Discovery of an orally available, brain penetrant BACE1 inhibitor that affords robust CNS Aβ reduction

Article abstract of DOI:10.1021/ml3001165

Inhibition of BACE1 to prevent brain Aβ peptide formation is a potential disease-modifying approach to the treatment of Alzheimer's disease. Despite over a decade of drug discovery efforts, the identification of brain-penetrant BACE1 inhibitors that substantially lower CNS Aβ levels following systemic administration remains challenging. In this report we describe structure-based optimization of a series of brain-penetrant BACE1 inhibitors derived from an iminopyrimidinone scaffold. Application of structure-based design in tandem with control of physicochemical properties culminated in the discovery of compound 16, which potently reduced cortex and CSF Aβ40 levels when administered orally to rats.

Full text of DOI:10.1021/ml3001165

Letter
pubs.acs.org/acsmedchemlett
Discovery of an Orally Available, Brain Penetrant BACE1 Inhibitor
That Affords Robust CNS Aβ Reduction
Andrew W. Stamford,*,†,△ Jack D. Scott,† Sarah W. Li,† Suresh Babu,‡ Dawit Tadesse,‡ Rachael Hunter,‡
Yusheng Wu,† Jeffrey Misiaszek,† Jared N. Cumming,† Eric J. Gilbert,† Chunli Huang,†
Brian A. McKittrick,† Liwu Hong,† Tao Guo,‡ Zhaoning Zhu,† Corey Strickland,# Peter Orth,#
Johannes H. Voigt,# Matthew E. Kennedy,§ Xia Chen,§ Reshma Kuvelkar,§ Robert Hodgson,§
Lynn A. Hyde,§ Kathleen Cox, Leonard Favreau, Eric M. Parker,§ and William J. Greenlee†
§
#
†Departments of Medicinal Chemistry, Neurobiology, Drug Metabolism, and Global Structural Chemistry, Merck Research
Laboratories, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, United States
‡Ligand Pharmaceuticals, Inc., 3000 Eastpark Boulevard Cranbury, New Jersey 08512, United States
S
* Supporting Information
ABSTRACT: Inhibition of BACE1 to prevent brain Aβ peptide
formation is a potential disease-modifying approach to the treatment
of Alzheimer’s disease. Despite over a decade of drug discovery
efforts, the identification of brain-penetrant BACE1 inhibitors that
substantially lower CNS Aβ levels following systemic administration
remains challenging. In this report we describe structure-based
optimization of a series of brain-penetrant BACE1 inhibitors derived
from an iminopyrimidinone scaffold. Application of structure-based design in tandem with control of physicochemical properties
culminated in the discovery of compound 16, which potently reduced cortex and CSF Aβ40 levels when administered orally to
rats.
KEYWORDS: BACE1, inhibitor, Alzheimer’s disease, Aβ40, iminopyrimidinone, X-ray crystallography
lzheimer’s disease (AD) is a chronic neurodegenerative
Adisease and is now the sixth leading cause of death in the
Unites States.1 Current treatments of AD are palliative and only
transiently effective, and they do not impact disease
progression.2 Due to the progressive and debilitating nature
of the disease and the lack of disease-modifying therapeutic
agents, novel treatments that slow or halt disease progression
are urgently sought.
Neuropathological hallmarks of AD are the accumulation of
β-amyloid as extracellular neuritic plaques and the appearance
of intracellular neurofibrillary tangles comprised of hyper-
phosphorylated tau protein.3 Although the cause of AD remains
poorly understood, several lines of evidence suggest that
accumulation of β-amyloid is an underlying cause of neuronal
cell death.3−5 The amyloid peptides are produced from amyloid
precursor protein (APP) in two proteolytic steps, the first of
which is mediated by the aspartyl protease β-site APP cleaving
enzyme-1 (BACE1)6 to produce a C-terminal fragment that is
subsequently processed by γ-secretase to yield Aβ peptides of
36−43 amino acids. While Aβ40 is the most abundant species
formed, Aβ42 is more prone to aggregation and formation of
oligomeric neurotoxic species.7−9 BACE1 inhibition to prevent
Aβ peptide formation is considered to be a highly attractive
amyloid-lowering approach to a potentially disease-modifying
AD treatment. In this regard, BACE1 knockout mice do not
produce β-amyloid, demonstrating that BACE1 is necessary for
Aβ production in vivo.10−12 Furthermore, BACE1 knockout
mice do not exhibit overt abnormalities, displaying a moderate
peripheral nerve hypomyelination caused by loss of BACE1-
mediated processing of Neuregulin-1.13,14
For more than a decade, there have been intense efforts to
identify potent, selective BACE1 inhibitors that effectively
inhibit Aβ peptide production in the CNS of preclinical animal
models. Although substantial work has been carried out on
peptidomimetic inhibitors, this has met with limited success, as
such inhibitors tend to be Pgp substrates and suffer restricted
brain penetration.15 Nonpeptidic BACE1 inhibitors have been
reported by us16−18 and others,19−23 and they offer the most
promising avenue to the discovery of orally bioavailable, brain
penetrant inhibitors. A recent report of a nonpeptidic BACE1
inhibitor that produced substantial reductions in central Aβ
levels in preclinical species and in healthy volunteers confirms
the validity of this approach.24
We had previously reported the discovery of the potent
iminohydantoin BACE1 inhibitor 1 that was rationally designed
from fragment-based screening hits.16,17 In the course of lead
optimization, the identification of 1 was an important milestone
en route to the discovery of the highly ligand efficient
Special Issue: Alzheimer’s Disease
Received: May 30, 2012
Accepted: July 12, 2012
© XXXX American Chemical Society
A
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iminohydantoin BACE1 inhibitor 2 (Chart 1).18 Upon oral
administration to rats at the dose of 30 mg/kg, 2 acutely
order to maximize the probability of identifying inhibitors with
high CNS exposure, our optimization strategy was to adhere to
the design principles of maintaining or improving LE while
limiting MW, restricting cLogP in a desirable range (≤4), and
avoiding additional H-bond donors to minimize Pgp activity
and polar surface area.25 In accordance with this approach, we
pursued focused modifications of the iminoheterocyclic core
and of the biaryl motif that occupies the S1 and S3 pockets of
the enzyme.
Chart 1. Iminoheterocycle BACE1 Inhibitor Design
Ring expansion of the core to a six-membered iminopyr-
imidinone 3 was expected to have a significant impact on
physicochemical properties and conformation. In comparison
to the iminohydantoin core, the iminopyrimidinone is
predicted to be more basic.26 We reasoned that enhanced
basicity could increase cell potency by favoring partitioning to
the acidic intracellular compartment where BACE1-mediated
APP processing is operative. A second major design
consideration centered on conformational differences between
the five- and six-membered ring iminoheterocycle scaffolds.
The iminopyrimidinone ring differs from the planar iminohy-
dantoin core in that the additional sp3 center imparts a
significant pucker to the ring. Consequently, the docked pose of
the iminopyrimidinone 4 (Chart 1) overlaid with iminohy-
dantoin 1 places the S1 phenyl substituent and the amidine in a
similar location; however, the 6-methyl substituent has a
trajectory that diverges from that of its iminohydantoin
counterpart, projecting toward Ile118 (Figure 1).27 Based on
this analysis, it was hypothesized that substituents at C6 smaller
than phenyl projecting toward the prime-site might be
necessary in order to avoid unfavorable steric interactions
with the enzyme.
To test these hypotheses, targeted sets of iminopyrimidi-
nones 4−6 and 7−9 were prepared that differed in their
substitution at C6 of the iminopyrimidinone ring (Table 1). As
predicted by docking experiments, the 6-methyl iminopyr-
imidinones 4−6 had BACE1 affinities that were substantially
higher than those of their 6-phenyl counterparts 7−9 and were
comparable to that of the iminohydantoin 1. Also noteworthy
was the observation that the cell Aβ40 IC50 values of the
iminopyrimidinones 4−6 were minimally shifted (2−3-fold)
relative to their Ki values, in contrast to the cases of the
corresponding 5,5-diphenyl iminohydantoins represented by
1.18 Comparison of iminopyrimidinones 4−6 in a rat oral
pharmacokinetic screen demonstrated that cyanophenyl de-
rivative 5 had superior rat oral pharmacokinetic properties.
Compound 5 adminstered at a dose of 10 mg/kg afforded a
plasma AUC0−6 h of 8.6 μM·h compared to corresponding
values of <1 μM·h for 4 and 6 administered at the same dose.28
An X-ray cocrystal structure of the 6-methyl iminopyrimidi-
none derivative 4 and BACE1 was in close agreement with the
predicted binding mode (Figure 2).29 Notably, the bound
conformation of the iminopyrimidinone ring projects the C6
methyl group to within 3.6 Å of Ile118, consistent with a
favorable hydrophobic interaction. As expected, the biaryl motif
of 4 binds in the contiguous S1−S3 pockets, with the methoxyl
group projecting toward the S3 subpocket. Well-defined density
for the flap was observed, with the side-chain of Tyr71
positioned <4 Å away from the methylene of the iminopyr-
imidinone core.
lowered CSF Aβ40 by approximately 50% 3 h postdose relative
to vehicle-treated controls. While the ability of 2 to elicit CSF
Aβ reduction represented a significant advance in our efforts to
develop centrally active BACE1 inhibitors, 2 had a significant
IC50 shift in a cell-based Aβ40 assay relative to its BACE1 Ki
and limited brain penetration. Consequently, in order to
develop inhibitors with improved in vivo potency, concurrent
optimization efforts focused on the identification of inhibitors
with improved enzymatic/cellular potency shifts and brain
exposures. Toward this goal, we report herein the design and
optimization of a series of BACE1 inhibitors based on the
homologous iminopyrimidinone scaffold 3 that also evolved
from the lead iminohydantoin 1 (Chart 1).
An X-ray cocrystal structure of 1 bound to BACE1 revealed
that the guanidine functionality of 1 forms intricate H-bond
donor−acceptor interactions with Asp32 and Asp228 at the
BACE1 active-site, the methoxybiphenyl moiety occupies the
contiguous S1 and S3 pockets, and the remaining phenyl
substituent occupies space in the vicinity of S2′ (Figure 1). In
Given the encouraging results obtained with the 6-methyl
iminopyrimidinones 4−6, we next explored a wide range of
biaryl and heterobiaryl motifs that could bind in S1−S3. The
most productive of these was replacement of the proximal
Figure 1. X-ray cocrystal structure of BACE1 and 1 (blue)
superimposed with docked 4 (yellow).
B
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Table 1. BACE1 Affinity, Cell Activity, and Selectivity of Iminopyrimidinone Derivatives 3
a
b
BACE1 Ki and cell IC50 values are the average of a minimum of two independent determinations. Selectivity ratios were derived from cathepsin D
c
Ki values (average of a minimum of two independent determinations). Not determined.
derivative 5 (Table 1). This improvement in affinity also
translated to improved cell potency, with the 2,4 regioisomer
10 (Ki 57 nM; cell IC50 68 nM) exhibiting Ki and cell IC50
values of <100 nM.
Replacement of the 3-cyanophenyl moiety by the 3-prop-1-
ynyl-5-pyridyl group as in 2, analogs 13−15, resulted in further
improved BACE1 affinity and cell activity, with 13 (Ki 7.8 nM;
cell IC50 13 nM) achieving single-digit nanomolar affinity and a
cell potency shift of only 2-fold. A further gain of affinity could
be realized by incorporation of chloro substitution at the
thiophene 4-position of 14 to afford 16, which had a Ki value of
1.7 nM. Similar to the case of 16, chloro substitution at the
thiophene 5-position of 15 gave 17 with enhanced affinity. The
increased lipophilicity conferred by chloro substitution resulted
in only modestly increased cell potency shifts, with 16
maintaining good cell activity (IC50 11 nM).
The selectivities of the iminopyrimidinone inhibitors for
BACE1 versus the closely related aspartyl protease cathepsin D
ranged from 61- to 130-fold for the biphenyl derivatives 4−6
and the 2,4-thiophenyl analogs 10 and 13, and they were
generally somewhat decreased for the 2,5- and 3,5-thiophenyl
analogs (e.g., 11, 14−17). In addition to selectivity over
cathepsin D (21-fold; Table 1), 16 was profiled against other
Figure 2. Comparison of 4 (yellow) docked to BACE1 and the X-ray
cocrystal structure of 4 (green).
phenyl ring with the bioisosteric thiophenyl group. In exploring
this modification, distal (3-cyano)phenyl substitution was
chosen because of the superior pharmacokinetic properties
conferred by this substituent. Of the three possible thiophenyl
regioisomers 10−12 that maintain the iminopyrimidinone core
and 3-cyanophenyl substituent in a 1,3-relationship, we were
gratified to find that the affinities and LEs of the 2,4-thiophenyl
and 2,5-thiophenyl regioisomers 10 and 11 were significantly
improved compared to those of the corresponding biphenyl
C
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human aspartyl proteases. Compound 16 is not selective for
BACE1 over the close homologue BACE2 (Ki < 1 nM) but
exhibits good selectivity over cathepsin E (350×) and pepsin
(260×) and excellent selectivity over renin (>10,000×).
An X-ray cocrystal structure showed that 16 bound to
BACE1 in a mode highly consistent with 4, but it was notable
in two respects that may contribute to its higher affinity (Figure
3). First, the propynyl group of 16 binds deep in the S3
□
Figure 4. Dose response of 16 on CSF ( ; collected from the cisterna
●
magna) and cortex ( ; Aβ40 3 h postdose in Sprague−Dawley rats
(PO, formulated in 20% hydroxypropyl-β-cyclodextrin, 5 mL/kg).
Doses ≥3 mg/kg were significantly different from vehicle, p < 0.02,
Dunnett’s posthoc test. Dose dependent increase in plasma
▲
concentration ( ) of 16; brain/plasma ratios ranged from 2.4 to 4.2.
a
Scheme 1. Synthesis of Compound 16
Figure 3. X-ray cocrystal structure of 16 (blue) and BACE1.
subpocket where it is positioned to engage in a hydrophobic
interaction with Ala335, as previously reported for 2.18 Second,
in comparison to the cocrystal structure of 4, the flap is in a
more closed conformation, forming a pocket which accom-
modates the chloro substituent while positioning the side-chain
of flap Tyr71 in close proximity to the methylene of the
iminopyrimidinone core.
In Caco-2 cells, 16 exhibited good permeability (Papp 153
nm·s−1) and a low efflux ratio (AP/BL 2.4), suggesting that the
compound does not possess an overt human Pgp liability. In rat
and human hepatocytes, 16 had low intrinsic clearance (4.9 and
2.7 μL·min−1·106 cells, respectively), reflecting low metabolic
turnover, and displayed significant free fractions of 0.02 and
0.04, respectively, in rat and human plasma protein binding
assays. These in vitro parameters translated to favorable rat
pharmacokinetic properties, with 16 displaying high oral
bioavailability (69%) and plasma exposure, a plasma half-life
of 2.8 h, and an average brain/plasma ratio of 3.
Based on its excellent in vitro and rat pharmocokinetic
profiles, 16 was subjected to pharmacodynamic profiling in rats.
In a dose titration study, 16 elicited a robust reduction in CSF
and cortex Aβ40 levels with ED50 values of 4 and 6 mg/kg in
the respective compartments 3 h after oral administration
(Figure 4). As shown in Figure 4, plasma and brain exposures
increased in a relatively dose-proportional manner across the
dose range of 0.3 to 30 mg/kg.
Optimization of 1 to inhibitor 16 achieved a significant gain
in BACE1 affinity without concomitant increase in number of
H-bond donors, cLogP, polar surface area, or MW, as reflected
by the large increase in LE from 0.35 kcal·mol−1 to 0.48
kcal·mol−1. In addition to good solubility (200 μM, pH 7.4),
the measured physical properties of 16, including a pKa value of
7.5 and a log D value of 1.8, fall within CNS druglike ranges.30
The synthesis of 16 (Scheme 1) employed an Ellman
asymmetric aldol condensation to assemble the β-amino acid
subunit and set the stereochemistry of the quaternary center.31
Thus, addition of the enolate of methyl acetate to (R)-tert-
butylsulfinamide 19, derived from 1-(3-chlorothiophen-2-yl)-
ethanone 18, in the presence of chlorotitanium triisopropoxide
afforded the (S)-β-aminoester derivative 20 in a >99:1
a
Reagents and conditions: (i) (R)-2-methylpropane-2-sulfinamide,
Ti(OEt)4, THF, reflux; (ii) LDA, methyl acetate, ClTi(OiPr)3, THF,
−78 °C; (iii) HCl, 1,4-dioxane; (iv) EDCI, N-[(methylamino)-
thioxomethyl]-tert-butylcarbamate, iPr2NEt, THF; (v) NBS, DMF, 50
°C; (vi) (5-(prop-1-ynyl)pyridin-3-yl)boronic acid, 2 M Na2CO3,
PdCl2(dppf)·CH2Cl2, 1,4-dioxane, 65 °C; (vii) CF3COOH, CH2Cl2.
diastereomeric ratio. Acidic cleavage of the sulfinamide of 20
and coupling of the derived amine to tert-butyl N-
[(methylamino)-thioxomethyl]carbamate proceeded with con-
comitant intramolecular cyclization to give the iminopyrimidi-
none derivative 21. Bromination of the thiophene afforded 22,
which was subjected to sequential Suzuki coupling with (5-
(prop-1-ynyl)pyridin-3-yl)boronic acid and Boc deprotection
to give 16.
In conclusion, application of structure-based design with
simultaneous control of physicochemical parameters enabled
discovery of the iminopyrimidinone BACE1 inhibitor 16
starting from prototype iminohydantoin 1. The robust
reduction of CNS Aβ levels that was achieved following oral
administration of 16 to rats demonstrates the potential of the
iminoheterocycle class of BACE1 inhibitors to deliver agents
that could test the therapeutic utility of BACE1 inhibition in
clinical trials.
D
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ASSOCIATED CONTENT
■
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AUTHOR INFORMATION
■
Corresponding Author
*Tel: 732-594-1960. E-mail: andrew.stamford@merck.com.
Present Address
△Merck Research Laboratories, 126 E. Lincoln Ave., Rahway,
NJ 07065, United States.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Thomas Novak and Charles Ross for HRMS spectra,
and James Cuff for optical rotation measurements. This work is
based upon research conducted at the Cornell High Energy
Synchrotron Source (CHESS), which is supported by the
National Science Foundation and the National Institutes of
Health/National Institute of General Medical Sciences under
NSF award DMR-0936384, using the Macromolecular
Diffraction at CHESS (MacCHESS) facility, which is supported
by award GM103485 from the National Institutes of Health,
through its National Institute of General Medical Sciences.
(16) Wang, Y.-S.; Strickland, C.; Voigt, J. H.; Kennedy, M. E.; Beyer,
B. M.; Senior, M. M.; Smith, E. M.; Nechuta, T. L.; Madison, V. S.;
Czarniecki, M.; McKittrick, B. A.; Stamford, A. W.; Parker, E. M.;
Hunter, J. C.; Greenlee, W. J.; Wyss, D. F. Application of Fragment-
Based NMR Screening, X-ray Crystallography, Structure-Based
Design, and Focused Chemical Library Design to Identify Novel μM
Leads for the Development of nM BACE-1 (β-Site APP Cleaving
Enzyme 1) Inhibitors. J. Med. Chem. 2010, 53, 942−950.
ABBREVIATIONS
■
AD, Alzheimer’s disease; APP, amyloid precursor protein;
BACE1, β-site APP cleaving enzyme-1; CNS, central nervous
system; CSF, cerebrospinal fluid; Pgp, P-glycoprotein; LE,
ligand efficiency; CatD, cathepsin D
(17) Zhu, Z.; Sun, Z.-Y.; Ye, Y.; Voigt, J.; Strickland, C.; Smith, E. M.;
Cumming, J.; Wang, L.; Wong, J.; Wang, Y.-S.; Wyss, D. F.; Chen, X.;
Kuvelkar, R.; Kennedy, M. E.; Favreau, L.; Parker, E.; McKittrick, B.
A.; Stamford., A.; Czarniecki, M.; Greenlee, W.; Hunter, J. C.
Discovery of Cyclic Acylguanidines as Highly Potent and Selective β-
Site Amyloid Cleaving Enzyme (BACE) Inhibitors: Part I - Inhibitor
Design and Validation. J. Med. Chem. 2010, 53, 951−965.
(18) Cumming, J. N.; Smith, E. M.; Wang, L.; Misiaszek, J.; Durkin,
J.; Pan, J.; Iserloh, U.; Wu, Y.; Zhu, Z.; Strickland, C.; Voigt, J.; Chen,
X.; Kennedy, M. E.; Kuvelkar, R.; Hyde, L. A.; Cox, K.; Favreau, L.;
Czarniecki, M. F.; Greenlee, W. J.; McKittrick, B. A.; Parker, E. M.;
Stamford, A. W. Structure Based Design of Iminohydantoin BACE1
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̈
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