Design and synthesis of 5,5′-disubstituted aminohydantoins as potent and selective human β-secretase (BACE1) inhibitors

Article abstract of DOI:10.1021/jm901414e

The identification of small molecule aminohydantoins as potent and selective human β-secretase inhibitors is reported. These analogues exhibit low nannomolar potency for BACE1, show comparable activity in a cell-based (ELISA) assay, and demonstrate < 100× selectivity for the other structurally related aspartyl proteases BACE2, cathepsinD, renin, and pepsin. On the basis of the cocrystal structure of the HTS-hit 2 in the BACE1 active site and by use of a structure-based drug design approach, we methodically explored the comparatively large binding pocket of the BACE1 enzyme and identified key interactions between the ligand and the protein that contributed to the affinity. One of the more potent compounds, (S)-55, displayed an IC₅₀ value for BACE1 of 10 nM and exhibited comparable cellular activity (EC ₅₀ = 20 nM) in the ELISA assay. Acute oral administration of (S)-55 at 100 mg/kg resulted in a 69% reduction of plasma Aβ₄₀ at 8 h in a Tg2576 mouse (p < 0.001).

Full text of DOI:10.1021/jm901414e

1146 J. Med. Chem. 2010, 53, 1146–1158
DOI: 10.1021/jm901414e
Design and Synthesis of 5,50-Disubstituted Aminohydantoins as Potent and Selective Human
β-Secretase (BACE1) Inhibitors
Michael S. Malamas,*,† Jim Erdei,† Iwan Gunawan,† Jim Turner,‡ Yun Hu,‡ Erik Wagner,‡ Kristi Fan,† Rajiv Chopra,§
Andrea Olland,§ Jonathan Bard,‡ Steve Jacobsen,‡ Ronald L. Magolda,† Menelas Pangalos,‡ and Albert J. Robichaud†
†Department of Chemical Sciences, Wyeth Research, CN 8000, Princeton, New Jersey 08543-8000, ‡Neuroscience, Wyeth Research,
CN 8000, Princeton, New Jersey 08543-8000, and §Department of Chemical Sciences, Wyeth Research, 200 Cambridge Park Drive,
Cambridge, Massachusetts 02140
Received September 22, 2009
The identification of small molecule aminohydantoins as potent and selective human β-secretase
inhibitors is reported. These analogues exhibit low nannomolar potency for BACE1, show comparable
activity in a cell-based (ELISA) assay, and demonstrate >100ꢀ selectivity for the other structurally
related aspartyl proteases BACE2, cathepsinD, renin, and pepsin. On the basis of the cocrystal structure
of the HTS-hit 2 in the BACE1 active site and by use of a structure-based drug design approach, we
methodically explored the comparatively large binding pocket of the BACE1 enzyme and identified key
interactions between the ligand and the protein that contributed to the affinity. One of the more potent
compounds, (S)-55, displayed an IC50 value for BACE1 of 10 nM and exhibited comparable cellular
activity (EC50 = 20 nM) in the ELISA assay. Acute oral administration of (S)-55 at 100 mg/kg resulted
in a 69% reduction of plasma Aβ40 at 8 h in a Tg2576 mouse (p < 0.001).
Introduction
fragments result from the sequential cleavage of APP,
first at the N-terminus by β-secretase enzyme ( β-site APP
cleaving enzyme, BACE1),9-11 followed at the C-terminus by
one or more γ-secretase complexes (intramembrane aspartyl
proteases),12 as part of the β-amyloidogenic pathway. More
specifically, BACE1 cleaves APP to generate secreted peptide
sAPPβ and the membrane bound C99 fragment, which is then
cleaved by γ-secretase complexes to produce peptide Aβ40 as
the predominant product (90% abundance) and peptide Aβ42
as a minor product (9% abundance). Peptide Aβ42 is repor-
tedly the more highly pathogenic and the primary source
of plaque formation. Although the cause of AD remains
unknown, a large body of evidence is beginning to accumulate
that highlights the central role of Aβ in the pathogenesis of the
disease.13-17 Thus, processes that limit Aβ production and
deposition by preventing formation, inhibiting aggregation,
and/or enhancing clearance may offer effective treatments for
AD. Since β-secretase mediated cleavage of APP is the first
and rate-limiting step of the amyloidogenic processing path-
way, BACE1 inhibition is considered a prominent therapeutic
target for treatingADby diminishingAβ peptide formation in
AD patients.
Alzheimer’s disease (ADa) is a progressive, degenerative
disease of the brain and the leading cause of dementia. At
the early stage, AD is associated with gradual loss of cogni-
tion that leads to complete deterioration of cognitive and
behavioral functions and ultimately death. AD is a debilita-
ting disease that affects millions of elderly men and women
worldwide and has been recognized as a major global social
and financial burden.1,2 The prognosis of AD is poor, and
there is a great need for medical intervention of this disease.
Currently, the approved medical therapies consist of choli-
nesterase inhibitors and N-methyl D-aspartate (NMDA) anta-
gonists that reduce the symptomatology of the disease in the
initial phase but are not capable of curing or stopping its
progression.3,4 The pathological hallmarks of AD include the
extracellular deposition of β-amyloid peptide (Aβ), which
leads to aggregation and plaque formation, and the abnormal
hyperphosphorylation of tau protein, which leads to the intra-
cellular formation of neurofibrillary tangles.5,6 β-Amyloid
deposits are predominately aggregates of the Aβ peptides
(Aβ, 39-43 residues) resulting from the endoproteolysis
of the amyloid precursor protein (APP).7,8 These peptide
Since the identification of a BACE1 knockout mouse and
the subsequent characterization of its viability, the interest in
this enzyme has been significant among researchers in the AD
area. Over the past decade multiple groups have investigated a
variety of approaches to the design of BACE1 inhibitors. The
initial work by Tang and Ghosh was concentrated on peptidic
substrate transition-state mimic inhibitors.18 These ligands
showed excellent low nanomolar inhibitory potency for
BACE1but hadlimitedpotentialasoralCNS drug candidates
because of their poor pharmacokinetic properties. These
initial findings, together with a coordinated drug design
*To whom correspondence should be addressed. Telephone: 732-274-
4428. Facsimile: 732-274-4505. E-mail: malamam@wyeth.com.
a Abbreviations. AD, Alzheimer’s disease; Aβ, β-amyloid peptide;
APP, β-amyloid precursor protein; BACE, β-site APP cleaving enzyme;
FRET, fluorescence resonance energy transfer; NMDA, N-methyl
D-aspartate; Abz, o-aminobenzoic acid; Dpa, 3-(2,4-dinitrophenyl)-
L-2,3-diaminopropionic amide; Dnp, 6-(2,4-dinitrophenyl); MOCAc,
7-methoxycoumarin-4-yl; EDANS, 5-[(2-aminoethyl)amino]naphtha-
lene-1-sulfonic acid; DABCYL, 4,4-dimethylaminoazobenzene-40-car-
boxylic acid; ELISA, enzyme-linked immune sandwich assay; CHO,
Chinese hamster ovary.
r
pubs.acs.org/jmc
Published on Web 12/07/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1147
Figure 2. Crystal structure of BACE1 complexed with 2 highlight-
ing the three key hydrogen-bonding interactions between the cata-
lytic aspartic acids Asp32 and Asp228 and the aminoimidazole
moiety.
Figure 1
Chemistry
structure-based approach, have paved the way for the gene-
ration of less peptidic, second-generation inhibitors.19-25
The workofTang (OklahomaMedicalResearch Foundation)
and Ghosh (University of Illinois at Chicago/Purdue Uni-
versity) has pioneered the evolution of substrate-based inhi-
bitors centered on OM99-226 (1, Figure 1), a highly potent
peptide-derivedBACE1 inhibitor (IC50 = 1.6 nM). The X-ray
crystal of 1 in a complex with BACE1 has sparked the way
forward for many new classes of BACE1 inhibitors. These
efforts have produced low molecular weight BACE1 inhibi-
tors with little or no peptidic character.27-32 In addition,
recent reports have revealed the application of fragment-
based lead generation computational approaches as an alter-
native path to the design of potent small-molecule BACE1
inhibitors.33-36 The discovery of low molecular weight
BACE1 inhibitors has also led to the improvement of the
physicochemical properties of the compounds, as demon-
strated by their high-permeability in cell-based assays.37a-m
In this paper, we report the design and synthesis of
potent and selective BACE1 inhibitors, which originated
from the HTS hit 2 (Figure 1). As we have previously
reported, lead compound 2 was discovered through a
FRET assay screen of our in-house sample collection.
It possessed moderate in vitro potency for the BACE1
enzyme with an IC50 of 38 μM and demonstrated similar
cell-based ELISA potency with an IC50 of 27 μM. The
crystal structure of 2 in a complex with BACE1 was
The compounds needed to delineate the SAR for this study
were prepared according to Schemes 1-3. An important step
for the preparation of the advanced aminohydantoins 10a,b
(Scheme 1) was the treatment of 1,2 disubstituted diketones
8a,b with appropriately functionalized 1-alkylguanidines 9
in the presence of base (Na2CO3 or K2CO3) to produce
the desired products in excellent yields. Working in reverse
fashion, the required diketones 8a,b for this transformation
were either commercially available or prepared straightfor-
wardly via several synthetic routes described in Schemes 1
and 2. In general, two routes were used for the formation of
thesediketones8a,b. Inroutea, commerciallyavailablebenzyl
bromides or chlorides 4 were converted to phosphonium
salts 5 upon treatment with triphenylphospine. These phos-
phonium salts 5 were initially treated with a suitable base
(n-butyllithium or potassium tert-butoxide) and then with the
requisite chlorides 6a,b to furnish ylides 7a,b. The required
acid chlorides 6a,b were either commercially available or
prepared from the corresponding carboxylic acids upon
treatment with oxalyl chloride. Oxidation of ylides 7a,b
with potassium permanganate in the presence of magnesium
sulfate produced diketones 8a,b. Following route b, diethyl
oxalate was treated with a suitable Grignard reagent 11 to
produce keto esters 12, which upon hydrolysis to the corre-
sponding carboxylic acids and further treatment with oxalyl
chloride afforded glycolic chlorides 13. A second treatment of
chlorides 13 with a diverse Grignard reagent 14 furnished
variably functionalized diketones 8a in excellent yields.
An alternative synthetic approach to the diketones 8a,b
is described in Scheme 2, where commercially available
benzaldehydes 15 were treated with triethyl phosphite
and chlorotrimethylsilane to produce silyl ethers 16.
These were then converted to silyloxy ethers 18a,b upon
sequential treatment with lithium diisopropylamide and
suitable acid chlorides 17a,b. Hydrolysis of the silyloxy
ethers 18a,b with sodium bicarbonate afforded the dike-
tones 8a,b, which were converted to the aminohydantoins
10a,b, as before.
˚
resolved to 2.3 A (Figure 2), defining the three-dimensional
structure of the enzyme, and revealed that compound 2
occupies the center of the rather large BACE1 binding
pocket (S1, S20 regions) in an orientation where the imida-
zole ring of the ligand directly interacts with both catalytic-
site aspartic acids (Asp32 and Asp228) via a hydrogen-
bonding network. Further examination shows that the six-
member tetrahydropyrimidine ring points toward solvent
space and makes no apparent beneficial contact with
residues of the enzyme’s backbone. The ensuing truncation
of this ring led to the more compact aminohydantoin
3 (Figure 1), which possessed a 10-fold enhancement in
potency (IC50 = 3.4 μM) as a consequence. The structure-
activity relationship studies around compound 3 are the
central focus of this report.
The synthetic approach of Scheme 3 was utilized for the
preparation of highly functionalized aminohydantoin deriva-
tives contained in Tables 3-6. Here again, the approach
utilizes two alternative strategies to increase the diversity

1148 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Malamas et al.
Scheme 1a
a Reagents: (a) PPh3, toluene; (b) Ar-COCl, tert-BuOK, THF; (c) KMnO4, acetone; (d) (CO2Et)2, THF; (e) aq K2CO3, EtOH; (f ) SOCl2 or (COCl)2;
(g) Ar-MgBr, CuBr, LiBr; (h) RNH(CdNH)NH2, K2CO3, dioxane, H2O.
Scheme 2a
cleavage of peptide substrate Abz-SEVNLDAEFR-Dpa
(Swedish substrate), while peptide substrate MOCAc-
GKPILFFRLK (Dnp)-D-R-NH2 was used for cathepsin
D
and pepsin, and peptide substrate RE(EDANS)-
IHPFHLVIHTK(DABCYL)-R for renin. Kinetic rates were
calculated, and IC50 values were determined by fitting the %
inhibition as a functionofcompoundconcentrationtotheHill
equation (y = ((B ꢀ Kn) þ (100 ꢀ xn))/(Kn þ xn).
We have routinely screened all prepared compounds for
BACE1, BACE2, and cathepsin D inhibition, and only
selected compounds were assayed in the pepsin and renin
screens, based on their meeting the screening protocol for
affinity to the target, and are reported in Tables 1-7.
The cell-based Aβ inhibition (Aβ40 or Aβ42) of the potential
inhibitors was assessedinanenzyme-linkedimmunesandwich
assay (ELISA) in Chinese hamster ovary (CHO) cells, recom-
binantly expressing human wild-type APP (CHO-wt). The
concentration at which the cellular production of Aβ40 or
Aβ42 was reduced by 50% (EC50) was determined and re-
ported in the tables. Potential compound toxicity was assessed
via mitochondrial function using MTS readout (MTS kit
from Promega); values are represented as LD50 or the dose
of compound that resulted in 50% of control signal. MTS
data will be discussed only for compounds indicating toxicity
concerns.
a Reagents: (a) P(OEt)3, ClSiMe3, toluene; (b) LDA, THF, A-COCl;
(c) NaHCO3, H2O; (d) RNH(CdNH)NH2, Na2CO3, EtOH, H2O.
and efficiency in preparation of multiple analogues in this
series. In route a, palladium-catalyzed cross-coupling reaction
of key diketones 8a with any number of heteroarylboronic
acids (Suzuki coupling38) or heteroaryl trialykl/triaryl stan-
nanes (Stille coupling39) in the presence of Pd(0) or Pd(II)
catalysts under a variety of well-known conditions produced
the desired products 20. Subsequent condensation of dike-
tones 20 with suitable 1-alkylguanidines 10, as before, fur-
nished aminohydantoins 21. In route b, the reverse order of
these two reactions is utilized to broaden the scope and
breadth of the SAR investigation. In this case, we first
converted diketones 8 to aminohydantoins 22 and then utilize
the Suzuki or Stille palladium-catalyzed cross-coupling pro-
tocols to obtain the desired products 21.
As previously described,41 high-throughput screening hit
imidazole 2 (Figure 1) was successfully cocrystallized with
˚
BACE1 enzyme to 2.3 A resolution (Figure 2). There are
several notable features of the ligand binding interactions.
First, the X-ray structure shows that the amino group of the
ligand interacts with both aspartic acids (Asp32 and Asp228)
and the N3 nitrogenof the imidazolering interacts with Asp32
via a hydrogen-bonding network. Furthermore, the BACE1/2
structure indicates that the opposing aryl groups of the ligand
extend into the S1 and S20 regions, making important but
suboptimal interactions with several residues of the binding
pocket. Finally, the six-member tetrahydropyrimidine ring
appears to point toward solvent space and makes no apparent
Results and Discussion
The primary screening assay utilized for the program was a
homogeneous, continuous fluorescence resonance energy
transfer(FRET) protocol, representingcompetitiveinhibition
for BACE1, BACE2, cathepsin D, pepsin, and renin acti-
vities.40 The BACE1 and BACE2 affinities were based on the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1149
Scheme 3a
a Reagents: (a) arylboronic acid or heteroarylboronic acid or stannates, (PPh3)4, or PdCl2[(o-tolyl)3P]2, toluene or 1,2-diethoxyethane, PPh3, toluene;
(b) RNH(CdNH)NH2, K2CO3 or Na2CO3, EtOH or dioxane or DMF, H2O.
Table 1. Diphenylaminohydantoins
IC50 (μM)a
EC50 (μM)a
ELISA
compd
R
R3
R4
BACE1
BACE2
cathepsin D
3
Me
Et
H
H
3.44 ( 0.29
25% at 5 μM
14% at 5 μM
3.47 ( 0.93
2.50 ( 0.37
3.10
0.77 ( 0.14
5.16 ( 1.95
87.19
36% at 100 μM
33% at 100 μM
13% at 100 μM
46% at 100 μM
69.18
3.4 ( 2.04
8.13 ( 2.9
23.5 ( 2.2
5.9 ( 2.9
6.97 ( 3.3
3.35 ( 1.24
2.13 ( 0.67
23
24
25
26
27
28
H
H
H
H
H
Me
Me
Me
Me
OMe
Cl
H
H
1.19 ( 0.66
0.42
1.72
H
OMe
Cl
3% at 100 μM
38.18 ( 1.13
H
2.25 ( 0.03
1.84 ( 0.03
a IC50 and EC50 values are the mean values of at least two experiments ( SD. Values without SD are for a single determination only.
Table 2. Adamantylaminohydantoins
IC50 (μM)a
EC50 (μM)a
ELISA
compd
R3
R4
BACE1
BACE2
cathepsin D
29
30
H
H
1.53 ( 0.06
0.16 ( 0.02
0.07 ( 0.02
50% at 5 μM
0.33 ( 0.07
0.87 ( 0.14
0.47 ( 0.1
0.27 ( 0.07
0.17 ( 0.04
45
0.83 ( 0.24
0.68 ( 0.16
0.23 ( 0.03
50% at 25 μM
2.53 ( 0.6
5.55 ( 0.56
3.87 ( 0.9
6.29 ( 2.2
4.02 ( 0.5
73.58
27.28
0.93 ( 0.2
0.044 ( 0.039
0.07 ( 0.003
1.2 ( 0.22
0.38 ( 0.3
0.57 ( 0.12
0.9 ( 0.4
0.23 ( 0.07
0.13 ( 0.01
1.9 ( 0.04
0.76 ( 0.2
0.19 ( 0.08
H
OMe
OMe
OMe
OEt
15.32 ( 2.6
13.37 ( 0.2
20.78
(S)-30
(R)-30
31
H
H
H
11.53
32
33
H
O-n-Bu
OCF3
OMe
OMe
OMe
OMe
OMe
8.4 ( 1.78
6.22 ( 1.7
11.1 ( 4
9.21
H
34
Me
Me
Me
Et
(S)-34
(R)-34
35
13.14
14.10
0.28
0.27 ( 0.04
4.33
9.59 ( 0.84
36
OMe
36.6 ( 14
a IC50 and EC50 values are the mean values of at least two experiments ( SD. Values without SD are for a single determination only.

1150 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Table 3. Pyridineaminohydantoins
Malamas et al.
IC50 (μM)a
EC50 (μM)a
ELISA
0
compd
R3
R4
BACE1
BACE2
cathepsin D
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Me
H
0.04 ( 0.01
0.09 ( 0.04
0.05 ( 0.01
0.05 ( 0.03
0.05 ( 0.04
0.06 ( 0.04
0.07 ( 0.01
0.12 ( 0.02
0.05 ( 0.01
0.036 ( 0.01
0.04 ( 0.01
0.15 ( 0.01
0.13 ( 0.1
0.78 ( 0.26
4.17 ( 1.4
0.6 ( 0.02
0.2 ( 0.1
0.17 ( 0.01
1.84 ( 0.9
0.3 ( 0.07
0.96 ( 0.09
0.52 ( 0.1
0.56
12.5 ( 3.3
23.1 ( 7.3
10.3 ( 1.9
10.1 ( 1.7
8.18 ( 1.5
12.2 ( 1.4
8.3 ( 1.1
1.87 ( 0.13
5.2 ( 1.2
6.28
0.12 ( 0.05
0.9 ( 0.4
OMe
OEt
OPr
OBu
H
H
0.52 ( 0.08
0.68 ( 0.3
0.67 ( 0.2
0.87 ( 0.3
0.67 ( 0.1
0.63 ( 0.3
0.42 ( 0.15
0.19 ( 0.05
0.29 ( 0.56
4.5 ( 0.51
0.23 ( 0.06
1.75 ( 0.3
H
H
O-i-Pr
O-cyclopentyl
cyclopentyl
H
H
H
F
Cl
H
H
CF3
CN
Me
Me
H
1.0 ( 0.01
0.55 ( 0.2
0.96 ( 0.2
2.32 ( 0.08
3.51 ( 0.09
7.3 ( 1.4
2.47 ( 0.29
2.19 ( 0.18
H
Me
CN
0.63 ( 0.08
a IC50 and EC50 values are the mean values of at least two experiments ( SD. Values without SD are for a single determination only.
Table 4. Bicyclic Aminohydantoins
Armed with this important structure based design data, we
investigated the ligand/protein interactions proximal to the
catalytic region of the protein. Replacing the N-methyl
group of the aminohydantoin 3 with the bulkier ethyl group
(entry 23, Table 1) resulted in a noticeable decrease in ligand
potency. The amino group of ligand 3 participates in a key
interaction with the catalytic-site aspartic acid Asp228, which
appears to have been adversely affected by the bulkier ethyl
group in this very important pocket and as a result has
affected the ligand potency. However, replacement of the
N-methyl group with the smaller hydrogen nucleus (24)
also resulted in significant loss of potency. Compound 24
would most likely exist predominantly in the enol form
(2-amino-4H-imidazol-5-ol), as opposed to the N-methylated
derivative, which would occupy a more ketomeric form of the
tautomeric pair. This keto-enol shift would have profound
implications on not only the pKa of the heterocycle but also
the hydrogen bonding ability of the moiety, resulting in a less
favorable interaction.
A significant effort was launched to examine the effects of
the substituents on the phenyl moiety that projects to the S20
a IC50 and EC50 values are the mean values of at least two experiments
pocket. Toward that end, the introduction of either electron
( SD. Values without SD are for a single determination only.
donating or withdrawing groups at the meta- and/or para-
significant contacts with residues of the binding pocket. It
was quickly discovered that truncation of the tetrahydropyri-
midine, which led to the aminohydantoin 3 (Figure 1), pos-
position of this phenyl moiety resulted in only a marginal
improvement of potency (25-28).
Concomitant with these investigations, we undertook opti-
mization of the S1 pocket binding affinity. Close examination
of the BACE1/2 crystalstructure revealed thatthe S1 pocket is
highly hydrophobicandapproximately sphericalinshape. We
hypothesized that increasing the ligand size and lipophilicity
to more completely occupy this region would result in in-
creased ligand affinity. To that end, replacement of the S1
phenyl moiety with the bulkier and more lipophilic adamantyl
group (30, Table 2) resulted in a nearly 20-fold increase in
potency (30 vs 27) supporting this hypothesis. As will be
discussed below, the X-ray structures of ligands 29 and 34
bound to the enzyme have confirmed the orientation of the
sessed a 10-fold enhancement in potency at the target (IC50
=
3.4 μM). The subsequent docking of ligand 3 to the X-ray
crystal structure of 2 bound to BACE1 revealed an excellent
superposition between these two structures, with both ligands
making similar contacts with residues of the binding pocket.
The increased affinity of ligand 3 is most likely attributed
to the increased acidity of the aminoimidazolone pharmaco-
phore of ligand 3 (3 pKa = 5.7 vs 2 pKa = 7.6), which we
hypothesize affects the strength of the hydrogen-bonding
interactions between the ligand and catalytic-site aspartic
acids.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1151
Table 5. Substituted Pyridineaminohydantoins
IC50 (μM)a
EC50 (μM)a
ELISA
0
00
00
R6
compd
R3
R4
R4
R2
BACE1
BACE2
cathepsin D
54
55
Me
Me
Me
Me
H
OMe
OMe
OMe
OMe
OCF3
OCF3
OCF3
H
F
F
H
0.02 ( 0.002
0.04 ( 0.02
0.01 ( 0.002
0.56 ( 0.2
0.06
0.78 ( 0.17
1.84 ( 0.13
0.81 ( 0.23
1.64 ( 0.12
2.48
2.58 ( 0.83
2.41 ( 0.17
0.82 ( 0.17
2.71 ( 0.23
3.15
0.11 ( 0.01
0.06 ( 0.01
0.02 ( 0.01
1.2 ( 0.3
0.37 ( 0.08
2.57 ( 0.9
4.84 ( 1.8
F
F
F
F
H
H
H
H
H
(S)-55
(R)-55
56
F
F
H
H
H
57
58
H
OMe
H
0.52 ( 0.08
0.9 ( 0.16
5.99 ( 0.5
2.51 ( 0.9
2.05 ( 0.6
4.43 ( 0.4
H
OMe
a IC50 and EC50 values are the mean values of at least two experiments ( SD. Values without SD are for a single determination only.
Table 6. Pyrimidineaminohydantoins
IC50 (μM)a
EC50 (μM)a
ELISA
0
0
0
R5
compd
R3
R4
R2
R4
BACE1
BACE2
cathepsin D
59
60
Me
Me
H
OMe
OMe
H
H
H
H
H
H
F
H
F
H
H
H
H
H
F
0.03 ( 0.01
0.02 ( 0.001
0.04 ( 0.01
0.02 ( 0.004
1.39
1.59 ( 0.6
2.8 ( 0.7
9.69 ( 2.0
7.54 ( 0.7
9.97
14.3 ( 5.4
6.7 ( 1.6
4.18 ( 0.8
2.33 ( 0.4
11.57
0.08 ( 0.04
0.04 ( 0.01
0.27 ( 0.04
0.07 ( 0.02
2.8 ( 0.07
61
OCF3
OCF3
OCF3
OCF3
OCF3
F
(R)-61
(S)-61
62
H
F
H
F
H
H
H
0.32 ( 0.07
1.36 ( 0.08
1.26 ( 0.07
5.04 ( 0.4
8.55 ( 0.9
26.18
63
H
H
2.83 ( 0.6
a IC50 and EC50 values are the mean values of at least two experiments ( SD. Values without SD are for a single determination only.
the S20-pocket by employing multiple substituents at the
phenyl moiety that project into the pocket. While disubstitu-
tions at the meta- and para-position of the phenyl moiety have
resulted in only a slight decrease in ligand potency, this has
produced an unexpected increase (∼20- to 30-fold) of the
ligand’s selectivity against BACE2 (34-36 vs 30). This intri-
guing finding prompted us to cocrystallize compounds 29 and
34 with BACE1 in an attempt to explain the role of these
substitutions on the ligand’s selectivity against BACE2. Over-
lapping of the X-ray crystal structures of compounds 29 and
34 bound to the enzyme indicated a nearly perfect super-
imposition of these two ligands (Figure 3). Close examination
of the overlapping structures, however, has revealed a shift of
the “flap” loop region of the protein backbone of the enzyme,
Table 7. Pepsin and Renin Inhibition of Representative Compounds
IC50 (μM)a
compd
pepsin
renin
3
11.1 ( 0.4
14.4 ( 0.3
15.0 ( 1.4
19.6 ( 1.7
66.3 ( 1.2
33.3 ( 1.4
35.3 ( 1.1
35.1 ( 0.7
9.6 ( 1.8
30
34
37
55
59
60
62
53.3 ( 2.3
59.5 ( 2.3
93.4 ( 1.4
23.2 ( 1.3
42.3 ( 3.6
57.6 ( 1.6
13.8 ( 0.7
a IC50 values are the mean values of at least two experiments ( SD.
adamantyl moiety at the enzyme’s S1 pocket, as intended. The
well-defined orientation of the adamantyl-bearing ligand
within the binding pocket, and new insights gained from this
structure, directed us to re-evaluate the effects of substituting
the phenyl ring of 30, described earlier, that projects into the
S20 pocket. Increasing the chain length of the para-substituent
on the phenyl moiety resulted in 2- to 5-fold potency loss
(31, 32 vs 30). Furthermore, the more electronegative tri-
fluoromethoxy group had weaker activity (33 vs 30), giving us
additional insights into the requirements for binding to the
S20-pocket. Next, we further probed the restrictions on size in
˚
orienting Tyr71 at an open position (approximately 5 A shift
vs apo-position) for the more selective compound 34. With the
unsubstituted ligand 29, the loop is in a more closed orienta-
tion (apo-position), which may explain the selectivity versus
BACE2. The open orientation of the “flap” region in the
BACE1/34 structure is perhaps attributed to the steric influ-
ence exerted by the meta-methyl group of the S20-phenyl
moiety. The question then became, “How does this substitu-
ent interact with the loop in the BACE 2 enzyme structure?”
Examinationof the proteinaminoacid sequences ofthe “flap”

1152 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Malamas et al.
to improve ligand affinity as was suggested by the BACE1/34
X-ray crystal structure and molecular modeling calculations.
However, functionalizing the adamantyl nucleus in such a
way was synthetically challenging, and thus, we had decided
to change strategy and use a diphenyl aminohydantoin, such
as 27, as the starting point to explore this objective. The
docking of ligand 27 to the X-ray crystal structure of the
adamantyl 34 bound to the enzyme indicated that building off
the meta-position of the phenyl moiety that projects into the
S1-pocket would allow for projection directly toward the
unoccupied S3 region (Figure 4). Additionally, X-ray struc-
tures together with modeling studies have proposed that a
pyridine and/or pyrimidine moiety “hinged” into the polar
groove (Gly11, Gly230, and Ser229) located near the catalytic
site may favorably interact with the BACE enzyme backbone
and improve ligand potency. To that end, we had introduced
the pyridine nucleus at the meta-position of the phenyl moiety
that led to 70-fold improvement in potency (37 vs 27), con-
firming this hypothesis. To further support our modeling
calculations, 37 was then cocrystallized with BACE1, and
as shown in Figure 5, the pyridine moiety projects deep
into the S3 pocket and makes a water-bridge contact with
Ser229 through the buried water found near the catalytic site
of the enzyme. This water-bridge contact together with the
additional van der Walls contacts between the ligand and
enzyme backbone at the S3 region can possibly explain the
ligand’s increased potency. In addition, the crystal structure
shows that the ligand makes several other key hydrogen-
bonding interactions with residues (Trp76, Asp32, and
Asp228) in the S20 and catalytic-site pockets. Taken together,
this constellation of contacts between the ligand and enzyme
effectively “locks” it in place within the rather large BACE1
binding pocket. This potent ligand with multiple contacts
offers the opportunity to further refine the aspects of the
ligand binding. Interestingly, the para-pyridine analogue
(4-pyridyl derivative of 37) was some 17-fold weaker than the
corresponding meta-substituted ligand 37. While it is difficult
to rationalize such a steep reduction of the ligand’s potency by
the loss of a single water-bridge contact, re-examination of the
BACE1/37 structure revealed an unfavorable electrostatic
repulsion involving the electron rich nitrogen of the pyridine
nucleus of 4-pyridyl nucleus (when docked into the enzyme site)
and the carbonyl oxygen of Gly11 at the enzyme’s backbone.
Further exploration of this pocket (i.e., electron withdrawing or
electron donating groups, multiple examples) had resulted in
only marginal improvements in ligand potency, with the ex-
ception of the methoxy groups adjacent to pyridine’s nitrogen,
which showed about a 5-fold decrease in potency (57 and 58 vs
56, Table 5). It is hypothesized that the bulkier methoxy group
could have adversely affected the water-bridge interaction
between the pyridine and residue Ser229 of the S3 pocket.
Also, molecular modeling studies (not shown) indicated that
the bulkier methoxy group could not be accommodated in this
space very well, thus affecting the ligand’s potency. Wholesale
replacement of the pyridine nucleus of ligand 37 with a phenyl
nucleus (not shown) resulted in about 5-fold reduction in ligand
potency, indicative of this crucial role of the water-bridge
interaction between the ligand and Asp229.
Figure 3. Crystal structures of BACE1 complexed with 29 (shown
in yellow) and (S)-34 (shown in white) are overlaid. Movement of
Tyr71 from the closed position in complex 29 to the open orienta-
tion in complex with (S)-34 is shown. Key hydrogen bonding
interactions between ligand and protein at the catalytic aspartic
acids Asp32 and Asp228 and at Trp76 of the S20 region are high-
lighted with yellow dashed lines.
loops of theBACE1 andBACE2 proteins(overlayofBACE1/
2 complex with BACE2 protein homology model not shown)
revealed a single amino acid difference (Pro70 in BACE1 is
replaced with Lys70 in BACE2) in proximity to the bound
ligand. This amino acid difference together with the dynamic
motion of the “flap” loop upon ligand engagement apparently
influences the ligand/protein contacts and impedes the li-
gand’s affinity for the BACE2 site, thus resulting in increased
selectivity.
In addition, the apparent hydrogen-bonding interaction
between the para-methoxy group of the phenyl moiety
of 34 projecting into the S20 pocket and residue Trp67
of the BACE1 enzyme backbone is illustrated in the X-ray
crystal structure. This key ligand/protein contact can
possibly explain the near 10-fold increase in potency of the
methoxy-substituted ligand 30 compared to the unsubstituted
analogue 29.
Furthermore, an investigation of the chiral preference
of the racemic mixtures of this class of compounds, once
separated, showed a clearpreference for theS-enantiomer (see
(S)-30 vs (R)-30). This was apparent as the crystal structures
and modeling studies bore this out. For the less active
enantiomer, one could envision keeping the adamantyl and
aryl groups in their designed pockets, but in so doing, the
aminohydantoin would be flipped into an unfavorable con-
firmation and pressed into the backbone of the active site of
the enzyme. An alternative binding pose of ligand 30 within
the binding pocket by flipping the respective P1 and P20 side
chains while retaining the interactions of the aminohydantoin
moiety with the catalytic aspartic acids would result in much
lessinteractions withthe sitecomparedto(S)-30. As a result, it
could be reflected in the 31-fold potency loss for the (R)-30
enantiomer.
Finally, the cell-based activity of the adamantyl analogues
also tracked well with their increased molecular binding with
EC50 values of 7-200 nM in the ELISA assay. Noteworthy
also is thatmostadamantyl compoundsexhibitedlowpotency
for cathepsin D with IC50 > 10 μM.
Confident that ligands such as 37 are anchored in place by
the multiple hydrogen-bonding interactions and the biaryl
moiety occupying the S1-S3 pocket, we focused our efforts
to further refine the ligand’s binding interactions at the S20
and S1 regions. To that end, we examined the substitu-
tion of electron withdrawing or donating groups at either
The next approach in the SAR study was to build off the
adamantyl moiety directly toward the unoccupied S3 pocket

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1153
decrease in ligand potency. This decrease in potency is most
certainly due to the steric conflict between the ligand and the
enzyme backbone residues of the S1 pocket. This hypothesis is
not without structure based support, as molecular modeling
studies and the BACE1/37 X-ray crystal structure are in
alignment. Noteworthy is that the cell-based activity of the
pyridine analogues also tracked well with their increased
molecular binding, with EC50 values in the range of 20-
300 nM in the requisite ELISA assay.
To further expand the scope and breadth of the
SAR described already, the pyrimidine analogues (59-63,
Table 6) were prepared. It is quite reasonable to consider that
the pyridine ring located in the S3 region (i.e., 37) can undergo
free rotation placing the pyridine nitrogen at a position
opposite to the buried water located at the S3 pocket. If this
were to happen, the favored water-bridge interaction with
Ser229 would be lost. By substituting the pyrimidine ring, we
have effectively prevented that occurrence. However, the
pyrimidine analogues prepared were only slightly better than
the analogous pyridine analogues. Nevertheless, the examples
had an unexpectedly improved selectivity against both the
highly conserved BACE2 and cathepsin D enzymes. Retro-
spective examination shows that there are two amino acid
differences in the S3 region between BACE1 and BACE2
(BACE1 Asn295 is replaced by Leu in BACE2, and BACE1
Gln11 is replaced by Arg in BACE2). In addition, the S3
pocket of the cathepsin D enzyme is smaller than that of
BACE1 and overlaps, in part, with the region occupied by the
conserved S3-water of the BACE1 enzyme (not shown).
Even though both the pyrimidine and pyridine groups can
be considered equal in size, their distinct electronic makeup
together with the residue differences at this region of the
enzymes could account for the ligand’s affinity divergence.
Finally, we have briefly examined the effect on the intro-
duction of fluorine atoms at positions 2, 4, and 5 of the phenyl
moiety located at the S1 pocket. The potential for improved
potency was a nascent consideration, as wewere attempting to
identify compounds with improved pharmacokinetic profiles,
as this ring was the focus of significant cytochrome P450
metabolism. The details of this investigation will not be
discussed here other than to say that significantly improved
ligands were realized with this approach. From a potency
perspective, while a fluorine group at position 4 only slightly
affected the ligand affinity (60 vs 59), substitutions at posi-
tions 2 and 5 had produced marked reductions (10- to 19-fold)
in BACE1 affinity (62 and 63 vs 61). Considering that fluoro
substituents do not significantly alter the size of the molecule,
electrostatic repulsions involving the fluorine groups are the
likely cause for the loss of potency. Pyrimidine (R)-61 showed
excellent selectivity against BACE2 (>370-fold). To further
explore this finding, ligand (R)-61 was cocrystallized with
BACE1 (Figure 6). The examination of the BACE1/(R)-61
structure revealed that the pyrimidine moiety of the ligand
makes a water-bridge contact with Ser229, similar to that
of the pyridine moiety, while the ligand’s phenyl moiety that
projects into the S20 pocket makes hydrogen-bonding-like
contacts between the fluorine atoms of the trifluoromethyl
group and the enzyme’s backbone residues Arg128 and
Asn37. The“flap” loop region of the proteinbackboneorients
at a closed position similar to the apo structure. Examination
of the amino acid sequences of both BACE1 and BACE2 at
this region revealed an amino acid difference between BACE1
and BACE2 (BACE1 Arg128 is replaced by Lys in BACE2)
that might play a critical role in the ligand’s affinity.
Figure 4. Crystal structure of BACE1 complexed with (S)-34
(shown in white) and 27 (shown in magenta) are overlaid. Key
hydrogen bonding interactions between ligand and protein at the
catalytic aspartic acids Asp32 and Asp228 and at Trp76 of the S20
region are highlighted with yellow dashed lines.
Figure 5. Crystal structure of BACE1 complexed to 37 highlighting
the key hydrogen-bonding interactions between the catalytic aspa-
rtic acids Asp32 and Asp228, Trp76 at the S20 region, and water-
bridge to Ser229.
the meta- or para-position of the phenyl group located in the
S20 pocket. In all examples, the ligand potency was only
minimally affected (∼2ꢀ) (entries 38-48, Table 3). However,
in stark contrast to the BACE1 enzyme SAR, the ligand
affinity for BACE2 with these substituted derivatives was
more variable. The dimethoxy analogue (38) exhibited the
highest selectivity (46-fold) against BACE2, while the longer-
chained groups, whichextended deepinto the S20 pocket, were
the least selective compounds (39-44). These findings are in
agreement with the observations described earlier for the
adamantyl aminohydantoins. In an attempt to further im-
prove the potency of these 3,4-disubstituted S20 analogues, we
prepared several examples of derivatives that possess a some-
what rigidified substituent pattern that extends into the S20
region. Compounds 51-53 (Table 4), were found to be
(2-6)ꢀ less potent than 37, showing how intolerant the S20
pocket can be to modest changes.
Focusing our attention once again back to the unprime side
of the scissile bond, specifically the S1 pocket, it was observed
that substitutions at the para-position of the proximal phenyl
moiety occupying this region with either a methyl (49) or
cyano(50) group haveresultedinanapproximate 3- to16-fold

1154 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Malamas et al.
(S)-55, displayed an IC50 value for BACE1 of 10 nM, cellular
EC50 activity of 20 nM, and >80-fold selectivity over the
other tested aspartyl proteases. Acute oral administration of
(S)-55 at 100 mg/kg resulted in a 69% reduction of plasma
Aβ40 at 8 h in a Tg2576 mouse (p < 0.001).
Experimental Section
Chemistry. Melting points were determined in open capillary
tubes on a Mel-Temp-II apparatus, and reported uncorrected.
1H NMR spectra were determined in the cited solvent on a
Varian Unity or Varian Inova (300-400 MHz) instrument, with
tetramethylsilane as an internal standard. Chemical shifts are
given in ppm, and coupling constants are in hertz. Splitting
patterns are designated as follows: s, singlet; br s, broad singlet;
d, doublet; t, triplet; q, quartet; m, multiplet. Mass spectra were
recorded on a Micromass LCT, Waters spectrometer. Elemental
analyses (C, H, N) were performed on a Perkin-Elmer 240
analyzer, and all compounds are within (0.4% of theory unless
otherwise indicated. HPLC techniques and high resolution mass
spectrometry were used to determine the purity of compounds
outside the range of the elemental analysis assessment. Purity of
all final products was >96% as determined by HPLC and/or
combustion analysis. Purity was determined by HPLC analysis
using the following protocols. Method A: mobile phase A,
10 mM ammonium formate in water (pH 3.5); solvent B,
50:50 ACN/MeOH; solvent gradient 85/15 to 5/95 A/B in 2
min, hold 1.25 min, re-equilibrate 0.5 min; flow rate 1.1 mL/min;
column, Agilent SB C18 1.8 μM, 3.0 mm ꢀ 50 mm; temperature
45 °C; detection at 210-370 nm. Method B: mobile phase B,
10 mM ammonium carbonate in water (pH 9.5); solvent B,
60:50 ACN/MeOH; solvent gradient 85/15 to 5/95 A/B in 2 min,
hold 1.25 min, re-equilibrate 0.5 min; flow rate 1.1 mL/min;
column Waters Xbridge C18 2.5 μM, 3.0 mm ꢀ 50 mm;
temperature 45 °C; detection at 210-370 nm. All products,
unless otherwise noted, were purified by “flash chromatogra-
phy” with use of 220-400 mesh silica gel. Thin-layer chroma-
tography was done on silica gel 60 F-254 (0.25 mm thickness)
plates. Visualization was accomplished with UV light and/or
10% phosphomolybdic acid in ethanol. The hydration was
determined by the Karl Fischer titration, using a Mitsubishi
moisture meter model CA-05. Unless otherwise noted, all
materials were obtained commercially and used without further
purification. All reactions were carried out under an atmosphere
of dried argon or nitrogen.
Figure 6. Crystal structures of BACE 1 complexed with (R)-61
highlighting the key hydrogen bonding interactions between ligand
and protein at the catalytic aspartic acids Asp32 and Asp228, Trp76
and Arg128 of the S20 region, Ille110 of the S1 region, and the water-
bridge with Ser229. The FLAP sits in the closed position. Water’s
position is an approximate depiction.
To confirm the specificity of the aminohydantoins for the
BACE1 enzyme, a representative set of compounds was
evaluated for inhibition of the closely related renin and pepsin
aspartyl proteases. As shown in Table 7, all compounds tested
demonstrated weak inhibition for both of these targets.
To further elucidate the ability of these ligands to reduce
Aβ, an advanced example from this class, (S)-55, was eval-
uated in the Tg2576 mice in vivo model for lowering plasma
and brain Aβ40.42 Acuteadministration of (S)-55 at100 mg/kg
po resulted in a significant 69% reduction of plasma Aβ40
measured at the 8 h time point (p < 0.001). Significant
reduction of brain Aβ40 was not observed at this dose because
of the limited brain exposure of this compound. Significant
focus has been dedicated to improving the central exposure of
these ligands while maintaining the robust affinity for the
enzyme site demonstrated in this report. Detailed studies
exploring and expanding the potential of this class of BACE1
inhibitors will be the subject of a later disclosure.
Representative synthetic protocols of the aminohydantoins
shown in Schemes 1-3 are described below.
Summary
Exemplified Analogues of Scheme 1. Route a. Preparation of
2-Amino-5-(4-methoxyphenyl)-3-methyl-5-tricyclo[3.3.1.13,7]dec-
1-yl-3,5-dihydro-4H-imidazol-4-one (10b, A = 1-Adamantyl,
R = Me, R3 = H, R4 = OMe). Step b. Lithium bis(trimethyl-
silyl)amide in THF (1.0 M, 52.3 mL, 52.3 mmol) was added
into a stirred suspension of (4-methoxybenzyl)triphenylpho-
sphonium chloride (21.9 g, 52.3 mmol) in THF (100 mL).
The mixture was stirred for 15 min at room temperature,
cooled to -5 °C, and treated with a solution of tricyclo-
[3.3.1.13,7]decane-1-carbonyl chloride (9.44 g, 47.5 mmol) in
THF (20 mL) and stirred for an additional 2 h, while the mixture
temperature was gradually raised to room temperature. The
mixture was then treated with water (50 mL) and sodium
periodate (11.18 g, 52.3 mmol), stirred at 50 °C for 17 h, cooled
to room temperature, and diluted with ethyl acetate. The organic
phase was separated and washed sequentially with water and
brine, dried over sodium sulfate, filtered, and concentrated.
Purification of the resultant residue by flash chromatography
(silica gel, EtOAc/hexanes, 1/9) afforded 1-(4-methoxyphenyl)-
2-tricyclo[3.3.1.13,7]dec-1-ylethane-1,2-dione (6.85 g, 48%) as a
yellow solid. MS m/e 299 (M þ H)þ; 1H NMR (300 MHz,
CDCl3) δ 2.05-1.55 (m, 15H), 3.89 (s, 3H), 6.96 (d, J = 8.9 Hz,
2H, 7.78 (d, J = 8.9 Hz, 2H).
In this report, we have described for the first time the
detailed exploration of the 5,5-disubstituted aminohydantoin
scaffold that led to the discovery of highly potent and selective
BACE1 inhibitors. Key analogues demonstrate low nano-
molar potency for BACE1 in a FRET assay and exhibit
comparable activity in a cell-based (ELISA) assay. In addi-
tion, these aminohydantoins show >100ꢀ selectivity for
the other structurally related aspartyl proteases BACE2,
cathepsin D, renin, and pepsin.
Our design strategy followed a traditional SAR approach
and was supported by molecular modeling studies based on
the cocrystal structure of the HTS-hit 3 in the BACE1 active
site. This approach enabled us to rapidly explore the large
ligand binding pocket of BACE1 and identify three key
protein-ligand interactions at the S20 and S3 pockets and
with the catalytic binding domain (Asp32 and Asp228) that
contributes to the ligand potency. Two distinct binding modes
between the ligand andthe target enzymewere also recognized
to contribute to their selectivity against the related BACE2
and cathepsin D aspartyl proteases. An optimized derivative,

Article
Step h.
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1155
A
mixture of 1-(4-methoxyphenyl)-2-tricyclo-
Preparation of 3-Methyl-4-methoxybenzyltriphenylphosphine
Chloride (5, R3 = Me, R4 = OMe). Triphenylphosphine (23.29 g,
87.9 mmol) was dissolved in 135 mL of toluene, and 3-methyl-
4-methoxybenzyl chloride (15 g, 11.4 mmol) was then added. The
mixture was heated to reflux for 3 h and then cooled to room
temperature. The solids were collected and washed with a small
amount of ether and dried. A white solid (23 g, 66% yield) was
recovered. MS m/e (M)þ 397; 1H NMR (DMSO-d6, 300 MHz)
δ 1.9 (s, 3H), 3.72 (s, 3H), 5.3 (d, 2H), 6.55 (s, 1H), 6.82 (m, 2H),
7.65 (m, 6H), 7.75 (m, 6H), 7.92 (m, 3H).
Exemplified Analogues of Scheme 2. Preparation of 2-Amino-
3-methyl-5-phenyl-5-tricyclo[3.3.1.13,7]dec-1-yl-3,5-dihydro-4H-
imidazol-4-one (10b, A = 1-Adamantyl, R = Me, R3 = H, R4 =
OMe). Step a. To a cold (0 °C) solution of benzaldehyde
(10.15 mL, 0.1 mol) and triethylphosphite (19.1 mL, 0.1 mol)
was added dropwise chlorotrimethylsilane (12.6 mL, 0.1 mol)
over a 10 min period. After the addition was complete, the ice
bath was removed and the reaction mixture was warmed up to
120 °C for 8 h and distilled (180 °C, 10.0 mmHg) to afford
diethyl 1-phenyl-1-(trimethylsilyloxy)methane phosphonate as
a colorless oil (25 g, 79% of yield): MS m/e (M þ H)þ 317; 1H
NMR (400 MHz, CDCl3) δ 0.08 (s, 9H), 1.22 (m, 6H), 4.01 (m,
4H), 4.97 (d, 1H), 7.32 (m, 3H), 7.44 (m, 2H).
[3.3.1.13,7]dec-1-ylethane-1,2-dione (6.71 g, 22.5 mmol) and
1-methylguanidine hydrochloride (11.1 g, 101 mmol) in dioxane
(75 mL) and ethanol (100 mL) was stirred at room temperature
for 5 min, treated with an aqueous solution of sodium carbonate
(10.7 g, 101 mmol; 10 mL of water), heated at 85 °C with stirring
for 3.5 h, cooled to room temperature, and concentrated
in vacuo. Purification of the resultant residue by flash chro-
matography (silica, 95:5:0.5 methylene chloride/methanol/
concentrated ammonium hydroxide) afforded 2-amino-5-
(4-methoxyphenyl)-3-methyl-5-tricyclo[3.3.1.13,7]dec-1-yl-3,5-
dihydro-4H-imidazol-4-one as a white solid: 3.41 g (43%
yield), mp 150-155 °C; MS m/e 354 (M þ H)þ; 1H NMR
(300 MHz, CDCl3) and 1H NMR (400 MHz, DMSO-d6) δ 1.38
(m, 6H), 1.55 (m, 3H), 1.65 (m, 3H), 1.82 (bs,3H), 2.81 (s, 3H),
3.67 (s, 3H), 6.26 (brs, 2H), 6.79 (d, J = 8.85 Hz, 2H), 7.55 (m,
J = 8.85 Hz, 2H). Anal. (C17H17N3O2 0.2H2O) C, H, N.
3
Generation of the Enantiomers of 10b (R = Me, R3 = H, R4 =
OMe) Using Chiral HPLC Techniques. Racemic mixture of
2-amino-5-(4-methoxyphenyl)-3-methyl-5-tricyclo[3.3.1.13,7]dec-
1-yl-3,5-dihydro-4H-imidazol-4-one was separated to its two
enantiomers by HPLC on Chiralcel AD, 0.46 cm ꢀ 25 cm using
mobile phase EtOH/hexane (1:9 with 0.1% DEA) and a flow rate
of 1.0 mL/min to afford the (R)- and (S)-compounds.
Step b. To a cold (-78 °C) solution of diethyl 1-phenyl-1-
(trimethylsilyloxy)methane phosphonate (3.16 g, 10 mmol) in
THF was added dropwise lithium diisopropylamide (2 M, 5.25
mL) over a 10 min period. The reaction mixture was stirred for
another 30 min, treated with tricyclo[3.3.1.13,7]decane-1-carbo-
nyl chloride (2.09 g, 10 mmol) in THF, and heated slowly
to room temperature overnight. Under cooling the reaction
mixture was poured into saturated NH4Cl solution and extracted
with ethyl ether. The extracts were combined, dried over MgSO4,
and concentrated in vacuo. The resultant residue was purified
by flash chromatography on silica gel (hexanes/EtOAc, 95/5)
to afford diethyl {2-oxo-2-phenyl-1-tricyclo[3.3.1.13,7]dec-1-yl-1-
[(trimethylsilyl)oxy]ethyl}phosphonate as a colorless oil (2.1 g,
(S)-2-amino-5-(4-methoxyphenyl)-3-methyl-5-tricyclo[3.3.1.13,7]-
dec-1-yl-3,5-dihydro-4H-imidazol-4-one (A):mp 215 °C; [R]25 -17.4
(c 1%, MeOH); MS m/e 352 (M - H)-; H NMR (400 MHz,
1
DMSO-d6) δ1.38(m,6H),1.55(m,3H),1.65(m,3H),1.82(bs,3H),
2.81 (s, 3H), 3.67 (s, 3H), 6.26 (brs, 2H), 6.79 (d, J = 8.85 Hz, 2H),
7.55 (d, J = 8.85 Hz, 2H). Anal. (C17H17N3O2 0.7H2O) C, H, N.
3
(R)-2-Amino-5-(4-methoxyphenyl)-3-methyl-5-tricyclo[3.3.1.13,7]-
dec-1-yl-3,5-dihydro-4H-imidazol-4-one: mp 215 °C; [R]25 þ17.6
(c 1%, MeOH); MS m/e (M - H)- 352; H NMR (400 MHz,
1
DMSO-d6) δ1.38(m, 6H), 1.55 (m, 3H), 1.65(m, 3H), 1.82 (bs,3H),
2.81 (s, 3H), 3.67 (s, 3H), 6.26 (brs, 2H), 6.79 (d, J = 8.85 Hz, 2H),
7.55 (d, J = 8.85 Hz, 2H). Anal. (C17H17N3O2 0.3H2O) C, H, N.
1
43% yield, mp 136 °C): MS m/e (M þ H)þ 479.1; H NMR
3
Preparation of 2-Amino-5-(4-methoxyphenyl)-3-methyl-5-phenyl-
3,5-dihydro-4H-imidazol-4-one (10a, A =Ph, R3 = H, R4 = OMe,
R = Me). Step h. To a mixture of 1-(4-methoxyphenyl)-2-pheny-
lethane-1,2-dione (0.30 g, 1.2 mmol), ethanol (15 mL), and water
(4 mL) were added 1-methylguanidine hydrochloride (0.09 g,
1.2 mmol) and K2CO3 (0.49 g, 3.6 mmol). The mixture was refluxed
for 3 h and then cooled to room temperature, and the volatiles were
removed under reduced pressure. The residue was taken in water
(50 mL) and extracted with CHCl3. The organic extracts were dried
over MgSO4. Evaporation and purification by flash chromatogra-
phy on silica gel (ethyl acetate as the eluting solvent) produced
2-amino-5-(4-methoxyphenyl)-3-methyl-5-phenyl-3,5-dihydro-
4H-imidazol-4-one as a white solid (0.29 g, 80% yield). MS
(400 MHz, DMSO-d6) δ 0.21 (s, 9H), 1.13 (t, J = 7.6 Hz, 6H),
1.45-1.82 (m, 15H), 3.91 (dq, J= 7.6 Hz, 4H), 7.30-7.43(m, 5H).
Step c. A mixture of diethyl {2-oxo-2-phenyl-1-tricyclo-
[3.3.1.13,7]dec-1-yl-1-[(trimethylsilyl)oxy]ethyl}phosphonate
(2.1 g, 4.38 mmol), aqueous saturated NaHCO3 (1.5 mL), and
MeOH (7 mL) was heated at reflux temperature for 2 h,
cooled, acidified with HCl (2.5 mL, 2 N), and extracted with
ether. The ether extracts were combined, dried over MgSO4,
and concentrated in vacuo. Purification of this residue by
flash chromatography on silica gel (hexane/EtOAc, 95/5)
gave 1-phenyl-2-tricyclo[3.3.1.13,7]dec-1-ylethane-1,2-dione
as a yellow oil (0.21 g, 18% yield); MS m/e (M þ H)þ
268.15; 1H NMR (400 MHz, DMSO-d6) δ 1.70 (m, 6H),
1.90 (bs, 6H), 2.02 (m, 3H), 7.62 (m, 2H,), 7.79 (m, 3H).
Step d. A mixture of 1-phenyl-2-tricyclo[3.3.1.13,7]dec-1-
ylethane-1,2-dione (0.21 g, 0.78 mmol), Na2CO3 (0.25 g, 2.34
mmol), 1-methylguanidine hydrochloride (0.12 g, 1,01 mmol),
and H2O (0.70 mL) in dioxane (3.5 mL) and EtOH (3.5 mL) was
stirred at 80 °C for 18 h and concentrated in vacuo. The resultant
residue was dissolved in CHCl3, washed with water, dried over
K2CO3, and evaporated to dryness. Purification of this residue
by flash chromatography on silica gel (EtOAc/CH2Cl2/Et3N,
7.5/2/0.5) gave 5-(1-adamantyl)-2-amino-3-methyl-5-phenyl-
3,5-dihydro-4H-imidazol-4-one as a white solid (0.11 g, 43%
yield, mp 250 °C); MS m/e (M þ H)þ 324; 1H NMR (400 MHz,
DMSO-d6) δ 1.40 (m, 6H), 1.50 (m, 3H), 1.70 (m, 3H), 1.85 (bs,
3H), 2.85 (s, 3H), 6.40 (bs, 2H), 7.22 (m, 3H), 7.62 (m, 2H). Anal.
m/e 296 (M þ H)þ; H NMR (DMSO-d6, 300 MHz) δ 2.97
1
(s, 3H), 3.71 (s, 3H), 6.59 (b, 2H), 6.84 (d, J = 8.4 Hz, 2H), 7.21
(m, 1H), 7.26-7.35 (m, 4H), 7.41 (d, J = 8.4 Hz, 2H). Anal.
(C17H17N3O2 0.2H2O) C, H, N.
3
Route b. Preparation of 1-(4-Methoxy-3-methylphenyl)-2-
phenylethane-1,2-dione (8a, A = Ph, R, R3 = Me, R4 = OMe).
Steps f and g. A mixture of oxo(phenyl)acetic acid (2 g, 13.32
mmol), oxalyl chloride (3.89 g, 30.63 mmol), CH2Cl2 (30 mL), and
N,N-dimethylformamide (0.005 mL) was stirred at room tempera-
ture for 3 h. The volatiles were removed in vacuo, and the residue
was taken in THF (20 mL) and added into a cold (0 °C) mixture of
bromo(4-methoxy-3-methylphenyl)magnesium (1 M, 15.05 mL,
15.05 mmol), CuBr (1.08 g, 7.51 mmol), LiBr (1.3 g, 15.02 mmol),
and THF (30 mL). The mixture was stirred for 30 min, poured into
cold HCl (1 N), and extracted with ethyl ether. The organic
extracts were dried over MgSO4. Evaporation and purification
by flash chromatography (5% EtOAc/hexanes) gave a yellow solid
(C20H25N3O 0.4H2O) C, H, N.
3
Exemplified Analogues of Scheme 3. Route a. Preparation of
4-[2-Amino-4-(4-methoxy-3-methylphenyl)-1-methyl-5-oxo-4,5-
dihydro-1H-imidazol-4-yl]-2-pyridin-3-ylbenzonitrile (Stille Coupling,
20, R3 =Me,R4 =OMe,R2 =4-CN,R5,R6 =H,Y=C). Stepa.
Dichlorobis(tri-o-tolylphosphine)palladium(II) (39.6 mg, 0.05 mmol)
(0.92 g, 33% yield): MS m/e 254 (M)þ; H NMR (300 MHz,
1
DMSO-d6): δ 2.21 (s, 3H), 3.92 (s, 3H), 7.16 (d, J = 8.3 Hz, 1H),
7.62 (m, 2H), 7.76-7.81 (m, 3H), 7.9 (d, J = 8.24 Hz, 1H).

1156 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Malamas et al.
was added into a mixture of 2-bromo-4-[(4-methoxy-3-methyl-
phenyl)(oxo)acetyl]benzonitrile (200 mg, 0.56 mmol), 3-(tributyl-
stannyl)pyridine (268 mg, 0.73 mmol), and 1,2-diethoxyethane (6 mL).
The reaction flask was immersed into an oil bath of 145 °C, and the
mixture was stirred for 30 min and filtered to remove the catalyst.
Evaporation and purification by flash chromatography (hexanes/
EtOAc, 2/1), and crystallization from ethyl ether/hexanes gave
4-[(4-methoxy-3-methylphenyl)(oxo)acetyl]-2-pyridin-3-ylbenzo-
nitrile as a yellow solid (181 mg, 91% yield): MS m/e 357 (M þ
H)þ; 1H NMR (400 MHz, DMSO-d6) δ 2.21 (s, 3H), 3.93 (s, 3H),
7.18 (d, J = 8.69 Hz, 1H), 7.61 (m, 1H), 7.82 (d, J = 8.54, 1H),
7.84 (dd, J = 8.54, 1.99 Hz, 1 H), 8.07 (dd, J = 7.76, 1.67 Hz, 1H),
8.12 (m, J= 1.38Hz, 2H), 8.25(d, J = 8.08 Hz, 1H), 8.74 (m, 1H),
8.85 (d, 1.68 Hz, 1H).
(m, 3H), 6.83 (d, J = 8.7 Hz, 1H), 3.80 (s, 3H), 3.10 (s, 3H),
2.30 (s, 3H).
Step a. A mixture of 2-amino-5-(3-bromo-4-fluorophenyl)-
5-(4-methoxy-3-methylphenyl)-3-methyl-3,5-dihydro-4H-imi-
dazol-4-one (4.50 g, 11.1 mmol), 2-fluoropyridine-3-boronic
acid (2.34 g, 16.6 mmol), sodium carbonate (3.52 g, 33.2 mmol),
bis(triphenylphosphino)palladium(II) dichloride (0.389 g,
0.554 mmol), and triphenylphosphine (0.291 g, 1.11 mmol) in
1:1 toluene/EtOH (200 mL) was degassed and heated at 110 °C
for 2.5 h. The mixture was cooled to room temperature,
concentrated, and purified by flash chromatography (silica,
97:3:0.25 methylene chloride/methanol/concentrated ammo-
nium hydroxide) to afford a 1.78 g batch of 2-amino-5-[4-
fluoro-3-(2-fluoropyridin-3-yl)-phenyl]-5-(4-methoxy-3-methyl-
phenyl)-3-methyl-3,5-dihydroimidazol-4-one (2.43 g, 52%) as a
white solid: (95:5:0.25 methylene chloride/methanol/concen-
trated ammonium hydroxide): mp 75-80 °C; MS m/e 423
(M þ H)þ; 1H NMR (300 MHz, CD3OD) δ 8.23 (dt, J = 5.0,
1.0 Hz, 1H), 7.93 (dt, J = 7.5, 1.5 Hz, 1H), 7.51-7.34 (m, 3H),
7.21-7.08 (m, 3H), 6.84 (d, J = 8.0 Hz, 1H), 3.80 (s, 3H), 3.10 (s,
Step b. Sodium carbonate (214 mg, 2.02 mmol) in water
(2 mL) was added into a mixture of 4-[(4-methoxy-3-methyl-
phenyl)(oxo)acetyl]-2-pyridin-3-ylbenzonitrile (160 mg, 0.45
mmol), 1-methylguanidine hydrochloride (222 mg, 2.02 mmol),
dioxane (7 mL), and ethyl alcohol (9 mL). The reaction mixture
was stirred at 85 °C for 3 h. The volatiles were removed in vacuo,
and the residue was taken in chloroform and washed with water.
The organic extracts were dried over MgSO4. Evaporation and
purification by flash chromatography (EtOAc/MeOH, 15/1)
and crystallization from CHCl3/hexanes gave 4-[2-amino-4-(4-
methoxy-3-methylphenyl)-1-methyl-5-oxo-4,5-dihydro-1H-imi-
dazol-4-yl]-2-pyridin-3-ylbenzonitrile as a white solid (138 mg,
75% yield): MS m/e 412(M þ H)þ; 1H NMR (400 MHz, DMSO-
d6) δ 2.09 (s, 3H), 2.98 (s, 3H), 3.74 (s, 3H), 6.74 (brs, 2H), 6.86 (d,
J = 8.69 Hz, 1H), 7.22 (s, 1H), 7.26 (d, J = 8.54 Hz, 1H),
7.58 (m, 1H), 7.69 (s, 1H), 7.70 (s, 1H), 7.9-8.0 (m, 2H), 8.7
3H), 2.01 (s, 3H). Anal. (C23H20F2N4O2 0.3H2O) C, H, N.
3
Biological Methods. FRET-Based Peptide Cleavage Assays. A
homogeneous, continuous fluorescence resonance energy trans-
fer (FRET) was used to assess compound inhibition for BACE1,
BACE2, cathepsin D, pepsin, and renin activities.41 The BACE1
and BACE2 activities were based on the cleavage of peptide
substrate Abz-SEVNLDAEFR-Dpa (Swedish substrate), while
peptide substrate MOCAc-GKPILFFRLK (Dnp)-D-R-NH2
was used for cathepsin D and pepsin, and peptide substrate
RE(EDANS)-IHPFHLVIHTK(DABCYL)-R was used for
renin. Kinetic rates were calculated, and IC50 values were
determined by fitting the % inhibition as a function of
compound concentration to the Hill equation (y = ((B ꢀ Kn) þ
(100 ꢀ xn))/(Kn þ xn).
(m, 2H). Anal. (C24H21N5O2 0.8H2O) C, H, N.
3
Preparation of 2-Amino-5-(4-methoxy-3-methylphenyl)-
5-[3-(pyridin-3-yl)phenyl]-3,5-dihydro-4H-imidazol-4-one (Suzuki
Coupling, 21, R, R3 = Me, R4 = OMe, R2, R5, R6 = H, Y =
C). Step b. 1-(3-Bromophenyl)-2-(4-methoxy-3-methylphenyl)-
ethane-1,2-dione (0.4 g, 1.2 mmol) was dissolved in 1,4-dioxane
(10 m) and water (2 mL). To this was then added 3-pyridine-
boronic acid (0.18 g, 1.44 mmol) followed by K2CO3 (0.38 g,
2.76 mmol). The mixture was heated to 70 °C, and triphenylpho-
sphine (0.018 g, 0.069 mmol) was added followed by palladium
acetate (0.0083 g, 0.037 mmol) addition. The mixture was stirred
at 100 °C for 16 h, cooled to room temperature, and extracted
with CHCl3. The organics washed with 1 N NaOH and water
and dried over MgSO4. Evaporation and purification by flash
chromatography on silica gel (2:1 ethyl acetate/hexane) gave
1-(4-methoxy-3-methylphenyl)-2-(3-pyridin-3-ylphenyl)ethane-
1,2-dione as a yellow solid (0.3 g, 75% yield): MS m/e (M)þ 332;
1H NMR (DMSO-d6 300 MHz) δ 2.22 (s, 3H), 3.92 (s, 3H), 7.15
(d, J = 8.39 Hz, 1H), 7.53 (dd, J = 7.93, 3.2 Hz, 1H), 7.75 (d, J =
7.78 Hz, 1H), 7.82 (m, 2H), 7.91 (d, J = 7.78 Hz, 1H), 8.15
(m, 2H), 8.2 (s, 1H), 8.64 (m, 1H), 8.94 (d, J = 1.98 Hz, 1H).
Cell-Based Aβ Inhibition Assay. CHO-K1 cells recombinantly
expressing human wild-type APP (CHO-WT) were grown to
confluence and then treated with serum-free medium
(Ultraculture) supplemented with test compound in DMSO or
DMSO alone (vehicle) at a final [DMSO] of 0.1% (v/v). Con-
ditioned medium was harvested at 24 h and assayed using
streptavidin MSD plates, an electrochemiluminescent immu-
noassay with biotinylated mouse monoclonal antibody 6E10
(Signet, Dedham, MA) was used as capture, and rabbit anti-
Aβ40 or Aβ42 antibodies (Biosource, Camarillo, CA) were used
as detection antibodies, with a secondary of MSD ECL tagged
sheep antirabbit used for electrochemiluminescent amplification.
Data analysis of the MSD assay was performed by fitting the
percent inhibition as a function of compound concentration to a
four-parameter logistic curve, and the concentration at which
the cellular production of Aβ40 or Aβ42 was reduced by 50%
(EC50) was determined. Compound toxicity was assessed via
mitochondrial function using an MTS readout (MTS kit from
Promega); values are represented as LD50 or the dose of
compound that resulted in 50% of control signal (e.g., 50% of
the highest MTS signal).
X-ray Crystallography. Cloning/Expression of BACE1. A
human BACE1 secreting mammalian secreting cell line (CHO
cell line) was made by expressing a construct in which the
prodomain plus ectodomain of human BACE1 (residues
22-454) was fused to the honeybee melittin secretory leader
sequence at the 50 end, and the Fc region of IgG, separated from
the BACE sequence via an enterokinase cleavage site, was fused
at the 30 end of BACE1. The BACE1/Fc fusion protein was
affinity purified by protein-A sepharose, and removal of the Fc
domain was achieved with enterokinase cleavage. Purified
BACE1 protein was accomplished via sequential size-exclusion
chromatography. E. coli-derived expression material was
used for cocrystallographic studies. A codon-derived BACE1
E. coli expression construct was fused to a carboxy-terminal
6ꢀ HIS tag. After scale-up, inclusion bodies were purified and
Anal. (C23H22N4O2 0.5H2O) C, H, N.
3
Route b. Preparation of 2-Amino-5-[4-fluoro-3-(2-fluoropyri-
din-3-yl)phenyl]-5-(4-methoxy-3-methylphenyl)-3-methyl-3,5-di-
hydroimidazol-4-one (21, R, R3 = Me, R4 = OMe, R2 = 4-F,
R5 = 2-F, R6 = H, Y = C). Step b. A mixture of 1-(3-bromo-
4-fluorophenyl)-2-(4-methoxy-3-methylphenyl)ethane-1,2-dione
(10.4 g, 29.5 mmol) and 1-methylguanidine hydrochloride
(14.6 g, 133 mmol) in dioxane (154 mL) and ethanol (154 mL)
was stirred at room temperature for 5 min. A solution of
sodium carbonate (14.0 g, 133 mmol) in water (62 mL) was then
added and the mixture immersed into an oil bath at 85 °C
and stirred for 45 min. The reaction mixture was cooled to room
temperature and concentrated. Purification by flash chromato-
graphy (silica, 95:5:0.5 methylene chloride/methanol/concen-
trated ammonium hydroxide) afforded 2-amino-5-(3-bromo-4-
fluorophenyl)-5-(4-methoxy-3-methylphenyl)-3-methyl-3,5-di-
hydro-4H-imidazol-4-one (10.9 g, 90%) as a white solid: MS m/e
406 (M þ H)þ; 1H NMR (300 MHz, CD3OD) δ 7.59 (dd, J =
6.6, 2.4 Hz, 1H), 7.38 (ddd, J = 8.7, 4.8, 2.4 Hz, 1H), 7.18-7.03

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1157
protein was refolded and dialyzed. The prodomain was cleaved
with furin, and BACE1 was further purified by size exclusion
chromatography. The refolded E. coli derived BACE1 showed
the same enzymatic activity as the CHO-derived material (data
not shown).
Crystallization and X-ray Diffraction Analysis of BACE1.
Crystals were grown by hanging drop vapor diffusion at 18 °C
in drops containing 1.0 μL of protein stock solution (200 μM
protein, 20 mM Tris-HCl, pH 7.5, 250 mM NaCl, and 240 μM
compound of interested diluted from a 100 mM stock in DMSO)
mixed with 1.0 μL of well solution (6% PEG 3350, 0.1 M sodium
acetate, pH 5.4) and equilibrated against 0.5 mL of well solu-
tion. Rod shaped crystals grew in several days.
Data Collection. Crystals were drawn through a solution of
25% glycerol and 75% well solution and cooled rapidly in liquid
nitrogen. Diffraction data were recorded at the ALS beamline
5.0.1 on a q-210 CCD camera. Intensities were integrated and
scaled using the programs Denzo and Scalepack.43
Phasing, Model Building and Refinement. Structures were
determined by molecular replacement using AmorE44 and
the apo structure of BACE1 (PDB code 1W50) as the search
model. The final structures were obtained after several iterative
cycles of refinement using Phenix45 and model improvement in
Coot.46
Data Deposition. The atomic coordinates of the BACE1
crystal structure for compounds 2 (3IGB),41 29 (3IND), (S)-34
(3INE), 37 (3INF), and (R)-61 (3INH) have been deposited in
the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, New Brunswick, NJ.
Molecular Modeling. Docking calculations were performed
using the QXP software package.47 Once the X-ray ligands were
minimized in the active site, constrained simulated annealing
calculations were performed to relieve any artifacts of the X-ray
refinement perceived as unfavorable interactions or strain by the
modified AMBER force field with QXP. The X-ray ligand was
then redocked to confirm that the lowest energy pose is in
agreement with the X-ray structure. Once the binding site model
was generated, docking of analogues was performed using the
QXP Monte Carlo docking algorithm MCDOCK in combina-
tion with CombiDOCK. Visualization of X-ray structures and
docking results was performed using the Insight II software
package (www.accelrys.com, Accelrys, Inc., San Diego, CA).
Conformational analysis was performed using Macromodel
(Macromodel 8.0; Schrodinger, LLC, Portland, OR), with the
OPLS-AA force fields, and a GBSA solvation model.
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Supporting Information Available: X-ray crystallographic
data (collection details, refinement statistics), 1H NMR and
analytical data of intermediates and final compounds not listed
in the Experimental Section, and supplemental information of
biological assays. This material is available free of charge via the
Internet at http://pubs.acs.org.
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