Multitargeted drug development: Discovery and profiling of dihydroxy substituted 1-aza-9-oxafluorenes as lead compounds targeting Alzheimer disease relevant kinases

Article abstract of DOI:10.1016/j.bmcl.2012.09.006

Alzheimer disease (AD) turned out to be a multifactorial process leading to neuronal decay. So far merely single target structures which attribute to the AD progression have been considered to develop specific drugs. However, such drug developments have been disappointing in clinical stages. Multitargeting of more than one target structure determines recent studies of developing novel lead compounds. Protein kinases have been identified to contribute to the neuronal decay with CDK1, GSK-3β and CDK5/p25 being involved in a pathological tau protein hyperphosphorylation. We discovered novel lead structures of the dihydroxy-1-aza-9-oxafluorene type with nanomolar activities against CDK1, GSK-3β and CDK5/p25. Structure-activity relationships (SAR) of the protein kinase inhibition are discussed within our first compound series. One nanomolar active compound profiled as selective protein kinase inhibitor. Bioanalysis of a harmless cellular toxicity and of the inhibition of tau protein phosphorylation qualifies the compound for further studies.

Full text of DOI:10.1016/j.bmcl.2012.09.006

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6914–6918
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Multitargeted drug development: Discovery and profiling of dihydroxy
substituted 1-aza-9-oxafluorenes as lead compounds targeting Alzheimer
disease relevant kinases
Volkmar Tell a, Max Holzer b, Lydia Herrmann b, Kazem Ahmed Mahmoud a, Christoph Schächtele c,
Frank Totzke c, Andreas Hilgeroth a,
⇑
a Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle, Germany
b University of Leipzig, Paul-Flechsig Institute of Brain Research, Jahnallee 59, 04109 Leipzig, Germany
c ProQinase GmbH, Breisacher Straße 117, 79106 Freiburg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 May 2012
Revised 3 September 2012
Accepted 5 September 2012
Available online 13 September 2012
Alzheimer disease (AD) turned out to be a multifactorial process leading to neuronal decay. So far merely
single target structures which attribute to the AD progression have been considered to develop specific
drugs. However, such drug developments have been disappointing in clinical stages. Multitargeting of
more than one target structure determines recent studies of developing novel lead compounds. Protein
kinases have been identified to contribute to the neuronal decay with CDK1, GSK-3b and CDK5/p25 being
involved in a pathological tau protein hyperphosphorylation. We discovered novel lead structures of the
dihydroxy-1-aza-9-oxafluorene type with nanomolar activities against CDK1, GSK-3b and CDK5/p25.
Structure–activity relationships (SAR) of the protein kinase inhibition are discussed within our first com-
pound series. One nanomolar active compound profiled as selective protein kinase inhibitor. Bioanalysis
of a harmless cellular toxicity and of the inhibition of tau protein phosphorylation qualifies the com-
pound for further studies.
Keywords:
Multitargeting
Inhibitors
Alzheimer disease
Structure–activity relationships
Tau protein
Ó 2012 Published by Elsevier Ltd.
Alzheimer disease (AD) is the presently most widespread
dementia disease.1 The possibilities to lower the observed neuro-
nal decay are limited to some acetylcholine esterase (ACE) inhibi-
AD patients.9 P25 constitutively activates CDK5 and thus p25 is
made responsible for the tau protein hyperphosphorylation medi-
ated by CDK5.9 The AD progression of the neuronal decay is known
to be a multifactorial process.10,11 Past efforts to develop specifi-
cally acting drugs were disappointing in clinical studies because
the patient benefit in the improvement of cognition or memory
was poor.11–13 Such drugs have been ACE inhibitors or secretase
inhibitors which prevent the formation of the amyloide-b proteins
by the inhibition of their cleavage from the amyloide precursor
protein (APP).
A novel strategy in the development of perspective AD thera-
peutics concentrates on the development of drugs which target
more than one AD relevant structure which contribute to the neu-
ronal decay.14,15 So recent studies tried to optimize a combined ef-
fect of the multitargeting drug Memoquin 1 as ACE inhibitor with
amyloide-b aggregation inhibiting properties.16
tors and the N-methyl
D-aspartat (NMDA) receptor antagonist
memantine. The therapeutical benefit of the ACE inhibitors is poor
and the NMDA receptor antagonist memantine is supposed to
cause neurotoxic effects.2,3 AD histopathology is characterized by
the formation of protein deposites in brain, namely the extracellu-
lar, insoluble amyloide plaques (APs) and the intracellular neurofi-
brillary tangles (NFTs) which consist of hyperphosphorylated tau
protein.4–6 The insoluble APs are discussed to be nontoxic. How-
ever, recent studies suggest an influence of the soluble oligomers
of the APs precursor the amyloide-b protein on the NFTs formation.
The hyperphosphorylation of the tau protein is known to be trig-
gered by the glycogen synthase kinase (GSK) 3-b which is found
being overexpressed in neuronal cells with NFTs.7 Another kinase
contributes to the tau protein hyperphosphorylation, namley the
cycline dependent kinase (CDK) 5 which is physiologically con-
trolled by the protein p35.8 High concentrations of p25 which is
a truncated subunit of p35 were found in the neuronal cells of
Inhibitors of GSK-3b have been reported but they all suffered
from a limited protein kinase selectivity. Only one inhibitor from
the thiadiazolidinone type 2 with micromolar activities reached
clinical trials, but nothing has been reported so far about its selec-
tivity (Fig. 1).17
Flavoperidol 3 as a nonselective CDK inhibitor with micromolar
activities has been investigated in clinical trials but toxic problems
occurred due to its nonselectivity of protein kinase inhibition.18
⇑
Corresponding author. Tel.: +49 345 55 25168; fax: +49 345 55 27207.
E-mail address: andreas.hilgeroth@pharmazie.uni-halle.de (A. Hilgeroth).
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.09.006

V. Tell et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6914–6918
6915
OMe
O
H
N
N
Et
N
Et
N
H
O
OMe
1
OH
O
Cl
HO
O
HO
COMe
OH
N
O
O
N
S
N
N
O
H
Me
3
elbfluorene
2
HO
OMe
N
O
4
Figure 1. Structures of Memoquin 1 and of protein kinase inhibitors in AD relevant clinical trials, namely thiadiazolidinone 2 and flavoperidol 3, including elbfluorene and
our 1-aza-9-oxafluorene starting structure 4.
Recent studies proved a certain influence of CDK1 on the decay
of neurons in AD brains. CDK1 is found being overexpressed in
such vulnerable neurons which reenter the cell cycle and die.1,19,20
We tried to develop a multifactorially acting protein kinase
inhibitor addressing the AD target structures CDK1, GSK-3b and
CDK5/p25 which contribute to the tau protein hyperphosphoryla-
tion and to the neuronal death by the later formed NFTs.
none until no more of this intermediate was detectable by tlc.25
The debenzylation of 13 followed the procedure of the formation
of compound 6.26
Micromolar activities in the inhibition of CDK5/p25 were re-
ported for our starting compound 4.27 We actually determined
the affinity data of 4 towards CDK1, CDK5/p25 und GSK-3b and
the two related serine/threonine kinases CDK2/E and CDK4/D
which both are also discussed to contribute to the tau protein
hyperphosphorylation.1,19,20 The Ki values determined in our pres-
ent assay system are shown in Table 1. We found micromolar affin-
ity data towards CDK1/B and GSK-3b. However, the compound was
completely inactive as CDK5/p25 inhibitor. The determined affini-
ties towards CDK2/E and CDK4/D were poor. While the 3-methoxy
function in 4 may serve as hydrogen bond acceptor function, the
contribution of the 6-hydroxy function to the protein kinase affin-
ity was not clear. Therefore we methylated this functional group. A
hydroxyl function may basically serve as hydrogen bond donator
or as hydrogen bond acceptor function. Compound 7 as twice-
methoxylated derivative showed a complete loss of affinity to all
the considered protein kinases. We concluded that the 6-hydroxy
function may play a central role in the binding to the protein back-
bone of the respective protein kinases. A replacement of the 3-
methoxy function in derivative 7 with a hydroxy function did not
restore the protein kinase affinity as shown with derivative 9
which was also inactive as protein kinase inhibitor. So a methyla-
tion of the 6-hydroxy function turned out as unfavourable to in-
Early 1-aza-9-oxafluorenes with a 3-carbonyl function like elb-
fluorene showed some CDK1 inhibiting properties.21
We started our search from the 3-methoxy-4-phenyl-1-aza-9-
oxafluorene 4 and consequently varied the substitution patterns
in both the 3- and the 6-position of the molecular scaffold with a
first benzo-annelated compound included. The synthesis of all
derivatives is shown in Scheme 1.
The varying 1-aza-9-oxafluorenes 5–14 have been individually
prepared. The 3,6-dihydroxy derivative 6 was given after treat-
ment of the 3-benzyloxy precursor 5 with hydrogen at a pressure
of 2 bar using palladium on charcoal (10%).22 The 3,6-dimethoxy
substituted derivative 7 was yielded by a methylation reaction of
compound 6 in a small volume of dried THF at room temperature
by the use of two equivalents of methyl iodide.24 Similar reaction
conditions were used to yield the 6-methoxy substituted deriva-
tive 8 from the 3-benzyloxy derivative 5. The used methyl iodide
excess was only the half of the previous reaction to yield 7. Finally,
the 3-hydroxy-6-methoxy compound 9 was given by the reaction
of the 3-benzyloxy derivative 8 with hydrogen and palladium on
charcoal similar to the formation of 6.
crease the protein kinase affinities of our compounds.
A
The methyl-substituted and the benzo-annelated 1-aza-9-oxa-
fluorenes 12 and 13 were prepared by the reaction of N-acetyl-3-
benzyloxy-1,4-dihydropyridine 10 with methylbenzoquinone or
naphthoquinone in dried dioxane under perchloric acid catalysis
(6%). The non-isolated primary formed tetrahydro derivative 11
was oxidized by the addition of portions of the corresponding qui-
replacement of the 3-methoxy group of derivative 4 with a 3-ben-
zyloxy function resulted in similar affinities of the resulting com-
pound 5 towards CDK1/B and GSK-b. Obviously, the more bulky
3-benzyloxy substituent is well tolerated if compared to the 3-
methoxy substituent because of some slight affinity improvements
especially towards CDK2/E with a micromolar affinity. The 6-meth-

6916
V. Tell et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6914–6918
HO
OBn
HO
OH
MeO
OMe
a
b
N
N
N
O
O
O
5
6
7
c
MeO
OBn
MeO
OH
a
N
N
O
O
8
9
HO
R2
O
R1
R2
OBn
R1
OBn
d
+
O
N
N
O
O
Me
Me
O
10
11
HO
R2
OH
R1
R2
R1
OBn
+
e
O
N
12, R1 = Me, R2 = H
13, R1 = -CH-CH=CH-CH-R2
OH
HO
OH
e
13
O
14
N
Scheme 1. Reagents and conditions for the preparation of compounds: (a) H2, Pd/C, MeOH, 2 bar, rt; (b) 2 equiv MeI, THF, 0 °C-rt; (c) 1 equiv MeI, THF, 0 °C-rt; (d) perchloric
acid (6%), dioxane, rt; (e) methylbenzoquinone or naphthoquinone, perchloric acid (6%), dioxane, rt; (f) H2, Pd/C, THF/MeOH, 2 bar, rt.
oxy derivative 8 showed a significant improvement of the GSK-3b
affinity to nanomolar ranges. A loss of CDK1/B affinity was accom-
panied by a CDK5/p25 activity. While a 6-methoxy function in the
compounds with a smaller either methoxy or hydroxyl function in
the 3-position was unfavourable with respect to the protein kinase
affinity, a 6-methoxy function in derivative 8 with the bulky 3-ben-
zyloxy substitution turned out as favourable. So a different binding
mode towards the protein kinases especially to GSK-3b was sug-
gested because of the main increase in affinity to nanomolar
ranges.
inhibitor with nanomolar affinities towards CDK1/B and GSK-3b
and submicromolar affinities towards CDK5/p25 and CDK2/E. The
increased affinities towards CDK5/p25 meant an improvement in
activity by a factor of 200 if compared to the first CDK5/p25 inhib-
itor 8 of our series. Thus we received the first multitargeting can-
didate for a further evaluation as will be demonstrated below.
We then investigated the influence of an additional 1-aza-9-
oxafluorene skeleton substitution by introducing a methyl group
into the 7-position. Compound 12 showed similar affinities to-
wards CDK1/B and GSK-3b than the non-methylated derivative 5.
However, some CDK5/p25 affinities were measured. We increased
the skeleton substitution by the annelation of a phenyl ring as a
more space demanding substituent in derivative 13. The additional
benzo-substitution led to main increases in the CDK1/B affinities
The 6-hydroxy function was favourable for a protein kinase
affinity of our derivative with a 3-methoxy function, so that we re-
placed the 3-methoxy function in derivative 4 with an additional
hydroxyl group. Compound 6 turned out to be a good multikinase

V. Tell et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6914–6918
6917
Table 1
Serine/threonine kinase inhibition profiles of target compounds 4–14
Ki valuesa,b
[lM]
CDK1/B
CDK2/E
CDK4/D
CDK5/p25
217
GSK-3b
4
5
6
7
8
9
12
13
14
5.3
2.3
24.0
6.4
0.60
147
93.6
36.6
18.5
14.8
5.8
c
n.a.
0.01
0.11
0.02
n.a.c
n.a.
n.a.
n.a.
n.a.
c
c
c
c
c
c
n.a.
n.a.
n.a.
n.a.
n.d.
n.d.
26.1
0.02
c
c
c
c
144
n.d.
n.d.
n.d.
n.a.
n.a.
d
d
1.3
0.09
0.70
25.3
2.10
0.07
5.8
1.60
0.01
d
d
d
d
n.d.
a
Ki values have been calculated from determined IC50 values of kinase inhibition
following described protocols.31,32
b
Standard errors of the Ki values are typically below 20%. In many cases standard
errors below 10% are found.
c
n.a., not active (Ki >1000).
n.d., not determined.
d
Figure 3. Decrease of phosphorylation of tau amino acids serine 202 and threonine
205 in transfected N2A neuroblastoma cells with increased concentrations of
compound 6.
and micromolar CDK5/p25 affinities were reached if compared to
the non-annelated derivative 5 which was completely inactive as
CDK5/p25 inhibitor. Moreover, the GSK-3b affinities improved.
The debenzylation of derivative 13 finally led the the 3- and 6-
dihydroxy substituted derivative 14 with nanomolar affinities to-
wards CDK5/p25 and GSK-3b and yet submicromolar affinities to-
wards CDK1/B. The additional benzo-annelation in compound 14
proved to be slightly less favourable with respect to the CDK1
affinities, but increased the CDK5/p25 and the GSK-3b affinities if
compared to the non-annelated derivative 6.
to concentrations of 90
as shown in Figure 2.
The membrane integrity was additionally examined in the lac-
tate dehydrogenase assay (LDH) by determination of the extracel-
lular activity of this enzyme.28 Also in this assay system
concentrations up to 90 lM showed no cell-toxic effects if com-
pared to the compound-free cells.
We then investigated our nontoxic compound 6 to inhibit pro-
tein kinases from various protein kinase families of the human ki-
nome. We determined Ki values to PKC isoforms of the PKA family
lM if compared to the compound-free cells
So both 3,6-dihydroxy substituted 1-aza-9-oxafluorenes 6 and
14 are excellent multikinase inhibitors of AD relevant kinases with
their nanomolar affinities.
(PKC-a, -c, -e and -iota), to kinases of the receptor tyrosine kinase
family (EGFR, VEGFR2, ERBB2 and TIE2), to the related CDK6/D1
Compound 6 was our best CDK1/B and CDK2/E inhibitor. Beside
the benefit of affecting also these kinases in a multitargeted AD
therapy we investigated possible cell toxic effects of this selected
and to kinases of the Casein kinase (Ck) family (WEE1 and Ck1-
a
1). Derivate 6 was completely inactive as inhibitor of all these ki-
nase with Ki values >1000 M. The Ki value towards CDK6/D1 was
little below 1000 M with a Ki value of 773 M and the Ki value to-
M which meant less than a residual
l
compound which may be caused by
influencing.
a possible cell cycle
l
l
wards EGFR was about 296
l
Cell toxic effects have been determined in various assay sys-
tems using concentrations up to 90 M to exclude any possible
activity.
l
A final bioanalysis was carried out with compound 6 as poten-
tial AD relevant kinases multitargeting inhibitor in N2A neuroblas-
toma cells to prove the ability of a cellular influence on the tau
protein phosphorylation. The neuroblastoma cells have been stably
transfected with a 3-repeat tau protein construct.29 The inhibition
of relevant amino acid phosphorylations of tau by the inhibitor has
been determined with respective antibodies of the representing
toxic problems. We investigated the effects in neuronal-like N2A
neuroblastoma cells. The mitochondrial function was checked in
the MTT assay by determination of the formed formazan crystals
by mitochondrial reduction via dehydrogenases.28 The formed for-
mazan amounts would mainly decrease in the case of a cellular
toxicity. We observed no decrease in the formazan formation up
Figure 2. Cellular toxicity of compound 6 in the lactate dehydrogenase (LDH) assay measured as increased LDH activity displayed left and in the MTT assay measured as
reduced formazan formation displayed right.

6918
V. Tell et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6914–6918
23. Krug, M.; Voigt, B.; Baumert, C.; Lüpken, R.; Hilgeroth, A. Eur. J. Med. Chem.
2010, 45, 2683.
phosphorylated amino acids serine 202 and threonine 205 as
shown in Figure 3.
24. Representing procedure for the methylation of compounds
5 and 6: 3,6-
Main reductions of the cellular tau protein amino acid phos-
phorylation of both serine 202 and threonine 205 were observed
at the lowest concentrations. Thus a first proof-of-concept could
be given for effective cellular activities of reducing tau protein
phosphorylation by the AD relevant protein kinases CDK5/p25
und GSK-3b.
Finally, we succeeded in a lead optimization with the discovery
of nanomolar active CDK1, CDK5/p25 and GSK-3b inhibitors of the
3,6-dihydroxy-1-aza-9-oxafluorene type 6 and 14. We demon-
strated nontoxic properties of the selective derivative 6.
Thus, first multikinase inhibitors for a perspective AD multitar-
geted therapy were found and will be investigated in further pre-
clinically directed studies. Moreover, our selective inhibitors will
help to understand the effect of tau phosphorylation on AD
progression.
dimethoxy-4-phenylbenzo[4,5]furo[2,3-b]pyridine (7). Compound 5 (0.010 g,
0.036 mmol) was dissolved in dried THF (1 mL) under argon atmosphere. The
solution was cooled down to 0 °C in an ice bath. Then a 14-molar excess of
sodium hydride (0.012 g, 0.5 mmol) in paraffin oil (60%) was added. After
stirring for 15 min the suspension was warmed-up to rt and stirring continued.
Then a 6-molar excess of methyl iodide (0.015 g, 0.11 mmol) with reference to
both hydroxy functions was added. After stirring for 9 h at rt the reaction
mixture was poured into ice water (2 mL) and extracted with chloroform (10/
10/5/5 mL) for four times. After drying over sodium sulfate the solvent was
removed in vacuum and the remaining oil was purified by column
chromatography over silica gel using an eluent mixture of cyclohexane and
ethyl acetate (60/40). The collected fractions were evaporated to dryness and
the residual oil was dissolved in ethyl ether from which 7 cyrstallized. Yield
0.006 g (54%); mp 143–145 °C; IR
m = 2927, 2836, 1587, 1477, 1435, 1272,
1189, 1089, 1033 cmꢀ1 1H NMR (CDCl3) d = 8.17 (s, 1H), 7.59–7.51 (m, 5H),
;
7.47 (d, J = 9.0 Hz, 1H), 7.02 (dd, J = 9.0, 2.7 Hz, 1H), 6.68 (d, J = 2.7 Hz, 1H), 3.88
(s, 3H), 3,63 (s, 3H); m/z (ESI) 306 (M+H+). Elemental Anal. Calcd. (%) for
C
19H15NO3: C, 74.74; H, 4.95; N 4.59. Found: C, 74.56; H, 4.68; N 4.22.
25. Procedure for the formation of the 3-benzyloxy-4-
phenylnaphtho[10,20:4,5]furo[2,3-b]pyridine-6-ol (13). 0.85 g of the N-acetyl
1,4-dihydropyridine 10 (2.8 mmol)23 and 0.53 g of the naphthoquinone
(3.4 mmol) were dissolved in a minimum volume of dried dioxane. Then a
mixture of ten parts of dried dioxane and one part of perchloric acid (70%) were
added reaching a final solution reaction volume of 75 mL. After 24 h of stirring
at rt the added naphthoquinone partly disappeared according to tlc analysis of
the reaction mixture and additional 0.17 g of naphthoquinone (1.1 mmol) were
added. The addition of portions of the naphthoquinone continued until no
more tlc-detectable tetrahydro derivative 11 was present in the reaction
mixture. Then ice water was added (15 mL) and the pH of the solution was
adjusted to pH = 9 using a sodium hydroxide solution (1 M). Then the reaction
mixture was extracted four times with each 50 mL of chloroform. The organic
layers were unified and dried over sodium sulfate. After filtration and
evaporation the remaining oil was purified by column chromatography over
silica gel using an eluent mixture of cyclohexane / ethyl acetate (80/20). The
collected compound-containing fractions were evaporated yielding compound
Acknowledgment
Financial support of the work was given from the AFI (Alzhei-
mer Forschung Initiative e.V.) to Max Holzer, Lydia Herrmann
and Andreas Hilgeroth and from the DFG (German Research Foun-
dation) to Volkmar Tell, Max Holzer and Andreas Hilgeroth.
References and notes
1. Neve, R. L.; McPhie, D. L. Pharmacol. Ther. 2006, 111, 99.
2. Takeda, A.; Loveman, E.; Clegg, A.; Kirby, J.; Picot, J.; Payne, E.; Green, C. Int. J.
Geriatr. Psychiatry 2006, 21, 17.
3. McShane, R.; Areo Sasastre, A.; Minakaran, N. Cochrane Database Syst. Rev. 2006,
19, CD003154.
13 as a brownish powder. Yield 0.18 g (15%); mp 219–222 °C; IR (KBr)
m = 3170,
3056, 3028, 2982, 2917, 1596, 1583, 1374, 1260, 1228 cmꢀ1 1H NMR (DMSO-
;
d6) d = 10.06 (s, 1H), 8.36 (s, 1H), 8.30 (d, J = 8.3 Hz, 1H), 8.21 (d, J = 8.3 Hz, 1H),
7.71 (t, J = 8.3 Hz, 2H), 7.63–7.59 (m, 5H), 7.32–7.28 (m, 5H), 6.63 (s, 1H), 5.20
(s, 2H); m/z (ESI) 418 [M+H+]. Elemental Anal. Calcd. (%) for C28H19NO3: C,
80.49; H, 4.59; N 3.36. Found: C, 80.13; H, 4.25; N 2.99.
4. Selkoe, D. J. Annu. Rev. Cell. Biol. 1994, 10, 373.
5. Vetrivel, K. S.; Thinakran, G. Neurology 2006, 66, S69.
6. Wippold, F. J.; Cairns, N.; Vo, K.; Holtzman, D. M.; Morris, J. C. Am. J. Neuroradiol.
2008, 29, 18.
7. Sun, W.; Qureshi, H. Y.; Cafferty, P. W.; Sobue, K.; Agarwal-Mawal, A.; Neufield,
K. D.; Paudel, H. K. J. Biol. Chem. 2002, 277, 11933.
8. Cruz, J. C.; Tseng, H. C.; Goldman, J. A.; Shih, H.; Tsai, L. H. Neuron 2003, 40, 471.
9. Kanungo, J.; Zheng, Y.-L.; Amin, N. D.; Pant, H. C. Cell. Mol. Neurobiol. 2009, 29,
1073.
10. Francotte, P.; Graindorge, E.; Boverie, S.; de Tullio, P.; Pirotte, B. Curr. Med.
Chem. 2004, 11, 1757.
26. Spectroscopical data of 4-phenylnaphtho[10,20:4,5]furo[2,3-b]pyridine-3,6-diol
(14): mp 258–260 °C; IR (KBr)
m
= 3337, 2977, 1640, 1599, 1582, 1444, 1369,
1256, 1212, 1172, 1070, 1037, 1028 cmꢀ1
;
1H NMR (acetone-d6) d = 9.04 (s,
1H), 8.49 (s, 1H), 8.36 (d, J = 8.3 Hz, 1H), 8.33 (d, J = 8.3 Hz, 1H), 8.16 (s, 1H),
7.74–7.70 (m, 1H), 7.67–7.57 (m, 6H), 6.71 (s, 1H); m/z (ESI) 328 [M+H].
Elemental Anal. Calcd. (%) for C21H13NO3: C, 77.05.49; H, 4.00; N 4.28. Found: C,
76.85; H, 4.09; N 4.22.
27. Voigt, B.; Meijer, L.; Lozach, O.; Schächtele, C.; Totzke, F.; Hilgeroth, A. Bioorg.
Med. Chem. Lett. 2005, 15, 823.
28. LDH-assay was performed according to the manufacturer´s recommendations
11. Wollen, K. A. Altern. Med. Rev. 2010, 15, 223.
12. Ethell, D. W. Neuroscientist 2010, 16, 614.
(Roche Diagnostics) in triplicate. 50 lL Medium was complemented with 50 lL
13. Kramp, P.; Herrling, P. Neurodegener. Dis. 2010, 8, 44.
14. Bolognesi, M. L.; Matera, R.; Minarini, A.; Rosini, M.; Melchiorre, C. Curr. Opin.
Chem. Biol. 2009, 13, 303.
15. Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.; Tumiatti, V.; Recanatini,
M.; Melchiorre, C. J. Med. Chem. 2008, 51, 347.
16. Bolognesi, M. L.; Bartolini, M.; Tarozzi, A.; Morroni, F.; Lizzi, F.; Milelli, A.;
Minarini, A.; Rosini, M.; Hrelia, P.; Andrisano, V.; Melchiorre, C. Bioorg. Med.
Chem. Lett. 2011, 21, 2655.
17. Castro, A.; Encinas, A.; Gil, C.; Brase, S.; Porcal, W.; Perez, C.; Moreneo, F. J.;
Martinez, A. Bioorg. Med. Chem. 2008, 16, 495.
18. Fischer, P. M.; Gianella-Borradori, A. Expert Opin. Investig. Drugs 2005, 14, 457.
19. Vincent, I.; Pae, C. I.; Hallows, J. L. Prog. Cell Cycle Res. 2003, 5, 31.
20. Manaco, E. A., III; Vallano, M. L. Front. Biosci. 2005, 10, 143.
21. Brachwitz, K.; Voigt, B.; Meijer, L.; Lozach, O.; Schächtele, C.; Monár, J.;
Hilgeroth, A. J. Med. Chem. 2003, 46, 876.
reagent mixture and absorbance values were measured at 492 nm using a HT3
microtiter plate reader after 20 min. MTT was added in triplicate to cells to a
final concentration of 1 mg/mL and cells were incubated for 2 h. After
incubation cell culture medium with MTT was removed, 200
l
L DMSO and
´
25
lL Soerensens glycine buffer (0.1 M Glycine, pH 10.5, 0.1 M NaCl) per well
were added and absorbance values were measured at 550 nm with the
microtiter plate reader. Absorbance values of N2A cells cultures treated with
compound 6 were compared to cells treated with 1% Triton X-100 (100% cell
death) and untreated cells.
29. Tau phosphorylation was quantified using N2A mouse neuroblastoma cells
stably transfected with an 3-repeat tau expression construct. For the
incubation of N2A cells with the protein kinase inhibitor cells were
´
maintained in a 96-well plate in DMEM/HAMs F-12 (pH 7.4) supplemented
with 5% (v/v) fetal bovine serum, 2 mM L-Glutamine, 50 g/mL gentamicin in a
5% CO2 environment at 37 °C for 24 h. Afterwards cells were maintained for
16 h in 150 L of the described media with the protein kinase inhibitor at
l
22. Representative procedure for the debenzylation of compounds
phenylbenzo[4,5]furo[2,3-b]pyridine-3,6-diol (6). Compound
5
5
and 8: 4-
(0.025 g,
l
different concentrations diluted from a stock of 25 mM in DMSO. The final
DMSO concentration in medium did not exceed 0.4% and was also included in
the control treatment group. Extracted proteins were separated by 8% SDS–
PAGE and then transferred onto PVDF membrane overnight using a tank blot
system. Immunodetection of phosphorylated tau protein was performed as
described.30
0.068 mmol)23 was dissolved in dried methanol (20 mL). Palladium on
charcoal (0.020 g, 10%) was added and the suspension was shaken under
hydrogen atmosphere at
a pressure of 2 bar for 3 h. After filtration and
evaporation to dryness the residue was purified by column chromatography
over silica gel using an eluent mixture of cyclohexane and ethyl acetate (60/
40). The collected fractions were unified, the eluent was evaporated and 5
crystallized from diethyl ether overnight in a refrigerator. Yield 0.018 g (93%);
30. Holzer, M.; Rödel, L.; Seeger, G.; Gärtner, U.; Narz, F.; Janke, C.; Heumann, R.;
Arendt, T. Neuroscience 2001, 105, 1041.
31. Krug, M.; Erlenkamp, G.; Sippl, W.; Schächtele, C.; Totzke, F.; Hilgeroth, A.
Bioorg. Med. Chem. Lett. 2010, 20, 6915.
32. Krug, M.; Wichapong, K.; Erlenkamp, G.; Sippl, W.; Schächtele, C.; Totzke, F.;
Hilgeroth, A. ChemMedChem 2011, 6, 63.
mp 214–217 °C; IR (KBr)
m ;
= 3349, 2965, 1466, 1377, 1277, 1250, 1183 cmꢀ1
1H NMR (DMSO-d6) d = 9.72 (br s, 1H), 9.29 (br s, 1H), 8.07 (s, 1H), 7.61–7.51
(m, 5H), 7.46 (d, J = 8.9 Hz, 1H), 6.89 (dd, J = 8.9, 2.6 Hz, 1H), 6.54 (d, J = 2.6 Hz,
1H); m/z (ESI) 278 (M+H+). Elemental Anal. Calcd. (%) for C17H11NO3: C, 73.64;
H, 4.00; N 5.05. Found: C, 73.38; H, 3.85; N 4.77.