Article

Design and Synthesis of β-Site Amyloid Precursor Protein Cleaving Enzyme (BACE1) Inhibitors with in Vivo Brain Reduction of β-Amyloid Peptides

Department of Medicinal Chemistry, ‡Department of Neuroscience, and §Department of Drug Metabolism and Pharmacokinetics (DMPK), AstraZeneca R&D Södertälje , SE-151 85, Södertälje, Sweden.
Journal of Medicinal Chemistry (Impact Factor: 5.45). 08/2012; 55(21). DOI: 10.1021/jm3009025
Source: PubMed
ABSTRACT
The evaluation of a series of aminoisoindoles as β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors and the discovery of a clinical candidate drug for Alzheimer's disease, (S)-32 (AZD3839), are described. The improvement in permeability properties by the introduction of fluorine adjacent to the amidine moiety, resulting in in vivo brain reduction of Aβ40, is discussed. Due to the basic nature of these compounds, they displayed affinity for the human ether-a-go-go related gene (hERG) ion channel. Different ways to reduce hERG inhibition and increase hERG margins for this series are described, culminating in (S)-16 and (R)-41 showing large in vitro margins with BACE1 cell IC(50) values of 8.6 and 0.16 nM, respectively, and hERG IC(50) values of 16 and 2.8 μM, respectively. Several compounds were advanced into pharmacodynamic studies and demonstrated significant reduction of β-amyloid peptides in mouse brain following oral dosing.

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Available from: Fredrik von Kieseritzky, Oct 20, 2015
Design and Synthesis of βSite Amyloid Precursor Protein Cleaving
Enzyme (BACE1) Inhibitors with in Vivo Brain Reduction of βAmyloid
Peptides
Britt-Marie Swahn,*
,
Karin Kolmodin,
Soa Karlstro
̈
m,
Stefan von Berg,
Peter So
̈
derman,
Jo
̈
rg Holenz,
Stefan Berg,
Johan Lindstro
̈
m,
Marie Sundstro
̈
m,
Dominika Turek,
Jacob Kihlstro
̈
m,
Can Slivo,
Lars Andersson,
David Pyring,
Didier Rotticci,
Liselotte O
̈
hberg,
Annika Kers,
Krisztian Bogar,
Fredrik von Kieseritzky,
Margareta Bergh,
Lise-Lotte Olsson,
Juliette Janson,
§
Susanna Eketja
̈
ll,
Biljana Georgievska,
Fredrik Jeppsson,
and Johanna Fa
̈
lting
Department of Medicinal Chemistry,
Department of Neuroscience, and
§
Department of Drug Metabolism and Pharmacokinetics
(DMPK), AstraZeneca R&D So
̈
derta
̈
lje, SE-151 85, So
̈
derta
̈
lje, Sweden
Discovery Sciences, AstraZeneca R&D Mo
̈
lndal, SE-43183 Mo
̈
lndal, Sweden
*
S
Supporting Information
ABSTRACT: The evaluation of a series of aminoisoindoles as
β-site amyloid precursor protein cleaving enzyme 1 (BACE1)
inhibitors and the discovery of a clinical candidate drug for
Alzheimers disease, (S)-32 (AZD3839), are described. The
improvement in permeability properties by the introduction of
uorine adjacent to the amidine moiety, resulting in in vivo
brain reduction of Aβ40, is discussed. Due to the basic nature
of these compounds, they displayed anity for the human
ether-a-go-go related gene (hERG) ion channel. Dierent ways to reduce hERG inhibition and increase hERG margins for this
series are described, culminating in (S)-16 and (R)-41 showing large in vitro margins with BACE1 cell IC
50
values of 8.6 and 0.16
nM, respectively, and hERG IC
50
values of 16 and 2.8 μM, respectively. Several compounds were advanced into
pharmacodynamic studies and demonstrated signicant reduction of β-amyloid peptides in mouse brain following oral dosing.
INTRODUCTION
Alzheimers disease (AD) is a neurodegenerative brain disorder
characterized clinically by progressive decline of cognitive
function, resulting ultimately in death. AD is currently the
leading cause of dementia in the elderly and represents a major
unmet medical need.
1
Pathologically, AD is characterized by
amyloid plaques containing A β peptides
2
and by neurobrillary
tangles (NFTs) containing hyperphosphorylated τ protein. Aβ
peptides are produced from membrane-bound amyloid
precursor protein (APP) by sequential proteolytic cleavage by
two aspartyl proteases, β- and γ-secretase. β-Secretase (β-site
APP cleaving enzyme, BACE1) has been identied as the
enzyme responsible for the initial processing of APP, generating
the secreted amino-terminal part of APP (sAPPβ) and the
membrane-bound carboxy-terminal part C99.
3
The C 99
fragment is subsequently cleaved by γ-secretase, leading to
toxic Aβ peptides. The generation of C99 and that of Aβ were
found to be blocked in BACE1 knockout mice.
4
Processes that
limit the accumulation of neurotoxic Aβ peptides are seen to be
prospective treatments of AD. Thus, inhibition of BACE1
represents a strategy for the development of disease-modifying
therapeutics for the treatment of AD.
5
Many of the earlier BACE1 inhibitor classes were generated
by rational structure-based desig n of pepti domimetics.
Transition-state isosteres such as statine, homostatine, and
hydroxyethylene (HE) were developed into cell-permeable
hydroxyethylamine (HEA) i sosteres . However, the major
drawbacks of these transition-state analogues are high MW,
high conformational exibility, and polar character limiting
their ability to cross the bloodbrain barrier. Even so, there
have recently been reports on HEA isosteres achieving brain Aβ
lowering in animal models.
6
We have found it to be exceedingly
dicult to nd BACE1 inhibitor leads using high-throughput
screening (HTS), but several compound classes such as
acylguanidines,
7
aminoimidazoles,
8
and amino-3,4-dihydroqui-
nazolines
9
have been discovered using this technique. New
BACE1 inhibitor leads have also evolved from fragment-based
lead generation approaches, resulting in compound classes such
as aminohydantoins,
10
2-aminopyridines,
11
dihydroisocyto-
sine,
12
and 2-aminoquinolines.
13
Despite the large eorts
during the past decade to develop BACE1 inhibitors, there
are few reports on compounds reducing brain Aβ peptides in
animal models,
6,13,14
and so far only one report has described
Special Issue: Alzheimer's Disease
Received: June 27, 2012
Published: August 27, 2012
Article
pubs.acs.org/jmc
© 2012 American Chemical Society 9346 dx.doi.org/10.1021/jm3009025 | J. Med. Chem. 2012, 55, 93469361
Page 1
successful BACE1 inhibition in human resulting in reduction of
Aβ levels in lumbar CSF.
15
In this paper, we report the design and synthesis of a potent
BACE1 inhibitor series with in vivo brain ecacy. As previously
described, fragment-based lead generation resulted in the
BACE1 inhibitor lead dihydroisocytosine 1.
12
This lead was
used as a starting point for scaold hopping into other series
such as aminohydantoins 2,
10
aminoimidazoles 3,
16
and
aminoisoindoles 4 as depicted in Figure 1. The aminoisoindoles
4 (R = H) were extensively investigated to build SAR
information, but the properties of these compounds rendered
it not possible to achieve robust in vivo brain ecacy. Herein,
we will discuss properties that are important for achieving in
vivo brain eects and describe the eorts to improve
aminoisoindole derivatives 4 to attain reduction of β-amyloid
peptides in the brain.
We were able to pinpoint the reason for the lack of in vivo
eects in the brain to the characteristics of the amidine moiety.
The properties of the amidine group could be modulated via
the introduction of a substituent (R = F) ortho to the amidine
group, resulting in sev eral novel BACE1 inhibitors with
enhanced permeability properties and potential to reduce the
Aβ peptide levels in the mouse brain. The BACE1 potency of
this uor oaminoisoindole series 4 (R = F) was further
improved and reached subnanomolar levels. These ba sic
compounds also displayed anity for the human ether-a-go-
go related gene (hERG)-encoded potassium ion channel, which
is involved in cardiac repolarization. Inhibition of the hERG
current may cause QT interval prolongation. Drug-induced QT
prolongation increases the likelihood of a polymorphous
ventricular arrhythmia known as Torsades de Pointes (TdP),
which may evolve into ventricular brillation and sudden
death.
17
A number of drugs have been withdrawn from late-
stage clinical trials and the market due to these cardiotoxic
eects; therefore, it is important to identify hERG inhibitors
early in drug discovery.
18
Dierent approaches to avoid hERG
side eects for the uoroaminoisoindole series are discussed.
CHEMISTRY
The syntheses of the compounds disclosed in this paper follow
the descriptions summarized in Schemes 14. The majority of
compounds were synthesized using method A (Scheme 1),
where the A-ring (as dened in Scheme 1) and a sulnimide
were reacted in the cyclization step. Commercially available
phenylzinc(II) iodide 5 was coupled with acid chloride 6 using
a palladium catalyst to yield the ketone 7. The ketone was
converted into the corresponding sulnimide 8 using titanium
ethoxide and 2-methyl-2-propanesulnamide. It was found
crucial to lithiate the corresponding pyridine (A-ring) and add
the sulnimide 8 to the lithiate d species at very low
temperatures to retrieve reasonable yields of the cyclized
compound 9.Inthenal synthetic step, the (C-ring)
pyrimidine was introduced by a palladium-catalyzed Suzuki
19
reaction to give racemate 10. The most active BACE1
enantiomer (S)-10 was isolated by chiral preparative super-
critical uid chromatography (SFC).
In some cases the lithiation of pyridines failed, due to base-
labile substituents, and method B shown in Scheme 2 was used.
The diuorophenylzinc(II) bromide reagent was prepared by
treating bromide 11 with Rieke zinc. Subsequent addition of
the reagent to the acid chloride 12 in the presence of CuCN
and LiCl produced the ketone 13. The ketone was converted
into the corresponding sulnimide 14 using titanium ethoxide
and 2-methyl-2-propanesulnamide. 1,3-Dibromobenzene was
lithiated with n-butyllithium and added to the sulnimide 14 at
low temperature to yield cyclized intermediate 15. A palladium-
catalyzed Suzuki coupling with 5-pyrimidinylboronic acid gave
racemate 16, which was separated using chiral chromatography
to yield the enantiomerically pure (S)-16.
Figure 1. Scaold hopping from dihydroisocytosines 1 to amino-
hydantoins 2, aminoimidazoles 3, and aminoisoindoles 4.
Scheme 1. General Synthesis Method A
a
a
Reagents and conditions: (a) tetrakis(triphenylphosphine)palladium(0), THF, 0 °C; (b) Ti(OEt)
4
, THF, 2-methyl-2-propanesulnamide, reux;
(c) t-BuLi (1.6 M in pentane), THF, 100 °C , 4-bromo -2-(triuoromethyl)pyridine; (d) 5-pyrimidinylboronic a cid, DMF, 90 °C,
Pd
II
Cl
2
dppf·CH
2
Cl
2
, aqueous K
2
CO
3
; (e) Chiralpak AD column (21.2 × 250 mm), eluent IPA (0.1% DEA)/CO
2
(20:80).
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Page 2
We found that it was possible to obtain chiral induction in
the cyclization step in Schemes 1 and 2 by using enantiomeri-
cally pure sulnimides. The commercially available (S)-2-
methyl-2-propanesulnamide was used to prepare enantiomeri-
cally enriched sulnimide (S)-17, applying the same procedure
as described for 8 and 14. The palladium-catalyzed coupling of
(S)-17 with ethynylcyclopropane to yield intermediate (S)-18
was performed b efore the cyclization step (Scheme 3).
Cautious treatment of 5-bromo-1-ethyl -3-methylpyridin-
2(1H)-one with butyllithium and butylmagnesium chloride
followed by subsequent addition of intermediate (S)-18 gave
(R)-19 with an enantiomeric purity of 70%. The pure (R)-19
enantiomer was isolated after chiral HPLC chromatography.
(S)-17 was also used as a starting material in the synthesis of
the amide derivative (R)-22 (Scheme 4). Addition of (S)-17 to
metalated 5-b romo -1-ethyl-3-methylp yridin-2 (1H)-on e gave
Scheme 2. General Synthesis Method B
a
a
Reagents and conditions: (a) Rieke zinc, THF, CuCN, LiCl, rt; (b) Ti(OEt)
4
, 2-methylpropane-2-sulnimide, THF, 60 °C; (c) n-BuLi, 1,3-
dibromobenzene, THF, 78 °C ; (d) 5-pyrimidinylboronic acid, PdCl
2
(PPh
3
)
2
, DME/EtOH/H
2
O (3:2:1), K
2
CO
3
, 100 °C; (e) Chiralpak AD
column (21.2 × 250 mm), eluent IPA (0.1% DEA)/CO
2
(15:85).
Scheme 3. Synthesis of Alkyne Derivative (R)-19
a
a
Reagents and conditions: (a) copper(I) iodide, bis(triphenylphosphine)palladium(II) chloride, 2-methyltetrahydrofuran, ethynylcyclopropane,
Et
3
N, 60 °C; (b) n-BuLi (2.5 M in hexanes), butylmagnesium chloride (2 M in THF), 5-bromo-1-ethyl-3-methylpyridin-2(1H)-one, THF, 40 °C;
(c) Chiralpak AD column (21.2 × 250 mm) eluent IPA (0.1% DEA)/n-heptane (10:90).
Scheme 4. Synthesis of Amide Analogue (R)-22
a
a
Reagents and conditions: (a) n-BuLi, n-butylmagnesium chloride, 5-bromo-1-ethyl-3-methylpyridin-2(1H)-one, THF, 25 °C; (b) trans-4-hydroxy-
L-proline, copper(I) iodide, K
2
CO
3
,DMSO,NH
3
(32% in H
2
O), microwave reactor, 110 °C; (c) 5-chloropicolinic acid, 1-(3-
(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride, DCM, DMF, 0 °C.
Journal of Medicinal Chemistry Article
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Page 3
enantiomerically enriched (R)-20 which was further puried by
chiral chromatography. Treatment of the bromide (R)-20 with
copper iodide and ammonia yielded the corresponding amine
(R)-21. Standard amide coupling reaction with 5-chloropico-
linic acid and (R)-21 aorded the corresponding amide (R)-22.
RESULTS AND DISCUSSION
The synthesized compounds were evaluated for BACE1
inhibition in a uorescence resonance energy transfer
(FRET) protocol using the soluble part of the human β-
secretase (aa1aa460) and substrate (europium)-
CEVNLDAEFK(Qsy7). The cell-based assay for BACE1
inhibition used specic antibodies to monitor reduction of
sAPPβ release from human neuronal-derived SH-SY5Y cells.
Measured properties such as Caco-2 permeability, eux, and
metabolic stability, which are important for the selection of
candidates to be evaluated in vivo, are shown in Tables 1 and 2.
The disclosed compounds are divided into two tables mainly
due to the dierent properties emanating from the A-rings (as
dened in Scheme 1), but also illustrating the development
history of this series.
As previously described, we have been able to soak dierent
inhibitors into crystals of BACE1.
16
The crystal structure of
compound 10 bound in BACE1 was rened to 1.79 Å
resolution (Figure 2). The X-ray structure shows that the
amidine group of the ligand interacts with residues Asp32 and
Asp228, in analogy with related aminoimidazole and amino-
hydantoin structures.
16,20
The A-ring is located close to the S2
region and lls the pocket occupied by Tyr71 in the peptide-
bound, ap-closed conformation of BACE1.
21
A hydrogen
bond acceptor in the para position of the A-ring, such as a
pyridine nitrogen or a methoxy substituent, makes an important
interaction with Trp76 in this open conformation. A
substituent ortho to the acceptor may either ll a subpocket
in the enzyme or prefer to be oriented toward the exposed side
of the ring, facing the ap. A triuoromethyl group ortho to the
pyridine nitrogen as in 10 lls the subpocket and displaces a
conserved water molecule normally coordinated by the
backbone of Asn37 and the side chain of Ser35. The B-ring
(dened in Scheme 1) is placed in the hydrophobic S1 pocket,
and the B-ring p-uoro substituent is small enough to t into S1
close to residues Phe108Ile110. The C-ring (dened in
Scheme 1) occupies the S3 pocket, and both pyrimidine
nitrogens coordinate one water molecule each. One of these
waters is buried in the S3 subpocket and interacts with Ser229,
thereby bridging the interaction between the protein and the
inhibitor. The enantiomeric preference for compound 10 would
be the (S )-enantiomer as apparent from the crystal structure
(Figure 2) and in line with discussions by Malamas et al.
8,20
The cell IC
50
value was determined to be 22 nM for (S)-10 and
7000 nM for (R)-10.
A uoro substitiuent ortho to the amidine in the isoindole
core leads to formation of a weak internal H-bond and reduced
pK
a
as shown for 23 compared to 25 (Table 1). Calculations
also show that the solvation energy is less negative for the o-
uoro-substituted compound, as compared to uoro in the
meta or para position (data not shown). The combination of
electronic and steric eects results in shielding of the polar
exocyclic nitrogen from the solvent. In practice, less than two
H-bond donors are seen by the environment and permeability
Table 1. Fluoroaminoisoindoles with Pyridine as the Trp76 Hydrogen Bond Acceptor
compd R1 R2
R3, R4,
R5
C-
ring
IC
50
(FRET)
a
(nM)
IC
50
(cell
sAPPβ)
a
(nM)
P
app
b
(10
6
cm/s)
eux
ratio
c
IC
50
(hERG)
d
(μM)
CL
int
e
(μL/
min/10
6
cells) pK
a
f
elogD
g
23 H H H, H, H C1 500 90.3 3.4 12 16 11.7 8.4 0.7
24 CF
3
H H, H, H C1 134 2.10 0.13 >10 5.5 5.2 nd
h
2.3
25 H H F, H, H C1 158 11.4 12 3.1 5.7 16 7.1 0.9
(S)-25 H H F, H, H C1 124 8.25 22 1.9 3.1 13.7 7.2 1.1
26 H H F, H, H C2 137 25.6 24 0.7 1.6 14.1 7.1 2.0
27 CF
3
H F, H, H C1 241 43.1 39 0.6 11 10.6 6.9 2.7
28 CF
3
H F, H, H C3 253 29.9 16 0.8 1.7 5.2 nd 3.7
10 CF
3
H F, H, F C1 198 29.3 25 1.0 16 6.9 6.4 3.0
(S)-10 CF
3
H F, H, F C1 125 22.1 30 0.9 7.9 10.0 nd 3.0
29 CH
3
CH
3
F, H, H C1 1380 10.7 19 2.2 6.4 5.6 8.0 1.4
30 cyclopropyl H F, H, H C1 86.4 18.4 42 0.5 3.5 27.6 7.4 2.1
31 OCH
3
H F, H, H C1 401 21.5 21 1.2 2.2 42.0 7.1 2.0
32 CF
2
H F, H, H C1 42.8 17.0 27 1.2 7.2 27.5 6.8 2.0
(S)-32 CF
2
H F, H, H C1 35.1 16.7 34 0.9 4.8 20.9 7.1 2.0
(S)-16 CF
2
H F, F, H C1 36.2 8.64 37 0.9 19 40.4 6.2 2.3
33 CF
2
H F, H, H C4 6.49 2.97 4.7 2.3 2.9 19.8 nd 3.9
a
IC
50
values are the means of at least two experiments.
b
P
app
is the measured permeability (apical (A) to basolateral (B)) through Caco-2 cells.
c
The
eux ratio is P
app
(BA)/P
app
(AB) in Caco-2 cells.
d
Measured in hERG-expressing CHO cells using IonWorks technology.
26
e
Metabolic stability in
rat hepatocytes.
27
f
Determined by pressure-assisted capillary electrophoresis.
28
g
Determined by reversed-phase liquid chromatography.
29
h
nd = not
determined.
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Page 4
and eux are improved, as evident when comparing
compounds 23 with 25 and compounds 24 with 27. The
introduction of the o-uoro substituent was well tolerated, and
for compound 25 compared to 23, the potency was increased
in the FRET assay from 500 to 158 nM. The uoro substituent
is nicely pentacoordinated by water and side chain oxygen
atoms in the X-ray structure. The OF distances range from
2.9 to 3.3 Å.
The racemic compound 25 was separated into its
enantiomers. Interestingly, the most active BACE1 enantiomer
(S)-25 displayed improved permeability and eux properties.
This trend seemed to be general and can be noticed when
comparing compound 10 with (S)-10 and 32 with (S)-32.
Unfortunately, anity for the hERG ion channel also increased.
For the most active BACE1 enantiomers (S)-10, (S)-25, and
(S)-32, the hERG IC
50
values were reduced by a factor of 2
compared to those of the racemates. Thus, enantiomer
separation resulted in a larger eect on hERG anity than
on BACE1 anity.
The two most common ways of reducing hERG anity are
to lower pK
a
or lip ophilicity.
22
The introduction of a
triuoromethyl group as in 27 reduced the pK
a
and resulted
Table 2. Fluoroaminoisoindoles with Methoxy or Pyridone as the Trp76 Hydrogen Bond Acceptor
a
IC
50
values are the means of at least two experiments.
b
P
app
is the measured permeability (apical (A) to basolateral (B)) through Caco-2 cells.
c
The
eux ratio is P
app
(BA)/P
app
(AB) in Caco-2 cells.
d
Measured in hERG-expressing CHO cells using IonWorks technology.
26
e
Metabolic stability in
rat hepatocytes.
27
f
Determined by pressure-assisted capillary electrophoresis.
28
g
Determined by reversed-phase liquid chromatography.
29
h
nd = not
determined.
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Page 5
in lower hERG anity as evident by comparing compound 25
with 27, but the BACE1 anity was also reduced. Lipophilicity
was experimentally determined as elogD values in a method
using reversed-phase liquid chromatography to estimate log D
on the basis of the compound retention time.
29
In the
uoroaminoisoindole series we noticed that an elogD value
below 1.5 increa sed the eux ratios, which would be
detrimental for reaching high brain concentrations. Thus, we
concentrated on designing compounds with a predicted log D
of above 1.5 but also below 3.5 to avoid issues associated with
high lipophilicity.
23
To improve BACE1 potency of the
(triuoromethyl)pyridine 27, we investigated if the 5-cyano-3-
pyridinyl C-ring previously found to increase anity would be
useful. The BACE1 inhibition in human neuronal-derived SH-
SY5Y cells was increased comparing compound 28 with 27;
however, hERG anity was increased even more. Another C-
ring previously indicating increased anity was the 5-uoro-3-
pyridinyl, but as evident from compound 26 it did not display
increased cell potency in this series.
The 4-pyridinyl analogue (S)-25 with a BACE1 potency in
the cell assay of 8.25 nM was a potent CYP inhibitor, which is a
common issue for ortho-unsubstituted 4-pyridinyls.
24
On the
other hand, the (triuoromethyl)pyridinyl compound 27 with
reduced CYP anity displayed good properties for Caco-2
permeability, eux, and metabolic stability in rat hepatocytes.
We therefore decided to synthesize 10 with the additional
uoro substituent in the para position of the B-ring. We
envisioned that the uorine could t into a small subpocket,
and indeed, the BACE1 cell potency increased to an IC
50
value
of 29 nM and hERG anity decreased to an IC
50
value of 16
μM.
Other substituents on the pyridine A-ring were examined,
and both the cyclopropyl 30 and methoxy 31 compounds were
more potent than the triuoromethyl analogue 10 in the cell
assay. However, the pK
a
was increased, which resulted in
unfavorable hERG anity. The dimethyl compound 29 was
even more potent in the cell assay, but here we could notice a
Figure 2. Crystal structure of compound 10 in complex with BACE1.
Key interactions between inhibitor (yellow), protein amino acid
residues (yellow), N (blue), O (red), F (light blue), and water
molecules (red spheres) are highlighted with dashed lines. The protein
surface is depicted in gray (residues 7273 in the ap region are not
shown for clarity). The data collection and renement statistics are
summarized in Table 3.
Table 3. X-ray Crystallography Data Collection and Renement Statistics
BACE1 complex with 10 BACE1 complex with (R)-41
Data Collection
space group P2
1
2
1
2
1
P2
1
2
1
2
1
unit cell dimensions a = 47.54 Å, b = 76.39 Å, c = 104.34 Å a = 47.96 Å, b = 75.91 Å, c = 104.65 Å
α = 90.0°, β = 90.0°, γ = 90.0° α = 90.0°, β = 90.0°, γ = 90.0°
resolution range
a
(Å) 35.871.79 (1.841.79) 43.081.83 (1.931.83)
no. of observations 125330 101856
no. of unique rens 36601 34330
data redundancy
a
3.4 (3.4) 3.0 (2.9)
data completeness (%)
a
99.9 (100.0) 99.6 (99.8)
I/σ(I)
a
20.6 (4.5) 15.0 (2.1)
R
merge
a
(%) 4.0 (25.7) 5.1 (45.2)
Renement
resolution range
a
(Å) 35.871.79 (1.841.79) 43.081.83 (1.891.83)
R
work
a
(%) 17.6 (26.4) 17.3 (24.0)
R
free
a
(%) 21.0 (31.4) 22.6 (27.7)
Wilson B-factor
2
) 22.0 23.0
overall mean B-factor
2
) 25.1 26.6
no. of atoms
protein atoms 2983 2974
heterogen atoms 449 416
solvent atoms 402 364
rmsd values
bond lengths (Å) 0.010 0.010
bond angles (deg) 1.12 1.14
Ramachandran statistics (%) (PROCHECK)
33
most favored + add. allowed 99.7 100
generously allowed 0.3 0
a
Numbers in parentheses refer to the highest resolution shell.
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Page 6
very large increase in potency in going from FRET to cell assay.
This increase in potency is a common feature for the more
basic inhibitors (pK
a
> 6),
25
but for 29 there could be
additional factors involved.
A closer examination of the crystal structure of compound 10
revealed that the triuoromethyl group seemed to be too large
for the S2 subpocket as one of the uorine atoms apparently
clashed with the BACE1 protein surface. The cyclopropyl and
methoxy groups t better, but the methoxy compound 31
showed poor metabolic stability and the cyclopropyl compound
30 displayed unfavorable hERG anity. Instead, we decided to
try a diuoromethyl group to keep the pK
a
low and at the same
time achieve a better t in the S2 subpocket. In particular, the
IC
50
value in the FRET assay improved from 241 to 43 nM
when replacing the triuoromethyl substituent in 27 with the
diuoromethyl group in 32. Compound 32 was separated into
its enantiomers, and (S)-32 showed promising in vitro
properties for BACE1 inhibition in the cell assay with an
IC
50
value of 16.7 nM, resulting in a 280-fold margin to the
hERG IC
50
(4.8 μM). The permeability and eux values were
good, i ndicating that high brain/plasma ratios could be
achieved. Compound (S)-32, with an oral bioavailability in
mice of 73% and an oral half-life of 1.5 h, was selected as a
preclinical candidate drug and therefore extensively studied in
animal models for Aβ40 reduction and for evaluation of
margins to hERG side eects. Blockade of the human hERG
channel and QT prolongation have become surrogate markers
of potential cardiotoxicity of pharmaceutical compounds. In
preclinical studies, the hERG assay is widely used as a rst
indication for a potential eect of compounds on IKr
conduction. However, even though a safety margin can be
predicted from these values, it is dicult to quantitatively
predict the actual level of QT prolongation expected in vivo in
preclinical studies and in human. Thus, these ndings need to
be followed up in an in vivo setting such as in the guinea pig
monophasic action potential (MAP) assay and/or in vivo dog
electrocardiogram (ECG) recordings. Compound (S)-32 was
designated clinical candidate AZD3839 and progressed to
further in vivo preclinical and clinical testing (a detailed account
of these studies will be published elsewhere by Fa
̈
lting et al.).
We wanted to explore if the in vitro hERG margins could be
improved, and the most promising way would be to reduce pK
a
for these pyridine analogues. An additional uoro substituent
para to the amidine moiety in the isoindole core structure was
predicted to result in decreased pK
a
, and compound (S)-16
displayed the lowest hERG anity in this series with an IC
50
value of 19 μM and a pK
a
of 6.2. The BACE1 cell potency was
increased to an IC
50
value of 8.6 nM, resulting in a 2200-fold
margin to the hERG IC
50
.
The compounds with methoxy as the hydrogen bond
acceptor interacting with Trp76 were potent BACE1 inhibitors
(Table 2). This indicated that the geometric constraints of the
BACE1 protein and the inhibitor govern the anity rather than
the hydrogen bond acceptor strength of the substituent on the
inhibitor. The pyridine analogue 25 was slightly less potent in
the FRET assay compared to methoxy analogue 34 (Table 2)
in contrast to what could be expected when comparing the
hydrogen bond acceptor strengths of these groups.
30
The p-
methoxy ortho-disubstituted A-rings displayed high BACE1
anity, and compounds 35 and 36 with cell IC
50
values of 2.9
and 3.4 nM, respectively, achieved high margins to hERG. The
permeability and eux properties were good for the methoxy
analogues 34, 35, and 36, but the methoxy group was a
metabolic weak spot and contributed to CYP inhibition.
Another opti on to increase margins to hERG besides
decreasing anity for hERG would be to increase anity for
BACE1, and we anticipated that increasing the hydrogen bond
acceptor strengths of the hydrogen acceptors on or in the A-
ring should result in increased BACE1 inhibition. Thus,
pyridone was tried as a replacement for the methoxyphenyl
A-ring. Compound 37 was reasonably potent, but the
desolvation energy cost of the pyridone moiety probably
reduced some of the expected increase in anity. This group
displayed reduced permeability and increased eux, but
metabolic stability was considerably improved compared to
those of the methoxyphenyl compounds 34, 35, and 36. The
improved metabolic stability of the pyridones was also
translated into excellent bioavailability in mouse and rat.
Elongating the methyl into ethyl as in 38 improved the FRET
potency considerably. We sought improved permeability and
eux properties, and since the pyridones 37 and 38 were quite
polar with elogD values of 0.9 and 1.3, respectively, we decided
to explore more lipophilic C-rings. Both the 5-uoro-3-
pyridinyl and 5-cyano-3-pyridinyl compounds 39 and 40
increased BACE1 inhibition in the FRET and cell assays, but
the eux ratio was still high (>1.5).
The 5-propynyl-3-pyridinyl analogue (R)-41 with addition-
ally increased lipophilicity displayed reduced eux and
reasonable permeability in Caco-2 cells. (R)-41 was very
potent in the BACE1 cell assay with an IC
50
value of 0.16 nM,
resulting in a high (>10000-fold) margin to the hERG IC
50
.
(R)-41 displayed excellent in vivo pharmacokinetic (PK)
properties, with an oral bioavailability in mice of 100% and
per os (po) half-life of 2.3 h, and was extensively studied in
animal models for Aβ40 reduction. A detailed account of these
studies will be published elsewhere by Fa
̈
lting et al.
The introduction of a uoro substituent para to the amidine
group as in compound (R)-42 did not result in reduced anity
for hERG and increased BACE1 potency, a result contrary to
what was expected when comparing this compound to
compound (S)-16.
The crystal structure of compound (R)-41 bound to BACE1
was rened at 1.83 Å resolution (Figure 3). The amidine
interacts with the two aspartic acids as with compound 10 in
Figure 2. The carbonyl of the pyridone ring interacts with
Trp76 via a hydrogen bond and is further coordinated by a
water molecule, forming a novel network of proteinwater
interactions as compared to the pyridinyls and methoxy-
substituted phenyls.
16,20,31
The methyl substituent next to the
carbonyl is oriented into the protein, whereas the ethyl group is
solvent exposed. In general, the orientation of the A-ring has
been dicult to predict by molecular modeling, and thus, X-ray
crystallography has been an important technique to support the
design of new inhibitors. The C-ring propynyl protrudes into
the S3 subpocket and displaces the water observed in the
BACE110 complex (Figure 2). This in turn induces a
conformational change of the so-called 10s loop dened by
residues 914.
32
The 5-propynyl-3-pyridinyl C-ring with its unique binding
into the S3 subpocket was also explored in the pyridinyl
subseries. Compound 33 (Table 1) was synthesized, and
indeed, it enhanced BACE1 potency in this subseries, but
unfortunately, permeability properties deteriorated and hERG
anity increased.
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Page 7
Substituents other than aromatic C-rings could be used to
reduce the molecular weight and polar surface area, resulting in
improved permeability propertiesaswehavepreviously
described.
16
A representative example for these analogues is
(R)-19, but in the pyridone subseries the eect on permeability
was at most minor when comparing (R)-41 with (R)-19. The
cyclopropylethynyl compound (R)-19 was found to be
surprisingly potent in the BACE1 cell assay with an IC
50
value of 0.76 nM, indicating that the aromatic C-rings were
probably not the optimal binding component in the S3 pocket.
This assumption was also evident when comparing compound
(R)-22 with 39 or 40, where the elongated compound (R)-22
displayed about a 10-fold increase in potency in the BACE1 cell
assay, reaching an IC
50
value of 0.15 nM. When examining the
crystal structure of an analogue closely related to (R)-22 in
complex wit h BACE1, we could see a hydrogen bond
interaction between the amide NH and the backbone carbonyl
of Gly230. This H-bond interaction probably explained the
increase in anity for (R)-22.
Both BACE1 and γ-secretase mediate cleavage of many
substrates involved in cell signaling, and it is crucial to sustain
these pathways while altering the Aβ formation. For γ-secretase
inhibitors, a major liability has been interference with the Notch
signaling pathway, critical in proliferative signaling during
neurogenesis. All uoroaminoisoindole compounds tested in a
Notch assay (not shown) are inactive, and thus, these
compounds are not believed to interfere with γ-secretase-
related signaling. The uoroaminoisoindoles were also tested
for selectivity against BACE2, and data for representative
compounds are shown in Table 4.
(S)-25 was the rst synthesized compound in our in-house
chemical series that produced a robust a nd statistically
signicant lowering of β-amyloid peptides in vivo. The
incorporation of a uoro adjacent to the amidine moiety had
a large impact on the ability of the series to reduce Aβ40 in the
brain. The reason for the in vivo eect is most likely explained
by the improved permeability and eux properties of the
uoroaminoisoindoles. Several compounds from the uoroa-
minoisoindole series were evaluated in vivo, and they all
displayed the ability to lower the Aβ40 levels in the brain of
C57BL/6 mice, as shown in Table 4. Furthermore, all
uoroaminoisoindoles tested in vivo reduced the levels of
Aβ42 and sAPPβ in brain at a magnitude similar to that of
Aβ40 (not shown). One representative compound from Table
1is(S)-10, and the dose- and time-dependent reduction of
Aβ40 in brain is shown in Figure 4. The maximum reduction
(50%) was seen 1.5 h after a 300 μmol/kg dose of (S)-10. The
free plasma and brain concentrations (C
u
(plasma) and
C
u
(brain), respectively)
34
1.5 h after dosing are summarized
in Table 4. Pharmacokinetic/pharmacodynamic (PK/PD)
modeling of (S)-10 time- and dose-response eect data in
vivo, using an indirect response model with inhibition on the
Aβ production rate, estimated the unbound brain concentration
giving 20% inhibition from baseline to 114 nM for (S)-10. The
fraction unbound (f
u
) in brain was less than that in plasma, and
the fraction unbound in brain was steeply correlated to
lipophilicity (measured as elogD) in this series. The inhibition
of Aβ40 in mouse primary cortical neurons was determined
(Table 4), and for compounds studied in the in vivo PD model,
we found a good correlation between measured in vitro IC
50
values and Aβ40 reduction in the mouse brain.
For the compounds with methoxy or pyridone as the
hydrogen bond acceptor (Table 2), a large variation of the in
vivo ecacy was observed, mainly due to a larger variability in
Caco-2 permeability, eux, and brain fraction unbound values.
One representative compound is (R)-19, and the dose- and
time-dependent reduction of brain Aβ40 levels is shown in
Figure 5. The maximum reduction (40%) was seen 1.5 h after a
200 μmol/kg dose of (R)-19. The free plasma and brain
concentrations 1.5 h after dosing are summarized in Table 4.
Figure 3. Crystal structure of compound (R)-41 in complex with
BACE1. Key interactions between inhibitor (yellow), protein amino
acid residues (yellow), N (blue), O (red), F (light blue), and water
molecules (red spheres) are highlighted with dashed lines. The protein
surface is shown in gray (residues 7273 in the ap region are not
shown for clarity). The data collection and renement statistics are
summarized in Table 3.
Table 4. Fluoroaminoisoindoles Reducing Aβ40 in the Brain of C57BL/6 Mice
compd
BACE2 FRET K
i
a
(nM)
IC
50
(primary neurons)
b
(nM)
dose
c
(μmol/kg)
Aβ40 reduction at 1.5
h, %
C
u
(plasma)
(nM)
f
u
(plasma)
(%)
C
u
(brain)
(nM)
f
u
(brain)
(%)
(S)-25 790 68 300 61 4118 8.9 445 8.8
27 nd
d
436 300 48 1221 4.1 883 5.1
(S)-10 nd 204 300 50 795 4.4 406 2.5
(S)-32 370 51 80 31 170 2.7 119 7.9
(S)-16 770 53 125 40 883 11 73 4.6
35 1700 40 75 50 237 9.6 13 1.1
37 740 30 300 32 16267 52 113 16
(R)-19 78 3.4 200 40 123 1.8 14 0.6
(R)-41 7.5 2.6 50 36 196 1.5 3 1.0
a
K
i
values are the means of at least two experiments.
b
IC
50
values are the means of at least two experiments.
c
Oral administration.
d
nd = not
determined.
Journal of Medicinal Chemistry Article
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Page 8
(R)-19 demonstrated a very low fraction unbound (f
u
) in brain
and showed reduced access to the brain. The reduced brain
access of (R)-19 could probably be explained by P-gp-induced
eux of the compound. The eux ratio was later determined
to be 5.1 in an MDCK-MDR1 cell-line assay. Even though this
compound had a predicted low metabolic stability as evident
from the intrinsic clearance (CL
int
) value in rat hepatocytes and
in vivo mouse PK analysis (CL = 150 mL/min/kg, po half-life
0.65 h), it still displayed a prolonged Aβ lowering eect in the
in vivo PD experiments. Thus, compound (R)-19 displayed a
better in vivo PD prole than would be expected from the in
vitro and in vivo PK properties. PK/PD modeling of the time-
and dose-response eect data in vivo estimated the unbound
brain concentration giving 20% inhibition from baseline to 6
nM for (R)-19.
When comparing the results for dierent compounds in the
in vivo PD experiments (Table 4) together with a combination
of compound properties, it was evident that compound (S)-32
showed a superior prole. The concentration unbound brain/
plasma ratio was determined to be 0.7, the highest in this
compound series. The low lipophilicity of this compound was
probably the explanation of why the fraction unbound in brain
was higher than that in plasma for this compound. Compounds
displaying large in vitro margins to hERG such as (S)-16 and
(R)-41 were found to have unfavorable unbound brain/plasma
ratios, and thus, (S)-32 turned out to be the compound with
the overall best properties for clinical evaluation.
CONCLUSION
In this paper we have disclosed an aminoisoindole series as
BACE1 inhibitors. Introducing a ourine adjacent to the
amidine moiety improved the permeability properties of the
series and made it possible to achieve in vivo brain ecacy.
Crystal structure information was used for structure-based
design of new potent BACE1 inhibitors. Due to the basic
nature of these compounds, they also displayed hERG anity.
Both lipophilicity and pK
a
were used as means for reducing
hERG inhibition, resulting in compound (S)-16 with an hERG
IC
50
value of 16 μM and BACE1 cell IC
50
value of 8.6 nM. The
BACE1 anity of this series was furth er increased to
subnanomolar levels, and compound (R)-41 with a cell IC
50
value of 0.16 nM gave a high (>10000-fold) margin to the
hERG IC
50
. Several compounds were evaluated in vivo for
reduction of the Aβ40 level in the brain of C57BL/6 mice.
Compound (S)-32 showed a superior prole in the in vivo PD
experiments with a concentration unbound brain/plasma ratio
of 0.7. Despite the very good in vitro hERG margins, the in vivo
margins to hERG side eects still remain a challenge for this
series.
EXPERIMENTAL SECTION
hBACE1 and hBACE2 TR-FRET Assay. The soluble part of
human β-secretase (recombinant hBACE1 enzyme, aa1aa460, or
hBACE2 enzyme, aa1aa473) diluted in reaction buer (sodium
acetate, CHAPS, Triton X-100, EDTA, pH 4.5) was mixed with the
test compound diluted in DMSO. After a preincubation period of 10
min, substrate, (europium)CEVNLDAEFK(Qsy7), was added and the
reaction allowed to proceed for 15 min at room temperature (rt). The
reaction was stopped by addition of 7 μL of sodium acetate, pH 9. The
uorescence 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 nal concentration of the enzyme was 2.7 μg/mL (or
54 ng/mL); the nal concentration of substrate was 100 nM. Reported
values are means of n 2 determinations, standard deviation 10%.
Cell sAPPβ Release Assay. SH-SY5Y cells (human neuroblastoma
cell line) were cultured in DMEM/F-12 with Glutamax, 10% FCS, and
1% nonessential amino acids. The test compound was incubated with
cells for 16 h at 37 °C, 5% CO
2
. Meso Scale Discovery (MSD) plates
were used for the detection of sAPPβ release; MSD sAPPβ plates were
blocked in 3% BSA in Tris wash buer for 1 h at rt and washed four
times in Tris buer. After incubation, 20 μL of medium was transferred
Figure 4. Reduction of Aβ40 levels in the brain of C57BL/6 mice after oral dosing of compound (S)-10. (A) Signicant eects were seen 1.5 h after
dosing with (S)-10 at 125 and 300 μmol/kg. (B) In the timeresponse study, signicant eects were seen 1.53 h after dosing (300 μmol/kg). The
free concentrations of (S)-10 were measured at dierent time points after dosing. Data are presented as mean values ± SEM (**, P < 0.01; ***, P <
0.001; compared to vehicle).
Figure 5. Reduction of Aβ40 levels in the brain of C57BL/6 mice after
oral dosing of compound (R)-19, as shown in a dose and time
response study (75 and 200 μmol/kg). At the higher dose (200 μmol/
kg) signicant reductions were seen 1.516 h after dosing, and
signicant eects were seen 34.5 h after the 75 μmol/kg dose. The
free concentrations of (R)-19 in the brain were measured at dierent
time points after dosing. Data are presented as mean values ± SEM (*,
P < 0.05; **, P < 0.01; ***, P < 0.001; compared to vehicle).
Journal of Medicinal Chemistry Article
dx.doi.org/10.1021/jm3009025 | J. Med. Chem. 2012, 55, 934693619354
Page 9
to the preblocked and washed 384-well MSD sAPPβ microplate and
incubated with shaking at rt for 2 h followed by washing four times in
Tris buer. A 10 μL volume of detection antibody was added (1 nM)
followed by incubation with shaking at rt for 2 h followed by washing
four times in Tris buer. A 40 μL volume of read buer was added per
well, and the plates were read in a SECTOR imager. In addition, the
cells incubated with test compound were further lysed and analyzed
for any cytotoxic eects of the compounds using the ViaLight Plus cell
proliferation/cytotoxicity kit (Cambrex BioScience) according to the
manufacturers instructions. Reported values are means of n 2
determinations, standard deviation 10%.
Mouse Primary Neuron Aβ40 Release Assay. Primary cortical
cells were isolated from fetal C57/BL6 mice (E16). The cortices were
kept in calcium- and magnesium-free Earle's balanced salt solution
(CMF-EBSS) containing 0.25% trypsin and 2 U/mL DNase for 1 h at
37 °C and 5% CO
2
. The cortices were washed in warm CMF-EBSS
and gently triturated with ame-polished pipets to separate the cells.
The cell solution was transferred to a 50 mL Falcon tube containing
medium (10% HamsF12, 10% fetal bovine serum, 1% 10 mM Hepes,
1% 2 mM
L-glutamine, 0.5% 50 U/0.5 mg penicillinstreptomycin,
and 77.5% DMEM with 4.5 g/L glucose) and ltered through a 100
μm cell strainer (BD Falcon). The cells were counted and plated onto
96-well poly-
D-lysine-coated plates at a density of 200 000 cells/200
μL/well. After ve DIV at 37 °C and 5% CO
2
, the medium was
exchanged to medium containing the compounds a t a nal
concentration of 1% DMSO and incubated overnight. The amount
of released Aβ40 in the extracellular medium was measured using
Invitrogen Biosource enzyme-linked immunosorbent assay (ELISA)
strips (KMB3481) according to the manufacturer s instructions. The
strips were read using a Spektramax microplate reader (Molecular
Devic es). The cytotoxic eect of the compounds was directly
evaluated on the cell plates utilizing a commercial cell proliferation/
cytotoxicity kit based on luciferase reaction on ATP released by cells.
Protein Crystallography. The BACE1 protein used for structure
determination was expressed and puried as previously described by
Patel et al.
32
and crystallized according to Swahn et al.
16
Crystallo-
graphic data of BACE1 in complex with 10 were collected at 100 K on
a Rigaku FR-E generator equipped with a Rigaku HTC detector to
1.79 Å resolution and processed with MOSFLM
35
and SCALA.
36
For
the (R)- 41 soaked crystal, data were collected at 100 K to 1.83 Å on a
Rigaku FR-E+ generator with a Rigaku Saturn A200 charge-coupled
device (CCD) detector and processed with autoPROC
37
from Global
Phasing utilizing XDS
38
and SCALA.
36
The crystals belong to space
group P2
1
2
1
2
1
, with one complex per asymmetric unit. A total of 5% of
the reections were used to calculate R
free
. The structures were solved
by rigid body renement using Refmac5
39
and a previously determined
BACE1 structure based on the published 1FKN structure.
21
The
ligands were well-dened in the dierence electron density. Refmac5
39
and AUTOBUSTER
40
were used for crystallographic renement, and
Coot
41
was used for model building. Data collection and renement
statistics are listed in Table 3. The coordinates for the crystal
structures of 10 and (R)-41 with BACE1 have been deposited with the
RCSB Protein Data Bank. All gures showing structural representa-
tions were prepared using PyMOL.
42
Permeability Assay. Caco-2 cells were grown for 14 21 days to
achieve conuency and polarization before being used for transport
experiments. For both apical to basolateral (AB) and basolateral to
apical (BA) transport directions, the pH was adjusted to 7.4. All
compounds were investigated at a concentration of 10 μM. Buer
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 cell monolayer was
monitored by measuring the passive transmembrane diusion of
[
14
C]mannitol. Concentrations of compounds in donor and receiver
samples were analyzed by liquid chromatographytandem mass
spectrometry. Liquid scintillation was used for analysis of [
14
C]-
mannitol. The apparent permeability coecient (P
app
) was calculated
according to P
app
=(dQ/dt)/(AC
0
), 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 C
0
is the starting concentration.
The eux ratio is the ratio P
app
(BA)/P
app
(AB).
In Vivo Pharmacodynamic Assay. Female C57BL/6 mice
(Harlan, The Netherlands), 814 weeks of age (n =612 per
group) received vehicle (0.3 M gluconic acid) or BACE1 inhibitor as a
single dose (50300 μmol/kg) vi a oral gavage. Animals were
anaesthetized 0.524 h after administration, and blood was collected
by heart puncture into prechilled microtainer tubes containing EDTA.
Plasma samples were prepared by centrifugation for 10 min at
approximately 3000g at 4 °C, and the samples were then stored at 70
°C until exposure and Aβ40 analysis. After blood sampling, the animals
were sacriced by decapitation, and the brains were dissected out, the
cerebellum and olfactory bulbs were removed, and the cerebrum was
divided into left and right hemispheres. Both hemispheres were
weighed, snap-frozen, and stored at 70 °C until exposure and Aβ40
analysis, respectively. Prior to analysis, the left hemispheres were
homogenized in 0.2% diethylamine (DEA) with 50 mM NaCl,
followed by ultracentrifugation. Recovered supernatants were neutral-
ized to pH 8.0 with 2 M TrisHCl, snap-frozen on dry ice, and stored
at 70 ° C. Aβ40 levels in brain extracts and plasma were analyzed
using a highly specic commercial Aβ
140
ELISA (KMB3481,
Invitrogen, Camarillo, CA). Drug concentrations in brain (right
hemisphere) and plasma samples were determined by reversed-phase
liquid chromatography and electrospray tandem mass spectrometry.
Analysis of Aβ data was performed using Prism 4 (GraphPad, La Jolla,
CA), with one-way analysis of variance (ANOVA) followed by
Dunnetts multiple comparison test or Bonferronismultiple
comparison test. The level of signicance was set at P < 0.05.
Chemistry. All solvents used were commercially available and were
used without further purication. Reactions were typically run using
anhydrous solvents under an inert atmosphere of nitrogen or argon.
Starting materials used were available from commercial sources or
prepared as described in the Supporting Information. Room
temperature refers to 2025 °C. Microwave heating was performed
in a Biotage Initiator microwave synthesizer at the indicated
temperature in the recommended microwave tubes.
1
H NMR spectra were recorded in the indicated deuterated solvent
at 400 MHz, and the spectra were obtained unless stated otherwise
using a Bruker av400 NMR spectrometer equipped with a 3 mm ow
injection SEI
1
H/D
13
C probe head with Z-gradients using a BEST
215 liquid handler for sample injection or using a Bruker DPX400
NMR spectrometer equipped with a four-nucleus probehead (
19
F)
with Z-gradients. Spectra (500 MHz) were recorded using a Bruker
500 MHz Avance III NMR spectrometer. Chemical shifts are given in
parts per million down- and upeld from TMS. Resonance
multiplicities are denoted s, d, t, q, m, and br for singlet, doublet,
triplet, quartet, multiplet, and broad, respectively.
Preparative HPLC was performed on a Waters Auto purication
HPLCUV system with a diode array detector using a Waters XTerra
MS C
8
column (19 × 300 mm, 7 μm), and a linear gradient of mobile
phase B was applied. Mobile phase A was 0.1 M ammonium acetate in
water/acetonitrile (95:5), and mobile phase B was acetonitrile. The
ow rate was 20 mL/min. Flash chromatography was performed using
Merck silica gel 60 (0.0400.063 mm) or employing a Combi Flash
Companion system using RediSep normal-phase ash columns.
LCMS analyses were performed on an LCMS system consisting
of a Waters sample manager 2777C, a Waters 1525 μ binary pump, a
Waters 1500 column oven, a Waters ZQ single-quadrupole mass
spectrometer, a Waters PDA 2996 diode array detector, and a Sedex
85 ELS detector. The mass spectrometer was equipped with an
electrospray (ES) ion source operated in positive and negative ion
modes. For separation a linear gradient was applied starting at 100%
(0.1% NH
3
in Milli-Q water) and ending at 100% methanol. The
column used was an XBridge C18, 3.0 × 50 mm, 5 μm, which was run
at a ow rate of 2 mL/min. Alternatively, an LCMS system
consisting of a Waters Alliance 2795 HPLC instrument, a Waters PDA
2996 diode array detector, a Sedex 85 ELS detector, and a ZQ single-
quadrupole mass spectrometer was used. The mass spectrometer was
equipped with an ES ion source operated in positive and negative ion
modes. Separation was performed on an XBridge C18 column, 3.0 ×
Journal of Medicinal Chemistry Article
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Page 10
50 mm, 3.5 μm, run at a ow rate of 1 mL/min. A linear gradient was
applied starting at 100% (0.1% NH
3
in Milli-Q water) and ending at
100% methanol.
Purity analyses were performed on an Agilent HP1100 system
consisting of a G1322A Micro vacuum degasser, a G1312A binary
pump, a G1367A well-plate autosampler, a G1316A thermostated
column compartment, a G1315C diode array detector, and a 6120,
G1978B mass spectrometer. The mass spectrometer was congured
with an atmospheric pressure chemical ionization (APCI) ion source
operated in positive and negative ion modes. The column used was an
XBridge C18, 3.0 × 100, 3 μm, run at a ow rate of 1.0 mL/min. A
linear gradient was used for both the blank and the sample, starting at
100% 10 mM NH
4
OAc in 5% CH
3
CN and ending at 95% CH
3
CN.
The blank run was subtracted from the sample run. All tested
compounds were puried to >95% purity as determined by reversed-
phase HPLC.
SFC purity analysis was run on an SFC Berger Analytix system with
an Agilent 1100 photodiode array (PDA) detector. The column was a
Chiralpak AD-H, 5 μm, 4.6 × 250 mm. The column temperature was
set to 50 °C. An isocratic condition of 2030% (methanol + 0.1%
DEA) and 7080% CO
2
was applied at a ow rate of 3.0 mL/min.
The PDA was scanned from 190 to 600 nm, and 220 nm was extracted
for purity determination. All tested compounds were puried to >99%
enantiomeric purity as determined by SFC.
SFC preparative chromatography was run on an SFC Berger
Multigram II system with a Knauer K-2501 UV detector. The column
was a Chiralpak AD-H, 5 μm, 21.2 × 250 mm. The column
temperature was set to 35 °C. An isocratic condition of 2030%
(methanol + 0.1% DEA) and 7080% CO
2
was applied at a ow rate
of 50.0 mL/min. The UV detector scanned at 220 nm. The UV signal
determined the fraction collection.
2-(3-Bromo-4-uorobenzoyl)-6-uorobenzonitrile (7). To a
solution of (2-cyano-3-uorophenyl)zinc(II) iodide (80.0 mL, 40.0
mmol, 0.5 M in THF) was added tetrakis(triphenylphosphine)-
palladium(0) (2.3 g, 2.0 mmol) in small portions at 0 °C. A solution of
3-bromo-4-uorobenzoyl chloride (10.0 g, 42.1 mmol) in anhydrous
THF (20 mL) was then added dropwise, and the reaction mixture was
stirred at 0 °C for 1 h. The reaction was quenched by addition of water
(150 mL), and the resulting mixture was extracted with ethyl acetate
(2 × 150 mL). The combined organic extracts were washed with brine,
dried over sodium sulfate, and concentrated in vacuo. The product was
puried by ash chromatography to aord the title compound (7.90 g,
61% yield):
1
H NMR (500 MHz, DMSO-d
6
) δ (ppm) 7.587.64 (m,
2 H) 7.83 (t, J = 8.91 Hz, 1 H) 7.88 (ddd, J = 8.55, 4.77, 2.13 Hz, 1 H)
7.93 (td, J = 8.12, 5.67 Hz, 1 H) 8.14 (dd, J = 6.70, 2.13 Hz, 1 H);
19
F
NMR (400 MHz, CDCl
3
) δ (ppm) 97.16, 103.63; MS (ESI,
positive ion) m/z 322, 324 (M + 1).
N-((3-Bromo-4-uorophenyl)(2-cyano-3-uorophenyl)-
methylene)-2-methylpropane-2-sulnamide (8). 2-(3-Bromo-4-
uorobenzoyl)-6-uorobenzonitrile (7.9 g, 24.5 mmol) dissolved in
dry THF (40 mL) was added to a solution of titanium(IV) ethoxide
(12.7 mL, 61.3 mmol) in dry THF (30 mL) at rt. 2-Methyl-2-
propanesulnamide (3.57 g, 29.4 mmol) was added, and the resulting
mixture was heated at reux temperature for 22 h. The reaction
mixture was cooled to rt, and then methanol (120 mL) was added,
followed by addition of saturated aqueous Na
2
CO
3
(12 mL). The
resulting suspension was ltered through a pad of sodium sulfate, and
the solids were washed thoroughly with ethyl acetate. The ltrate was
concentrated in vacuo and puried by ash chromatography to aord
the title compound (5.7 g, 55% yield):
1
H NMR (400 MHz, CDCl
3
) δ
(ppm) 1.37 (s, 9 H), 7.19 (t, J = 8.2 Hz, 1 H), 7.22 (m, 1 H), 7.36
7.32 (m, 1 H), 7.48 (m, 1 H), 7.727.67 (m, 1 H), 7.81 (m, 1 H);
19
F
NMR (400 MHz, CDCl
3
) δ (ppm) 99.65, 100.18, 104.55,
105.17; MS (ESI, positive ion) m/z 424, 426 (M + 1).
1-(3-Bromo-4-uorophenyl)-4-uoro-1-(2-(triuoromethyl)-
pyridin-4-yl)-1H-isoindol-3-amine (9). tert-Butyllithium (1.6 M in
pentane) (6.08 mL, 9.73 mmol) was slowly added to dry THF (40
mL) at 100 °C under an argon atmosphere. 4-Bromo-2-
(triuoromethyl)pyridine (1.1 g, 4.87 mmol) in dry THF (8.0 mL)
was added dropwise. After 10 min N-((3-bromo-4-uorophenyl)(2-
cyano-3-uorophenyl)methylene)-2-methylpropa ne-2-sulnamide
(2.07 g, 4.87 mmol) in dry THF (8.0 mL) was added dropwise, and
the resulting reaction mixture was stirred at 100 °C for 30 min and
then allowed to reach rt. The reaction was quenched by addition of
water and extracted with ethyl acetate (3×). The combined organic
extracts were washed (brine), dried (Na
2
SO
4
), and concentrated in
vacuo. The residue was dissolved in methanol (40 mL), and
hydrochloric acid (2.0 M in diethyl ether) (7.3 mL, 14.6 mmol) was
added. The mixture was stirred at rt overnight and subsequently
concentrated in vacuo. The residue was partitioned between saturated
aqueous Na
2
CO
3
and dichloromethane (3×), and the combined
organic layers were dried (Na
2
SO
4
), ltered, and concentrated in
vacuo. Purication by ash chromatography gave the title compound
(1.14 g, 50% yield):
1
H NMR (500 MHz, DMSO-d
6
) δ (ppm) 6.86
(br s, 1 H), 7.277.42 (m, 3 H), 7.537.63 (m, 2 H), 7.64 (ddd, J =
5.24, 0.91, 0.79 Hz, 1 H), 7.67 (d, J = 0.79 Hz, 1 H), 7.76 (d, J = 7.57
Hz, 1 H), 8.70 (d, J = 5.52 Hz, 1 H); MS (ESI, positive ion) m/z 468,
470 (M + 1).
4-Fluoro-1-(4-uoro-3-(pyrimidin-5-yl)phenyl)-1-(2-
(triuoromethyl)pyridin-4-yl)-1H-isoindol-3-amine (10). 1-(3-
Bromo-4-uorophenyl)-4-uoro-1-(2-(triuoromethyl)pyr idin-4-yl)-
1H-isoindol-3-amine (0.30 g, 0.64 mmol) and 5-pyrimidinylboronic
acid (0.104 g, 0.84 mmol) in DMF (5.0 mL) were heated to 90 °C
under an argon atmosphere. Pd
II
Cl
2
dppf·CH
2
Cl
2
(0.039 g, 0.05 mmol)
and aqueous K
2
CO
3
(2.0 M) (0.96 mL, 1.92 mmol) were added, and
the resulting mixture was stirred at 90 °C for 1 h. The reaction mixture
was cooled to rt and then puried by preparative HPLC to give the
title compound (0.16 g, 51% yield):
1
H NMR (500 MHz, DMSO-d
6
)
δ (ppm) 6.83 (br s, 2 H), 7.31 (dd, J = 9.38, 8.43 Hz, 1 H), 7.37 (dd, J
= 10.25, 8.83 Hz, 1 H), 7.51 (ddd, J = 8.67, 4.89, 2.36 Hz, 1 H), 7.54
7.62 (m, 2 H), 7.68 (dd, J = 5.04, 1.42 Hz, 1 H), 7.71 (d, J = 0.95 Hz, 1
H), 7.86 (d, J = 7.57 Hz, 1 H), 8.70 (d, J = 5.20 Hz, 1 H), 8.95 (d, J =
1.26 Hz, 2 H), 9.21 (s, 1 H); MS (ESI, positive ion) m/z 468 (M + 1).
(S) -4-Fluoro-1-( 4-uoro-3-(py rimidin-5-yl)phenyl)- 1-(2-
(triuoromethyl)pyridin-4-yl)-1H-isoindol-3-amine ((S)-10). 4-
Fluoro-1-(4-uoro-3-(pyrimidin-5-yl)phenyl)-1-(2-(triuoromethyl)-
pyridin-4-yl)-1H-isoindol-3-amine (0.5 g, 1.1 mmol) was dissolved in
methanol (20 mL), and the resulting solution was injected (20 stacked
injections) on a Chiralpak AD column (21.2 × 250 mm) using IPA
(0.1% DEA)/CO
2
(20:80) as the eluent. The title compound was
collected and concentrated in vacuo to yield 0.2 g, >99% enantiomeri-
cally pure:
1
H NMR (500 MHz, DMSO-d
6
) δ (ppm) 6.83 (br s, 1 H),
7.32 (t, J = 8.83 Hz, 1 H), 7.37 (t, J = 9.46 Hz, 1 H), 7.487.54 (m, 1
H), 7.557.62 (m, 2 H), 7.68 (d, J = 5.20 Hz, 1 H), 7.71 (s, 1 H), 7.86
(d, J = 7.57 Hz, 1 H), 8.70 (d, J = 5.04 Hz, 1 H), 8.95 (dd, J = 0.95,
0.47 Hz, 2 H), 9.21 (s, 1 H); MS (ESI, positive ion) m/z 468 (M + 1).
2-((2-(Diuoromethyl)pyridin-4-yl)car bonyl)-4,6-diuoro-
benzonitrile (13). LiCl (244 mg, 5.75 mmol) was added to a solution
of 2-(diuoromethyl)isonicotinic acid (1.0 g, 5.77 mmol) in anhydrous
acetonitrile (30 mL), and the mixture was stirred at 55 °C for 30 min.
The mixture was cooled to rt, and DMF (0.09 mL, 1.16 mmol) was
added followed by dropwise addition of oxalyl chloride (0.54 mL, 6.29
mmol). The mixture was stirred for 1 h at rt and then concentrated
slowly over 1 h at 45 ° C to a 10 mL volume. The mixture was cooled
to rt, and CuCN (103 mg, 1.15 mmol) and (2-cyano-3,5-
diuorophenyl)zinc bromide (0.1 M in THF) (69 mL, 6.9 mmol)
were added. The mixture was stirred at rt for 2 h and then
concentrated in vacuo. The residue was treated with 10%
L-ascorbic
acid, and the aqueous phase was extracted with DCM and with EtOAc.
The combined extracts were washed with s odium phosphate
monobasic (10% solution), dried over MgSO
4
, and concentrated.
The residue was puried by ash chromatography to give the title
compound (310 mg, 18% yield):
1
H NMR (400 MHz, CDCl
3
) δ
(ppm) 6.74 (t, J
HF
= 56 Hz, 1H), 7.187.22 (m, 1 H), 7.277.31 (m,
1 H), 7.74 (d, J = 5.08 Hz, 1 H), 7.92 (s, 1 H), 8.94 (d, J = 5.08 Hz, 1
H); MS (ESI, positive ion) m/z 295 (M + 1).
N-((2-Cyano-3,5-diuorophenyl)(2-(diuoromethyl)-4-
pyridyl)methylene)-2-methylpropane-2-sulnamide (14). Ti-
(OEt)
4
(0.54 mL, 2.57 mmol) and 2-methylpropane-2-sulnimide
(198 mg, 1.62 mmol) were added to a stirred solution of 2-((2-
Journal of Medicinal Chemistry Article
dx.doi.org/10.1021/jm3009025 | J. Med. Chem. 2012, 55, 934693619356
Page 11
(diuoromethyl)pyridin -4-yl)carbonyl)-4,6-diuorobenzonitril e (300
mg, 1.01 mmol) in dry THF (30 mL). The reaction mixture was
heated at 60 °C for 36 h and then cooled to rt. MeOH (2 mL) and
saturated aqueous NaHCO
3
(0.5 mL) were added. The mixture was
stirred for 2 h at rt and then ltered through a pad of Celite and
MgSO
4
. The solids were washed with THF (60 mL), and the ltrate
was concentrated in vacuo. The residue was puried by ash
chromatography to give the title compound (260 mg, 64% yield):
1
H NMR (400 MHz, CDCl
3
) δ (ppm) 1.43 (s, 9 H), 6.79 (t, J
HF
=56
Hz, 1H), 6.746.80 (m, 1 H), 6.94 (dd, J = 8.99, 1.95 Hz, 1 H), 7.56
(d, J = 4.30 Hz, 1 H), 7.79 (s, 1 H), 8.67 (d, J = 5.47 Hz, 1 H); MS
(ESI, positive ion) m/z 398 (M + 1).
3-(3-Bromophenyl)-3-(2-(diu orome thyl) pyri din-4 -yl) -5,7-
diuoro-3H-isoindol-1-amine (15). n-BuLi (0.5 mL, 1.25 mmol)
was added to a solution of 1,3-dibromobenzene (243 mg, 1.03 mmol)
in dry THF (50 mL) at 78 °C. The mixture was stirred for 30 min
and then added to a solution of N-((2-cyano-3,5-diuorophenyl)(2-
(diuoromethyl)-4-pyridyl) methylene)-2-met hylpropane-2-sul na-
mide (205 mg, 0.51 mmol) in THF (5 mL) at 78 °C. The mixture
was stirred at 78 °C for 15 min and then at 0 °C for 30 min. The
reaction was quenched by addition of methanolic HCl (1.25 M, 6 mL),
neutralized using saturated aqueous NaHCO
3
solution, and extracted
with EtOAc. The combined organic extracts were dried over MgSO
4
and concentrated in vacuo. The residue was puried by ash
chromatography to aord the title compound (180 mg, 78% yield):
1
H NMR (400 MHz, CDCl
3
) δ (ppm) 6.79 (t, J
HF
= 56 Hz, 1H), 6.92
(td, J = 9.09, 1.76 Hz, 1 H), 7.06 (dd, J = 7.42, 1.95 Hz, 1 H), 7.16 (s,
1 H), 7.177.22 (m, 1 H), 7.32 (d, J = 5.08 Hz, 1 H), 7.39 (d, J = 1.95
Hz, 1 H), 7.44 (d, J = 7.82 Hz, 1 H), 7.52 (s, 1 H), 8.58 (d, J = 5.47
Hz, 1 H); MS (ESI, positive ion) m/z 450, 452 (M + 1).
1-[2-(Diuoromethyl)pyridin-4-yl]-4,6-diuoro-1-(3-pyrimi-
din-5-ylphenyl)-1H-isoindol-3-amine (16). K
2
CO
3
(83 mg, 0.60
mmol) and pyrimidin-5-ylboronic acid (37 mg, 0.29 mmol) were
added to a solution of 3-(3-bromophenyl)-3-(2-(diuoromethyl)-
pyridin-4-yl)-5,7-diuoro-3H-isoindol-1-ylamine (90 mg, 0.20 mmol)
in a DME/EtOH/H
2
O mixture (3:2:1, 12 mL). The mixture was
degassed for 30 min, and then PdCl
2
(PPh
3
)
2
(14 mg, 0.02 mmol) was
added. The reaction mixture was heated at 100 °C for 3 h, cooled to
room temperature, ltered, and concentrated in vacuo. The residue
was puried by ash chromatography to give the title compound (24
mg, 26% yield):
1
H NMR (400 MHz, CDCl
3
) δ (ppm) 6.76 (t, J
HF
=
56 Hz, 1H), 7.08 (t, J = 8.79 Hz, 1 H) 7.16 (dd, J = 7.23, 1.76 Hz, 1
H), 7.31 (d, J = 8.21 Hz, 1 H), 7.41 (d, J = 4.69 Hz, 1 H), 7.49 (s, 1
H), 7.527.57 (m, 2 H), 7.587.63 (m, 1 H), 8.67 (d, J = 5.08 Hz, 1
H), 8.92 (s, 2 H), 9.22 (s, 1 H);
19
F NMR (376 MHz, CDCl
3
) δ
(ppm) 76.4, 116.2; MS (ESI, positive ion) m/z 450 (M + 1).
(S)-1-[2-(Diuoro methyl)pyridin-4-yl]-4,6-diuoro-1-(3-pyri-
midin-5-ylphenyl)-1H-isoindol-3-amine ((S)-16). 1-[2-
(Diuoromethyl)pyridin-4-yl]-4,6-diuoro-1-(3-pyrimidin-5-ylphen-
yl)-1H-isoindol-3-amine was subjected to enatiomer separation using
SFC preparative chromatography to yield the title compound with an
enantiomeric purity of 99.7%:
1
H NMR (400 MHz, CDCl
3
) δ (ppm)
6.76 (t, J
HF
= 56 Hz, 1H), 7.08 (t, J = 8.79 Hz, 1 H) 7.16 (dd, J = 7.23,
1.76 Hz, 1 H), 7.31 (d, J = 8.21 Hz, 1 H), 7.41 (d, J = 4.69 Hz, 1 H),
7.49 (s, 1 H), 7.527.57 (m, 2 H), 7.587.63 (m, 1 H), 8.67 (d, J =
5.08 Hz, 1 H), 8.92 (s, 2 H), 9.22 (s, 1 H);
19
F NMR (376 MHz,
CDCl
3
) δ (ppm) 76.4, 116.2; MS (ESI, positive ion) m /z 450 (M
+ 1).
2-(3-Bromobenzoyl)-6-uorobenzonitrile (17a). A solution of
copper(I) cyanide (4.7 g, 52.5 mmol) and lithium bromide (2.63 mL,
105.0 mmol) in THF (65 mL) was added to (2-cyano-3-
uorophenyl)zinc(II) iodide (100 mL, 50 mmol) at 78 °C under
an argon atmosphere. The mixture was stirred at rt for 1 h and then
cooled to 78 °C. 3-Bromobenzoyl chloride (6.94 mL, 52.5 mmol)
was added dropwise, and the mixture was stirred at rt for 4 h. Then
aqueous NH
4
Cl (50 mL) was added followed by water (50 mL). The
THF was removed in vacuo, and the aqueous residue was diluted with
water (100 mL) and DCM (150 mL). A precipitate was ltered o,
and the ltrate was added to a separation funnel. The organic layer was
separated and the water phase extracted with DCM (100 mL). The
combined organics were washed with brine (150 mL), dried over
MgSO
4
, concentrated, and puried with ash chromatography to give
the title compound (13.4 g, 88% yield):
1
H NMR (600 MHz, DMSO-
d
6
) δ (ppm) 7.56 (t, J = 8.11 Hz, 1 H), 7.607.65 (m, 1 H), 7.777.86
(m, 2 H), 7.907.94 (m, 1 H), 7.957.99 (m, 2 H); MS (ESI, positive
ion) m/z 304, 306 (M + 1).
N-((3-Bromophenyl)(2-cyano-3-uorophenyl)methylene)-2-
methylpropane-2-sulnamide (17). Titanium ethoxide (7.13 mL,
34.6 mmol), 2-methyl-2-propanesulnamide (2.73 g, 22.5 mmol), and
2-(3-bromobenzoyl)-6-uorobenzonitrile (5.26 g, 17.3 mmol) in dry
THF (57 mL) were reuxed overnight under an argon atmosphere.
The solution was allowed to cool to rt and was then transferred to an
open ask containing MeOH (150 mL), aqueous NaHCO
3
(30 mL),
and EtOAc (600 mL). The resulting mixture was stirred for 25 min
and was then ltered through a mixture of Celite and Na
2
SO
4
and
concentrated. The product was puried by ash chromatography to
give the title compound (5.96 g, 85% yield):
1
H NMR (500 MHz,
DMSO-d
6
) δ (ppm) 1.28 (br s, 9 H), 7.447.55 (m, 3 H), 7.67 (m, 1
H), 7.717.76 (m, 1 H), 7.817.97 (m, 2 H); MS (ESI, positive ion)
m/z 407, 409 (M + 1).
(S )-N -((3-Bromophenyl)(2-cyano-3- uorophenyl)-
methylene)-2-methylpropane-2-sulnamide (( S)-17). Following
the procedure described for 17 using commercially available (S)-2-
methyl-2-propanesulnamide gave the corresponding enantiomerically
pure (S)-N-((3-bromophenyl)(2-cyano-3-uorophenyl)methylene)-2-
methylpropane-2-sulnamide.
(S)-N-((2-Cyano-3-uorophenyl)(3-(cyclopropylethynyl)-
phenyl)methylene)-2-methylpropane-2-sulnamide ((S)-18).
(S)-N-((3-Bromophenyl)(2-cyano-3-uorophenyl)methylene)-2-
methylpropane-2-sulnamide (27.7 g, 68.1 mmol), bis-
(triphenylphosphine)palladium(II) chloride (2.39 g, 3.4 mmol), and
copper(I) iodide (1.3 g, 6.8 mmol) were dissolved in dry 2-
methyltetrahydrofuran (70 mL) at rt under a nitrogen atmosphere.
Ethynylcyclopropane (11.5 mL, 136 mmol) and triethylamine (33 mL,
238 mmol) were added, and the resulting mixture was stirred at 60 °C
overnight. Additional copper(I) iodide (1.3 g, 6.8 mmol), bis-
(triphenylphosphine)palladium(II) chloride (1.4 g, 2.0 mmol), and
ethynylcyclopropane (5.7 mL, 68.0 mmol) were added, and the
mixture was heated at 60 °C for 6 h. After the mixture was cooled to rt,
ethyl acetate was added followed by water and saturated aqueous
NaHCO
3
. The organic layer was collected, activated charcoal was
added, and the mixture was stirred for 3 min, after which it was ltered
through Celite and concentrated. Purication by ash chromatography
gave the title compound (22.6 g, 84% yield):
1
H NMR (500 MHz,
DMSO-d
6
) δ (ppm) 0.74 (m, 2 H), 0.850.92 (m, 2 H), 1.27 (br s, 9
H), 1.54 (m, 1 H), 7.327.71 (m, 6 H), 7.88 (br s, 1 H); MS (ESI,
positive ion) m/z 393 (M + 1).
(R)-5-(3-Amino-1-(3-(cyclopropylethynyl)phenyl)-4-uoro-
1H-isoindol-1-yl)-1-ethyl-3- methylpyridin-2(1H)-one ((R)-19).
n-BuLi (2.5 M in hexanes) (1.76 mL, 4.4 mmol) was added to THF
(3 mL) in a dry bottle at 40 °C (external temperature).
Butylmagnesium chloride (2 M in THF) (1.03 mL, 2.05 mmol) was
added dropwise over 3 min, and the resulting mixture was stirred for
30 min. Then 5-bromo-1-ethyl-3-methylpyridin-2(1H)-one (1.27 g,
5.8 mmol) in THF (5 mL) was added dropwise, and after 1 min, (S)-
N-((2-cyano-3-uorophenyl)(3-(cyclopropylethynyl)phenyl)-
methylene)-2-methylpropane-2-sulnamide (1.15 g, 2.9 mmol) in
THF (5 mL) was added. The mixture was allowed to reach rt and was
then stirred for 1 h. EDTA (0.43 g, 1.5 mmol) in water (5 mL) was
added followed by 6 M HCl (1.5 mL, 8.8 mmol). After the mixture
was stirred for 10 min, aqueous NaHCO
3
was added, and the mixture
was extracted with ethyl acetate. The organic phase was dried over
MgSO
4
, ltered, and concentrated. The residue was dissolved in THF
(3 mL), and 1.25 M HCl in methanol (11.7 mL, 14.7 mmol) was
added. After the mixture was stirred overnight, aqueous NaHCO
3
was
added, and the mixture was extracted with ethyl acetate. The organic
phase was dried over MgSO
4
and concentrated. Purication by ash
chromatography gave the title compound (1.13 g, 91% yield) with a
chiral purity of 70%. This material was subjected to enatiomer
separation using SFC preparative chromatography to yield the title
Journal of Medicinal Chemistry Article
dx.doi.org/10.1021/jm3009025 | J. Med. Chem. 2012, 55, 934693619357
Page 12
compound with an enantiomeric purity of over 99.5%:
1
H NMR (500
MHz, DMSO-d
6
) δ (ppm) 0.660.72 (m, 2 H), 0.810.88 (m, 2 H),
1.101.17 (m, 3 H), 1.46 1.54 (m, 1 H), 1.93 (s, 3 H), 3.763.89
(m, 2 H), 6.55 (br s, 2 H), 7.17 7.29 (m, 7 H), 7.51 (td, J = 7.88, 4.73
Hz, 1 H), 7.557.60 (m, 1 H); MS (ESI, positive ion) m/z 426 (M +
1).
(R)-5-(3-Amino-1-(3-bromophenyl)-4-uoro-1H-isoindol-1-
yl)-1-ethyl-3-methylpyridin-2(1H)-on e ((R)- 20). n-Butyl lithium
(53.4 mL, 133.4 mmol) and THF (100 mL) were added to a dry
reactor. After the mixture was cooled to an inner temperature of 25
°C, n-butylmagnesium chloride (39.0 mL, 66.7 mmol) was added over
20 min. After 45 min, 5-bromo-1-ethyl-3-methylpyridin-2(1H)-one
(39.9 g, 184.7 mmol) in THF (100 mL) was added over 30 min. After
the mixture was stir red for an additional 30 min, (S)-N-((3-
bromophenyl)(2-cyano-3-uorophenyl)methylene)-2-methylpropane-
2-sulnamide (41.8 g, 102.6 mmol) dissolved in THF (100 mL) was
added. The mixture was allowed to reach rt over 45 min, and then the
mixture was stirred at rt for 2 h. After the mixture was cooled to 20
°C, EDTA (1.42 g) was added, followed by a mixture consisting of
ammonium chloride (25.6 g) and water (150 mL). The mixture was
allowed to reach rt over 50 min. After extractive workup with isopropyl
acetate and DCM, the organic phases were dried (Mg
2
SO
4
), ltered,
concentrated in vacuo to give the title compound with an enantiomeric
purity of 73% (24 g, 53% yield). This material was subjected to
enantiomer separation on a Chiralpak AD-H column (50 × 300 mm)
using 80% n-heptane/20% (EtOH + 0.1% DEA) as the eluent. The
second eluting enantiomer was collected (11 g, 99.8% enantiomeric
purity):
1
H NMR (600 MHz, DMSO-d
6
) δ (ppm) 1.14 (t, J = 7.12 Hz,
3 H) 1.94 (s, 3 H) 3.83 (m, 2 H) 6.60 (br s, 2 H) 7.25 (m, 4 H) 7.33
(d, J = 7.97 Hz, 1 H) 7.42 (m, 2 H) 7.52 (td, J = 7.79, 4.77 Hz, 1 H)
7.60 (d, J = 7.54 Hz, 1 H); MS (ESI, positive ion) m/z 440, 442 (M +
1).
(R)-5-(3-Amino-1- (3-amin ophenyl)-4- uoro-1H-isoindol-1-
yl)-1-ethyl-3-methylpyridin-2(1H)-one ((R)-21). A solution of
(R)-5-(3-amino-1-(3-bromophenyl)-4-uoro-1H-isoindol-1-yl)-1-
ethyl-3-methylpyridin-2(1H)-one (1.0 g, 2.27 mmol), trans-4-hydroxy-
L-proline (0.3 g, 2.27 mmol), copper(I) iodide (0.22 g, 1.14 mmol),
and potassium carbonate (0.94 g, 6.8 mmol) was placed in a
microwave vial. Dimethyl sulfoxide (8 mL) was added, and the mixture
was stirred at rt for 1 h. Then ammonia (32% in H
2
O) (3.5 mL, 66.1
mmol) was added via a syringe. The reaction mixture was heated at
110 °C for 3 h in a microwave reactor. The reaction mixture was
diluted with brine and extracted with ethyl acetate. The organic phase
was dried over MgSO
4
, ltered, and concentrated in vacuo. The
product was puried by ash chromatograpy to give the title
compound (176 mg, 21%):
1
H NMR (500 MHz, DMSO-d
6
) δ
(ppm) 1.051.18 (m, 4 H), 1.94 (s, 3 H), 3.17 (d, J = 5.04 Hz, 2 H),
5.01 (br s, 2 H), 6.236.67 (m, 5 H), 6.89 (t, J = 7.72 Hz, 1 H), 7.15
7.30 (m, 3 H), 7.447.55 (m, 2 H); MS (ESI, positive ion) m/z 377
(M + 1).
(R)-N-(3-(3-Amino-1-(1-ethyl-5-methyl-6-oxo-1,6-dihydro-
pyridin-3-yl)-4-uoro-1H-isoindol-1-yl)phenyl)-5-chloropicoli-
namide ((R)-22). 5-Chloropicolinic acid (0.047 g, 0.30 mmol) and 1-
(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (0.06
g, 0.32 mmol) were added to a dry vial. DCM (2 mL) was added, and
the mixture was stirred for 5 min. The reaction mixture was then
added dropwise to a cold (0 °C) solution of (R)-5-(3-amino-1-(3-
aminophenyl)-4-uoro-1H-isoindol-1-yl)-1-ethyl-3-methylpyridin-
2(1H)-one (0.09 g, 0.23 mmol) in N,N-dimethylformamide (2 mL)
and hydrochloric acid (3 M) (0.07 mL, 0.21 mmol). The reaction
mixture was stirred at 0 °C for 1 h, then allowed to reach rt, and stirred
overnight. The reaction was quenched by addition of MeOH (1 mL),
and stirring was continued for 1 h. The reaction mixture was
partitioned between saturated aqueous Na
2
CO
3
solution and ethyl
acetate, and the organic phase was dried over MgSO
4
, ltered, and
concentrated in vacuo. Purication performed by preparative HPLC
gave the title compound (0.07 g, 57% yield):
1
H NMR (500 MHz,
DMSO-d
6
) δ (ppm) 1.081.20 (m, 3 H), 1.94 (s, 3 H), 3.84 (q, J =
7.25 Hz, 2 H), 6.52 (br s, 2 H), 7.05 (d, J = 7.88 Hz, 1 H), 7.217.29
(m, 5 H), 7.51 (td, J = 7.80, 4.89 Hz, 1 H), 7.557.60 (m, 1 H), 7.79
(d, J = 7.88 Hz, 1 H), 7.82 (s, 1 H), 8.108.15 (m, 1 H), 8.168.22
(m, 1 H), 8.75 (d, J = 2.21 Hz, 1 H), 10.62 (s, 1 H); MS (ESI, positive
ion) m/z 516 (M + 1).
(R)-5-(3-Amino-4-uoro-1- (3-(5-prop-1-ynylpyridin-3-yl)-
phenyl)-1H-isoindol-1-yl)-1-ethyl-3-methylpyridin-2(1H)-one
((R)-41). (R)-20 (143 mg, 0.32 mmol), (5-prop-1-ynylpyridin-3-
yl)boronic acid (105 mg, 0.65 mmol), Pd
II
Cl
2
dppf·CH
2
Cl
2
(13 mg,
0.02 mmol), 2 M aqueous K
2
CO
3
(0.49 mL, 0.97 mmol), and dioxane
(3 mL) were mixed in a vial and heated in a microwave reactor at 130
°C for 15 min. The mixture was concentrated, and the resulting
residue was dissolved in DCM (3 mL) and water (2 mL). The organic
phase was separated, concentrated, and puried by preparative HPLC
to aord the title compound (91 mg, 59% yield, >99% enantiomeri-
cally pure):
1
H NMR (400 MHz, DMSO-d
6
) δ (ppm) 1.14 (t, J = 7.07
Hz, 3 H), 1.93 (s, 3 H), 2.11 (s, 3 H), 3.763.91 (m, 2 H), 6.58 (br s,
2 H), 7.217.31 (m, 3 H), 7.397.46 (m, 2 H), 7.52 (td, J = 7.89, 4.93
Hz, 1 H), 7.567.63 (m, 2 H), 7.72 (d, J = 7.58 Hz, 1 H), 7.95 (t, J =
2.02 Hz, 1 H), 8.56 (d, J = 1.77 Hz, 1 H), 8.70 (d, J = 2.27 Hz, 1 H);
MS (ESI, positive ion) m/z 477 (M + 1).
ASSOCIATED CONTENT
*
S
Supporting Information
Synthetic procedures and analytical data for test compounds
23, 24, 25,(S)-25, 26, 27, 28, 29, 30 , 31, 32,(S)-32, 33, 34, 35 ,
36, 37, 38, 39, 40, and (R)-42 and synthetic procedures for
intermediates. This material is available free of charge via the
Internet at http://pubs.acs.org.
Accession Codes
New protein/ligand coordinates for 10 and (R)-41 have been
deposited in the PDB with IDs 4azy and 4b00, respectively.
AUTHOR INFORMATION
Corresponding Author
*Phone: +46 (0) 70 290 72 01. E-mail: brittmarieswahn@
gmail.com.
Notes
The authors declare no competing nancial interest.
ACKNOWLEDGMENTS
We specially thank NAEJA Pharmaceuticals Inc., Canada, for
the supply of synthesized intermediates. We thank the
Physiochemical Characterization Team at the Department of
Medicinal Chemistry at AstraZeneca So
̈
derta
̈
lje for physi-
ochemical chara cterization, the Analytical & Purication
Sciences Team for enantiomer separations and purity
determinations, and the Safety Assessment screening center at
AstraZeneca Alderly Park for hERG assessments. We thank
Martin Ekman and Martin Levin for FRET and sAPPβ
measurements and Ann-Cathrin Radesa
̈
ter for measurements
in primary neurons. We thank Carolina Sandman and Carrie
Tsoi for Caco-2 measurements, Eva Floby and Anneli
Bengtsson for rat hepatocyte CL
int
values, Eva Spennare and
Jenny Johansson for determination of the fraction unbound in
brain and plasma protein binding, Stefan Martinsson, Jessie
Dahlstro
̈
m, and Sveinn Briem for bioanalysis, Anna Aagaard for
crystallization, and Madeleine Åhman for pK
a
measurements. In
addition, we thank the people involved in performing the in
vivo experiments, Kristina Eliason, Daniel Bergstro
̈
m, Gunilla
Ericsson, Anna Bogstedt, Ann Staund, Anette Sta
̊
lebring
Lo
̈
wstedt, Susanne Gustavsson, and Carina Stephan.
ABBREVIATIONS USED
BACE, β-site APP cleaving enzyme; AD, Alzheimers disease;
Aβ, β-amyloid; APP, amyloid-β precursor protein; sAPP β,
Journal of Medicinal Chemistry Article
dx.doi.org/10.1021/jm3009025 | J. Med. Chem. 2012, 55, 934693619358
Page 13
soluble amyloid-β precursor protein; SAR, structureactivity
relationship; PSA, polar surface area; ER, eux ratio; PD,
pharmacodynamic; C
u
, concentration unbound; f
u
, fraction
unbound; DCM, dichloromethane; DEA, diethylamine; IPA,
isopropyl alcohol; TEA, triethylamine; SFC, supercritical uid
chromatography; rt, room temperature
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NOTE ADDED AFTER ASAP PUBLICATION
After this paper was published online September 17, 2012,
author Fredrik von Kieseritzky was added to the author list.
The corrected version was reposted October 3, 2012.
Journal of Medicinal Chemistry Article
dx.doi.org/10.1021/jm3009025 | J. Med. Chem. 2012, 55, 934693619361
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  • Source
    • "The dissociation-rate constant (k off ) was calculated using the 1:1 dissociation model and then recalculated to a half-life (t 1/2 = ln 2/k off ) for the complex. This method has been suitable for comparative experiments with AZD3839 [17, 26] and other similarly fast dissociating compounds. However, for slowly dissociating compounds like AZD3293, dissociation could not be distinguished from the background drift in the signal. "
    [Show abstract] [Hide abstract] ABSTRACT: A growing body of pathological, biomarker, genetic, and mechanistic data suggests that amyloid accumulation, as a result of changes in production, processing, and/or clearance of brain amyloid-β peptide (Aβ) concentrations, plays a key role in the pathogenesis of Alzheimer's disease (AD). Beta-secretase 1 (BACE1) mediates the first step in the processing of amyloid-β protein precursor (AβPP) to Aβ peptides, with the soluble N terminal fragment of AβPP (sAβPPβ) as a direct product, and BACE1 inhibition is an attractive target for therapeutic intervention to reduce the production of Aβ. Here, we report the in vitro and in vivo pharmacological profile of AZD3293, a potent, highly permeable, orally active, blood-brain barrier (BBB) penetrating, BACE1 inhibitor with unique slow off-rate kinetics. The in vitro potency of AZD3293 was demonstrated in several cellular models, including primary cortical neurons. In vivo in mice, guinea pigs, and dogs, AZD3293 displayed significant dose- and time-dependent reductions in plasma, cerebrospinal fluid, and brain concentrations of Aβ40, Aβ42, and sAβPPβ. The in vitro potency of AZD3293 in mouse and guinea pig primary cortical neuronal cells was correlated to the in vivo potency expressed as free AZD3293 concentrations in mouse and guinea pig brains. In mice and dogs, the slow off-rate from BACE1 may have translated into a prolongation of the observed effect beyond the turnover rate of Aβ. The preclinical data strongly support the clinical development of AZD3293, and patients with AD are currently being recruited into a combined Phase 2/3 study to test the disease-modifying properties of AZD3293.
    Full-text · Article · Feb 2016 · Journal of Alzheimer's disease: JAD
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    • "The two b-secretase inhibitors (S)-25 and AZD3839, belonging to the aminoisoindolseries (Swahn et al., 2012b), were evaluated with regard to their biotransformation in rats and humans. The main focus is on the biotransformation of (S)-25, but AZD3839 is also discussed, because it showed a different metabolic pattern despite its similar structure. "
    [Show abstract] [Hide abstract] ABSTRACT: Recently, the discovery of the aminoisoindoles as potent and selective inhibitors of β-secretase was reported [Swahn et al., J. Med. Chem. 2012. DOI: 10.1021/jm3009025] including the close structural analogues compound (S)-25 and AZD3839, the latter being recently progressed into the clinic. The biotransformation of (S)-25 was investigated in vitro and in vivo in rat, rabbit and human and compared to AZD3839 to further understand the metabolic fate of these compounds. In vitro, CYP3A4 was the major responsible enzyme and metabolized both compounds to a large extent in the commonly shared pyridine and pyrimidine rings. The main proposed metabolic pathways in various in vitro systems were N-oxidation of the pyridine and/or pyrimidine ring as well as conversion to 4-pyrimidone and pyrimidine-2,4-dione. Both compounds were extensively metabolized and more than 90% was excreted in feces following i.v. dosing of radiolabeled compound to the rat. Here, the main pathways were N-oxidation of the pyridine and/or pyrimidine ring, and a ring contraction of the pyrimidine ring into an imidazole ring. Ring-contracted metabolites accounted for 25% of the total metabolism in the rat for (S)-25, whereas the contribution was much smaller for AZD3839. This metabolic pathway was not foreseen based on the obtained in vitro data. In conclusion, we have discovered an unusual metabolic pathway of aryl-pyrimidine-containing compounds by a ring-opening reaction followed by elimination of a carbon atom and a ring closure to form an imidazole ring.
    Full-text · Article · Mar 2013 · Drug metabolism and disposition: the biological fate of chemicals
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    [Show abstract] [Hide abstract] ABSTRACT: Amino-2H-imidazoles are described as a new class of BACE-1 inhibitors for the treatment of Alzheimer's disease. Synthetic methods, crystal structures, and structure-activity relationships for target activity, permeability, and hERG activity are reported and discussed. Compound (S)-1m was one of the most promising compounds in this report, with high potency in the cellular assay and a good overall profile. When guinea pigs were treated with compound (S)-1m, a concentration and time dependent decrease in Aβ40 and Aβ42 levels in plasma, brain, and CSF was observed. The maximum reduction of brain Aβ was 40-50%, 1.5 h after oral dosing (100 μmol/kg). The results presented highlight the potential of this new class of BACE-1 inhibitors with good target potency and with low effect on hERG, in combination with a fair CNS exposure in vivo.
    Full-text · Article · Sep 2012 · Journal of Medicinal Chemistry
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