Design of an orally efficacious hydroxyethylamine (HEA) BACE-1 inhibitor in a preclinical animal model

Article abstract of DOI:10.1016/j.bmcl.2010.08.102

In this Letter, we describe our efforts to design HEA BACE-1 inhibitors that are highly permeable coupled with negligible levels of permeability- glycoprotein activity. These efforts culminate in producing 16 which lowers Αβ by 28% and 32% in the cortex and CSF, respectively, in the preclinical wild type Hartley guinea pig animal model when dosed orally at 30 mpk BID for 2.5 days.

Full text of DOI:10.1016/j.bmcl.2010.08.102

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6231–6236
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design of an orally efficacious hydroxyethylamine (HEA) BACE-1 inhibitor in
a preclinical animal model
Anh P. Truong a, Gergley Tóth b, Gary D. Probst a, , Jennifer M. Sealy a, Simeon Bowers a, David W. G. Wone a,
⇑
Darren Dressen a, Roy K. Hom a, Andrei W. Konradi a, Hing L. Sham a,c, Jing Wu c, Brian T. Peterson c,
Lany Ruslim d, Michael P. Bova d, Dora Kholodenko d, Ruth N. Motter d, Frédérique Bard d, Pamela Santiago e,
Huifang Ni e, David Chian e, Ferdie Soriano e, Tracy Cole e, Elizabeth F. Brigham e, Karina Wong f,
Wes Zmolek f, Erich Goldbach f, Bhushan Samant f, Linda Chen f, Hongbing Zhang f, David F. Nakamura f,
Kevin P. Quinn f, Ted A. Yednock a,b,c,d,e,f, John-Michael Sauer f
a Department of Medicinal Chemistry, Elan Pharmaceuticals, 180 Oyster Point Boulevard, South San Francisco, CA 94080, United States
b Department of Molecular Design, Elan Pharmaceuticals, 180 Oyster Point Boulevard, South San Francisco, CA 94080, United States
c Department of Process and Analytical Chemistry, 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 In Vivo Pharmacology, 800 Gateway Boulevard, South San Francisco, CA 94080, United States
f 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:
In this Letter, we describe our efforts to design HEA BACE-1 inhibitors that are highly permeable coupled
with negligible levels of permeability-glycoprotein activity. These efforts culminate in producing 16
which lowers Fb by 28% and 32% in the cortex and CSF, respectively, in the preclinical wild type Hartley
guinea pig animal model when dosed orally at 30 mpk BID for 2.5 days.
Received 3 August 2010
Revised 18 August 2010
Accepted 19 August 2010
Available online 24 August 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
BACE-1 inhibitor
Hydroxyethylamine (HEA)
Alzheimer’s disease
Fluorine
Alzheimer’s disease (AD) is a form of senile dementia, charac-
terized by a progressive loss of memory and cognitive ability,
affecting more than 35 million elderly people worldwide with
skyrocketing healthcare costs far exceeding $100 billion annually.1
The pathology of this neurodegenerative disorder uniquely mani-
fests itself with the presence of extraneuronal aggregation of pla-
ques composed of b-amyloid peptides (Ab).2 Intracellular
neurofibrillary tangles (NFTs), aggregates of aberrantly hyperphos-
phorylated tau proteins, are also a component of the pathology but
not exclusive to AD.3 Ab-peptides are derived from the sequential
proteolytic cleavage of the b-amyloid precursor protein (b-APP)
Between these two proteases, b-secretase (BACE-1) may be a
more alluring therapeutic target based on the following distinc-
tions. c
-Secretase processes a myriad of substrates,8 such as Notch,
raising concerns about mechanism-based side-effects due to a defi-
ciency in selectivity.9 Conversely, gene deletion of BACE-1 in mice
produced no consistent phenotypic differences between their wild
type counterparts.10 These knockout mice are without compensa-
tory activity, thus devoid of the ability to generate Ab in the
brain.11 Modest reductions of BACE-1 activity resulted in
significant reduction of plaque burden.12 Furthermore, cleavage
of b-APP by BACE-1 is the rate-limiting step in Ab-peptide produc-
tion4 and releases the soluble N-terminal APP fragment which
initiates a cascade event ultimately leading to apoptosis.13 Addi-
tionally, soluble oligomeric Ab42 indirectly signals for hyper-
phosphorylation of tau at AD-specific epitopes producing NFTs
that lead to neurotoxicity.14 Moderately abating the production
of Ab monomers results in a disproportionate reduction of syna-
ptotoxic soluble Ab oligomers15 leading to improved long-term
potentiation,16 a measure of memory and synaptic plasticity. Some
reports suggest that the severity of cognitive deficits in AD patients
by two aspartic acid proteases, referred to as b- and c-secretase,
respectively.4 Inhibition of either protease5 has been demonstrated
to result in reduction of brain Ab-peptide in preclinical studies.6
Inhibitors of either protease offer attractive candidates as poten-
tially disease-modifying, rather than palliative, treatments for peo-
ple afflicted with this debilitating malady.7
⇑
Corresponding author. Tel.: +1 650 616 2684; fax: +1 650 877 7486.
E-mail address: gary.probst@elan.com (G.D. Probst).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.08.102

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A. P. Truong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6231–6236
correlates better with brain levels of soluble Ab than fibrillized
Ab.17 These data validates BACE-1 and presents its clear advantages
S1'
F
O
S1
S2'
over
c-secretase as a therapeutic target, and buttresses the
HN
amyloid cascade hypothesis.18
F
N
19a
Previously, we reported our research concerning the S10
and
H
OH
S2019b pockets as well as the aryl linker19c of our hydroxyethylamine
(HEA) peptidomimetics. Permeability-glycoprotein (P-gp) efflux
activity has been the dominant impediment for the HEA template
to penetrate into the parenchyma of the brain and lower Ab. Herein,
we will describe the properties of the HEAs that led to our working
hypothesis concerning the pharmacokinetic deficiencies, and the
strategy to design highly permeable BACE-1 inhibitors devoid of
P-gp liability. The terminology used to classify the permeability
and recovery of the compounds is outlined in Table 1. The recovery
is a measure of the inhibitors affinity to embed in the membrane
(low recovery equates to high membrane affinity) vide infra.
The binding mode of our HEA BACE-1 inhibitors is as follows:
the difluoroaryl, cyclohexyl, and tert-butyl substituents occupy
the S1, S10, and S20 pockets, respectively (Fig. 1).19 The protonated
amine and hydroxyl group binds at the scissile site to Asp32 and
Asp228, respectively. The carbonyl of Gly34 forms a hydrogen
bond with the ionized amine and the hydrogen on the aryl linker
flanked by the two alkyl groups.20 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.
Compound 1, while potent, suffers from poor permeability
(7 nm/s) and low recovery (11%) in the in vitro Madin-Darby canine
kidney (MDCK) cell assay (Table 2). Highly lipophilic molecules that
possess a basic amine exhibit a pronounced affinity towards mem-
branes.21 The measured log P and pKa of 1 is 4.8 and 8.1, respectively.
Our working hypothesis involved the basic amine binding to the
polar head and the greasy portion partitioning into the lipophilic tail
of the membrane (Fig. 2). To overcome this liability, we sought to
lower the log P on the prime side and modulate the pKa of the amine
below the pH (7.4) of the medium. The P10 pyran 2 has a measured
log P of 4.3 and pKa of 7.2 which imparts a moderate rate of perme-
ability and an improved recovery which indicates a reduction in
membrane affinity. The pyrimidine 3 has a log P and pKa of 3.7 and
6.7, respectively, and these features led to excellent permeability
and recovery with heterocyclic aryl linkers in general. Similarly, a
pattern of increased permeability and recovery arose with a polar
P20 (4 and 5). Another trend emerged when a polar P10 combined
with a polar P20, 6 and 7, led to reduced permeability, relative to
2–5, in conjunction with high levels of recovery implying that
compounds of this archetype had capitulated membrane affinity.
Elevated P-gp efflux activity was also observed for 6. These results
indicated that a fine balance of physicochemical properties was
necessary to penetrate the blood brain-barrier (BBB). P-gp efflux
activity remained a significant obstacle as it was unaffected or exac-
erbated by these perturbations.
1
aryl linker
Figure 1. Binding mode of the HEAs.
Table 2
SAR of the cyclohexyl prime side binding regiona
F
O
HN
2
R
F
N
H
OH
#
R
BACE-1 IC50 (nM)
cell ED50 (nM)
Cat-D IC50 (nM)
Papp (nm/s)
% recovery
P-gp efflux ratio
log P @
pH =7.4
pKa
47
17
25
7 nm/s
11%
19
4.8
8.1
1
2
61
15
230
81 nm/s
46%
18
4.3
7.2
O
2400
350
250
265 nm/s
112%
20
3.7
6.7
3
N
N
59
4.0
22
72 nm/s
54%
22
4.0
7.9
N
N
4
88
7.1
140
100 nm/s
64%
31
4.0
8.1
O
5b
110
13
160
48 nm/s
77%
48
2.9
na
O
O
N
N
6
290
28
150
12 nm/s
77%
na
2.8
na
O
7b
a
See Ref. 19a for experimental and synthetic details.
1:1 mixture of epimers.
b
F
F
Compound 4 was examined in vivo for correlation with the
in vitro assays. Hartley guinea pigs, a wild type preclinical animal
model, were dosed orally up to 100 mpk. At the highest dose no
reduction of central Ab was detected while the plasma Ab levels
were reduced by 82%. Inspection of the time course profile of the
brain and plasma levels of 4 (Fig. 3) revealed a peculiarity: the
NH
HO
H
H
O
Polar Head
N
Polar Head
Table 1
1
Classification of permeability and recovery
Figure 2. Schematic of proposed HEA anchoring into the cell membrane.
Class
Papp (nm/s)
% recovery
Poor
Low
Moderate
Good
High
625 nm/s
610%
26 nm/s 6 50 nm/s
51 nm/s 6 99 nm/s
—
11% 6 30%
31% 6 50%
51% 6 70%
P71%
brain levels did not track with the plasma levels, but instead
remained constant throughout the study. This implied that 4
embedded into the extensive network of endothelial cells that
constitute the BBB,22 and did not penetrate into the parenchyma
P100 nm/s

A. P. Truong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6231–6236
6233
Table 3
of the brain despite the improvements to both the permeability
SAR of the prime side binding regiona
and recovery in the in vitro MDCK assay. The desired profile of
the inhibitor should have the brain levels mirror those of the plas-
ma, an in-and-out profile.
F
O
HN
2
R
A search for compounds with an in vivo in-and-out profile and a
method to ameliorate the P-gp liability was initiated. Modulating
the pKa of the amine on the HEA template to reduce the P-gp liabil-
ity was critical.6a,19b,23 A P10 cyclopropyl was used to attenuate the
basicity of the amine in place of heteroatoms which induced P-gp
activity. Compound 10 had a low rate of forward flux (50 nm/s) and
recovery (27%) indicating a high degree of membrane affinity
(Table 3). Interestingly, 10 had a negligible P-gp efflux ratio of
1.3. This demonstrated that the HEA template was not inherently
flawed with insurmountable P-gp liability. Employing the previous
tactics to create permeability through the use of a polar aryl linker
or P20 once again led to high rates of permeability with good to
high levels of recovery, but untenable levels of P-gp efflux
remained albeit at significantly depressed levels relative to their
cyclohexyl counterparts. A gambit to create polarity without using
heteroatoms was needed. From the viewpoint that polarity was
simply a dipole moment, then halogens could be utilized to create
polarity and do so in a manner that P-gp would be unable to recog-
nize. Fluorine reduces the log P of and generates a dipole moment
on hydrocarbons.24 To expedite the testing of the hypothesis a
readily accessible fluorinated P20, 14, was prepared via a Suzuki-
Miyaura cross-coupling reaction25 between 3-cyanophenylboronic
acid and 2-bromo-3,3,3-trifluoroprop-1-ene followed by chemistry
analogous to that of Scheme 1. Gratifyingly, 14 afforded a high rate
of permeability (103 nm/s) with good recovery (66%), and most
importantly, 14 was devoid of P-gp liability. Increased enzymatic
activity against BACE-1 could be attained by increasing the branch-
ing at the benzylic position of the P20 motif.19b Furthermore,
molecular modeling indicated the possibility of the newly intro-
duced P20 fluoride(s) forming a hydrogen bond with the side chain
of the Arg128 and/or Tyr198 which could lead to an improvement
in potency relative to 10.20 To this end, several fluoroalkyls 15–17
were synthesized (Schemes 1–3) to probe for an increase in
potency. As expected, these perturbations (15–17) enhanced the
potency in comparison to 14, maintained high rates of permeabil-
ity coupled with good to high levels of recovery, and had diminu-
tive levels of P-gp efflux activity (1.1–1.7).
F
N
H
OH
#
R
BACE-1 IC50 (nM)
cell ED50 (nM)
Cat-D IC50 (nM)
Papp (nm/s)
% recovery
P-gp efflux ratio
log P@
pH = 7.4
pKa
1300
84
>10,000
13 nm/s
25%
na
4.1
na
8
200
17
610
7 nm/s
20%
na
4.4
na
9
140
51
220
50 nm/s
27%
1.3
4.6
6.6
10
350
54
720
102 nm/s
67%
11
3.0
7.1
11
N
N
980
230
750
143 nm/s
67%
6.0
3.1
6.7
N
N
12
760
97
480
132 nm/s
71%
8.5
3.1
na
O
13b
1300
620
1200
103 nm/s
66%
0.78
4.6
na
14
15
16
CF3
F
290
25
480
186 nm/s
64%
1.7
4.1
na
230
26
490
122 nm/s
54%
1.1
4.0
6.7
F
F
F
390
21
500
168 nm/s
78%
1.5
3.7
na
F
17
610
110
380
3 nm/s
17%
na
4.6
7.8
18
1700
130
na
25 nm/s
26%
2.6
4.4
na
19c
F
F
The carbon framework of the P20 was installed via a palladium
catalyzed enolate cross-coupling26 between 20 and 21 (Scheme 1).
a
b
c
See Ref. 19a for experimental details.
1:1 mixture of epimers.
Most active of the four diastereomers.
100
Plasma
Brain
NC
Br
+
a-c
F
NC
HN
OH d-f
CO2Me
10
20
21
22
1
0.1
Boc
O
g-i
F
+
15
H2N
F
23
24
Scheme 1. Reagents and conditions: (a) n-BuLi, Cy2NH, Pd2dba3–CHCl3, P(tert-Bu)3-
HBF4, PhMe, 58%; (b) aq NaOH, dioxane, MeOH; (c) BH3-DMS, THF, 80% over two
steps; (d) Tf2O, pyridine, CH2Cl2, 0 °C; (e) TBAF, MeCN, 80 °C, 23% over two steps; (f)
Ti(O-iPr)4, EtMgBr, Et2O, 0 °C to ambient temperature then BF3-OEt2, 48%; (g) N,N-
diisopropylethylamine, isopropanol, reflux, 50%; (h) 4 N HCl in dioxane; (i)
Ac2NOMe, triethylamine, CH2Cl2, 65% over two steps.
0.01
0.001
0
6
12
18
24
Time (hours)
Saponification of the ester followed by borane reduction gave the
alcohol 22. A two-step transformation of the alcohol into a fluoride
followed by a modified Kulinkovich reaction27 on the nitrile
Figure 3. Twenty-four hour time course profile of the brain (blue) and plasma (red)
levels of 4 after oral dosing in guinea pigs at 100 mpk.

6234
A. P. Truong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6231–6236
F
100
10
a-c
F
22
H2N
+
H2N
F
Plasma
Brain
F
26
25
Scheme 2. Reagents and conditions: (a) DMP, NaHCO3, CH2Cl2, 72%; (b) DAST,
CH2Cl2, 62%; (c) Ti(O-iPr)4, EtMgBr, Et2O, 0 °C to ambient temperature then
BF3-OEt2, 63% 25:26 1:2.
1
OBn
0.1
NC
c-e
a-b
CO2Me
NC
CO2Me
0.01
0.001
27
28
H2N
OBn
F
f-i
F
NC
F
29
30
0
6
12
18
24
Time (hours)
Scheme 3. Reagents and conditions: (a) LiHMDS, MeI, THF, ꢀ78 °C, 91%;
(b) NaHMDS, BOM-Cl, THF, ꢀ78 °C, 82%; (c) LiBH4, THF, 40 °C, 82%; (d) Tf2O, pyridine,
CH2Cl2, 0 °C; (e) TBAF, MeCN, 80 °C, 77% over two steps; (f) FeCl3, CH2Cl2, 91%;
(g) Tf2O, pyridine, CH2Cl2, 0 °C; (h) TBAF, MeCN, 80 °C, 63% over two steps;
(i) Ti(O-iPr)4, EtMgBr, Et2O, 0 °C to ambient temperature then BF3-OEt2, 55%.
Figure 4. Twenty-four hour time course profile of the brain (blue) and plasma (red)
levels of 16 in guinea pigs after dosing orally at 10 mpk. The dotted line is the
cellular ED50
.
furnished the cyclopropylamine 23. Epoxide ring opening of 2428
with the amine 23, deprotection, and acetylation29 of the primary
amine afforded 15.
125
100
75
50
25
0
The alcohol 22 was oxidized with Dess–Martin periodinane30
followed by treatment of the aldehyde with DAST (Scheme 2).
The nitrile was converted to the cyclopropyl amine 25 via the mod-
ified Kulinkovich reaction. The cyclopropylamine 26 originated
from a rearrangement during the DAST reaction as the major prod-
uct. The aforementioned chemistry (vide supra) was employed to
complete the synthesis of 16.31
Sequential alkylation of 27 with iodomethane and BOM-Cl
afforded 28 (Scheme 3). Reduction of the ester yielded the alcohol,
which was converted to the fluoride 29. Debenzylation of 29 with
ferric chloride32 afforded the alcohol that was transformed into a
fluoride followed by conversion of the nitrile into the cyclopropyl-
amine 30. Standard chemistry (vide supra) was used to finish the
synthesis of 17.
Vehicle
30 mg/kg
Vehicle
30 mg/kg
The geminal di-fluoride 16 was administered orally at 10, 30, and
100 mpk to wildtype Hartley guinea pigs. No acute reduction of cor-
tical Ab was observed at 10 mpk, but a modest 12% and robust 42%
reduction of cortical Ab was observed at 30 and 100 mpk, respec-
tively, 6 h post administration. Analysis of the time course profile
of 16 revealed that the brain levels did not mirror those of the plas-
ma, but they did decrease over time giving rise to a fast-in-slow-out
profile (Fig. 4). Some portion of 16 was quitelikely being sequestered
in the BBB since the recovery from the MDCK assay was only 54%.
The time course profile for the 30 mpk dose is analogous to Figure 4.
We sought to exploit the fast-in-slow-out profile of 16 by driv-
ing the brain levels higher via a BID dosing paradigm at 10 and
30 mpk. Gratifyingly, dosing 16 at 30 mpk BID over a period of
2.5 days led to a reduction in cortical and CSF Fb of 28% and 32%,
respectively (Fig. 5). Central Ab was unaffected after administering
16 orally BID at 10 mpk for 2.5 days. The mean brain levels at 10
Brain Aβ
CSF Aβ
Figure 5. Reduction of cortical and CSF Ab1-x by 16 in Hartley guinea pigs 6 h post
final dose. Each circle or square represents one animal.33
16 proved to be culpable of this undesirable attribute. Preincuba-
tion 16 with and without NADPH led to an IC50 of 0.004 and
0.046
In addition to being a TDI, 16 is also a competitive inhibitor of
CYP3A4 with an IC50 of 0.11 M.
l
M, respectively, giving an 11-fold shift against CYP3A4.35
l
To further improve the potency and abrogate the time-depen-
dent inhibition, we explored increasing the lipophilicity in the
P10. Cyclobutylamines undergo a similar P450 catalyzed ring open-
ing36 but do not inhibit the CYPs in a time-dependent fashion due
to the slower rate of ring opening.37Unfortunately, the P10 cyclobu-
tyl moiety 18 lacked the desired potency. Additionally, this method
suffers from limitations since even slight increases to the lipophil-
icity, 19, are detrimental to both the permeability and recovery.
In conclusion, we have designed highly permeable HEAs with
very low levels of P-gp liability and this translated into an orally
efficacious inhibitor in the wildtype preclinical guinea pig animal
model. The decisive element for success was to focus on engineer-
ing in the appropriate pharmacokinetic parameters, not the
potency, of the inhibitor. This was accomplished by modulating
and 30 mpk following the final dose were 0.46 and 7.85 lM,
respectively, with brain-to-plasma ratios of 0.63 and 0.71, respec-
tively. This nonlinear correlation compelled us to reexamine the
time course profile of the compound.
Inspection of the time course profile of 16 administered orally
at 100 mpk revealed that the brain levels had flat lined, but the
plasma levels also plateaued (Fig. 6). This phenomenon is indica-
tive of saturating the clearance mechanism. Cyclopropylamines
are known to be time-dependant CYP inhibitors (TDI),34 and indeed

A. P. Truong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6231–6236
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0
6
12
18
24
Time (hours)
Figure 6. Twenty-four hour time course profile of the brain (blue) and plasma (red)
levels of 16 in guinea pigs after dosing orally at 100 mpk. The dotted line is the
cellular ED50
.
the pKa of the amine with a cyclopropyl moiety, and generating
polarity and/or lowering the log P on the P20 hydrocarbon with
fluorine. The cellular activity may be a more pertinent measure-
ment with which to judge an inhibitor, rather than enzymatic po-
tency, since 16 is 26 nM in the cell and only 230 nM in the
biochemical assay. The cell-to-enzyme ratios were excellent possi-
bly due to compartmentalization,38 which could result in higher lo-
cal concentrations of the inhibitor relative to the BACE-1
enzymatic assay. Although this class of inhibitors had limitations,
this research served as a springboard for future efforts.
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Acknowledgment
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