Design and synthesis of cell potent BACE-1 inhibitors: Structure-activity relationship of P1′ substituents

Article abstract of DOI:10.1016/j.bmcl.2009.09.061

Using structure-guided design, hydroxyethylamine BACE-1 inhibitors were optimized to nanomolar Aβ cellular inhibition with selectivity against cathepsin-D. X-ray crystallography illuminated the S1′ residues critical to this effort, which culminated in compounds 56 and 57 that exhibited potency and selectivity but poor permeability and high P-gp efflux.

Full text of DOI:10.1016/j.bmcl.2009.09.061

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6386–6391
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of cell potent BACE-1 inhibitors: Structure–activity
relationship of P10 substituents
a
a,
*
Jennifer M. Sealy a, Anh P. Truong a, Luke Tso a, Gary D. Probst a, , Jose Aquino , Roy K. Hom
,
*
Barbara M. Jagodzinska a, Darren Dressen a, David W. G. Wone a, Louis Brogley a, Varghese John a,
Jay S. Tung a, Michael A. Pleiss a, John A. Tucker a, Andrei W. Konradi a, Michael S. Dappen b,
Gergely Toth c, Hu Pan c, Lany Ruslim d, Jim Miller d, Michael P. Bova d, Sukanto Sinha d, Kevin P. Quinn e,
John-Michael Sauer e
a Department of Medicinal Chemistry, Elan Pharmaceuticals, 180 Oyster Point Boulevard, South San Francisco, CA 94080, United States
b Department of Process and Analytical Chemistry, Elan Pharmaceuticals, 180 Oyster Point Boulevard, South San Francisco, CA 94080, United States
c Department of Molecular Design, Elan Pharmaceuticals, 180 Oyster Point Boulevard, South San Francisco, CA 94080, United States
d Department of Biology, Elan Pharmaceuticals, 1000 Gateway Boulevard, South San Francisco, CA 94080, United States
e Department of Lead Finding, Drug Disposition, and Safety Evaluation, Elan Pharmaceuticals, 800 Gateway Boulevard, South San Francisco, CA 94080, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Using structure-guided design, hydroxyethylamine BACE-1 inhibitors were optimized to nanomolar Ab
cellular inhibition with selectivity against cathepsin-D. X-ray crystallography illuminated the S10 residues
critical to this effort, which culminated in compounds 56 and 57 that exhibited potency and selectivity
but poor permeability and high P-gp efflux.
Received 8 August 2009
Revised 14 September 2009
Accepted 16 September 2009
Available online 19 September 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Alzheimer’s disease
b-Secretase (BACE-1) inhibitor
Hydroxyethylamine (HEA)
Alzheimer’s disease (AD) is a form of senile dementia, charac-
terized by a progressive loss of memory and cognitive ability,
affecting 24 million elderly people worldwide.1 The pathology of
this neurodegenerative disorder uniquely manifests itself with
the presence of extraneuronal aggregation of plaques composed
of b-amyloid peptides (Ab).2 Ab-peptides are derived from the
sequential proteolytic cleavage of the b-amyloid precursor protein
produced no consistent phenotypic differences between their wild
type counterparts.8 These knockout mice are without compensa-
tory activity, thus devoid of the ability to generate Ab in the brain.9
Furthermore, modest reductions of BACE-1 activity have resulted
in significant reductions in plaque burden.10 Additionally, cleavage
of b-APP by BACE-1 is the rate-limiting step in Ab-peptide produc-
tion3 and releases the soluble N-terminal APP fragment (sAPP-b)
which triggers a cascade event ultimately leading to neurodegen-
eration.11 This validates BACE-1 and presents its clear advantages
(b-APP) by two aspartic acid proteases, referred to as b- and
c-
secretase, respectively.3 Inhibition of either protease has been
demonstrated to result in reduction of brain Ab-peptide in preclin-
ical studies.4 Inhibitors of either protease offer attractive candi-
dates as disease-modifying treatments for people afflicted with
this debilitating malady.5
over c-secretase as a therapeutic target.
Our goal was to develop cell potent BACE-1 inhibitors that were
selective over closely related aspartyl proteases such as cathepsin-
D (cat-D) due to potential toxicity.12 To effect this, we imple-
mented a strategy to exploit the sequence differences between
BACE-1 and cat-D in the S10 pocket.13 Herein, we describe a general
binding mode of hydroxyethylamines (HEA) to BACE-1, and specif-
ically the amino acid residues that comprise the S10 pocket. From
these differences arose the strategy toward selectivity while
enhancing cell potency.
Between these two proteases, b-secretase (BACE-1) is the more
alluring therapeutic target based on the following distinctions. c-
Secretase processes a myriad of substrates,6 such as Notch, raising
concerns about mechanism-based side-effects due to a deficiency
in selectivity.7 Conversely, gene deletion of BACE-1 in mice
The X-ray crystal structure of BACE-1 and compound 114 illus-
trates the active site binding mode (Figs. 1 and 2). The difluoroaryl,
cyclohexyl, and tert-butyl substituents occupy the S1, S10, and S20
pockets, respectively, as shown in Figures 1 and 2. The S10 pocket
* Corresponding authors. Tel.: +1 650616 2684; fax: +1 650794 2387 (G.D.P.),
tel.: +1 650616 5061; fax: +1 650794 2458 (R.K.H.).
E-mail addresses: gary.probst@elan.com (G.D. Probst), roy.hom@elan.com
(R.K. Hom).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.061

J. M. Sealy et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6386–6391
6387
Table 1
SAR of the P10 ring sizes and oxygen containing heterocycles
F
O
HN
6
F
R
OH
a
b
c
#
R
BACE IC50 (nM)
Cat D IC50 (nM)
Cell ED50 (nM)
1
7
8
47
25
22
17
N
H
N
N
59
61
4.0
N
H
O
230
170
130
4900
15
N
H
O
N
N
9d
10
270
220
2400
91
N
H
>100
>1000
N
H
O
N
N
11d
N
H
a
b
c
See Ref. 17.
See Ref. 18.
See Ref. 19.
1:1 mixture of epimers.
d
S1'
F
O
S1
HN
S2'
F
N
H
OH
1
aryllinker
Figure 1. Binding mode of the HEAs to BACE-1.
is amphiphilic in nature. The ‘backside’ is bordered by two hydro-
phobic residues Val332 and Ile226. Two hydrophilic residues
Lys227 and Thr329 constitute the ‘top’, and the ‘right side’ consists
of Tyr198 and Gly34 (Fig. 3). The ‘left side’ contains Arg235 and
Thr72 which is part of the flap, while Asp228 and Thr231 compose
the ‘bottom’ (Fig. 4). The protonated amino and hydroxyl group
binds at the scissle site to Asp32 and Asp228, respectively (not
shown). The carbonyl of Gly34 also forms a hydrogen bond with
the ionized amine and hydrogen on the aryl linker flanked by the
two alkyl groups (not shown).15 The acetamide functions as a bi-
dentate ligand with the carbonyl forming a hydrogen bond with
the flap residue Gln73 while the NH interacts with Gly230 (not
shown).
Figure 2. Crystal structure of 1 binding to truncated (56–455) human BACE-1 (1.8 Å
resolution). The PBD deposition code is 3ivh.
from 3 and added to a variety of cyclic ketones to produce tertiary
alcohols which were converted to azides followed by subsequent
reduction to amine 4. Alkylation of the epoxide 516 by the amine
4, deprotection, and acetylation of the primary amine afforded
the desired compound 1.
An exemplary synthetic route employed to construct the HEA
inhibitors is outlined in Scheme 1. An aryl lithium was generated

6388
J. M. Sealy et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6386–6391
We also examined the SAR of two series of nitrogen containing
heterocycles (Tables 2 and 3). The results from both series of pip-
erdines were disappointing except for the hydroxyl amine 19,
which displayed a similar cellular potency and a slight selectivity
for BACE-1 over cat-D relative to 1. The d-lactams 15 and 16 were
significantly more selective for BACE-1 over cat-D compared to 1
and slightly more potent against BACE-1 but with similar cellular
activity.
The d-lactam 23 in Table 3 was synthesized via the route out-
lined in Scheme 2. A palladium-catalyzed arylation20 between 24
and 3, followed by Michael addition with ethyl acrylate, and reduc-
tion of the nitrile afforded the d-lactam. Installation of the amine
Table 2
SAR of nitrogen containing heterocycles at the 4-position
Figure 3. The S10 pocket: the ‘backside’, ‘top’, and ‘right side’ residues.
R
N
F
O
X
HN
12
F
N
H
OH
a
b
c
#
X
R
BACE IC50 (nM) Cat D IC50 (nM) Cell ED50 (nM)
13 CH2
H
4700
>10,000
4300
330
710
>1000
21
14 CH2 CONH2 380
15d CO
16d CO
H
H
18
19
1100
23
a
b
c
See Ref. 17.
See Ref. 18.
See Ref. 19.
d
Separated single diastereomer.
Table 3
SAR of nitrogen containing heterocycles at the 3-position
F
O
Figure 4. The S10 pocket: the ‘left side’ and ‘bottom’ residues.
X
R
N
HN
17
F
N
During the course of our studies, we discovered that occupying
space in the S10 pocket resulted in a significant increase in potency
with six-membered rings being the most potent amongst the var-
ious ring sizes (e.g., 1 vs 10) (see Table 1). To further increase po-
tency and selectivity we sought to establish contact with the
hydrophilic residues, Lys227 and/or Thr329, at the ‘top’ of the S10
pocket by placing an oxygen atom in the ring at the position distal
to the amine. The pyran 8 did not enhance potency relative to 1 but
it did reverse the selectivity bestowing a modest fourfold increase
in selectivity for BACE-1 over cat-D. Molecular modeling suggested
that compounds 9 and 11 could make contact with the phenol of
Tyr198, but both modifications failed to increase potency.
H
OH
a
#
X
R
BACE IC50b (nM) Cat D IC50c (nM) Cell ED50d (nM)
18 CH2
19 CH2 OH
H
>10,000
400
2327
670
>1000
45
O
20 CH2
860
3.3
780
O
21 CH2 COMe
22 CH2 CONH2
23 CO
1300
330
47
410
90
180
560
210
76
H
a
b
c
All compounds are 1:1 mixture of epimers.
See Ref. 17.
See Ref. 18.
See Ref. 19.
F
d
O
a-c
I
Boc
O
+
HN
+
F
H2N
2
3
4
5
O
F
O
NH
a-g
NC CO2Et
d-f
HN
1
H2N
F
N
24
H
OH
25
Scheme 1. Reagents and conditions: (a) n-BuLi, THF, ꢀ78 °C, 65%; (b) TMSN3, BF3–
OEt2, CH2Cl2, reflux or TFA, NaN3, CH2Cl2, reflux, 45%; (c) LiAlH4, Et2O, 0 °C or 10%
Pd/C, H2, EtOAc, 100%; (d) N,N-diisopropylethylamine, isopropanol, reflux, 60%; (e)
4 N HCl in dioxane; (f) Ac2NOMe, triethylamine, CH2Cl2, 90% over two steps.
Scheme 2. Reagents and conditions: (a) 3, Pd2(dba)3–CHCl3, P(tert-Bu)3–HBF4,
Na3PO4, PhMe, 100 °C, 57%; (b) ethyl acrylate, EtONa, EtOH, 71%; (c) Raney Ni, H2,
EtOH, then 75 °C, 69%; (d) NH2NH2–H2O, EtOH; (e) NaNO2, AcOH, H2O; (f) 2-
trimethylsilylethanol, PhMe, reflux, 66% over three steps; (g) TBAF, THF, reflux, 78%.

J. M. Sealy et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6386–6391
6389
chemistry (Scheme 3). This ketone proved to be a versatile inter-
mediate to derivatize (e.g., reductive aminations). Compound 29
was constructed by standard chemistry previously discussed in
this paper and served as a versatile intermediate that led to the
synthesis of 34, 36, and 37 (Scheme 4 and Table 4).
O
O
O
a-c
d-f
H2N
O
F
O
F
26
27
O
Substituents trans to the amine were invariably more active
than the corresponding cis isomer (e.g., 33 vs 32, respectively) (Ta-
ble 4). The trans alcohol 33 was twice as potent as 1, had similar
cellular activity, and displayed a selectivity of 58-fold over cat-D.
In general, the BACE-1 SAR remained relatively flat with respect
to lipophilic or hydrophilic substituents placed at the 4-position
(33–34 and 36–39) until a breakthrough discovery with the ketox-
ime 43. This functional group simultaneously donates and accepts
hydrogen bonds which led to a fivefold increase in potency, nearly
2 orders of magnitude of improvement in selectivity over cat-D,
and a 19-fold enhancement in cellular activity relative to 1. This
moiety was hydrolytically unstable, so the search for other do-
nor/acceptors groups was initiated. Only the hydroxylamine 44,
O
HN
28
N
H
OH
Scheme 3. Reagents and conditions: (a) tert-butylsulfinamide, Ti(OEt)4, THF, 60%;
(b) 3, n-BuLi, THF, ꢀ78 °C, 47%; (c) HCl in Et2O, 95%; (d) 5, N,N-diisopropylethyl-
amine, isopropanol, reflux, 62%; (e) 4 N HCl in dioxane; (f) Ac2NOMe, triethylamine,
CH2Cl2, 71% over two steps.
OH
O
a-d
e-f
which may not be
a pharmaceutically acceptable functional
N3
group,21 was comparable to 43 in the enzymatic and cellular as-
says, and exhibited greater than 200-fold selectivity for BACE-1
over cat-D. Several other donor/acceptor functionalities resulted
in reduced potencies of 100–400 nM against BACE-1, but still
exhibited excellent cellular activities of 1.3–16 nM (45–49). An
N-aryl series (51–52) of inhibitors attained results comparable to
1 but were significantly more selective for BACE-1 over cat-D.
We hypothesized that an aromatic ring would be a chemically
stable surrogate for the ketoxime 43. The amine in intermediate
27 was protected as the carbamate, Bredereck’s reagent was uti-
lized to generate the vinylogous amide 54 which served as the
key intermediate for the rapid synthesis of a diverse set of hetero-
cycles such as the pyrazole 55 (Scheme 5).
H2N
30
O
O
26
29
Scheme 4. Reagents and conditions: (a) n-BuLi, Ph3PCH3Br, THF, 0 °C, 79%; (b) 10%
aq HCl, THF, 88%; (c) 3, n-BuLi, THF, ꢀ78 °C, 81%; (d) TMSN3, BF3–OEt2, CH2Cl2,
reflux, 37%; (e) 9-BBN, THF, then aq NaOH, 30% H2O2, 0 °C, 3:1 trans:cis 79%; (f) 10%
Pd/C, H2, EtOAc, 100%.
via a Curtius rearrangement followed by deprotection afforded 25.
Standard chemistry (vide supra) completed the synthesis of 23.
Next, our attention turned to the exploration of the substituents
at the 4-position of the cyclohexyl ring. Condensation of the ketone
26 with tert-butylsulfinamide followed by addition of an aryl lith-
ium, and subsequent removal of the sulfinyl group afforded inter-
mediate 27 which in turn produced 28 via previously described
Gratifyingly, the pyrazole 56 and 2-aminopyrimidine 57 was
nearly as potent as 43 in both the biochemical and cellular assays,
Table 4
SAR of substituents distal to the amine
R1 R2
F
O
HN
31
F
N
H
OH
R1
R2
BACE IC50 (nM)
Cat D IC50 (nM)
Cell ED50 (nM)
a
b
c
#
28
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
—
OH
H
H
H
—
H
H
H
H
H
H
—
H
H
H
H
H
H
H
H
H
H
@O
H
OH
CH2OH
CH2CH2OH
@CH2
Me
SMe
120
9200
24
38
>10,000
48
49
24
33
140
380
210
8.8
450
5.6
870
11
31
>1000
15
17
7.5
54
13
37
100
0.89
0.96
7.6
16
2.4
5.9
1.3
48
>10,000
1400
330
>10,000
22
80
84
28
130
640
2300
430
3900
530
2000
>10,000
7200
2300
>10,000
4400
3100
9800
CF3
OMe
CN
SO2Me
@NOH
NHOH
NHOMe
NHCO2Me
NHCHO
NHSO2Me
N(OH)Ac
NHAc
19
100
240
210
400
240
350
73
NH-2-pyridine
NH-2-thiazole
NH-3-pyridine
21
18
na
85
500
a
b
c
See Ref. 17.
See Ref. 18.
See Ref. 19.

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J. M. Sealy et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6386–6391
and exhibited selectivity over cat-D of 25- and 15-fold, respectively
(Table 5). Furthermore, both 56 and 57 exhibited excellent cell
ED50 of 2 nM. The co-crystal structure of the active diastereomer
of 56 (Fig. 5) clearly shows the pyrazole functioning as a bi-dentate
ligand hydrogen bonding to both Thr329 and Lys227 residues at a
distance of 2.9 Å (see Fig. 5).
NH
O
NMe2
N
O
O
a-c
d-e
HN
H2N
H2N
Boc
54
27
55
Scheme 5. Reagents and conditions: (a) (Boc)2O, 1,2-DCE, 60 °C, 85%; (b) AcOH,
H2O, 75 °C, 98%; (c) tert-butoxybis(dimethylamino)methane, 80 °C; (d) NH2NH2–
H2O, glacial AcOH, EtOH, 74% over two steps; (e) 4 N HCl in dioxane 100%.
Unfortunately, the permeability for all of these compounds was
poor except for the pyran 8, which displayed moderate permeabil-
ity (Table 6). More importantly from a drug properties perspective,
Table 6
Table 5
Permeability and P-gp efflux of selected inhibitors
SAR of appended aromatic rings
F
O
F
O
HN
6
F
R
HN
6
OH
F
R
OH
a
b
#
R
BACE IC50 (nM) Papp (nm/s) P-gp efflux ratioc
a
b
#
R
BACE IC50
(nM)
Cat D IC50c (nM) Cell ED50d (nM)
1
47
61
24
12
7
81
3
19
18
51
10
N
H
NH
N
56
57
58
12
14
93
300
210
910
2.1
1.8
N
H
O
8
NH2
N
N
H
N
OH
33
N
H
N
H
NH
N
N
N
56d
16
15
N
H
N
H
a
b
c
See Ref. 17.
See Ref. 22.
See Ref. 23.
1:1 mixture of epimers.
a
b
c
All compounds are 1:1 mixture of epimers.
See Ref. 17.
See Ref. 18.
See Ref. 19.
d
d
all of these compounds showed an unacceptable Permeability-gly-
coprotein (P-gp) efflux liability. This combination of poor perme-
ability and high efflux was not conducive to penetration into the
brain parenchyma to exert the desired pharmacodynamic effect.
In conclusion, via structure-guided design we synthesized cell
potent nanomolar BACE-1 inhibitors with good selectivity over
cat-D. The cell-to-enzyme ratios were excellent possibly due to
compartmentalization,24 resulting in higher local concentrations
relative to the BACE-1 assay. Unfortunately, the low compound
permeability coupled with unacceptable P-gp efflux liability led
to a lack of efficacy in wild type animal models that express P-gp
at their blood–brain barrier for acute reductions of brain Ab follow-
ing oral dosing. Subsequent efforts to address these liabilities will
be reported in due course.
Acknowledgements
We would like to thank Ronald Morgan, Wes Zmolek, Jackie
Kwong, Lee Latimer, Jill Labbe, Nancy Jewett, Pam Keim, Chris Fos-
ter, Christine Olivero, Heather Runes, and Colin Lorentzen for their
contributions to this work.
Figure 5. Crystal structure of truncated (56–455) human BACE-1 bound to the
more active diastereomer of 56 in green (2.2 Å resolution) overlaid with compound
1 in magenta. The pyrazole is depicted hydrogen bonding to the ‘top’ residues,
Lys227 and Thr329. The PBD deposition code is 3ivi.

J. M. Sealy et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6386–6391
6391
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17. Compounds serially diluted in DMSO were added to recombinant BACE
purified from E. coli in 100 mM sodium acetate buffer containing 0.001%
Tween-20 at pH 4.5 and allowed to incubate for 20 min at room temperature. A
biotinylated peptide substrate, based on the Swedish mutant APP sequence,
containing an Oregon Green moiety at the C-terminus, was added to initiate
the reaction which was allowed to proceed for 3 h at 37 °C. The reaction was
quenched by the addition of a fivefold volume excess of 100 mM sodium
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.09.061.
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