Acylguanidines as small-molecule β-secretase inhibitors

Article abstract of DOI:10.1021/jm0607451

BACE1 is an aspartyl protease responsible for cleaving amyloid precursor protein to liberate Aβ, which aggregates leading to plaque deposits implicated in Alzheimer's disease. We have identified small-molecule acylguanidine inhibitors of BACE1. Crystallographic studies show that these compounds form unique hydrogen-bonding interactions with the catalytic site aspartic acids and stabilize the protein in a flap-open conformation. Structure-based optimization led to the identification of potent analogs, such as 10d (BACE1 IC₅₀ = 110 nM).

Full text of DOI:10.1021/jm0607451

6158
J. Med. Chem. 2006, 49, 6158-6161
BACE2 (memapsin1, Asp1) also cleaves APP at the â-site but
cleaves more efficiently after Phe19 and Phe20 of Aâ and is
poorly expressed in the CNS.14
Acylguanidines as Small-Molecule â-Secretase
Inhibitors
The first crystal structure of BACE1 with the heptapeptide 1
(Ki ) 1.6 nM) (Figure 1) containing the Leu-Ala hydroxyeth-
ylene isostere as the transition state mimic15 was published by
Hong et al.16 Structure-based optimization of this peptide
mimetic led to the identification of potent small molecular
weight hydroxyethylene inhibitors such as 2 (IC50 ) 30 nM)17
as well as a series of constrained analogues, e.g., 3 (IC50 ) 10
nM).18 Others have replaced the hydroxyethylene isostere with
the statin scaffold, leading to inhibitors such as 4 (IC50 ) 91
nM).19
Like other aspartic proteases,20-23 BACE1 consists of two
structural domains, the N- and C-terminus, which constitute the
active site and a â-hairpin loop which forms the flap region.
The flap opens to allow the substrate to enter and then closes
down on the substrate during the catalysis step and reopens to
release the hydrolyzed products. The design of the transition
state based inhibitors described above is based on replacing the
cleavage site of the peptide substrate. Not unexpectedly, it has
been observed in crystallographic studies that these peptidomi-
metic inhibitors bind with BACE1 in a closed-flap form.
High-throughput screening (HTS) of the Wyeth Corporate
compound library using a FRET assay identified the acylguani-
dine inhibitor, 7a (Scheme 1). Acylguanidines have not previ-
ously been described as aspartic acid protease inhibitors, and
this discovery of a modestly potent, low molecular weight
inhibitor represents a new direction in the design of BACE1
inhibitors. Herein, we describe the characterization, cocrystal
structure, and optimization of this lead using structure-based
design.
Compound 7a (BACE1 IC50 ) 3.7 µM) was demonstrated
to bind to BACE1 by NMR experiments, had a Kd of 2.8 µM
by isothermal titration calorimetry, and inhibited Aâtotal forma-
tion in a cellular assay with an IC50 of 8.9 µM. Significantly,
in a radiolabeled immunoprecipitation cellular assay (RICA),
7a caused a dose-dependent reduction of âCTF and Aâ levels
without affecting secreted R-secretase amyloid precursor protein
(R-sAPP) levels, consistent with a â-secretase mediated inhibi-
tion mechanism.
An X-ray crystal structure of 7a complexed with the BACE1
catalytic domain was solved at 2.4 Å resolution. In this structure
the acylguanidine moiety forms four key hydrogen-bonding
interactions with the catalytic aspartic acids Asp32 and Asp228
(Figure 2). Important structural changes were observed in
BACE1 upon binding of 7a (Figure 3). In contrast to complexes
with peptidomimetic inhibitors in which the flap region closes
in over the bound inhibitor,16 in the 7a complex the flap adopts
an “open conformation” to make room for the diarylpyrrole
portion of the inhibitor, with a total movement of 5.5 Å at the
tip of the hairpin turn and a movement of 7.5 Å for the Tyr71
hydroxyl moiety. In fact, the diarylpyrrole moiety of 7a occupies
the space occupied by Tyr71 in the peptidomimetic structures.
π-Edge stacking between the pyrrole and the phenyl ring of
Tyr71 appears to contribute to the stabilization of the flap in
this configuration. Stabilization of the flap region in an open
position has previously been observed crystallographically for
the enzyme/inhibitor complexes of renin24 and pepsin,25 and the
implications of these conformations for rational drug design have
been reviewed by Bursavich and Rich.26
Derek C. Cole,*,† Eric S. Manas,‡,§ Joseph R. Stock,†
Jeffrey S. Condon,# Lee D. Jennings,† Ann Aulabaugh,†
Rajiv Chopra,#,| Rebecca Cowling,† John W. Ellingboe,†
Kristi Y. Fan, Boyd L. Harrison, Yun Hu,∞
Steve Jacobsen,∞ Guixan Jin,† Laura Lin,#
Frank E. Lovering,# Michael S. Malamas, Mark L. Stahl,#
James Strand,# Mohani N. Sukhdeo,† Kristine Svenson,#
M. James Turner,∞ Erik Wagner,∞ Junjun Wu,#
Ping Zhou, and Jonathan Bard∞
Chemical and Screening Sciences, Wyeth Research,
401 N. Middletown Road, Pearl RiVer, New York 10965,
Chemical and Screening Sciences, Wyeth Research,
200 Cambridge Park DriVe, Cambridge, Masssachusetts 02139,
Chemical and Screening Sciences, Wyeth Research,
Princeton, New Jersey 08852, DiscoVery Neuroscience,
Wyeth Research, Princeton, New Jersey 08852, and Chemical and
Screening Sciences, Wyeth Research, 500 Arcola Road,
CollegeVille, PennsylVania 19426
ReceiVed June 22, 2006
Abstract: BACE1 is an aspartyl protease responsible for cleaving
amyloid precursor protein to liberate Aâ, which aggregates leading to
plaque deposits implicated in Alzheimer’s disease. We have identified
small-molecule acylguanidine inhibitors of BACE1. Crystallographic
studies show that these compounds form unique hydrogen-bonding
interactions with the catalytic site aspartic acids and stabilize the protein
in a flap-open conformation. Structure-based optimization led to the
identification of potent analogs, such as 10d (BACE1 IC50 ) 110 nM).
Alzheimer’s (AD) is a progressive, degenerative disease of
the brain and the most common form of dementia. A variety of
therapeutic strategies for modulating the progression or preven-
tion of AD are currently being investigated. Although the exact
pathological mechanism remains unclear, overwhelming evi-
dence implicates amyloid â-peptide (Aâ, 39-43 residues) (most
likely in multimeric forms such as oligomers) in the neuronal
dysfunction and death that causes the cognitive decline seen in
AD.1 Aâ is produced by the sequential proteolytic cleavage of
amyloid precursor protein (APP) via the action of two proteases,
â- and γ-secretase. Specifically, â-secretase (also called BACE1,
memapsin 2, and Asp2) mediates the primary cleavage of APP,2
generating the membrane-bound C-terminal APP fragment
(âCTF/C99), which in turn is cleaved by γ-secretase liberating
the Aâ peptide.3,4 BACE1 is a membrane-bound aspartyl
protease highly expressed in the central nervous system
(CNS).5-9 BACE1 knockout mice have been shown to be
healthy with an absence of Aâ production,10-13 indicating that
BACE1 is the key enzyme responsible for Aâ production. Thus,
BACE1 is a therapeutic target for inhibitors of Aâ production
for the treatment and prevention of AD. The close homologue
* To whom correspondence should be addressed. Phone: 845-602-4619.
Fax: 845-602-5561. E-mail: coledc@wyeth.com.
† Chemical and Screening Sciences, Pearl River.
‡ Chemical and Screening Sciences, Collegeville.
§ Current address: TransForm Pharmaceuticals, Inc., 29 Hartwell Avenue,
Lexington, MA 02421.
# Chemical and Screening Sciences, Cambridge.
| Current address: Novartis Institute of Biomedical Research, 250
Massachusetts Avenue, Cambridge, MA 02139.
Chemical and Screening Sciences, Princeton.
∞ Discovery Neuroscience, Princeton.
10.1021/jm0607451 CCC: $33.50 © 2006 American Chemical Society
Published on Web 09/15/2006

Letters
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21 6159
7a-c. Direct alkylation of the acylguanidines 7 proved unsat-
isfactory, so the desired substituted acylguanidines 10 were
prepared by reaction of 6 with 1H-pyrazole-1-carboxamidine
using CDI activation followed by displacement of the pyrazole
leaving group with 3-amino-1-propanol.
Compounds were tested for inhibition of isolated BACE1 in
an in vitro peptide cleavage FRET assay. Compound 7b, in
which one of the phenyl groups on 7a is replaced with the more
spherical adamantyl group, inhibited BACE1 activity with an
IC50 of 0.6 µM, 6-fold more potently than the initial lead. A
cocrystal structure of 7b complexed with BACE1 (not shown)
confirmed that the adamantyl group occupies the S1 pocket as
intended. Analogues were also made to project functional groups
into the S3 pocket from the P1 phenyl. One of the most potent
compounds in this initial library was 7c (BACE1 IC50 ) 160
nM) in which a 4-acetoxyphenoxy group is attached in the para
position of the P1 phenyl substituent. Again, a cocrystal structure
(not shown) provided conclusive proof that the 4-(4-acetox-
yphenoxy)phenyl group extends through the S1 pocket into the
S3 pocket as predicted by our models. The 2-chloro group on
the P2′ phenyl on this molecule (7c) is directed toward the back
of the S2′ pocket, but on the basis of the interactions it makes
with the protein and our SAR studies, this P1′ substitution does
not appear to contribute significantly to potency. As mentioned
above, the S2′ pocket presents Tyr198 and Arg128 in the
immediate vicinity of the ligand and contains several buried
water molecules. Attempts to take advantage of opportunities
for improved interactions, and possible displacement, of these
buried water molecules through the attachment of polar sub-
stituents on the 2-, 3-, and 4-position of P2′ phenyl ring were
unsuccessful.
Figure 1. BACE1 peptidomimetic inhibitors: hydroxyethylene based
heptapeptide 1,16 optimized hydroxyethylene based inhibitor 2,17
conformationally constrained analogue 3,18 and statine based tetrapeptide
4.19
Further examination of the BACE1/7a complex indicates that
the two aryl groups extend into the S1 and S2′ pockets. The
large lipophilic S1 pocket is approximately spherical and appears
suboptimally occupied by the flat phenyl ring. In addition, the
para position of the P1 phenyl group projects directly toward
the unoccupied S3 pocket, indicating an opportunity to add
substituents to the P1 phenyl extending into the S3 pocket and
thereby potentially increasing binding affinity. The phenyl
occupying S2′ is involved in a π-edge stacking interaction with
Trp76. In contrast to the S1-S3 pocket, the S2′ pocket provides
access to more polar/charged groups (e.g., Trp76, Tyr198, and
Arg128) in the immediate vicinity of the ligand and contains
several buried water molecules, offering the potential for
analogues of 7a to form additional hydrogen bonds directly with
BACE1 or through tightly held water molecules.
While two of the nitrogen groups on the acylguanidine moiety
are intimately involved in key hydrogen-bonding interactions
with the catalytic aspartic acids, the third nitrogen faces away
from the catalytic residues toward the S1′ pocket. Although this
nitrogen appears to be involved in an intramolecular hydrogen
bond with the adjacent carbonyl group, it does not make any
significant interactions with the protein. Thus, we felt substitu-
tion on this nitrogen might allow access to the unoccupied S1′
pocket, a pocket that accommodates an Asp residue in the
natural substrate and is flanked by Arg235, Lys224, and Thr329,
thus presenting potential opportunities to form polar and/or
hydrogen-bonding interactions.
The synthetic pathway to the desired analogues is shown in
Scheme 1. The 1,4-diarylbutane-1,4-diones 5a and 5c,d were
prepared in one step by coupling enolizable methyl ketones and
R-bromomethyl ketones under the action of ZnCl2, t-BuOH, and
Et2NH, using the procedure described by Kulinkovich.27 1-Aryl-
4-adamantylbutane-1,4-dione 5b was prepared by the reaction
of an ethylbenzoyl acetate with 1-adamantyl bromomethyl
ketone to give the ethyl 2-aryl-4-oxo-4-alkylbutanoate 8, which
was subjected to ester hydrolysis and decarboxylation to give
the 1,4-dione 5b. The 1,4-diones were condensed with glycine
in refluxing acetic acid or with glycine methyl ester in toluene
followed by ester hydrolysis to give the different 2-(2,5-
disubstituted-1H-pyrrol-1-yl)acetic acids 6. Activation of the
acid with 1,1′-carbonyldiimidazole (CDI) and reaction with
guanidine hydrochloride gave the unsubstituted acylguanidines
Substitutions of the guanidine nitrogen terminating in polar
functionalities were expected to access the S1′ pocket and
anticipated to pick up additional hydrogen bonds with, or
displace, bound water molecules in this pocket. Substitution on
this nitrogen with a 3-propanol group yielded 10b, which had
a BACE1 IC50 of 240 nM, approximately 2.5-fold higher
potency than the corresponding unsubstituted acylguanidine 7b.
Continued exploration of the S1-S3 region of the molecules
led to the discovery that the large phenyl is not required for
optimal S3 pocket binding; in fact, the much smaller compound
10d with the p-n-propyloxyphenyl group extending from S1-
S3 (BACE1 IC50 ) 110 nM) achieves similar or greater potency.
A 2.4 Å resolution cocrystal structure of BACE1 with
inhibitor 10b and modeled structure of 10d (Figure 4) highlight
many of the new interactions with these optimized inhibitors.
As with 7a, the acylguanidine moiety forms hydrogen bonds
with the two catalytic aspartic acid residues. Substitution of the
acylguanidine nitrogen extends into the S1′ pocket, forming
hydrogen-bonding interactions with Arg235 and Thr329 via
bridging water molecules. The p-n-propyloxyphenyl group
extends into the S1-S3 pocket with minimal strain, as was
intended.
The acylguanidine inhibitors were tested for their inhibition
of the closely related BACE2 enzyme and more distant aspartic
proteases cathepsin-D and pepsin (Table 1). In general, these
compounds are highly selective for BACE1 over cathepsin
(>50-fold, except 7a which is 16-fold) and pepsin (IC50 > 50
µM). Compounds with diaryl substituents on the pyrrole
generally have lower selectivity for BACE1 over BACE2 (3-
to 20-fold), while those with the adamantyl substituent show
greater selectivity (14- to 20-fold). Substitution on the guanidine
nitrogen also leads to lower BACE1/BACE2 selectivity. Several

6160 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 21
Letters
Scheme 1a
a (a) Et2NH, t-BuOH, ZnCl2, toluene, room temp, 2-5 days; (b) glycine, p-TSA, EtOH, 80 °C, 3 days; or (i) Gly-OMe, p-TSA, toluene, 110 °C, 2 days;
(ii) LiOH, THF, room temp, 2 days; (c) (i) CDI, DMF, room temp, 1 h; (ii) guanidine‚HCl, Et3N, room temp, 5 h; (d) NaH, THF, room temp, 4 h; (e) 3 N
NaOH, EtOH, 80 °C, 16 h; (f) CDI, DCM, room temp, 5 h, then 1H-pyrazole-1-carboximidamide, TEA, DMAP, room temp, 18 h; (g) 3-amino-1-propanol,
DIEA, DCM, room temp, 48 h.
Figure 4. X-ray structure of BACE1 complexed with 10b (colored
by atom type with carbon in green, oxygen in red, nitrogen in blue),
overlaid with a modeled structure of 10d (colored in magenta).
Figure 2. Crystal structure of BACE1 complexed with 7a highlighting
Interactions between the hydroxypropyl group and bound water
the four key hydrogen-bonding interactions between the catalytic
molecules in the S1′ pocket are indicated as light-blue dashed lines.
aspartic acids Asp32 and Asp228 and the acylguanidine moiety.
Table 1. IC50 Valuesa of Inhibition of BACE1 and Related Aspartyl
Proteases by Compounds 7 and 10
BACE1
IC50, µM
BACE2
IC50, µM
cathD
IC50, µM
pepsin
IC50, µM
cellular Aâtotal
ED50, µM
7a
7b
7c
3.7 ( 1.0
0.6 ( 0.2
>50
12.9 ( 2.8
60.0 ( 11.4
30.2 ( 6.8
8.2 ( 1.0
>50
>50
>50
>50
>50
8.9 ( 1.6
3.1 ( 1.9
8.6 ( 3.8
1.8 ( 0.6
ND
0.16 ( 0.01 3.2 ( 0.2
10b 0.24 ( 0.05 3.5 (0.6
10d 0.11 ( 0.01 0.34 ( 0.03 5.9 ( 0.3
19.4 ( 0.9
a IC50 values are reported as the median and standard deviation of at
least three individual determinations.
a flap-open conformation. Structure-based design was used to
optimize the series, leading to potent analogues such as 10d
(BACE1 IC50 ) 110 nM).
Figure 3. Crystal structure of BACE1 complexed with 7a (colored
by atom type with carbon in green, oxygen in red, nitrogen in blue)
overlayed with the published structure of BACE1 complexed with 116
(colored in magenta). The yellow arrow highlights the movement of
Tyr71 from the closed position in complex with 1 (Tyr71 shown in
magenta) to the open conformation in complex with 7a (Tyr71 shown
in green, blue, and red). A solid Connolly surface of the BACE1/7a
binding site is shown, with polar/charged regions of the pocket colored
blue and lipophilic regions colored red.
Supporting Information Available: Experimental details and
characterization data for 5-10, BACE1, BACE2, cathepsin-D, and
pepsin enzyme; RICA and Aâ cell assay conditions. This material
is available free of charge via the Internet at http://pubs.acs.org.
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JM0607451