N-Substituted carbazolyloxyacetic acids modulate Alzheimer associated γ-secretase

Article abstract of DOI:10.1016/j.bmcl.2006.09.061

N-Sulfonylated and N-alkylated carbazolyloxyacetic acids were investigated for the inhibition and modulation of the Alzheimer's disease associated γ-secretase. The introduction of a lipophilic substituent, which may vary from arylsulfone to alkyl, turned

Full text of DOI:10.1016/j.bmcl.2006.09.061

Bioorganic & Medicinal Chemistry Letters 17 (2007) 176–182
N-Substituted carbazolyloxyacetic acids modulate Alzheimer
associated c-secretase
a
b
c
c
´
Rajeshwar Narlawar, Blanca I. Perez Revuelta, Karlheinz Baumann, Robert Schubenel,
Christian Haass,b Harald Steinerb and Boris Schmidta,*
aClemens Scho¨pf-Institute of Chemistry and Biochemistry, Darmstadt University of Technology,
Petersenstr. 22, D-64287 Darmstadt, Germany
bLaboratory for Alzheimer’s and Parkinson’s Disease Research, Ludwig Maximilian University Mu¨nchen,
Schillerstr. 44, D-80336 Mu¨nchen, Germany
cF. Hoffmann-La Roche Ltd, Pharmaceuticals Division, Preclinical Research CNS, Bldg. 70/345, CH-4070 Basel, Switzerland
Received 28 August 2006; revised 20 September 2006; accepted 21 September 2006
Available online 10 October 2006
Abstract—N-Sulfonylated and N-alkylated carbazolyloxyacetic acids were investigated for the inhibition and modulation of the Alz-
heimer’s disease associated c-secretase. The introduction of a lipophilic substituent, which may vary from arylsulfone to alkyl,
turned 2-carbazolyloxyacetic acids into potent c-secretase modulators. This resulted in the selective reduction of Ab42 and an
increase of the less aggregatory Ab38 fragment by several compounds (e.g., 7d and 8c). Introduction of an electron donating group
at position 6 and 8 of N-substituted carbazolyloxyacetic acids either decreased the activity or inversed modulation. The most active
compounds displayed activity on amyloid precursor protein (APP) overexpressing cell lines in the low micromolar range and little or
no effect on the c-secretase cleavage at the e-site.
Ó 2006 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is a devastating neurodegen-
erative disorder that causes the most common form of
dementia, affecting approximately 5% of the popula-
tion over the age of 65 years in Europe. Short-term
memory impairment, disorientation, aphasia and a
general cognitive decline are common symptoms early
in disease development. As the disease progresses, spa-
tial and motor abilities are affected and the patient be-
comes bedridden and completely dependent on the
caretaker. According to the World Health Organiza-
tion, an estimated 37 million people worldwide cur-
rently have dementia; AD affects about 18 million of
them.1
tase. Cleavage at the Ab N-terminus is executed by
the b-site cleaving enzyme (BACE).2,3 The following
intramembrane cleavage by c-secretase occurs with lit-
tle sequence specificity, resulting in Ab fragments of
different length, predominantly Ab40, Ab42 and some
Ab38. Ab42 being the most aggregatory. The C-terminal
fragments generated by a- and b-secretase cleavage,
C83 and C99, are cleaved within their transmembrane
domains to produce the p3 peptide. The functional
c-secretase complex requires the correct assembly of
at least five components: presenilin-1 (PS1), nicastrin,
anterior pharynx defective-1 (Aph-1), presenilin
enhancer-2 (Pen-2) and the substrate.4–10 PS1, a cata-
lytic subunit of the c-secretase complex, harbours two
aspartates in the transmembrane domains 6 and 7 that
define a novel active site closely resembling that of
other recently identified aspartic proteases.11–14 PS1
acts as a heterodimer of N- and C-terminal fragments
(NTF, CTF) that derive from autoproteolysis. c-Secre-
tase activity can be controlled by the inhibition of the
active site of PS1 or by interference with complex
assembly or substrate recognition, the latter resulting
in allosteric modulation or inhibition. The allosteric
mechanisms are particularly attractive targets for drug
development as they may control the ratio of the Ab
Pathological lesions and plaques consisting of the amy-
loid b-peptide (Ab) are found in the brains of the AD
patients. Ab is heterogeneously produced by the
sequential cleavages of amyloid precursor protein
(APP) by the two aspartic proteases: b- and c-secre-
Keywords: Secretase; Alzheimer dementia; Modulator; Aspartic
protease.
*
Corresponding author. Tel.: +49 6151 163075; fax: +49 6151
163278; e-mail: schmidt_boris@t-online.de
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.061

R. Narlawar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 176–182
177
fragments: Ab38, Ab40, and Ab42, while retaining the
cleavage of other substrates. Most of the reported
and confirmed c-secretase inhibitors are not substrate
specific and inhibit the processing of other c-secretase
substrates equally,15 for example, Notch and other.16
Epidemiological studies indicated an association be-
tween nonsteroidal anti-inflammatory drugs (NSAIDs)
O
O
OH
OH
OH
O
Cl
Cl
O
O
O
O
N
Decyl
N
a, b
H
N
N
R
1 Carprofen
H
2 BSc3041
6
8
O
OH
F
Entry
Compound
R
CO2H
Cl
1
2
3
4
8a
Octyl
F
F
N
O
O S
N
O
S
O
8b
8c
8d
Nonyl
Decyl
H
Cl
4 BMS-299897
3 BSc2842
Scheme 3. Synthesis of N-alkylated carbazolyloxyacetic acids and 8d.
Reagents and conditions: (a) KOtBu, RX, THF, 0 °C rt, 2–8 h, 80–
91%; (b) NaOH, MeOH/H2O (1:1) rt, 2 h, 92–97%.
Scheme 1. Carprofen, the most active carprofen derivatives and BMS-
299897.
O
O
OH
O
a
N
H
N
H
6
5
OH
O
O
b, c
N
O
S
O
R
7
Entry
Compound
R
7a
7b
7c
7d
7e
7f
4-methylphenyl
1
2
3,5-bis(trifluoromethyl)phenyl
4-biphenyl
3
4
2,4,6-tri-iso-propylphenyl
4-n-propylphenyl
5
6
Octyl
7
7g
7h
7i
(1S)-1-(methyl)-7,7-dimethylbicyclo[2.2.1]heptan-2-one
4-chlorophenyl
8
9
3,5-difluorophenyl
10
11
2-bromophenyl
7j
3-nitrophenyl
7k
t
Scheme 2. Synthesis of N-sulfonylated carbazolyloxyacetic acid derivatives. Reagents and conditions: (a) ClCH2CO2 Bu, K2CO3, acetone, 60–70 °C,
16 h, 100%; (b) NaH, RSO2Cl, THF, 0 °C rt, 2–8 h, 80–100%; (c) 20% TFA in DCM, 1–4 h, 80–100%.

178
R. Narlawar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 176–182
use and a reduced risk for the development of
AD.17–20 Recently, we reported N-sulfonylated and N-
alkylated derivatives (2, 3) of carprofen (Scheme 1) that
modulate c-secretase and selectively reduce Ab42.21,22
This is accompanied by an increase of the less aggrega-
tory Ab38 species. Carprofen 1 is a COX-2 inhibitor
(COX = cyclooxygenase) approved for the use in dogs,
cows and horses as ImadylÒ or RimadylÒ. The selectiv-
ity of carprofen versus COX-2canine and COX-1canine is
greater than 100:1 (COX-2canine IC50/R/S-carprofen
102 nM, R-carprofen 5.97 lM, S-carprofen 37 nM).23
Original carprofen (as isolated from RimadylÒ tablets
500 mg) was found to be a weak inhibitor of c-secretase
and reduced Ab38, Ab40 and Ab42 at high concentra-
tions. Knowing about the relevance of the carboxylic
acid and N-substitution of carprofen, we decided to
investigate scaffolds not included in the patent applica-
tions and publications by Koo et al.24 or Stock,25 or
Imbimbo26 and others.27,28 Carprofen’s similarity to
the c-secretase inhibitor BMS-299897 (Scheme 1)
prompted us to investigate N-sulfonylated and N-alkyl-
ated 2-hydroxy carbazolyloxyacetic acids. The corre-
sponding sulfonamides 7a–k were readily accessible by
straightforward synthesis.
OPMB
OPMB
OH
a
b
Cl
Cl
NO2
11
NO2
10
NO2
O
9
A series of 2-hydroxy carbazole N-sulfonamide deriv-
atives was prepared as outlined in Scheme 2. 2-
Hydroxycarbazole 5 was alkylated using anhydrous
OPMB
OPMB
O
t
c
K2CO3 and ClCH2CO2 Bu in acetone at 60–70 °C to
N
H
N
give compound 6. N-Sulfonylation of 6 was carried
out using NaH, various sulfonyl chlorides in THF
and subsequent t-butyl deprotection by 20% trifluoro-
acetic acid in dichloromethane furnished the desired
compound 7.
H
O
12b
12a
Scheme 4. Synthesis of 6-methoxy and 8-methoxy carbazoles.
Reagents and conditions: (a) PMBCl, K2CO3, acetone, reflux, 16 h,
97%; (b) [(Ph3P)4Pd], 3-methoxybenzene-boronic acid, K2CO3, H2O,
toluene, reflux, 18 h, 67% (c) Ph3P, o-dichlorobenzene, reflux, 16 h,
60%.
N-Alkylated carbazolyloxyacetic acids were prepared to
evaluate the contribution of the sulfonamide moiety in
O
R1
O
O
R1
OPMB
a, b
N
N
H
H
R2
R2
12
13
OH
O
R1
O
c, d
N
R2
O
S
O
14
R3
Entry Compound
R1
R2
R3
1
2
3
4
5
6
14a
14b
14c
14d
14e
14f
OMe
OMe
OMe
OMe
H
H
H
H
H
3,5-(bis-trifluoro)phenyl
4-chlorophenyl
4-biphenyl
2-bromophenyl
OMe
OMe
4-chlorophenyl
4-biphenyl
H
Scheme 5. Synthesis of 6-methoxy and 8-methoxy N-sulfonylated carbazolyloxyacetic acids. Reagents and conditions: (a) 10% Pd–C, H2, MeOH,
60 psi, rt, 12 h, 81%; (b) ClCH2CO2 Bu, K2CO3, acetone, reflux, 16–18 h, 82–88%; (c) R3SO2Cl, NaH, THF, 0 °C rt, 1–3 h, 55–71%; (d) 20% TFA in
DCM, rt, 2 h, 80–94%.
t

R. Narlawar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 176–182
179
Table 1. Activity report of N-alkylated and N-sulfonylated carbazol-
yloxyacetic acids
20% TFA in DCM furnished the desired compound
14.
Entry Compound Cell toxicityd(lM)
IC50 (lM)
a
Compounds 7a–k and 8a–c turned out to be effective
modulators of c-secretase. They affected the cleavage
at the c38, c40 and c42 sites to a different extent,
and particularly reduced the formation of Ab42, while
increasing the formation of Ab38 (see Table 1 and
Fig. 1).33 The most potent inhibitors of Ab42 were
compounds 7d, 7g, and 8c. Compounds 14a–c showed
modulatory activity but required very high concentra-
tions. Compounds 14e and 14f showed some indica-
tions to be inverse modulators, they increased Ab42
formation and reduced Ab38 formation at very high
concentrations (see Fig. 2). The necessary levels for
IC50/EC50 determination were not reached at 40 lM.
The effect of the most potent compounds on c-secre-
tase cleavage at the e-site was assessed using an in vi-
tro assay that monitors the de novo generation of
b-amyloid precursor protein intracellular domain
(AICD).29,30 The formation of AICD was affected by
the compounds to various extent (Fig. 3). However,
much higher compound concentrations than those
determined to be modulatory were required to inhibit
the e-cleavage. One of the most active N-sulfonylated
carbazolyloxyacetic acids (7d) was selected for evalua-
tion in COX-1 and COX-2 assays to rule out COX-1
or COX-2 mediated effects at the concentrations nec-
essary for c-secretase modulation. The assays were
performed at CEREP (www.cerep.com) using indo-
methacin as a standard for COX-1 and NS398 for
COX-2. The compound 7d displayed no activity on
COX-1 and COX-2 at 10 lM concentration. Only a
few compounds within this structural class displayed
cell toxicity in H4 cells at the highest concentration
tested (40 lM). It is very likely that this toxicity is
caused by unique structural properties of single
compounds.34,35
Ab38 Ab40 Ab42
1
2
7a
7b
7c
7d
7e
7f
7g
7h
7i
20
20
13
>40 39
>40 >40
>40
13
3
20
20
40
19
24
7.5
4
7.8
5
>40
>40
40
NDb >40 38
NDb >40 37
6
7
14
32
13
>40
16
24
30
>40 20
>40 32
>40 >40
>40 >40
>40 >40
>40 19
>40 17
8
40
40
9
10
11
12
13
14
15
16
17
18
7j
40
40
7k
8a
8b
8c
8d
2
>40
>40
>40
>40
—
NDb >40 11
>40
5.8
9.3
—
>40 >40
>40 2.9
>20 8.5
3
4c
—
—
—
0.0071
a EC50 values are displayed for Ab38
.
b Maximum effect on Ab38 not observed at the highest concentration
tested.
c IC50 value is displayed for total Ab.
d Significant cellular toxicity observed at this concentration. Viability
reduction > 20%.
7g and the most active derivate 7d, where the sulfon-
amide is shielded by isopropyl substituents. The N-alkyl-
ated carbazolyloxyacetic acids were synthesized as
shown in Scheme 3. Compound 6 was alkylated using
KOtBu and alkyl halide in dry THF. Subsequent depro-
tection of t-butyl group by base hydrolysis provided de-
sired N-alkylated carbazolyloxyacetic acid 8.
We synthesized the 2-hydroxy-6-methoxy and 2-hy-
droxy-8-methoxy N-sulfonylated carbazolyloxyacetic
acids to investigate the impact of an electron donating
substituent on the activity. We commenced the synthesis
of 6-methoxy and 8-methoxy-2-hydroxy carbazoles
from the commercially available 4-chloro-3-nitrophenol
9 as outlined in Scheme 4. The –OH functionality of 9
was protected as a para-methoxybenzyl (PMB) ether
10. Suzuki–Miyaura coupling of 10 with 3-methoxyben-
zeneboronic acid using the catalyst [(PPh3)4]Pd gave
biphenyl 11, which was then cyclized to its correspond-
ing carbazole by refluxing it in o-dichlorobenzene with
PPh3. The PPh3 mediated cyclization resulted in two
regioisomers, 6-methoxy carbazole, 12a and 8-methoxy
carbazole, 12b which were separated by flash column
chromatography.
The introduction of a single lipophilic substituent,
which may vary from arylsulfone to alkyl, turns the
N-substituted carbazolyloxyacetic acids into a potent
c-secretase modulator. Some in silico parameters of
these lead candidates are close to the range of drug-like
compounds,31 but the lipophilic substituents cause a
dramatic increase of the clogP. The carboxylic
acid may interfere with uptake, but the total polar sur-
2
˚
face area of 8c is just 62.9 A , this compares to
2
COX189 (lumiracoxib, tPSA = 58 A ).The more polar
˚
compound 7c displays similar properties (clogP =
2
6.426, tPSA = 107.5 A ) as the carboxylic acid BMS-
˚
2
299897 (clogP = 5.92, tPSA = 93.4 A ).
27
˚
Further
increases in chain length will worsen this property,
but the determination of the maximum length may pro-
vide information on drug localisation or trafficking.
We speculate that the lipophilic substituent anchors
the N-substituted carbazolyloxyacetic acid in the re-
quired orientation within the membrane, thus the max-
imum tolerated length should be similar to natural
phospholipids. This hypothesis will be explored in the
next series. The desired selective reductions of Ab42
and the accompanying increases of the less aggregatory
Ab38 fragment are displayed by several compounds
N-Sulfonylated derivatives of 2-hydroxy-6-methoxy
and 2-hydroxy-8-methoxy carbazolyloxyacetic acids
were synthesized as depicted in Scheme 5. The
PMB group of 12 was deprotected by hydrogenation
at 60 psi and the free –OH was alkylated using
t
anhydrous K2CO3 and ClCH2CO2 Bu in acetone at
60–70 °C to yield alkylated 13. N-Sulfonylation of
13 was carried out using NaH and R3SO2Cl in dry
THF and subsequent t-butyl group removal by

180
R. Narlawar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 176–182
Figure 3. Dose–response curves for the most active N-substituted
carbazolyloxyacetic acids (7d, 7c, 8c, and 7g) on in vitro AICD
generation. Results are the average of three independent experiments.
Error bars indicate the standard error of the mean.
(e.g., 7d and 8c). The introduction of an electron
donating substituent at 6- and 8-position of
N-substituted carbazolyloxyacetic acids either de-
creased the modulatory activity (14a–14c) or inversed
modulatory activity (14d–14f). If affected at all, the
e-cleavage of c-secretase was inhibited at much higher
compound concentrations than those determined to
be modulatory at the c-site (Fig. 3). The compounds
are thus expected to have little or no impact on
c-secretase-mediated signalling via the AICD or via
intracellular domains (ICDs) of other c-secretase
substrates.
The present data qualify the scaffold as a lead structure
for c-secretase modulation, but do not justify extensive
investigation of the current compounds in animals.
Improvements of potency, solubility and the investiga-
tion in neuronal cells are required prior to the investiga-
tion of in vivo activity.
Figure 1. Dose–response curves for the most active N-substituted
carbazolyloxyacetic acid derivatives; Ab (% of control). (A) Com-
pound 7d. (B) Compound 7g. (C) Compound 8c.
Figure 2. Activity of the compounds 14a–14f.

R. Narlawar et al. / Bioorg. Med. Chem. Lett. 17 (2007) 176–182
181
24. Koo, E. H. M.; Golde, T. E.; Galasko, D. R. WO
2001078721, 2001.
Acknowledgments
25. Stock, N.; Munoz, B.; Wrigley, J. D. J.; Shearman, M. S.;
Beher, D.; Peachey, J.; Williamson, T. L.; Bain, G.; Chen,
W.; Jiang, X.; St-Jacques, R.; Prasit, P. Bioorg. Med.
Chem. Lett. 2005, 16, 2219.
26. Peretto, I.; Radaelli, S.; Parini, C.; Zandi, M.; Raveglia, L.
F.; Dondio, G.; Fontanella, L.; Misiano, P.; Bigogno, C.;
Rizzi, A.; Riccardi, B.; Biscaioli, M.; Marchetti, S.;
Puccini, P.; Catinella, S.; Rondelli, I.; Cenacchi, V.;
Bolzoni, P. T.; Caruso, P.; Villetti, G.; Facchinetti, F.;
DelGiudice, E.; Moretto, N.; Imbimbo, B. P. J. Med.
Chem. 2005, 48, 5705.
27. Barten, D. M.; Guss, V. L.; Corsa, J. A.; Loo, A.; Hansel,
S. B.; Zheng, M.; Munoz, B.; Srinivasan, K.; Wang, B.;
Robertson, B. J.; Polson, C. T.; Wang, J.; Roberts, S. B.;
Hendrick, J. P.; Anderson, J. J.; Loy, J. K.; Denton, R.;
Verdoorn, T. A.; Smith, D. W.; Felsenstein, K. M.
J. Pharmacol. Exp. Ther. 2005, 312, 635.
We thank the DFG (B.S. and R.N., SPP1085
(SCHM1012-3-1/2); C.H. and H.S., SFB596), the EU
[C.H., B.S., and R.N., APOPIS (Contract LSHM-CT-
2003-503330)] and the BMBF (C.H., NGFN) for sup-
port of this work.
References and notes
1. Mount, C.; Downton, C. Nat. Med. 2006, 12, 780.
2. Schmidt, B. ChemBioChem 2003, 4, 366.
3. Schmidt, B.; Braun, H. A.; Narlawar, R. Curr. Med.
Chem. 2005, 12, 1677.
4. Chung, H.-M.; Struhl, G. Nat. Cell Biol. 2001, 3, 1129.
5. Goutte, C.; Tsunozaki, M.; Hale, V. A.; Priess, J. R. Proc.
Natl. Acad. Sci. U.S.A. 2002, 99, 775.
28. Beher, D.; Bettati, M.; Checksfield, G. D.; Churcher, I.;
Doughty, V. A.; Oakley, P. J.; Quddus, A.; Teall, M. R.;
Wrigley, J. D. WO 2005013985, 2005.
29. Sastre, M.; Steiner, H.; Fuchs, K.; Capell, A.; Multhaup,
G.; Condron, M. M.; Teplow, D. B.; Haass, C. EMBO
2001, 2, 835.
6. Francis, R.; McGrath, G.; Zhang, J.; Ruddy, D. A.; Sym,
M.; Apfeld, J.; Nicoll, M.; Maxwell, M.; Hai, B.; Ellis, M.
C.; Parks, A. L.; Xu, W.; Li, J.; Gurney, M.; Myers, R. L.;
Himes, C. S.; Hiebsch, R.; Ruble, C.; Nye, J. S.; Curtis, D.
Dev. Cell 2002, 3, 85.
7. Zhou, S.; Zhou, H.; Walian, P. J.; Jap, B. K. Proc. Natl.
Acad. Sci. U.S.A. 2005, 102, 7499.
8. Gu, Y.; Sanjo, N.; Chen, F.; Hasegawa, H.; Petit, A.;
Ruan, X.; Li, W.; Shier, C.; Kawarai, T.; Schmitt-Ulms,
G.; Westaway, D.; St. George-Hyslop, P.; Fraser, P. E. J.
Biol. Chem. 2004, 279, 31329.
30. Larbig, G.; Zall, A.; Schmidt, B. Helv. Chim. Acta 2004,
87, 2334.
31. Boehm, H.-J.; Schneider, G. Virtual Screening for Bioac-
tive Molecules; Wiley-VCH: Weinheim/New York, 2000.
32. Brockhaus, M.; Grunberg, J.; Rohrig, S.; Loetscher, H.;
Wittenburg, N.; Baumeister, R.; Jacobsen, H.; Haass, C.
Neuroreport 1998, 9, 1481.
9. Takasugi, N.; Tomita, T.; Hayashi, I.; Tsuruoka, M.;
Niimura, M.; Takahashi, Y.; Thinakaran, G.; Iwatsubo,
T. Nature 2003, 422, 438.
33. Ab liquid phase electrochemiluminescence (LPECL) and
AICD assays. To evaluate the compounds for their
potency in modulating c-secretase activity, we used the
LPECL assay to measure Ab isoforms. APP-overexpress-
ing cell lines were generated by stably transfecting human
neuroglioma H4 cells obtained from ATCC (Accession
No. CRL-1573 or HTB-148) with human APP695 in
vector pcDNA3.1. The biotinylated Ab specific antibodies
6E10 or 4G8 were used (Signet Laboratories) as capture
antibodies. The C-terminal specific Ab antibodies BAP15,
BAP24 and BAP29 were generated as described previous-
ly.32 Antibodies were labelled with TAG electro-chemilu-
minescent label according to manufacturer’s protocol
(Bioveris). Labelled antibodies were purified from unin-
corporated label using a PD-10 column (Pharmacia) and
stored in phosphate-buffered saline (PBS) containing 0.1%
sodium azide at 4 °C for several weeks or at À80 °C for
long-term storage. Cells were plated in 96-well plates (30–
60,000 cells/200 lL/well) and allowed to adhere for 2 h.
Compounds were dissolved in DMSO (vehicle) and
further diluted in cell culture medium to the desired
concentration. Hundred microlitres of compound con-
taining media was added to the cells and incubated for 20–
24 h at 37 °C. Before use, M-280 paramagnetic beads
(Bioveris) were diluted with assay buffer (50 mM Tris,
60 mM NaCl, 0.5% BSA and 1% Tween 20, pH 7.4). Fifty
microlitres of conditioned culture medium from the plated
cells were incubated with 50 lL of beads and 25 lL of each
labelled antibody (6E10-bio and BAP29-TAG for detec-
tion of Ab1-38, 6E10-bio and BAP24-TAG for detection of
Ab40, 6E10-bio and BAP15-TAG for detection of Ab42;
for detection of total Ab 4G8-bio and 6E10-TAG were
used) in a final volume of 250 lL for 3 h at room
temperature with gentle shaking. Synthetic Ab38, Ab40
and Ab42 peptides (Bachem) were used to generate
standard curves. These Ab peptides were solubilised in
DMSO at a concentration of 1 mg/mL, aliquoted and
10. Kimberly, W. T.; LaVoie, M. J.; Ostaszewski, B. L.; Ye,
W.; Wolfe, M. S.; Selkoe, D. J. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 6382.
11. De Strooper, B.; Annaert, W.; Cupers, P.; Saftig, P.;
Craessaerts, K.; Mumm, J. S.; Schroeter, E. H.; Schrijvers,
V.; Wolfe, M. S.; Ray, W. J.; Goate, A.; Kopan, R. Nature
1999, 398, 518.
12. Wolfe, M. S.; Xia, W.; Ostaszewski, B. L.; Diehl, T. S.;
Kimberly, W. T.; Selkoe, D. J. Nature 1999, 398, 513.
13. Esler, W. P.; Kimberly, W. T.; Ostaszewski, B. L.; Diehl,
T. S.; Moore, C. L.; Tsai, J.-Y.; Rahmati, T.; Xia, W.;
Selkoe, D. J.; Wolfe, M. S. Nat. Cell Biol. 2000, 2, 428.
14. Steiner, H.; Kostka, M.; Romig, H.; Basset, G.; Pesold, B.;
Hardy, J.; Capell, A.; Meyn, L.; Grim, M. L.; Baumeister,
R.; Fechteler, K.; Haass, C. Nat. Cell Biol. 2000, 2, 848.
15. Churcher, I.; Beher, D. Curr. Pharm. Des. 2005, 11, 3363.
16. Evin, G.; Sernee, M. F.; Masters, C. L. CNS Drugs 2006,
20, 351.
17. in’t Veld, B. A.; Ruitenberg, A.; Hofman, A.; Launer, L.
J.; van Duijn, C. M.; Stijnen, T.; Breteler, M. M. B.;
Stricker, B. H. C. N. Engl. J. Med. 2001, 345, 1515.
18. McGeer, P. L.; Schulzer, M.; McGeer, E. G. Neurology
1996, 47, 425.
19. Breitner, J. C.; Welsh, K. A.; Helms, M. J.; Gaskell, P. C.;
Gau, B. A.; Roses, A. D.; Pericak-Vance, M. A.; Saun-
ders, A. M. Neurobiol. Aging 1995, 16, 523.
20. Aisen, P. S.; Luddy, A.; Durner, M.; Reinhard, J. F., Jr.;
Pasinetti, G. M. J. Neurol. Sci. 1998, 161, 66.
21. Schmidt, B.; Baumann, S.; Narlawar, R.; Braun, H. A.;
Larbig, G. Neurodegen. Dis. 2006, 3, in press, doi:10.1159/
000095269.
22. Narlawar, R.; Blanca, P. R.; Steiner, H.; Haass, C.;
Schmidt, B.; Baumann, K. J. Med. Chem. 2006, 49,
accepted for publication.
23. Ricketts, A. P.; Lundy, K. M.; Seibel, S. B. Am. J. Vet.
Res. 1998, 59, 1441.

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stored frozen at À80 °C. Immediately before use, they
assays by a colourimetric cell proliferation assay (CellTiter
96TM AQ assay, Promega) utilizing the bioreduction of
MTS (Owen’s reagent) to formazan according to the
manufacturer’s instructions. Briefly, after removal of the
supernatant for Ab assays 80 lL of fresh cell culture
medium was added to each well and incubated for 1 h
before 20 lL MTS/PES solution was added. The optical
density was recorded at 490 nm after 1 h incubation at
37 °C. Cell viability was expressed as % of untreated
control.
were diluted in culture media to 16–2000 pg/mL. Electro-
chemiluminescence was quantified using an M-Series M8
analyzer (Bioveris). IC50/EC50 values were calculated using
GraphPad Prism ver4 software. EC50 values can only be
calculated when the dose–response curve had reached a
stable plateau at the maximum applied concentration.
c-Secretase activity was monitored by de novo production
of AICD (APP intracellular domain) in vitro by the
previously reported assay.30
34. Cell toxicity assay. Cell viability was measured in the
corresponding cells after removal of the media for the Ab
35. Barltrop, J. A.; Owen, T. C.; Cory, A. H.; Cory, J. C.
Bioorg. Med. Chem. Lett. 1991, 1, 611.