Discovery of an orally efficaceous 4-phenoxypyrrolidine-based BACE-1 inhibitor

Article abstract of DOI:10.1016/j.bmcl.2007.10.053

Based on a lead compound identified from the patent literature, we developed patentably novel BACE-1 inhibitors by introducing a cyclic amine scaffold as embodied by 1a and 1b. Extensive SAR studies assessed a variety of isophthalamide replacements including substituted pyrrolidinones and ultimately led to the identification of 11. Due to its favorable overall profile, 11 has been extensively profiled in various in vivo settings.

Full text of DOI:10.1016/j.bmcl.2007.10.053

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 418–422
Discovery of an orally efficaceous 4-phenoxypyrrolidine-based
BACE-1 inhibitor
U. Iserloh,a,* J. Pan,a A. W. Stamford,a M. E. Kennedy,b Q. Zhang,b L. Zhang,b
E. M. Parker,b N. A. McHugh,b L. Favreau,c C. Stricklanda and J. Voigta
aDepartment of Chemical Research, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
bDepartment of Neurobiology, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
cDepartment of Drug Metabolism and Pharmacokinetics, Schering-Plough Research Institute, 2015 Galloping Hill Road,
Kenilworth, NJ 07033, USA
Received 23 July 2007; revised 2 October 2007; accepted 5 October 2007
Available online 18 October 2007
Abstract—Based on a lead compound identified from the patent literature, we developed patentably novel BACE-1 inhibitors by
introducing a cyclic amine scaffold as embodied by 1a and 1b. Extensive SAR studies assessed a variety of isophthalamide replace-
ments including substituted pyrrolidinones and ultimately led to the identification of 11. Due to its favorable overall profile, 11 has
been extensively profiled in various in vivo settings.
Ó 2007 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD)1 is a progressive and ulti-
mately fatal neurodegenerative disorder, for which no
effective treatment is currently available. One of the ma-
jor pathological hallmarks of AD is the abnormal depo-
sition of amyloid plaques comprised of Ab40,42 peptides
in the brain of AD patients. These peptides arise from
sequential cleavage of the amyloid precursor protein
(APP) by b-amyloid cleaving enzyme-1 (BACE-1)2 and
c-secretase.3 As BACE-1 catalyzes the first committed
step in the synthesis of b-amyloid, inhibitors of
BACE-1 are expected to be useful treatments for AD.
exhibited insufficient pharmacokinetic properties in rats
as evidenced by low plasma levels following oral dosing.
We now report on studies of various isophthalamide
replacements to improve the bioactivity, pharmacoki-
netic parameters as well as ancillary profile of our cyclic
amine BACE-1 inhibitors, ultimately resulting in the dis-
covery of 11.
F
R
In the preceding paper in this issue,4 we described the
discovery of conformationally constrained versions of
the hydroxyethylamine (HEA) motif found in many
peptidomimetic inhibitors.5 Based on a lead structure
identified from the patent literature,6 we developed
4-benzyloxypyrrolidine and 4-phenoxypyrrolidine con-
taining inhibitors 1a and 1b with good in vitro potency
(5 nM and 3 nM, respectively) although with low cellu-
lar activity and modest selectivity against other human
aspartyl proteases (Fig. 1). In addition, both inhibitors
F
HN
N
O
H
OH
N
O
1a OBn
OPh
1b
Assaya
1a (OBn)
1b (OPh)
BACE-1 IC50
BACE-2 IC50
Cell IC50 (HEK 293)
Cathepsin D Ki
Cathepsin E Ki
Pepsin Ki
5 nM
45 nM
150 nM
36 nM
3 nM
54 nM
165 nM
291 nM
24 nM
5 nM
158 nM
162 nM*hr
232 nM
227 nM*hr
Keywords: Alzheimer’s disease; b-Secretase inhibitors; Aspartyl
proteases.
AUC0-6h (rat, 10 mpk po)
*
Figure 1. Profile of pyrrolidine-containing BACE-1 inhibitors 1a and
1b. aSee Ref. 7 for details of in vitro and cell assays.
Corresponding author. Tel.: +1 908 740 7375; fax: +1 908 740
7152; e-mail: ulrich.iserloh@spcorp.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.053

U. Iserloh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 418–422
419
Inspection of the X-ray structure for 4-benzyloxypyrr-
olidine-based inhibitor 1a bound to BACE-1 revealed
two hydrogen-bonds from the isophthalamide car-
bonyl-groups to the peptide backbone NH-groups of
Thr232 (S3 subsite) and Thr72 (flap), along with hydro-
phobic contacts within the S3 and S2 enzyme subsites
(Fig. 2).8 As part of our plan to identify isophthalamide
replacements, we decided to retain the internal amide
with its key interaction to the flap Thr72 while broadly
screening for more appropriate S2–S3 moieties. Synthet-
ically, this would entail amide formation from our key
intermediate, aminoalcohol 2,4 followed by Boc-depro-
tection (Scheme 1).
Considering that pyrrolidinones have a larger molecular
volume than azetidinones and display the pyrrolidine
N-substituent differently with respect to Thr232 and
the S3-enzyme subsite, we decided to investigate
BACE-1 inhibitors containing N-substituted pyrrolidi-
nones 99 (Tables 1 and 2).
The modest potency of the prototypical inhibitor 9a
with its small N-ethyl group (BACE-1 IC50 = 7 lM)
underlines the importance of hydrophobic interactions
in the S3 enzyme subsite (Table 1). With increasing
chain length (9b, 9d–9f), in vitro potencies greatly im-
proved reaching a maximum of 45 nM for 9e. While fur-
ther branching was tolerated (9c, 9g, and 9h),
incorporation of a less hydrophobic oxygen atom re-
sulted in diminished activity (9i–9l).
Several dozen inhibitors including aliphatic amides (3),
benzamides (4), arylacetamides (5), and cinnamides (6)
were synthesized yet elicited uniformly poor bioactivity
(BACE-1 IC50: 10–100 lM) and were not further pro-
filed (Scheme 1). Amides (7) and sulfonamides (8) incor-
A screen of benzyl groups (9m–9s) revealed the incom-
patibility of para-substitution—even a small fluoro-sub-
stituent (9n) led to a 10-fold loss of activity (vs. N-benzyl
9m). Meta- and ortho-substitution was tolerated with
BACE-1 IC50s similar to the parent compound, though
cellular activity was diminished. To moderate the cellu-
lar potency, we also screened a variety of heteroaryl
groups (9t–9y). Despite maintaining in vitro potency
for all but 9y (BACE-1 IC50: 219–312 nM), cellular
activities were largely unimproved.
porating
azetidine-3-carboxylic
acid
exhibited
reasonable potency (BACE-1 IC50 = 1–10 lM) yet dem-
onstrated a fairly flat SAR most likely due to reduced
hydrophobic contacts in the S2 subsite and suboptimal
interactions with Thr232.
Since X-ray crystallography of 9d and 9e suggested that
stereodefined 2-substituted pyrrolidinones may yield
additional binding affinity, we evaluated inhibitors
10a–10h (Table 2). Although substituting the C2-posi-
tion of 9d with alkyl or benzyl groups (10a–10d) moder-
ately improved the in vitro and cellular activity, we did
not observe a similar improvement for the N-pentyl ser-
ies (10e–10h).
Taken together, these studies revealed that the pyrrolid-
inone scaffold appeared inferior to the isophthalamide
moiety, prompting us to revisit further modifications
of 1a and 1b.
Figure 2. X-ray structure of 1a bound to BACE-1, with enzyme flap
shown in green. Key hydrogen-bonds from 1a to Thr232 and flap
Thr72 are indicated by dotted lines.
Another isophthalamide moiety widely adopted
throughout the patent literature10 forms the basis for
BACE-1 inhibitor 11 and its benzyloxypyrrolidine con-
gener (Fig. 3).8 Replacement of the N,N-dipropylamide
present in 1b with a 2-(R)-methoxymethylpyrrolidine
amide resulted in a marked increase in cellular potency
(BACE-1 Ki = 0.7 nM, cell IC50 = 21 nM), while main-
taining good selectivity over related human aspartyl
proteases such as cathepsin D, cathepsin E, and renin.
F
RCO2H,
HOAt/HATU;
2
OBn
3-11
F
TFA
H2N
N
Boc
OH
When tested in rats (10 mpk po dose), 11 exhibited im-
proved pharmacokinetic properties with reasonable
AUC (841 nMÆh) though a low brain/plasma ratio of
less then 0.1. In a study with a 5 mpk iv dose, a rat plas-
ma half-life of 3.2 h was determined, with a high volume
of distribution (Vd = 15 L/kg) and clearance (Fig. 3).
BACE-1 inhibitor 11 has a good ancillary profile and
did not inhibit the tested CYP enzymes (>30 lM). The
compound has high human and mouse plasma protein
binding, reasonable kinetic solubility and no significant
inhibition in a rubidium-efflux hERG assay. Although
O
O
O
O
Ar
R
R
Ar
O
R
3
4
5
6
O
OBn
3,5-F2Ph
N
R
N
N
H
N
S
H
8
7
OH
O
O
O
Scheme 1. Synthesis of BACE-1 inhibitors 3–11; IC50 (BACE-1)
>10 lM (3–6), >1 lM (7–8).

420
U. Iserloh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 418–422
Table 1. SAR for N-substituted pyrrolidinone BACE-1 inhibitors
Table 1 (continued)
Compound
R
BACE-1
a
IC50 (nM)
Cell
IC50 (nM)
a
OBn
Ar
O
O
9v
9x
9y
225
303
1070
N
H
N
O
H
OH
N
R
9a - 9y
S
S
1350
6200
Compound
R
BACE-1
a
IC50 (nM)
Cell
IC50 (nM)
a
1200
N
9a
9b
9c
9d
9e
9f
Ethyl
Propyl
7000
900
3000
370
45
5000
Ar = 3,5-difluorophenyl.
a See Ref. 7 for details of in vitro and cell assays.
Isopropyl
Butyl
2300
610
Pentyl
Hexyl
100
582
moderate permeability was observed in the Caco-2 P-gp
assay, the efflux ratio was high (B–A/A–P = 174) indi-
cating that 11 is most likely a substrate for the P-gp
transporter. This may account for the low brain/plasma
ratio observed in the rat PK studies.
9g
9h
71
1200
795
2
155
Due to its favorable overall profile, inhibitor 11 was
extensively profiled in the transgenic CRND8 mouse
model (Fig. 4).11 Upon oral dosing to 6-week old pre-
plaque CRND8 mice at 10, 30, and 100 mpk, plasma
Ab40 was reduced by 4%, 25%, and 70% relative to the
vehicle group.
9i
2000
2950
270
2000
O
O
9j
Subcutaneous dosing further improved exposure levels
resulting in even greater reduction of plasma Ab40
(10 mpk: À58%; 30 mpk: À77%; 100 mpk: À88%). De-
spite whole brain concentrations in excess of 50-fold
its cellular IC50, 11 had no effect on cortical Ab40 levels.
9k
9l
1200
1500
1700
5150
O
O
850
In conclusion, replacement of the isophthalamide por-
tion of BACE-1 inhibitor 1a with various truncated
amides and N-substituted pyrrolidinones did not im-
prove in vitro or cellular potency. However, incorpora-
9m
9n
290
2800
F
9o
9p
18800
46000
Cl
Table 2. SAR for enantiomer 2-substituted pyrrolidinone BACE-1
inhibitors
OBn
Ar
R2
O
CF3
N
N
O
H
H
OH
N
R
9q
9r
338
350
2130
2150
10a - 10h
F
Compound
R
R2
H
BACE-1
IC50 (nM)
Cell
IC50 (nM)
9d
Butyl
370
142
110
57
2300
400
10a
10b
10c
10d
9e
Ethyl
Propyl
330
260
F
9s
9t
9u
365
312
219
1400
3600
5600
Isobutyl
Benzyl
78
45
1200
610
Pentyl
H
N
10e
10f
10g
10h
Cyclopropyl
Cyclopentyl
Isobutyl
Benzyl
570
295
365
293
2400
4250
2600
4000
N
Ar = 3,5-difluorophenyl.

U. Iserloh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 418–422
421
ous dosing, demonstrating the utility of 11 as a tool
compound suitable for in vivo studies. Further SAR
studies will be reported in due course.
F
OPh
F
O
HN
N
O
H
OH
N
O
Acknowledgments
11
The authors thank Drs. A. Buevich and T.-M. Chan for
NMR analyses, R. Kuvelkar and X. Chen for Ki and
IC50 determinations, and Dr. William Greenlee for con-
tinued support. Use of the IMCA-CAT beamline 17-ID
at the Advanced Photon Source was supported by the
companies of the Industrial Macromolecular Crystallog-
raphy Association through a contract with the Center
for Advanced Radiation Sources at the University of
Chicago. Use of the Advanced Photon Source was sup-
ported by the US Department of Energy, Office of Sci-
ence, Office of Basic Energy Sciences, under Contract
No. W-31-109-Eng-38.
Assay
Assay
BACE-1 Ki
BACE-2 Ki
Cell IC50 (HEK)
Cathepsin D
Cathepsin E
Renin
0.7 nM
20 nM
21 nM
2525 nM
170 nM
500 nM
CYP 2D6
CYP 3A4
CYP 2C9
>30 uM
>30 uM
>30 uM
PPBc (human)
PPBc (mouse)
Solubility
96.0%
97.7%
175 uM
12%
a
Rat AUC0-6h
C6h
841 nM*hr hERG Rb Efflux
101 ng/mL (Inh. at 5 ug/mL)
a
Brain/plasmaa
<0.1
3.2 hr
Caco-2 Efflux ratio
(B-A/A-B)
x 174
b
t1/2
Clearanceb
Vdb
147 mL/min/kg
15 L/kg
a
Figure 3. Detailed profile for novel BACE-1 inhibitor 11. 10 mpk po
References and notes
c
dose (rat). b5 mpk iv dose (rat). Protein plasma binding.
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2. BACE-1 is also known as b-secretase. Recent efforts
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Durham, T. B.; Shepherd, T. A. Curr. Opin. Drug Discov.
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Cumming, J. N.; Iserloh, U.; Kennedy, M. E. Curr. Opin.
Drug Discov. Devel. 2004, 7, 536.
tion of a methoxymethylpyrrolidine isophthalamide
achieved a significant improvement in cellular activity,
prompting us to broadly profile 11 in the CRND8
mouse model. Here, dose-dependent reduction of plas-
ma Ab40 was observed both upon oral and subcutane-
3. Kimberly, W. T.; Wolfe, M. S. J. Neurosci. Res. 2003, 74,
353.
4. Iserloh, U.; Wu, Y.; Cumming, J. N.; Pan, J.; Wang, L.
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5. Leung, D.; Abbenante, G.; Fairlie, D. P. J. Med. Chem.
2000, 43, 305.
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closed by Elan/Pharmacia/Pfizer: Maillaird, M.; Hom, R.;
Gailunas, A.; Jagodzinska, B.; Fang, L. Y.; John, V.;
Freskos, J. N.; Pulley, S.R.; Beck, J. P.; Tenbrink, R. E.
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7. BACE-1 enzymatic inhibition was determined using an
APP-derived peptide containing the Swedish mutant: (a)
Kennedy, M. E.; Wang, W.; Song, L.; Lee, J.; Zhang, L.;
Wong, G.; Wang, L.; Parker, E. Anal. Biochem. 2003, 319,
49; Compound IC50s for inhibition of Ab40 production
were determined by incubating HEK-293 cells, stably
transfected with the human APP cDNA containing both
Swedish and London FAD mutations, with increasing
concentrations of BACE inhibitors. Ab40 levels were
measured in the cell culture media using an Ab1À40
specific ELISA: (b) Zhang, L.; Song, L.; Terracina, G.;
Figure 4. Dose-dependent plasma Ab40 lowering in 6-week-old
CRND8 mice at 3 h post-dosing relative to vehicle group. Top panel
shows results from oral dosing, bottom panel highlights efficacy upon
s.c. dosing. NTg: non-transgenic mice devoid of APP mutations.

422
U. Iserloh et al. / Bioorg. Med. Chem. Lett. 18 (2008) 418–422
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8. X-ray coordinates for the complex of BACE-1 with
inhibitors 1a, 1b, and 11 have been deposited in the
Protein Data Bank (www.rcsb.org), and can be accessed
under PDB ID 2QMD, 2QMF, and 2QMG.
9. Racemic pyrrolidinone-3-carboxylic acids were obtained
by condensation of primary amines with dimethyl
itaconate; diastereomers were separated after coupling
to 2: (a) Felluga, F.; Pitacco, G.; Prodan, M.; Pricl,
S.; Visintin, M.; Valentin, E. Tetrahedron Asymmetr.
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Upjohn: Varghese, J.; Maillard, M.; Jagodzinska, B.;
Beck, J.P.; Gailunas, A.; Fang, L.; Sealy, J.; Tenbrink, R.;
Freskos, J.; Mickelson, J.; Samala, L.; Hom, R. PCT Int.
Appl., WO 2003040096, 2003.
11. CRND8-APP mice are an aggressive model of amyloid
pathology that utilize a prion promoter to drive expression
of human APP containing the Swedish and Indiana
mutations which enhance b-secretase cleavage and Ab42
production, respectively.