Discovery of novel phenoxazinone derivatives as DKK1/LRP6 interaction inhibitors: Synthesis, biological evaluation and structure-activity relationships

Article abstract of DOI:10.1016/j.bmc.2016.01.025

Amino derivatives of NCI8642 were synthesized and evaluated as inhibitors of DKK1/LRP6 interactions. The new inhibitors were able to activate the Wnt signaling pathway as indicated by the increased levels of β-catenin, and decrease the DKK1-induced Tau ph

Full text of DOI:10.1016/j.bmc.2016.01.025

Accepted Manuscript
Discovery of Novel Phenoxazinone Derivatives as DKK1/LRP6 Interaction In-
hibitors: Synthesis, Biological evaluation and Structure-Activity Relationships
Savvas Thysiadis, Spyros Mpousis, Nicolaos Avramidis, Sotirios Katsamakas,
Athanasios Balomenos, Rosaria Remelli, Spyros Efthimiopoulos, Vasiliki Sarli
PII:
S0968-0896(16)30025-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.01.025
BMC 12768
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 December 2015
11 January 2016
14 January 2016
Please cite this article as: Thysiadis, S., Mpousis, S., Avramidis, N., Katsamakas, S., Balomenos, A., Remelli, R.,
Efthimiopoulos, S., Sarli, V., Discovery of Novel Phenoxazinone Derivatives as DKK1/LRP6 Interaction Inhibitors:
Synthesis, Biological evaluation and Structure-Activity Relationships, Bioorganic & Medicinal Chemistry (2016),
doi: http://dx.doi.org/10.1016/j.bmc.2016.01.025
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Discovery of Novel Phenoxazinone
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Derivatives as DKK1/LRP6 Interaction
Inhibitors: Synthesis, Biological evaluation
and Structure-Activity Relationships
Savvas Thysiadisa, Spyros Mpousisa, Nicolaos Avramidisb, Sotirios Katsamakasc, Athanasios Balomenosb,
Rosaria Remellid, Spyros Efthimiopoulosb,, Vasiliki Sarlia,
a Department of Chemistry, Aristotle University of Thessaloniki, University Campus, 54124, Thessaloniki,
Greece
bDivision of Animal and Human Physiology, Department of Biology, National & Kapodistrian University of
Athens, Panepistimiopolis, Ilisia, 15784, Greece
cDepartment of Pharmacy, Aristotle University of Thessaloniki, University Campus, 54124, Thessaloniki,
Greece
dVIB Discovery Sciences Bio-incubator Leuven, Gaston Geenslan 1, 3001, Leuven, Belgium

Bioorganic & Medicinal Chemistry
Discovery of Novel Phenoxazinone Derivatives as DKK1/LRP6 Interaction
Inhibitors: Synthesis, Biological evaluation and Structure-Activity Relationships
Savvas Thysiadisa, Spyros Mpousisa, Nicolaos Avramidisb, Sotirios Katsamakasc, Athanasios Balomenosb,
Rosaria Remellid, Spyros Efthimiopoulosb,, Vasiliki Sarlia, 
a Department of Chemistry, Aristotle University of Thessaloniki, University Campus, 54124, Thessaloniki, Greece
bDivision of Animal and Human Physiology, Department of Biology, National & Kapodistrian University of Athens, Panepistimiopolis, Ilisia, 15784, Greece
cDepartment of Pharmacy, Aristotle University of Thessaloniki, University Campus, 54124, Thessaloniki, Greece
dVIB Discovery Sciences Bio-incubator Leuven, Gaston Geenslan 1, 3001, Leuven, Belgium
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Amino derivatives of NCI8642 were synthesized and evaluated as inhibitors of DKK1/LRP6
interactions. The new inhibitors were able to activate the Wnt signaling pathway as indicated by
the increased levels of β-catenin, and decrease the DKK1-induced Tau phosphorylation at serine
396.
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
DKK1/LRP6 interaction inhibitors
phenoxazinone
Tau phosphorylation inhibitors
NCI864
Wnt signaling
1. Introduction
neurons challenged with Aβ-peptide, as well as in neurons
subjected to excitotoxic or ischemic insults.6,7 Additional findings
Senile plaques and neurofibrillary tangles (NFT) are the
pathologic hallmarks of Alzheimer's disease (AD).1 Senile
plaques are composed mainly of beta-amyloid peptide (Aβ) and
NFTs of hyperphosphorylated microtubule-associated protein
Tau protein.2 Under normal physiological conditions Tau protein
plays a crucial role in the structure of the neuron by stabilizing
microtubules. Extensive evidence supports that Wnt signaling
and DKK1 protein are associated with the pathophysiology of
neuronal degeneration in AD.3 Dickkopf related protein 1
(DKK1) is a chemokine that belongs to the DKK protein family,
which also includes DKK2, DKK3 and DKK4.4 DΚΚ1, 2, and 4
function as antagonists of canonical Wnt signaling by either
forming inhibitory complexes with LDL receptor-related proteins
5 and 6 (LRP5 and LRP6) or by binding to Kremen-1 or -2 and
triggering the internalization of LRP-5/6.5 LPR5 and LPR6 are
single-pass transmembrane proteins that appear to act as co-
receptors for Wnt ligands involved in the Wnt/β-catenin
signaling. DKK1 protein has been shown to be induced in
reveal that DKK1 induces Tau hyperphosphorylation and NFT
formation after Wnt inhibition, due to the dysregulation of
GSK3β and PP2A activities.8 DKK1 up-regulates GSK3β
activity, which regulates Tau by phosphorylation.9 In another
study, Purro and coworkers demonstrated that acute exposure to
Aβ oligomers induces DKK1 expression together with the loss of
synaptic sites and that the use of DKK1-neutralizing antibodies
suppresses Aβ-induced synapse loss in mouse brain slices.10,11
Collectively, all these studies indicate that regulation of Wnt
signaling pathway by targeting the DKK/LRP interactions may
offer a promising approach to AD therapy. In fact, the
DKK1/LRP5 interaction has been recently targeted with
monoclonal anti-DKK1 antibodies for peripheral indications.12,13
However, since antibodies and are too large to cross the blood
brain barrier and enter the brain, their application for the
treatment of CNS diseases is limited.
NCI8642 (gallocyanine) was identified as the first small
molecule inhibitor of DKK1/LRP6 interactions.14 Surface
plasmon resonance studies showed that it interacts with the
degenerating neurons from Alzheimer patients, in cultured
———
 Corresponding author. Tel.: +0-000-000-0000; fax: +0-000-000-0000; e-mail: author@university.edu
 Corresponding author. Tel.: +0-000-000-0000; fax: +0-000-000-0000; e-mail: author@university.edu

immobilized LRP6 with a reproducible and concentration-
dependent binding in the absence of DKK1. NCI8642 inhibits
DKK1 binding with an IC50 of about 3 μM and effectively
regulates Wnt/β-catenin signaling. It also reduces basal blood-
glucose concentrations and improves glucose tolerance in mice.15
NCI8642 belongs to the family of phenoxazines, which display
important biological properties such as antitumor, antiviral, anti-
inflammatory and antiparasitic.16, 17
on these studies, several compounds were carefully chosen for
synthesis.
2.1. Chemical properties and predicted ADME properties
In order to obtain an inhibitor with improved pharmacokinetic
(PK) profile, several PK properties were also calculated and
considered for the inhibitor design (Table S1, see Supporting
Information). Although there is an outlined tendency of a
correlation between their Fsp3 and PSA values, no secure results
can be generated in terms of their PK descriptor values, due to
the small number compounds under consideration. All the
compounds tested do not have any Lipinski's ‘rule of five’
violations and their overall drug-like profile indicates a good
partitioning and distribution coefficients. Generally, the PK
descriptors vary from 96 to 118 on the PSA, 1 to 3 HBD, 7 to 9
HBA and 0.14 to 0.41 on their flatness indicator (Fsp3). The
flatness levels are entirely contributed from the substituents
present, since the parent compound poses a value of just 0.13
Fsp3.
In a recent publication, our group has performed the synthesis
and biological evaluation of a series of NCI8642 derivatives as
inhibitors of DKK1/LRP6 interactions.18 It was demonstrated that
the phenoxazine is a privileged scaffold for LRP6, due to a
versatile interaction either through the nitrogen or oxygen atom
with Tyr875 of interface A of LRP6. Based on these results, key
groups on the phenoxazine important for inhibition has been
recognized (Figure 1), while changes on the length of alkyl ester
groups were not essential. In this work, we further tested another
collection of gallocyanine derivatives, that arise from the 1,4-
amine addition on gallocyanine methyl ester and oxidation. The
cytotoxicity and the ability of the synthesized compounds to
increase the levels of β-catenin and to inhibit DKK1-induced Tau
phosphorylation was also demonstrated.
2.2. Synthesis of gallocyanine derivatives
The synthesis of the amino derivatives was performed
according to Martinek and coworkers as depicted in scheme 1.21
Initially, the amine is added to the unsaturated esters 2a and 2b
producing phenols of type 3, which are readily oxidized to
phenoxazinones 4a-k in air.
Scheme 1. Synthesis of phenoxazine derivatives
2.3. NCI8642 derivatives inhibit binding of DKK1 to LRP6
The
synthesized
compounds
were
tested
by
immunocytochemistry for their potency to inhibit binding of
DKK1 as described by Iozzi and coworkers.14 The HEK293 cells
overexpressing LRP6 were incubated with DKK1 plus DMSO,
NCI8642 or NCI8642 derivatives for 2 hours at 4 °C. After
washing, fixation and blocking, the cells were incubated with a
primary anti-DKK1 antibody and a fluorescent secondary
antibody. Fluorescence images were analyzed using ImageJ
software and quantified results are shown in Figure 2. The
procedure was applied using different concentrations of each
compound (0.1, 0.5, 1, 10, 50 and 100 μM) in order to estimate
the IC50 values for the inhibition of DKK1/LRP6 interactions.
The estimated IC50 values (in μM) for all the tested compounds
are presented in Table 1.
Figure 1. View of the heterotetrameric LRP6 structure in gold and grey
color per subunit and the interacting DKK1 module in purple color. The
figure was obtained by PyMol version 1.4.1.19
2. Results and discussion
Based on our previous studies the phenoxazinone core
structure has been characterized as a privileged scaffold,18
oriented towards Tyr875A on a hydrophobic pocket of LRP6,
which is occupied by Leu231 of the DKK1c. An extensive in-
house formed library of amino-gallocyanine derivatives was
screened and analyzed over the heterotetramer structure of the
PDB entry 3S8V4.20 In an effort to generate novel protein-small
molecule interactions, this library involved compounds that
maintained general key structural features of NCI8642, such as
the dimethylamino and methyl ester groups, while varying alkyl
and aryl amines of diverse structure on the phenoxazinone. In the
Supporting Information section, the docking score from the top
scoring pose of selected compounds is presented Table S2. Based

adjacent hydrophobic pocket formed by His834A, Phe836B,
Trp850A, Trp850B and Met877B (Figure 3, A). In the case of
compound 4f, the introduction of an extra carbon atom between
the ring and the amine, results in an increase of the substituent’s
flexibility. Thus, the usual observed binding mode change and 4f
seems to locate to a different polar pocket formed of the residues
Arg638B, Arg639B, Arg853A, Asp874B and Tyr875B. This
binding mode (Figure 3, B) becomes more probable when butyl
ester was used, a change that results to the top scoring function of
the compound 4k (Table S2). The docking studies of 4k provided
an extra potential HB formation with Arg638B in comparison to
the docking solutions of the other compounds tested. It should be
noted that all inhibitors reported herein, occupy a space that is
normally filled by residue Leu231 of the DKK1 protein.
Figure 2. The effect of different concentrations of NCI8642 and related
derivatives on the binding of DKK1 to the surface of HEK-293 cells
overexpressing LRP6 (HEK-293LRP6). HEK-293LRP6 cells were incubated
with DKK1 conditioned medium (DKK1-CM) plus DMSO, NCI8642 or
NCI8642 derivatives for 2 hours at 4 °C. Immunofluorescence images were
used to quantify DKK1 binding at the cell surface using Zen software. The
results are expressed as percentage of DKK1 binding. The experiment was
repeated three times in duplicates and 750 cells were analyzed in each case.
The differences are statistically significant (pvalue<0.01).
A)
Table 1. IC50 values for the inhibition of DKK1/LRP6 interactions
compound
R1
R2
IC50 (μM)
6.38
5.23
6.16
3.77
4.71
4.87
4.33
5.5
H
H
NCI8642
B)
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OBu
amino
4a
4b
4c
4d
4e
4f
phenylamino
4-Me-phenylamino
4-MeO-phenylamino
3,4-dichlorophenylamino
4-MeO-benzylamino
piperidino
4g
4h
4i
cyclohexylamino
morpholino
4.06
3.74
4.61
3.38
cyclopentylamino
4-MeO-benzylamino
4j
Figure 3. Schematic representation of the hydrogen bonding network with
green dashed lines between compounds 4b (Figure 3A), 4k (Figure 3B) and
the neighboring residues (left), and the predicted docking conformations of
4b (Figure 3A), and 4k (Figure 3B) in the X-ray structure of LRP6 (right).
The cavity surface is depicted as the grey mesh. Drawings were done by
means of the software PYMOL version 1.4.1.19
4k
The results show that all the amino derivatives were
equipotent or more potent than NCI8642. The introduction of
solely the amino group, compound 4a in regard to the parent
compound NCI8642 showed an 18% drop in activity. Both alkyl
and arylamines provided active compounds with 4k to be the
most active with IC50 of 3.38 μM. The 4-Me-phenylamino (4c)
and morpholino, (4i) compounds were also potent with IC50
values of 3.77 and 3.74 μM respectively.
2.4. Cytotoxicity of the compounds
The synthesized compounds were screened for their cytotoxic
effects in SH-SY5Y cells using the colorimetric MTT assay.22
The NCI8642 derivatives were not found to be neurotoxic at
concentrations of 10 and 100 μM (See Supporting Information
Figure S1).
The insertion of saturated (e.g. compounds 4h and 4j) or
aromatic cyclic amines (e.g. compounds 4b, 4c, 4d and 4e) that
lacked a carbon linker and/or when the nitrogen participated in an
heterocyclic ring (e.g. compounds 4g and 4i) despite the ring
substituents seems to maintain the binding mode of gallocyanine
in our docking solutions. The compounds reside entirely inside
the pocket formed at DKK1/LRP6 interface with no immediate
polar contacts forming, owing to the present residues on the
2.5. The effect of NCI8642 derivatives on the levels of β-catenin
The levels of β-catenin are regulated by the Wnt signaling
pathway.23 Specifically, activation of the Wnt signal transduction
pathway (Figure 4) results in reduced levels of GSK3β-mediated
phosphorylation of β-catenin and reduced β-catenin catabolism
and, as a consequence, increased β-catenin levels.24,25 Inhibition

of the Wnt pathway by DKK1 leads finally to decreased levels of
β-catenin (Figures 4 and S2). Thus, the levels of β-catenin
consists an indicator of the activation of the Wnt signal
transduction pathway and can be used to evaluate the effect of
NCI8642 on the function of this pathway.
levels of phosphorylated Tau in comparison to cells treated with
DKK1 alone. Compound 4e was the most potent in this assay.
Figure 6. NCI8642 and NCI8642 derivatives decrease Tau
phosphorylation at serine 396. SH-SY5Y cells were treated with DKK1-CM
plus DMSO or DKK1-CM plus NCI8642 or NCI8642 derivatives at 10 μΜ
concentration. Cell lysates were analyzed by western blot using antibodies
against Tau phosphorylated at serine 396 or actin. Quantification of the
results obtained from three independent experiments. The differences are
statistically significant (pvalue<0.01).
3. Conclusions
Amino derivatives of gallocyanine methylester were
synthesized and evaluated as inhibitors of DKK1/LRP6
interactions. The compounds tested provided active compounds
with IC50 values in the range of 3.38-5.5 μΜ. Although the
potency was not significantly improved compared to the parent
compound NCI8642, the new compounds activated Wnt
signaling pathway as indicated by the increased levels of β-
catenin. Interestingly, the new inhibitors were slightly more
potent than NCI8642 in inhibiting DKK1-induced Tau
phosphorylation at serine 396. Collectively, the NCI8642
derivatives remain a subject of further research for future
applications in the development of new treatment for
Alzheimer’s disease.
Figure 4. Simplified overview of the Wnt signaling pathway.
For this purpose, SY5Y cells were incubated for 1 hour at 37
oC with DKK1, DKK1 plus NCI8642 or DKK1 plus NCI8642
derivatives at a concentration of 10 μΜ. Western blot analysis for
the levels of β-catenin followed by quantification of the data
(Figure 5) indicated that NCI8642 and its derivatives caused a
significant increase in the levels of β-catenin in comparison to
cells treated with DKK1 alone. DKK1 decreased the levels of β-
catenin by 121% relative to the respective value of untreated cells
(Data not shown).
4. Experimental section
General Experimental Details. All reactions were carried out
under an atmosphere of Ar unless otherwise specified.
Commercial reagents of high purity were purchased and used
without further purification, unless otherwise noted. Reactions
were monitored by TLC and using UV light as a visualizing
agent and aqueous ceric sulfate/phosphomolybdic acid, ethanolic
p-anisaldehyde solution, potassium permanganate solution, and
1
heat as developing agents. The H and 13C NMR spectra were
recorded at 500 and 125 MHz, and tetramethylsilane was used as
an internal standard. Chemical shifts are indicated in δ values
Figure 5. NCI8642 and NCI8642 derivatives increase the levels of β-
1
(ppm) from internal reference peaks (TMS H 0.00; CDCl3 1H
o
catenin. SH-SY5Y cells were incubated for 1 hour at 37 C with DKK1-CM
7.26, 13C 77.00; DMSO-d6 1H 2.50, 13C 39.51, pyridine-d5 1H
8.74, 7.58, 7.22; 13C 150.35, 135.91, 123.87). Melting points
(mp) are uncorrected. High-resolution mass spectra (HRMS)
were recorded by direct injection of 2 μl of a 2 μM solution of the
compounds in water–acetonitrile (1/1; v/v) and 0.1% formic acid
plus DMSO, DKK1-CM plus NCI8642 or DKK1-CM plus NCI8642
derivatives at a concentration of 10 μΜ. Protein extracts were analysed by
western blot using an antibody against β-catenin. The intensity of the bands
was quantified by densitometry and the results of 3 independent experiments
are presented. The differences are statistically significant (pvalue<0.01).
on
a mass spectrometer (hybrid ion trap-orbitrap mass
2.6. The effect of NCI8642 derivatives on Tau phosphorylation
spectrometer) equipped with an electrospray ion source in
positive mode (source voltage 3.5 kV, sheath gas flow 10,
capillary temperature 275 °C) with resolution R = 60.000 at m/z
= 400 (mass range = 150–2000) and dioctylphthalate (m/z =
391.28428) as the “lock mass”.
Next, the effect of NCI8642 derivatives on DKK1-induced tau
phosphorylation at serine 396 was analyzed. SY5Y cells were
o
incubated for 1 hour at 37 C with DKK1-CM to induce Tau
phosphorylation, DKK1-CN plus NCI8642 or DKK1-CM plus
NCI8642 derivatives at 10 μΜ. Western blot analysis for the
levels of Tau phosphorylated at serine 396 followed by
quantification of the data (Figures 6 and S3) indicated that
NCI8642 and its derivatives caused a significant decrease in the
The polyclonal/monoclonal Rabbit Anti-Mouse antibody was
kindly provided from Dr. Luc Buee (Insern, Fac de Medicine,
Univ. Lille 2, Lille France) and is directed against the N-terminus
of Tau. The polyclonal Rabbit Anti-Mouse antibody detecting tau

phosphorylated at serine396 were purchased from Acris
Antibodies GmbH (Germany). Goat Anti-Rabbit IgG-HRP
0.318 mmol) and p-toluidine (170 mg, 1.59 mmol) were heated
under reflux in ethanol (23 mL) for 24 hours. The solvent was
evaporated at half volume and the reaction mixture was then
secondary
antibody
was
purchased
from
Jackson
o
ImmunoResearch (Baltimore Pike, USA). The monoclonal
Mouse antibody used to detect actin by immunoblot was
purchased from Merck Millipore (Darmstatd, Germany). Goat
Anti-Mouse IgG-HRP secondary antibody was purchased from
Santa Cruz Biotechnology (Dallas/Texas, USA). The polyclonal
Rabbit Anti-Human antibody against DKK1 was obtained from
Santa Cruz Biotechnology (Dallas/Texas, USA). Goat anti-
Rabbit IgG (H+L) Alexa Fluor 488-conjugated and/or Goat anti-
Rabbit IgG (H+L) Cy 3-conjugated secondary was purchased
from Jackson ImmunoResearch (Baltimore Pike, USA). B27 and
Neurobasal used to culture mouse primary neuronal culture were
purchased from Life Technologies (Grand Island/NY, USA).
Common chemicals were purchased from Sigma Aldrich.
stored at 8 C for 24 hours. The precipitate was collected by
filtration to obtain 4c as a dark green iridescent solid (64 mg). To
increase the yield the filtrate was concentrated and the residue
was purified by flash column chromatography (eluent;
hexane/ethyl acetate = 1/1) to obtain additional 38 mg of 4c (total
1
yield= 77%). 4c: mp = 270-273 oC; H NMR (500 MHz, CDCl3)
δ 7.61 (d, J = 9.1 Hz, 1H, H-9), 7.48 (s, 1H, OH), 7.15 (d, J = 8.2
Hz, 2H, ArH), 7.08 (d, J = 8.2 Hz, 2H, ArH), 6.74 (dd, J = 9.1,
2.7 Hz, 1H, H-8), 6.67 (d, J = 2.7 Hz, 1H, H-6), 6.36 (s, 1H,
NH), 3.26 (s, 3H, OCH3), 3.13 (s, 6H, NCH3), 2.34 (s, 3H,
ArCH3); 13C NMR (126 MHz, CDCl3) δ 172.5 (C=O), 165.7
(C=O), 152.3 (C=C), 144.9 (C=C), 139.4 (C=C), 137.4 (C=C),
136.5 (C=C), 135.2 (C=C), 131.6 (C=C), 131.1 (C=C), 130.1
(C=C), 129.6 (C=C), 127.2 (C=C), 123.7 (C=C), 110.7 (C=C),
104.2 (C=C), 96.6 (C=C), 51.6 (OCH3), 40.4 (NCH3), 21.0
(ArCH3); FT-IR (KBr): 3266, 2940, 2921, 2850, 2803, 1724,
1617, 1598, 1560, 1522, 1420, 1355, 1312, 1280, 1199, 1177,
1128, 1051, 1003; HRMS m/z for C23H22N3O5 [M + H]+ calcd
420.1559 found 420.1560.
4.1. Chemical synthesis
Methyl 2-amino-7-(dimethylamino)-4-hydroxy-3-oxo-3H-
phenoxazine-1-carboxylate, (4a): Methyl 7-(dimethylamino)-4-
hydroxy-3-oxo-3H-phenoxazine-1-carboxylate (prune, 2a) (100
mg, 0.318 mmol) and ammonia (7N in methanol, 1.59 mmol)
o
were heated at 90 C in ethanol (16 mL) for 24 hours. The
Methyl
7-(dimethylamino)-4-hydroxy-2-((4-methoxy
(4d):
solvent was evaporated at half volume and the reaction mixture
was then stored at 8 oC for 24 hours. The precipitate was
collected by filtration to obtain 4a as a dark blue-black solid (61
mg). To increase the yield the filtrate was evaporated,
dichloromethane was added and the solid was removed by
filtration. The filtrate was concentrated to give additional 30 mg
of 4a (total yield = 87%). 4a: mp > 320 oC; 1H NMR (500 MHz,
CDCl3) δ 7.66 (d, J = 9.1 Hz, 1H, H-9), 7.18 (s, 1H, OH), 6.78
(dd, J = 9.1, 2.6 Hz, 1H, H-8), 6.67 (d, J = 2.6 Hz, 1H, H-6), 3.99
(s, 3H, OCH3), 3.13 (s, 6H, NCH3); 13C NMR (126 MHz, CDCl3)
δ 171.0 (C=O), 168.7 (C=O), 152.2 (C=C), 146.9 (C=C), 144.3
(C=C), 139.4 (C=C), 132.4 (C=C), 131.1 (C=C), 130.8 (C=C),
(1C is missing due to overlapping), 126.6 (C=C), 110.9 (C=C),
96.5 (C=C), 52.0 (OCH3), 40.4 (NCH3); FT-IR (KBr): 3319,
2923, 2850, 1616, 1559, 1458, 1374, 1316, 1263, 1120; HRMS
m/z for C16H16N3O5 [M + H]+ calcd 330.1090, found 330.1084.
phenyl)amino)-3-oxo-3H-phenoxazine-1-carboxylate,
Prune 2a (100 mg, 0.318 mmol) and p-anisidine (196 mg, 1.59
mmol) were heated under reflux in ethanol (23 mL) for 24 hours.
The solvent was evaporated at half volume and the reaction
mixture was then stored at 8 oC for 24 hours. The precipitate was
collected by filtration to obtain 4d as a dark green iridescent solid
(54 mg). To increase the yield the filtrate was concentrated and
the residue was purified by flash column chromatography
(eluent; hexane/ethyl acetate = 1/1) to obtain additional 13 mg of
4d (total yield= 50%). 4d: mp = 279-282 oC; 1H NMR (500 MHz,
CDCl3) δ 7.60 (d, J = 9.1 Hz, 1H, H-9), 7.40 (s, 1H, OH), 7.14
(d, J = 8.8 Hz, 2H, ArH), 6.88 (d, J = 8.8 Hz, 2H, ArH), 6.73 (dd,
J = 9.1, 2.6 Hz, 1H, H-8), 6.67 (d, J = 2.6 Hz, 1H, H-6), 6.35 (s,
1H, NH), 3.82 (s, 3H, OCH3), 3.30 (s, 3H, OCH3), 3.13 (s, 6H,
NCH3); 13C NMR (126 MHz, CDCl3) δ 172.5 (C=O), 165.8
(C=O), 157.6 (C=C), 152.3 (C=C), 144.9 (C=C), 139.5 (C=C),
137.6 (C=C), 131.8 (C=C), 131.5 (C=C), 131.1 (C=C), 130.1
(C=C), 127.1 (C=C), 126.1 (C=C), 114.2 (C=C), 110.7 (C=C),
104.0 (C=C), 96.6 (C=C), 55.5 (OCH3), 51.7 (OCH3), 40.4
(NCH3); FT-IR (KBr): 3259, 2928,2850, 1723, 1618, 1599,
1582,1561, 1526, 1416, 1357, 1313, 1240, 1179, 1129; HRMS
m/z for C23H22N3O6 [M + H]+ calcd 436.1508 found 436.1502.
Methyl 7-(dimethylamino)-4-hydroxy-3-oxo-2-(phenylamino)
-3H-phenoxazine-1-carboxylate, (4b): Prune 2a (150 mg, 0.478
mmol) and aniline (222 mg, 2.39 mmol) were heated under reflux
in ethanol (35 mL) for 18 hours. The solvent was evaporated at
o
half volume and the reaction mixture was then stored at 8 C for
24 hours. The precipitate was collected by filtration. The filtrate
was concentrated and the residue was purified by flash column
chromatography (eluent; hexane/ethyl acetate = 1/1) to afford 4b
as dark green iridescent solid (74 mg). To increase the yield the
filtrate was concentrated and the residue was purified by flash
column chromatography (eluent; hexane/ethyl acetate = 1/1) to
obtain additional 103 mg of 4b (total yield= 91%). 4b: mp = 271-
Methyl 2-((3,4-dichlorophenyl)amino)-7-(dimethylamino)-
4-hydroxy-3-oxo-3H-phenoxazine-1-carboxylate, (4e): Prune
2a (100 mg, 0.318 mmol) and 3,4-dichloroaniline (258 mg, 1.59
mmol) were heated under reflux in ethanol (23 mL) for 24 hours.
The solvent was evaporated at half volume and the reaction
mixture was then stored at 8 oC for 24 hours. The precipitate was
collected by filtration to obtain 4e as green iridescent solid (28
mg). To increase the yield the filtrate was concentrated and the
residue was purified by flash column chromatography (eluent;
hexane/ethyl acetate = 1/1) to obtain 31 mg of 4e (total
yield=41%). 4e: mp = 264-266 oC; 1H NMR (500 MHz, CDCl3) δ
7.62 (d, J = 9.1 Hz, 1H, H-9), 7.44 (s, 1H, OH), 7.39 (d, J = 8.5
Hz, 1H, ArH), 7.29 (overlapping with CDCl3, 1H, ArH), 7.02 (d,
J = 7.3 Hz, 1H, ArH), 6.76 (dd, J = 9.1, 2.4 Hz, 1H, H-8), 6.67
(d, J = 2.4 Hz, 1H, H-6), 6.36 (s, 1H, NH), 3.49 (s, 3H, OCH3),
3.14 (s, 6H, NCH3); 13C NMR (126 MHz, CDCl3) δ 171.9 (C=O),
165.6 (C=O), 152.9 (C=C), 145.2 (C=C), 139.1 (C=C), 138.6
(C=C), 136.0 (C=C), 132.8 (C=C), 131.8 (C=C), 131.5 (C=C),
130.7 (C=C), 130.3 (C=C), 128.3 (C=C), 127.4 (C=C), 124.3
(C=C), 122.2 (C=C), 111.1 (C=C), 106.6 (C=C), 96.5 (C=C),
o
274 C; 1H NMR (500 MHz, CDCl3) δ 7.62 (d, J = 9.1 Hz, 1H,
H-9), 7.54 (s, 1H, OH), 7.34 (t, J = 7.8 Hz, 2H, ArH), 7.23 – 7.13
(m, 3H, ArH), 6.74 (dd, J = 9.1, 2.7 Hz, 1H, H-8), 6.68 (d, J =
2.7 Hz, 1H, H-6), 3.24 (s, 3H, OCH3), 3.13 (s, 6H, NCH3); 13C
NMR (126 MHz, CDCl3) δ 172.4 (C=O), 165.6 (C=O), 152.5
(C=C), 145.0 (C=C), 139.3 (C=C), 137.1 (C=C), 131.7 (C=C),
131.3 (C=C), 130.2 (C=C), 129.3 (C=C), 129.1 (C=C), 127.2
(C=C), 125.3 (C=C), 123.4 (C=C), 115.1 (C=C), 110.8 (C=C),
96.6 (C=C), 51.6 (OCH3), 40.4 (NCH3); FT-IR (KBr): 3267,
2923, 2847, 2803, 1724, 1603, 1617, 1580, 1561, 1518, 1420,
1356, 1314, 1198, 1177, 1127; HRMS m/z for C22H20N3O5 [M +
H]+ calcd 406.1403, found 406.1403.
Methyl
7-(dimethylamino)-4-hydroxy-3-oxo-2-(p-tolyl
amino)-3H-phenoxazine-1-carboxylate, 4c: Prune 2a (100 mg,

40.4 (OCH3), 30.9 (NCH3); FT-IR (KBr): 3252, 2924, 2853,
1724, 1654, 1617, 1560, 1508, 1458, 1420, 1355, 1176, 1127;
HRMS m/z for C22H18Cl2N3O5 [M + H]+ calcd 474.0623 found
474.0624.
(NCH), 40.4 (NCH3), 33.9 (CH2), 25.5 (CH2), 24.9 (CH2); FT-IR
(KBr): 3346, 3280, 2928, 2855, 1727, 1655, 1618, 1555, 1500,
1423, 1361, 1347, 1320, 1199, 1172, 1154, 1132, 1056, 1043;
HRMS m/z for C22H26N3O5 [M + H]+ calcd 412.1872 found
412.1872.
Methyl 7-(dimethylamino)-4-hydroxy-2-((4-methoxybenzyl)
amino)-3-oxo-3H-phenoxazine-1-carboxylate, (4f): Prune 2a
(100 mg, 0.318 mmol) and p-methoxy-benzylamine (218 mg,
1.59 mmol) were heated under reflux in ethanol (23 mL) for 24
hours. The solvent was evaporated at half volume and the
Methyl 7-(dimethylamino)-4-hydroxy-2-morpholino-3-oxo-
3H-phenoxazine-1-carboxylate, (4i): Prune 2a (100 mg, 0.318
mmol) and morpholine (139 mg, 1.59 mmol) were heated under
reflux in ethanol (23 mL) for 24 hours. The solvent was
evaporated at half volume and the reaction mixture was then
o
reaction mixture was then stored at 8 C for 24 hours. The
o
precipitate was collected by filtration to obtain 4f as a dark green
stored at 8 C for 24 hours. The precipitate was collected by
1
iridescent solid (114 mg, yield 80%). 4f: mp = 195-197 oC; H
filtration to obtain 4i as a dark green iridescent solid (50 mg). To
increase the yield the filtrate was concentrated and the residue
was purified by flash column chromatography (eluent;
hexane/ethyl acetate = 1/1) to obtain 50 mg of 4i (total
yield=79%). 4i: mp = 245-248 oC; 1H NMR (500 MHz, CDCl3) δ
7.55 (d, J = 9.1 Hz, 1H, H-9), 6.69 (dd, J = 9.1, 2.7 Hz, 1H, H-8),
6.60 (m, 2H, H-6, OH), 3.99 (s, 3H, OCH3), 3.91 – 3.75 (m, 4H,
OCH2), 3.52 – 3.31 (m, 4H, NCH2), 3.13 (s, 6H, NCH3); 13C
NMR (126 MHz, CDCl3) δ 175.3 (C=O), 167.5 (C=O), 153.0
(C=C), 145.3 (C=C), 143.3 (C=C), 139.5 (C=C), 132.8 (C=C),
131.5 (C=C), 129.0 (C=C), 126.8 (C=C), 118.8 (C=C), 110.4
(C=C), 96.7 (C=C), 67.4 (OCH2), 52.6 (OCH3), 50.4 (OCH2),
40.4 (NCH3); FT-IR (KBr): 3278, 2946, 2918, 2850, 1732, 1723,
1618, 1585, 1549, 1422, 1449, 1434, 1357, 1312, 1268, 1229,
1207, 1175, 1129, 1116, 1050, 1024; HRMS m/z for C20H22N3O6
[M + H]+ calcd 400.1508 found 400.1502.
NMR (500 MHz, CDCl3) δ 7.57 (d, J = 9.0 Hz, 1H, H-9), 7.27 (d,
J = 8.6 Hz, 2H, ArH), 6.90 (d, J = 8.6 Hz, 2H, ArH), 6.72 (dd, J
= 9.0, 2.6 Hz, 1H, H-8), 6.66 (d, J = 2.6 Hz, 1H, H-6), 6.02 (t, J =
5.3 Hz, 1H, NH), 4.39 (d, J = 5.3 Hz, 2H, CH2Ar), 3.91 (s, 3H,
OCH3), 3.81 (s, 3H, OCH3), 3.11 (s, 6H, NCH3); 13C NMR (126
MHz, CDCl3) δ 172.5 (C=O), 168.4 (C=O), 159.4 (C=C), 151.9
(C=C), 144.8 (C=C), 140.1 (C=C), 138.3 (C=C), 131.2 (C=C),
130.6 (C=C), 129.8 (C=C), 129.4 (C=C), 129.4 (C=C), 126.6
(C=C), 114.2 (C=C), 110.5 (C=C), 102.5 (C=C), 96.8 (C=C),
55.3 (OCH3), 52.4 (OCH3), 47.4 (NHCH2), 40.4 (NCH3); FT-IR
(KBr): 3333, 3238, 3003, 2946, 2921, 1720, 1651, 1618, 1551,
1508, 1424, 1361, 1313, 1246,1217, 1174, 1131, 1044, 1022;
HRMS m/z for C24H24N3O6 [M + H]+ calcd 450.1665 found
450.1664.
Methyl 7-(dimethylamino)-4-hydroxy-3-oxo-2-(piperidin-
1-yl)-3H-phenoxazine-1-carboxylate, (4g): Prune 2a (100 mg,
0.318 mmol) and piperidine (135 mg, 1.59 mmol) were heated
under reflux in ethanol (23 mL) for 24 hours. The solvent was
evaporated and the crude mixture was purified by flash column
chromatography (eluent; hexane/ethyl acetate = 1/1) to afford
100 mg of 4g as dark blue solid in 80% yield. 4g: mp =170-173
oC; 1H NMR (500 MHz, CDCl3) δ 7.54 (d, J = 9.0 Hz, 1H, H-9),
6.67 (dd, J = 9.0, 2.6 Hz, 1H, H-8), 6.60 (d, J = 2.6 Hz, 1H, H-6),
3.99 (s, 3H, OCH3), 3.46 – 3.25 (m, 4H, NCH2), 3.11 (s, 6H,
NCH3), 1.71-1.72 (m, 4H, CH2), 1.65 (m, 2H, CH2); 13C NMR
(126 MHz, CDCl3) δ 175.8 (C=O), 167.9 (C=O), 152.6 (C=C),
145.1 (C=C), 145.0 (C=C), 140.1 (C=C), 132.8 (C=C), 131.2
(C=C), 128.8 (C=C), 126.6 (C=C), 116.7 (C=C), 110.2 (C=C),
96.8 (C=C), 52.6 (OCH3), 51.5 (NCH2), 40.4 (NCH3), 26.6
(CH2), 24.2 (CH2); FT-IR (KBr): 3337, 2920, 2851, 2815, 1719,
1621, 1573, 1527, 1484, 1450, 1419, 1355, 1314, 1278, 1237,
1194, 1160, 1116, 1041, 1006; HRMS m/z for C21H24N3O5 [M +
H]+ calcd 398.1716 found 398.1710.
Methyl 2-(cyclopentylamino)-7-(dimethylamino)-4-hydro
xy-3-oxo-3H-phenoxazine-1-carboxylate, (4j): Prune 2a (100
mg, 0.318 mmol) and cyclopentylamine (135 mg, 1.59 mmol)
were heated under reflux in ethanol (23 mL) for 24 hours. The
solvent was evaporated at half volume and the reaction mixture
was then stored at 8 oC for 24 hours. The precipitate was
collected by filtration to obtain 4j as a dark green iridescent solid
(85 mg). To increase yield the filtrate was concentrated and the
residue was purified by flash column chromatography (eluent;
hexane/ethyl acetate = 1/1) to obtain 15 mg of 4j (total
yield=79%). 4j: mp > 320oC; 1H NMR (500 MHz, CDCl3) δ 7.61
(d, J = 8.7 Hz, 1H, H-9), 6.74 (dd, J = 8.7, 1.8 Hz, 1H, H-8), 6.68
(d, J = 1.8 Hz, 1H, H-6), 6.27 (s, 1H, NH), 4.05 (m, 1H, OCH),
3.99 (s, 3H, OCH3), 3.11 (s, 6H, NCH3), 1.97 (m, 2H, CH2), 1.75
(m, 2H, CH2), 1.62 (m, 4H, CH2); 13C NMR (126 MHz, DMSO-
d6) δ 172.7 (C=O), 167.7 (C=O), 151.3 (C=C), 144.3 (C=C),
140.4 (C=C), 137.4 (C=C), 132.0 (C=C), 130.2 (C=C), 129.2
(C=C), 125.1 (C=C), 110.2 (C=C), 101.1 (C=C), 96.4 (C=C),
53.8 (OCH3), 52.0 (CHN), 45.6 (NCH3), 33.3 (CH2), 23.3 (CH2);
FT-IR (KBr): 3333, 3259, 2959, 2921, 2850, 1731, 1618, 1552,
1503, 1418, 1353, 1320, 1194, 1172, 1132, 1046; HRMS m/z for
C21H24N3O5 [M + H]+ calcd 398.1716 found 398.1716.
Methyl 2-(cyclohexylamino)-7-(dimethylamino)-4-hydroxy
-3-oxo-3H-phenoxazine-1-carboxylate, (4h): Prune 2a (100
mg, 0.318 mmol) and cyclohexylamine (158 mg, 1.59 mmol)
were heated under reflux in ethanol (23 mL) for 24 hours. The
solvent was evaporated at half volume and the reaction mixture
was then stored at 8 oC for 24 hours. The precipitate was
collected by filtration to obtain 4h as a dark green iridescent solid
(79 mg). To increase the yield the filtrate was concentrated and
the residue was purified by flash column chromatography
(eluent; hexane/ethyl acetate = 1/1) to obtain 50 mg of 4h (total
Butyl 7-(dimethylamino)-4-hydroxy-2-((4-methoxybenzyl)
amino)-3-oxo-3H-phenoxazine-1-carboxylate, (4k): Butyl 7-
(dimethylamino)-4-hydroxy-3-oxo-3H-phenoxazine-1-carboxyla
te (50 mg, 0.14 mmol) and p-methoxybenzylamine (92 μL, 0.702
mmol) were heated under reflux in ethanol (9.8 mL) for 24 hours.
The solvent was evaporated at half volume and the reaction
mixture was then stored at 8 oC for 24 hours. The precipitate was
collected by filtration to obtain 31 mg of 4k as a dark green
1
yield=98%). 4h: mp = 178-179 oC; H NMR (500 MHz, CDCl3)
δ 7.56 (d, J = 9.1 Hz, 1H, H-9), 6.71 (dd, J = 9.1, 2.5 Hz, 1H, H-
8), 6.65 (d, J = 2.5 Hz, 1H, H-6), 6.25 (s, 1H, OH), 5.94 (d, J =
9.5 Hz, 1H, NH), 3.98 (s, 3H, OCH3), 3.48 – 3.33 (m, 1H, NCH),
3.10 (s, 6H, (NCH3), 2.05 (m, 2H, CH2), 1.79 (m, 2H, CH2), 1.28
(m, 6H, CH2); 13C NMR (126 MHz, CDCl3) δ 172.9 (C=O),
168.6 (C=O), 151.8 (C=C), 144.7 (C=C), 140.3 (C=C), 137.3
(C=C), 131.1 (C=C), 130.5 (C=C), 129.6 (C=C), 126.7 (C=C),
110.4 (C=C), 101.9 (C=C), 96.8 (C=C), 52.5 (OCH3), 52.0
o
1
iridescent solid in 46% yield. 4k: mp = 185-188 C; H NMR
(500 MHz, CDCl3) δ 7.56 (d, J = 8.8 Hz, 1H, H-9), 7.26 (m, 2H,
ArH, overlapping with solvent), 6.90 (d, J = 8.0 Hz, 2H, ArH),
6.76 (d, J = 8.8 Hz, 1H, H-8), 6.70 (s, 1H, H-6), 6.31 (s, 1H,
NH), 4.44 (s, 2H, OCH2), 4.34 (t, J = 6.3 Hz, 2H, NCH2), 3.81 (s,
3H, OCH3), 3.11 (s, 6H, NCH3), 1.80 – 1.67 (m, 2H, CH2), 1.49
(dd, J = 14.7, 7.4 Hz, 2H, CH2), 0.96 (t, J = 7.3 Hz, 3H, CH3);

13C NMR (126 MHz, CDCl3) δ 172.7 (C=O), 168.1 (C=O), 159.4
(C=C), 151.8 (C=C), 144.7 (C=C), 140.3 (C=C), 138.2 (C=C),
131.2 (C=C), 130.4 (C=C, 1C is missing due to overlapping),
129.7 (C=C), 129.5 (C=C), 129.4 (C=C), 129.3 (C=C), 114.3
(C=C), 110.5 (C=C), 96.9 (C=C), 65.6 (OCH2), 55.3 (OCH3),
47.5 (NCH2), 40.4 (NCH3), 30.6 (CH2), 19.3 (CH2), 13.7 (CH3);
FT-IR (KBr): 3447, 2961, 2923, 2847, 1718, 1618, 1559, 1508,
1425, 1358, 1130; HRMS m/z for C27H30N3O6 [M + H]+ calcd
492.2134 found 492.2135.
HEK-293 human embryonic kidney cells overexpressing LRP6
(HEK-293LRP6) or DKK1 (HEK-293DKK1) and SH-SY5Y
human neuroblastoma cells were cultured in a 5% CO2
o
atmosphere at 37 C in Dulbecco's Modified Eagle's Medium
(DMEM) (Sigma Aldrich) supplemented with 10% heat
inactivated fetal bovine serum (FBS) (PAA Laboratories GmbH,
Linz, Austria), 1% penicillin/streptomycin (Sigma Aldrich) and
10 μg/ml Blasticidine for HEK-293DKK1 and 100 μg/ml
Zeocine for HEK-293LRP6. Confluent HEK293 cells
overexpressing DKK1 (HEK293-DKK1) cells were cultured for
72 hours and the conditioned medium was collected, cleared
from cell debris and floating cells by centrifugation and stored at
-80 °C until use.
4.2. Computational chemistry
Several novel 1,4-Michael adduct compounds based on parent
NCI864226 were designed and rationalized in silico prior to
synthesis. These compounds were compiled into a smile
formatted library (*.smi) utilizing the freely available program
Open Babel.27 Thereafter, conformers were generated with the
use of Omega v. 2.5.1.4 software (OpenEye Scientific Software,
Inc., Santa Fe, NM, USA; www.eyesopen.com).28 All the docking
experiments took place on a typical desktop pc running Windows
7 64-bit operating system (Dual Core 3,2 GHz Pentium CPU, 8
GB RAM, 500 GB HDD), using the program OEDocking suite v.
3.0.1 (OpenEye Scientific Software, Inc., Santa Fe, NM, USA;
www.eyesopen.com).29 Visualization states of the resulted
docking solutions are provided by the software PyMol v. 1.4.1.19
4.5. DKK1 binding assay
Binding of DKK1 was performed as previously described by
Iozzi and coworkers.14 Briefly, HEK-293-LRP6 cells were
seeded onto poly-D-lysine-coated coverslips 24 hours before the
experiment and were incubated with DKK1 conditioned media
(DKK1-CM) plus DMSO, DKK1-CM plus NCI8642 (0.1 μM,
0.5 μM, 1 μM, 10 μM, 50 μM, 100 μM) or DKK1-CM plus
NCI8642 derivatives (0.1 μM, 0.5 μM, 1 μM, 10 μM, 50 μM, 100
μM) for 2 hours at 4 °C. After washing, the cells was fixed in 3%
paraformaldehyde (15 min, room temperature), blocked with
PBS/0.1% BSA, and incubated with the primary rabbit-anti-
DKK1 antibody, and fluorescent secondary antibody Alexa488
diluted into blocking solution. This antibody which emits
fluorescence at 519 nM was very carefully chosen following the
observation that some of the NCI8642 derivatives emit
fluorescence at 570 nM. Cell nuclei were counterstained with
DAPI. Images were collected using a fluorescence microscope
and 750 cells were analyzed each time using ZEN software.
Several physico-chemical (PK) descriptor values were
calculated for the above library, by filling their profile as drug
candidates (Table S1). Among these are the sp3 factor (Fsp3)
providing the compound saturation levels (flatness),30 Lipinski's
rule of five
descriptors (where calculated logarithmic
lipophilicity "clogPMB" was achieved with the use of program
Marvin Beans v. 14.9.29) and spatial characteristics calculated
over the web with the freely available Molinspiration property
calculation service (www.molinspiration.com).
4.6. Cell treatments and cell lysis
4.3. Docking experiments
SH-SY5Y cells grown in Dulbecco’s Modified Eagle’s
Medium (DMEM) that contained 10% fetal bovine serum, 1% of
penicillin, and streptomycin in a 37°C humidified incubator
consisting of 5% CO2 were treated with DKK1-CM plus DMSO,
DKK1-CM plus NCI8642 (10 μM) or DKK1-CM plus NCI8642
derivatives (10 μM) for 1 hour or 24 hours. Following treatment,
cells were lysed at 4 °C in lysis buffer [50 mM Tris–HCl, 150
mM NaCl, 2 mM EDTA, pH 7.6, 1% Triton X-100 (v/v)]
supplemented with complete protease inhibitor cocktail and
phosphatase inhibitor cocktail (Roche Applied Science,
Manheim, Germany). Cell lysates were left on ice for 30 minutes
and then centrifuged at 14,000 g for 12 min at 4 °C. Protein
concentrations in supernatants were determined by using the
Bradford protein assay.
Ligand and Protein Preparation. The protein complex, PDB
entry 3S8V, was used in this study.20 DKK1 module was
removed from the data and with the use of OEDocking suite
program MAKE RECEPTOR v. 3.0.1 (OpenEye Scientific
Software, Inc., Santa Fe, NM, USA; www.eyesopen.com)29 the
experiment was parameterized based on our previously reported
methodology.18 The search space was centered over subunits A
and B interface with a wider area covering subunit B E3, since
this domain shows a more extended interaction surface on DKK1
module. This, generated an initial 40,025 Å3 box which resulted
after a balanced site-shape potential implementation to an outer
contour docking space of 13,515 Å3, narrowed by an inner
contour docking space of 10,559 Å3. No further residue
modifications took place in the protein and no constraints were
present. By utilizing the program Open Babel, 27 we compiled an
extended *.smi formatted library of NCI8642 derivatives
followed by their conformer generation with the use of Omega v.
2.5.1.4 software (OpenEye Scientific Software, Inc., Santa Fe,
NM, USA; www.eyesopen.com).29 Docking experiments were
performed using OEDocking suite program FRED v. 3.0.1
(OpenEye Scientific Software, Inc., Santa Fe, NM, USA;
www.eyesopen.com),29 utilizing an Exhaustive Search
Algorithm. A refinement of all the resulted solutions using the
OEDocking suite program FRED rescore v. 3.0.1 (OpenEye
4.7. Western blot and densitometry
Appropriate protein concentrations of cell lysates were prepared
in Laemmli solubilisation buffer containing β-mercaptoethanol
and analyzed by sodium dodecyl sulfate polyacrylamide (SDS-
PAGE) gel electrophoresis. Separated proteins were transferred
to polyvinyldiene difluoride membranes (Roth GmbH, Karlsruhe,
Germany) and immunoblotted with the appropriate dilutions of
the primary antibody overnight at 4 °C. Primary antibodies were:
Polyclonal rabbit antibody detecting mouse tau phosphorylated at
serine396 (1:1000) purchased from Acris Antibodies GmbH
(Herford, Germany), a polyclonal antibody directed against the
N-terminus of tau (1:1000) that was a kind gift of Dr. Luc Buee
(Inserm, Lille, France) and a polyclonal antibody against β-
catenin Acris Antibodies GmbH (Herford, Germany). The
membranes were washed with Tris-buffered Saline-Tween 20
(TBS-T) three times for 5min and incubated with a 1:5000
Scientific
Software,
Inc.,
Santa
Fe,
NM,
USA;
www.eyesopen.com)29 provided sorting of the poses based on its
standard options.
4.4. Cell culture and preparation of mouse primary cortical
neurons

dilution of goat anti-rabbit or anti-mouse horseradish peroxidise-
conjugated secondary antibodies for 1 h at room temperature.
Following washing with TBS-T, proteins were detected by
chemiluminescence using the enhanced chemiluminescence
system (GenScript Piscataway, NJ, USA) on a Fluorochem 8800
imaging station (Alpha Innotech, CA, USA). TIFF files of the
images were viewed in ImageJ and band intensity was quantified
with ImageJ software. The intensity measurements of the bands
were normalized to the levels of actin. The results from three
independent experiments were presented in graphs as mean±SD.
containing various concentrations (10 μΜ or 100 μM) of
NCI8642 and NCI8642 derivatives and incubated for 24 h at 37
°C. In all cases the final concentration of DMSO was ˂ 0.1 %. 20
μl of MTT reagent (2.5 mg/ml MTT in PBS) was added to each
well and incubated for 4 h. The resulting formazan dye was
extracted with 100 μl isopropanol/HCl (100 ml isopropanol + 833
μl HCl) and the absorbance was measured spectrophotometrically
at a wavelength of 545 nm.
4.9. Statistical analysis
All experiments were repeated three times. One-way ANOVA
with Bonferroni’s Multiple Comparison Test was used to
evaluate the statistical significance of the differences. Statistical
4.8. MTT assay
SH-SY5Y cells were seeded in 96-well plates at a density of
25000 cells/well and 15000 cells/well and grown at 37 °C in an
atmosphere of 5% CO2. Then, medium was changed to that
significance
was
defined
as
p<0.05.
12. Glantschnig, H.; Hampton, R. A.; Lu, P.; Zhao, J. Z.; Vitelli, S.;
Huang, L.; Haytko, P.; Cusick, T.; Ireland, C.; Jarantow, S. W.;
Ernst, R.; Wei, N.; Nantermet, P.; Scott, K. R.; Fisher, J. E.;
Talamo, F.; Orsatti, L.; Reszka, A. A.; Sandhu, P.; Kimmel, D.;
Flores, O.; Strohl, W.; An, Z.; Wang, F. J. Biol Chem. 2010, 285,
40135–40147.
13. Yaccoby, S.; Ling, W.; Zhan, F.; Walker, R.; Barlogie, B.;
Shaughnessy, J. D., Jr. Blood, 2007, 109, 2106–2111.
14. Iozzi, S.; Remelli, R.; Lelli, B.; Diamanti, D.; Pileri, S.; Bracci, L.;
Roncarati, R.; Caricasole, A.; Bernocco S. Functional ISRN
Acknowledgments
The authors would like to thank Openeye Scientific Software,
Inc., Santa Fe, NM, USA; www.eyesopen.com, for providing an
academic license of their programs. This programme was
implemented within the framework of the Operational
Programme «Education and Lifelong Learning» of NSRF:
ARISTEIA II. It was co-funded by the European Union and
national resources.
Molecular
Biology,
2012,
Article
ID
823875,
doi:10.5402/2012/823875
15. Li, X.; Shan, J.; Chang, W.; Kim, I.; Bao, J.; Lee, H. J.; Zhang, X.;
Samuel, V. T.; Shulman, G. I.; Liu, D.; Zheng, J. J.; Wu, D. Proc
Natl Acad Sci U S A. 2012, 109, 11402-11407.
16. a) Hara, K.; Okamoto, M.; Aki, T.; Yagita, H.; Tanaka, H.;
Mizukami, Y.; Nakamura, H.; Tomoda, A.; Hamasaki, N.; Kang,
D. Mol Cancer Ther. 2005, 4, 1121-1127. b) Yang, Y.; Gao, P.;
Liu, Y.; Ji, X.; Gan, M.; Guan, Y.; Hao, X.; Li, Z.; Xiao, C.;
Bioorg Med Chem Lett. 2011, 21, 3943-3946. c) Prinz, H.;
Chamasmani, B.; Vogel, K.; Böhm, K. J.; Aicher, B.; Gerlach, M.;
Günther, E. G.; Amon, P.; Ivanov, I.; Müller, K. J Med Chem.
2011, 54, 4247-4263.
17. a) Ge, J. F.; Arai, C.; Yang, M.; Bakar, Md. A.; Lu, J.; Ismail, N.
S.; Wittlin, S.; Kaiser, M.; Brun, R.; Charman, S. A.; Nguyen, T.;
Morizzi, J.; Itoh, I.; Ihara, M. ACS Med. Chem. Lett. 2010, 1, 360–
364. b) Marcu, A.; Schurigt, U.; Müller, K.; Moll, H.; Krauth-
Siegel, R. L.; Prinz, H. Eur J Med Chem. 2015, 108, 436-443. c)
Frade, V. H.; Sousa, M. J.; Moura, J. C.; Gonçalves, M. S. Bioorg
Med Chem. 2008, 16, 3274-3282.
18. Mpousis, S.; Thysiadis, S.; Avramidis, N.; Katsamakas, S.;
Efthimiopoulos, S.; Sarli, V. Eur J Med Chem, 2015, 108, 28-38.
19. The PyMOL Molecular Graphics System, Version 1.4.1,
Schrödinger, LLC.
20. Cheng, Z.; Biechele, T.; Wei, Z.; Morrone, S.; Moon, R. T.;
Wang, L.; Xu, W. Nat Struct Mol Biol 2011, 18, 1204-1210.
21. Martinek, M.; Kotouček, M.; Ružička E. Monatshefte für Chemie
und verwandte Teile anderer Wissenschaften, 1967, 98, 1532-
1536.
22. Mosmann, T. J Immunol Methods, 1983, 65, 55–63.
23. Liu, C.; Li, Y.; Semenov, M.; Han, C.; Baeg, G. H.; Tan, Y.;
Zhang, Z.; Lin, X.; He, X. Cell. 2002, 108, 837-47.
24. Aberle, H.; Bauer, A.; Stappert, J.; Kispert, A.; Kemler, R. EMBO
J. 1997, 16, 3797-3804.
25. Li, V. S.; Ng, S. S.; Boersema, P. J.; Low, T. Y.; Karthaus, W. R.;
Gerlach, J. P.; Mohammed, S.; Heck, A. J.; Maurice, M. M.;
Mahmoudi, T.; Clevers, H. Cell 2012, 149, 1245-1256.
26. Wu, D.; Zhang, Y.; Liu, P.; Li, X.; Zhang, J.; Shan, J.; Engelhardt,
D. European Patent 2012, EP1758562 B1, 19.
27. O'Boyle, N. M.; Banck, M.; James, C. A.; Morley, C.;
Vandermeersch, T.; Hutchison, G. R., J Cheminform 2011, 3, 33.
28. (a) Hawkins, P. C.; Nicholls, A. J Chem Inf Model 2012, 52,
2919-2936; (b) Hawkins, P. C.; Skillman, A. G.; Warren, G. L.;
Ellingson, B. A.; Stahl, M. T. J Chem Inf Model 2010, 50, 572-
584.
References and notes
1.
a) Goedert, M.; Spillantini, M. G.; Jakes, R.; Rutherford, D.;
Crowther, R. A. Neuron, 1989, 3, 519–526. b) Hardy, J.; Selkoe,
D. J. Science 2002, 297, 353–356. c) Grundke-Iqbal, I.; Iqbal, K.;
Quinlan, M.; Tung, Y. C.; Zaidi, M. S.; Wisniewski, H. M. J Biol
Chem. 1986, 261, 6084–6089.
2.
a) Kosik, K. S.; Joachim, C. L.; Selkoe, D. J. Proc Natl Acad Sci
USA. 1986, 83, 4044–4048. b) Alonso, A.; Zaidi, T.; Novak, M.;
Grundke-Iqbal, I.; Iqbal. K. Proc Natl Acad Sci U S A. 2001, 98,
6923–6928. c) Thangavel, R.; Stolmeier, D.; Yang, X.;
Anantharam, P.; Zaheer, A. Neuropathol Appl Neurobiol. 2011,
38, 572–581.
3.
a) Selkoe, D. J. Cold Spring Harb Perspect Biol 2011, doi:
10.1101/ cshper spect.a004457 b) da Cruz e Silva, O. A.;
Henriques, A. G.; Domingues, S. C.; da Cruz e Silva, E. F. CNS
Neurol Disord Drug Targets. 2010, 9, 720-726. c) Killick, R..;
Ribe, E. M.; Al-Shawi, R.; Malik, B.; Hooper, C.; Fernandes, C.;
Dobson, R.; Nolan, P. M.; Lourdusamy, A.; Furney, S.; Lin, K.;
Breen, G.; Wroe, R.; To, A. W.; Leroy, K.; Causevic, M.; Usardi,
A.; Robinson, M.; Noble, W.; Williamson, R.; Lunnon, K.; Kellie,
S.; Reynolds, C. H.; Bazenet, C.; Hodges, A.; Brion, J. P.;
Stephenson, J.; Simons, J. P.; Lovestone, S. Mol Psychiatry. 2014
19, 88-98. d) Bruggink, K. A.; Kuiperij, H. B.; Gloerich, J.; Otte-
Höller, I.; Rozemuller, A. J.; Claassen, J. A.; Küsters, B.;
Verbeek, M. M.; J Neurochem. 2015, 134, 1152-1162. e) Wan,
W.; Xia, S.; Kalionis, B.; Liu, L.; Li, Y. Biomed Res Int. 2014,
2014, 301575.
4.
5.
6.
Kawano, Y.; Kypta, R. J. Cell Sci. 2003, 116, 2627–2634.
Brott, B. K.; Sokol, S. Y. Mol Cell Biol. 2002, 22, 6100–6110.
Caricasole, A.; Copani, A.; Caraci, F.; Aronica, E.; Rozemuller,
A. J.; Caruso, A.; Storto, M.; Gaviraghi, G.; Terstappen, G. C.;
Nicoletti, F. J. Neurosci. 2004, 24, 6021–6027.
7.
Cappuccio, I.; Calderone, A.; Busceti, C. L.; Biagioni, F.;
Pontarelli, F.; Bruno, V.; Storto, M.; Terstappen, G. T.; Gaviraghi,
G.; Fornai, F.; Battaglia, G.; Melchiorri, D.; Zukin, R. S.; Nicoletti
F.; Caricasole, A. J. Neurosci. 2005, 25, 2647–2657.
8.
9.
Salcedo-Tello, P.; Hernández-Ortega, K.; Arias, C. J Alzheimers
Dis. 2014, 39, 775-785.
Doble, B. W.; Woodgett, J. R. J Cell Sci. 2003, 116, 1175–1186.
29. (a) McGann, M. J Chem Inf Model 2011, 51, 578-96; (b)
McGaughey, G. B.; Sheridan, R. P.; Bayly, C. I.; Culberson, J. C.;
Kreatsoulas, C.; Lindsley, S.; Maiorov, V.; Truchon, J. F.;
Cornell, W. D. J Chem Inf Model 2007, 47, 1504-19; (c) McGann,
10. Purro, S. A.; Dickins, E. M.; Salinas P. C. J Neurosci. 2012, 32,
3492-3498.
11. Purro, S. A.; Galli, S.; Salinas, P. C. J Mol Cell Biol. 2014, 6, 75-
80.

M. R.; Almond, H. R.; Nicholls, A.; Grant, J. A.; Brown, F. K..
Biopolymers 2003, 68, 76-90.
Supplementary Material
30. Lovering, F.; Bikker, J.; Humblet, C., J Med Chem 2009, 52,
6752-6756.
Characterization data of the described compounds (1H NMR
and 13C NMR spectra), predicted pharmacokinetic properties and
docking results. This material is available free of charge via the
Internet at….