Aminoimidazoles as BACE-1 inhibitors: The challenge to achieve in vivo brain efficacy

Article abstract of DOI:10.1016/j.bmcl.2012.01.079

The evaluation of a series of bicyclic aminoimidazoles as potent BACE-1 inhibitors is described. The crystal structures of compounds 14 and 23 in complex with BACE-1 reveal hydrogen bond interactions with the protein important for achieving potent inhibit

Full text of DOI:10.1016/j.bmcl.2012.01.079

Bioorganic & Medicinal Chemistry Letters 22 (2012) 1854–1859
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Aminoimidazoles as BACE-1 inhibitors: The challenge to achieve in vivo
brain efficacy
Britt-Marie Swahn a, , Jörg Holenz a, Jacob Kihlström a, Karin Kolmodin a, Johan Lindström a,
⇑
Niklas Plobeck a, Didier Rotticci a, Fernando Sehgelmeble a, Marie Sundström a, Stefan von Berg a,
Johanna Fälting b, Biljana Georgievska b, Susanne Gustavsson b, Jan Neelissen c, Margareta Ek d,
Lise-Lotte Olsson d, Stefan Berg a,
⇑
a Department of Medicinal Chemistry, AstraZeneca R&D Södertälje, SE-15185 Södertälje, Sweden
b Department of Neuroscience, AstraZeneca R&D Södertälje, SE-15185 Södertälje, Sweden
c Department of DMPK, AstraZeneca R&D Södertälje, SE-15185 Södertälje, Sweden
d Discovery Sciences, AstraZeneca R&D Mölndal, SE-43183 Mölndal, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
The evaluation of a series of bicyclic aminoimidazoles as potent BACE-1 inhibitors is described. The crys-
tal structures of compounds 14 and 23 in complex with BACE-1 reveal hydrogen bond interactions with
the protein important for achieving potent inhibition. The optimization of permeability and efflux prop-
erties of the compounds is discussed as well as the importance of these properties for attaining in vivo
brain efficacy. Compound (R)-25 was selected for evaluation in vivo in wild type mice and 1.5 h after oral
Received 9 December 2011
Revised 20 January 2012
Accepted 21 January 2012
Available online 28 January 2012
co-administration of 300
by 17% and the plasma Ab40 level by 76%.
lmol/kg (R)-25 and efflux inhibitor GF120918 the brain Ab40 level was reduced
Keywords:
Alzheimer’s disease (AD)
b-Secretase
Ó 2012 Elsevier Ltd. All rights reserved.
BACE-1 inhibitors
Aminoimidazoles
Efflux
In vivo brain efficacy
Alzheimer’s disease (AD) is a neurodegenerative brain disorder
characterized clinically by progressive decline of cognitive func-
tion. Pathologically, AD is characterized by amyloid plaques,1 con-
taining Ab peptide(s), and by neurofibrillary tangles (NFTs)
containing hyper-phosphorylated tau protein. Ab peptides are pro-
duced from membrane-bound b-amyloid precursor protein (APP)
by the sequential proteolytic cleavage of two aspartyl proteases,
the bicyclic ring of 3. Thus independently from others5 we em-
barked on investigating this compound class. In this paper we will
discuss the properties important for in vivo brain efficacy and de-
scribe the effort to improve bicyclic aminoimidazole derivatives 3
towards achieving in vivo brain efficacy. We disclose novel BACE-1
inhibitors with enhanced permeability properties, culminating in
the design of R-(25) displaying Ab40 lowering effects in mice brain.
Within the first scoping activities of this series different
aromatic rings as well as aromatic ring substituents were evalu-
ated for potency and ADME properties.
b- and
c-secretase. b-Secretase (b-site APP cleaving enzyme,
BACE-1), has been identified as the enzyme responsible for the ini-
tial processing of APP.2 Processes that limit the accumulation of
neurotoxic Ab peptides could offer effective treatments of AD. Thus
inhibition of BACE-1 represents a strategy for the development of
disease-modifying therapeutics for the treatment of AD.3
The original BACE-1 inhibitor lead dihydroisocytosine 1, ema-
nating from fragment based lead generation,4 was the starting
point for scaffold hopping via the aminohydantoin 2 into the bicy-
clic aminoimidazoles 3 as depicted in Figure 1. One of the reasons
for selecting the bicyclic lead 3 was the possibility to fine-tune the
properties of the amidine moiety by introducing R substituents on
R
H2N
H2N
H2N
N
R
O
N
N
N
N
N
O
N
1
2
3
⇑
Corresponding authors.
E-mail address: britt-marie.swahn@astrazeneca.com (B.-M. Swahn).
Figure 1. Scaffold hopping from dihydroisocytosines to aminohydantoins to
aminoimidazoles.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.079

B.-M. Swahn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1854–1859
1855
NH2
with ammonia and tert-butylhydroperoxide in methanol to give
the 2,3,4,8-tetrahydroimidazo[1,5-a]pyrimidine-6-amine (THIP)
derivative 9. In the final step, the 2-fluoro-3-methoxyphenyl group
was introduced by a palladium catalyzed microwave assisted
Suzuki7 reaction to give compound 10.
The in vitro inhibition of BACE-1 was determined using a fluo-
rescence energy transfer FRET-based screen.8 The pIC50 values
and the Caco-2 permeability9 values for selected compounds are
shown in Table 1. Potent BACE-1 inhibition can be attained in this
series, as for example, shown for compound 13, but low Caco-2
permeability values as exemplified for 10–12 were limiting their
use as pharmacological probes. High Caco-2 values (>10) are an
indicator for good blood brain permeability properties and an
improvement for this series was needed.
S
Br
N
d
a, b, c
N
Br
N
Br
4
5
6
Br
S
S
S
N
HN
S
HN
N
g
f
e
N
N
7
Br
8
Br
The polar surface area (PSA) of the compounds was in an
acceptable range for passing the blood brain barrier (BBB), so we
reasoned that the low permeability could be due to the high basi-
city of the aminoimidazole group (pKa estimated to be >8) and that
it is the fraction of non-protonated species that permeates the
membrane. To increase the amount of the neutral form a reduction
of the pKa seemed reasonable. Therefore, a di-F moiety was intro-
duced in the bicyclic ring to allow for a lowering of the pKa. The
synthesis of these compounds followed the same procedure as de-
scribed for 10, but the diamine in the ring forming reaction
(Scheme 1, step f) was replaced with 2-di-F-propane-1,3-
diamine.10
H2N
H2N
N
N
h
N
N
N
N
N
N
Br
9
F
10
O
Scheme 1. Reagents and conditions: (a) BuLi, THF, À78 °C; (b) 4-cyanopyridine; (c)
NaBH4, MeOH, rt, 12 h, 62%; (d) (im)2CS, CH2Cl2, ꢀ100%; (e) CS2, t-BuOK, THF, À78
to 0 °C, ꢀ100%; (f) propylenediamine, EtOH, 80 °C, 2 h, 89%; (g) TBHP, NH4OH,
MeOH, 40 °C, 12 h, ꢀ100%; (h) 2-fluoro-3-methoxyphenyl boronic acid, Pd(dppf)Cl2,
CsCO3, DME–H2O–EtOH 6:3:1, MW 130 °C, 45%.
The di-F substituted tetrahydroimidazopyrimidines generally
displayed increased Caco-2 permeability together with decreased
11
pKa as shown in Table 2. In some instances, as for 15 and 16,
the permeability was still low and apparently other factors than
pKa were also contributing to the compounds permeability proper-
ties. We suspected that transporters could be involved and there-
fore we started to assess efflux in the Caco-2 assay.9 The
measured efflux values as shown in Table 2 also confirmed that
the compounds are substrates for transporters.
The pyridine containing tetrahydroimidazopyrimidine ana-
logues6 were synthesized as exemplified in Scheme 1. The
procedure was modified especially in the first step, compared to
the published synthesis,5 to allow for the introduction of pyridine.
1,3-Dibromobenzene 4 was treated with 1 equiv of butyl lithium in
THF at À78 °C, followed by reaction with 4-cyanopyridine to give
the imine which was reduced to amine 5 by reaction with sodium
borohydride in methanol at room temperature over night. Treat-
ment of the amine with thiocarbonyldiimidazole in dichlorometh-
ane quantitatively gave the isothiocyanate 6 which was reacted
with potassium tert-butoxide and carbon disulfide in THF at low
temperature to give the thiazolidine-2,5-dithione derivative 7.
Reaction with propylenediamine gave 3,4,7,8-tetrahydroimi-
dazo[1,5-a]pyrimidine-6-thione derivative 8 which was treated
The compounds were also assessed for their ability to inhibit
the formation of sAPPb in a cell-based assay.12 There was generally
a good correlation between the pIC50 values in the FRET and the
cell assay, except when the pKa of the compounds were below
ꢀ6. In these cases, a drop-off in potency in the cell assay could
be observed, probably due to the smaller fraction of inhibitors
being protonated by the catalytic aspartates. A pKa below 6 was
measured or predicted for pyridines as in examples 14, 15 and
20. Both 15 and 20 display a drop-off that can be explained by
pKa but the lack of drop-off for 14 is not fully understood.
One of the more potent analogues (14) was subjected to crystal-
lization in BACE-1 protein and the structure of the complex was
determined at 1.75 Å resolution (Fig. 2).13 The aminoimidazole
moiety of compound 14 interacts via a hydrogen bond network
to the two catalytic residues Asp32 and Asp228, as previously
shown for BACE-1 inhibitors containing an aminoheterocycle moi-
ety.4 We hypothesize that a proton is shared between Asp32 and
the aminoimidazole resulting in a formal charge of À1 for the cat-
alytic residues and the compound together. This would be the
same charge state as in the catalytically active enzyme–substrate
complex where one of the aspartic residues is believed to be pro-
tonated and the peptide bond together with the nucleophilic water
is neutral.14 However, it cannot be excluded that an additional pro-
ton is shared between Asp32 and Asp228 in this complex as the
closest carboxyl oxygens are only 3.1 Å apart. Compound 14 binds
to a protein conformation where the so called flap is open. This
allows the R1 substituted aryl to interact with Trp76. In the case
of 14 the nitrogen of the 4-pyridyl ring accepts a hydrogen bond
from Trp76. The di-F substitution on the tetrahydroimidazopyrim-
idine is completely solvated and does not interfere with any parts
of the protein.
Table 1
Biological activities of tetrahydroimidazopyrimidines
H2N
X
N
R1
H
Ar
N
10
2-F, 3-OCH3-Ph
N
N
11
N
H
5-pyrimidinyl
12
C
4-OCH3
5-pyrimidinyl
X
R1
13
C
4-OCH3
3,5-di-Cl-Ph
Ar
Caco-2 (10À6 cm/s)
pKa
PSA (Å)
a
Compound
pIC50
10
11
125
13
7.05
6.58
7.04
7.65
1.0
0.6
0.6
nd
nd
7.6
nd
nd
68
79
78
57
nd; not determined.
a
Values are means of n ꢁ 2 determinations, absolute value of standard deviation
ꢂ10%.

1856
B.-M. Swahn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1854–1859
Table 2
Biological activities of di-F-tetrahydroimidazopyrimidines
X
N
Y
H
R1
H
Ar
F
H2N
N
14
2-F, 3-OCH3-Ph
F
Y
15
N
H
H
5-pyrimidinyl
16
C
H
4-OCH3
5-pyrimidinyl
N
N
17
18
19
20
21
C
C
C
N
C
H
H
H
H
F
4-OCHF2
4-OCHF2
4-OCHF2
H
5-pyrimidinyl
5-OCH3, 3-pyridinyl
2-F, 3-pyridinyl
2-F, 5-OCH3-Ph
3-pyridinyl
X
R1
Ar
4-OCH3 ,3-CH3
pIC50 cella sAPPb
Caco-2 (10À6 cm/s)
Efflux ratio
pKa
a
Compound
pIC50
14
(R)-14
15
16
17
18
19
20
21
7.39
7.69
6.97
7.00
7.63
7.86
7.52
7.54
7.43
7.27
7.51
5.92
6.74
7.71
7.85
7.90
6.55
7.35
7.0
6.7
0.6
1.1
4.1
2.4
6.6
8.0
3.3
7.7
8.1
nd
nd
nd
7.5
nd
6.3
nd
nd
5.3
nd
6.8
6.1
nd
nd
5.9
nd
nd; not determined.
a
Values are means of n ꢁ 2 determinations, absolute value of standard deviation ꢂ10%.
The difluoromethoxy substituent as in analogue 17, compared
to methoxy 16 and pyridine 15, increased the potency both in
the FRET and in the cell-based assay. A similar trend was observed
in compounds 18 and 19, both displaying good potencies. Com-
pound 20 with the highest permeability displayed a drop-off in po-
tency when going from the FRET to the cell assay. We also
investigated if an electron withdrawing substituent such as fluoro
in the aryl ring examplified by 21 could have a positive effect on
the permeability. This effect was minor and the permeability was
equal for Ar such as 16 and best permeability was demonstrated
by compound 21.
When examining some Br-intermediates, we found that a com-
pound without the biaryl motif as in 22 (Table 3) showed better
permeability and efflux properties when compared to the corre-
sponding biaryl analogues. We decided to explore this further by
introducing aryl group replacements and a few examples are
shown in Table 3. The alkynes 23 and 24 were potent BACE-1
inhibitors in the cell assay and the F-propyl ether analogue 25
Figure 2. Crystal structure of compound 14 bound to BACE-1, PDB code 4acu. Key
interactions between inhibitor (yellow), protein (gray), N (blue), O (red) and water
molecules (red balls) are highlighted with dashed lines. Figures made using
PyMOL.15
Table 3
Biological activities of non-biaryl di-F-tetrahydro-imidazopyrimidines
F
H2N
R2
R1
F
N
N
Br
22
23
24
25
4-OCH3
N
CCCH2OCH3
4-OCHF2
4-OCHF2
4-OCHF2
CCCH2CH2OCH3
OCH2CH2CH2F
R1
R2
pIC50 cella sAPPb
Caco-2 (10À6 cm/s)
Efflux ratio
pKa
a
Compound
pIC50
22
23
24
25
5.47
7.11
7.25
7.23
7.50
nd
11
8.4
9.5
11
8
1.2
3.5
3.6
3.2
0.8
nd
nd
nd
6.5
nd
7.46
7.87
7.83
8.10
(R)-25
nd; not determined.
a
Values are means of n ꢁ 2 determinations, absolute value of standard deviation ꢂ10%.

B.-M. Swahn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1854–1859
1857
The crystal structure of compound 23 in complex with BACE-1
has been refined to 2.0 Å resolution and is shown in Figure 3.13
Compound 23 overlaps well with the structure of compound 14.
However, in this case the oxygen of the R1 substituent forms the
hydrogen bond interaction with Trp76. The R2 alkyne extends to-
wards the S3 pocket.
Compound 25 was synthesized following a somewhat modified
procedure, compared to Scheme 1, starting from the F-propyloxy
substituted benzaldehyde 26 as shown in Scheme 2. The aldehyde
was converted into the sulfinimine 27 which was subsequently
treated with a modified Grignard reagent made from 1-bromo-4-
difluoromethoxybenzene to yield derivative 28. The sulfinamide
was then hydrolyzed to amine 29 with HCl in Et2O. Treatment of
the amine with thiophosgene in dichloromethane quantitatively
gave the isothiocyanate 30 which was reacted with potassium
tert-butoxide and carbon disulfide in THF at low temperature to
give the thiazolidine-2,5-dithione derivative 31. Reaction with
2.2-difluoropropane-1,3-diamine at elevated temperature over
night yielded compound 32 which was treated with ammonia
and tert-butylhydroperoxide in methanol to give the desired di-
F-tetrahydroimidazopyrimidine derivative 25.
Figure 3. Crystal structure of compound 23 in complex with BACE-1, PDB code
4acx. Key interactions between inhibitor (yellow), protein (gray), N (blue), O (red)
and water molecules (red balls) are highlighted with dashed lines.
displaying the best permeability value was selected for enantio-
meric HPLC separation. The more active enantiomer of 25, (R)-25,
showed a better efflux ratio than the racemate.
Alkynes 23 and 24 were synthesized using the general method
as described in Scheme 1, but the last reaction (Scheme 1, step h)
was replaced by a Pd catalysed coupling of the bromides using the
Sonogashira protocol and the appropriately substituted
acetylenes.16
The F-propyl ether (R)-25 being a low nM inhibitor of BACE-1 in
the cell assay, displaying reasonable permeability and efflux ratio,
was selected for in vivo studies. The plasma and brain concentra-
tions of (R)-25 were examined and it was found that the compound
had a low brain/plasma ratio as shown in Figure 4. However, when
a Pgp/BCRP inhibitor (GF120918)17 was dosed before (R)-25 the
O
O
S
N
S
O
NH
H
H
a
b
c
O
O
O
O
F
S
F
26
27
28
F
F
F
S
NH2
S
N
HN
S
d
e
O
O
O
O
O
F
F
O
F
F
F
F
F
29
31
30
F
F
F
S
F
H2N
N
F
N
F
HN
N
O
N
g
f
N
O
O
O
F
F
F
F
32
25
F
F
Scheme 2. Reagents and conditions: (a) 2-methyl-2-propanesulfinamide, Ti(IV)ethoxide, THF, 78%; (b) (1) n-BuLi (2.5 M in hexane), iPrMgBr (1 M in THF), THF, 0 °C; (2) 1-
bromo-4-(difluoromethoxy)benzene, THF, À65 °C, 97%; (c) HCl (1.0 M in Et2O), MeOH, 12 h, 45%; (d) Cl2CS, Na2CO3 (satd), CH2Cl2, 0 °C, ꢀ100%; (e) CS2, t-BuOK, THF, À78 °C,
ꢀ100%; (f) 2,2-difluoropropane-1,3-diamine dihydrochloride, N,N-diisopropylethylamine, EtOH, 70 °C, 55%; (g) TBHP, NH4OH, MeOH, 12 h, 46%.

1858
B.-M. Swahn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1854–1859
Acknowledgments
The authors would like to thank Torben Pedersen for skillful
synthetic contributions as well as Margareta Bergh and the Physio-
chemical Characterization Team at the Department of Medicinal
Chemistry for compound solubility/purity assessments. We thank
Carolina Sandman and Carrie Tsoi for Caco-2 measurement, Stefan
Martinsson and Olle Gissberg for bioanalysis, Anna Aagaard for
crystallization. We thank Martin Ekman and Martin Levin for FRET
and SHSY-5Y sAPPb measurements. In addition we thank the peo-
ple involved in performing the in vivo experiment Kristina Eliason,
Daniel Bergström, Ann Staflund, Anette Stålebring Löwstedt, Anja
Finn and Carina Stephan.
Supplementary data
Figure 4. In vivo plasma and brain concentration in C57BL/6 mice of compound (R)-
25 compared to plasma and brain concentration with pre-dosed Pgp/BCRP inhibitor
GF120918.
Supplementary data (the crystallization, X-ray data collection
and structure) associated with this article can be found, in the on-
line version, at doi:10.1016/j.bmcl.2012.01.079.
References and notes
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John, V. Nature 1999, 402, 537; (b) Vassar, R.; Bennett, B. D.; Babu-Khan, S.;
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R.; Luo, Y.; Fisher, S.; Fuller, J.; Edenson, S.; Lile, J.; Jarosinski, M. A.; Biere, A. L.;
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Science 1999, 286, 735.
3. (a) Hills, I. D.; Vacca, J. P. Curr. Opin. Drug Discov. Dev. 2007, 10, 383; (b)
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Cutler, L.; Demont, E.; Dingwall, C.; Dunsdon, R.; Hawkins, J.; Howes, C.;
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D.; Walter, D.; Wayne, G. Bioorg. Med. Chem. Lett. 2009, 19, 3674; (e) Stachel, S.
J.; Coburn, C. A.; Rush, D.; Jones, K. L. G.; Zhu, H.; Rajapakse, H.; Graham, S. L.;
Simon, A.; Holloway, K. M.; 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; (f) Zhou, P.; Li, Y.; Fan, Y.; Wang, Z.; Chopra, R.; Olland, A.; Hu,
Y.; Magolda, R. L.; Pangalos, M.; Reinhart, P. H.; Turner, M. J.; Bard, J.; Malamas,
M. S.; Robichaud, A. J. Bioorg. Med. Chem. Lett. 2010, 20, 2326; (g) Malamas, M.
S.; Robichaud, A.; Erdei, J.; Quagliato, D.; Solvibile, W.; Zhou, P.; Morris, K.;
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Pangalos, M. Bioorg. Med. Chem. Lett. 2010, 20, 6597.
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Figure 5. Reduction of Ab40 levels in brain and plasma of C57BL/6 mice by
compound (R)-25 dosed together with Pgp/BCRP inhibitor GF120918.
brain/plasma ratio was increased considerably from 0.18 to 2.3.18
Even though the compound displayed minor efflux in Caco-2 cells
this did not fully reflect the efflux properties of transporters ex-
pressed at the BBB, since inhibiting Pgp/BCRP increased the Cbr/
Cpl ratios >10Â. Apparently the effect of transporters like Pgp
can not be fully evaluated in Caco-2 cells and the efflux ratio of
(R)-25 in MDCK-MDR1 cells was later evaluated to be 19. The total
brain concentration was as shown ꢀ4
lM but the fraction unbound
in brain was small 0.57% indicating that these amidines display
large non-specific binding to brain tissue.19 The free brain concen-
tration of (R)-25 was determined and was judged to be sufficient to
show a decrease in brain Ab40 level if dosed together with the Pgp/
BCRP inhibitor. The brain Ab40 level was reduced by 17% in wild
type mice 1.5 h after oral co-administration of Pgp/BCRP inhibitor
6. (a) Berg, S.; Högdin, K.; Kihlström, J.; Plobeck, N.; Sehgelmeble, F.; Wirstam, M.
WO 2007/145568.; (b) Berg, S.; Holenz, J.; Högdin, K.; Kihlström, J.; Kolmodin,
K.; Lindström, J.; Plobeck, N.; Rotticci, D.; Sehgelmeble, F.; Wirstam, M. WO
2007/145569.
7. Miyaura, N.; Suzuki, A. Chem. Rev. 1995, 95, 2457.
(90
l
mol/kg) and (R)-25 (300
l
mol/kg), as shown in Figure 5, and
8. BACE-1 TR-FRET assay: Soluble part of the human b-Secretase (AA 1–AA 460)
and substrate (Europium)CEVNLDAEFK(Qsy7) was mixed in reaction buffer
(Na-acetate, chaps, triton X-100, EDTA pH 4.5). b-Secretase were mixed with
compound in DMSO and pre-incubated for 10 min. Substrate was added and
the reaction allowed to proceed for 15 min at rt. The reaction was stopped with
the plasma Ab40 level by 76%.18
In summary, during the effort to optimize the aminoimidazole
series towards in vivo brain efficacy we found a way to consider-
ably improve the Caco-2 permeability properties by converting
them into the di-F-tetrahydroimidazopyrimidine scaffold. In spite
of reasonable Caco-2 permeability properties they still exhibited
large efflux. A way to reduce efflux, as described, was to move
away from the bi-aryl motif by replacing the second aryl with a lin-
ear chain. Compound (R)-25 with low nM affinity for BACE-1 and
low efflux in Caco-2 was examined in vivo, with co-administration
of GF120918 (R)-25 displayed a statistically significant Ab40 low-
ering effect in mice brain.
7
l
L Na-acetate, pH 9. The fluorescence of the product was measured on a
Victor II plate reader with an excitation wavelength of 340 nm and an emission
wavelength of 615 nm. The final concentration of the enzyme was 2.7 g/mL;
l
the final concentration of substrate is 100 nM. Reported values are means of
n ꢁ 2 determinations, standard deviation ꢂ 10%.
9. Caco-2 cells grown for 14–21 days were used for the transport experiments.
For both apical to basolateral (A–B) and basolateral to apical (B–A) transport
directions the pH was adjusted to 7.4 with HBSS-HEPES. All compounds were
investigated at a concentration of 10 lM. Buffer volumes in the 24-well plates
were 0.20 mL on the apical side and 0.80 mL on the basolateral side. Samples
were withdrawn after 60 min from both sides. The integrity of the epithelial

B.-M. Swahn et al. / Bioorg. Med. Chem. Lett. 22 (2012) 1854–1859
1859
cell monolayer was monitored by measuring the passive transmembrane
diffusion of [14C]mannitol. Concentrations of compounds in donor and receiver
samples were analyzed by liquid chromatography tandem mass spectrometry.
23, respectively, have been deposited in the Protein Data Bank (www.rcsb.org)
under accession codes 4acu and 4acx.
14. Pearl, L.; Blundell, T. FEBS Lett. 1984, 174, 96.
15. Delano, W. L. The PyMOL Molecular Graphis System; Delano Scientific: Palo Alto,
CA, USA, 2002.
16. Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron Lett. 1975, 50, 4467.
17. Jonker, J. W.; Smit, J. W.; Brinkhuis, R. F.; Maliepaard, M.; Beijnen, J. H.;
Schellens, J. H. M.; Schinkel, A. H. J. Natl. Cancer Inst. 2000, 92, 1651.
18. Female 9-week old C57BL/6 mice (n = 12/group) received the Pgp/BCRP
Liquid scintillation was used for analysis of
[
14C]mannitol. The apparent
permeability coefficient Papp was calculated according to Papp = (dQ/dt)/
(A * C0), where dQ/dt is the slope at 60 min of the graph of the cumulative
amount transported vs time, A is the surface area of the membrane, and C0 is
the starting concentration. The efflux ratio is the ratio Papp BA/Papp AB.
10. (a) Berg, S.; Holenz, J.; Högdin, K.; Kolmodin, K.; Plobeck, N.; Rotticci, R.;
Sehgelmeble, F. WO 2007/145570.; (b) Berg, S.; Holenz, J.; Högdin, K.;
Kolmodin, K.; Plobeck, N.; Rotticci, D.; Sehgelmeble, F.; Wirstam, M. WO
2007/145571.
11. (a) Wan, H.; Holmén, A.; Wang, Y. D.; Lindberg, W.; Englund, M.; Någård, M.;
Thompson, R. Rapid Commun. Mass Spectrom. 2003, 1, 2639; (b) Wan, H.;
Holmén, A.; Någård, M.; Lindberg, W. J. Chromatogr., A 2002, 979, 369.
12. sAPPb release assay: SH-SY5Y cells were cultured in DMEM/F-12 with
Glutamax, 10% FCS and 1% non-essential amino acids. Compound was
incubated with cells for 16 h at 37 °C, 5% CO2. Meso Scale Discovery (MSD)
plates were used for the detection of sAPPb release. MSD sAPPb plates were
blocked in 3% BSA in Tris wash buffer for 1 h in rt and washed four times in Tris
inhibitor GF120918 (90
lmol/kg, po) 15 min prior to administration of
vehicle or the BACE-1 inhibitor (R)-25, given at 300
lmol/kg as a single dose
via oral gavage. Animals were anaesthetized 1.5 h after final administration of
vehicle or (R)-25. Blood samples were collected and brains dissected for
analysis of compound exposure and level of Ab40 in plasma and brain. The
blood was collected by heart puncture into pre-chilled microtainer tubes
containing EDTA. Plasma samples were prepared by centrifugation for 10 min
at approximately 3000 g at 4 °C within 20 min from sampling and stored
frozen at À70 °C until analysis. After blood sampling, the animals were
sacrificed by decapitation and brains were dissected. Cerebellum and olfactory
bulbs were removed and cerebrum was divided into left and right
hemispheres. The right hemisphere was weighed snap-frozen in liquid
nitrogen and stored at À70 °C until exposure analysis. The left hemisphere
was weighed snap-frozen in liquid nitrogen and stored at À70 °C until Ab40
analysis. Prior to analysis, soluble Ab was extracted according to a standardized
method using diethylamine. Drug concentration in brain samples was
determined by reversed-phase liquid chromatography and electro spray
tandem mass spectrometry. Ab40 levels in brain and plasma were analysed
using commercial ELISA.
buffer. After incubation, 20
and washed 384 well MSD sAPPb microplate, incubated with shaking in rt for
2 h followed by washing four times in Tris buffer. 10 L detection antibody was
added (1 nM) followed by incubation with shaking in rt for 2 h followed by
washing four times in Tris buffer. 40 L Read Buffer was added per well and the
plates were read in a SECTOR Imager. In addition, 20 L medium from the cell
lL of medium was transferred to the pre-blocked
l
l
l
plates were used to analyse cytotoxicity using the ViaLight™ Plus cell
proliferation/cytotoxicity kit (Cambrex BioScience) according to the
manufacturer’s instructions. Reported values are means of n P 2
determinations, standard deviation 6 10.
19. Fridén, M.; Gupta, A.; Antonsson, M.; Bredberg, U.; Hammarlund-Udenaes, M.
Drug Metab. Dispos. 2007, 35, 1711.
13. The experimental details are described in the attached Supplementary data.
The coordinates and structure factors for the complexes of BACE-1 with 14 and