The marine natural-derived inhibitors of glycogen synthase kinase-3β phenylmethylene hydantoins: In vitro and in vivo activities and pharmacophore modeling

Article abstract of DOI:10.1016/j.bmc.2009.06.054

The Red Sea sponge Hemimycale arabica afforded the known (Z)-5-(4-hydroxybenzylidene)-hydantoin (1). This natural phenylmethylene hydantoin (PMH) 1 and the synthetic (Z)-5-(4-(ethylthio)benzylidene)-hydantoin (2) showed potent in vitro and in vivo anti-gr

Full text of DOI:10.1016/j.bmc.2009.06.054

Bioorganic & Medicinal Chemistry 17 (2009) 6032–6039
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
The marine natural-derived inhibitors of glycogen synthase kinase-3b
phenylmethylene hydantoins: In vitro and in vivo activities
and pharmacophore modeling
*
Mohammad A. Khanfar, Bilal Abu Asal, Mudit Mudit, Amal Kaddoumi, Khalid A. El Sayed
Department of Basic Pharmaceutical Sciences, College of Pharmacy, University of Louisiana at Monroe, 1800 Bienville Drive, Monroe, LA 71201, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The Red Sea sponge Hemimycale arabica afforded the known (Z)-5-(4-hydroxybenzylidene)-hydantoin
(1). This natural phenylmethylene hydantoin (PMH) 1 and the synthetic (Z)-5-(4-(ethylthio)benzyli-
dene)-hydantoin (2) showed potent in vitro and in vivo anti-growth and anti-invasive properties against
PC-3M prostate cancer cells in MTT, spheroid disaggregation, and in mice models. To explore a possible
molecular target of PMHs, the most potent synthetic analogue 2 has been virtually screened against var-
ious protein kinases. Molecular modeling study has shown that 2 can be successfully docked within the
binding pocket of glycogen synthase kinase-3b (GSK-3b) similar to the well-known GSK-3b inhibitor I-5.
Received 30 April 2009
Revised 22 June 2009
Accepted 23 June 2009
Available online 27 June 2009
Keywords:
Marine natural products
Phenylmethylene hydantoins
Synthase kinase-3b (GSK-3b)inhibitors
Pharmacophore modeling
Molecular modeling
Cancer
Several PMHs showed potent in vitro GSK-3b inhibitory activity with an IC50 range of 4–20 lM. The most
potent analogue 3 showed a significant increase in liver glycogen level at the 5, 15, and 25 mg/kg dose
levels, in vivo. Pharmacophore model was built and validated using in-house database of active and inac-
tive GSK-3b inhibitors. The GSK-3b inhibitory activity of PMHs entitles them to be potential leads for the
treatment of cancer, Alzheimer’s disease, bipolar disorders, stroke, different tau pathologies, and type-2
diabetes.
Alzheimer’s disease
Type-2 diabetes
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
tion, anti-arrhythmic and antihypertensive, anti-diabetic, neuro-
protective, HDL/cholesterol modulating, antiviral, and growth
The sponge genus Hemimycale (family Mycalidae) is well known
for its bioactive secondary metabolites especially biogenetically
complex guanidine alkaloids.1–3 Ptilomycalin A1 has a unique poly-
cyclic guanidine skeleton with a spermidine group linked to a 16-
hydroxyhexadecanoic acid moiety.1–3 The ethanolic extract of the
abundant shallow water Red Sea sponge Hemimycale arabica was
targeted because it inhibited the proliferation and invasion of the
highly metastatic human prostate cancer PC-3M cell line.4,5 The
natural (Z)-5-(4-hydroxybenzylidene)-hydantoin (PMH, 1) and
the synthetic (Z)-5-(4-(ethylthio)benzylidene)-hydantoin (2)
showed potent in vitro anti-growth and anti-invasive properties
against PC-3M prostate cancer cells in MTT, and spheroid disaggre-
gation assay.4,5 They decreased the orthotopic tumor growth and
inhibited the formation of tumor micrometastases in distant or-
gans without apparent cytotoxic effects at the test doses.5
hormone secretagogue.7
GSK-3b, also called tau phosphorylating kinase I is a serine/
threonine kinase implicated in the control of several regulatory
proteins.8,9 It was first discovered by virtue of its ability to phos-
phorylate and inactivate glycogen synthase, the regulatory enzyme
of mammalian glycogen synthesis.10 Its pleiotropic but unique
activities have made GSK-3b a favorite target for the treatment of
several human diseases such as type-2 diabetes,11 Alzheimer’s dis-
ease (AD),12 CNS disorders like manic depressive disorder and neu-
rodegenerative diseases,13 and chronic inflammatory disorders.14
The search for GSK-3b inhibitors became a very active research
trend for academic centers and pharmaceutical industry. Several
structurally diverse compounds were reported to inhibit GSK-3b.
Examples of these are thiadiazolidindiones (TDZD), hydantoins, tri-
azoles, thiazoles, maleimides, dithiazolidindiones, and pyrazole-
pyridines.15–26
Marine natural products from sponges, ascidians, and gastropod
mollusks have played a central role in providing novel GSK-3b
inhibitors. Examples of these active marine-derived compounds
are palinurin, tricantin, hymenialdisine, meridianines, manzamine
A, and indirubines.22–24 The marine environment represents an
enormous resource for the discovery of potential GSK-3b inhibi-
tors.22–24
The cyclic imide hydantoins are well investigated for their anti-
convulsant activity.6,7 Various pharmacological activities were re-
ported for hydantoins including fungicidal, herbicidal, anti-
inflammatory, anti-HIV, analgesic, cannabinoid receptor-1 (CB-1),
5HT, purine P-2X receptor antagonism, platelet aggregation inhibi-
* Corresponding author. Tel.: +1 318 342 1725; fax: +1 318 342 1737.
E-mail address: elsayed@ulm.edu (K.A. El Sayed).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.054

M. A. Khanfar et al. / Bioorg. Med. Chem. 17 (2009) 6032–6039
6033
NH
O
The well-known protein substrate of GSK-3b is the microtu-
bule-associated protein tau. GSK-3b phosphorylates tau at the spe-
cific sites; serine residue 199 and serine residue 396.26
Deregulation of GSK-3b activity is implicated in the pathophysiol-
ogy of AD by regulating microtubule stability and phosphorylating
the microtubule associating protein, tau.27,28 Abnormal hyper-
phosphorylation of tau and not its aggregation into filaments con-
sidered an essential key step in neurofibrillary degeneration.27,28
GSK-3b has been reported to play a role in the toxic effect medi-
ated by b-amyloid, a protein that aggregates as extracellular amy-
loid plaques in brain of AD patients.29 Exposure of cortical and
hippocampal primary neuronal cultures to b-amyloid induces acti-
vation of GSK-3b, tau hyperphosphorylation, and cell death.30
Therefore, inhibition of GSK-3b controls tau phosphorylation level
in cells overexpressing tau protein in a dose-dependent manner.31
Blockade of GSK-3b expression by antisense oligonucleotides or its
activity by lithium addition inhibits b-amyloid-induced neurode-
generation of cortical and hippocampal primary cultures.32–37
Therefore, GSK-3b inhibition is therapeutically important for sev-
eral neurodegenerative diseases including AD.
O
O
H
O
NH
O
HN
NaHCO3 /Aq. EtOH
Ethanolamine
HN
+
R
R
Reflux
H
Scheme 1. General synthetic scheme of phenylmethylene hydantoins.6
8 showed the stretching vibration bands at a higher frequency re-
gion (1660–1675 cmꢀ1) characteristic for Z-oriented C@C bonds,
which are distinguishable from those expected for the E isomers
(1630–1640 cmꢀ1).6 The chemical shift of the most diagnostic ole-
finic proton H-6 of 1–8 was downfield shifted in the 1H NMR spec-
tra (dH 6.40–7.00), which further confirmed the Z-orientation of the
D5,6 system. The expected chemical shift of H-6 in the E isomers
should be 6.20–6.30 ppm.6 The downfield shift of H-6 in the Z-iso-
mer is attributed to the anisotropic effect of the spatially nearby C-
4 carbonyl group.6 Different electronic (
r) and lipophilic (p) sub-
stituents were varied at the aromatic ring of PMH to probe the
chemical space around the aromatic ring and its correlation with
the activity (Table 1).
In an effort to explore possible molecular target(s) of PMHs,
compound 2, the most potent anti-metastatic analogue against
PC-3M, has been virtually screened versus several kinases (AKB,
CDK1, CDK2, CDK5, PKA, PKC, EGFR, PDGFR, ERK, MEK1, PI-3 ki-
nase, MAPKK, and GSK-3b) available at the RCSB Protein Data Bank
(PDB) using SURFLEX DOCK Program implanted in SYBYL 8.0 package.
Interestingly, 2 shows structure similarity and binding orientation
to I-5 (Fig. 1), a potent and selective GSK-3b inhibitor.38,39 Both I-5
and 2 showed high binding scores at the GSK-3b’s ATP binding site.
Therefore, various PMH analogues were selected for in silico
screening to explore their possible GSK-3b binding affinity. In vitro
and in vivo testing further supported the in silico data. Conse-
quently, a pharmacophore model was established to understand
the essential features required for GSK-3b binding and to discover
and design new analogues that could be used therapeutically. This
study reports the identification and characterization of PMHs as a
potent class of GSK-3b inhibitors.
The HREIMS data of 5 showed a molecular ion peak at m/z
234.0463, suggesting the molecular formula C11H10N2O2S. Analysis
of 1H and 13C NMR data (Section 4) indicated that 5 is (Z)-5-(4-
(methylthio)benzylidene)-hydantoin. PMH 5 is closely related to
the known 2 with the replacement of the S-ethyl group with S-
methyl.4,5 The methyl singlet at d 2.48 was assigned as the S-
methyl functionality. This was based on its 3J-HMBC correlation
with the quaternary aromatic C-10 (d 139.7). Meanwhile, the aro-
matic doublet H-8/H-12 (d 7.56) showed 3J-HMBC correlations
with C-10 and with olefinic methane carbon C-6 (d 108.7). Detailed
1H, and 13C NMR, and other analytical data for PMHs 1–4 and 6–8
are included in the Supplementary data (Tables S1–S3).
2.2. Molecular docking studies
PMHs were docked into the ATP binding site of GSK-3b (PDB
code 1q41) using SURFLEX DOCK interface implemented into SYBYL
8.0.40–42 Surflex is a fully automatic flexible molecular docking
algorithm that combines the scoring function from the Hammer-
head docking system with a search engine that relies on a sur-
face-based molecular similarity method as a mean to rapidly
generate suitable putative poses for molecular fragments.40,41
2. Results and discussion
2.1. Chemistry
Six known (2–4 and 6–8) and one new (5) PMHs were synthe-
sized using Scheme 1.4,6 This scheme includes base-catalyzed con-
densation of hydantoin with substituted benzaldehydes.6
Geometrical isomerism (E/Z isomers) was possible due to the re-
stricted rotation around the exocyclic C@C double bond of the
PMHs, however this method regioselectively afforded the Z geom-
etry as confirmed by the analytical data. The IR spectra of PMHs 1–
Table 1
PMH structures and their in vitro GSK-3b inhibitory activities
H
N
O
3
O
HN1
6
8
A
B
12
R
10
H
N
O
No.
R
IC50 (lM) SEM
O
1
2
4-OH
4-SCH2CH3
13.7 1.2
7.8 0.7
4.2 0.4
14.1 1.9
6.9 0.9
13.5 1.1
6.4 0.6
18.2 2.0
12.3 1.3
HN
3
4
5
6
7
8
4-N(CH2CH3)2
3-OCH3
4-SCH3
2-Thiophene
4-Thiophene
4-Cl
Cl
HO2C
Cl
I-5
Manzamine Aa
—
Figure 1. (A) Molecular structure of I-5. (B) Compound 2 binds similarly to I-5 into
the ATP-binding side of GSK-3b.
Manzamine A was used as a positive control in the in vitro assay.24
a

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M. A. Khanfar et al. / Bioorg. Med. Chem. 17 (2009) 6032–6039
The corresponding interacting amino acids within the binding
using CHARMM. Molecular dynamic simulation using AMBER7 FF02
site of GSK-3b with structure I-5 are shown in Figure 2A. Com-
pound 3 showed the highest docking score. PMH 3 forms strong
interactions with the hinge region of GSK-3b; carbonyl oxygen at
position 2 form a H-bonding with backbone nitrogen of Val 135
and the NH at position 3 to the carbonyl oxygen of Asp 133
(Fig. 2B). The hydantoin ring was sandwiched between Ala 83, on
top, and Leu 188, on the bottom. The aromatic ring is rotated out
of plane from the hydantoin plane, allowing extensive interactions
with the nucleotide-binding loop. Furthermore, the aniline nitro-
gen of 3 builds a H-bonding interaction with the guanidine moiety
of Arg 141. Interestingly, these interactions are the same hot spots
provided by the co-crystallized ligand of I-5, potent and selective
inhibitor of GSK-3b (Fig. 2A and C). Moreover, the phenylmethyl-
ene moiety occupies a hydrophobic pocket assembled from Ile
62, Glu 63, and Val 70.
force field implemented in SYBYL 8.0 was subsequently conducted
in the presence of explicit solvent to investigate the stability of
H-bonding interactions described above. Figure 2D shows the opti-
mized 3-GSK-3b complex maintaining H-bonding interactions
with Asp 133, Val 135, and Arg 141 at optimum distance of 2.11,
1.67, and 3.13 Å, respectively. Although the later H-binding inter-
action is weaker than the electrostatic interaction created with I-
5, targeting Arg 141 is important to improve the activity in the pro-
cess of designing new derivatives because it is considered the
selectivity residue for GSK-3b.43,44 Many other kinases have either
neutral or negatively charged residues rather than the positively
charged guanidine at the same position.44 In CDK2, a kinase that
share 33% amino acid identity with GSK-3b, although it bears a
positively charged Lys 86 at that position but it is oriented away
from the ligand.45 Accordingly, Arg 141 is unique in GSK-3b and
provides an interesting model to explain the high GSK-3b selectiv-
ity observed for many inhibitors.45 This unique binding feature is
Since the receptor is fixed in docking, the generated PMH 3-
GSK-3b complex from docking simulation was pre-minimized
Figure 2. (A) Detailed view of the co-crystallized structure I-5 and the corresponding interacting amino acids within the binding site of GSK-3b. (B) Detailed view of the
docked 3 structure and the corresponding interacting amino acid moieties within the binding site of GSK-3b. (C) Compound 3 aligned with I-5 in the ATP binding site of GSK-
3b. (D) Detailed view of the docked 3 structure in different view angle in the ATP binding site of GSK-3b after the AMBER7 FF02 optimization.

M. A. Khanfar et al. / Bioorg. Med. Chem. 17 (2009) 6032–6039
6035
formed in the docked pose of 3, which may justify a possible high
PMH selectivity for GSK-3b versus other kinases.
sis of glycogen from
3b inhibitors may have a great therapeutic potential for lowering
blood glucose levels.
The most in vitro active PMH 3 was tested in Sprague Dawley
rat model to evaluate its in vivo potency by measuring the hepatic
glycogen disposition. Figure 3 shows the significant increase in
rats’ liver glycogen content at the three dose levels of 3 used in
the study (5, 15, and 25 mg/kg) compared to the vehicle control
group (P <0.05) in a dose-dependent manner.
D
-glucose.46 These findings indicate that GSK-
The validation for docking–scoring procedure was performed
through employing the same conditions to dock I-5 into the bind-
ing pocket of this enzyme (Fig. 2A). The docking simulation re-
sulted in a close model to the crystallographic structure (Fig. S3,
Supplementary data) highlights the potential and selectivity of
PMHs as GSK-3b inhibitors.
2.3. Monitoring GSK-3b activity using a tau [pS396]
phosphoELISA Kit
Compound 3 has been previously tested in vivo for its anticon-
vulsant activity using maximal electroshock seizure (MES) assay
for up to 200 mg/kg and did not show CNS-depressant effects.6 In
addition, a previous study showed the lack of cytotoxicity of 3 even
TM
A well-known protein substrate of GSK-3b is the microtubule-
associated protein tau. GSK-3b phosphorylates tau at the specific
sites: serine residue 199 and serine residue 396.26 BioSource has
at 200 l
M in vitro against prostate cancer cells.4 These in vitro and
in vivo assays provide a preliminary indication of the safety and
tolerability of this compound.
TM
recently introduced phosphoELISA kits for monitoring the phos-
phorylation of tau at serine 396 that have utility in monitoring
the activity of GSK-3b (BioSource; KHB7031). The Invitrogen Hu-
man Tau [pS396] kit is a solid phase sandwich Enzyme Linked-Im-
muno-Sorbent Assay (ELISA). To evaluate the inhibitory effect of
PMHs against GSK-3b, an in vitro GSK-3b inhibitory assay was con-
ducted. In this inhibitory assay, the concentration of PMH that
inhibits 50% of the enzyme, IC50, was measured. Table 1 shows
the in vitro GSK-3b inhibitory activities of PMH analogues. Consis-
tent with the in silico studies, PMH 3 shows the most potent inhi-
2.5. Pharmacophore model generation
3D pharmacophore mapping methodology based on distance
comparison technique is built for the three most active analogues
TM
(2, 3, and 7) using DISCOtech module implemented in SYBYL
8.0.47,48 DISCOtech is a well-established module in designing
TM
pharmacophoric map and in the process of virtual screening to dis-
cover new leads.49,50 Given a set of molecules that are related by
TM
bition with an IC50 value of 4.2
lM. The validity of the test was
their ability to bind to same protein receptor, DISCOtech identifies
established by testing the inhibitory action of the marine-derived
features that could be elements in a pharmacophore model.47,48
TM
GSK-3b inhibitor, manzamine, which showed an IC50 value of
DISCOtech operates in distance space and can perform clique
12.3
l
M that was comparable to the published data.24 Based on
detection to generate pharmacophore hypotheses on up to 300
conformers per molecule.47,48 Therefore, DISCOtech can be effi-
TM
the abovementioned results, PMH 3 was selected for in vivo activ-
ity evaluation in rat model for ability to enhance glycogen disposi-
tion in liver as a subsequent inhibition of GSK-3b.
ciently used with as low as 3–5 compounds to generate validated
pharmacophore models.49–52
These diverse conformers are used in DISCOtech’s clique detec-
tion routine to find 3D alignments of the pharmacophore features
in different molecules.47,48 A clique is a subgraph in which every
2.4. In vivo Sprague Dawley rat model. Determination of
hepatic glycogen contents
node is connected to each other’s node.47,48 DISCOtech reduces
TM
GSK-3b plays an important role in controlling the hepatic and
muscular glycogen content and blood glucose level.35,36 There are
different mechanisms by which GSK-3b accomplish this critical
function. GSK-3b phosphorylates and inactivates glycogen syn-
thase, the enzyme which converts excess glucose residues to glyco-
gen for storage. Knockdown action, by insulin or small inhibitors,
on GSK-3b results in increased glycogen storage in muscle and li-
ver.20–24 A previous study showed that inhibition of GSK-3b selec-
tively reduces glucose-6-posphatase and phosphoenolpyruvate
carboxykinase gene expression.46 Consequently, these effects re-
sult in the reduction of glucose output and increase of the synthe-
the conformers to sets of pharmacophore features (nodes) and
interfeature distances (connections).47,48
The criterion for the validation of the developed pharmaco-
phoric queries was based on in-house database of active and inac-
tive GSK-3b inhibitors (100 each). Representative examples from
both sets are included in the Supplementary data (Figs. S1 and
S2). Compound is considered active as GSK-3b inhibitor with
IC50 6 20 l
M.16–21 Model that filters the active and excludes inac-
tive compounds was selected and then optimized. Several initial
DISCO runs were performed by varying the tolerance and range
of required features. Table 2 shows the results of the initial efforts.
6
5
4
3
2
1
0
Control
5
15
25
Dose (mg/Kg)
Figure 3. Effect of 5, 15, and 25 mg/kg doses of 3 on the liver glycogen storage in Sprague Dawley rats (n = 3/dose). Error bars indicate the SEM of n = 3/dose.

6036
M. A. Khanfar et al. / Bioorg. Med. Chem. 17 (2009) 6032–6039
Table 2
Number of models obtained along with the pharmacophoric features and tolerance values for each of the DISCO pharmacophoric run
*
Selected by the pharmacophore model showing the highest score. The highlighted model used for further modification.
Except Run-2(b), all the models turned out to be nonspecific for
two reasons; either they have few (ꢁ4–5) features as in Run-
1(a)–Run-1(c) or high tolerance limit as in Run-1(c) and Run-
2(c). The highest score query generated from Run-2(b) and named
Mod-2(b)-1, which shows a slight selectivity, was selected for fur-
ther refinement (Fig. 4).
Mod-2(b)-1was optimized by adding and deleting some specific
features (Table 3). Adding donor atom feature (DA3) through
UNITY interface at the aniline N and removing DA1 and AA2 gener-
ated Mod-7 (Fig. 5). This query picked more number of GSK-3b
inhibitors and only a few numbers of inactive GSK-3b inhibitors.
This model is selective enough to pick a complex structure like
staurosporine, a potent natural GSK-3b inhibitor (IC50 50 nM), from
our in-house database (Fig. 5B). Mod-7 has the characteristic fea-
tures required for an ideal pharmacophoric query, because it pos-
sessed the important interactions required for this series of GSK-
3b inhibitors, worked consistently with published GSK-3b pharma-
cophore model, and performed satisfactorily with the in-house
database.20,51,52
with benzyl or phenethyl at C-12 can afford potent and selective
GSK-3b inhibitors.
3. Conclusion
Experimental in vitro and in vivo GSK-3b inhibitory activities of
PMHs were documented. The feasible, cost effective, and regiose-
lective synthesis of this class bode well for their future develop-
ment as potential therapeutics for cancers, Alzheimer’s disease,
Table 3
Models generated by modifying Mod-2(b)-1 along with percentage of hits picked by
each model from the in-house database
Model no.
Pharmacophoric
featuresa,b
Number of
active
Number of
inactive
GSK-3b hits (%)
GSK-3b hits (%)
Mod-1
Mod-2
Mod-3
Mod-4
Mod-5
Mod-6
Mod-7
Mod-8
DA2, AA1, AA2, AA3, HD1, HD2
DA1, DA2, AA2, AA3, HD1, HD2
DA1, AA1, AA2, AA3, HD1, HD2
DA1, DA2,AA1, AA3, HD1, HD2
DA2, AA2, AA3, HD1, HD2
AA1, AA2, AA3, HD1, HD2
78
63
61
79
72
79
89
58
11
15
11
10
8
8
3
14
Based on the results above, future design of potent and selective
GSK-3b inhibitors should consider the following important hot
spots: (i) H-bonding interaction with the hinge region of Asp 133
and Val 135, (ii) targeting Arg 141 and Gln 185 amino acids, and
(iii) filling the Val 70, Lys 85, and Cys 99 hydrophobic pocket. For
example, keeping the hydantoin ring, and placing carboxylate or
other negatively charged moiety at C-9 or C-10 positions, along
DA2, AA1, AA3, HD1, HD2
DA1, AA2, AA3, HD1, HD2
a
AA—acceptor atom, DA—donor atom, HYD—hydrophobic center.
The corresponding acceptor and donor sites are not tabulated.
b
TM
Figure 4. Pharmacophoric features of Mod-2(b)-1and their distance relation generated by DISCOtech module. AA—acceptor atom, DA—donor atom, HD—hydrophobic center.

M. A. Khanfar et al. / Bioorg. Med. Chem. 17 (2009) 6032–6039
6037
TM
Figure 5. The pharmacophoric features of Mod-7 generated by DISCOtech module along with PMH 3 (A) and with staurosporine (B), the potent GSK-3b inhibitor.
AA—acceptor atom, DA—donor atom, HD—hydrophobic center, DS—donor site, AS—acceptor site.
bipolar disorders, stroke, different tau pathologies, and type-2 dia-
betes. The validated pharmacophore model of PMH-derived GSK-
3b inhibitors provides a powerful tool to design new leads and to
discover virtual screening based-GSK-3b inhibitors from available
online databases.
iments were conducted at the University of Michigan on a
Micromass LCT spectrometer.
4.2. Preparation of phenylmethylene hydantoins
Hydantoin (1.0 g) was dissolved in 10 mL H2O while heating at
70 °C on oil bath with continuous stirring (Scheme 1).6 The pH was
adjusted to 7.0 using saturated NaHCO3 solution after complete
dissolution. The temperature was then raised to 90 °C after the
addition of 0.9 mL ethanolamine.6 Equimolar quantity of the corre-
sponding aldehyde solution in 2–5 mL EtOH was then added drop-
wise with continuous stirring.6 The reaction was kept under reflux
for approximately 5–8 h. The reaction was monitored by TLC every
hour till a yellow or white precipitate was formed. After complete
depletion of the starting aldehyde, the mixture was cooled and the
precipitate was filtered and washed with EtOH/H2O (1:5) before
recrystallization from EtOH.6 Reaction yield range from 60% to
90%, based on the nature of the used aldehyde. Generally with
few exceptions, it has been noticed that aromatic substitutions
4. Experimental
4.1. General experimental procedures
Melting points were determined using Fisher digital melting
point apparatus. IR spectra were recorded on a Varian 800 FT-IR
spectrophotometer. Optical rotation measurements were carried
out on a Rudolph Research Analytical Autopol III polarimeter. TLC
analyses were carried out on precoated Silica Gel 60 F254 500 lm
TLC plates, using MeOH/CHCl3 (1:9) as a developing solvent. The
1H and 13C NMR spectra were recorded in DMSO-d6, using TMS
as an internal standard, on a JEOL Eclipse NMR spectrometer oper-
ating at 400 MHz for 1H and 100 MHz for 13C. The HREIMS exper-

6038
M. A. Khanfar et al. / Bioorg. Med. Chem. 17 (2009) 6032–6039
with electron donating groups enhance the yield, unlike electron
withdrawing functionalities, which decrease the overall yield. Bulk
groups at the ortho positions can also diminish the product yield.6
4.4.2. Determination of the inhibitory IC50 values
Recombinant GSK-3b (Invitrogen) was dissolved in a buffer
solution (pH 7.2) containing the following: 40 mM HEPES, 5 mM
MgCl2, 5 mM EDTA, 100
l
M ATP, and 50
lg/mL heparin to reach
4.2.1. (Z)-5-(4-(Methylthio)benzylidene)-hydantoin (5)
Amorphous yellow powder. Mp 252–254 °C, 1H NMR (DMSO-d6,
400 MHz) d 2.48 (s, 3H), 6.38 (s, 1H), 7.25 (d, 2H, J = 8.4), 7.56 (d,
2H, J = 8.1), 10.70 (s, 1H), 11.14 (s, 1H). 13C NMR (DMSO-d6,
100 MHz) d 14.8 (CH3), 108.7 (CH), 126.2 (2CH), 127.8 (2CH),
129.9 (qC), 130.4 (CH), 139.7 (qC), 156.2 (qC), 166.1 (qC). IR mmax
(CHCl3) 3384, 1722, 1648, 1590 cmꢀ1; HREIMS m/z 234.0463
[M]+ (calcd for C11H10N2O2S, 234.0463).
a final enzymatic solution of 10 pg/mL. Subsequently, 50
quots of the enzymatic solution were pipetted into 1.5 mL vials.
l
L ali-
Thereafter, appropriate volumes of the compounds’ stock solu-
tions were pipetted into the enzymatic solution to yield 20
10 M, 1 M, and 100 nM of each hit after completion to 75
with the buffer solution. The hits were incubated with the enzyme
over 30 min at room temperature. Then about 25 L of 2000 pg/mL
lM,
lL
l
l
l
tau protein solution in HEPES was added to give a final tau protein
concentration of 500 pg/mL. This mixture was incubated over 1 h
at room temperature.
4.3. Molecular modeling
The detection of tau phosphorylation was performed as follows:
the GSK-3b reaction mixtures were diluted 1:1 with sodium azide
aqueous solution (15 mM) to achieve a final tau protein concentra-
Three-dimensional structure building and all modeling were
performed using the SYBYL program package, version 8.0, installed
on DELL desktop workstations equipped with a dual 2.0 GHz IntelÒ
XeonÒ processor running the Red Hat Enterprise Linux (version 5)
operating system.25 Conformations of each compound were gener-
tion of 250 pg/mL.57 Then 100
lL aliquots of this solution were
pipetted into the wells of the tau [pS396] phosphoELISA kit (Invit-
rogen, Carlsbad, California). Subsequently, the wells were incu-
bated for 2 h at room temperature. Then they were aspirated and
washed (with the washing solution provided in the kit). Thereafter,
100 lL aliquots of rabbit detector antibody solution were pipetted
in the wells and incubated for 1 h at room temperature. The wells
TM
ated using Confort conformational analysis. Energy minimizations
were performed using the Tripos force field with a distance-depen-
dent dielectric and the Powell conjugate gradient algorithm with a
convergence criterion of 0.01 kcal/(mol A).53 Partial atomic charges
were calculated using the semiempirical program MOPAC 6.0 and
applying the AM1.54
were then aspirated and washed with the washing buffer. About
100 lL aliquots of goat (polyclonal) anti-rabbit IgG-HRP were then
added to the wells and incubated for 30 min at room temperature.
4.3.1. Molecular docking
Subsequently, the wells were aspirated and washed with the wash-
SURFLEX DOCK program version 2.0 interfaced with SYBYL 8.0 was
used to dock the compounds to the ATP binding site of GSK-3b.42
SURFLEX DOCK employs an idealized active site ligand (protomol) as
a target to generate putative poses of molecules or molecular frag-
ments.55,56 These putative poses were scored using the Hammer-
head scoring function.55,56 The 3D structure was taken from the
Brookhaven Protein Databank (PDB code: 1q4 l).9
ing buffer. Finally, a total of 100 lL of TMB substrate chromogen
solution aliquots was added to each well and incubated for 20–
30 min. After the termination of the HRP reaction in each well,
the solution’s absorbance was measured spectrophotometrically
at k of 450 nm. The marine-derived b-carboline alkaloid GSK-3b
inhibitor manzamine A was employed as positive control.24 The
IC50 for each compound was calculated using nonlinear regression
(curve fit) of log concentration versus percent of inhibition imple-
mented in GraphPad Prism 5.0.
4.3.2. Pharmacophore generation
The three most active analogues 2, 3, and 7, were used to build
TM
the pharmacophoric map using DISCOtech module. The structure
4.5. In vivo evaluation
of these compounds were constructed manually using SYBYL 8.0,
minimized using the Tripos force field to obtain a local minimum,
and partial atomic charges were calculated using the semiempiri-
cal program MOPAC 6.0 and applying the AM1. Diverse conformers
4.5.1. Preparation of drug hits
The effect of 3 on liver glycogen content of Sprague Dawley rats
was investigated. The sodium salt of 3 was prepared by the addi-
tion of equimolar concentration of 1 N NaOH solution and the
formed salt was then dissolved in PBS (pH 7.2). Three doses of 3
were used; 5, 15, and 25 mg/kg. The drug was dissolved to a con-
centration (mg/mL) that yielded the desired final dosage (mg/kg)
when injected at 0.5 mL/rat.
TM
were generated for each structure using the Confort conforma-
tional analysis tool in SYBYL. Derivation of the pharmacophore mod-
TM
el was undertaken using DISCOtech . Assignment of the initial
pharmacophore features for the DISCO-based pharmacophore
mapping was conducted using the following features: aromatic
and aliphatic ring centroids as hydrophobic centers, hydrogen
bond donors and acceptors, and external site points representing
receptor-associated hydrogen bond acceptor sites and donor sites.
Validation of the pharmacophoric models was carried out using in-
house database search. This database contained a total of 200 com-
pounds including 100 GSK-3b inhibitors and 100 inactive mole-
cules. An acceptable pharmacophoric query should be able to
pick up active GSK-3b inhibitors (IC50 6 20) and should omit the
inactive molecules (IC50 >20).
4.5.2. Determination of liver glycogen
Six-week old female Sprague Dawley female rats with average
weight of 200 g were used for this investigation. The animals were
randomized and fed ad libitum with standard food and water ex-
cept when fasting was needed in the course of the study. All ani-
mals were housed in the same conditions and separated
randomly to four groups. Three groups (three rats/group) used to
investigate compound 3 were injected ip with the three escalating
doses of PMH 3 (mentioned earlier) and one group was injected ip
with PBS as negative control (n = 3). On the day of the experiment,
food and water were removed 6 h before the injection. The animals
were sacrificed by a pentobarbital overdose, and their livers were
immediately removed for glycogen determination. Liver glycogen
content was determined quantitatively following a reported proce-
dure.58 Briefly, livers were removed immediately after the animals
sacrificed and were homogenized using a homogenizer (IKA-T8 Ul-
tra-Turrax, Germany) with appropriate volume of 5% trichloroace-
4.4. In vitro GSK-3b inhibitory activity assay using a tau [pS396]
TM
phosphoELISA
4.4.1. Preparation of compounds for in vitro enzymatic assay
Each compound was dissolved in DMSO to give a 10 mM solu-
tion. Subsequently, 1
l
L was transferred to 100
lL HEPES buffer
(pH 7.2) to give a final stock solution of 100
lM used for subse-
quent enzymatic assays.

M. A. Khanfar et al. / Bioorg. Med. Chem. 17 (2009) 6032–6039
6039
tic acid over 5 min.58 The homogenate was centrifuged (Eppendorf
centrifuge 5804 R, Germany) at 3000 rpm for 5 min. The superna-
tant fluid was taken and filtered using acid-washed filter paper,
and the residues were homogenized again with another volume
of 5% trichloroacetic over 1–3 min to ensure better extraction of
glycogen. The glycogen of 1.0 mL of this filtrate was precipitated
using ethanol (95%, 5 mL), incubated in water bath at 37–40 °C
for 3 h, and centrifuged at 3000 rpm for 15 min. The clear liquid
is gently decanted from the packed glycogen, and the tubes were
allowed to drain in an inverted position for 10 min. The glycogen
was dissolved in distilled water (2 mL) and mixed with 10 mL of
the anthrone reagent (0.05% anthrone, 1.0% thiourea in 72%
H2SO4). The mixture was incubated in boiling water over 30 min,
and subsequently, the absorbance was spectrophotometerically
measured at 620 nm by a Spectroscan 80D UV–vis spectrophotom-
eter. Blank and standard solutions were prepared by adding 10 mL
of anthrone reagent to 2 mL of water and to 2 mL of glucose solu-
tion containing 0.1 mg of glucose in saturated benzoic acid, respec-
tively. The liver glycogen content is estimated using the following
formula:
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¼ ðDU=DSÞ ꢂ ðVolume of Extract ðmLÞ=
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Acknowledgments
This study was partially supported by the Louisiana Biomedical
Research Network.
Supplementary data
Supplementary data associated with this article can be found, in
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