Novel protein kinase inhibitors related to tau pathology modulate tau protein-self interaction using a luciferase complementation assay

Article abstract of DOI:10.3390/molecules23092335

The current number of drugs available for the treatment of Alzheimer's disease (AD) is strongly limited and their benefit for therapy is given only in the early state of the disease. An effective therapy should affect those processes which mainly contribu

Full text of DOI:10.3390/molecules23092335

molecules
Communication
Novel Protein Kinase Inhibitors Related to Tau
Pathology Modulate Tau Protein-Self Interaction
Using a Luciferase Complementation Assay
Max Holzer 1 , Nico Schade 2, Ansgar Opitz 2, Isabel Hilbrich 1, Jens Stieler 1, Tim Vogel 1,
Valentina Neukel 1, Moritz Oberstadt 3, Frank Totzke 4, Christoph Schächtele 4,
Wolfgang Sippl 2 and Andreas Hilgeroth 2,
*
1
Department for Molecular and Cellular Mechanisms of Neurodegeneration,
Paul Flechsig Institute for Brain Research, University of Leipzig, Liebigstraße 19, 04103 Leipzig, Germany;
max.holzer@medizin.uni-leipzig.de (M.H.); isabel.hilbrich@medizin.uni-leipzig.de (I.H.);
jens.stieler@medizin.uni-leipzig.de (J.S.); tim.vogel.le@gmail.com (T.V.);
valentina.neukel@hs-furtwangen.de (V.N.)
Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, 06120 Halle, Germany;
nico.schade@pharmazie.uni-halle.de (N.S.); ansgar.opitz@pharmazie.uni-halle.de (A.O.);
wolfgang.sippl@pharmazie.uni-halle.de (W.S.)
Department of Neurology, University of Leipzig, Liebigstraße 20, 04103 Leipzig, Germany;
moritz.oberstadt@medizin.uni-leipzig.de
ProQinase GmbH Freiburg, Breisacher Straße 117, 79106 Freiburg, Germany;
f.totzke@proqinase.com (F.T.); c.schaechtele@proqinase.com (C.S.)
2
3
4
*
Correspondence: andreas.hilgeroth@pharmazie.uni-halle.de; Tel.: +49-345-55-25168
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 27 June 2018; Accepted: 8 September 2018; Published: 12 September 2018
Abstract: The current number of drugs available for the treatment of Alzheimer’s disease (AD)
is strongly limited and their benefit for therapy is given only in the early state of the disease.
An effective therapy should affect those processes which mainly contribute to the neuronal decay.
There have been many approaches for a reduction of toxic Aβ peptides which mostly failed to
halt cognitive deterioration in patients. The formation of neurofibrillary tangles (NFT) and its
precursor tau oligomers have been suggested as main cause of neuronal degeneration because of a
direct correlation of their density to the degree of dementia. Reducing of tau aggregation may be
a viable approach for the treatment of AD. NFT consist of hyperphosphorylated tau protein and
tau hyperphosphorylation reduces microtubule binding. Several protein kinases are discussed to
be involved in tau hyperphosphorylation. We developed novel inhibitors of three protein kinases
(gsk-3
tau–tau interaction as a nucleation stage of a tau aggregation in cells. Strongest effects were observed
for those inhibitors with effects on all the three kinases with emphasis on gsk-3 in nanomolar ranges.
β, cdk5, and cdk1) and discussed their activity in relation to tau phosphorylation and on
β
Keywords: AD drug discovery; synthesis; derivatives; structure-activity; lead structure
1. Introduction
Alzheimer’s disease (AD) is a worldwide disease affecting more and more people preferably in
the higher years of age [
The progressive neurodegenerative disease is characterized by deteriorating cognitive functions and a
loss of memory [ ]. Presently, the disease cannot be cured even if it is recognized at an early state.
The clinically available therapeutics are acetylcholine esterase inhibitors that increase the neuronal
1,2]. It is predicted that by 2050 one of 85 people globally will have AD [3,4].
1,5
availability of the lowered acetylcholine levels [
1
,6,
7]. Memantine is used as N-methyl-D-aspartate
Molecules 2018, 23, 2335; doi:10.3390/molecules23092335
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2335
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glutamate receptor antagonist to prevent an ongoing stimulation of excitatoric neurons [
8]. However,
those drugs cannot combat the cause of the disease. Thus, the drugs’ benefit is limited to just the early
stage of disease.
A
β
peptides processed from the amyloid precursor protein (APP) by the activity of
-secretase form neurotoxic oligomers by aggregation [ ]. Secretase inhibitors were partly
disappointing in clinical trials so far with an observed worsening of cognitive functions [ 11]. Other
-related strategies to prevent amyloid aggregation or remove amyloid by immunotherapeutics
failed so far to halt cognitive deterioration [ 12]. Reasons for that have been critical side effects or a
β- and
γ
1,6
6,9–
A
β
6,9,
doubtful efficiency for a perspective clinical outcome [6,13].
Another key element in AD is hyperphosphorylated tau protein that forms tau filaments
which further assemble to aggregates named neurophil threads and neurofibrillary tangles [6,14,15].
The density of those aggregates is well correlated with the degree of dementia in most studies of AD
patients [16]. Therefore, a reasonable strategy has been to identify effective tau aggregation blockers.
Although first experimental results have been promising, no compounds progressed into clinical
practice so far [1,17,18].
Another strategy may be the prevention of tau hyperphosphorylation by the inhibition of tau
kinases. Tau protein with an abnormal high degree of phosphorylation is hindered from binding
to microtubules increasing free tau protein concentration and is missorted to the somatodendritic
compartment. A major tau kinase is the glycogen synthase kinase-3
β
(gsk-3β) [19]. Early inhibitors
such as indirubins [20], maleimides [21] and thiazoles [22] of gsk-3
β
have been ATP-competitive,
but did not reach clinical trials due to a limited specificity and toxic effects [23]. Lithium as almost
weak and noncompetitive inhibitor showed promising effects in used mouse models of tauopathies
to prevent the development of tau pathology in the early stage of the disease [24]. From other
noncompetitive inhibitors such as the thiadiazolidindiones (TDZDs) and the alkaloid manzamine
with an allosteric binding mode tideglusip as TDZD reached clinical trials with a so far disappointing
efficiency [25
developed which are phosphorylated by gsk-3
mouse model, they showed a lowered A load with reduced A
Irreversible gsk-3 inhibitors of the halomethylketones presently showed only weak inhibitory
activity [25]. One published multitarget inhibitor of gsk-3 of the 2,4-dihydropyrano[2,3-c]pyrazole
–
27]. Alternatively, substrate-competitive inhibitors with a peptide scaffold have been
and then react as inhibitors [25 28]. In an AD
aggregates and fibrils detected [28].
β
,
β
β
β
β
family showed inducing in vitro effects on antioxidant and anti-inflammatory enzymes which result
from the inhibition of gsk-3β at micromolar concentrations [29].
We wondered whether a kinase inhibitor with a broader activity towards tau-relevant kinases such
as cdk5 and cdk1 may be a promising alternative for a perspective AD drug development. In addition,
we tested whether such inhibitors not only affect tau phosphorylation but as a functional read-out
also attenuate tau protein self-interaction because tau-dimer formation is the rate-limiting step in tau
aggregation [30].
We developed novel small-molecule inhibitors with a benzofuropyridine scaffold and evaluated
their properties to inhibit three most prominent kinases related to tau pathology. One earlier
benzofuropyridine showed some cdk1 inhibitory properties beside a residual gsk-3β affinity [31].
Compound structure-dependent effects on kinase inhibition are discussed and first lead compounds
could be identified. A preliminary proof-of-concept for a perspective reduced tau aggregation was
evaluated in a tau interaction assay system to reflect a correlation of the compounds potency of kinase
inhibition and a reduced tau interaction. Here, the roles of both the number and the single kinase
potency are discussed related to the assay aggregation results.

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2. Results and Discussion
2.1. Synthesis of the Benzofuropyridines
First, the 3-alkoxy pyridines 1a and
b
were synthesized from 3-hydroxypyridine by the use of the
corresponding alkyl bromides in a strong alkaline THF solution and tetra-n-butyl ammonium bromide
as phase transfer catalyst (Scheme 1).
R
OH
a
N
N
1a, R = OEt
1b, R = OBn
R
R
R
c
b
+
N
N
N
Cl-
1a, b
1c, R = OMe
1d, R = F
O
O
2a-d
d
e
HO
R
HO
O
N
R
3a-d
O
N
4a, b
f
3b, 4a
3e, 4c
Scheme 1. Synthesis of the benzofuropyridines 3a
12 h reflux; ( ) CuI, Me2S, MeCOCl, THF, 15 min
) p-benzoquinone, HClO4, dioxane; ( ) 1,4-naphthoquinone, HClO4, dioxane; and (
MeOH, 2 bar.
–
e
and 4a
−
–
c
: (
) PhMgCl, THF, 15 min
) H2, Pd/C,
a
) KOH, nBu4NBr, alkyl Br, THF,
b
20 ◦C; (
c
−
20 ◦C;
(
d
e
f
The pyridine compounds 1a
sulphide addition. After cooling down the temperature to
–
d
were then dissolved in THF under copper(I) iodide and dimethyl
◦
−
20 C, acetyl chloride was added dropwise
and the reaction mixture was stirred for 15 min at the low temperature. Then, phenyl magnesium
chloride was added as solution in THF and stirring continued for additional 15 min. After work up of
the mixture, the 1,4-dihydropyridines 2a–d were yielded from a methanol solution under cooling.
A double set of proton signals was found in the compound spectra due to the existence of two
sets of isomers A and B, one with the acetyl function on the right site of the molecule as isomer A
and one with that acetyl function on the left side of molecule as isomer B. The isomers result from
a hindered rotability of the C-acetyl-N-pyridine bond due to a double bond character of that bond
typical for such amide bonds.

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The 1,4-dihydropyridines were then dissolved in dry dioxane containing 10% perchloric acid
and the corresponding quinones were added to give either the benzoquinone compounds 3a
or the naphthoquinone compounds 4a The reaction mechanism is suggested to start with
–
d
,b.
a primary Michael addition of the electrophilic protonated quinone to the nucleophilic C-5 of
the 1,4-dihydropyridine. Then, one quinone oxygen might have attacked the C-6 atom of the
dihydropyridine to form a tetrahydro cycloaddition product. Excess of quinone then aromatized
the scaffold under removing the acetyl residue to give the benzofuropyridine target compounds.
Finally, the benzyl residues in the 3-benzyloxy compounds 3b and 4a were cleaved in methanol using
hydrogen and palladium/charcoal as catalyst and a pressure of 2 bar to give the 3-hydroxy compounds
3e and 4c, respectively.
Spectroscopical target compound characteristics have been a low field shift of the 5-protons next
to the hydroxyl function to about 6.5 ppm resulting from a shielding effect of the 4-phenyl residue that
does not lie in a plane with the molecular scaffold due to the 3-substituents.
2.2. Protein Kinase Inhibition
Tau protein hyperphosphorylation is mediated by an imbalance of protein kinases and
phosphatase activity. Most prominent kinase has been gsk-3β that is discussed to interfere additionally
with A pathology [32]. A constitutive activation of the cyclin dependent kinase 5 (cdk5) by p25 as
β
the truncated cdk5-regulative subunit p35 is discussed as causative for the cdk5 overactivation in
AD brains, thus contributing to the tau hyperphosphorylation [33]. Moreover, recent studies prove
that overactive cdk5 induces cell cycle regulating kinase cdk1 [34]. Such an aberrant cdk1 activation
triggers neuronal death and thus potentiates the AD pathology. Thus, an inhibition of those kinases
may have a strong benefit to prevent the tau-mediated AD pathology.
The protein kinase inhibitory activity has been determined in an assay system using the respective
kinases, the kinase substrates and ATP with incorporated radioactive phosphorus P33 together with
increasing inhibitor concentrations. The degree of the reduced substrate phosphorylation has been
determined by scintillation counting and the inhibitory compound activity has been calculated
accordingly. The results are shown in Table 1.
Table 1. Protein kinase affinities of target compounds 3a–e and 4a–c.
Protein Kinase Affinities (µM) a
Cpd.
R
cdk1
cdk5/p25
gsk-3β
3a
3b
3c
3d
3e
4a
4b
4c
OEt
OBn
OMe
F
OH
OBn
F
0.17 ± 0.031
2.30 ± 0.33
0.46 ± 0.022
0.083 ± 0.034
5.80 ± 1.20
0.062 ± 0.023
0.38 ± 0.93
0.024 ± 0.0021
1.60 ± 0.23
190 ± 37
b
n.a.
0.14 ± 0.023
1.40 ± 0.24
0.51 ± 0.012
b
n.a.
0.013 ± 0.0031
0.091 ± 0.012
1.06 ± 0.63
0.11 ± 0.043
2.10 ± 0.24
160 ± 34
0.073 ± 0.012
b
OH
0.77 ± 0.062
0.012 ± 0.013
a
Mean of three determinations. not active.
The 3-ethoxy function in compound 3a resulted in a submicromolar affinity towards cdk1 and
cdk5 and a nanomolar one towards gsk-3 . The replacement of the ethyl with a benzyl function in
β
compound 3b was less tolerated by all the kinases with decreases in activity and a best micromolar
activity towards cdk1. The 3-methoxy function in compound 3c resulted in similar affinities towards all
the three kinases than the 3-ethoxy substitution. If the 3-alkoxy function was replaced with a hydroxyl
function in derivative 3e we found best activities towards all the kinases reaching nanomolar ranges
for both cdk1 and gsk-3
β
and a lowest submicromolar affinity towards cdk5. A oxygen-free 3-fluoro
in
function in compound 3d was less favourable. However, we found better affinities towards gsk-3
β

Molecules 2018, 23, 2335
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submicromolar and towards cdk1 in low micromolar ranges than those determined for the 3-benzyloxy
substituted compound 3b.
Next, we evaluated the benzo-annelated compounds 4a–c. The benzo-annelated compound 4a
with the 3-benzyloxy substitution showed an overall improved kinase inhibitory activity compared to
compound 3b without the additional phenyl ring. The cdk1 inhibitory activity reached nanomolar
ranges, the gsk-3
4b with the additional phenyl ring showed a slightly improved cdk1 affinity compared to compound
3d without the attached phenyl residue. The gsk-3 affinity was just residual similar to a determined
β affinity increased and a micromolar cdk5 activity was found. The 3-fluoro compound
β
cdk5 activity that was not found for compound 3d. The attached phenyl residue in derivative 4c
with the 3-hydroxy function resulted in almost unchanged activities if compared to the derivative 3e
without that phenyl substituent. However, best nanomolar affinities towards cdk5 and gsk-3
β
were
reached for compound 4c.
Overall, it can be stated that the 3-hydroxy function in both compound types
best kinase inhibitory activities.
3 and 4 resulted in
Docking studies have been carried out to demonstrate the binding mode and explain observed
activities of respective compounds. The 3-hydroxy compounds 3e and 4c are able to form three
hydrogen bridge bonds to amino acid residues of the protein backbone in the ATP binding pocket
as demonstrated for compound 3e in the case of gsk-3β with bonds of the 3-hydroxy function to
asparagine200 via a localized water molecule, the 6-hydroxy function with a direct bond to the NH
function of arginine141 and, finally, the pyridine nitrogen with a bond to the NH function of valine135
of the catalytic site (see Figure S1 in the Supplementary Materials). If the 3-hydroxy function is replaced
with a benzyloxy residue only two hydrogen bridge bonds are observed for compound 4a to explain the
lowered activity. Docking to both cdk1 and cdk5 shows each hydrogen bridge bonds of the 6-hydroxy
function of 4a to the carbonyl function of isoleucine10 of each enzyme and the pyridine nitrogen to
the respective amino acid of the catalytic site of cdk1 with leucine83 and of cdk5 with cysteine83 (see
Figure S2a,b in the Supplementary Materials). The benzyloxy residue shows hydrophobic interactions
with each phenyl residue of phenylalanine80 as gatekeeper amino acid. In compound 4b with a fluoro
substitution such interactions are not possible and thus a decrease in activity was observed.
A first protein kinase profiling has been carried out with compound 3e which was evaluated to
inhibit kinases of five kinase families. We found no activity towards kinases of the PKA family (PKC-
, - and -iota), towards kinases of the receptor tyrosine kinase family (VEGFR2, ErBB2 and TIE2),
and towards WEE1 and CK1- of the casein kinase family. Towards cdk6 of the family of the cyclin
dependant kinases the activity was 829 M and towards EGFR of the receptor tyrosine kinases 523 M.
Finally, the activity towards Src of the SRC family was evaluated with 230 µM.
Next, we investigated three of our compounds 3b 4a and 3a to inhibit the tau phosphorylation in
α,
-
γ ε
α
µ
µ
,
tau transfected COS-7 cells. They showed increasing activities towards the kinases from 3b to 3a as
discussed and thus were expected to result in a compound-dependent reduced tau phosphorylation
related to a lowered activity. Moreover, they were selected with a favourite solubility in the assay
system. We used AT180 monoclonal antibody to detect the tau phosphorylation of threonine 231 and
serine 235 as relevant amino acids for tau phosphorylation by the respective kinases using Western
blot technique. The results are shown in Figure 1.
Compound 3b with a just micromolar affinity towards two kinases (cdk1, gsk-3β) resulted in a
reduction of amino acid phosphorylation of 19% compared to the untreated control. Compound 4a
with an additional nanomolar affinity towards cdk1 reduced tau amino acid phosphorylation by 55%.
Finally, compound 3a with an increased submicromolar affinity towards two kinases (cdk5, gsk-3β)
reduced the tau phosphorylation by 61%. The data demonstrate an expected correlation of the degree of
protein kinase inhibition with the tau phosphorylation in living cells of AD-relevant phosphorylation.

Molecules 2018, 23, 2335
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Figure 1. Quantification of threonine 231 und serine 235 tau phosphorylation after 24 h inhibitor
treatment at 8 M concentration of 2N4R-tau transfected COS-7 cells by Western blot analysis.
µ
Phospho-dependent immunoreactivity was normalized to total tau immunoreactivity and depicted as
mean ± SD.
2.3. Tau Protein Interaction Assay
An overall increase in tau phosphorylation results in a reduced microtubule binding and
increased pool of free tau protein, which is more prone to aggregation by laws of mass action [35].
Protein complementation assays have recently been employed to visualize and measure protein
oligomerization and aggregate propagation of amyloidogenic proteins such as tau protein, SOD1,
TDP43 or alpha synuclein in cultured cells [36–40].
In our tau protein interaction assay in living cells, the intracellular luciferase activity is
reconstituted, when two or more tau molecules oligomerize and come in close contact, thereby aligning
the two split luciferase fragment in close proximity. Two different NanoLuc luciferase subunits (large
Bit (lgBit), an 18 kDa polypeptide, and small bit (smBit), a 1.3 kDa peptide) have been fused to
the N-terminal and C-terminal site of 441aa 2N4R human tau isoform, respectively. This optimal
configuration (N-terminal or C-terminal fusion) has been tested. The NanoBit subunits only weakly
associate by themselves, so that the reconstitution of luciferase activity is only dependent on tau protein
self-interaction. A human liver cell line (HuH-7), devoid of endogenous tau protein was transfected
with the two fusion constructs and after 24 h post-transfection cells were subcultured in a 384-well plate
for inhibitor treatment and luminescence detection. Further, to detect off-target effects of compounds
such as toxicity or interference with luciferase reaction, protein homeostasis and thymidine kinase
promoter activity, we applied the same compounds onto HuH-7 cells transfected with a construct
expressing full length constitutively-active NanoLuc luciferase driven by the same promoter like the
tau-fusion constructs. Therefore, results of tau protein self-interaction were normalized with respect to
full length NanoLuc activity after inhibitor treatment to even rule out such side effects. Toxicity at
10
µM inhibitor concentration has additionally been measured using the WST-8 assay as described [39]
and has been found negligible.
The assay is a straightforward method to determine the effects on tau protein self-interaction in a
short time-frame of 60 min, where effects are mainly caused by direct effects on tau phosphorylation
or indirect by affecting tau scaffolding proteins and can be related to the kinase inhibitors potency.
The luminescence signal determined for vehicle control (0.1% DMSO) is set to 100% and increase or
decrease of interaction is given in percent of vehicle control.
Thus, an observed correlation of the protein kinase inhibitory potential of our compounds with the
reduction of tau self-interaction would be a proof-of-concept that lesser phosphorylated tau regained

Molecules 2018, 23, 2335
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microtubule binding and/or has a reduced propensity for self-interaction, which reduces the pool of
free tau protein prone to tau aggregation.
The two 3-hydroxy substituted compounds 3e and 4c with nanomolar protein kinase inhibitory
activities towards two kinases and highest gsk-3
luminescence of 38% and 29% at the lower concentration of 1
β
inhibitor potency showed a reduction of
M (Figure 2a, above), which resulted in
µ
a higher inhibition of 71% and 84% at the 10 µM inhibitor concentration, respectively (Figure 2b).
Figure 2. Modulation of tau interaction by protein kinase inhibitors. Tau protein self-interaction was
assessed using a split-luciferase assay. Luminescence readings were related to vehicle control (Co) and
normalized to the full length constitutively-active NanoLuc luciferase and depicted as mean
) Inhibitor concentration of 1 M and ( ) inhibitor concentration of 10 M. * p < 0.05; ** p < 0.01,
*** p < 0.001.
±
SD.
(
a
µ
b
µ
Both 3-alkoxy substituted compounds 3a and 3c caused only a mild inhibition of tau interaction
at a 1 M concentration of 7% for the 3-methoxy substituted derivative 3c and of 22% for the 3-ethoxy
substituted of compound 3a. Tau interaction is inhibited by 65% (3a) and 75% (3c) at the higher
concentration of 10 M that may be related to a partly reduced protein kinase inhibitory activity in
nanomolar ranges towards just one kinase compared to best compounds 3e and 4c. The 3-benzyloxy
µ
µ

Molecules 2018, 23, 2335
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substitution of compound 3b with just micromolar affinities towards two kinases resulted in a weak
inhibitory activity of 15% at 1 M and with a just 38% reduced luminescence at 10 M, similar to the
µ
µ
3-fluoro substitution of compound 3d with a 12% luminescence reduction at the lower and 40% at the
higher inhibitor concentration and a reduced compound activity towards one kinase in submicromolar
and one in micromolar ranges. The benzo-annelated compound 4b at a 1 µM concentration caused a
luminescence inhibition of 23% compared to a 12% inhibition of compound 3d without that phenyl
substitution. Using the benzo-annelated compound 4c, we found an increased luminescence inhibition
compared to compound 3e at 10 µM.
Summarizing our results, it can be stated that the split luciferase interaction assay correlates
with the differential protein kinase inhibitory activity of the compounds. Kinase inhibitors with a
nanomolar Ki towards gsk-3β in combination with a nanomolar inhibition of a second kinase cdk5
or cdk1 proved the best inhibitors of tau self-interaction. Only compound 4a showed an unexpected
low influence in the tau interaction that may be plausible with a partial observed precipitation of the
compound in the assay system. Cellular compound toxicity studies using the WST-8 assay [39] have
been carried out to demonstrate a non-toxicity of the compounds, so that a reduced luminescence
under compound application is not influenced by a cellular compound toxicity (data not shown).
As proof-of-concept it can be stated that a kinase inhibitor-induced reduced tau phosphorylation leads
to a lowered tau interaction and may diminish tau pathology by influencing tau aggregation.
3. Material and Methods
3.1. Chemical Reagents and Instruments
Commercial reagents were used without further purification. The 1H-NMR spectra (400 MHz)
were measured using tetramethylsilane as internal standard. TLC was performed on E. Merck 5554
silica gel plates. The high-resolution mass spectra were recorded on a Finnigan LCQ Classic mass
spectrometer. Elemental analysis indicated by the symbols of the elements was within
theoretical values and was performed using a Leco CHNS-932 apparatus.
±
0.4% of the
3.2. General Procedure for the Synthesis of Compounds 1
Five grams (52.6 mmol) 3-hydroxypyridine, 5.9 g (110 mmol) pulverized potassium hydroxide
and 0.85 g (2.6 mmol) nBu4NBr were dissolved in 150 mL of dried THF. After addition of 84 mmol of
the respective alkyl halogenide the mixture was stirred for 12 h under reflux. The reaction was finished
with the addition of 200 mL water and extraction with each 100 mL of hydrochloric acid (10%) for
two times. The unified water phases were alkalined with a 10 M sodium hydroxide solution and then
extracted with each 75 mL of chloroform for three times. After removal of the solvent of the unified
organic layers in vacuum, the raw product was purified using column chromatography technique over
silica gel and an eluent mixture of cyclohexane and ethyl acetate in a relation of 1:1.
1
3-Ethoxypyridine (1a). Yield 78%; IR (ATR)
ν
= 1260 (C-O-C) cm−1; H-NMR (CDCl3)
δ
= 8.26 (dd,
J = 2.7, 0.8 Hz, 1H, 2-H), 8.16 (dd, J = 4.4, 1.7 Hz, 1H, 6-H), 7.16 (ddd, J = 8.4, 4.2, 0.8 Hz, 5-H), 7.12
(ddd, J = 8.4, 2.7, 1.7 Hz, 1H, 4-H), 4.03 (q, J = 7 Hz, 2H, OC
H2CH3), 1.39 (t, J = 7 Hz, 3H, OCH2CH3);
m/z (ESI) 124.16 (M + H+).
3-Benzyloxypyridine (1b). Yield 59%; IR (ATR)
ν δ = 8.37 (d,
= 1260 (C-O-C) cm−1; 1H-NMR (acetone-d6)
J = 2.9, Hz, 1H, 2-H), 8.18 (dd, J = 4.6, 1.3 Hz, 1H, 6-H), 7.57–7.5 (m, 2H, 20-, 60-H), 7.41–7.31 (m, 4H,
4-H, 30-, 40-, 50-H), 7.26 (ddd, J = 8.4, 4.6, 0.6 Hz, 1H, 5-H), 5.18 (s, 2H, OC
H2C6H5); m/z (ESI) 186.28
(M + H+).
3.3. General Procedure for the Synthesis of Compounds 2
First, 10 mmol of the respective pyridine were dissolved in 50 mL of dried THF. After addition
of 0.5 mmol of copper(I) iodide and 3 mL of dimethyl sulphide, the mixture was stirred at room

Molecules 2018, 23, 2335
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◦
20 C. After 15 min, 10 mmol
temperature until a clear solution resulted. Then, it was cooled down to
−
of acetyl chloride were added dropwise and stirring continued for 15 min. Then, 10 mmol of phenyl
magnesium chloride were added and stirring followed for additional 15 min. Then, the mixture
warmed up to room temperature. Then, 50 mL of an ammonium chloride solution (20%) were added
and extraction with diethyl ether followed with portions of 50, 25 and again 25 mL. The unified organic
layer was extracted with each 50 mL of a solution of ammonium chloride in ammonia for two times,
with 50 mL water, hydrochloric acid (10%) for two times, water and finally a saturated solution of
sodium chloride in water. The organic layer was then dried over sodium sulphate, filtered and finally
removed under reduced pressure to give an oily substance that partially crystallized from methanol.
◦
1-(3-Ethoxy-4-phenyl-4H-pyridine-1-yl)ethanone (2a). Yield 56%; mp 124–129 C: IR (ATR)
ν = 1669
1
(
COCH3), 1633 cm−1; H-NMR (CDCl3)
δ
= 7.35–7.28 (m, 5H, 20-, 60-H of isomer A and B, 6-H of
isomer A), 7.23–7.19 (m, 6H, 30-, 40-, 50-H of isomer A and B), 6.86 (d, J = 1,2 Hz, 1H, 2-H of isomer B),
6.64 (dt, J = 8.2, 1.2 Hz, 1H, 6-H of isomer B), 6.06 (d, J = 1.2 Hz, 1H, 2-H of isomer A), 5.13 (dd, J = 8.2,
4.4 Hz, 1H, 5-H of isomer A), 5.04 (dd, J = 8.2, 4.4 Hz, 1H, 5-H of isomer B), 4.21 (d, J = 4.4 Hz, 2H, 4-H
of isomer A and B), 3.74 (ABX3, J = 9.4, 7.0 Hz, 2H, OC
2H, OC 2CH3 of isomer A), 2.25 (s, 3H, COCH3 of isomer B), 2.23 (s, 3H, COC
(AB 3, J = 6.9 Hz, 3H, OCH2CH3 of isomer A), 1.17 (AB
m/z (ESI) 244.31 (M + H+).
H
2CH3 of isomer B), 3.68 (ABX3, J = 9.0, 6.9 Hz,
H
H
3 of isomer A), 1.20
X
X3, J = 7.0 Hz, 3H, OCH2CH3 of isomer B);
◦
1-(3-Benzyloxy-4-phenyl-4H-pyridine-1-yl)ethanone (2b). Yield 58%; mp 78–82 C: IR (ATR)
ν = 1672
1
(
COCH3), 1635 cm−1; H-NMR (CDCl3)
δ
= 7.33–7.20 (m, 17H, OCH2C6H5 of isomer A and B, 30-,
40-, 50-H of isomer A and B, 6-H of isomer A), 7.13–7.10 (m, 4H, 20-, 60-H of isomer A and B), 6.98 (d,
J = 1,3 Hz, 1H, 2-H of isomer B), 6.67 (dt, J = 8.2, 1.3 Hz, 1H, 6-H of isomer B), 6.10 (d, J = 1.3 Hz, 1H,
2-H of isomer A), 5.15 (dd, J = 8.2, 4.3 Hz, 1H, 5-H of isomer A), 5.07 (dd, J = 8.2, 4.3 Hz, 1H, 5-H of
isomer B), 4.77 (AB, J = 11.8 Hz, 2H, OCH2C6H5 of isomer B), 4.74 (AB, J = 11.9 Hz, 2H, OCH2C6H5 of
isomer A), 4.31 (d br, J = 4.3 Hz, 2H, 4-H of isomer A and B), 2.26 (s, 3H, COCH3 of isomer B), 2.16 (s,
3H, COCH3 of isomer A); m/z (ESI) 306.38 (M + H+).
◦
1-(3-Methoxy-4-phenyl-4H-pyridine-1-yl)ethanone (2c). Yield 40%; mp 50–70 C: IR (ATR)
(
ν = 1674
COCH3), 1633 cm−1; 1H-NMR (CDCl3)
δ = 7.34 (dt, J = 8.2, 1.3 Hz, 1H, 6-H of isomer A), 7.34–7.28 (m,
4H, 20-, 60-H of isomer A and B), 7.24–7.20 (m, 6H, 30-, 40-, 50-H of isomer A and B), 6.90 (d, J = 1,3 Hz,
1H, 2-H of isomer B), 6.64 (dt, J = 8.2, 1.3 Hz, 1H, 6-H of isomer B), 6.07 (d, J = 1.3 Hz, 1H, 2-H of isomer
A), 5.12 (dd, J = 8.2, 4.3 Hz, 1H, 5-H of isomer A), 5.03 (dd, J = 8.2, 4.3 Hz, 1H, 5-H of isomer B), 4.23
(dd, J = 4.3, 1.3 Hz, 2H, 4-H of isomer A and B), 3.55 (s, 3H, OCH3 of isomer B), 3.52 (s, 3H, OCH3 of
isomer A), 2.27 (s spl, 6H, COCH3 of isomer A and B); m/z (ESI) 230.28 (M + H+).
◦
1-(3-Fluoro-4-phenyl-4H-pyridine-1-yl)ethanone (2d). Yield 61%; mp 69–70 C: IR (KBr)
ν
= 1674 (COCH3),
1638 cm−1; 1H-NMR (CDCl3)
δ = 7.32 (dd, J = 2.7, 1.2 Hz, 1H, 2-H of isomer B), 7.35 (dd, J = 2.7, 1.2 Hz,
1H, 2-H of isomer A), 7.34–7.22 (m, 10H, C6H5 of isomer A and B), 6.69 (dd, J = 8.2, 1,6 Hz, 1H, 6-H of
isomer A), 6.62 (dd, J = 8.2, 1.6 Hz, 1H, 6-H of isomer B), 5.18–5.11 (m, 1H, 5-H of isomer A), 5.09–5.02
(m, 1H, 5-H of isomer B), 4.44 (s br, 2H, 4-H of isomer A and B), 2.25 (s, 3H, COCH3 of isomer B), 2.21
(s, 3H, COCH3 of isomer A); m/z (ESI) 218.25 (M + H+).
3.4. General Procedure for the Synthesis of Compounds 3 and 4
First, 3.3 mmol of the respective compound
2 was dissolved in a minimum volume of freshly
distilled dried dioxane. After addition of 4.1 mmol of the respective quinone, either 1,4-benzoquinone
or 1,4-naphthoquinone, a mixture of dioxane and perchloric acid (70%) in a ratio of 22:1 (v:v) was
added until a final solution volume of 75 mL was reached. The reaction proceeding was followed by
thin layer chromatography (tlc). After 48 h of reaction 1.4 mmol of the quinone was added until no
more starting or intermediate compounds were detectable by tlc. Then, 30 mL of water were added
and the pH of the solution was adjusted to 8–9. Then, extraction with chloroform followed for three

Molecules 2018, 23, 2335
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times using each half of the volume of the water phase. The unified organic layer was dried over
sodium sulphate and filtered. The oily residue was purified by column chromatography over silica gel
using an eluent mixture of cyclohexane and ethyl acetate in a ratio of 4:1 (v:v). After evaporation of the
collected compound fractions, the target compounds crystallized from diethyl ether.
3-Ethoxy-4-phenylbenzo[4,5]furo[2,3-b]pyridine-6-ol (3a). Yield 10%; mp 231–235 ◦C; IR (KBr)
(OH), 1277 (C-O-C) cm−1; 1H-NMR (DMDO-d6)
= 9.33 (s, 1H, OH), 8.25 (s, 1H, 2-H), 7.59–7.51 (m,
5H, C6H5), 7.49 (d, J = 8.9 Hz, 1H, 8-H), 6.91 (dd, J = 8.9, 2.5 Hz, 1H, 7-H), 6.51 (d, J = 2.5 Hz, 1H, 5-H),
4.09 (q, J = 7.0 Hz, 2H, OC 2CH3), 1.18 (t, J = 7.0 Hz, 3H, OC
2CH3); m/z (ESI) 306.35 (M + H+). Anal.
(C19H15NO3) Calc. C 74.74, H 4.95, N 4.59; Found C 74.99, H 4.81, N 4.70.
ν = 3435
δ
H
H
◦
3-Benzyloxy-4-phenylbenzo[4,5]furo[2,3-b]pyridine-6-ol (3b). Yield 13%; mp 220–224 C; IR (KBr)
(OH), 1276 (C-O-C) cm−1; 1H-NMR (DMDO-d6)
= 9.34 (s, 1H, OH), 8.32 (s, 1H, 2-H), 7.60–7.54 (m,
5H, C4-C6H5), 7.49 (d, J = 8.9 Hz, 1H, 8-H), 7.31–7.26 (m, 5H, OCH2C6 5), 6.92 (dd, J = 8.9, 2.7 Hz,
1H, 7-H), 6.51 (d, J = 2.7 Hz, 1H, 5-H), 5.17 (s, 2H, OC
2C6H5); m/z (ESI) 368.42 (M + H+). Anal.
ν = 3401
δ
H
H
(C24H17NO3) Calc. C 78.46, H 4.66, N 3.81; Found C 78.95, H 4.59, N 3.76.
3-Methoxy-4-phenylbenzo[4,5]furo[2,3-b]pyridine-6-ol (3c). Yield 8%; mp 240–245 ◦C; IR (KBr)
ν = 3434
(OH), 1275 (C-O-C) cm−1; 1H-NMR (DMDO-d6)
δ = 9.34 (s, 1H, OH), 8.25 (s, 1H, 2-H), 7.61–7.53 (m,
3H, 30-, 40-, 50-H), 7.51-7.49 (m, 2H, 20-, 60-H), 7.49 (d, J = 8.8 Hz, 1H, 8-H), 6.91 (dd, J = 8.8, 2.5 Hz, 1H,
7-H), 6.47 (d, J = 2.5 Hz, 1H, 5-H), 3.84 (s, 3H, OCH3); m/z (ESI) 292.32 (M + H+). Anal. (C18H15NO3)
Calc. C 74.22, H 4.50, N 4.81; Found C 73.89, H 4.45, N 4.95.
◦
3-Fluoro-4-phenylbenzo[4,5]furo[2,3-b]pyridine-6-ol (3d). Yield 6%; mp 266–274 C; IR (KBr)
ν = 3162
1
(OH), 1185 (C-O-C) cm−1; H-NMR (DMDO-d6)
δ = 9.48 (s, 1H, OH), 8.51 (d, J = 2.3 Hz, 1H, 2-H),
7.68–7.66 (m, 5H, C4-C6H5), 7.59 (d, J = 8.9 Hz, 1H, 8-H), 7.00 (dd, J = 8.9, 2.6 Hz, 1H, 7-H), 6.70 (d,
J = 2.6 Hz, 1H, 5-H); m/z (ESI) 280.27 (M + H+). Anal. (C17H10FNO2) Calc. C 73.11, H 3.61, N 5.02;
Found C 72.97, H 3.69, N 4.97.
3-Benzyloxy-4-phenylnaphtho[10,20:4,5]furo[2,3-b]pyridine-6-ol (4a). Yield 6%; mp 219–222 ◦C; IR (KBr)
= 3176 (OH), 1261 (C-O-C) cm−1; 1H-NMR (DMDO-d6)
= 10.10 (s, 1H, OH), 8.37 (s, 1H, 2-H), 8.32
(d, J = 8.2 Hz, 1H, 10-H), 8.23 (d, J = 8.4 Hz, 1H, 7-H), 7.72 (t, J = 7.5 Hz, 1H, 9-H), 7.66–7.57 (m, 6H,
C4-C6H5, 8-H), 7.38–7.25 (m, 5H, OCH2C6 5), 6.64 (s, 1H, 5-H), 5.20 (s, 2H, OC 2C6H5); m/z (ESI)
418.47 (M + H+). Anal. (C28H19NO3) Calc. C 80.56, H 4.59, N 3.36; Found C 80.23, H 4.35, N 3.25.
ν
δ
H
H
◦
3-Fluoro-4-phenylnaphtho[10,20:4,5]furo[2,3-b]pyridine-6-ol (4b). Yield 2%; mp 246–248 C; IR (KBr)
1
ν
= 3140 (OH), 1178 (C-O-C) cm−1; H-NMR (DMDO-d6)
δ
= 9.63 (s, 1H, OH), 8.87–8.82 (m, 2H,
2-, 10-H), 8.80 (d, J = 8.4 Hz, 1H, 7-H), 8.23-8.08 (m, 7H, C4-C6H5, 8-, 9-H), 7.27 (s, 1H, 5-H); m/z (ESI)
330.32 (M + H+). Anal. (C21H12FNO2) Calc. C 76.59, H 3.67, N 4.25; Found C 76.25, H 3.42, N 4.15.
3.5. General Procedure for the Synthesis of 3-Hydroxy Compounds 3e and 4c
One equivalent of the respective 3-benzyloxy compound 3b or 4a was dissolved in 20 mL of
distilled dried methanol at room temperature and 0.3 equivalent of palladium on charcoal (10%)
were added. The solution was shaken under hydrogen atmosphere with a pressure of 2 bar. After
2–3 h, the reaction was stopped and the solution filtered. After removal of the solvent in vacuum,
the 3-hydroxy compounds crystallized from diethyl ether.
4-Phenylbenzo[4,5]furo[2,3-b]pyridine-3,6-diol (3e). Yield 91%; mp 214–217 ◦C; IR (KBr)
1183 (C-O-C) cm−1; 1H-NMR (DMDO-d6)
= 9.72 (s br, 1H, OH), 9.29 (s br, 1H, OH), 8.07 (s, 1H, 2-H),
ν = 3350 (OH),
δ
7.61–7.51 (m, 5H, C4-C6H5), 7.46 (d, J = 8.9 Hz, 1H, 8-H), 6.89 (dd, J = 8.9, 2.6 Hz, 1H, 7-H), 6.54 (d,
J = 2.6 Hz, 1H, 5-H); m/z (ESI) 278.25 (M + H+). Anal. (C17H11NO3) Calc. C 73.64, H 4.00, N 5.05;
Found C 73.38, H 3.85, N 4.99.

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4-Phenylnaphtho[10,20:4,5]furo[2,3-b]pyridine-3,6-diol (4c). Yield 91%; mp 252–255 ◦C; IR (KBr)
ν
= 3337
(OH), 1172 (C-O-C) cm−1; 1H-NMR (acetone-d6)
δ = 9.04 (s, 1H, OH), 8.49 (s, 1H, OH), 8.36 (d, J = 8.3 Hz,
1H, 10-H), 8.33 (d, J = 8.3 Hz, 1H, 7-H), 8.16 (s, 1H, 2-H), 7.74–7.70 (m, 1H, 9-H), 7.67–7.57 (m, 6H,
C4-C6H5, 8-H), 6.71 (s, 1H, 5-H); m/z (ESI) 328.39 (M + H+). Anal. (C21H13NO3) Calc. C 77.05, H 4.00,
N 4.28; Found C 76.79, H 4.12, N 4.21.
3.6. Protein Kinase Assay
The protein kinases were all expressed in Sf9 insect cells as human recombinant GST fusion
proteins and purified by affinity chromatography using GSH-agarose. The kinase purity was finally
checked by SDS-PAGE/Coomassie staining.
The measuring of protein kinase activity was performed in 96-well FlashPlatesTM in a 50
µL
reaction volume. The reaction mixture consisted of 20
µL of assay buffer solution, 5
µL of ATP solution
in water, 5 L of used test compound in a 10% DMSO solution and finally a premixture of each 10
µ
µL
of used substrate and enzyme solutions. The assay buffer solution contained 70 mM of HEPES-NAOH,
each 3 mM of magnesium chloride and manganese(II) chloride, 3 M of sodium orthovanadate, 1.2 mM
of DTT, 50 g/mL of PEG20000 and finally 15 M of [
-33P]-ATP making approximately 1.2 106
cpm per well.
The final kinase concentrations have been 3.5 nM for cdk1/cycline B, 3.3 nM for cdk5/p25,
and finally 13.1 nM for gsk-3 . The substrate was RBER-CHKtide using amounts of 2000 ng/50 L in
µ
µ
µ
γ
×
β
µ
the case of cdk1/cycline B and 1000 ng/50 µL in the case of cdk5/p25 and gsk-3β.
3.7. Tau Protein Phosphorylation Assay
COS-7 cells transfected with pDSRed2-humanTau (2N4R isoform) and cultured in DMEM/5%
fetal calf serum for 24 h in 12-well culture plates were incubated with the respective compounds
◦
each 8
10 min at 4 ◦C with 300
with 300
L PBS, 1% Triton X-100 including protease and phosphatase inhibitors (cOmpleteTM
µ
M or 0.1% DMSO as vehicle control for 12 h at 37 C. Cells were scraped, centrifuged for
×
g, washed with cooled PBS and centrifuged again. Proteins were extracted
µ
,
PhosSTOPTM, Roche Life Science, (Germany, Penzberg). Protein content was calculated by the
Pierce 660 nm protein assay (Thermo Scientific, Rockford, IL, USA). Ten micrograms of protein
from each sample were run as duplicates on 8% SDS-PAGE and transferred to a PVDF membrane.
Total tau protein was labelled with a polyclonal total tau antibody (1:4000, v:v, #A0024, Dako, Glostrup,
Denmark) and phospho-tau (pThr231, pSer235) was labelled with the monoclonal antibody AT180
(1:500, v:v, MN1040, Thermo Scientific). Western blots were imaged after incubation with anti-rabbit
HRP/anti-mouse HRP (1:10,000, v:v NA934, NA931, GE Healthcare, Little Chalfont, UK) using the
DNR Chembis ECL station and immunoreactive bands were quantified by densitometry using Tina
2.09 software (Raytest, Straubenhardt, Germany). Phospho-tau immunoreactivity was normalized to
total tau immunoreactivity.
3.8. Tau Protein Interaction Assay
Cell Culture and Transfection
Human liver cell line (HuH-7) was maintained in Dulbecco’s modified Eagle’s minimum
essential medium (DMEM/F-12) containing 9% foetal bovine serum. Transfection of the plasmids
pFN33_tau and pFC36_tau (human 441aa 2N4R tau isoform fused to split luciferase fragments LgBit
and SmBit, respectively) or pNL1.1.TK[Nluc/TK] vector (full length constitutively-active NanoLuc
luciferase, Promega, Mannheim, Germany) in HuH-7 cells was performed in 6-well plates using the
Lipofectamine® 2000 Transfection reagent (ThermoFisher Scientific, Schwerte, Germany) according to
manufacturer’s recommendation. Briefly, 2.5
6.2
L of Lipofectamine® 2000 in 250 L Opti-MEMTM incubated for 20 min and added to the HuH-7
µg DNA in 250 µ
L Opti-MEMTM have been mixed with
µ
µ

Molecules 2018, 23, 2335
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cells per well. After 24 h incubation period, Huh-7 cells were plated into 384-well plates (3000 cells per
well, µclear/white #781093 greiner bio-one) for inhibitor treatment and luciferase assay.
NanoBiT® Luciferase Assay
The assay is based on complementation of a split luciferase nanoluc by the interaction of the
tested proteins. For evaluation of the best configuration of the split-luciferase and tau fusion proteins,
N-terminal and C-terminal fusion have been tested. Plasmid pFN33_tau and pFC36_tau gave the
best signal to noise ratio of all combinations tested and are orientated antiparallel with regard
luciferase large bit (17.6 kDa) and small bit (11 amino acids) fusions. Tau-Fusion protein expression is
governed by the relatively weak HSV-thymidine kinase promoter, and has been verified by Western
blotting (data not shown). After 16 h overnight incubation in serum-containing DMEM growth
medium, medium was replaced with 30
DMSO as vehicle control. Inhibitor treatment for 60 min at 37 ◦C was followed by addition of 5
µ µM, 10 µM inhibitors or 0.1%
L Opti-MEMTM comprising 1
µL
luciferase substrate furimazine (2-furanylmethyl-deoxy-coelenterazine #N1130, 1:400 (v:v) diluted in
Optimem, Promega). The luminescence signal has been measured immediately using Mithras LB
940 Multimode Microplate Reader (Berthold Technologies, Bad Wildbad, Germany). Background
luciferase activity from nontransfected cells was subtracted and luminescence was normalized to
vehicle control (0.1% DMSO). A 384-well plate was run in parallel were HuH-7 cells were transfected
with pNL1.1.TK[Nluc/TK] vector expressing a constitutive active luciferase as a reference and inhibitor
treatment was done accordingly. Luciferase readings from the tau interaction assay were normalized
to the constitutive luciferase readings to remove unspecific effects [39]. All inhibitor treatments were
repeated 8-fold.
4. Conclusions
The presently used drugs in AD therapy show a temporary benefit only in the early stage of the
disease. A causative cure of AD is not possible with those drugs, as they allow only a symptomatic
treatment. A novel approach for a causative therapy may be a reduction of the formation of harmful
tau aggregation intermediates and tau filaments. A hyperphosphorylation of the tau protein that is
discussed to contribute to the tau aggregate formation may be prevented by the use of protein kinase
inhibitors. Within our first compound series, we identified nanomolar inhibitors of three kinases with
relevance to tau pathology. Structural features which favour such activities are 3-hydroxy substitutions
and an annelated phenyl residue attached to the benzofuropyridine scaffold. A reduction of tau
protein phosphorylation could be demonstrated depending from the respective kinase activity of three
relevant compounds with different activities towards the respective kinases. We could demonstrate a
direct correlation between the structure-dependent compound ability to inhibit the relevant protein
kinases and a reduced tau interaction in a luminescence based protein complementation assay.
Thus, the identified lead compounds may be a perspective tool for further preclinical tests
to reduce tau aggregation based on a combination of diminished tau interaction and reduced
tau phosphorylation.
Supplementary Materials: The following are available online, Figure S1: Predicted binding mode of compound
3e (colored orange) at gsk-3β, Figure S2: Predicted binding mode of compound 4a (colored magenta) at cdk1
and cdk5.
Author Contributions: M.H. designed the biological studies; N.S. and A.O. provided the compounds by synthesis;
I.H. conducted the tau-phosphorylation experiments; J.S. was responsible for cell culture and transfection
experiments; T.V. was responsible for the establishing and standardisation of the tau interaction assay; V.N. carried
out the cloning and sequencing of the split luciferase vectors and the vector testing; M.O. helped with plasmide
preparation and financial support; F.T. and C.S. carried out the kinase measurements; W.S. made the docking
studies; and A.H. designed the synthetic experiments and wrote the paper.
Funding: We acknowledge the financial support of the Sächsische Aufbaubank (SAB)/European Union (EFRE;
#100240945) to Max Holzer.
Acknowledgments: We are grateful to Reinhild Prange for providing the Huh-7 cell line.

Molecules 2018, 23, 2335
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Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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