Rational design and synthesis of aminopiperazinones as β-secretase (BACE) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.10.050

Aminopiperazinone inhibitors of BACE were identified by rational design. Structure based design guided idea prioritization and initial racemic hit 18a showed good activity. Modification in decoration and chiral separation resulted in the 40 nM inhibitor,

Full text of DOI:10.1016/j.bmcl.2011.10.050

Bioorganic & Medicinal Chemistry Letters 21 (2011) 7255–7260
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Rational design and synthesis of aminopiperazinones as b-secretase
(BACE) inhibitors
Gary Tresadern a, , Francisca Delgado b, Oscar Delgado b, Harrie Gijsen c, Gregor J. Macdonald c,
⇑
Dieder Moechars d, Frederik Rombouts c, Richard Alexander e, John Spurlino e, Michiel Van Gool b,
Juan Antonio Vega b, Andrés A. Trabanco b,
⇑
a Research Informatics & Integrative Genomics, Calle Jarama 75, Poligono Industrial, Toledo 45007, Spain
b Neuroscience Medicinal Chemistry, Janssen Pharmaceutical Research & Development, Calle Jarama 75, Poligono Industrial, Toledo 45007, Spain
c Neuroscience Medicinal Chemistry, Janssen Pharmaceutica N.V., Turnhoutsweg 30, B-2340 Beerse, Belgium
d Neuroscience Biology, Janssen Research & Development, Janssen Pharmaceutica N.V., Turnhoutsweg 30, B-2340 Beerse, Belgium
e Structural Biology, Janssen Research & Development, Welsh and McKean Rd., Spring House, PA 19477,USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Aminopiperazinone inhibitors of BACE were identified by rational design. Structure based design guided
idea prioritization and initial racemic hit 18a showed good activity. Modification in decoration and chiral
separation resulted in the 40 nM inhibitor, (À)-37, which showed in vivo reduction of amyloid beta pep-
tides. The crystal structure of 18a showed a binding mode driven by interaction with the catalytic aspar-
tate dyad and distribution of the biaryl amide decoration towards S1 and S3 pockets.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 22 September 2011
Revised 13 October 2011
Accepted 14 October 2011
Available online 20 October 2011
Keywords:
BACE
Alzheimer’s disease
Amyloid
b-Secretase inhibitor
Neurodegeneration
Amyloid beta 42 (Ab42)
Memapsin
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder and the most common form of dementia. The societal bur-
den of AD is vast: the cost of treatment, care and loss of productiv-
ity is estimated at US$300 billion per year; in 2010 there were
454000 new cases of Alzheimer’s in the US alone and the number
of people with dementia is expected to double in the next
20 years.1 Current treatment with either acetylcholinesterase
therefore represent a potential disease modifying treatment for
AD.5
Despite the attractiveness of attenuating BACE mediated APP
proteolysis there have been difficulties identifying low molecular
weight, cell and brain penetrant BACE inhibitors.6 Early BACE
inhibitors were high affinity peptidomimetics such as OM99-2
(1)7 (Fig. 1) which has been crystallized with BACE.8 Lower MW
series were subsequently identified such as isonicotinamides (2)9
and hydroxy ethylamines (3).10 Lead optimization improved cellu-
lar activity and P-glycoprotein susceptibility,11 however, they re-
tained traits of their peptidic origin and were large, flexible and
polar. X-ray structures revealed these molecules form interactions
between the amino-alcohol group and the aspartic acid dyad in the
enzyme active site, Figure 2.12 Meanwhile, series containing ami-
dino and guanidino substructures were identified which interact
with the same aspartic acids. Acylguanidines (4)13, aminoimidaz-
oles (5 and 6)14,15 as well as 2-amino-3,4-dihydroquinazolines
(7) from our laboratories16 were all reported. In addition, amino-
pyrimidone hits were found from NMR fragment screening which
resulted in the attractive CNS lead, 8.17 BACE functions optimally
at acidic pH and is believed to be located in acidic intracellular
compartments, pH ꢀ5. At such pH amidino and guanidino motifs
inhibitors or N-methyl D-aspartate (NMDA) antagonists is mainly
symptomatic and does not cure or reverse progression of the dis-
ease.2 The cause of AD is unclear but Tau fibrils and amyloid beta
(Ab) deposits are characteristic neuropathological hallmarks and
may be associated with disease pathogenesis.3 The insoluble pla-
ques are predominantly aggregates of Ab peptides of 39–43 amino
acids formed via the sequential cleavage of b-amyloid precursor
protein (APP) by aspartyl proteases, b- and c
-secretase.4 The inhi-
bition of b-secretase (BACE, b-site APP cleaving enzyme) may
⇑
Corresponding authors. Tel.: +34 925 245782 (G.T.); tel.: +34 925 245792
(A.A.T.).
E-mail addresses: gtresade@its.jnj.com (G. Tresadern), atrabanc@its.jnj.com (A.A.
Trabanco).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.050

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G. Tresadern et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7255–7260
9 2IQG). Docking used the GOLD software (version 4.11)21 with
slow ‘most accurate’ approach, the GoldScore fitness function and
ten genetic algorithm runs per ligand. Protein structures were pre-
pared in MOE,22 the protonate 3D tool was used to ionise amino
acids, hydrogens were added, charges calculated, and the ligand re-
O
O
O
O
H
H
N
H2N
N
OH
N
H
N
H
N
H
N
H
O
OH
O
O
O
moved. A 14 Å radius around Asp228 (a-carbon) was used and all
H2N
O
OH
O
OH
waters were retained. There is debate about the ionisation state of
Asp32 and Asp228 in the active site.23,24 Here, both acids were
deprotonated which delivered good results for re-docking of the
crystallized ligands and avoided the need for placement restraints
which enforce ligand-protein interactions. Inhibitors were ionised
according to their calculated pKa at pH 5 with the ACD25 soft-
ware.26 This program performs well compared to other ap-
1, OM99-2
O
NH2
S
N
O
O
O
S
N
O
O
N
H
CF3
N
N
N
H
H
OH
NH
proaches.27
A 3D conformation with correct chirality was
NH
O
generated with MMFF94x force field minimisation in MOE and
used as input for docking. The best ranked docking solutions com-
pared to the experimental structures, for each of the five protein li-
gand complexes are shown in Figure 3. The protocol performed
well at predicting the binding mode of the distinct chemical series
when docked into their accompanying protein structure.
2
3
NH2
N
N
NH2
O2N
N
O
N
N
The BACE active site is flexible and adopts different conforma-
tions depending on the inhibitor.28,29 Docking performance is
dependent on the protein structure used.30 Using a protein struc-
ture solved with an inhibitor from a significantly different struc-
tural class is problematic as most approaches are unable to
predict the required enzyme flexibility. For the assessment of our
proposed ideas this was overcome by using different enzyme
structures for the virtual screening. Given that most ideas were
either amidino/guanidino heterocycles or were similar to our 2-
amino-3,4-dihydroquinazolines the 2Q11 and 2VA7 crystal struc-
tures were both used. The ideas were small scaffold-like motifs
and needed to be converted into virtual molecules in a consistent
way to allow fair comparison of each. Enumeration with sidechains
such as biphenyl, bisarylamide and biphenethyl, as seen in 8, gen-
erated over 300 virtual molecules of similar size and decoration.
N
N
NH2
O
NH2
F
4
5
6
O
N
O
O
N
N
N
OMe
H2N
N
NH2
7
8
Figure 1. BACE inhibitors 1–8.
H
O
N+
H
N
O
NH
O
H
O
O
N
N+
H
O
H
O
H
Asp228
O
O
Asp228
O
Asp32
Asp32
Figure 2. Schematic view of interaction between hydroxy ethylamine and amidino/
guanidino substructures and the catalytic aspartic acid dyad in the BACE active site.
5, 3H0B
6, 3IGB
8, 2VA7
are protonated and form a salt bridge interaction to the aspartate
as well as a sophisticated series of hydrogen bonds, Figure 2. More
recently there have been further reports of aminohydantoins18 and
aminopyridines19 which also adopt a similar binding mode.
We pursued a strategy to design new amidino and guanidino
containing heterocycles with the potential to bind at the aspartic
acid dyad in the BACE active site. It had been recognized that some
molecules from our series of lipophilic 2-amino-3,4-dihydroqui-
nazolines had a particularly high basic pKa. An experimental pKa
of 10.6 was measured for one example and was typical of other
compounds in the series. The combination of high lipophilicity
and high basicity is detrimental for a CNS lead.20 Therefore, given
the need for cellular penetration and CNS activity we sought to tar-
get new species with lower basicity. De-novo ideas were generated
and in silico virtual screening with docking and pKa calculations
was used for prioritization.
7, 2Q11
N
O
F
O
HN
I
F
N
H
OH
9
9, 2IQG
Firstly a docking protocol was validated which could be used to
assess and prioritize ideas. The aim was to reproduce five experi-
mental ligand-BACE complexes (5 3H0B, 6 3IGB, 7 2Q11, 8 2VA7,
Figure 3. Docking validation performed by re-docking known inhibitors into their
corresponding BACE X-ray structures. Docked pose is shown in atom-type coloring
and original X-ray coordinates are in magenta.

G. Tresadern et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7255–7260
7257
O
Docking of the virtual molecules was performed into both crys-
O
H2N CN
H2N CO2Me
N
tal structures and the best scoring were inspected by eye. Piperaz-
inone 10 was amongst the best solutions from docking into the
2VA7 structure, Figure 4. The amidine of the piperazinone formed
the salt bridge interaction and the H-bond network with the aspar-
tate dyad. The trial molecule contained a biaryl amide at a quater-
nary centre. The NH of the amide formed an H-bond interaction
with the backbone carbonyl of Gly230. This interaction was both
attractive to target and plausible as it had been seen in a series
of non-aspartate inhibitors.31 The calculated basic pKa with ACD
was predicted to be 6.3 or 8.0 depending on the tautomeric form
of the amidine. Either would be mainly protonated at pH 5 whilst
being less basic than our previous series. Synthesis was initiated to
target similar piperazinone molecules, pursuing two regioisomers
(Type I and II) of the piperazinone with biaryl amide decoration
at the chiral centre.
(iv), (v)
HN
(i)
(ii), (iii)
O
NO2
NO2
NO2
NO2
11
12
13
14
H2N
S
H2N
N
N
N
(viii)
(vi)
(vii)
N
HN
N
O
O
O
NH2
NO2
NO2
17
15
16
O
N
18a: X = CH, R = Cl
18b: X = CH, R = CN
18c: X = CH, R = CF3
N
H2N
(ix)
O
The synthesis of the final 5-amino-3,6-dihydro-1H-pyrazin-2-
N
X
18d: X = N; R = Me
one derivatives (Type I) is depicted in Scheme 1. The
a-aminoni-
N
H
trile 12 was prepared from the corresponding 1-(3-nitro-phenyl)-
ethanone (11) in 82% yield by the Strecker reaction. Hydrolysis of
the nitrile with hydrochloric acid followed by esterification gave
R
Scheme 1. Reagents and conditions: (i) TMSCN, NH4Cl, NH3 (7 N in MeOH), rt,
4 days, 82%; (ii) HCl (6 N), reflux, 18 h, 63%; (iii) SOCl2, MeOH, 0 °C, 15 min, then
reflux, 18 h, 63%; (iv) 2-chloroacetylchloride, Et3N, CH2Cl2, 0° C, 1 h, 99%; (v) NH2Me
(33% in EtOH), EtOH, 70 °C, 3 h, 99%; (vi) Lawesson’s reagent, pyridine, toluene,
90 °C, 18 h, 16%; (vii) NH3 (7 N in MeOH), NH3 (33% aqueous), 67 °C, 4 h, 37%; (viii)
H2, Pd/C, EtOH/AcOEt, rt, 12 h, 99%; (ix) ArCOOH, HATU, PhN(CH3)2, CH2Cl2, rt, 3 h,
30–67%.
the
a-aminoester 13 which was converted into the piperazine-
2,5-dione 14 by treatment with 2-chloroacetylchloride and subse-
quent cyclization by reaction with methylamine. Setup of the ami-
dine moiety was done via conversion of amide to thioamide,
followed by treatment with NH3. The thioamide intermediate 15
was prepared by reaction of 14 with Lawesson’s reagent in toluene
at 90 °C in the presence of base. The use of pyridine as a base was
crucial for the regioselectivity in the reaction, otherwise mixtures
of products were obtained. Transformation of the thioamide 15
into amidine 16 using a mixture of NH3 (7 N in MeOH) and NH3
(33% aqueous) and subsequent hydrogenation of the nitro group
in the phenyl ring gave the key intermediate 17. Finally, reaction
of 17 with a selected set of carboxylic acids using HATU and
PhN(CH3)2, in dry CH2Cl2 gave compounds 18a–18d.32
O
O
O
O
HO
H2N
HN CHO
HN
N
H
(i)
(ii), (iii)
(iv)
(v), (vi)
13
NO2
NO2
NO2
19
20
21
O
O
O
O
Type II inhibitors, 3-amino-5,6-dihydro-1H-pyrazin-2-one
derivatives, were prepared following the reaction sequences
O
O
H2N
H2N
N
N
N
N
(vii)
(viii)
(ix)
HN
N
N
N
showed in Schemes 2 and 3. Reduction of
a-aminoester 13 with
NaBH4 followed by reaction with (Boc)2O and subsequent oxida-
tion of the hydroxyl group with Dess–Martin periodinane fur-
nished aldehyde 20. Next, the N-methyl group was introduced by
reductive amination to give intermediate 21. Acylation with ethyl
oxalyl chloride and subsequent deprotection of the N-Boc amino
group gave piperazine-2,3-dione 22 by in situ cyclization of the
aminoester intermediate. The conversion of amide 22 to amidine
24 via a thioamide intermediate, analogous to synthesis of 16,
proved problematic. Alternatively, the piperazine-dione 22 was
O-methylated using Me3OBF4 to afford 23 which was converted
to amidine 24 after treatment with NH3 in the presence of NH4Cl.
NO2
NO2
NO2
NH2
22
23
24
25
O
N
N
H2N
(x)
O
N
N
H
Cl
26
Scheme 2. Reagents and conditions: (i) NaBH4, EtOH, rt, 3 h, 87%; (ii) Boc2O,
NaHCO3, THF, 0 °C to rt, 99%; (iii) Dess-Martin periodinane, CH2Cl2, 0 °C to rt, 1 h,
95%; (iv) (a) MeNH2 (2 M in THF), CH2Cl2/AcOH, rt, 1 h; (b) NaBH(OAc)3, rt, 2 h, 95%;
(v) ethyl oxalyl chloride, DIPEA, CH2Cl2, 0° C, 3 h, 98%; (vi) HCl (4 M in 1,4-dioxane),
rt, 1 h, 83%; (vii) Me3OBF4, CH2Cl2, rt, 3 days, 79%; (viii) NH4Cl, NH3 (2 M in EtOH),
75 °C, 18 h, 49%; (ix) H2, Pd/C, EtOH/AcOEt, rt, 12 h, 98%; (x) ArCOOH, HATU,
PhN(CH3)2, CH2Cl2, rt, 3 h, 52%.
O
N
O
N
N
N
H2N
H
Hydrogenation of the nitro group to the aniline 25 followed by
acylation gave the final product 26.
10
3-Amino-5,6-dihydro-1H-pyrazin-2-one derivatives bearing a
fluorine atom in 2 position of the phenyl ring were prepared start-
ing from 1-(5-bromo-2-fluoro-phenyl)-ethanone 27, Scheme 3. A
bromo substituent instead of nitro as used in Schemes 1 and 2
was chosen to avoid SNAr type side-reactions expected with the
use of the highly electron deficient 1-(5-nitro-2-fluoro-phenyl)-
ethanone. Commercially available 27 was transformed into
N
N
O
O
N
H2N
H2N
R
R
Type I
Type II
Figure 4. Docked pose for piperazinone idea molecule 10 docked into the BACE
crystal structure 2VA7.

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G. Tresadern et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7255–7260
HO
expected lower pKa of this cyclic amidine. Chiral separation of
O
F
H2N
H2N CN
H2N CO2Me
18a revealed (À)-18a to be the active stereoisomer. Molecules
18b, 18c and 18d show some SAR for variation in the distal aryl
on the amide. More polar aryls, cyano pyridyl 18b and methyl pyr-
azine 18d were less active compared to chloro and CF3 pyridyls 18a
and 18c respectively. In the Type II aminopiperazinones, 26 had
comparable decoration to 18a and showed similar activity, 0.55
Br
Br
Br
Br
(iv)
(i)
(ii), (iii)
F
F
F
27
28
29
30
O
F
O
O
O
O
O
N
HN
HN CHO
HN
and 0.107 lM in enzymatic and cellular hAbTOT assays respectively.
The experimental basic pKa of 26 was 7.6. Again chiral separation
N
Br
Br
Br
H
(viii), (ix)
(vii)
(v), (vi)
F
F
revealed one stereoisomer to be responsible for the activity, (À)-
31
32
33
26 0.295 and 0.085 lM in enzymatic and cellular hAbTOT assays.
Interestingly, molecule 37 demonstrates that introduction of fluo-
O
O
O
O
H2N
N
H2N
rine on the anilinic phenyl results in improved activity, 0.04 and
0.025 lM in the enzymatic and cellular assays.
N
N
N
(x)
(xi)
(xii)
N
N
Br
Br
H2N
The crystal structure of (À)-18a with BACE was solved at 2.2 Å
resolution, Figure 5. The resultant binding mode was similar to that
predicted from the docking virtual screening. The piperazinone
amidine interacts with the catalytic aspartate dyad as expected
and the biaryl amide is directed to sub-pockets S1 and S3. The
amide NH is 2.3 Å from the carbonyl oxygen of Gly230 and forms
a favorable interaction although it is not as well aligned as ex-
pected from the docking. The chloro substituent on the distal pyr-
idyl extends deep into the S3 pocket and contributes to affinity.
The X-ray structure confirmed the stereochemistry of (À)-18a to
be R as predicted by the docking studies.
Molecules 18a, 26 and (À)-37 were assessed for their ability to
reach the brain. A 30 mg/kg dose of 18a and 26 was administered
subcutaneously (sc) to mice, and brain and plasma levels were
measured at 1, 2 and 4 h. Although 18a displayed high plasma lev-
els, 4633, 1712 and 439 ng/ml at 1, 2 and 4 h respectively, the cor-
responding brain levels were low, 184, 123 and 90 ng/g. Molecule
26 showed plasma levels of 993, 461 and 200 ng/ml and brain lev-
els of 614, 157 and 20.5 ng/g. A higher 60 mg/kg sc dose of (À)-37
resulted in plasma levels of 7145, 2455 and 270 ng/ml at 1, 2 and
4 h, which in turn led to relatively high brain exposure 8078, 3342
and 347 ng/g. These molecules showed a rapid decrease in plasma
concentration levels, likely due to low metabolic stability.35 The
three similar molecules had calculated logP of 1.7, 2.3 and 2.5 for
18a, 26 and (À)-37 respectively. 18a and 26 had comparable
experimental basic pKa, 7.8 and 7.6. The trend in brain exposure
may be driven by gradual increase in log P, but remains to be
understood in more detail.
F
F
F
34
35
36
O
N
N
(xiii)
H2N
F
O
N
N
H
Cl
37
Scheme 3. Reagents and conditions: (i) TMSCN, NH4Cl, NH3 (7 N in MeOH), rt,
4 days, 98%; (ii) HCl (6 N), reflux, 18 h, 62%; (iii) H2SO4 (conc.), MeOH, reflux, 48 h,
95%; (iv) NaBH4, EtOH, rt, 3 h, 87%; (v) Boc2O, NaHCO3, THF, 0 °C, 15 h, quant. (vi)
Dess–Martin periodinane, CH2Cl2, 0 °C to rt, 1 h, 83%; (vii) (a) MeNH2 (2 M in THF),
CH2Cl2/AcOH, rt, 1 h; (b) NaBH(OAc)3, rt, 2 h, 64%; (viii) ethyl oxalyl chloride, DIPEA,
CH2Cl2, 0° C, 3 h, 93%; (ix) HCl (4 M in 1,4-dioxane), rt, 1 h, 99%; (x) Me3OBF4,
CH2Cl2, rt, 3 days, 73%; (xi) NH4Cl, NH3 (2 M in EtOH), 75 °C, 18 h, 55%; (xii) NaN3,
CuI, Na2CO3, DMSO, N,N’-dimethylethylenediamine, 110 °C, 1 h, 95%; (ix) DMTMM,
MeOH, 0 °C, 3 h, 25%.
Table 1
BACE inhibitory activity of aminopiperazinonesa
Compds
Enzymatic
Cellular
BACE IC50
(
l
M)a
hAb42 IC50
(
l
M)a
hAbTOT IC50(l
M)a
18a
0.447
>30
0.098
>10
0.078
>10
(S)-(+)-18a
(R)-(À)-18a
18b
18c
18d
0.324
0.813
1.380
7.079
0.550
>30
0.295
>30
0.040
0.036
0.186
0.068
1.259
0.115
>10
0.083
>10
0.028
0.037
0.151
0.079
1.148
0.107
>10
0.085
>10
0.025
26
(+)-26
(À)-26
(+)-37
(À)-37
a
Data presented from enzymatic and cellular assays.37
amidine 35 following a reaction sequence identical to Scheme 2.
Copper catalyzed amination of the bromoderivative 35 led to inter-
mediate 36 in excellent yield. Finally, selective acylation of the ani-
line 36 with the corresponding carboxylic acid was achieved using
DMTMM33 as coupling agent in MeOH to afford the final product
37.
All synthesized inhibitors34 were screened against BACE to
determine IC50 values in both enzymatic and cellular assays35, Ta-
ble 1. For the type I aminopiperazinones the first example, race-
mate 18a, showed 0.447 and 0.078 lM activity in the enzymatic
and cellular hAbTOT assays respectively. There were no difficulties
with cell permeation as this and other compounds in the series
all show good cellular activity. The basic pKa of racemate 18a
was experimentally determined as 7.8. This is acceptable for a
CNS lead and commensurate with the calculated value and
Figure 5. Crystal structure of (À)-18a with BACE (pdb ID code: 3U6A). Key
interactions between the ligand and Asp32 and Asp228 are shown as well as the
location of Tyr71 in the active site flap.

G. Tresadern et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7255–7260
7259
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Molecules 26 and (À)-37 were tested for their ability to reduce
Ab peptides in vivo.36 A 30 mg/kg sc dose of 26 resulted in a 34%
reduction in AbTOT levels at 1 h, with no effect seen at 2 and 4 h
due to the low amounts in brain. The higher brain levels of (À)-
37 following the 60 mg/kg sc dose resulted in a more pronounced
reduction of AbTOT by 48%, 67% and 73% at 1, 2 and 4 h respectively.
The results demonstrate that in vivo reduction of Ab peptides is
tractable for this series.
Overall, aminopiperazinones show moderate basicity (pKa <8.0),
relatively low lipophilicity, (log P 2.5 for (À)-37) and low MW (390
for (À)-37). The series demonstrate good cellular activity and
examples show in vivo reduction of Ab peptides following subcuta-
neous administration. Future work will focus on reducing clear-
ance by improvement of metabolic stability.
Acknowledgments
The authors thank Daniele Bemporad for input in the early pro-
ject and Jeroen Van De Ven and Geert Van Hecke for generating the
in vitro data. Crystals were grown and soaked by Shanghai Medic-
ilon, Inc. and Medicilon Preclinical Research (Shanghai) LLC. The X-
ray experiments were performed on the PX1 BEAMLINE at the
Swiss Light Source, Paul Scherrer Institut, Villigen, Switzerland.
Data were collected by Expose GmbH.
20. Wager, T. T.; Chandrasekaran, R. Y.; Hou, X.; Troutman, M. D.; Verhoest, P. R.;
Villalobos, A.; Will, Y. ACS Chem. Neurosci. 2010, 1, 420.
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33. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride.
34. Racemic compounds 18a, 26 and 37 were separated into their corresponding
enantiomers by chiral SFC (CHIRALPAK AD-H 5
35. Molecules 26 and (À)-37 were 99% and 100% metabolised respectively after
15 min incubation with mouse liver microsomes at 1 M concentration.
lm 250 Â 20 mm).
l
36. For in vivo Ab peptide levels detection mice were analyzed. In brief mice (CD1,
male, 22-28gr) treated with the compounds were examined and compared to
those untreated or treated with vehicle. Compounds were formulated in 20%
hydroxypropyl b cyclodextrin and administered as a single subcutaneous dose
to overnight fasted animals. After the indicated time animals were sacrificed
and the left hemisphere was resuspended in
8 volumes of 0.4% DEA
(diethylamine)/50 mM NaCl containing protease inhibitors (Roche-
11873580001 or 04693159001) per gram of tissue. All samples were
homogenized in the FastPrep-24 system (MP Biomedicals) using lysing
matrix D (MPBio #6913-100) at 6 m/s for 20 seconds. Homogenates were
centrifuged at 221.300Âg for 50 min. The resulting high speed supernatants
were then transferred to fresh eppendorf tubes. Nine parts of supernatant were
neutralized with 1 part 0.5 M Tris–HCl pH 6.8 and used to determine Ab levels.
To quantify the amount of AbTOT in the soluble fraction of the brain
homogenates, ELIZA was used. Briefly, the standards (a dilution of synthetic
Ab1-40, Bachem) were prepared with final concentrations ranging from 10000
to 0.3 pg/ml. The samples and standards were co-incubated with the
biotinylated mid-domain antibody 4G8. 50 ll of conjugate/sample or
conjugate/standards mixtures were then added to the antibody-coated plate
(antibody JRF/rAb/2). The plate was allowed to incubate overnight at 4 °C in
order to allow formation of the antibody-amyloid complex. A Streptavidine–
Peroxidase-Conjugate was added, followed 60 min later by an additional wash
step and addition of Quanta Blu fluorogenic peroxidase substrate according to
the manufacturer’s instructions (Pierce Corp., Rockford, Il). A reading was
performed after 10–15 min (excitation 320 nm /emission 420 nm).
14. Stachel, S. J.; Coburn, C. A.; Rush, D.; Jones, K. L. G.; Zhu, H.; Rajapakse, H.;
Graham, S. L.; Simon, A.; Holloway, M. K.; Allison, T. J.; Munshi, S. K.; Espeseth,
A. S.; Zuck, P.; Colussi, D.; Wolfe, A.; Pietrak, B. L.; Lai, M. –T.; Vacca, J. P. Bioorg.
Med. Chem. Lett. 2009, 19, 2977.
15. Malamas, M. S.; Erdei, J.; Gunawan, I.; Barnes, K.; Johnson, M.; Hui, Y.; Turner,
J.; Hu, Y.; Wagner, E.; Fan, K.; Olland, A.; Bard, J.; Robichaud, A. J. J. Med. Chem.
2009, 52, 6314.

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G. Tresadern et al. / Bioorg. Med. Chem. Lett. 21 (2011) 7255–7260
37. Enzymatic assay: BACE1 was assayed fluorimetrically on Monaco Safas
spektrofluorometer flx. The wavelength for excitation was 328 nm and for
emission 400 nm. The reactions were followed at 25 °C over 20 min. A human
recombinant BACE1 (Enzo Life Sciences, Lörrach, Germany) stock solution of
five different inhibitor concentrations and IC50 values obtained. Cellular Assay:
In an lisa assays the levels of AbTOT produced and secreted into the medium of
human neuroblastoma SKNBE2 cells were quantified. The assay is based on the
human neuroblastoma SKNBE2 expressing the wild type Amyloid Precursor
Protein (hAPP695). The compounds were diluted and added to the cells,
a
500 lg/mL was diluted 1:10 with assay buffer (20 mM sodium acetate pH 4.5,
0.1% CHAPS, 0.1% Top Block). Inhibitor stock solutions were prepared with
DMSO. A 1 mM stock solution of the substrate Mca-Ser-Glu-Val-Asn-Leu-Asp-
Ala-Glu-Phe-Lys(Dnp)-OH (Bachem, Bubendorf, Switzerland) was diluted 1:10
with 50% DMSO and 1:3 with ammonium acetate buffer (10 mM, pH 7.4). The
final concentration of DMSO was 2.4%, and the final concentration of the
incubated for 18 h and then AbTOT was measured by sandwich alisa. alisa is a
sandwich assay using biotinylated antibody AbN/25 attached to streptavidin
coated beads and antibody Ab4G8 for the detection of AbTOT. In the presence of
AbTOT, the beads come into close proximity. The excitation of the Donor beads
provokes the release of singlet oxygen molecules that triggers a cascade of
energy transfer in the Acceptor beads, resulting in light emission. Light
emission is measured after 1 hour incubation (excitation at 650 nm and
emission at 615 nm). A best-fit curve was fitted to the plot of % Controlmin
versus compound concentration and an IC50 value was obtained.
substrate was 2.5
0.5 g/mL of BACE1. In a cuvette 905
DMSO in a total volume of 10 L and 10
thoroughly mixed and incubated for 1 h at 25 °C. The reaction was initiated by
adding 75 L of the substrate. Experiments were performed in duplicate with
l
M. Assays were performed with a final concentration of
L assay buffer, inhibitor solution and
L BACE1 solution were added,
l
l
l
l
l