Lead optimization and modulation of hERG activity in a series of aminooxazoline xanthene β-site amyloid precursor protein cleaving enzyme (bace1) inhibitors

Article abstract of DOI:10.1021/jm501266w

The optimization of a series of aminooxazoline xanthene inhibitors of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is described. An early lead compound showed robust Aβ lowering activity in a rat pharmacodynamic model, but advancement was precluded by a low therapeutic window to QTc prolongation in cardiovascular models consistent with in vitro activity on the hERG ion channel. While the introduction of polar groups was effective in reducing hERG binding affinity, this came at the expense of higher than desired Pgp-mediated efflux. A balance of low Pgp efflux and hERG activity was achieved by lowering the polar surface area of the P3 substituent while retaining polarity in the P2′ side chain. The introduction of a fluorine in position 4 of the xanthene ring improved BACE1 potency (5-10-fold). The combination of these optimized fragments resulted in identification of compound 40, which showed robust Aβ reduction in a rat pharmacodynamic model (78% Aβ reduction in CSF at 10 mg/kg po) and also showed acceptable cardiovascular safety in vivo.

Full text of DOI:10.1021/jm501266w

Article
pubs.acs.org/jmc
Lead Optimization and Modulation of hERG Activity in a Series of
Aminooxazoline Xanthene β‑Site Amyloid Precursor Protein Cleaving
Enzyme (BACE1) Inhibitors
Oleg Epstein,*,† Marian C. Bryan,†,# Alan C. Cheng,§ Katayoun Derakhchan,⊥ Thomas A. Dineen,†
Dean Hickman,∥ Zihao Hua,† Jason B. Human,† Charles Kreiman,† Isaac E. Marx,† Matthew M. Weiss,†
Robert C. Wahl,§ Paul H. Wen,‡ Douglas A. Whittington,§ Stephen Wood,‡ Xiao Mei Zheng,†
Robert T. Fremeau, Jr.,‡ Ryan D. White,† and Vinod F. Patel†,∇
‡
§
∥
†Departments of Therapeutic Discovery, Neuroscience, Molecular Structure and Characterization, Pharmacokinetics and Drug
Metabolism, and ⊥Comparative Biology and Safety Sciences, Amgen Inc., 360 Binney Street, Cambridge, Massachusetts 02142, One
Amgen Center Drive, Thousand Oaks, California 91320, and 1120 Veterans Boulevard, South San Francisco, California 94080,
United States
S
* Supporting Information
ABSTRACT: The optimization of a series of aminooxazoline
xanthene inhibitors of β-site amyloid precursor protein
cleaving enzyme 1 (BACE1) is described. An early lead
compound showed robust Aβ lowering activity in a rat
pharmacodynamic model, but advancement was precluded by
a low therapeutic window to QTc prolongation in
cardiovascular models consistent with in vitro activity on the
hERG ion channel. While the introduction of polar groups was
effective in reducing hERG binding affinity, this came at the
expense of higher than desired Pgp-mediated efflux. A balance of low Pgp efflux and hERG activity was achieved by lowering the
polar surface area of the P3 substituent while retaining polarity in the P2′ side chain. The introduction of a fluorine in position 4
of the xanthene ring improved BACE1 potency (5−10-fold). The combination of these optimized fragments resulted in
identification of compound 40, which showed robust Aβ reduction in a rat pharmacodynamic model (78% Aβ reduction in CSF
at 10 mg/kg po) and also showed acceptable cardiovascular safety in vivo.
off-target selectivity.7,8 More recent efforts have largely
transitioned to scaffolds utilizing a cyclic amidine to engage the
catalytic dyad that also have lower molecular weight with less
conformational flexibility and increased success in avoiding efflux
INTRODUCTION
■
Alzheimer’s disease (AD) is a chronic and ultimately fatal
neurodegenerative disease that directly or indirectly affects an
increasing number of people.1 AD represents a significant, unmet
transporters (i.e., Pgp) to achieve acceptable exposure in the
medical need worldwide as current treatments do not impact
brain.9,10 Importantly, multiple groups have reported robust
disease progression.2 Although the cause of AD remains unclear,
lowering of Aβ in the CSF and brain in preclinical models as well
much evidence suggests that accumulation of β-amyloid (Aβ)
as in humans with chemical matter from these scaffolds.11,12
peptides is a key contributor to disease progression.3 In
We recently reported the discovery of aminooxazoline
particular, human genetic evidence strongly implicates activating
xanthenes as orally efficacious BACE1 inhibitors.13 Initial
mutations that lead to increased Aβ production with early onset
(familial) AD4 while decreased Aβ production rate correlates
with decreased incidence of neurodegeneration and AD.5
Leading approaches to develop a disease modifying treatment
for AD have focused on the inhibition of Aβ production.3 Aβ
peptides arise from the amyloid precursor protein (APP)
through two proteolytic steps, the first of which is mediated by
the aspartyl protease β-site APP cleaving enzyme (BACE1).6
Since BACE1 was characterized more than a decade ago,
significant effort has been expended to identify orally available,
small-molecule inhibitors that are able to effectively decrease Aβ
production in the CNS of preclinical models. Unfortunately,
early efforts focused on peptidomimetic inhibitors that failed to
achieve meaningful target coverage in the CNS and/or adequate
optimization efforts demonstrated that the S2′ pocket of
BACE1 enzyme could be exploited for improved potency and
led to identification of compound 1. This compound was a
promising lead with good cellular potency combined with
properties necessary for brain exposure. After brief exploration of
close-in analogues, we found that homologation of the
isobutyloxy P2′ substituent14 to the larger neopentyloxy afforded
compound 2 with improved potency and overall favorable
properties where a significant pharmacodynamic (PD) effect in
rats was observed (Table 1). A single oral dose (30 mg/kg)
Received: August 18, 2014
© XXXX American Chemical Society
A
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Table 1. In Vitro Profiles and PD Results for 1 and 2
PD at 30 mg/kg, % Aβ
a
f
BACE1 IC50 (nM)
reduction
b
c
d
e
compd
P2′
enzyme
HEK
hER
hERG Ki (μM)
HLM (μL/(min·mg))
CSF
brain
1
2
OCH2i-Pr
OCH2t-Bu
8
2
0.3
1
36
25
7
8
0.9
1.5
1.84
19
21
76
80
45
61
0.66
a
b
c
IC50 values were averaged values determined by at least two independent experiments. Human embryonic kidney cells. Efflux measured in LLC-
d
e
PK1 cells transfected with human MDR1 and are reported as a ratio of (B to A)/(A to B). From [3H]-dofetilide binding assay. Human liver
microsomal (HLM) clearance. Compound concentration = 1 μM. Microsomal protein concentration = 250 μg/mL. Pharmacodynamic assay
f
̈
measuring Aβ reduction in CSF and brain after 4 h postdose of test compound in naıve male Sprague−Dawley rats compared with vehicle treated
animals.
a
Scheme 1. Preparation of Aminooxazoline Xanthenes 6a−f
a
Reagents: (a) (i) Cs2CO3, CuOTf (cat.), EtOAc (cat.), toluene, 90 °C, then HCl, (ii) H2SO4 or PPA, 60−110 °C, 26−85%; (b) (i) MeMgCl, THF,
−10 °C, then PPTS or HCl, or TMSCH2Li, THF, −78 °C, then AcCl, (ii) I2, AgOCN, THF, 0 °C to rt, then NH3, THF, 23−53%.
resulted in 61% brain Aβ reduction after 4 h. A dose response
study from 0.3 to 30 mg/kg gave an EC50,u of 0.025 μM.15
Unfortunately, the increased lipophilicity of 2 was associated
with higher inhibitory activity of the hERG ion channel.16
Assessment in an ex vivo isolated rabbit heart (IRH) model as
well as an in vivo anesthetized dog model indicated an
unacceptable margin for QTc prolongation that precluded
further development of 2.17 Consequently, we sought to improve
the cardiovascular (CV) safety profile in this series. Herein we
describe the strategy and design considerations in our efforts to
simultaneously minimize both hERG inhibition and efflux
transporter recognition (i.e., Pgp) while maintaining BACE1
potency for robust CNS effect.
Avoiding inhibition of the hERG ion channel while
simultaneously minimizing Pgp recognition and achieving potent
BACE1 inhibition was considered a significant challenge given
that the most straightforward strategies to address each
parameter did not align toward a common physiochemical
property profile. For example, reducing the basicity and overall
lipophilicity of target molecules are common approaches for
reducing hERG affinity.18−20 However, for BACE1, it is known
that decreasing basicity of the Asp-binding element typically
decreases cellular BACE1 potency,21−23 while introduction of
polarity increases the total polar surface area (PSA), which is
strongly correlated with increased Pgp recognition, leads to
decreased exposure in the central compartment.24 We sought to
determine if it would be possible within the context of the
xanthene series to balance hERG, Pgp efflux, and BACE1
potency by identifying regions of the molecule where polarity
could be incorporated to minimize hERG activity without
leading to significant efflux. The aminooxazoline warhead would
initially be maintained (pKa = 6.7)25 and increased BACE1
potency explored by optimizing other key interactions observed
in compound 2 while trying to limit further molecular weight
increase in an already “large” core (∼420 Da).
CHEMISTRY
■
For the preparation of aminooxazoline xanthenes, we followed
our previously reported approach13 depicted in Scheme 1.
Suitably substituted phenols 3a,f were arylated with 2-
halobenzoic acids 4a−f using Cu-catalyzed conditions reported
by Buchwald and co-workers,26 and the resulting 2-arylox-
ybenzoic acids underwent intramolecular Friedel−Crafts cycliza-
tion under acidic conditions to afford substituted xanthenones
5a−f.27 The carbonyl group was then converted to an
exomethylene using MeMgCl followed by dehydration of the
transient tertiary alcohol or through Peterson olefination by
treatment with TMSCH2Li followed by acetyl chloride work-
up.28 Reaction of the olefin with iodine isocyanate generated
from iodine and silver cyanate followed by treatment with
ammonia afforded racemic aminooxazoline xanthenes 6a−f.29
B
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a
Scheme 2. Preparation of Compounds 7, 8, 10, and 11
a
Reagents: (a) 5-pyrimidylboronic acid, Pd(PPh3)4 (cat.), Na2CO3, dioxane/water, 22%; (b) (i) t-butylacetylene, PdCl2(PPh3)2 (cat.), t-Bu3P·HBF4,
DBU, Cs2CO3, DMF, 150 °C, 49%, (ii) SFC separation; (c) H2, Pd/C, 63%; (d) (i) 2-methyl-3-butyn-2-ol, Pd(PPh3)4 (cat.) CuI (cat.), i-PrNH2,
97%, (ii) 5-pyrimidylboronic acid, Pd(PPh3)4 (cat.), Na2CO3, DME/water, 62%, (iii) SFC separation; (e) MsOH, MeOH, 35%.
a
Scheme 3. Preparation of Compounds 12−14
a
Reagents: (a) 5-pyrimidylboronic acid, Pd(PPh3)4, Na2CO3, DMF/water, 62%; (b) 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane, Pd(Amphos)Cl2, K2CO3, dioxane/water, 65%; (c) morpholine or 2,2-dimethylmorpholine, X-Phos Palladacycle,33 X-Phos, LiHMDS,
THF, 17% for 13 and 19% for 14.
a
Scheme 4. Preparation of O-Linked Compounds 16−21
a
Reagents: (a) BBr3, DCM, 74%; (b) 5-pyrimidylboronic acid, Pd(PPh3)4, Na2CO3, DMF/water, 99%; (c) alkyl halide or alkyl triflate, Cs2CO3,
DMF, 22−82%.
Initially, P3 and P2′ substituents were introduced into the
racemic aminooxazoline core and enantiomerically pure final
products were obtained after supercritical fluid chromatography
(SFC) separation using a chiral stationary phase. For example, to
prepare compound 7 which contained a t-butylacetylene P2′
group and 5-pyrimidinyl P3 group, the 2-chloro-7-bromo-
derivative 6a was used (Scheme 2). Selective Suzuki−Miyaura
coupling30 allowed the introduction of the P3 aryl substituents
first, followed by installation of the P2′ alkyne moiety through a
modified Sonogashira coupling.31 The triple bond was then
hydrogenated in the presence of palladium catalyst to afford 8
which contained a saturated alkyl P2′ substituent. The harsh
conditions used for Sonogashira coupling of the aryl chlorides
were not suitable for more sensitive alkynes such as propargylic
alcohols, and the corresponding 2-iodo-7-bromo derivative 6b
was used instead for reversing the order of introduction of the
P2′ and P3 substituents onto the xanthene core. Highly selective
Sonogashira coupling of 2-iodo-7-bromo-substituted amino-
oxazoline 6b afforded aryl alkyne 9. Suzuki coupling with 5-
pyrimidylboronic acid followed by chiral SFC separation
provided compound 10. Treatment of the propargylic alcohol
10 with methanesulfonic acid in methanol afforded methoxy
alkyne 11.
2-Iodo-7-bromo aminoooxazoline xanthene 6b was recog-
nized as an exceptionally convenient template for the
introduction of different substituents onto the xanthene core
through selective palladium-catalyzed C−C and C−N coupling
reactions. After chiral separation, both enantiomers of 6b could
be used for sequential installation of P2′ and P3 substituents. For
example, compounds 12, 13, and 14 were prepared by selective
Suzuki coupling of intermediate (R)-6b with 5-pyrimidyl boronic
acid followed by another Suzuki coupling32 with 4-dihydropyr-
anyl boronate ester, resulting in 12 or Pd-catalyzed amination33
to afford morpholines 13 and 14 (Scheme 3).
For the synthesis of oxygen-linked P2′ substituents, we
primarily utilized the 7-bromo-2-methoxy substituted xanthene
core (6c). Chiral separation of 6c using SFC afforded
enantiomerically pure (>99% ee) intermediate (R)-6c. Boron
tribromide-mediated demethylation resulted in phenol 15 as a
useful building block for further functionalization. Thus,
introduction of 5-pyrimidyl P3 group followed by alkylation of
C
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the phenol represented a convenient way to efficiently prepare
compounds with O-linked P2′ groups (16−21) (Scheme 4).
For screening of P3 substituents (compounds in Table 3), the
neopentyloxy P2′ substituent was installed first and heteroaryl
groups were introduced via Pd-catalyzed coupling of the aryl
bromide 22 with commercially available boronic acids/boronate
esters or organotin compounds to give 24−31 (Scheme 5). In
the phenol 45 with 2-fluoro-3-pyridylboronic acid gave 46.
Phenol 46 could be alkylated to afford alkoxy-substituted
compounds 37 and 38. Activation of the phenol was achieved
through aryl triflate formation, and the triflate 47 was
subsequently used in Pd-catalyzed C−C and C−N coupling
reactions to install a variety of P2′ groups.
RESULTS AND DISCUSSION
■
a
Scheme 5. Preparation of Compounds 24−32
We began optimization by exploring S2′pocket while maintain-
ing the P314 pyrimidyl substituent (Table 2). As expected,
introduction of lipophilic groups resulted in compounds with a
significant increase in hERG inhibitory activity, especially when
cLogP >3.5, (compounds 7, 8, and 16, Table 2). Introduction of
polarity into the P2′ group to afford compounds with cLogP in a
lower range (1.7−3) gave more promising results. For example,
introduction of a polar hydroxyl group (10 and 17) significantly
reduced hERG inhibitory activity compared to compound 2 but
at the expense of high Pgp-mediated efflux, likely due to the
presence of an additional hydrogen bond donor. Substitution to a
cyano group or oxetane (20 and 21) also led to compounds
which demonstrated high efflux, presumably a result of increased
total PSA. Incorporating a fluorine atom into the P2′ group (19)
lowered hERG activity to 5 μM with only a minor effect on Pgp
efflux. Masking PSA from Pgp recognition by the addition of
neighboring lipophilic groups was effective in lowering efflux but
overall led to increased microsomal turnover consistent with the
increase in cLogP (cf. 10 and 11, 17 and 18, 13 and 14). A
desirable combination of BACE1 potency, balanced with low
hERG activity (>10 μM) and a relatively low efflux ratio, was
found in compound 12, containing a 4-dihydropyranyl P2′
substituent.
a
Reagents: (a) P3-B(OH)2, Pd(PPh3)4, Na2CO3, dioxane/water, 6−
77%; (b) 2-(tributylstannyl)pyridine, Pd(PPh3)4, DMF, 24%; (c)
B2Pin2, Pd(dppf)Cl2, KOAc, dioxane; (d) 2,4-difluoro-3-iodopyridine,
Pd(Amphos)Cl2, KOAc, BuOH/water, 53%.
cases when heteroaryl halides were commercially available,
aminooxazoline intermediate 22 was converted to boronic ester
23, which was used in Suzuki coupling reactions to prepare
compound 32 (Table 3).
Preparation of the BACE1 inhibitors included in Table 4 was
achieved by similar methods using fluorinated aminooxazoline
xanthenes 6c−e as described in Scheme 4 through sequential
introduction of heteroaryl P3 substituents via Suzuki coupling
followed by alkylation of the corresponding phenols with 3-
(bromomethyl)-3-methyloxetane.
The preparation of 4-fluoro-substituted aminooxazoline
xanthenes 37−44 (Table 5) was streamlined using a single
intermediate (S)-6e. As described in Scheme 6, demethylation of
bromomethoxy derivative (S)-6e followed by Suzuki coupling of
With the initial goal of changing the distribution of PSA
around the molecule as a means of potentially better separating
BACE1 potency from hERG and Pgp efflux, we performed an
additional screen of P3 groups while keeping the neopentyloxy
P2′group constant. As can be seen from selected data shown in
Table 3, the position of the nitrogen atom in the P3 substituent is
important for maintaining high BACE1 enzymatic activity (3-
pyridinyl (26), being superior to 2- and 4-pyridinyl (24 and 25)).
Fluorine substitution was well tolerated around 3-pyridyl ring
(27, 29, 31, and 32). Substitution at the 3-position of the
pyridine ring was well tolerated (28−30), although introduction
a
Scheme 6. Preparation of 4-Fluoro-Substituted Aminooxazoline Xanthenes 37−44
a
Reagents: (a) BBr3, DCM, 96%; (b) 2-fluoro-3-pyridylboronic acid, Pd(PPh3)4, Na2CO3, DMF/water, 87%; (c) 3-(bromomethyl)-3-methyloxetane
or 2-fluoro-2-methylpropyl trifluoromethanesulfonate, Cs2CO3, NaI, DMF, 35% for 37 and 60% for 38; (d) N-(5-chloro-2-pyridyl)bis-
(trifluoromethanesulfonimide) (Commins’ reagent), Et3N, DCM, 84%; (e) RB(OH)2, Pd(PPh3)4, Na2CO3, dioxane/water; (f) amine, Pd2dba3,
DavePhos, LiHMDS, THF, 11−61%; (g) 2-pyridylzinc chloride, Pd(PPh3)4, THF, 72%.
D
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Table 2. Selected SAR of P2′ Substituents
a
b
c
IC50 values were averaged values determined by at least two independent experiments. Human embryonic kidney cells. Apparent permeability
measured in parental LLC-PK1 cells. Values are an average of apical to basolateral (A to B) and basolateral to apical (B to A) velocities and are
d
e
reported as 10−6 cm/s. Efflux measured in LLC-PK1 cells transfected with human MDR1 and are reported as a ratio of (B to A)/(A to B). Human
f
liver microsomal (HLM) clearance. Compound concentration = 1 μM. Microsomal protein concentration = 250 μg/mL. From [3H]-dofetilide
binding assay.
of a methoxy group in 30 led to poor metabolic stability. Out of
this set of P3 groups, 2-fluoro-3-pyridinyl and 2,4-difluoropyr-
idinyl substituents led to increased BACE1 inhibition. Moreover,
the two compounds containing these P3 groups (31 and 32) did
not show significant hERG inhibition despite cLogP values ∼5.
This suggests that overall lipophilicity of the molecules is not
solely responsible for activity on hERG channel, and subtle
structural modifications could disrupt interactions that affect
hERG affinity. Considering potential complications arising from
chemical reactivity of the 2-fluoro-substituted pyridine moiety,
compounds containing these groups were incubated in buffered
solutions in the presence of glutathione (GSH), as well as with
hepatocytes, and only the compounds with 2,4-difluoro-3-
pyridine group showed formation of a GSH-conjugate (data
not shown). On the basis of these findings and overall profile of
compounds with the 2-fluoro-3-pyridinyl group, this P3 group
was preferred for further optimization in this series.
Changing focus from decreasing hERG activity to improving
BACE1 potency, we carefully examined a crystal structure of 2
with BACE1 (Figure 1). This cocrystal suggested that
E
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Table 3. Selected SAR of P3 Substituents
a
b
c
IC50 values were averaged values determined by at least two independent experiments. Human embryonic kidney cells. Apparent permeability
measured in parental LLC-PK1 cells. Values are an average of apical to basolateral (A to B) and basolateral to apical (B to A) velocities and are
d
reported as 10−6 cm/s. Efflux measured in LLC-PK1 cells transfected with either rat MDR1A/1B or human MDR1 and are reported as a ratio of (B
e
to A)/(A to B). Human liver microsomal (HLM) clearance. Compound concentration = 1 μM. Microsomal protein concentration = 250 μg/mL.
f
From [3H]-dofetilide binding assay.
introducing substituents in the xanthene core may result in
additional favorable interactions. Particularly intruiguing was the
potential to interact with Trp 76 via positions 3 and/or 4 of the
xanthene ring. We hypothesized that a heteroatom (N or F) at
either of these positions could lead to a beneficial interaction with
the protein to improve binding affinity.34 Similar interactions of
fluorine, oxygen, and nitrogen atoms with the NH of Trp76 in
crystal structures of inhibitors with the BACE1 protein have been
reported to lead to increased BACE1 potency.18,22,35,36
To explore this possibility, we initially examined fluorination at
different positions of the xanthene ring in combination with
preferred P3 and P2′ groups. Selected results of the initial screen
are shown in Table 4. Introduction of a fluorine atom at the 3
position led to marginal improvements of both enzyme and cell
potency compared to the unsubstituted core. A fluorine atom at
the 4 position resulted in a 7-fold improvement in enzymatic
potency and a ∼4-fold improvement in cellular activity. Fluorine
Figure 1. Crystal structure of compound 2 in complex with BACE1.
F
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Table 4. Screen of F-Substitution on the Xanthenes Ring
metabolic stability
a
e
BACE1 IC50 (nM)
(μL/(min·mg))
b
c
d
d
compd
xanthene core
P3
3-Py
enzyme
HEK
Papp (10−6 cm/s)
hER
rER
RLM
HLM
33
34
35
36
37
all-H
3-F
4-F
5-F
4-F
7
4
1
2
15
12
4
2
8.8
13.5
15.2
7.1
4.1
1.4
2
5.1
2.9
3.3
6.3
1.8
67
67
67
71
40
47
91
3-Py
0.8
0.08
7
3-Py
0.3
6
129
82
3-Py
71 15
178
5.2
1.3
2-F-3-Py
0.4 0.1
3
1
12.7
108
a
b
c
IC50 values were averaged values determined by at least two independent experiments. Human embryonic kidney cells. Apparent permeability
measured in parental LLC-PK1 cells. Values are an average of apical to basolateral (A to B) and basolateral to apical (B to A) velocities and are
d
reported as 10−6 cm/s. Efflux measured in LLC-PK1 cells transfected with either rat MDR1A/1B or human MDR1 and are reported as a ratio of (B
e
to A)/(A to B). Rat liver microsomal (RLM) and human liver microsomal (HLM) clearance. Compound concentration = 1 μM. Microsomal
protein concentration = μg/mL.
substitution at the 5-position was not well tolerated, with a ∼10-
fold drop in BACE1 activity. As previously observed in our P3
group screen, the 2-fluoro-3-pyridyl P3 group was superior to 3-
pyridyl and brought additional potency in the enzymatic and
cellular assays (37).
EC50 and cellular IC50 (4 nM), minimal impairment at the
blood−brain barrier due to Pgp efflux was observed and
consistent with low measured Pgp efflux.38
The crystal structure of compound 40 bound to BACE1 was
refined to 1.78 Å (Figure 2). As seen in previous structures of
aminooxazoline xanthene compounds, the amidine moiety
interacts with the two catalytic aspartic acids and Tyr71 engages
in a π-stacking interaction with the xanthene core. As anticipated,
the fluorine atom in position 4 of the xanthene ring interacts with
Trp76 via a hydrogen bond,39 likely the reason for the increased
BACE1 inhibitory activity. The fluoropyridine side chain
occupies the S3 pocket of BACE1, with the nitrogen atom
involved in a water-mediated H-bonding interaction with the
backbone carbonyl of Ser229, and the dihydropyran unit resides
in the S2′ pocket of the protein where the oxygen atom is within
hydrogen bond distance to Arg128.
Compound 40 was evaluated further in both time-course and
dose−response rat PD model studies. Pharmacodynamic studies
in rats at oral doses of 1, 3, 10, and 30 mg/kg showed dose-and
exposure-dependent reduction of Aβ level at the 4 h time point.
Unbound plasma concentrations to achieve 50% Aβ reduction
were determined to be 0.005 and 0.010 μM for CSF and brain
effects, respectively (Figure 3).
As a next step, we combined our findings from the xanthene
core substitution (4-fluoro-) and P3 group (2-fluoro-3-pyridyl)
and performed an additional screen of P2′ substituents including
groups that were identified in our initial optimization to lower the
hERG affinity of pyrimidine P3 derivatives (Table 5). We were
pleased to find that the previously described SAR translated well
to the 4-F xanthenes, affording compounds with favorable in
vitro properties. Compounds from this series showed single-digit
nanomolar BACE1 inhibitory activity in the cellular assay and
permeability and efflux properties expected to allow for CNS
exposure. Notably, the 3-dihydropyranyl P2′ group provided the
same level of BACE1 activity as the 4-dihydropyranyl isomer (39
and 40, respectively) although with elevated liver microsomal
turnover, while morpholine 41 and 3-fluoro-substituted
pyrrolidine 42 were also well tolerated. Selected 5- and 6-
membered ring heterocyles (43 and 44) also represent suitable
P2′ substitution, resulting in compounds with low hERG
inhibition and desirable in vitro profiles.
In vivo reduction of Aβ levels was initially examined using
naive Sprague−Dawley (SD) rats that were dosed orally at 10
Time-dependent reduction of Aβ with compound 40 was also
studied at a 30 mg/kg dose using 1.5, 4, 7, 10, 16, and 24 h time
points. Maximum Aβ lowering in both the brain and CSF was
observed between 4 and 10 h (Figures 4 and 5), with levels
returning to baseline after 24 h. Pharmacokinetic/pharmacody-
namic (PK/PD) modeling of 40 using an indirect response
model that accounts for the formation and clearance of Aβ in
CSF40 estimated the in vivo plasma IC50, unbound for CSF and
brain effects of 40 to be 0.017 and 0.013 μM, respectively.
Importantly, the QT prolongation issues associated with 2
were not observed with 40. Although the in vitro margin between
hERG inhibition (3.28 μM) and BACE1 cell IC50 (4 nM) for 40
was large (820-fold), the compound was assessed further in ex
vivo and in vivo CV safety models. In the ex vivo IRH model,
hearts (n = 3) were perfused with five concentrations of 40 at
increasing doses of 0.1, 0.3, 1, 3, and 10 μM. Each dose was
applied for 20 min with measurements collected continuously.
No effect on QTc interval was observed at any of the doses. In an
anesthetized dog CV model, anesthetized dogs (n = 2) were
̈
mg/kg. After a single dose, compound plasma exposure and Aβ
levels (both CSF and brain) were measured at the 4 h time point.
Significant inhibition of Aβ production was observed for most of
the compounds with high BACE1 cellular potency and good
microsomal stability. Despite favorable in vitro properties, low
PD response was observed for oxetane-containing compound 37
as a result of poor in vivo target coverage (exposure multiple
Cplasma,u/cell IC50 = 0.5). Compounds were rank ordered using
estimates from single-point experiment unbound plasma
concentrations to achieve 50% Aβ lowering in brain.37 In
general, compounds with low efflux showed little shift between
the cellular IC50 and in vivo PD EC50 determined from a time-
course experiment or estimated from the single-point screen.
Compound 40 showed a very robust in vivo PD response at 10
mg/kg dose (78% in CSF and 67% in brain). The unbound
plasma EC50 for brain effect of 40 in this single dose PD
experiment was 13 nM. Given the small difference between PD
G
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Table 5. Selected SAR of 4-Fluoro Xanthene Series
a
b
c
IC50 values were averaged values determined by at least two independent experiments. Human embryonic kidney cells. Apparent permeability
measured in parental LLC-PK1 cells. Values are an average of apical to basolateral (A to B) and basolateral to apical (B to A) velocities and are
d
reported as 10−6 cm/s. Efflux measured in LLC-PK1 cells transfected with either rat MDR1A/1B or human MDR1 and are reported as a ratio of (B
e
f
to A)/(A to B). From [3H]-dofetilide binding assay. Rat liver microsomal (RLM) and human liver microsomal (HLM) clearance. Compound
g
concentration = 1 μM. Microsomal protein concentration = 250 μg/mL. Pharmacodynamic assay measuring Aβ reduction in CSF and brain after 4
h postdose of test compound in naıve male Sprague−Dawley rats comparing with vehicle treated animals. Estimated unbound plasma EC50 for
brain effect from a single dose experiment at 4 h time point.
h
̈
treated intravenously with 30 min infusions of 40 at doses of 3.3,
11, and 35 mg/kg. Average unbound plasma concentrations of
0.049, 0.356, and 0.654 μM were achieved where only 11%
increase in QTc interval in one subject was observed at the
highest dose, affording a 65-fold margin over the plasma
EC50,unbound for brain Aβ reduction in rats and a 50-fold margin
over the in vivo IC50,unbound as determined by the indirect
response model.
inhibitors with lower hERG binding affinity without sacrificing
brain exposure and metabolic stability. Introduction of a fluorine
in the 4-position of the xanthene ring increased BACE1 enzyme
and cellular potency resulting in compounds that produced
̈
robust reductions in CSF and brain Aβ levels in a naive rat
pharmacodynamic model. Compound 40 was selected for
further advancement based on its excellent in vitro profile and
in vivo PD response. Profiling of this compound in a dog
cardiovascular safety model showed significant improvement to 2
with only minor effects on the QTc interval at exposures
CONCLUSION
■
approximately 50-fold over the in vivo IC50,unbound
.
In conclusion, optimization of a series of aminooxazoline
xanthenes was performed with an emphasis on mitigating the
hERG-mediated QTc prolongation observed in an anesthetized
dog CV safety model. It was determined that introduction of
polarity in the P2′ region of the inhibitor while simultaneously
removing polarity from P3 enabled the identification of
EXPERIMENTAL SECTION
Chemistry. Unless otherwise noted, all materials were obtained from
commercial suppliers and used without further purification. Anhydrous
solvents were obtained from Aldrich or EM Science and used directly.
■
H
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Figure 2. Cocrystal structure of 40 with BACE1. (a) Interactions of 40 with the catalytic aspartic acids of BACE1. (b) Interactions of fluorine atom with
Trp76 and the dihydropyran moiety with Arg128 in P2′.
Figure 3. Dose dependent reduction of Aβ in a rat pharmacodynamic model at 4 h after oral dose of 40.
Figure 4. Time dependent reduction of Aβ in rat pharmacodynamic model after 30 mg/kg oral dose of 40.
All reactions involving air- or moisture-sensitive reagents were
performed under a nitrogen or argon atmosphere. All microwave
assisted reactions were conducted with a Smith synthesizer from
Personal Chemistry, Uppsala, Sweden. Silica gel chromatography was
performed using prepacked silica gel cartridges (Biotage or ISCO). 1H
NMR spectra were recorded on a Bruker AV-400 (400 MHz)
spectrometer at ambient temperature. Chemical shifts are reported in
parts per million (ppm, δ units) downfield from tetramethylsilane. Data
are reported as follows: chemical shift, multiplicity (s = singlet, d =
doublet, t = triplet, q = quartet, dd = double of doublets, dt doublet of
triplets, etc., br = broad, m = multiplet), coupling constant(s), and
number of protons. Purity for final compounds was greater than 95%
unless otherwise noted and was measured using Agilent 1100 series high
performance liquid chromatography (HPLC) systems with UV
I
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mL, 17.7 mmol) was added in four portions every 20 min and stirred for
an additional 30 min. The reaction was monitored by TLC (3:1
hexanes:EtOAc, starting material Rf = 0.5, product Rf = 0.7). The
solution was cooled to RT and slowly added via addition funnel to a
prestirred mixture of silver cyanate (39.8 g, 265 mmol) and iodine (23.6
g, 93 mmol) in 400 mL of THF that was cooled to −30 °C (dry ice
acetonitrile bath) and had been stirring for 30 min in a 2 L three-neck
flask equipped with an overhead stirrer. The mixture was stirred for
another 30 min at −30 °C after the addition was complete (addition
over 10 min). Ammonia (2 M solution in 2-propanol) (221 mL, 442
mmol) was then added, and the dark mixture was stirred at room
temperature for 20 h. The mixture was filtered and concentrated in
vacuo to ca. 100 mL. The solution was diluted with EtOAc and washed
with aqueous sodium thiosulfate, water, and brine, then dried over
sodium sulfate and concentrated. The semisolid was diluted with ether
(ca. 100 mL) and toluene (100 mL). Precipitated solid was collected by
filtration to afford 20.0 g of crude product. The mother liquors were
concentrated and purified by silica gel chromoatography using 2−6%
MeOH in DCM with 1% NH4OH to a material (4.0 g). Total yield of
racemic 6e: 24.0 g (53%). Racemic material was separated by SFC on 25
mm × 200 mm Chiralpak AD-H column using 25% MeOH (+0.1%
diethylamine)/CO2 to afford 8.9 g (19% yield) of first eluting peak ((R)-
enantiomer) and 9.0 g (20% yield) of second eluting peak ((S)-
enantiomer, desired). 1H NMR (400 MHz, DMSO-d6) δ ppm 7.51 (dd,
J = 8.7, 2.5 Hz, 1 H), 7.40 (d, J = 2.5 Hz, 1 H), 7.19 (d, J = 8.7 Hz, 1 H),
7.00 (dd, J = 12.5, 3.0 Hz, 1 H), 6.63 (dd, J = 3.0, 1.7 Hz, 1 H), 6.60 (br s,
2 H), 4.10−4.16 (m, 2 H), 3.75 (s, 3 H). MS m/z = 379.0 [M + H]+.
(S)-2-Amino-7′-bromo-4′-fluoro-5H-spiro[oxazole-4,9′-xanthen]-
2′-ol (45). To a solution of (S)-7′-bromo-4′-fluoro-2′-methoxy-5H-
spiro[oxazole-4,9′-xanthen]-2-amine ((S)-6e) (15.72 g, 41.5 mmol) in
DCM (200 mL), boron tribromide (9.80 mL, 104 mmol) was added
dropwise at room temp. The solution was stirred for 30 min, and then
the flask was cooled with ice−water bath and carefully quenched by
addition of satd NaHCO3 solution (∼50 mL). Most of DCM was
removed under reduced pressure, and the precipitate was neutralized by
further stirring with saturated NaHCO3 solution (∼200 mL) for 1 h,
then filtered off, washed twice with water, and dried on air for 2 days.
Yield: 14.59 g (96%). 1H NMR (400 MHz, DMSO-d6) δ ppm 9.69 (br s,
1 H), 7.48 (dd, J = 8.7, 2.4 Hz, 1 H), 7.35−7.42 (m, 1 H), 7.15 (d, J = 8.7
Hz, 1 H), 6.66 (dd, J = 12.3, 2.8 Hz, 1 H), 6.49−6.59 (m, 3 H), 4.10 (s, 2
H). MS m/z = 365.0 [M + H]+.
(S)-2-Amino-4′-fluoro-7′-(2-fluoropyridin-3-yl)-5H-spiro[oxazole-
4,9′-xanthen]-2′-ol (46). A round-bottom flask was charged with (S)-2-
amino-7′-bromo-4′-fluoro-5H-spiro[oxazole-4,9′-xanthen]-2′-ol (45)
(10.00 g, 27.4 mmol), 2-fluoropyridin-3-ylboronic acid (6.95 g, 49.3
mmol), and sodium carbonate (8.71 g, 82 mmol). DMF (80 mL) was
added, and the mixture was stirred for 1 min. Tetrakis-
(triphenylphosphine) palladium(0) (1.582 g, 1.369 mmol) and water
(30 mL) were added, the mixture was capped with argon, the flask was
equipped with reflux condenser with argon inlet, and the mixture was
stirred at 85 °C for 16 h. The mixture was cooled to room temperature,
and yellow precipitate was filtered off and washed with DMF. The
filtrate was poured into a stirred semisaturated ammonium chloride
solution (300 mL), and the mixture was stirred for 2 h at room
temperature. The precipitated solid was filtered off and was washed with
water and dried overnight to afford (S)-2-amino-4′-fluoro-7′-(2-
fluoropyridin-3-yl)-5H-spiro[oxazole-4,9′-xanthen]-2′-ol (46) (9.09 g,
23.84 mmol, 87% yield) as a tan solid which was used without further
purification. Purity by HPLC 90%. 1H NMR (400 MHz, DMSO-d6) δ
ppm 9.71 (br s, 1 H), 8.24 (dt, J = 4.8, 1.6 Hz, 1 H), 8.10 (ddd, J = 10.3,
7.3, 2.0 Hz, 1 H), 7.54−7.62 (m, 2 H), 7.48 (ddd, J = 7.3, 5.0, 2.0 Hz, 1
H), 7.28−7.36 (m, 1 H), 6.69 (dd, J = 12.3, 2.8 Hz, 1 H), 6.60 (dd, J =
2.8, 1.5 Hz, 1 H), 6.52 (br s, 2 H), 4.10−4.21 (m, 2 H). MS m/z = 382.0
[M + H]+.
Figure 5. Reduction of brain Aβ levels in rats after 30 mg/kg oral dose of
40.
detection at 254 nm (system A, Agilent Zorbax Eclipse XDB-C8 4.6 mm
× 150 mm, 5 μm, 5−100% CH3CN in H2O with 0.1% TFA for 15 min at
1.5 mL/min; system B, Waters Xterra 4.6 mm × 150 mm, 3.5 μm, 5−
95% CH3CN in H2O with 0.1% TFA for 15 min at 1.0 mL/min).
7-Bromo-4-fluoro-2-methoxy-9H-xanthen-9-one (5e). Step 1: A 5
L round-bottom flask equipped with a reflux condenser and overhead
stirrer was charged with 2-fluoro-4-methoxyphenol (140 g, 982 mmol),
2,5-dibromobenzoic acid (250 g, 893 mmol), ethyl acetate (4.4 mL, 44.7
mmol), and toluene (2500 mL). Copper(I) triflate toluene complex
(2:1) (4.75 g, 22.3 mmol) was added, and then cesium carbonate (582 g,
1786 mmol) was carefully added in portions. The mixture was stirred for
10 min at room temperature then heated at 95 °C for 3 h. The toluene
was distilled off under vacuum. The residue was dissolved in 2 L of water
and washed with 500 mL of EtOAc. The aqueous layer was acidified with
concentrated HCl to pH 2, and the precipitate was collected by filtration
and washed with water two times and dried on air. The resulting solid
was washed with a 4:1 hexane/ethyl acetate twice and then dried under
vacuum to afford 5-bromo-2-(2-fluoro-4-methoxyphenoxy)benzoic acid
1
(235 g, 689 mmol, 77% yield) as a brown solid. H NMR (400 MHz,
DMSO-d6) δ ppm 13.24 (br s, 1 H), 7.89 (d, J = 2.5 Hz, 1 H), 7.65 (dd, J
= 8.9, 2.5 Hz, 1 H), 7.08−7.16 (m, 1 H), 7.04 (dd, J = 12.7, 2.9 Hz, 1 H),
6.80 (ddd, J = 9.0, 2.9, 1.4 Hz, 1 H), 6.75 (dd, J = 9.0, 0.6 Hz, 1 H), 3.78
(s, 3H). MS m/z = 341.0 [M + H]+.
Step 2: To polyphosphoric acid (1130 g, 1150 mmol) in a 2 L round-
bottom flask equipped with an overhead stirrer was added crude 5-
bromo-2-(2-fluoro-4-methoxyphenoxy)benzoic acid (197 g, 577 mmol)
from the previous step, and the viscous mixture was stirred at 125 °C for
2 h. The viscous solution was cooled slightly and poured into well stirred
(overhead stirrer) ice−water (3 L). After 45 min of stirring, the solids
were collected by filtration on a glass filter and washed sequentially with
water, 1N NaOH, and then twice with water. The filter cake was dried
for 18 h on the filter under vacuum, and the solids were then further
dried by azeotroping with toluene to afford 7-bromo-4-fluoro-2-
methoxy-9H-xanthen-9-one (5e) (160 g, 495 mmol, 86% yield) as an
off-white solid. 1H NMR (400 MHz, DMSO-d6) δ ppm 8.25 (d, J = 2.6
Hz, 1 H), 8.04 (dd, J = 9.0, 2.6 Hz, 1 H), 7.73 (d, J = 9.0 Hz, 1 H), 7.61
(dd, J = 12.3, 3.0 Hz, 1 H), 7.38 (dd, J = 3.0, 1.6 Hz, 1 H), 3.89 (s, 3 H).
MS m/z = 323.0 [M + H]+.
(S)-7′-Bromo-4′-fluoro-2′-methoxy-5H-spiro[oxazole-4,9′-xanth-
en]-2-amine ((S)-6e). Step 1: 7-Bromo-4-fluoro-2-methoxy-9H-xanth-
en-9-one (39.5 g, 122 mmol) was dissolved in 580 mL of THF and
cooled to −30 °C in a dry ice acetonitrile bath. Methylmagnesium
chloride (3 M in THF) (82 mL, 245 mmol) was added via addition
funnel over 30 min while maintaining temp < −15 °C. After 1 h, the
mixture was allowed to warm to 0 °C. The mixture became
homogeneous after 2.5 h. The reaction was carefully quenched with
saturated NH4Cl (100 mL) and diluted with brine (200 mL). The
aqueous layer was extracted with ethyl acetate, and the combined
organic layers were washed with brine and dried over anhydrous sodium
sulfate. Concentration of the mixture in vacuo afforded crude 7-bromo-
4-fluoro-2-methoxy-9-methyl-9H-xanthen-9-ol (42.0 g) as a tan semi-
solid and was advanced without further purification.
(S)-2-Amino-5′-fluoro-2′-(2-fluoropyridin-3-yl)-5H-spiro[oxazole-
4,9′-xanthene]-7′-yl trifluoromethanesulfonate (47). To a solution of
(S)-2-amino-4′-fluoro-7′-(2-fluoropyridin-3-yl)-5H-spiro[oxazole-4,9′-
xanthen]-2′-ol (46) (2.62 g, 6.87 mmol) in DCM (35 mL) was added
triethylamine (1.39 g, 13.74 mmol), and the mixture was cooled to 0 °C.
N-(5-Chloro-2-pyridyl)bis(trifluoromethanesulfonamide) (2.97 g, 7.56
Step 2: To a 1 L round-bottom flask charged with 7-bromo-4-fluoro-
2-methoxy-9-methyl-9H-xanthen-9-ol (30.0 g, 88 mmol) in THF (200
mL), and the mixture was warmed to 40 °C. HCl (4 M in dioxane) (4.4
J
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mmol) was added, and the mixture was stirred for 24 h at room
temperature. The mixture was washed with 1N NaOH solution twice,
filtered through Celite, and concentrated. The residue was purified by
silica gel chromatography (0−50% DCM/MeOH/NH4OH 90:10:1 in
DCM) to afford (S)-2-amino-5′-fluoro-2′-(2-fluoropyridin-3-yl)-5H-
spiro[oxazole-4,9′-xanthene]-7′-yl trifluoromethanesulfonate (47)
(S)-2-amino-5′-fluoro-2′-(2-fluoropyridin-3-yl)-5H-spiro[oxazole-4,9′-
xanthene]-7′-yl trifluoromethanesulfonate (47) (800 mg, 1.558 mmol),
as described for 49 using 2-(5,6-dihydro-2H-pyran-3-yl)-4,4,5,5-
tetramethyl-1,3,2-dioxaborolane (655 mg, 3.12 mmol) and tetrakis-
(triphenylphosphine)palladium(0) (90 mg, 0.078 mmol) in DMF (5
mL) and 5 M sodium carbonate solution (1.56 mL, 7.79 mmol). Yield:
185 mg (27%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.25 (dd, J = 4.8,
1.4 Hz, 1 H), 8.08−8.15 (m, 1 H), 7.57−7.64 (m, 2 H), 7.49 (ddd, J =
7.3, 5.0, 1.8 Hz, 1 H), 7.34−7.40 (m, 2 H), 7.11 (s, 1 H), 6.51 (br s, 2 H),
6.26−6.31 (m, 1 H), 4.31−4.49 (m, 2 H), 4.23 (s, 2 H), 3.74 (t, J = 5.5
Hz, 2 H), 2.20−2.32 (m, 2 H). m/z = 448.0 [M + H]+.
1
(2.98 g, 5.80 mmol, 84% yield) as yellow foam. H NMR (400 MHz,
CDCl3) δ ppm 8.20 (d, J = 4.5 Hz, 1 H), 7.75−7.86 (m, 1 H), 7.59 (s, 1
H), 7.54 (d, J = 8.6 Hz, 1 H), 7.28−7.31 (m, 1 H), 6.96−7.19 (m, 4 H),
6.73 (d, J = 8.0 Hz, 1 H), 6.28 (br s, 2 H), 4.42 (d, J = 8.8 Hz, 1 H), 4.36
(d, J = 8.8 Hz, 1 H). MS m/z = 513.8 [M + H]+.
General Procedure for Pd-Catalyzed Amination of Intermediate
47: (S)-4′-Fluoro-7′-(2-fluoropyridin-3-yl)-2′-morpholino-5H-spiro-
[oxazole-4,9′-xanthen]-2-amine (41). A resealable vial was charged
with (S)-2-amino-5′-fluoro-2′-(2-fluoropyridin-3-yl)-5H-spiro[oxazole-
4,9′-xanthene]-7′-yl trifluoromethanesulfonate (47) (200 mg, 0.390
mmol), Pd2dba3 (18 mg, 0.019 mmol), biphenyl-2-yldi-tert-butylphos-
phine (14 mg, 0.047 mmol), and morpholine (85 mg, 0.974 mmol).
LiHMDS (1 M in THF) (1.17 mL, 1.17 mmol) was added, and the
mixture was heated in microwave reactor for 1 h at 110 °C. The reaction
mixture was diluted with water and extracted with ethyl acetate. The
organic layer was washed with water and brine and concentrated in
vacuo. The residue was purified via preparative HPLC (gradient elution
20−90% MeCN/H2O, 0.1% TFA) followed by silica gel column
chromatography using 10−90% 90/10/1 DCM/MeOH/NH4OH in
DCM to afford (S)-4′-fluoro-7′-(2-fluoropyridin-3-yl)-2′-morpholino-
5H-spiro[oxazole-4,9′-xanthen]-2-amine (41) (20 mg, 0.044 mmol,
Typical Procedure for Preparation of Compounds with O-Linked
P2′ Groups: (S)-4′-Fluoro-2′-(2-fluoro-2-methylpropoxy)-7′-(2-fluo-
ropyridin-3-yl)-5H-spiro[oxazole-4,9′-xanthen]-2-amine (38). To a
round-bottom flask were added 2-fluoro-2-methylpropyl trifluorome-
thanesulfonate (176 mg, 0.787 mmol), (S)-2-amino-4′-fluoro-7′-(2-
fluoropyridin-3-yl)-5H-spiro[oxazole-4,9′-xanthen]-2′-ol (46) (250
mg, 0.656 mmol), cesium carbonate (427 mg, 1.311 mmol), and
sodium iodide (44.2 mg, 0.295 mmol) in DMF (2 mL). The mixture was
purged with nitrogen and stirred at room temperature for 18 h. The
mixture was then diluted with water and extracted with EtOAc. The
organic layer was washed with brine, filtered through Celite, and
concentrated. The crude material was purified by silica gel
chromatography (0−25−50% DCM/MeOH/NH4OH (90:10:1) in
40% EtOAc in hexanes) to provide (S)-4′-fluoro-2′-(2-fluoro-2-
methylpropoxy)-7′-(2-fluoropyridin-3-yl)-5H-spiro[oxazole-4,9′-
xanthen]-2-amine (38) (179 mg, 0.393 mmol, 60% yield) as a white
1
1
11% yield) as a white solid. H NMR (400 MHz, DMSO-d6) δ ppm
solid. H NMR (400 MHz, DMSO-d6) δ ppm 8.23−8.26 (m, 1 H),
8.20−8.27 (m, 1 H), 8.05−8.13 (m, 1 H), 7.52−7.60 (m, 2 H), 7.48
(ddd, J = 7.3, 5.0, 1.9 Hz, 1 H), 7.32 (d, J = 8.5 Hz, 1 H), 6.99 (dd, J =
13.9, 2.8 Hz, 1 H), 6.64 (d, J = 1.7 Hz, 1 H), 6.48 (s, 2 H), 4.18 (s, 2 H),
3.74 (t, J = 4.6 Hz, 4 H), 3.01−3.12 (m, 4 H). MS m/z = 451.0 [M + H]+.
(S)-4′-Fluoro-7′-(2-fluoropyridin-3-yl)-2′-((S)-3-fluoropyrrolidin-1-
yl)-5H-spiro[oxazole-4,9′-xanthen]-2-amine (42). Prepared as de-
scribed for 41 using (S)-2-amino-5′-fluoro-2′-(2-fluoropyridin-3-yl)-
5H-spiro[oxazole-4,9′-xanthene]-7′-yl trifluoromethanesulfonate (47)
(300 mg, 0.584 mmol), (S)-3-fluoropyrrolidine hydrochloride (367 mg,
2.92 mmol), Pd2(dba)3 (27 mg, 0.029 mmol), biphenyl-2-yldi-tert-
butylphosphine (21 mg, 0.070 mmol), and LHMDS (1 M in THF)
8.07−8.13 (m, 1 H), 7.54−7.65 (m, 4 H), 7.49 (ddd, J = 7.3, 5.0, 1.9 Hz,
1 H), 7.34 (d, J = 8.4 Hz, 1 H), 7.08 (dd, J = 12.5, 2.9 Hz, 1 H), 6.70 (dd, J
= 2.8, 1.5 Hz, 1 H), 6.53 (s, 2 H), 4.16−4.24 (m, 2 H), 3.93−4.10 (m, 2
H), 1.39−1.47 (m, 6 H). MS m/z = 456.0 [M + H]+.
(S)-4′-Fluoro-7′-(2-fluoropyridin-3-yl)-2′-((3-methyloxetan-3-yl)-
methoxy)-5H-spiro[oxazole-4,9′-xanthen]-2-amine (37). Prepared
from (S)-2-amino-4′-fluoro-7′-(2-fluoropyridin-3-yl)-5H-spiro-
[oxazole-4,9′-xanthen]-2′-ol (46) (58 mg, 0.152 mmol)) as described
for 38 using 3-(bromomethyl)-3-methyloxetane (16 μL, 0.167 mmol)
and cesium carbonate (99 mg, 0.304 mmol) in 1 mL of DMF. Yield: 25
mg (35%). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.25 (d, J = 4.7 Hz, 1
H), 8.06−8.15 (m, 1 H), 7.55−7.63 (m, 2 H), 7.49 (t, J = 5.4 Hz, 1 H),
7.35 (d, J = 8.4 Hz, 1 H), 7.09 (dd, J = 12.6, 2.6 Hz, 1 H), 6.72 (br s, 1 H),
6.53 (br s, 1 H), 4.50 (d, J = 5.8 Hz, 2 H), 4.31 (d, J = 5.9 Hz, 2 H), 4.22
(d, J = 5.7 Hz, 2 H), 4.06 (q, J = 9.2 Hz, 2 H), 1.37 (s, 3 H). MS m/z =
466.0 [M + H]+.
General Procedure for Suzuki Coupling of Intermediate 63: (S)-2′-
(3,6-Dihydro-2H-pyran-4-yl)-4′-fluoro-7′-(2-fluoropyridin-3-yl)-5H-
spiro[oxazole-4,9′-xanthen]-2-amine (40). A 50 mL round-bottom
flask was charged with (S)-2-amino-5′-fluoro-2′-(2-fluoropyridin-3-yl)-
5H-spiro[oxazole-4,9′-xanthene]-7′-yl trifluoromethanesulfonate (47)
(1.966 g, 3.83 mmol), 2-(3,6-dihydro-2H-pyran-4-yl)-4,4,5,5-tetrameth-
yl-1,3,2-dioxaborolane (1.126 g, 5.36 mmol), tetrakis-
(triphenylphosphine)palladium(0) (0.443 g, 0.383 mmol), sodium
carbonate (1.624 g, 15.32 mmol), DMF (14 mL), and water (5 mL).
The mixture was heated at 85 °C overnight under positive pressure of
argon. The reaction mixture was cooled to rt and filtered, and the
precipitate was washed with water. The filtrate was diluted with water
(∼20 mL), and precipitated material was filtered and washed with water.
The solids were combined, redissolved in DCM, dried, and
concentrated. The crude material was purified by silica gel
chromatography (10−60% DCM/MeOH/NH4OH 90:10:1 in DCM)
to afford (S)-2′-(3,6-dihydro-2H-pyran-4-yl)-4′-fluoro-7′-(2-fluoropyr-
idin-3-yl)-5H-spiro[oxazole-4,9′-xanthen]-2-amine (40) (1.05 g, 2.347
mmol, 61% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm 8.25 (dt, J =
4.8, 1.5 Hz, 1 H), 8.12 (ddd, J = 10.3, 7.5, 2.0 Hz, 1 H), 7.57−7.65 (m, 2
H), 7.50 (ddd, J = 7.3, 5.0, 2.0 Hz, 1 H), 7.45 (dd, J = 12.4, 2.2 Hz, 1 H),
7.38 (d, J = 8.5 Hz, 1 H), 7.21 (s, 1 H), 6.52 (s, 2 H), 6.22−6.32 (m, 1 H),
4.25 (s, 4 H), 3.77−3.87 (m, 2 H), 2.36−2.47 (m, 2 H). MS m/z = 448.0
[M + H]+.
1
(4.97 mL, 4.97 mmol). Yield: 82 mg (31%). H NMR (400 MHz,
DMSO-d6) δ ppm 8.20−8.27 (m, 1 H), 8.10 (t, J = 8.2 Hz, 1 H), 7.58 (br
s, 2 H), 7.48 (ddd, J = 7.3, 5.0, 1.9 Hz, 1 H), 7.27−7.36 (m, 1 H), 6.37−
6.70 (m, 3 H), 6.29 (br s, 1 H), 5 (dm, J = 54.4 Hz 1 H), 4.21 (m, 2 H),
3.31−3.60 (m, 4 H), 2.08−2.30 (m, 2 H). MS m/z = 453.2 [M + H]+.
(S)-4′-Fluoro-7′-(2-fluoropyridin-3-yl)-2′-(1-methyl-1H-pyrazol-4-
yl)-5H-spiro[oxazole-4,9′-xanthen]-2-amine (43). Prepared as de-
scribed for 40 from (S)-2-amino-5′-fluoro-2′-(2-fluoropyridin-3-yl)-
5H-spiro[oxazole-4,9′-xanthene]-7′-yl trifluoromethanesulfonate (47)
(100 mg, 0.195 mmol), tetrakis(triphenylphosphine)palladium(0) (23
mg, 0.019 mmol), 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)-1H-pyrazole (81 mg, 0.380 mmol), and 5 M sodium carbonate
solution (0.2 mL, 0.974 mmol) in DMF (2 mL); isolated by preparative
HPLC (gradient elution 20−90% MeCN/H2O, 0.1% TFA) as TFA salt.
Yield 65 mg (61%). 1H NMR (400 MHz, DMSO-d6) δ ppm 11.12 (br s,
1 H), 9.32−9.69 (m, 2 H), 8.15−8.33 (m, 3 H), 7.98 (s, 1 H), 7.61−7.85
(m, 3 H), 7.44−7.57 (m, 2 H), 5.17 (m, 2 H), 3.88 (s, 3 H). MS m/z =
446.0 [M + H]+.
(S)-4′-Fluoro-7′-(2-fluoropyridin-3-yl)-2′-(pyridin-2-yl)-5H-spiro-
[oxazole-4,9′-xanthen]-2-amine (44). A resealable vial was charged
with (S)-2-amino-5′-fluoro-2′-(2-fluoropyridin-3-yl)-5H-spiro[oxazole-
4,9′-xanthene]-7′-yl trifluoromethanesulfonate (47) (100 mg, 0.195
mmol), tetrakis(triphenylphosphine)palladium(0) (23 mg, 0.019
mmol), and pyridin-2-ylzinc(II) bromide (0.5 M in THF) (1.95 mL,
0.974 mmol). The vial was sealed and heated at 70 °C for 5 h. The
reaction was quenched by addition of 1 mL of saturated aq NH4Cl and
extracted with ethyl acetate. The organic layer was concentrated, and the
residue was purified by silica gel chromatography (10−80% DCM/
MeOH/NH4OH 90:10:1 in DCM). Yield: 62 mg (72%). 1H NMR (400
MHz, DMSO-d6) δ ppm 8.67−8.71 (m, 1 H), 8.25 (dt, J = 4.8, 1.5 Hz, 1
H), 8.13 (ddd, J = 10.3, 7.5, 1.9 Hz, 1 H), 8.00−8.05 (m, 2 H), 7.95−7.99
(S)-2′-(5,6-Dihydro-2H-pyran-3-yl)-4′-fluoro-7′-(2-fluoropyridin-
3-yl)-5H-spiro[oxazole-4,9′-xanthen]-2-amine (39). Prepared from
K
dx.doi.org/10.1021/jm501266w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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(m, 1 H), 7.88−7.94 (m, 1 H), 7.60−7.66 (m, 2 H), 7.50 (ddd, J = 7.3,
5.0, 2.0 Hz, 1 H), 7.35−7.43 (m, 2 H), 6.55 (br s, 2 H), 4.30 (s, 2 H). MS
m/z = 443.0 [M + H]+.
BACE1 Enzymatic Assay. BACE1 enzymatic activity was
determined by the enhancement of fluorescence intensity upon
enzymatic cleavage of the fluorescence resonance energy transfer
substrate according to the previously published procedure.8d
Cell-Based Assay. BACE 1 cellular activity was determined in
Isolated Rabbit Heart Using Langendorff Perfusion. All rabbit
experiments were conducted in compliance with the Amgen Institu-
tional Animal Care and Use Committee and USDA regulations.
Isolated rabbit heart experiments were performed using procedure
described by Qu et al.42 using 0.3, 1, 3, and 10 μM solutions of
compound 2 or 0.1, 0.3, 1, 3, and 10 μM solutions of compound 40 were
tested under constant flow condition, with each concentration applied
for 20 min.
Cocrystallization of BACE1 Complexes. The extracellular
domain of BACE1 was expressed, purified, and crystallized according
to published procedures.43 Inhibitor complexes were obtained by
soaking apo BACE1 crystals in mother liquor solutions supplemented
with 0.5 mM compound 40 or 1 mM compound 2 for a period of ∼3 h.
Crystals were transferred briefly into a cryo solution consisting of 25%
(w/v) PEG 5000 MME, 0.1 M sodium citrate (pH 6.6), 0.2 M
ammonium iodide, and 20% (v/v) glycerol prior to being flash frozen in
liquid nitrogen. Diffraction data were collected either on an FR-E
rotating anode X-ray source equipped with an RAXIS IV++ detector
(compound 2) or at beamline 5.0.1 of the Advanced Light Source using
an ADSC Q315R CCD detector and λ = 0.97741 Å (compound 40).
Images were processed using the HKL suite of programs.44 The
structures were refined using REFMAC,45 and model building was
performed with COOT.46 Data collection and refinement statistics
appear in the Supporting Information.
human embryonic kidney cells (HEK293) stably expressing APPSW
.
After incubation overnight with the test compounds, the conditioned
media was collected and the Aβ40 levels were determined using a
sandwich ELISA as described previously.8d
Permeability assay was performed in the wild-type cell line LLC-PK1
(porcine renal epithelial cells, WT-LLC-PK1) transfected with human
MDR1 gene (hMDR1-LLC-PK1) and rat mdr1a gene (rMdr1a-LLC-
PK1) as described previously.8d
Microsomal Stability Assay. Compounds (1 μM) were incubated
with liver microsomes from human and rat for 30 min at 37 °C, and the
samples were analyzed according to published procedure.13
hERG Binding Assay. A stable HEK293 cell line expressing the
hERG channel was established in-house. Compounds were tested in the
[3H]-dofetilide binding assay with cell membranes prepared from this
cell line using method of Finlayson with some modifications.41 Briefly,
filtration assays were carried out in 194 μL of binding buffer (10 mM
HEPES, pH 7.4, 60 mM KCl, 71.5 mM NaCl, 1 mM CaCl2, 2 mM
MgCl2) with 10 μg/well membrane (based on membrane protein) and
[3H]-dofetilide (8 nM) and 6 μL of compound dissolved in 100%
DMSO. Nonspecific binding was determined by using 10 μM cold
dofetilide (∼1000-fold molar excess over hot ligand). The entire assay
was conducted in 96-well Whatman Unifilter plates at room temperature
for 90 min. The binding assay was terminated by washing the plates four
times on a Millipore Vacuum filtration manifold with 100 μL/well of ice
cold wash buffer (10 mM HEPES, pH 7.4, 131.5 mM NaCl, 1 mM
CaCl2, 2 mM MgCl2). The bound radioisotope was quantified using a
Packard TopCount NTS liquid scintillation counter with scintillation
fluid.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures and analytical data for test compounds and
synthetic procedures for intermediates, 1H and 13C NMR spectra
of compound 40, BACE1 enzyme data and calculated LogP and
PSA for compounds 37−44, determination of IC50, and data
collection and refinement statistics for cocrystals of 2 and 40 with
BACE1. This material is available free of charge via the Internet at
http://pubs.acs.org.
Accession Codes
New protein/ligand coordinates for 2 and 40 with BACE1 have
been deposited in the PDB with IDs 4RCE and 4RCF.
Rat Pharmacodynamic Assay. Male Sprague−Dawley rats (175−
200 g) were administered compound by oral gavage at the appropriate
dose. Samples of plasma, CSF, and brain were analyzed according to
reported procedures.13
AUTHOR INFORMATION
Corresponding Author
*Phone: 1-617-444-5353. E-mail: oepstein@amgen.com. Ad-
dress: Department of Medicinal Chemistry, Amgen Inc., 360
Binney Street, Cambridge, Massachusetts 02142, United States.
Dog Cardiovascular Safety Studies. Animal Preparation. Male
beagle dogs (10−12 kg) were anesthetized with 2 mg/kg morphine,
dosed subcutaneously, followed by a 110 mg/kg intravenous bolus of α-
chloralose. After induction, animals were intubated and ventilated at an
appropriate rate and volume with ambient air. Maintenance on surgical
plane was achieved with a constant infusion of 45 mg/kg/h of α-
chloralose. Animals were kept on a thermal blanket and heat lamp to
maintain normal body temperature and monitored throughout the study
by veterinary staff. The study was conducted with approval of the
Institutional Animal Care and Use Committee.
Cardiovascular Monitoring. Arterial pressure and heart rate were
measured directly from a femoral artery catheter and catheters were
placed in both femoral veins for infusion of drug and blood sampling,
using standard surgical techniques. ECG signals (l II and V) were
captured simultaneously from subcutaneous limb and precordial needle
electrodes, respectively. After surgery, the animals were allowed to
stabilize for a minimum of 45 min prior to collecting pretreatment
(baseline) data. After baseline values were collected, the test article was
administered in a rising-dose paradigm in which vehicle and escalating
doses were infused intravenously. Each dose (vehicle or test article) was
given over 30 min. Animals were euthanized at the conclusion of the
study.
■
Present Addresses
#For M. C. B.: Genentech, Inc., 1 DNA Way, South San
Francisco, California 94080, United States.
∇For V. F. P.: Sanofi-Aventis, 153 Second Avenue, Waltham,
Massachusetts 02451, United States.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Matthew Potter, Grace Bi, and Larry Miller for chiral
separation, Jie Yan and Michele Kubryk for help with resynthesis
of the lead compounds for safety studies, and Alexander M. Long
for purification of BACE1 protein. We also thank Kui Chen,
Steven Louie, Joel Esmay, and Yi Luo for in vitro screening of the
test compounds and Yusheng Qu, Mei Fang, BaoXi Gao, and
Shanti Amagasu for ex vivo safety studies.
Data Acquisition and Analysis. A computerized data acquisition
system (CARecorder; DISS, Inc.) was used to collect all raw input
signals. Arterial pressure, heart rate, and cardiac intervals were recorded
continuously throughout the study and averaged at 30 s intervals. All raw
data were converted with EMKA ecg-auto (version 2.5.1.30; Paris,
France) into *.d01 files for analysis purposes. In the tables, values were
compared to end of vehicle infusion. Data were analyzed using ecg-auto
v2.5.1.30 (EMKA Technologies).
ABBREVIATIONS USED
■
hERG, human ether-a-go-go-related gene; BACE1, β-site APP
cleaving enzyme; AD, Alzheimer’s disease; Pgp, P-glycoprotein;
Aβ, β-amyloid; APP, amyloid-β precursor protein; CNS, central
L
dx.doi.org/10.1021/jm501266w | J. Med. Chem. XXXX, XXX, XXX−XXX

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A.; Horne, D. B.; Huang, H.; Imbeah-Ampiah, R.; Judd, T.; Kaller, M. R.;
Kreiman, C. R.; La, D. S.; Li, V.; Lopez, P.; Louie, S.; Monenschein, H.;
Nguyen, T. T.; Pennington, L. D.; Rattan, C.; San Miguel, T.; Sickmier,
E. A.; Wahl, R. C.; Wen, P. H.; Wood, S.; Xue, Q.; Yang, B. H.; Patel, V.
F.; Zhong, W. Design and Preparation of a Potent Series of
Hydroxyethylamine Containing β-Secretase Inhibitors That Demon-
strate Robust Reduction of Central β-Amyloid. J. Med. Chem. 2012, 55,
9009−9024. (d) Dineen, T. A.; Weiss, M. M.; Williamson, T.; Acton, P.;
Babu-Khan, S.; Bartberger, M. D.; Brown, J.; Chen, K.; Cheng, Y.;
Citron, M.; Croghan, M. D.; Dunn, R. T.; Esmay, J.; Graceffa, R. F.;
Harried, S. S.; Hickman, D.; Hitchcock, S. A.; Horne, D. B.; Huang, H.;
Imbeah-Ampiah, R.; Judd, T.; Kaller, M. R.; Kreiman, C. R.; La, D. S.; Li,
V.; Lopez, P.; Louie, S.; Monenschein, H.; Nguyen, T. T.; Pennington,
L. D.; San Miguel, T.; Sickmier, E. A.; Vargas, H. M.; Wahl, R. C.; Wen,
P. H.; Whittington, D. A.; Wood, S.; Xue, Q.; Yang, B. H.; Patel, V. F.;
Zhong, W. Design and Synthesis of Potent, Orally Efficacious
Hydroxyethylamine Derived β-Site Amyloid Precursor Protein Cleaving
Enzyme (BACE1) Inhibitors. J. Med. Chem. 2012, 55, 9025−9044.
nervous system; PD, pharmacodynamic; IRH, isolated rabbit
heart; QTc, corrected QT interval; Ki, absolute inhibition
constant; DCM, dichloromethane; TLC, thin layer chromatog-
raphy
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