Improving the permeability of the hydroxyethylamine BACE-1 inhibitors: Structure-activity relationship of P2′ substituents

Article abstract of DOI:10.1016/j.bmcl.2010.06.112

Herein we describe further evolution of hydroxyethylamine inhibitors of BACE-1 with enhanced permeability characteristics necessary for CNS penetration. Variation at the P2′ position of the inhibitor with more polar substituents led to compounds 19 and 32

Full text of DOI:10.1016/j.bmcl.2010.06.112

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4789–4794
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Improving the permeability of the hydroxyethylamine BACE-1 inhibitors:
Structure–activity relationship of P20 substituents
a
a
a
a
Anh P. Truong a, Gary D. Probst a, , Jose Aquino , Larry Fang , Louis Brogley , Jennifer M. Sealy ,
*
a
a
a
a
a
Roy K. Hom a, , John A. Tucker , Varghese John , Jay S. Tung , Michael A. Pleiss , Andrei W. Konradi ,
Hing L. Sham a,b, Michael S. Dappen b, Gergley Tóth c, Nanhua Yao c, Eric Brecht c, Hu Pan c, Dean R. Artis c,
Lany Ruslim d, Michael P. Bova d, Sukanto Sinha d, Ted A. Yednock a,b,c,d,e, Wes Zmolek e, 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:
Herein we describe further evolution of hydroxyethylamine inhibitors of BACE-1 with enhanced perme-
ability characteristics necessary for CNS penetration. Variation at the P20 position of the inhibitor with
more polar substituents led to compounds 19 and 32, which retained the potency of more lipophilic ana-
log 1 but with much higher observed passive permeability in MDCK cellular assay.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 24 May 2010
Revised 14 June 2010
Accepted 21 June 2010
Available online 25 June 2010
Keywords:
BACE-1 inhibitor
Hydroxyethylamine (HEA)
Alzheimer’s disease
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 sky-
rocketing healthcare costs far exceeding 100 billion dollars annu-
ally.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 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)
Secretase processes a myriad of substrates,8 such as Notch, raising
concerns about mechanism-based side-effects due to a deficiency
in selectivity.9 Conversely, gene deletion of BACE-1 in mice pro-
duced 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 signifi-
cant reduction of plaque burden.12 Furthermore, cleavage of b-
APP by BACE-1 is the rate-limiting step in Ab-peptide production4
and releases the soluble N-terminal APP fragment which initiates a
cascade event ultimately leading to apoptosis.13 Additionally, solu-
ble oligomeric Ab42 indirectly signals for hyperphosphorylation of
tau at AD-specific epitopes producing NFTs that lead to neurotox-
icity.14 Moderately abating the production of Ab monomers results
in a disproportionate reduction of synaptotoxic soluble Ab oligo-
mers leading to improved synaptic plasticity.15 This validates
by two aspartic acid proteases, referred to as b- and c-secretase,
respectively.4 Inhibition of either protease has been demonstrated
to result in reduction of brain Ab-peptide in preclinical studies.5
Inhibitors of either protease offer attractive candidates as poten-
tially disease-modifying, rather than palliative, treatments for peo-
ple afflicted with this debilitating malady.6
BACE-1 and presents its clear advantages over
c-secretase as a
Between these two proteases, b-secretase (BACE-1)7 is the more
therapeutic target, and buttresses the amyloid cascade hypothesis.
Previously, we reported our research in the S10 pocket with our
hydroxyethylamine (HEA) peptidomimetics.16 Although our HEA
inhibitors were potent, they were deficient of the pharmacokinetic
parameters, most notably high permeability and low Permeability-
glycoprotein (P-gp) efflux, necessary for an efficacious central
alluring therapeutic target based on the following distinctions. c-
* Corresponding authors. Tel.: +1 650 616 2684; fax: +1 650 877 7486. (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 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.112

4790
A. P. Truong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4789–4794
Table 1
S1'
Classification of permeability and recovery
F
O
S1
Class
Papp (nm/s)
% Recovery
HN
S2'
Poor
Low
Moderate
Good
High
ꢀ 25 nm/s
ꢀ 10%
F
N
H
26 nm/s ꢀ 50 nm/s
51 nm/s ꢀ 99 nm/s
—
11% ꢀ 30%
31% ꢀ 50%
51% ꢀ 70%
ꢁ 71%
OH
1
aryl linker
ꢁ 100 nm/s
Figure 1. Binding mode of the HEAs.
nervous system drug. We opted to address these impediments in
an incremental fashion by first focusing on improving the passive
permeability of the inhibitors. Herein, we will describe the proper-
ties of the HEAs that generated our working hypothesis concerning
the pharmacokinetic issues, and the strategy to design highly
permeable and potent BACE-1 inhibitors. 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, vide infra.
The crystal structure of BACE-1 complexed with compound 116
illustrates the active site binding mode (Figs. 1 and 2). The diflu-
oroaryl, cyclohexyl, and tert-butyl substituents occupy the S1, S10,
and S20 pockets, respectively, as shown in Figures 1 and 2. The
S20 pocket is amphiphilic in nature. The ‘‘top” is composed of
Ser35, Ile126, and Tyr198 which forms the border between the
S10 and S20 (Fig. 3). Two hydrophilic residues Asn37 and Arg128
constitute the ‘‘rear” portion of the pocket. The ‘‘bottom” of the
pocket consists of Val69 and Pro70, and Tyr71 on the ‘‘left side.”
The protonated amine and hydroxyl group binds at the scissile 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.17
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.
Figure 2. Crystal structure of 1 binding to truncated (56-455) human BACE-1 (1.8 Å
resolution). The PDB deposition code is 3ivh.
The synthetic route employed to synthesize the HEA inhibitors
is outlined in Scheme 1. An aryl lithium generated from 3 was
added to cyclohexanone to produce a tertiary alcohol which in turn
was converted to the amine 4 via the azide. Epoxide ring opening
of 518 with the amine 4, deprotection, and acetylation19 of the pri-
mary amine afforded compound 6. The aryl iodide 6 served as a
versatile synthetic intermediate for transition metal catalyzed
cross-coupling reactions such as Suzuki–Miyaura,20 Stille,21 Negi-
shi,22 Heck,23 N-arylation,24 and enolate arylation.25 This approach
was highly effective and versatile for the synthesis of compounds
9–12, 19–26, and 30–44. Alternatively, 1 and 13–18 were prepared
with the same chemistry in Scheme 1, but with the P20 moiety al-
ready in place.16
Initially success in increasing the potency was achieved by the
placement of alkyl substituents in the S20 pocket (Table 2). The
tert-butyl group was optimal for enzymatic potency while reduced
branching resulted in a decrease in biochemical potency (8–10) as
did extension deeper into the pocket (11–13). The permeability of
these highly lipophilic inhibitors (1 and 9) was low, and in several
cases (11–13) the recovery of the compound from the permeability
assay was so poor (ꢀ10%) that the result was deemed to be unre-
liable, thus the compounds were considered un-testable.
The low recovery from the permeability assay indicated that the
compounds embedded into the membrane of the Madin-Darby ca-
nine kidney (MDCK) cells. Highly lipophilic molecules that possess
a basic amine exhibit a pronounced affinity towards membranes.26
Since compound 1 has a measured log P of 4.8 and pKa of 8.1, 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. 4). In order to overcome this liability, we envi-
sioned replacing the tert-butyl group with a polar moiety which
Figure 3. Crystal structure of 1 binding to BACE-1 with the S20 residues highlighted.
F
O
a-c
I
I
Boc
O
+
HN
+
I
I
H2N
F
2
3
F
4
5
O
d-f
HN
6
F
N
H
OH
Scheme 1. Reagents and conditions: (a) n-BuLi, THF, ꢂ78 °C, 50–70%; (b) TMSN3,
BF3-OEt2, CH2Cl2, reflux or TFA, NaN3, CH2Cl2, reflux, 30–50%; (c) PMe3, H2O, THF,
60 °C; (d) N,N-diisopropylethylamine, isopropanol, reflux, 60–70% over two steps;
(e) 4 N HCl in dioxane; (f) Ac2NOMe, triethylamine, CH2Cl2, 70–80% over two steps.
would lower the log P leading to reduced membrane affinity, there-
by generating the high rate of permeability required to penetrate

A. P. Truong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4789–4794
4791
Table 2
Table 4
SAR of alkyl groupsa
SAR of aromatic ringsa
F
O
F
O
HN
HN
7
R
7
R
F
N
F
N
H
H
OH
OH
#
R
BACE-1 IC50, nM
Cell ED50, nM
Papp, nm/s
%Recovery
#
R
BACE-1 IC50, nM
Cell ED50, nM
Papp, nm/s
%Recovery
47
17
7
11%
N
59
4.0
72
54%
1
19
N
6
8
9
–I
460
85
O
1100
110
Un-testable
Low recovery
20
21
22
23
na
–H
–Et
>10,000
>1000
31
40%
120
36
Un-testable
Low recovery
O
S
690
120
11
na
44
34
Un-testable
Low recovery
100
13
Un-testable
Low recovery
S
370
310
Un-testable
Low recovery
10
11
12
380
120
Un-testable
Low recovery
S
110
13
8
31%
24
25
N
130
200
Un-testable
Low recovery
1600
26
19
69%
N
N
810
230
Un-testable
Low recovery
13
N
7700
110
45
36%
N
26
N
a
See Ref. 16 for experimental details.
a
See Ref. 16 for experimental details.
F
F
by forming a hydrogen bond with one of the side chains of
Asn37, Arg128, or Tyr198 in the S20 pocket.
NH
A select group of examples illustrated in Table 3 demonstrate
that moieties more polar than tert-butyl afforded improved perme-
ability, but the potency was diminished. The dimethylaniline 14
had a low rate of permeability (39 nm/s) with a moderate recovery
(41%), while 15 and 16 had moderate rates of forward flux (58 and
91 nm/s) in conjunction with a further increase in recovery. The
alcohol 17 and sulfone 18 exhibited poor (20 nm/s) and low
(36 nm/s) rates permeability coupled with good (69%) and high
(82%) levels of recovery, respectively, indicating that the com-
pounds had become too polar, thus losing the membrane affinity
necessary for the requisite passive permeability. While these re-
sults were certainly less than ideal from a potency perspective,
they indicated that the direction of the research was on the correct
path to improve the permeability of the inhibitors. Also, these re-
sults underscored the fact that a fine balance of physiochemical
properties would be necessary to effectuate the requisite passive
permeability.
HO
H
H
O
Polar Head
N
Polar Head
1
Figure 4. Schematic of proposed HEA anchoring into the cell membrane.
Table 3
SAR of polar groupsa
F
O
HN
7
R
F
N
H
OH
Next, we examined aromatic rings as the P20 substituents with
representative examples in Table 4. The N-linked pyrazole 19
(measured log P = 4.0 and pKa = 7.8) is nearly equipotent to 1 in
an enzymatic assay and 4.0 nM in the cellular assay, but exhibited
a significant increase in both permeability and recovery. X-ray co-
crystal structure of 19 with BACE-1 shows a hydrogen bond (2.7 Å)
between the pyrazole in 19 and the phenolic hydrogen of Tyr198
(Fig. 5). Surprisingly, the 2-furan 20 lost nearly 20-fold in potency
while the 3-furan 21 was only twofold less active relative to 19, but
neither gave measurable permeability. Similarly, the 3-thienyl
moiety 22 has a comparable potency to 1 and 19, but not the 2-thi-
enyl compound 23. Neither 22 nor 23 were testable in the perme-
ability assay due to the low recovery. The thiazole 24 led to a
twofold decrease in potency with a substantial decrease in perme-
ability in comparison to 19. Imidazoles and triazoles, such as 25
and 26, were significantly less potent, and offered only marginal
improvements to the permeability relative to 1. A plethora of
#
R
BACE-1 IC50, nM
Cell ED50, nM
Papp, nm/s
%Recovery
410
44
39
41%
14
N
166
ꢀ40
6200
93
58
47%
15
16
CN
–CN
91
64%
260
42
20
69%
17
18
OH
–SO2Me
720
55
36
82%
a
See Ref. 16 for experimental details.
the blood–brain barrier. Concurrently, the potency could be re-
tained and/or improved with this newly introduced polar moiety

4792
A. P. Truong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4789–4794
Table 5
SAR of non-aromatic cyclic moieties with P1’ cyclohexyla
F
O
HN
7
R
F
N
H
OH
#
R
BACE-1 IC50, nM
Cell ED50, nM
Papp, nm/s
%Recovery
940
na
180
23
88
7.1
Un-testable
Low recovery
117
68%
100
30
O
31b
32b
O
64%
O
820
66
24
90%
33
34
35
N
Figure 5. Crystal structure of 19 binding to truncated (57-453) human BACE-1
(2.7 Å resolution). The PBD deposition code is 3N4L. The pyrazole is depicted as
hydrogen bonding to Tyr198 (2.7 Å).
O
1300
170
22
81%
O
N
O
1400
97
7
77%
NH
N
six-membered aromatic rings were synthesized but none had sub-
micromolar activity.
O
800
75
51
90%
36b
37
Cyclic non-aromatic moieties were synthesized to explore for
potency and examine their effect on permeability (Table 5). Rings
of different sizes and shapes combined with hydrogen bond donors
and/or acceptors offered different presentations to hydrogen bond
with one of the polar residues of BACE-1. In addition to cyclohexyl,
cyclopropyl was another commonly used P10 substituent (Table 6),
but as a general rule they were three-to-four times less potent than
their cyclohexyl counterparts (31 vs 46). An exemplary synthetic
route to these cyclopropyl analogs is outlined in Scheme 2. Starting
with the alkyne 27, formation of a vinyl bromide,27 removal of the
>10,000
>1000
Un-testable
Low recovery
O
340
25
31
93%
38c
N
O
N
O
N
O
250
20
11
79%
39
40
41
1300
110
8
84%
O
THP protecting group, and
a
Suzuki coupling20 with 3-cya-
1000
76
2
84%
nophenylboronic acid afforded compound 28. Iodo-etherification
of the olefin 28, reduction of the iodide, and a Kulinkovich reac-
tion28 gave the cyclopropylamine 29 with which the standard
chemistry (vide supra) was used to complete the synthesis. The
tetrahydrofurans, 31 and 32 (measured log P = 4.0), were 5- and
10-times more potent than the cyclopentyl analog 30, and highly
permeable (P 100 nm/s) with good recoveries (68% and 64%).
Molecular modeling indicated that the oxygen formed a hydrogen
bond with the Tyr198, and that both epimers of 31 and 32 had sim-
ilar binding energies. The 2-methyltetrahydrofuran 47 was de-
signed to fill space like the tert-butyl and hydrogen bond with
the Tyr198 but the potency was disappointing. Molecular modeling
indicates that the pyrans 48 and 49 form a hydrogen bond with
Tyr198. Carbonyls at the 2-position of five-membered rings did
not increase the binding affinity (33–36 vs 30) but did so for six-
membered rings (38–41 vs 37) by forming a hydrogen bond with
the Tyr198 as predicted by molecular modeling. The conformation
of the P20 ring appears to be an important factor for binding (37 vs
51). Seven-membered rings afforded sub-optimal potency (42–44).
The carbonyl derivatives (33–35 and 38–42) consistently resulted
in poor rates of permeability (625 nm/s) (except 38 at 31 nm/s)
in conjunction with high levels (P 71%) of recovery (except 41 at
55%) indicating that the substrates had lost membrane affinity.
The ketone 36 bucked this trend with a moderate permeability
(51 nm/s). Ethers (31–32, 43–44, and 46–50) consistently gener-
ated high rates of permeability coupled with good to high levels
of recovery indicating very good passive permeability. Reducing
the basicity of the amine (pKa = 6.5–6.7) and lipophilicity associ-
ated with the cyclopropyl P10, compared to the cyclohexyl P10,
did not affect the permeability or recovery with a polar P20 (31
vs 46), and an ether was still required to generate high rates of per-
meability (50 vs 51).
NH
N
O
N
750
63
5
55%
42
O
O
O
O
560
43
138
77%
43b
44
700
43
107
86%
N
a
b
c
See Ref. 16 for experimental details.
1:1 mixture of epimers.
1:1 mixture of diastereomers at the bridgehead positions.
Despite the considerable improvements to the rate of passive
permeability, the P-gp efflux of these inhibitors remained
unacceptably high precluding efficacy in wild type animal models
(Table 7). Reduced, but untenable, P-gp liability was a general
phenomena observed with cyclopropyl as the P10 (46) due to the
lower pKa of the amine.5a,29
In conclusion, via chemistry driven SAR with support from
molecular design substantial improvements to the permeability,
an important pharmacokinetic parameter, have been introduced
into the BACE-1 inhibitors by incorporating a polar P20 substituent
in place of a lipophilic group. The S20 pocket is highly selective
since only three of newly introduced P20 moieties maintained po-
tency comparable to compound 1. Ethers proved to be a functional
group that consistently produced desirable rates of permeability
and recoveries. The cell-to-enzyme ratios were excellent possibly
due to compartmentalization,30 which could result in higher
local concentrations of the inhibitor relative to the BACE-1
enzymatic assay. Also, none of these P20 perturbations enhanced

A. P. Truong et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4789–4794
4793
Table 6
SAR of non-aromatic cyclic moieties with P1’ cyclopropyla
Acknowledgements
F
O
We would like to thank Colin Lorentzen, Jackie Kwong, Jill Lab-
be, Lee Latimer, Jim Miller, and Nancy Jewett for their contribu-
tions to this work.
HN
45
R
F
N
H
OH
Supplementary data
#
R
BACE-1 IC50, nM
Cell ED50, nM
Papp, nm/s
%Recovery
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.06.112.
O
O
580
700
114
69%
46b
47b
48b
580
66
90
57%
References and notes
O
O
1700
300
111
67%
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1900
410
Un-testable
Low recovery
N
a
b
c
See Ref. 16 for experimental details.
1:1 mixture of epimers.
1:1 mixture of diastereomers at the bridgehead positions.
O
a-c
O
NC
OH
O
27
28
d-f
29
H2N
Scheme 2. Reagents and conditions: (a) 9-BBN-Br, CH2Cl2, 0 °C, then glacial AcOH;
(b) CSA, MeOH; (c) 3-cyanophenylboronic acid, Pd(PPh3)4, K2CO3, DME, H2O, 80 °C,
7% yield over three steps; (d) NIS, Et2O, reflux; (e) 10% Pd/c, EtOAc, H2, 24% over two
steps; (f) Ti(O-iPr)4, EtMgBr, Et2O, 0 °C to ambient temperature then BF3-OEt2, 30%.
Table 7
P-gp efflux ratios of potent inhibitorsa
F
O
HN
n
52
R
F
N
H
OH
#
n
R
BACE-1 IC50, nM
P-gp efflux ratio
Papp, nm/s
%Recovery
7
11%
72
54%
100
64%
114
69%
1
4
4
4
1
47
59
19
22
31
12
N
19
32b
46b
N
O
88
O
580
a
See Ref. 16 for experimental details.
1:1 mixture of epimers.
b
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