BACE-1 inhibition by a series of ψ[CH2NH] reduced amide isosteres

Article abstract of DOI:10.1016/j.bmcl.2006.04.076

A series of β-site amyloid precursor protein cleaving enzyme (BACE-1) inhibitors containing a ψ(CH₂NH) reduced amide bond were synthesized. Incorporation of this reduced amide isostere as a non-cleavable peptide surrogate afforded inhibitors possessing low nanomolar potencies in both an enzymatic and cell-based assay.

Full text of DOI:10.1016/j.bmcl.2006.04.076

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3635–3638
BACE-1 inhibition by a series of w[CH2NH]
reduced amide isosteres
Craig A. Coburn,a,* Shawn J. Stachel,a Kristen G. Jones,a Thomas G. Steele,a
Diane M. Rush,a Jillian DiMuzio,b Beth L. Pietrak,b Ming-Tain Lai,b Qian Huang,b
Janet Lineberger,b Lixia Jin,c Sanjeev Munshi,d M. Katharine Holloway,e Amy Espeseth,b
Adam Simon,b Daria Hazuda,b Samuel L. Grahama and Joseph P. Vaccaa
aDepartment of Medicinal Chemistry, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
bDepartment of Alzheimer’s Research, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
cDepartment of Drug Metabolism, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
dDepartment of Structural Biology, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
eDepartment of Molecular Systems, Merck Research Laboratories, PO Box 4, West Point, PA 19486, USA
Received 23 March 2006; revised 25 April 2006; accepted 25 April 2006
Available online 11 May 2006
Abstract—A series of b-site amyloid precursor protein cleaving enzyme (BACE-1) inhibitors containing a w(CH2NH) reduced amide
bond were synthesized. Incorporation of this reduced amide isostere as a non-cleavable peptide surrogate afforded inhibitors
possessing low nanomolar potencies in both an enzymatic and cell-based assay.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Alzheimer’s disease (AD) is a devastating neurodegen-
erative disease that accounts for the majority of
dementia diagnosed in the elderly. AD is characterized
clinically by a slow and progressive decline in cogni-
tive function that inevitably leads to incapacitation
and death.
b-Secretase (BACE-1) is an aspartyl protease that func-
tions as the rate limiting step in the generation of these
Ab peptide fragments. Given the central role of Ab in
AD pathology, as well as the rich history in the field
of aspartic acid protease inhibition, there has been much
support for the pursuit of BACE-1 as a target for AD
therapy.3
Current AD therapy relies primarily on palliative treat-
ment that can temporarily alleviate the symptoms but
fails to provide disease modifying amelioration. The pre-
dominant hypothesis that guides current research into
disease modifying therapies is based on the amyloid cas-
cade.1 This hypothesis states that the gradual and chron-
ic imbalance between the production and clearance of
Ab peptides results in their accumulation in the brain.
These secreted peptides, known as Ab40 and Ab42, are
the result of the proteolytic processing of the amyloid
precursor protein (APP) by two enzymes, b- and c-secre-
tase. Once formed, the Ab monomers can aggregate and
form neurotoxic oligomers that disrupt neuronal func-
tion and lead to cell death and memory loss that is the
phenotype of AD.2
Most of the inhibitors of aspartyl proteases (e.g., HIV
protease, renin, plasmepsin, and cathepsin D) that have
been reported in the literature contain a transition-state
isostere as the key binding element. In a similar fashion,
the majority of the BACE-1 inhibitors that have ap-
peared thus far are peptide-based analogs of the enzyme
substrate that replace the scissile amide bond with a
non-cleavable isostere. Typically the aspartate-bound
water molecule responsible for peptide cleavage is mim-
icked by an alcohol or amine and is contained within the
framework of an hydroxyethylamine (HEA), hydroxy-
ethylene (HE), statine, or norstatine pseudopeptide
backbone4 (Fig. 1).
The incorporation of a w[CH2NH] peptide bond isostere
into various peptide substrates is well documented and
has produced many important compounds for structure
activity investigations. In the context of aspartyl
Keywords: b-Secretase; Reduced amide; Isostere.
Corresponding author. Tel.: +1 215 652 5511; fax: +1 215 652
3971; e-mail: craig_coburn@merck.com
*
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.04.076

3636
C. A. Coburn et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3635–3638
O O
S
O O
amine 2b with the prime-side binding elements con-
S
N
N
tained in 3 and produced a w[CH2NH] reduced amide
inhibitor 4a (P10 ¼ H and P02 ¼ iBu), that showed po-
tent activity against BACE-1 in our enzymatic assay
(IC50 = 117 nM, Table 1). Although quite potent in
the enzyme assay, inhibitor 4a was less effective in
the cell culture assay that measured the production
of a secreted soluble N-terminal fragment of an APP
variant from cultured HEK 293T cells (sAPPb_NF;
IC50 = 3.7 lM).14
F
F
OH
Ph
H
H
H
H
H
N
N
N
N
N
X
O
O
O
O
Ph
1
2a; X =OH
2b; X = NH
OH P1'
P1'
H
H
H
H
N
N
N
N
N
P2'
H
O
O
O
O
Ph
Ph
In order to optimize activity in both assays, we delineat-
ed SAR at the P01 region of the inhibitor and found that
the addition of either small alkyl groups (4b–d) or longer
hydrophilic substituents (4e,f) led to inhibitors with
enhanced enzymatic potencies. Compounds that incor-
porated a small alkyl group at P01 also resulted in a dra-
matic increase in cellular potency and produced
inhibitors with IC50 values ranging from 17 to 22 nM.
While the more polar substituents maintained enzymatic
activity, this modification resulted in diminished cell-
based activity, and can most likely be attributed to the
lower cell permeability of these compounds. All of the
reduced amide derivatives showed some selectivity over
the homologous enzyme BACE-2 (5- to 10-fold) and the
IC50 values versus human renin were greater than
20 lM.
3
4
Figure 1. Structures of BACE inhibitors.
protease inhibition, the concept of the reduced amide
isostere was first exploited by Szelke et al. to generate
potent inhibitors of human renin.5 X-ray crystallograph-
ic analysis showed that these inhibitors interacted with
the enzyme in a manner similar to their statine and
HE analogs.6 Since then, this isostere has been incorpo-
rated into the skeleton of inhibitors of other aspartyl
proteases, such as rhizopuspepsin,7 Sap28 and HIV
protease.9
In the course of our research to develop inhibitors of
b-secretase, we investigated utilizing the reduced amide
isostere as a key binding element. Previously we report-
ed that HEA inhibitors such as 1, when incorporated
into an N-terminal isophthalamide scaffold, were potent
inhibitors of b-secretase.10 The 1,3,5-trisubstituted
aromatic scaffold used in this paradigm was designed
to function as a P3–P1 Val-Asn-Phe peptidomimetic of
an optimized b-secretase cleavage site of APP.11 In those
examples there was a two pronged engagement of the
catalytic dyad upon binding with the hydroxyl and
secondary amino groups and further binding energy
was gained as the cyclopropyl group filled the S01 binding
pocket.
Molecular modeling of compound 4b docked into the
BACE-1 active site revealed that the terminal amide ac-
cessed the S02 binding pocket. As such, we explored the
SAR in this region in order to optimize activity
(Table 2). We found that small alkyl, cycloalkyl and
benzyl groups were tolerated but only at the expense
of both cellular and enzymatic potency (4g–i). Also
apparent was a strict requirement for the presence of a
secondary NH group (cf. 4j) that functioned to properly
align the hydrogen bonding network between the
enzyme and the inhibitor.
The chemistry employed for the preparation of reduced
amide analogs 4 is detailed in Scheme 1. The synthesis
relied on the BOP-mediated coupling reaction between
carboxylic acid 810 and penultimate diamine 7.
To understand the limits of engaging the catalytic
dyad, compound 2a, which was devoid of any prime-
side binding elements, was synthesized. While a poten-
cy loss of 60-fold (IC50 = 630 nM) was observed from
truncation to the terminal primary alcohol, it was
noteworthy that this small molecule inhibitor displayed
even moderate activity since it occupied only three
sub-sites of the BACE-1 enzyme. More discouraging
was that an additional 10-fold loss in potency was ob-
served when the hydroxyl group was substituted with a
primary amine (2b; IC50 = 5.9 lM). A similar loss in
potency was observed when amino statine isosteres
were compared to the corresponding statine-derived
inhibitors of BACE-1.12 In an effort to regain this pre-
cipitous loss in potency, we targeted the alkylation of
the amine for extension toward the prime-side of the
enzyme.
Table 1. BACE-1 enzyme and cell inhibition for P01 derivatives
O
O
S
N
F
P1'
H
H
H
N
N
N
N
H
O
O
O
Ph
Compound
P10
BACE-1
a
IC50 (nM)
sAPPb_NF
IC50 (nM)
a
4a
4b
4c
4d
4e
4f
H
117
8
3710
22
Me
Et
7
20
17
n-Pr
–CH2CH2OH
–CH2CH2SO2Me
4
13
13
751
17,500
Early work in our laboratory detailed the SAR of a
series of micromolar, statine-derived BACE-1 inhibi-
tors 3.13 As such, we evaluated the elaboration of
a n = 3.

C. A. Coburn et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3635–3638
Table 2. BACE-1 enzyme and cell inhibition for P02 analogs
3637
O O
S
N
F
H
H
N
N
P2'
N
H
O
O
O
Ph
Compound
P20
BACE-1
a
IC50 (nM)
sAPPb_NF
IC50 (nM)
a
4g
4h
4i
NHEt
52
24
242
133
NHc-Pr
NHBn
Proline
139
17,200
731
>20,000
4j
a n = 3.
Intermediate 7 was synthesized by an amide coupling
reaction of isobutylamine to N-Boc-L-alanine. Depro-
tection with HCl gas and reductive amination of the
functionalized a-amino amide (6) with commercially
available N-Boc-L-Phe aldehyde afforded a mono-pro-
tected diamine precursor. Exposure of this intermediate
to HCl in EtOAc afforded the dihydrochloride salt 7.
Coupling to benzoic acid 8 afforded the final compounds
in good overall yield.
Figure 2. Overlay of HEA inhibitor 1 and reduced amide 4b.
O
Asn233
Ser325
HO
3.3
HN
Thr232
OH
O O
S
O Gln73
N
Gly34
NH
3.2
3.2
O
O
F
O
3.0
NH
2.9
NH
Ph
NH
3.2
NH
2.7
O
O
Owing to our success in obtaining a co-crystal structure
of HEA inhibitor 1, we were able to acquire an X-ray
O
3.0
O
O
Gly230
HN
15
˚
structure of 4b at 1.8 A resolution. When compared
to compound 1, both inhibitors were found to occupy
the same general space in the active site with a few key
differences (Fig. 2).
Asp228
Thr72
Figure 3. Hydrogen bonding network of inhibitor 4b.
The amide bond of the reduced amide subunit is
involved in two key interactions with the flap of the
enzyme that provided added potency. Namely, there is
In both series, the a-methylbenzamide and Phe-derived
P1 groups occupy the S3 and S1 binding pockets,
respectively and the SAR is conserved between the
two structural classes. In addition there are hydrogen
bonding interactions between both of the P1 and P3
benzamide NH groups and the carbonyl of Gly230
(Fig. 3).
˚
one interaction between the Gln73 NH (3.2 A) and the
P1 carbonyl and a second between the P20 isobutyl
˚
amide carbonyl and Thr72 NH (3.0 A) of BACE-1.
The carbonyl of Gly34 is in close proximity to the
˚
isobutyl amide NH (3.0 A).
The sulfonamide group present on the aromatic scaffold
of inhibitor 4b occupies the S2 site in a manner consis-
tent with that of compound 1. Each sulfonamide oxygen
makes important hydrogen bonding interactions with
the enzyme; one with the backbone NH of Asn233,
and the other with the OH of Ser325.
Further analysis of the crystal structure of inhibitor 4b
in complex with BACE-1 revealed that unlike the mode
of binding of typical transition state isosteres, where the
hydroxyl and/or amino group is in close contact with
both catalytic aspartates, the nitrogen atom at the scis-
sile bond of reduced amide 4b shared a hydrogen bond
H
H
a, b
OH
c,b
N
H2N
Ph
N
H2N
BocNH
N
H
O
O
O
7
5
6
O
S
O
O
S
O
N
N
F
F
d
H
N
H
O
N
O
H
O
OH
O
HN
Ph
N
N
H
O
4
8
Scheme 1. Reagents: (a) isobutylamine, BOP reagent, DIPEA, DCM, 88%; (b) HCl(g), EtOAc, 99%; (c) N-Boc-L-Phe-CHO, NaBH3CN, MeOH,
74%; (d) BOP reagent, amine 7, DIPEA, DMF, 78%.

3638
C. A. Coburn et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3635–3638
2. Lacor, P. N.; Buniel, M. C.; Chang, L.; Fernandez, S. J.;
O O
S
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N
O
H
N
3. Beher, D.; Graham, S. L. Exp. Opin. Invest. Drugs 2005,
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HN
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5. Szelke, M.; Leckie, B.; Hallett, A.; Jones, D. M.; Sueiras,
J.; Atrash, B.; Lever, A. F. Nature 1982, 299, 555.
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Figure 4. Reduced amide inhibitor 9.
with only half of the dyad, specifically Asp228. This
observation is consistent with the known binding prop-
erties of w[CH2NH]pseudopeptides to aspartic acid
proeteases.7–9
9. Weber, J.; Majer, P.; Litera, J.; Urban, J.; Soucek, M.;
Vondrasek, J.; Konvalinka, J.; Novek, P.; Sedlacek, J.;
Strop, P.; Kra¨usslich, H.-G.; Pichova, I. Arch. Biochem.
Biophys. 1997, 341, 62.
Cognizant of the fact that this motif introduced another
amide bond into the framework of the inhibitor, we
incorporated the reduced amide binding element into
one of our previously described non-amide P3 analogs.16
Gratifyingly, we substituted the P3 benzamide ligand of
inhibitor 4b with the isosteric cis vinylcyclopropane moi-
ety that also contained a trans methyl substituent
(Fig. 4). As such, we produced inhibitor 9 that was
equipotent in both the enzyme and cell-based assays
(BACE-1; IC50 = 20 nM; sAPPb_NF; IC50 = 23 nM).
This modification also improved the cell permeability
of the series (Papp = 24 · 10ꢀ6 cm/s) when compared to
either HEA inhibitor 1 (Papp = 0.6 · 10ꢀ6 cm/s) or re-
duced amide 4b (Papp = 10 · 10ꢀ6 cm/s). However, both
compounds were susceptible to P-glycoprotein transport.
10. Stachel, S. J.; Coburn, C. A.; Steele, T. G.; Jones, K. G.;
Loutzenhiser, E. F.; Gregro, A. R.; Rajapakse, H. A.; Lai,
M.-T.; Crouthamel, M.; Xu, M.; Tugusheva, K.; Lineber-
ger, J. E.; Pietrak, B. L.; Espeseth, A. S.; Shi, X.-P.; Chen-
Dodson, E.; Holloway, M. K.; Munshi, S.; Simon, A. J.;
Kuo, L.; Vacca, J. P. J. Med. Chem. 2004, 47, 6447.
11. Shi, X.-P.; Tugusheva, K.; Bruce, J. E.; Lucka, A.; Chen-
Dodson, E.; Hu, B.; Wu, G.-X.; Price, E.; Register, R. B.;
Lineberger, J.; Miller, R.; Tang, M.-J.; Espeseth, A.;
Kahana, J.; Wolfe, A.; Crouthamel, M.-C.; Sankaranaraya-
nan, S.; Simon, A.; Chen, L.; Lai, M.-T.; Pietrak, B.;
DiMuzio,J.;Li,Y.;Xu,M.;Huang,Q.;Garsky,V.;Sardana,
M. K.; Hazuda, D. J. J. Alzheimers Dis. 2005, 7, 139.
12. Yang, W.; Lu, W.; Lu, Y.; Zhong, M.; Sun, J.; Thomas, A.
E.; Wilkinson, J. M.; Fucini, R. V.; Lam, M.; Randal, M.;
Shi, X.-P.; Jacobs, J.; McDowell, R.; Gordon, E. M.;
Ballinger, M. D. J. Med. Chem. 2006, 49, 839.
13. Brady, S. F.; Singh, S.; Crouthamel, M.-C.; Holloway,
M. K.; Coburn, C. A.; Garsky, V. M.; Bogusky, M.;
Pennington, M. W.; Vacca, J. P.; Hazuda, D.; Lai,
M.-T. Bioorg. Med. Chem. Lett. 2004, 14, 601.
14. Pietrak, B. L.; Crouthamel, M.-C.; Tugusheva, K.; Line-
berger, J. E.; Xu, M.; DiMuzio, J. M.; Steele, T.; Espeseth,
A. S.; Stachel, S. J.; Coburn, C. A.; Graham, S. L.; Vacca,
J. P.; Shi, X.-P.; Simon, A. J.; Hazuda, D. J.; Lai, M.-T.
Anal. Biochem. 2005, 342, 144.
The reduced amide isostere represents a new application
of an established pharmacophore in the field of aspartyl
protease inhibition and an alternative structural class of
BACE-1 inhibitors. The incorporation of the 5-substi-
tuted isophthalamide scaffold at the N-terminal site of
this isostere resulted in potent compounds that display
impressive cellular IC50 values. Further modification of
this general template has paved the way toward more
potent and efficacious inhibitors with improved physical
properties. Additional results from these modifications
will be forthcoming.
15. X-ray coordinates have been deposited with the RCSB
Protein Data Bank (PDB) database.
16. Stachel, S. J.; Coburn, C. A.; Steele, T. G.; Crouthamel,
M.-C.; Pietrak, B. L.; Lai, M.-T.; Holloway, M. K.;
Munshi, S. K.; Graham, S. L.; Vacca, J. P. Bioorg. Med.
Chem. Lett. 2006, 16, 641.
References and notes
1. Golde, T. E. Brain Path. 2005, 15, 84.