Identification of dihydropyridines that reduce cellular tau levels

Article abstract of DOI:10.1039/c0cc02253e

A series of dihydropyridines were identified that have an effect on the accumulation of tau, an important target in Alzheimer's disease. The dihydropyridine collection was expanded using the Hantzsch multicomponent reaction to develop preliminary structur

Full text of DOI:10.1039/c0cc02253e

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Identification of dihydropyridines that reduce cellular tau levelsw
Christopher G. Evans,a Umesh K. Jinwal,b Leah N. Makley,a Chad A. Dickeyb and
Jason E. Gestwicki*a
Received 30th June 2010, Accepted 4th October 2010
DOI: 10.1039/c0cc02253e
A series of dihydropyridines were identified that have an effect
on the accumulation of tau, an important target in Alzheimer’s
disease. The dihydropyridine collection was expanded using the
Hantzsch multicomponent reaction to develop preliminary
structure–activity relationships.
Tau is a microtubule-binding protein that accumulates in a
number of neurodegenerative disorders, including fronto-
temporal dementia and Alzheimer’s disease (AD).1 These
diseases are collectively termed tauopathies and they are
characterized by aggregation of hyperphosphorylated tau.
The presence of abnormal tau correlates with neuron loss
and memory deficits.2 Therefore, selectively reducing its levels
might be an effective strategy.
Efforts towards that goal have largely focused on either
inhibitors of tau aggregation,3 inhibitors of its phosphoryla-
tion4 or compounds that stimulate its degradation.5 Each of
these strategies is potentially promising, but many of the
compounds identified to date have relatively modest activity.
For example, methylene blue (MB), which both inhibits tau
aggregation3 and stimulates its degradation through heat
shock protein 70 (Hsp70),5 has an EC50 value of approxi-
mately 10 mM. Other promising compounds, such as the
Hsp90 inhibitors 17-AAG and EC1012, reduce tau levels but
they also produce a robust stress response, which is expected
to diminish their long-term efficacy.5,6 Thus, new compounds
Scheme 1 Variation of the aldehyde in the Hantzsch reaction to
that counteract tau accumulation are still of interest.
expand the diversity of the dihydropyridine collection.
While conducting cell-based screens for small molecules that
impact tau levels, we identified the 1,4-dihydropyridine 4a
(data not shown). Based on this finding, we sought to synthe-
size a focused collection to facilitate characterization of
structure–activity relationships (SAR). Accordingly, we were
attracted to the Hantzsch multicomponent reaction because of
its high atom economy and suitability for combinatorial
synthesis. This reaction produces the dihydropyridine core
scaffold from an aldehyde, amine, and two 1,3-dicarbonyls
in a single step (Scheme 1). Also, it has good functional group
tolerance and there are known stereoselective routes.7
(1.5 equiv.), ethylacetoacetate 2 (1 equiv.), and Yb(OTf)3
(10 mol%) were mixed in acetonitrile. After stirring for
10 minutes, the aldehyde (1.0 equiv.) and ammonium acetate
(1.0 equiv.) were added. The reactions then proceeded for
3–5 hours, after which they were poured into saturated NaCl,
washed with ethylacetate and the products re-crystallized from
1 : 3 water : ethanol. Using this approach, compounds 4a–r
were obtained in moderate to good yields (69–94%).
To expand the diversity in this collection, we took
advantage of published methods8 to exchange the ester for a
thioester on compounds 4a and 4b. Briefly, these examples
were refluxed in toluene with 2.2 equivalents of Lawesson’s
reagent for 1 h. The resulting products, 5a and 5b, were filtered
through Celite and purified as above in good yield (Scheme 2).
To test whether modifications to the heterocyclic amine
could be tolerated, we combined dimedone with aryl or alkyl
amines in acetonitrile to form the enamine.9 After 30 minutes,
ethylacetoacetate (1.0 equiv.), 2,4-dicholoro benzaldehyde 3a
(1.0 equiv.) and 10% Yb(OTf)3 were added and the reaction
was allowed to proceed for an additional 4–5 hours. This
To generate a dihydropyridine collection, we first explored a
series of aldehydes that were functionalized with ether bulky
aromatics or smaller, alkyl groups (Scheme 1). To maintain
the general structure of the initial compound, dimedone 1
a Department of Pathology and the Life Sciences Institute, University
of Michigan, 210 Washtenaw Ave., Ann Arbor, MI 48109, USA.
E-mail: gestwick@umich.edu; Tel: +1 734-615-9537
b Department of Molecular Medicine, University of South Florida,
4001 E. Fletcher Ave., MDC 36, Tampa, FL 33613, USA.
E-mail: cdickey@health.usf.edu; Tel: +1 813-396-0639
w Electronic supplementary information (ESI) available: Synthesis
and characterization of compounds. See DOI: 10.1039/c0cc02253e
c
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Chem. Commun., 2011, 47, 529–531 529

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Scheme 2 Introduction of a thioester into the dihydropyridines.
procedure generated compounds 7a–d with yields ranging
from 71–82% (Scheme 3).
To further diversify the scaffold, we next varied the identity
of the 1,3-dicarbonyls (8 and 9; Scheme 4). Specifically, we
used indanedione and 2,4-pentanedione in place of dimedone
to produce derivatives 10a and 10b in good yields. On the
other side of the molecule, we substituted either methyl-
acetoacetate or benzylacetoacetate for ethylacetoacetate to
produce 10c and 10d in 82 and 85% yield, respectively.
Finally, to fully exploit the strengths of the Hantzsch
reaction we varied multiple components simultaneously.
Using the reaction conditions and starting materials employed
earlier, we made derivatives 11a–11k (Scheme 5). These
compounds include examples, such as 11b and 11c, which
contain b-ketoamides. Together, these efforts produced a
library of 39 functionalized dihydropyridines. At this stage,
no attempt was made to separate the enantiomers.
Scheme 4 Substitutions of 1,3-dicarbonyls to add diversity.
With this collection in hand, we treated cultured IMR32
neuroblastoma cells for 24 h with 100 mM compound and
measured endogenous tau levels by Western blot. Some of the
Scheme 5 Multiple components of the Hantzsch reaction were simulta-
Scheme 3 Substitutions of the amine in the dihydropyridine.
530 Chem. Commun., 2011, 47, 529–531
neously exchanged to create dihydropyridines with increased diversity.
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compounds, such as 11b–f, were found to be toxic under these
conditions and excluded from further analysis. We then
compared the remaining examples to the benchmark
compounds MB and 17-AAG, which reduced tau levels by
approximately 50 to 70% (Fig. 1).5,6 Based on those values, we
imposed an arbitrary threshold of 25% to focus on the most
active compounds in the dihydropyridine collection. This
analysis focused attention on compounds 4p, 11a and 11g,
which reduced tau levels by at least 25%. Interestingly, we also
identified examples, such as 4a–b, 4d, 10b–c, 11j and 11k,
which increased tau levels by at least 25%. Previous efforts
have also noted compounds that increase tau levels and both
types of molecules have been useful probes of tau biology.5a–b
An analysis of these results suggested some preliminary
SAR. Specifically, large substitutions on the aldehyde such
as naphthyl (4j) or p-diphenyl (4o) were not tolerated. Like-
wise, conversion of the ester to a thioester in 5a and 5b reduced
activity, as did any modification of the heterocyclic amine
(compounds 7a–d). Modest substitutions of an ethyl to methyl
group in the diketone (e.g. from 4a to 10c) had marginal effects
but larger groups, such as the benzyl ester in 10d, abolished
activity. Interestingly, minimally functionalized benzyls in the
aldehyde position, such as 4a, 4b and 4d, produced the most
potent stimulators of tau accumulation, while smaller alkyl
groups, such as in 4p, tended to produce compounds that
reduced tau levels. This finding suggests a way of converting a
compound from one that causes tau accumulation to one that
leads to reductions. However, the impact of the aldehyde
position also seemed to be influenced by the identity of the
1,3-diketone. For example, if dimedone was used, compounds
4a and 4b promoted tau accumulation, but replacing it for a
methyl diketone, as in 11a and 11g, produced relatively strong
inhibitors. Together, these findings reveal patterns of substitu-
tion that promote either tau degradation or accumulation.
Following this initial screening, we selected compound 11g
for in-depth studies. In dose dependence experiments, we
found that this compound reduced tau levels by B70% with
an IC50 of 7.0 Æ 1.5 mM (Fig. 1B), a potency comparable to
some of the best, known anti-tau compounds.5 To test whether
11g activates a cellular stress response, we examined the levels
of stress-inducible Hsp70 by Western blot and found that its
levels were not significantly increased, suggesting that 11g does
not act through this pathway (Fig. 1B). Next, we generated the
two enantiomers of 11g (11gS and 11gR), using an organo-
catalytic procedure.7a In the IMR32 cells, only 11gS had
activity, with an IC50 value of B 600 nM (Fig. 1C). The
cellular target of these dihydropyridines is currently unclear,
but the relatively potent activity of 11gS suggests that further
studies are warranted.
C.G.E. was supported by the NIH (GM008353). We also
acknowledge support from the Abe and Irene Pollin CBD
Fund, the Alzheimer’s Association (IRG-09-130689) and NIH
(NS059690 and AG031291).
Notes and references
1 (a) T. L. Spires-Jones, W. H. Stoothoff, A. de Calignon, P. B. Jones
and B. T. Hyman, Trends Neurosci., 2009, 32, 150;
(b) S. Jeganathan, M. von Bergen, E. M. Mandelkow and
E. Mandelkow, Biochemistry, 2008, 47, 10526.
2 H. Braak and E. Braak, Acta Neuropathol., 1991, 82, 239.
3 (a) S. Taniguchi, N. Suzuki, M. Masuda, S. Hisanaga, T. Iwatsubo,
M. Goedert and M. Hasegawa, J. Biol. Chem., 2005, 280, 7614;
(b) C. M. Wischik, P. C. Edwards, R. Y. Lai, M. Roth and
C. R. Harrington, Proc. Natl. Acad. Sci. U. S. A., 1996, 93, 11213.
4 M. P. Mazanetz and P. M. Fischer, Nat. Rev. Drug Discovery, 2007,
6, 464.
5 (a) J. Koren, 3rd, U. K. Jinwal, D. C. Lee, J. R. Jones, C. L. Shults,
A. G. Johnson, L. J. Anderson and C. A. Dickey, J. Cell. Mol.
Med., 2009, 13, 619; (b) U. K. Jinwal, Y. Miyata, J. Koren, 3rd,
J. R. Jones, J. H. Trotter, L. Chang, J. O’Leary, D. Morgan,
D. C. Lee, C. L. Shults, A. Rousaki, E. J. Weeber,
E. R. Zuiderweg, J. E. Gestwicki and C. A. Dickey, J. Neurosci.,
2009, 29, 12079; (c) S. Oddo, A. Caccamo, B. Tseng, D. Cheng,
V. Vasilevko, D. H. Cribbs and F. M. LaFerla, J. Neurosci., 2008,
28, 12163.
Fig. 1 Screening results for the dihydropyridines. (A) IMR32 cells
were treated for 24 h with 100 mM compound, followed by Western
blots for total tau. Quantification of these blots is shown versus a
vehicle control (1% DMSO). Also shown are two positive controls,
methylene blue (MB) and 17-AAG. Arbitrary activity cut-offs are
shown at Æ25% (dotted line). Inactive compounds are shown as open
symbols. Active compounds are shown in solid symbols and are
labeled. *Toxic compounds. (B) Dose response analysis of 11g. (C)
Testing of the enantiomers of 11g. The S enantiomer (open symbols)
had activity while the R enantiomer (closed symbols) was inactive.
Results are the average of triplicates and the error bars are SEM.
6 C. A. Dickey et al., J. Clin. Invest., 2007, 117, 648.
7 (a) C. G. Evans and J. E. Gestwicki, Org. Lett., 2009, 11, 2957;
(b) J. C. Legeay, J. Y. Goujon, J. J. Vanden Eynde, L. Toupet and
J. P. Bazureau, J. Comb. Chem., 2006, 8, 829; (c) A. M. Maurya and
R. A. Kumar, Tetrahedron, 2007, 63, 1946.
8 B. A. Vigante, Y. Y. Ozols, B. S. Chekavichus and G. Y. Dubur,
Khim. Geterotsikl. Soedin., 1988, 9, 1232.
9 (a) O. I. el-Sabbagh and H. M. Rady, Eur. J. Med. Chem., 2009, 44,
3680; (b) S. J. Tu, Y. Zhang, R. H. Jia, B. Jiang, Y. J. Zahang and
S. J. Ji, Tetrahedron Lett., 2006, 47, 6521.
c
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