Hydroxyethylamine-based inhibitors of BACE1: P1-P3 macrocyclization can improve potency, selectivity, and cell activity

Article abstract of DOI:10.1016/j.bmcl.2013.05.028

We describe a systematic study of how macrocyclization in the P ₁-P₃ region of hydroxyethylamine-based inhibitors of β-site amyloid precursor protein (APP)-cleaving enzyme (BACE1) modulates in vitro activity. This study reveals that in a number of instances macrocyclization of bis-terminal dienes leads to improved potency toward BACE1 and selectivity against cathepsin D (CatD), as well as greater amyloid β-peptide (Aβ)-lowering activity in HEK293T cells stably expressing APP_SW. However, for several closely related analogs the benefits of macrocyclization are attenuated by the effects of other structural features in different regions of the molecules. X-ray crystal structures of three of these novel macrocyclic inhibitors bound to BACE1 revealed their binding conformations and interactions with the enzyme.

Full text of DOI:10.1016/j.bmcl.2013.05.028

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4459–4464
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Hydroxyethylamine-based inhibitors of BACE1: P1–P3
macrocyclization can improve potency, selectivity, and cell activity
b
c
c
Lewis D. Pennington a, , Douglas A. Whittington , Michael D. Bartberger , Steven R. Jordan ,
Holger Monenschein d, , Thomas T. Nguyen a, Bryant H. Yang a, Qiufen M. Xue a, Filisaty Vounatsos e,
Robert C. Wahl c, Kui Chen c, Stephen Wood f, Martin Citron g, , Vinod F. Patel h, , Stephen A. Hitchcock d,
Wenge Zhong a
⇑
,
a Medicinal Chemistry, Amgen, One Amgen Center Drive, Thousand Oaks, CA 91320, USA
b Molecular Structure and Characterization, Amgen, 360 Binney Street, Cambridge, MA 02142, USA
c Molecular Structure and Characterization, Amgen, One Amgen Center Drive, Thousand Oaks, CA 91320, USA
d Takeda Pharmaceuticals, 10410 Science Center Drive, San Diego, CA 92121, USA
e Chemical Process R&D, Amgen, One Amgen Center Drive, Thousand Oaks, CA 91320, USA
f Neuroscience Research, Amgen, One Amgen Center Drive, Thousand Oaks, CA 91320, USA
g Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
h Sanofi-Aventis, 270 Albany Street, Cambridge, MA 02139, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe a systematic study of how macrocyclization in the P1–P3 region of hydroxyethylamine-based
inhibitors of b-site amyloid precursor protein (APP)-cleaving enzyme (BACE1) modulates in vitro activity.
This study reveals that in a number of instances macrocyclization of bis-terminal dienes leads to
improved potency toward BACE1 and selectivity against cathepsin D (CatD), as well as greater amyloid
b-peptide (Ab)-lowering activity in HEK293T cells stably expressing APPSW. However, for several closely
related analogs the benefits of macrocyclization are attenuated by the effects of other structural features
in different regions of the molecules. X-ray crystal structures of three of these novel macrocyclic inhib-
itors bound to BACE1 revealed their binding conformations and interactions with the enzyme.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 2 April 2013
Revised 1 May 2013
Accepted 7 May 2013
Available online 16 May 2013
Keywords:
Conformational constraint
Alzheimer’s disease
BACE1 inhibitor
Macrocycle
b-Secretase
Aloysius Alzheimer reported in 1907 a previously unrecognized
neurodegenerative disease clinically presented as a presenile and
progressive loss of cognitive function and dementia, and character-
ized by the presence of neurofibrillary tangles and amyloid plaques
in the brains of deceased patients.1,2 These biochemical hallmarks
of Alzheimer’s disease (AD) and their involvement in its etiology
have been studied extensively over the last 30 years.3 The amyloid
cascade hypothesis posits that the accumulation and aggregation
of amyloid b-peptide (Ab) is neurotoxic, triggering a variety of
pathogenic processes leading to cognitive impairment and neuro-
nal death.3,4 Since Alzheimer’s initial description of this epony-
mous disease, aged populations have increased globally and a
disease-modifying therapy has remained a critical unmet medical
need for this grievous and costly illness.5
and Ab42 was revealed in 1999 to be mediated by b-site APP-cleav-
ing enzyme (BACE1).6 Subsequently, gene-knockout studies in
mice7 and genome-wide association studies in humans8 have lent
further credence to the promise of this enzyme as a therapeutic
target for AD. Despite tremendous effort by the biomedical re-
search community, however, no inhibitor of BACE1 has yet been
approved for the treatment of AD.9 That BACE1 is an aspartyl
protease situated within neurons of the central nervous system
(CNS) and shielded by the blood–brain barrier (BBB) presents a
significant obstacle to this endeavor.9,10 Another challenge is
achieving selectivity against cathepsin D (CatD), a related aspartyl
protease with high sequence homology to BACE1 at the active site,
the inhibition of which may give rise to toxic side-effects.11,12
The first X-ray crystal structure of BACE1 was reported in 2000
as a complex with OM99-2, a potent peptide-based inhibitor incor-
The rate-limiting first step of the proteolysis cascade converting
b-amyloid precursor protein (APP) to the pathogenic peptides Ab40
porating
a
hydroxyethylene transition state isostere (BACE1
Ki = 0.0016
l
M).13,14 Over the last 13 years, myriad chemotypes
have been disclosed as BACE1 inhibitors, including peptides and
a wide variety of peptidomimetic and heterocyclic compounds.9
In patent applications published in December 2002, Pulley et al.
⇑
Corresponding author. Tel.: +1 805 447 0048; fax: +1 805 480 1337.
E-mail addresses: lewpenn@icloud.com, lewisp@amgen.com (L.D. Pennington).
Current address.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.028

4460
L. D. Pennington et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4459–4464
O
diene substrates was envisioned to allow ready comparison of both
NH
the different chain-length dienes and their cognate methylene-
linked macrocycles (e.g., 5: R1 = –CH2CHCH2, R2 = Me, R3 = –
[CH2]nCHCH2, X = CH; 6: n = 1–3, R2 = Me, X = CH).22 Though un-
clear at the outset of these studies what ring size and conformation
would be optimal, preliminary computational studies suggested
that 13- and 14-membered rings would be favored (i.e., 6: n = 2
or 3).23 Our group has also explored hydroxyethylamine-based
inhibitors containing a pyridone ring with a variety of substituents
penetrating into the S2 and S3 pockets of BACE1 (e.g., 7; BACE1
O
OH
H
N
HN
CF3
O
O
O
1
S
N
F
O
O
H
N
HN
N
H
O
IC50 = 0.076 lM; CatD IC50 >10 lM; Cell Ab40 IC50 = 0.21 lM;
Fig. 2).24 Anticipating that modification of the linker region of the
simple methylene-linked macrocycles depicted in Figure 2 might
be necessary to improve BACE1 potency and CatD selectivity, as
well as to modulate physicochemical properties, evaluation of
more complex macrocycles based on inhibitor 7 was also planned.
The results of the study are detailed in Table 1. Dienes 8, 9, and
10 exhibit similar potency toward BACE1 (BACE1 IC50 = 0.11, 0.045,
2
NH
O
OH
H
N
H
N
N
H
Ph
HN
H
N
O
O
O
O
O
3
O
N
OH
H
N
and 0.092 l
M, respectively)25,26 and similarly increased potency
HN
against CatD (CatD IC50 = 0.029, 0.0063, and 0.036 lM, respec-
O
tively).27 Interestingly, whereas dienes 8 and 9 display comparable
decreases in cell potency versus biochemical potency (Cell Ab40
4
IC50 = 1.5 and 0.81
lM, respectively; Cell Ab40 IC50/BACE1
Figure 1. A selection of early macrocyclic inhibitors of BACE1.
IC50 = 14 and 18, respectively), the more lipophilic diene 10 shows
a much greater cell shift (Cell Ab40 IC50 >10 lM; Cell Ab40 IC50/
BACE1 IC50 >110).28 Macrocycles 12, 13, and 14 display some anal-
ogous, and several notably different, bioassay trends from their
respective parental dienes 8, 9, and 10. Whereas cyclization of
diene 8 to 12-membered macrocycle 12 results in nearly identical
potency toward BACE1 (BACE1 IC50 = 0.12 lM), cyclization of 9 and
10 to 13- and 14-membered macrocycles 13 and 14, respectively,
R3
HN
O
O
OH
( )n
OH
P1−P3
H
N
H
N
HN
macrocyclization
R1
O
O
optimal ring size?
favorable conformation?
R2
X
R2
X
leads to improved BACE1 potency (BACE1 IC50 = 0.017 and
5
6
0.036
cycles 12, 13, and 14 exhibit decreased potency against CatD (CatD
IC50 = 0.23, 0.37, and 0.89 M, respectively). The CatD selectivity is
lM, respectively). In contrast to dienes 8, 9, and 10, macro-
P2
O
N
O
l
especially favorable for macrocycles 13 and 14 (CatD IC50/BACE1
IC50 = 22 and 25, respectively). Compared to parental dienes 8
and 9, macrocycles 12 and 13 also display a much smaller cell shift
(Cell Ab40 IC50 = 0.25 and 0.058 lM, respectively; Cell Ab40 IC50/
BACE1 IC50 = 2.1 and 3.4, respectively). Although a much greater
N
O
OH
H
N
HN
CF3
P3
P1
7
cell shift was observed for the more lipophilic macrocycle 14 (Cell
Ab40 IC50 = 0.96 lM; Cell Ab40 IC50/BACE1 IC50 = 27), the magnitude
Figure 2. Strategy for pursuing macrocyclization in the P1–P3 region of hydroxy-
ethylamine-based inhibitors of BACE1.
is still less than that observed for its diene precursor 10. As previ-
ous studies in our laboratory revealed that a neopentyl group at C6
of the chroman moiety increased BACE1 potency and N atom sub-
stitution at C8 of this ring system mitigated cell shifts in potency,
albeit with lower CatD selectivity (Fig. 2; e.g., 5: R1 = H, R2 = t-Bu,
revealed the first macrocyclic inhibitors of BACE1, including
hydroxyethylamine-based macro-bis-lactam 1 (Fig. 1; BACE1 IC50
<50
Stachel,16 Ghosh,17 and Machauer18 unveiled BACE1-inhibiting
macrocycles including (BACE1 IC50 = 0.0040 M), (BACE1
Ki = 0.025 M), and 4 (BACE1 IC50 = 0.0020 M), respectively. Since
l
M).15 Subsequent early reports from the laboratories of
R3 = Me, X = N; BACE1 IC50 = 0.0058
Cell Ab40 IC50 = 0.0031
l
M; CatD IC50 = 0.0045
lM;
M),21 we desired to incorporate these
2
l
3
l
l
l
structural motifs into the best diene/macrocycle pair (9/13).
Although the resultant diene 11 exhibits nearly 10-fold better
these seminal reports, the appeal of macrocycles19 as conforma-
tionally constrained congeners of existing acyclic chemotypes with
potentially improved potency, selectivity, and other drug-like
properties has led to several other disclosures of macrocyclic inhib-
itors of BACE1.20
potency toward BACE1 (BACE1 IC50 = 0.0047
increased potency against CatD and a large cell shift (CatD
IC50 = 0.00084 M; Cell Ab40 IC50 = 0.066 M; Cell Ab40 IC50/BACE1
IC50 = 14). Compared to macrocycle 13, macrocycle 15 shows a ꢀ3-
fold increase in potency toward BACE1 (BACE1 IC50 = 0.0061 M)
and negated selectivity against CatD (CatD IC50 = 0.0043 M);
pleasingly, 15 displays negligible cell shift (Cell Ab40
IC50 = 0.0059 M).
lM), it also displays
l
l
Our laboratory recently disclosed hydroxyethylamine-based
inhibitors of BACE1 represented by generic structure 5 (Fig. 2;
l
l
e.g., R1 = H, R2 = Me, R3 = Me, X = CH; BACE1 IC50 = 0.11
IC50 = 0.70 lM; Cell Ab40 IC50 = 0.37 l
l
M; CatD
a
M).21 Inspired by the initial
l
reports of macrocycles 1–4 in Figure 1 and the analysis of several
X-ray crystal structures of Amgen inhibitors bound to BACE1,21
we began our exploration of macrocyclization in late 2004. We
desired first to examine simple methylene-linked macrocycles clo-
sely related to 5 to allow rapid entry into this arena (e.g., 5 ? 6;
Fig. 2). A divergent synthesis hinging upon a late-stage ring-closing
methathesis (RCM)–reduction sequence of simple bis-terminal
An overlay of the X-ray crystal structures of 7, 13, and 14 bound
to BACE1 is depicted in Figure 3.29 As predicted,23 13 and 14 dis-
play similar binding conformations and key contacts with the en-
zyme, with only minor differences in the linker region. The
conformational constraints afforded upon macrocyclization of 9
and 10 may help to explain the increased BACE1 potency and
improved CatD selectivity of 13 and 14.30 The large number of

L. D. Pennington et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4459–4464
4461
Table 1
P1–P3 macrocyclization of hydroxyethylamine-based inhibitors of BACE1: effects on potency, selectivity, and cell activity
O
O
( )n
OH
( )n
OH
1. RCM
2. reduction
H
N
H
N
HN
HN
O
O
R2
X
R2
X
8 (n =1, R2 = Me, X = CH)
9 (n =2, R2 = Me, X = CH)
10 (n =3, R2 = Me, X = CH)
11 (n =2, R2 = t-Bu, X = N)
12 (n = 1, R2 = Me, X = CH)
13 (n = 2, R2 = Me, X = CH)
14 (n = 3, R2 = Me, X = CH)
15 (n = 2, R2 = t-Bu, X =N)
R1
R1
O
O
1. RCM
N
O
2. reduction
N
O
( )n
( )n
OH
R2
OH
R2
H
N
H
N
HN
HN
except for
22 and 23
O
O
X
X
16 (n =1, R1 = Br, R2 = Me, X = CH)
19 (n = 1, R1 = H, R2 = Me, X = CH)
17 (n =1, R1 = 2-pyridyl, R2 = Me, X = CH)
18 (n =1, R1 = N-pyrrolidinoyl, R2 = Me, X = CH)
20 (n = 1, R1 = 2-pyridyl, R2 = Me, X = CH)
21 (n = 1, R1 = N-pyrrolidinoyl, R2 = Me, X = CH)
22 (n = 1, R1 = H, R2 = t-Bu, X = N)
23 (n = 0, R1 = H, R2 = t-Bu, X = N)
a
b
c
Compd
n
R1
R2
X
BACE1 IC50
(l
M)
CatD IC50
(
lM)
Cell Ab40 IC50
(
lM)
clogPd
diene ? macro.
diene ? macro.
diene ? macro.
diene ? macro.
diene ? macro.
8 ? 12
9 ? 13
1
2
3
2
1
1
1
1
0
—
—
—
—
Me
Me
Me
t-Bu
Me
Me
Me
t-Bu
t-Bu
CH
CH
CH
N
CH
CH
CH
N
0.11 ? 0.12
0.029 ? 0.23
0.0063 ? 0.37
0.036 ? 0.89
0.00084 ? 0.0043
<0.0020 ? 0.072
0.078 ? 0.054
0.029 ? 0.0081
NDg ? <0.0020
NDg ? 0.0020
1.5 ? 0.25
0.81 ? 0.058
>10 ? 0.96
0.066 ? 0.0059
0.17 ? 0.054
0.21 ? 0.014
0.085 ? 0.0020
NDg ? 0.0051
NDg ? 0.0078
5.9 ? 5.6
6.4 ? 6.2
6.9 ? 6.7
6.7 ? 6.5
6.5 ? 5.0
6.4 ? 5.9
5.5 ? 5.0
NDg ? 5.4
NDg ? 4.8
0.045 ? 0.017
0.092 ? 0.036
0.0047 ? 0.0061
0.081 ? 0.0025
0.12 ? 0.0059
0.010 ? 0.0025
NDg ? 0.0097
NDg ? 0.0027
10 ? 14
11 ? 15
16 ? 19
17 ? 20
18 ? 21
NSf ? 22
NSf ? 23
Br ? He
2-pyridyl
N-pyrrolidinoyl
H
H
N
a
b
c
d
e
f
Data generally represents an average of at least two determinations, see Ref. 26.
Data generally represents an average of at least two determinations, see Ref. 27.
Data generally represents an average of at least two determinations, see Ref. 28.
Calculated using Daylight Chemical Information Systems software (web site: http://www.daylight.com).
The Br atom of 16 was cleaved to the H atom of 19 during the hydrogenation step of the RCM–reduction sequence.
NS: not synthesized.
ND: no data.
g
thermally accessible conformers available to untethered dienes 9
and 10 (the majority of which may be undesirable for BACE1 bind-
ing) are greatly reduced in constrained macrocycles 13 and 14,
thereby increasing the population of desired, bioactive conformer;
additionally, undesired low-energy conformers potentially capable
of inhibition of CatD are eliminated from the conformational
ensemble.30
Comparison of the X-ray crystal structures of 7 and 14 sug-
gested that a substituted pyridone ring might be readily accommo-
dated in the linker region as part of a 14-membered ring (Fig. 3). As
mentioned above, our laboratory has previously explored hydroxy-
ethylamine-based inhibitors containing a pyridone ring with a
variety of substituents penetrating into the S2 and S3 pockets of
BACE1 (e.g., 7; Fig. 2).24 For this focused effort incorporating the
pyridone moiety into macrocycles, we chose to examine pyridonyl
linkers bearing H-, 2-pyridyl-, and N-pyrrolidinoyl substituents at
the P2 position (Table 1). Compared to the identical chain-length
diene 10, bromo- and 2-pyridyl-substituted pyridonyl dienes 16
and 17 display similar potency toward BACE1 (BACE1
CatD (CatD IC50 = 0.078 and 0.029 lM, respectively). In contrast
to diene 10, dienes 16 and 17 display a minimal decrease in cell po-
tency versus biochemical potency (Cell Ab40 IC50 = 0.17 and
0.21
respectively); however, the less lipophilic diene 18 exhibits a siz-
able cell shift (Cell Ab40 IC50 = 0.085 M; Cell Ab40 IC50/BACE1
lM, respectively; Cell Ab40 IC50/BACE1 IC50 = 2.1 and 1.8,
l
IC50 = 8.5). The 14-membered pyridone-containing macrocycles
19, 20, and 21, derived from dienes 16, 17, and 18, respectively, ex-
hibit a few similar, and many quite divergent, bioactivity trends
from their parental dienes and the 14-membered methylene-
linked macrocycle 14.31 Like 14, macrocycles 19, 20, and 21 display
improved potency toward BACE1 versus their diene precursors
(BACE1 IC50 = 0.0025, 0.0059, and 0.0025 lM, respectively), with
the respective 32- and 20-fold improvement for 19 and 20 being
particularly dramatic.31 Although the selectivity against CatD is
quite pronounced for macrocycle 19 (CatD IC50 = 0.072
IC50/BACE1 IC50 = 29), the CatD selectivity for macrocycles 20 and
21 is greatly diminished (CatD IC50 = 0.054 and 0.0081 M, respec-
tively; CatD IC50/BACE1 IC50 = 9.2 and 3.2, respectively). Whereas
macrocycle 19 displays a large cell shift (Cell Ab40 IC50 = 0.054 M;
Cell Ab40 IC50/BACE1 IC50 = 22), that observed for macrocycles 20
and 21 is minimal (Cell Ab40 IC50 = 0.014 and 0.0020 M, respec-
lM; CatD
l
IC50 = 0.081 and 0.12
substituted pyridonyl diene 18 exhibits nearly 10-fold better
BACE1 potency (BACE1 IC50 = 0.010
M).31 Interestingly, whereas
diene 16 displays greatly increased CatD potency (CatD IC50
<0.0020 M), dienes 17 and 18 are roughly equipotent against
lM, respectively), whereas N-pyrrolidinoyl-
l
l
l
tively; Cell Ab40 IC50/BACE1 IC50 = 2.4 and 0.80, respectively). As
we did for the simple methylene-linked macrocycles discussed
l

4462
L. D. Pennington et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4459–4464
Figure 4. X-ray crystal structure of 19 bound to BACE 1 (cutaway surface view of
BACE1, colored by cavity depth). Flap residues not shown for clarity; key aspartate
residues colored in yellow.
Figure 3. Overlay of the X-ray crystal structures of 7 (ligand in transparent white;
aligned backbone of protein not shown for clarity), 13 (ligand and protein in
yellow), and 14 (ligand in blue and protein in green) bound to BACE1. Flap and
catalytic residues rendered in stick view with key H-bonding contacts indicated.
O
above, we wanted to evaluate pyridone-containing macrocycles
bearing a neopentyl group at C6 and N atom substitution at C8 of
the chroman moiety. Compared to the C6-ethyl/C8-CH group mac-
rocycle 19, the C6-neopentyl/C8-N atom macrocycle 22 displays
unexpectedly decreased potency toward BACE1, as well as unfa-
MgBr
+
O
a
b
O
Br
Br
MeO2C
24
25
26
vorably reversed selectivity against CatD (BACE1 IC50 = 0.0097
CatD IC50 <0.0020 M), but as anticipated, 22 exhibits a negligible
cell shift (Cell Ab40 IC50 = 0.0051 M). As observed for methylene-
lM;
H
N
O
O
O
BocHN
BocHN
l
c,d
e
l
Br
Br
linked macrocycle 15, pyridone-containing macrocycle 23 shows
excellent potency toward BACE1 with only a small cell shift, but
28
29
27
no selectivity against CatD (BACE1 IC50 = 0.0027
lM; CatD
OH
H
N
IC50 = 0.0020 M; Cell Ab40 IC50 = 0.0078 M). The X-ray crystal
l
l
H2N
BocHN
f
g,h
structure of macrocyclic inhibitor 19 bound to BACE1 is depicted
O
O
in Figure 4.29
The synthesis of diene 9 and macrocycle 13 is described in
Scheme 1 and exemplifies the general manner in which com-
pounds 8–21 in Table 1 were acquired. Commercially available
compounds 3-bromobenzylmagnesium bromide (24) and (R)-
methyl glycidate (25) were coupled at ꢁ78 °C to give ketone 26
in 57% yield.32 Reductive amination of 26 using allylamine and
tetramethylammonium triacetoxyborohydride afforded amine 27
in 60% yield as a 12:1 mixture of diastereomers favoring the
desired S isomer.33 The allyl group of 27 was cleaved by treatment
with (Ph3P)4Pd/1,3-dimethylbarbituric acid34 and the liberated
amine was re-protected as its tert-butyl carbamate 28. Suzuki
cross-coupling of 28 and commercially available allylboronic acid
pinacol ester provided 29 in 70% yield.35 Heating an ethanol solu-
tion of epoxide 29 and known chromanyl amine 3036 gave
hydroxyethylamine 31 in 46% yield. The Boc protecting group of
31 was removed by exposure to HCl and the resulting amine was
acylated with 5-hexenoic acid using O-(7-azabenzotriazol-1-yl)-
N,N,N0,N0-tetramethyluronium hexafluorophosphate (HATU) and
i-Pr2NEt to afford 9. Finally, ring-closing methathesis37 of bis-
terminal diene 9 was effected with Grubb’s II catalyst and hydroge-
nation of the crude RCM product (1 atm H2/10% Pd–C) afforded
macrocycle 13 in 13% yield for the two-step sequence.
30
31
O
O
OH
OH
H
N
H
N
HN
HN
i, j
O
O
9
13
Scheme 1. Chemical synthesis of diene 9 and macrocycle 13: (a) 1.5:1 Et2O–CH2Cl2,
ꢁ78 °C (57%); (b) allylamine, Me4N(AcO)3BH, 20:1 CH2Cl2–AcOH, 23 °C (60%;
dr = 12:1); (c) 1,3-dimethylbarbituric acid, (Ph3P)4Pd, CH2Cl2, 23 °C (70%); (d)
Boc2O, CH2Cl2, 23 °C (59%); (e) allylboronic acid pinacol ester, (Ph3P)4Pd, K2CO3,
THF, reflux (70%); (f) 29, EtOH, 70 °C (46%); (g) HCl, 4:1 CH2Cl2–dioxane, 23 °C; (h)
5-hexenoic acid, HATU, i-Pr2NEt, DMF, 23 °C (76%, 2 steps); (i) Grubb’s II RCM
catalyst, benzene, reflux; (j) H2, 10% Pd–C, EtOH, 23 °C (13%, 2 steps).
(34) was allylated using allyl bromide and K2CO3 to give pyridone
35 in excellent yield. Hydroboration of the alkene moiety of 35
with 9-BBN followed by Suzuki cross-coupling of the resulting
alkylborane with 33 (Pd(dppf)Cl2/Ph3As)38 gave 36 in 54% yield.
Sequential deprotection of 36 by cleavage of the carbamate
(TBSOTf/2,6-lutidene)39 and saponification of the ester (LiOH/
MeOH) gave the desired amino acid, which underwent macrolacta-
mization upon exposure to HATU/i-Pr2NEt to afford macrocycle 37
in 57% yield for the three-step process. Selective desilylation of 37
Macrocycles 22 and 23 were accessed by a different route, illus-
trated by the synthesis of 23 in Scheme 2. Known primary alcohol
3221 was silylated with TBSCl to afford bis-silyl ether 33. Commer-
cially available methyl 6-oxo-1,6-dihydropyridine-3-carboxylate

L. D. Pennington et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4459–4464
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OTBS
OH
OTBS
OTBS
BocHN
BocHN
a
Br
Br
32
33
CO2Me
CO2Me
O
N
OTBS
OTBS
c,d
BocHN
e,f,g
O
N
R
34: R = H
35: R = allyl
b
36
O
O
N
O
N
O
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h,i
N
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CHO
37
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O
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O
OH
H
N
H2N
HN
j
O
O
N
N
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39
23
Scheme 2. Chemical synthesis of macrocycle 23: (a) TBSCl, DMAP, i-Pr2NEt, CH2Cl2,
23 °C (84%); (b) allylbromide, K2CO3, DMF, 23 °C (92%); (c) 9-BBN, THF,
ꢁ10 ? 23 °C; (d) 33, Pd(dppf)Cl2, Ph3As, Cs2CO3, DMF, 70 °C (54%, 2 steps); (e)
TBSOTf, 2,6-lutidine, CH2Cl2, 23 °C; (f) 2.0 M aq LiOH, MeOH, 55 °C; (g) HATU, i-
Pr2NEt, DMF, 23 °C (57%, 3 steps); (h) HCl, 18:1 EtOH–dioxane, 0 °C (42%); (i) Dess–
Martin periodinane, NaHCO3, CH2Cl2, 23 °C; (j) 38, Na(AcO)3BH, trimethyl ortho-
formate, ClCH2CH2Cl, 23 °C; MeOH, 23 °C (48%, 2 steps).
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(HCl/EtOH/0 °C) provided the desired primary alcohol, which was
oxidized using Dess–Martin periodinane40 to afford aldehyde 38.
Finally, reductive amination of 38 with known aza-chromanyl
amine 3921 gave macrocycle 23 in 48% yield for the two-step
sequence.
In conclusion, this study demonstrates that for a number of
hydroxyethylamine-based inhibitors of BACE1, several in vitro bio-
activity improvements can be realized upon conversion of bis-ter-
minal dienes to the corresponding saturated P1–P3 macrocycles. In
general, the macrocycles give improved BACE1 potency, CatD
selectivity, and cell activity versus their parental dienes, with the
13-membered methylene-linked macrocycle 13 giving the best
balance of all these properties. Importantly, however, this study
also reveals that the benefits of macrocyclization can be attenuated
by the effects of other structural features in different regions of the
molecules.
Acknowledgments
21. (a) Monenschein, H.; Horne, D. B.; Bartberger, M. D.; Hitchcock, S. A.; Nguyen,
T. T.; Patel, V. F.; Pennington, L. D.; Zhong, W. Bioorg. Med. Chem. Lett. 2012, 22,
3607; (b) Dineen, T. A.; Weiss, M. M.; Williamson, T.; Acton, P.; Babu-Khan, S.;
Bartberger, M. D.; Brown, J.; Chen, K.; Cheng, Y.; Citron, M.; Croghan, M. D.;
Dunn, R. T.; Esmay, J.; Graceffa, R. F.; Harried, S. S.; Hickman, D.; Hitchcock, S.
A.; Horne, D. B.; Huang, H.; Imbeah-Ampiah, R.; Judd, T.; Kaller, M. R.; Kreiman,
C. R.; La, D. S.; Li, V.; Lopez, P.; Louie, S.; Monenschein, H.; Nguyen, T. T.;
Pennington, L. D.; San Miguel, T.; Sickmier, E. A.; Vargas, H. M.; Wahl, R. C.;
Wen, P. H.; Whittington, D. A.; Wood, S.; Xue, Q.; Yang, B. H.; Patel, V. F.; Zhong,
W. J. Med. Chem. 2012, 55, 9025; (c) Weiss, M. M.; Williamson, T.; Babu-Khan,
S.; Bartberger, M. D.; Brown, J.; Chen, K.; Cheng, Y.; Citron, M.; Croghan, M. D.;
Dineen, T. A.; Esmay, J.; Graceffa, R. F.; Harried, S. S.; Hickman, D.; Hitchcock, S.
A.; Horne, D. B.; Huang, H.; Imbeah-Ampiah, R.; Judd, T.; Kaller, M. R.; Kreiman,
C. R.; La, D. S.; Li, V.; Lopez, P.; Louie, S.; Monenschein, H.; Nguyen, T. T.;
Pennington, L. D.; Rattan, C.; San Miguel, T.; Sickmier, E. A.; Wahl, R. C.; Wen, P.
H.; Wood, S.; Xue, Q.; Yang, B. H.; Patel, V. F.; Zhong, W. J. Med. Chem. 2012, 55,
9009; (d) Kaller, M. R.; Harried, S. S.; Albrecht, B.; Amarante, P.; Babu-Khan, S.;
Bartberger, M. D.; Brown, J.; Brown, R.; Chen, K.; Cheng, Y.; Citron, M.; Croghan,
M. D.; Graceffa, R.; Hickman, D.; Judd, T.; Kriemen, C.; La, D.; Li, V.; Lopez, P.;
Luo, Y.; Masse, C.; Monenschein, H.; Nguyen, T.; Pennington, L. D.; San Miguel,
T.; Sickmier, E. A.; Wahl, R. C.; Weiss, M. M.; Wen, P. H.; Williamson, T.; Wood,
We thank Vivian Li and Alexander Long (Molecular Structure
and Characterization, Amgen) for providing the BACE1 protein
used in the structural studies. The Advanced Light Source is sup-
ported by the U.S. Department of Energy under Contract No. DE-
AC02-05CH11231.
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A
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22. We elected to reduce the crude RCM products and test only the saturated
derivatives to avoid potential complications arising from the possibility of
different alkene geometries.
23. Modeling of 6 (n = 2 or 3, R2 = Me, X = CH) suggested a reasonable alignment of
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Thr72 and Gly230) with co-crystal structures of potent acyclic inhibitors, while
retaining a low-energy conformation.
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a FRET substrate derived from
a
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