Research, Shanghai Institute of Materia
Medica) for assistance with the bioassay.
19. Kang, N. S.; Lee, G. N.; Kim, C. H.; Bae, M.; Kim, I.; Cho, Y. S. Bioorg. Med. Chem.
Lett. 2009, 19, 533.
20. Schneider, G. Nat. Rev. Drug Disc. 2010, 9, 273.
21. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
22. Meijer, L.; Skaltsounis, A. L.; Magiatis, P.; Polychronopoulos, P.; Knockaert, M.;
Leost, M.; Ryan, X. P.; Vonica, C. A.; Brivanlou, A.; Dajani, R., et al Chem. Biol.
2003, 10, 1255.
23. (a) Levai, A.; Duddeck, H. Pharmazie 1983, 38, 827; (b) Lancelot, J. C.; Letois, B.;
Saturnono, C.; De Caprariis, P.; Robba, M. Org. Prep. Proced. Int. 1992, 24, 204;
(c) Physical and spectroscopic data for compound 4j: white solid, yield: 75%;
mp 186.7–189.5 °C; 1H NMR (400 MHz, CDCl3) d 8.24–6.88 (m, 13H), 5.54 (d,
J = 25.7 Hz, 2H), 5.06–4.73 (m, 1H), 3.15–2.74 (m, 2H); 13C NMR (100 MHz,
CDCl3) d 171.0, 148.1, 145.8, 143.6, 136.8, 133.7, 132.7, 130.7, 129.6, 128.8,
128.0, 127.8, 127.4, 127.1, 126.1, 125.0, 123.4, 52.9, 50.9, 49.5, 42.1, 29.7; ESI-
MS: 390.9 (M+1)+.
Supplementary data
24. Polgár, T.; Baki, A.; Szendrei, G. I.; Keseruu, G. M. J. Med. Chem. 2005, 48, 7946.
The measurement of GSK-3b inhibition was performed in assay buffer using
transparent 96-well plates according to the Kinase-Glo assay method of Baki.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
09.043.
In
(dissolved in DMSO) was diluted by 14
enzyme solution were added to each well followed by 20
containing 12.5 M substrate and 4 M ATP. After 30 min of incubation at
30 °C, the enzymatic reaction was stopped with 40 L of Kinase-Glo reagent.
a
typical assay,
4
lL
of different concentration compound of interest
L of assay buffer, and 2 L (20 ng) of
L of assay buffer
l
l
l
l
l
References and notes
l
Glow-type luminescence was recorded after 10 min. 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 IC50 value was defined as the concentration of each
compound that reduces 50% the enzymatic activity with respect to that
without inhibitors..
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