Structure guided P1′ modifications of HEA derived β-secretase inhibitors for the treatment of Alzheimer's disease

Article abstract of DOI:10.1016/j.bmcl.2012.04.060

The synthesis and SAR of a series of BACE-1 hydroxyethyl amine inhibitors containing substitutions on a spirocyclobutyl moiety is described. Selectivity against cathepsin D, a related aspartyl protease with potential off target toxicity, and improved micr

Full text of DOI:10.1016/j.bmcl.2012.04.060

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3607–3611
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure guided P10 modifications of HEA derived b-secretase inhibitors
for the treatment of Alzheimer’s disease
Holger Monenschein c, , Daniel B. Horne a, , Michael D. Bartberger b, Stephen A. Hitchcock c,
,
⇑
Thomas T. Nguyen a, Vinod F. Patel d, , Lewis D. Pennington a, Wenge Zhong a
a Department of Chemistry Research and Discovery, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
b Department of Molecular Structure, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA
c Envoy Therapeutics, 555 Heritage Drive, Jupiter, FL 33458, USA
d 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:
Received 13 February 2012
Revised 5 April 2012
Accepted 10 April 2012
Available online 19 April 2012
The synthesis and SAR of a series of BACE-1 hydroxyethyl amine inhibitors containing substitutions on a
spirocyclobutyl moiety is described. Selectivity against cathepsin D, a related aspartyl protease with
potential off target toxicity, and improved microsomal stability is exemplified.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Ab amyloid
Alzheimer’s disease
Aspartyl protease
b-secretase
BACE inhibitors
Hydroxyethylene derivatives
Protease inhibitors
Alzheimer’s disease (AD) is a neurodegenerative disease charac-
terized by the progressive formation of insoluble amyloid plaques
and neurofibrillary tangles in the brain. AD currently afflicts more
than 27 million people worldwide, and the AD population is pre-
dicted to grow to 106 million by 2050,1 creating a major economic
and emotional burden on society. Currently only symptomatic
treatments with modest effectiveness are available to treat AD.
b-secretase (BACE-1) is a membrane bound aspartyl protease
that initiates the amyloid cascade by cleavage of amyloid precursor
protein (APP). The subsequent cleavage of the C-terminus of APP by
gamma-secretase results in the formation of beta-amyloid pep-
tides (Ab-40 and Ab-42), the principal components of amyloid pla-
ques.2 BACE-1 null mice are viable and do not produce amyloid
plaques upon aging, suggesting that small-molecule inhibitors of
BACE-1 would be potential disease modifying agents for AD.3
The identification of small-molecule inhibitors of BACE-1 has
been the focus of many pharmaceutical and academic groups
worldwide for the past 20 years.4 Recently, potent non-peptidome-
metic BACE-1 inhibitors with drug-like properties have been
described.5 However in large part, the successful design of
inhibitors has been hampered by the inherently poor pharmacoki-
netic properties of peptidomimetic compounds associated with
inhibitors of aspartyl proteases coupled with the stringent physico-
chemical properties required to attain penetration of small-mole-
cules across the blood–brain barrier.6 As an additional challenge,
selectivity against related aspartyl proteases is challenging due to
high sequence homology of the active site, and in particular
Cathepsin D (CatD) selectivity is desired in order to reduce poten-
tial off-target toxicity.7
We have previously reported our design of orally efficacious
hydroxyethylamine (HEA) inhibitors of BACE-1.7 During the course
of optimization of this series, we arrived at a potent series of pep-
tidomemetic HEA inhibitors containing a spirocyclobutyl moiety
(1–4, Table 1). Unfortunately, it was found that this series,
although potent on BACE-1 and orally efficacious in the rat
in vivo Ab lowering model, suffered from poor pharmacokinetics
leading to poor projected human dose, high microsomal turnover,
and had limited selectivity over CatD. Herein we describe our ef-
forts towards optimization of the P10 spirocylobutyl group to attain
enhanced microsomal stability (hopefully leading to enhanced PK
profiles), and superior CatD selectivity.
The in-house X-ray co-crystal structure8 of 2 demonstrated the
occupation of the S10 subsite by the cyclobutyl moiety.9 However,
it was observed that additional room existed for substitution in
⇑
Corresponding author.
E-mail address: dhorne@amgen.com (D.B. Horne).
Current address.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.060

3608
H. Monenschein et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3607–3611
Table 1
Selected rat and human microsomal stability data for spirocyclobutyl compounds 1–4 (S1–S20indicate BACE-1 enzyme binding domains)
R1
RLM/HLMa
(lL/min/mg)
BACE-1 IC50 (nM)
CatD IC50 (nM)
Selectivity (fold)
b
b
#
1
2
3
4
3-F-Ph
4-F-Ph
3,5-diF-Ph
CH@CH2
226/621
180/107
186/921
56/79
1
5
7
31
4
3
2
465
4.0
0.7
0.3
15.2
a
RLM = rat liver microsomal stability; HLM = human liver microsomal stability. In vitro microsomal stability measured in a high-throughput automated format. Compound
concentration = 1 M. Microsomal protein concentration = 250 g/mL.
Average IC50’s of at least two independent experiments.
l
l
b
Scheme 1. Reagents and conditions: (a) (i) allylMgBr, THF, rt; (ii) TBSOTf, DIEA,
CH2Cl2, rt; (b) (i) OsO4, NMO, t-BuOH/H2O/THF = 2/2/1, 6 h, rt; (ii) NaIO4 t-BuOH/
H2O/THF = 2/2/1, rt; (iii) CuSO4, (R)-2-methyl-2-propanesulfinamide, CH2Cl2, rt; (c)
LiTMP, 2-fluoro-5-neopentylpyridine, THF, ꢀ78 °C, then 7, ꢀ78 °C to rt; (d) (i) TBAF,
THF, rt, (ii) NaH, THF, 65 °C (iii) HCl (4 M in dioxane), THF.
Figure 1. Model of cis-hydroxy compound 40 (yellow) docked into the active site of
BACE-1 (cutaway surface view; flap residues not shown.) Key BACE-1 residues
labeled in green, with relevant Cathepsin D S10 residues (transparent grey) labeled
in white.
periodate to give an aldehyde which was condensed with Ellman’s
chiral sulfinamide12 to give sulfinimine 7. Diastereoselective addi-
tion of the aryl lithium typically gave >5:1 dr of the desired amine
8. TBAF deprotection of the hydroxyl followed by NaH-promoted
cyclization onto the fluoropyridine and deprotection of the amine
with HCl gave the desired 8-azachroman amines 9.
this region, giving rise to a potential avenue for additional BACE-1
potency and/or selectivity over CatD. In particular, the mismatched
steric and polar characteristics of S10 sites of BACE-1 versus CatD
were of note. Docking studies suggested that either van der Waals
interactions, or even hydrogen bonding contacts if desired, could
be made between substituents on the cyclobutyl ring and the near-
by Thr329 and Lys224 residues of BACE-1. On the other hand, anal-
ogous docking into CatD (using literature structure 1LYB)10
showed that such substitutions would be completely encapsulated
by the bulkier and purely hydrophobic, 4-residue array consisting
of Ile229, Ile311, Leu318, and Ile320 (Fig. 1) and thus polar and/
or hydrophilic substitutions might be particularly desirable. Fur-
thermore, since Met-ID studies suggested that the spirocyclobutyl
subunit is a major site of oxidative metabolism, SAR studies were
undertaken to attempt to improve stability of the inhibitors by
reducing lipophilicity or blocking these potential sites of
metabolism.
For the synthesis of 2- and 3-methyl substituted spirocyclobutyl
chromans we first required 2- and 3-methylcyclobutanone
(Scheme 2). The synthesis of 3-methylcyclobutanone 12 started
from commercially available 2-methylpropane-1,3-diol 10 which
was converted to a ditosylate, and then cyclized to dithiane 11 uti-
lizing conditions similar to those described for cyclization of
dibromoalkanes with methyl methylthiomethyl sulfoxide.13 Acid-
mediated cleavage afforded 3-methylcyclobutanone 12. 2-Meth-
ylcyclobutanone 16 was prepared by methanolysis of (1-ethoxycy-
clopropoxy)trimethylsilane14 14 followed by addition of
vinylmagnesium bromide to give vinyl cyclopropane 15. Acid-facil-
itated cyclopropane rearrangement14 gave 2-methylcyclobutanone
16. The cyclobutanones were then carried forward using the gener-
ic procedure above (Scheme 1) to provide the 2- and 3-methylspi-
rocyclobutylchromans 13 and 17.
The synthesis of the required compounds was undertaken uti-
lizing our established protocol for the synthesis of spirocyclobutyl
containing 8-aza chroman amines (Scheme 1).9,11 Allylation of
cyclobutanone 5 followed by protection of the secondary hydroxyl
with TBSOTf gave allylcyclobutane 6. The olefin was oxidatively
cleaved by OsO4 catalyzed dihydroxylation followed by sodium
For the 3-hydroxy and 3,3-difluoro analogs, the synthesis began
with the 2+2 cycloaddition of vinyl pivalate 18 with in situ gener-
ated dichloroketene to afford a 2,2-dichlorocyclobutanone, which
upon zinc mediated reduction gave cyclobutanone 19 (Scheme
3). Allylation with allyltrimethylsilane and TiCl4 and protection

H. Monenschein et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3607–3611
3609
Scheme 4. Reagents and conditions: (a) (diethylamino)sulfurtrifluoride, CH2Cl2,
75 °C, 71%.
Scheme 2. Reagents and conditions: (a) (i) TsCl, Py, rt, 12 h, 81%; (ii) methylsulfi-
nyl(methylthio)methane, n-BuLi; ꢀ10 °C to rt (b) HCl, distillation 110–120 °C, 60%
(c) (i) MeOH, rt, 62%; (ii) H2C@CHMgBr, 70%; (d) H2SO4, 0 °C, 55%.
Scheme 5. Reagents and conditions: (a) NaBH(OAc)3, CH(OEt)3, EtOH,(b) HCl,
MeOH/1,4-dioxane (c) acetylimidazole, i-Pr2NEt, DMF or EDCI, i-Pr2NEt,
R2CH2CO2H, DMF.
34 and 36 showed 4-10 fold less CatD selectivity versus the cis-iso-
mers. Unfortunately, average microsomal stability of the 3-methyl
analogs was significantly worse than the parent compounds.
As an alternative to introducing methyl groups to the spirocyc-
lobutyl ring, which are potential sites of metabolism, we switched
our attention towards blocking sites of metabolism with fluorine
atoms.15 On average however, none of the 3-fluoro 38–41 or 3,3-
difluoro 42 analogs offered significant stability improvement, and
only moderate CatD selectivity was observed (Table 4).
Scheme 3. Reagents and conditions: (a) (i) trichloroacetylchloride, Zn powder,
Et2O, rt, 100%; (ii) Zn powder, AcOH, 0 °C, 85%; (b) (i) allyltrimethylsilane, TiCl4, 0 °C
to rt, 95%; (ii) TBSOTf, iPr2NEt, CH2Cl2, rt, 77%; (c) DIBAL-H, CH2Cl2, ꢀ78 °C, 98% (d)
TBSOTf, iPr2NEt, CH2Cl2, rt, 82%; (e) (i) Dess–Martin preiodinane, NaHCO3, CH2Cl2,
91%; (ii) (diethylamino)sulfurtrifluoride, 10% EtOH, CH2Cl2, rt, 28%.
of the hydroxyl group as the tert-butyldimethylsilyl ether yielded
20a. Protecting group switch from the pivalate to the silyl ether
afforded disilylether 20c, which was carried on (Scheme 1) to give
the 2-hydroxyl chromans 21. The 3,3-difluoro analog was prepared
from 20a by reductive removal of the pivalate protecting group to
give 3-hydroxycyclobutane 20b. Oxidation of the secondary alco-
hol to the ketone and transformation of the ketone to the 3,3-di-
fluoro cyclobutane 22 was accomplished by DAST-mediated
fluorination. The cyclobutane then intercepted intermediate 6 in
the generic synthesis (Scheme 1) to give chroman 23.
We prepared the 3-fluoro analogs from the 3-hydroxy chroman
24 by DAST mediated fluorination. The diastereomeric fluoro com-
pounds were separated by silica gel chromatography to give 25 and
26 (Scheme 4).
With the required chromans in hand, the conversion to final
compounds was accomplished by means of the three step proce-
dure below (Scheme 5). Reductive amination with the chroman
27 and aldehyde 28, followed by global deprotection of the TBS
ether and the Boc amine with HCl, and subsequent installation of
the secondary amide with acetylimidazole (or EDCI and an acid)
gave the required HEA compounds 29.
2-methyl substituted analogs 30–33 were screened in BACE-1
and Cat D enzymatic assays and in vitro stability assessment was
performed. We found that both CatD selectivity and microsomal
stability in the 2-methyl substituted analogs 30–33 was disap-
pointing (Table 2). Interestingly compounds 31 and 33 were
substantially less active on BACE-1 showing a 2–3 order of magni-
tude loss of potency compared to the des-methyl compound 2.
We found that the introduction of the 3-methyl group, prefera-
bly in the cis configuration, relative to the oxygen, (35 and 37,
Table 3) significantly improved CatD selectivity when compared
to the parent compounds (2 and 3, Table 1). The trans-compounds
In order to address the metabolic stability issues, we explored
reducing lipophilicity while also blocking a potential site of metab-
olism. Thus we examined the 3-hydroxyl substituted analogs 43–
46 (Table 5). In contrast to the 3-methyl analogs we found that
the introduction of the 3-hydroxy group had a more pronounced
affect on CatD selectivity, in excess of 100 fold. The stereochemical
cis preference (cis-44 versus trans-43) tracked nicely with the pre-
viously established 3-methyl SAR. The selectivity came at a price
however, and microsomal stability in the series was still relatively
poor, particularly in RLM. One explanation for the decreased stabil-
ity could be that the sterically exposed 3-hydroxy group may be
prone to metabolic oxidation. In addition, examination of the P-
gp efflux potential of the molecules in P-gp expressing LLC-PK1
cells (pig kidney epithelial cells) showed that compound 44 was
poorly permeable. Compounds 45 and 46 were made in attempt
to shield the amide NH by an intramolecular hydrogen bond to in-
crease permeability,16 but did not result in better efflux properties.
Combination of the preferred cis-3-hydroxyspirocyclobutyl sub-
stitution with the moderately CatD selective and microsomally sta-
ble P1-allyl group compound 4 (Table 1) gave compound 47 which
had >8500 fold CatD selectivity (Table 5). Microsomal stability was
also significantly improved. However, efflux remained a liability
and the molecule was poorly permeable.
In light of the favorable profiles of compounds 44 and 47, in re-
gards to potency and CatD selectivity, they were advanced to
in vivo rat PD efficacy studies.17 The efficacy studies with 44 and
47 at 100 and 30 mg/kg doses (PO) showed negligible (<15%) low-
ering of Ab40 in cerebral spinal fluid in rat after 4 h and low plasma
concentrations of the parent compounds (44 = 0.012
lM, 100 mg/
kg, 4 h post dose; 47 = 0.009 M, 30 mg/kg, 4 h post dose) Subse-
l
quent profiling revealed that compound 44 suffered from poor
in vivo PK (rat IV clearance = 7.6 L/h/kg) as well as poor passive

3610
H. Monenschein et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3607–3611
Table 2
Effect of 2-methyl substitution on microsomal stability and CatD selectivity
b
b
#
RLM/HLMa
(lL/min/mg)
BACE-1 IC50 (nM)
CatD IC50 (nM)
Selectivity (fold)
30
31
32
33
236/232
399/829
227/68
7
5
0.7
0.7
1.6
—
30500
29
>40000
20500
46
>40000
399/590
a
RLM = rat liver microsomal stability; HLM = human liver microsomal stability. In vitro microsomal stability measured in a high-throughput automated format. Compound
concentration = 1 M. Microsomal protein concentration = 250 g/mL.
Average IC50’s of at least two independent experiments.
l
l
b
Table 3
Effect of 3-methyl substitution on microsomal stability and CatD selectivity
RLM/HLMa
(lL/min/mg)
BACE-1 IC50 (nM)
CatD IC50 (nM)
Selectivity (fold)
b
b
#
X
34
35
36
37
4-F
4-F
3,4-diF
3,4-diF
399/246
451/108
921/775
706/399
9
4
0.7
0.7
8
38
4
0.9
9.5
6.2
25
17
a
RLM = rat liver microsomal stability; HLM = human liver microsomal stability. In vitro microsomal stability measured in a high-throughput automated format. Compound
concentration = 1 M. Microsomal protein concentration = 250 g/mL.
Average IC50’s of at least two independent experiments.
l
l
b
Table 4
Effect of 3-fluoro and 3-gem-difluoro substitution on microsomal stability and CatD selectivity
RLM/HLMa
(lL/min/mg)
BACE-1 IC50 (nM)
CatD IC50 (nM)
Selectivity (fold)
b
b
#
X
38
39
40
41
42
4-F
4-F
3-F
3-F
4-F
370/223
411/240
492/829
447/706
399/408
9
6
2
2
8
8
0.9
2.3
6
11
1.4
14
12
22
11
a
RLM = rat liver microsomal stability; HLM = human liver microsomal stability. In vitro microsomal stability measured in a high-throughput automated format. Compound
concentration = 1 M. Microsomal protein concentration = 250 g/mL.
Average IC50’s of at least two independent experiments.
l
l
b
permeability (Papp A to B = 3.2 ꢁ 10ꢀ6 cm/sec). Although com-
pound 47 initially appeared promising due to the inherent micro-
somal stability compared to 44, the poor results in the rat PD
efficacy study and low plasma concentration likely also resulted
from poor pharmacokinetic properties. In addition, the poor
permeability of these compounds makes the prospect of these
inhibitors crossing the blood–brain barrier to inhibit BACE-1 in
the brain unlikely, providing further deemphasis of these particu-
lar compounds.
Despite a committed and focused effort addressing the inherent
low microsomal stability and the correlated unfavorable in vivo
pharmacokinetic profile showing high clearance and low oral bio-
availability of this chemical series, it proved to be rather difficult to
obtain analogs containing both high intrinsic stability coupled with
good selectivity over CatD simply by substitution of the cyclobutyl
ring. A notable exception to this was compound 47, which was sta-
ble in microsomes, potent, and selective, but suffered from high ef-
flux and poor PK properties in the rat in vivo Ab lowering model.

H. Monenschein et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3607–3611
3611
Table 5
Effect of 3-hydroxy substitution on microsomal stability and CatD selectivity
b
b,c
b
#
R2
metabolic stability RLM/HLMa
L/min/mg)
BACE-1 IC50
(nM)
HEK IC50
(nM)
CatD IC50
(nM)
CatD selectivity
(fold)
Pappd (ꢁ10ꢀ6 cm/
Pgp-Effluxe
Rat Human
(
l
sec)
43
44
45
46
47
H
H
OMe
F
H
228/90
706/34
682/115
775/65
56/<14
14
3
2
4
5
41
5
8
10
27
1650
577
695
347
>40000
115
184
285
81
2.6
4.3
6.5
6.4
<2
—
>50
—
—
>50
21.5
13.5
35.8
>50
>50
>8500
a
RLM = rat liver microsomal stability; HLM = human liver microsomal stability. In vitro microsomal stability measured in a high-throughput automated format. Compound
concentration = 1 M. Microsomal protein concentration = 250 g/mL.
Average IC50’s of at least two independent experiments.
l
l
b
c
d
e
Cell potency measured in human embryonic kidney (HEK293) cells stably expressing APP.
Apparent permeability measured in parental LLC-PK1 cells.
Efflux measured in LLC-PK1 cells transfected with rat MDR1A/1B or human MDR1 and reported as a ratio of (B to A)/(A to B).
Xue, M.; Yang, B.H.; Zhang, J.; Patel, V.F.; Zhong, W.; Hitchcock, S. Med. Chem.
Lett. 2012, in press.
Further efforts describing additional compounds with more exten-
sive substitutions and resulting in improved PK, and in vivo effi-
cacy than that described in this manuscript will be reported
subsequently.
10. Baldwin, E. T.; Bhat, T. N.; Gulnik, S.; Hosur, M. V.; Sowder, R. C.; Cachau, J.;
Collins, J.; Silva, A. M.; Erickson, J. W. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 6796.
11. (a) Zhong, W.; Hitchcock, S.; Patel, V.F.; Croghan, M.; Dineen, T.; Harried, S.;
Horne, D.; Judd, T.; Kaller, M.; Kreiman, C.; Lopez, P.; Monenschein, H.; Nguyen,
T.; Weiss, M.; Xue, Q.; Yang, B. PCT Int. Appl. WO2008147547, 2008.; (b) Zhong,
W.; Hitchcock, S.; Patel, V.F.; Croghan, M.; Dineen, T.; Horne, D.; Kaller, M.;
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obtained by centrifugation for drug exposures. Brains were removed and,
along with the CSF, immediately frozen on dry ice and stored at ꢀ80 °C until
use. The frozen brains were subsequently homogenized in 10 volumes of (w/v)
of 0.5% Triton X-100 in TBS with protease inhibitors. The homogenates were
centrifuged at 100,000 rpm for 30 min at 4 °C. The supernatants were analyzed
for Ab40 levels by immunoassay as follows: Meso Scale 96-well avidin plates
were coated with Biotin-4G8 (Covance) and detected with Ruthenium-labeled
Fab specific for Ab40. Plates were read in MSD Sector6000 imager according to
manufacturer’s recommended protocol (Meso Scale Discovery, Inc.). Ab40
concentrations were plotted using Graphpad Prism and analyzed by one-way
ANOVA followed by Dunnett’s multiple comparison analysis to compare drug-
treated animals to vehicle-treated controls.