A potent and orally efficacious, hydroxyethylamine-based inhibitor of β-Secretase

Article abstract of DOI:10.1021/ml3000148

β-Secretase inhibitors are potentially disease-modifying treatments for Alzheimer's disease. Previous efforts in our laboratory have resulted in hydroxyethylamine-derived inhibitors such as 1 with low nanomolar potency against β-site amyloid precursor pro

Full text of DOI:10.1021/ml3000148

Letter
pubs.acs.org/acsmedchemlett
A Potent and Orally Efficacious, Hydroxyethylamine-Based Inhibitor
of β-Secretase
Matthew R. Kaller,*,† Scott S. Harried,*,† Brian Albrecht,#,∇ Patricia Amarante,† Safura Babu-Khan,§
Michael D. Bartberger,‡ James Brown,† Ryan Brown,#,∞ Kui Chen,∥ Yuan Cheng,† Martin Citron,§,¥
Michael D. Croghan,† Russell Graceffa,# Dean Hickman,⊥ Ted Judd,† Chuck Kriemen,# Daniel La,#
Vivian Li,‡ Patricia Lopez,† Yi Luo,§ Craig Masse,#,○ Holger Monenschein,¶ Thomas Nguyen,†
Lewis D. Pennington,† Tisha San Miguel,∥ E. Allen Sickmier,‡ Robert C. Wahl,∥ Matthew M. Weiss,#
Paul H. Wen,§ Toni Williamson,§ Stephen Wood,§ May Xue,† Bryant Yang,† Jianhua Zhang,§
Vinod Patel,#,+ Wenge Zhong,† and Stephen Hitchcock†,¶
‡
§
∥
†Chemistry Research and Discovery, Department of Molecular Structure, Department of Neuroscience, Department of HTS and
⊥
Molecular Pharmacology, and Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., One Amgen Center Drive,
Thousand Oaks, California 91320, United States
#Chemistry Research and Discovery, Amgen Inc., One Kendall Square Building 1000, Cambridge, Massachusetts 02139, United States
S
* Supporting Information
ABSTRACT: β-Secretase inhibitors are potentially disease-modifying treatments for
Alzheimer's disease. Previous efforts in our laboratory have resulted in hydroxyethylamine-
derived inhibitors such as 1 with low nanomolar potency against β-site amyloid precursor
protein cleaving enzyme (BACE). When dosed intravenously, compound 1 was also shown to
significantly reduce Aβ40 levels in plasma, brain, and cerebral spinal fluid. Herein, we report
further optimizations that led to the discovery of inhibitor 16 as a novel, potent, and orally
efficacious BACE inhibitor.
KEYWORDS: β-site amyloid precursor protein cleaving enzyme (BACE), Alzheimer's disease (AD),
hydroxyethylamine (HEA) isostere
he need for a disease-modifying treatment for Alzheimer's
disease (AD) continues to grow. In the United States
the S2 and S3 binding pockets of BACE, and the 6-ethyl 2,2-
spirocyclobutylchromane engages in hydrophobic interactions
with the S2′ and S1′ binding pockets. Compound 1 exhibited
potent cellular activity (8.8 nM); however, it also displayed
poor metabolic stability (mouse liver microsomes = 568 μL/
min/mg), and its measured in vivo clearance was greater than
liver blood flow (mouse iv, 2 mg/kg, 5.6 L/h/kg). Despite
these liabilities, when dosed intravenously in Tg2576 transgenic
mice9 (30 mg/kg, 4 h time point), compound 1 significantly
reduced Aβ40 levels by 91, 58, and 57% in plasma, brain, and
cerebral spinal fluid (CSF), respectively.
Although compound 1 displays impressive enzymatic
inhibition, it is not an efficient inhibitor of BACE (BE =
0.24)10 and has a relatively high molecular weight of 647 amu.
It also has a large total polar surface area (PSA), high clog P,
and the number of H-bond donors and acceptors are not ideal
for a target located in the CNS.11 To optimize the properties of
1, guided by the well-established binding models, we reasoned
T
alone, the number of cases exceeds 5 million, and AD is the
seventh leading cause of death.1 The disease is characterized by
the progressive loss of cognitive ability, dementia, and
eventually death. Extracelullar amyloid plaques, largely
comprised of Aβ40−42 peptides, and intracellular neurofibrillary
tangles are the defining pathological characteristics of AD.
Aβ40−42 is a product of the cleavage of amyloid precursor
protein (APP) by β-secretase and γ-secretase. The cloning of
the β-site amyloid precursor protein cleaving enzyme (BACE)
enzyme and its critical role in the amyloid cascade2 have led to
the hypothesis that BACE inhibitors are potential disease-
modifying treatments for AD.3−5 During the past decade,
numerous pharmaceutical companies have committed consid-
erable resources to develop BACE inhibitors.6,7 In this letter,
we will describe our effort toward the design and synthesis of a
novel, potent, and orally efficacious BACE inhibitor.
Previous work in our laboratories revealed hydroxyethyl-
amine (HEA)-derived compound 1 (Figure 1) to be a potent
BACE inhibitor.8 On the basis of X-ray analysis, the HEA
transition state isostere binds to the two catalytic aspartyl
residues in the expected fashion. The N-cyclopentyl-3-(2-
pyridyl) pyridone amide moiety of 1 provides interactions with
Special Issue: Alzheimer's Disease
Received: January 13, 2012
Accepted: March 29, 2012
Published: March 29, 2012
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
compound intrinsic stability in liver microsomes as a means to
improve in vivo clearance in rodents. This communication will
describe our efforts in detail on further optimization of 4,
particularly modifications to the P1 substituent, leading
ultimately to the discovery of 16 as a novel, potent, and orally
bioavailable efficacious BACE inhibitor.
The synthesis of 4 and analogues 16−33 began with
commercially available cyclobutanone (Scheme 1). The
a
Scheme 1. Synthesis of Azachromane Intermediate 12
Figure 1. Truncating the amide of 1 and optimizing P2′ led to BACE
inhibitor 4 (S1−S2′ indicate BACE binding domains).
a
Reagents and conditions: (a) Allylmagnesium bromide, THF, −78
°C. (b) TBSOTf, TEA, DCM, 0 °C. (c) OsO4, NMO, H2O:t-
BuOH:THF (1:1:0.03). (d) NaIO4, THF:t-BuOH:H2O (1:1:2). (e)
CuSO4, (R)-2-methylpropane-2-sulfinamide, DCM. (f) nBuLi, 2,2,6,6-
tetramethylpiperidine, THF, −78 °C. (g) CsF, DMSO, 120 °C. (h) 4
M HCl, DCM.
that the HEA core had to be kept intact to properly engage the
catalytic aspartic residues of BACE. We decided to target the
pyridone and chromane moieties of 1 as the most likely regions
to provide meaningful changes to the physiochemical proper-
ties of 1, while maintaining high cellular potency against BACE.
A major advancement in our early structure−activity relation-
ship (SAR) was the identification of the highly truncated
acetamide analogue 2.12 Despite the removal of P2 and P3
interactions with the enzyme, acetamide 2 retained modest
activity but with higher binding efficiency (BE = 0.31), thanks
to the significant decrease in molecular weight (224 amu). In
addition, the apparent permeability of 2 was greatly improved
over 1 by 5-fold (14 × 10−6 cm/s, LLC-PK1), which was of
particular importance for CNS penetration.
To further improve enzyme potency, insights from X-ray
cocrystal structures and molecular modeling suggested that an
ethyl group in the highly hydrophobic S2′ binding pocket was
probably not optimal. Modifying 2 to better fill the S2′ pocket
with larger hydrophobic substituents led to the neopentyl
chromane derivative 3 as a potent BACE inhibitor (IC50 = 7.2
nM). Upon examination of modeled structures, we realized that
the 8-position of the chromane moiety is open to a solvent-
exposed area, suggesting that introduction of some polarity
would not be detrimental to enzyme potency but could
modulate physicochemical properties. We thus incorporated a
nitrogen atom into the chromane at the 8-position. Gratifyingly,
we found that such a subtle structural variation resulted in
compound 4, which not only was equipotent as compound 3 in
the enzyme assay but also displayed potent cellular activity (cell
IC50, 3.1 nM). We next turned our attention to improving
addition of allylmagnesium bromide to cyclobutanone, followed
by protection of the secondary alcohol with tert-butyldime-
thylsilyl triflate, gave the silyl ether 5. Dihydroxylation was
effected with osmium tetroxide and N-methylmorpholine-N-
oxide to give diol 6, which was then treated with sodium
periodate to afford aldehyde 7. The sulfinylimine 8 was formed
by condensation with Ellman's chiral tert-butylsulfinamide
auxiliary with copper sulfate.13 The fluoropyridine 9 was
produced via a palladium-catalyzed zinc cross-coupling between
commercially available reagents 2-fluoro-5-bromopyridine and
neopentylzinc chloride. Fluoropyridine 9 was regioselectively
deprotonated with lithium tetramethylpiperidide at the 3-
position and then treated with sulfinylimine 8 to give the
sulfinylamide adduct 10 in good yield with moderate
diastereoselectivity (2:1) in favor of the desired diastereomer.
Silyl ether deprotection of 10 with concurrent cyclization in the
presence of cesium fluoride in DMSO at 120 °C furnished
azachromane 11, which was readily separated from the minor
diasteromer via column chromotagraphy. The chiral auxiliary
was then smoothly cleaved under acidic conditions to give
enantiomerically pure azachromane amine 12.
We synthesized HEAs 2 and 3 (Figure 1) following well-
established literature procedures that utilize a nucleophilic
opening of an amino acid-derived epoxide with the appropriate
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ACS Medicinal Chemistry Letters
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chromane amine.8 We discovered that this methodology was
sluggish and inefficient when an 8-azachromane amine was used
as the nucleophile in this alkylation reaction. Several attempts
to improve this epoxide opening with azachromane 12,
including using Lewis acid additives, did not provide syntheti-
cally useful yields. Therefore, a reductive amination approach,
one that circumvents the poor nucleophilic reactivity of 12, was
developed to allow efficient coupling between the azachromane
amine 12 with a suitable aldehyde 14 (Scheme 2).14 To
microsomes (621 μL/mL/min). Moving the fluorine to the 2-
position (18) provided good potency, but this compound was
also not stable in human liver microsomes (342 μL/mL/min).
Several alternative substititions were tried at the 4-position of
the aryl ring, such as chlorine and trifluoromethoxy (19 and
20), but both were inferior to fluorine in terms of potency and
metabolic stability. Substituting at both the 3- and the 5-
positions with fluorine atoms (21) was consistent with single
substitution with good potency but poor metabolic stability.
Substituting trifluoromethyl for flourine (22) in the 3-position
of the P1 phenyl group provided improved potency over 16 but
suffered from poor human microsomal stability and reduced
permeability. Placing fluorine atoms at both the 2- and the 4-
positions (23) resulted in modest enzyme potency, but the
increased lipohpilicity led to a larger enzyme to cell shift.
Realizing that fluorine at the 4-position provided some stability
and that the 3-position provided some improved potency,
various substituents were tried at the 3-position in combination
with the 4-fluoro substituent (24−29). The compounds were
all comparable to 16 in potency, but almost all demonstrated
poor stability in human liver microsomes. Other substituents at
the 4-position (30−32) with fluorine at the 3-position generally
provided good potency but failed to improve the metabolic
stability of the molecules. These substituted P1 analogues have
good potency and good permeability but generally suffer from
poor in vitro microsomal stability (rat and human).
a
Scheme 2. Synthesis of BACE Inhibitors 16−32
On the basis of favorable permeability and moderate in vitro
microsomal stability, 16 was selected for further profiling.
Pharmacokinetic analysis of 16 showed that in vivo clearance in
rat was 1.2 L/h/kg, Vdss was 1.6 L/kg, and T1/2 was 7.2 h
following an iv dose (2 mg/kg). Oral bioavailability was
measured at 40% based on a 10 mg/kg dose.
a
Reagents and conditions: (a) Dess−Martin periodinane, Na2CO3,
Compound 16 was evaluated in a rat pharmacodynamic
(PD) model. The model allowed for the measurement of acute
reductions in soluble Aβ40 in plasma, brain, and CSF. The
compound was administered at 100 mg/kg orally as a
suspension in 2% HPMC, 1% Tween-80 at pH 2.0 to wild-
type male Sprague−Dawley rats. Samples were collected from
plasma, brain, and CSF 4 h after dosing.17 Compound 16
showed Aβ40 level reductions of 71, 32, and 35% in plasma,
brain, and CSF, respectively (Figures 2−4). Concentrations of
16 at this time point were measured at 4.3, 0.66, and 0.006 μM,
respectively, for the plasma, brain, and CSF. On the basis of the
rat plasma protein binding (98.5%) of 16, the estimated
unbound concentration was 64 nM in plasma and 10 nM in
brain. The estimated unbound brain concentration is similar to
the measured CSF concentration. The reduction in Aβ40 levels
was consistent with the exposure levels and the in vitro cellular
IC50.
The binding of mode of compound 16 was determined by X-
ray crystallography and is depicted in Figure 5.19 The hydroxyl
group and basic amine of the HEA core engage in the expected
contacts with the two catalytic aspartyl residues Asp32 and
Asp228, respectively. The latter amine−Asp interaction is
augmented by a hydrogen bond between the amine and the
carbonyl oxygen of Gly34 (not shown.) Hydrogen-bonding
interactions involving the acetamide moiety of the inhibitor and
both the underside CO of Gly230 and the backbone NH of
flap Thr72, along with a CH···O type contact (ca. 3.4 Å)
between C5 of the azachromane group and the CO of Gly34,
round out the additional, anchoring polar contacts between the
inhibitor and the active site of BACE. The 4-fluoro substituent
on the P1 aryl ring, although likely experiencing a somewhat
DCM. (b) Compound 12, NaBH(OAc)3, trimethylorthoformate,
DCM. (c) 4 M HCl, MeOH, 50 °C. (d) 1-(1H-Imidazol-1-
yl)ethanone, DIPEA, DMF.
complete the synthesis of inhibitors 4 and 16−32, the alcohol
1315 was oxidized with Dess−Martin periodinane to give
aldehyde 14. Sodium triacetoxyborohydride-promoted16 reduc-
tive amination gave a very smooth and highly efficient coupling
reaction to provide adduct 15 in excellent yields. It is
noteworthy that no epimerization of aldehyde 14 was observed
in either the oxidation or the reductive amination sequence.
Global deprotection of 15 was effected with HCl in methanol
to give the penultimate hydroxyl diamine, which was then
selectively acylated with acyl imidazole to give acetamides 4 and
16−32.
Metabolic identification studies of 4 in the presence of rat
liver microsomes revealed that oxidation on the P1 ring and P2′
side chain was the major sites of oxidative metabolism. We
surmised that the poor metabolic stability was one of the
sources of high in vivo clearance (rat iv, 2 mg/kg, 5.3 L/h/kg).
We explored modifications on the P1 aryl ring to address this
metabolic soft spot of inhibitor 4. The results are summarized
in Table 1.
In general, we found that a wide range of substituents on the
P1 aryl ring were well tolerated in terms of potency against the
BACE enzyme. The first modification was to substitute a
hydrogen atom with a single fluorine atom at different positions
around the aryl ring. Placing a fluorine atom at the 4-position of
the aryl ring (16) provided good potency and permeability.
Moving the fluorine to the meta position (17) improved the
potency 5-fold but was detrimental to stability in human liver
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ACS Medicinal Chemistry Letters
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a
Table 1. SAR of Phenyl Ring Substituents
d
μL/min/mg
b
c
a
compd
R1
R2
BACE IC50 (nM)
cell IC50 (nM)
RLM
HLM
LLC-PK1 Papp (×10−6 cm/s)
4
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
H
H
H
H
H
H
H
5.8 6.3
5.0 3.0
1.0 0.47
3.6 0.86
8.3 2.7
14 1.3
6.9 8.7
2.4 0.97
6.5 5.3
2.0
3.1 4.2
12 6.1
1.8 1.2
5.1 4.3
13 9.1
65 4.3
6.6 7.5
4.0 1.6
18 0.41
2.4 1.1
4.6 2.6
18 8.8
26 18
241
180
227
206
160
101
186
179
173
465
107
621
342
443
181
921
308
71
22
21
16
13
21
4-F
3-F
2-F
4-Cl
4-OCF3
3-F
5-F
15
12
3-CF3
4-F
H
2-F
4-F
3-F
16
15
12
8.6
4-F
3-CH3
3-CF3
3-OCF3
3-CN
3-OCH3
3-F
4.0 3.0
11 14
2.1
>399
116
220
185
165
155
258
121
829
134
360
188
628
>399
162
443
4-F
4-F
4-F
2.4 0.06
4.5 0.99
0.99 0.37
3.6 0.73
44 11
4.5 1.1
7.6 3.4
2.2 0.82
8.5 6.4
4-F
20
18
28
4-CH3
4-OCH3
4-CF3
3-F
c
32
3-F
160
16
a
b
1
All values lacking standard deviations were measured as single data points. All compounds were >95% pure by HPLC and characterized by H
c
d
NMR and HRMS. Compound is mixture of diastereomers (1:1, 2S,3R and 2R,3S). Compounds are incubated with microsomes for 30 min at a
concentration of 1 μM.
Figure 2. PD study of 16 in rat, plasma Aβ40 concentration (100 mg/
kg po, 4 h postdose, P < 0.05). A literature γ-secretase inhibitor18 was
used as a positive control (10 mg/kg, po).
Figure 3. PD study of 16 in rat, brain Aβ40 concentration (100 mg/kg
po, 4 h postdose, P < 0.05). A literature γ-secretase inhibitor18 was
used as a positive control (10 mg/kg, po).
repulsive interaction with the backbone carbonyl of Phe108,
interacts with the edge of Trp115 and also likely helps to
augment the attractive, edge-to-face interaction between the
fluorophenyl substituent and the electron-rich π face of flap
Tyr71. Interactions between the azachromane with Tyr198 (ca.
4.1 Å; not marked), occupation of S1′ by the spirocyclobutyl
moiety, and occupation of the large S2′ cavity by the
hydrophobic neopentyl group provide additional contacts
contributing to the high binding affinity of 16.
In summary, starting from 1, we have improved several key
parameters to help increase the likelihood of achieving CNS
penetration. Compound 16 has a lower molecular weight (163
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ACS Medicinal Chemistry Letters
Letter
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 805-447-8004. E-mail: mkaller@amgen.com (M.R.K.)
and harried@gmail.com (S.S.H.).
Present Addresses
∇Constellation Pharmaceuticals, 215 First Street, Suite 200,
Cambridge, Massachusetts 02142, United States.
○Nimbus Discovery, 25 First Street, Suite 303, Cambridge,
Massachusetts 02141, United States.
¶Envoy Therapeutics, 555 Heritage Drive, Jupiter, Florida
33458, United States.
+Sanofi-Aventis, 270 Albany Street, Cambridge, Massachusetts
02139, United States.
¥Eli Lilly and Company, Lilly Corporate Center, Indianapolis,
Indiana 46285, United States.
Notes
Figure 4. PD study of 16 in rat, CSF Aβ40 concentration (100 mg/kg
po, 4 h postdose, P < 0.05). A literature γ-secretase inhibitor18 was
used as a positive control (10 mg/kg, po).
The authors declare no competing financial interest.
∞Deceased.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the Advanced Light Source. The
Advanced Light Source is supported by the U.S. Department of
Energy under Contract No. DE-AC02-05CH11231.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Procedures and characterization for all newly prepared
compounds, coordinates for the cocrystal structure of inhibitor
16 with BACE, and experimental procedures for the enzyme
and cellular assays. This material is available free of charge via
the Internet at http://pubs.acs.org.
890
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