Design, synthesis, and crystal structure of hydroxyethyl secondary amine-based peptidomimetic inhibitors of human β-secretase

Article abstract of DOI:10.1021/jm061242y

The design and synthesis of a novel series of potent and cell permeable peptidomimetic inhibitors of the human β-secretase (BACE) are described. These inhibitors feature a hydroxyethyl secondary amine isostere and a novel aromatic ring replacement for the

Full text of DOI:10.1021/jm061242y

776
J. Med. Chem. 2007, 50, 776-781
Design, Synthesis, and Crystal Structure of Hydroxyethyl Secondary Amine-Based
Peptidomimetic Inhibitors of Human â-Secretase†
Michel C. Maillard,‡ Roy K. Hom,*,‡ Timothy E. Benson,*,§ Joseph B. Moon,§ Shumeye Mamo,‡ Michael Bienkowski,§
Alfredo G. Tomasselli,§ Danielle D. Woods,§ D. Bryan Prince,§ Donna J. Paddock,§ Thomas L. Emmons,§ John A. Tucker,‡
Michael S. Dappen,‡ Louis Brogley,‡ Eugene D. Thorsett,‡ Nancy Jewett,‡ Sukanto Sinha,‡ and Varghese John‡
Elan Pharmaceuticals, 800 Gateway BouleVard, South San Francisco, California 94080, and Pfizer, Inc., Chesterfield, Missouri 63017 and
Ann Arbor, Michigan 48105
ReceiVed October 23, 2006
The design and synthesis of a novel series of potent and cell permeable peptidomimetic inhibitors of the
human â-secretase (BACE) are described. These inhibitors feature a hydroxyethyl secondary amine isostere
and a novel aromatic ring replacement for the C-terminus. The crystal structure of BACE in complex with
this hydroxyethyl secondary amine isostere inhibitor is also presented.
Introduction
bioavailable, CNS-active BACE inhibitor, we report herein the
rational design of a new series of potent and cell permeable
inhibitors of BACE. Compound 6b represents an advanced
BACE inhibitor incorporating the hydroxyethyl secondary amine
(HEA) isostere and a nonpeptidic C-terminus. Furthermore, we
have determined the X-ray structure of a representative of this
series of inhibitors complexed with BACE at a resolution of
1.7 Å.
Alzheimer’s disease (AD) is a progressive and ultimately fatal
neurodegenerative disorder that afflicts nearly five million
people in the United States alone. Intensifying research efforts
have focused on understanding the role of AD’s neuropatho-
logical markers, plaques and tangles, in the disease process.1
The amyloid cascade hypothesis, which has been the dominant
research paradigm, centers around the amyloid â (Aâ) peptide,
which is the principal component of the amyloid plaques
detected in the brain of AD patients. Although the link between
Aâ, either in its partially soluble or aggregated forms, and AD
neuronal dysfunction is still controversial, there is, however,
compelling biochemical, pathological, and genetic evidence that
progressive accumulation of the Aâ peptide within the brain is
critically responsible for the neurodegeneration that occurs in
AD.2 Therefore, a strategy aimed at lowering the concentration
of neurotoxic Aâ represents an attractive avenue for clinical
intervention.3
The dominant anti-amyloid (Aâ lowering) approaches that
have been pursued are aimed at either increasing Aâ clearance
by immunization4 or decreasing Aâ production or aggregation.
The latter approach has focused primarily on two proteolytic
enzymes, â- and γ-secretases, that participate in the processing
of the amyloid precursor protein (APP).5 Our discovery program
has targeted one of these enzymes, â-secretase, because it plays
a crucial role in the first (rate-limiting) step of the amyloid
cascade.
Inhibitor Design and Results
We have previously reported on the development of potent
statine and hydroxyethylene (HE) transition state isostere (TSI)-
based inhibitors of BACE using the substrate-based approach.10
However, these compounds (such as 1) demonstrated unfavor-
able cell-to-enzyme (C/E) potency ratios (C/E ∼ 100), which
prevented further development of these series. In the search for
an alternate TSI compatible with favorable pharmacokinetics,
we became interested in the HEA isostere because of its success
in the HIV protease (HIV-PR) field.11 We targeted the hydroxy-
ethyl secondary amine TSI due to the strict preference of BACE
for acyclic residues at the S1′ subsite,12 in contrast to HIV-PR.13
This TSI, first exploited in the renin field,14 was especially
attractive considering that the majority of CNS drugs contain a
basic amine.15 Furthermore, structural data obtained for acyclic
HEA-based inhibitors complexed to HIV-PR revealed a compact
network of hydrogen bonds with the catalytic dyad.16
The data gathered from our previous studies, using potent
HE inhibitors of BACE, provided the basis for our current
exploration. The conversion of the HE inhibitor 1 into the HEA
inhibitors 2 and 3a can be envisioned by an insertion of a
secondary nitrogen between the methylene and the P1′ substitu-
ent in the hydroxyethylene TSI. We found that the preferred
configuration for the central Ψ (CH-OH) functionality was
opposite to the stereochemistry of other BACE TSIs that we
have previously described.1 For HEA inhibitors of BACE, the
(R) configuration of the alcohol afforded the most active
inhibitor 3a, while the (S) isomer 2 was 1000-fold less active
(Figure 1). These results are in agreement with that of HEA-
based inhibitors of this size in HIV protease17 and BACE.18
The cellular activity of compound 3a reinforced our initial
hopes for this series. The basic nitrogen did, in fact, appear to
favorably impart cellular potency, as similarly observed in
another amino-containing BACE inhibitor series.19 Compound
3a demonstrated a C/E ) 1, while the corresponding neutral
In 1999, we along with a number of other laboratories
reported on the isolation and cloning of BACE6-8 (â-site APP-
cleaving enzyme) and found that this enzyme has all the
functional properties and characteristics of â-secretase. The
identification of this promising pharmacological target along
with the results from the BACE knockout (BACE -/-) animals,
which are normal and devoid of the ability to generate Aâ, has
created new hope for the development of effective AD
therapeutics.9 As a first step toward the identification of an orally
† The authors dedicate this manuscript to the memory of Dr. Miguel A.
Ondetti who passed away on August 23, 2004.
* To whom correspondence should be addressed. Tel.: (650) 616-5061
(R.K.H.); (636) 247-9769 (T.E.B.). Fax: (650) 877-7486 (R.K.H.); (636)
247-5620
(T.E.B.).
E-mail:
roy.hom@elan.com
(R.K.H.);
timothy.e.benson@pfizer.com (T.E.B.).
‡ Elan Pharmaceuticals.
§ Pfizer, Inc.
10.1021/jm061242y CCC: $37.00 © 2007 American Chemical Society
Published on Web 01/10/2007

Peptidomimetic Inhibitors of Human â-Secretase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 777
Table 2. Structure of BACE Inhibitors and Enzyme and Cell Activity
a
b
IC50
EC50
cmpd
R1
(nM)
(nM)
6a
6b
6c
H
I
OMe
130
5
20
40
3
15
a BACE enzymatic inhibition was determined using MBPC125Swe
assay.8 b Cellular inhibition was determined in HEK-293 cells.28
Scheme 1. Synthesis of HEA Analogs 3c and 6b
Figure 1. Stereochemistry effects on activity of HEA BACE inhibitors
2 and 3a as compared with that of HE 1.
Table 1. Structure of BACE Inhibitors and Enzyme and Cell Activity
a
b
IC50
EC50
cmpd
R1
R2
R3
(nM)
(nM)
3a
3b
3c
3d
H
H
Me
Me
Me
H
Me
Me
H
Me
H
130
500
70
230
600
80
compounds 3c, suggesting that 6b may concentrate in the
intracellular compartments where BACE is located.
With the discovery of the nonpeptidic C-terminal benzyl
amine, a set of benzylamines were selected to prepare a follow-
up library. The most potent compound was the meta-iodo analog
6b, which was 25-fold more potent than the parent analog 6a.
A close analog, 6c, with a meta-OMe substituent was also very
potent and more drug-like. Thus, 6b and 6c were enzymatically
more active than the initial lead HE BACE inhibitor 1, and
reduction in the number of amide bonds and introduction of a
basic amine into the molecule gave rise to dramatically enhanced
cell potency.
Me
200
230
a BACE enzymatic inhibition was determined using MBPC125Swe
assay.8 b Cellular inhibition was determined in HEK-293 cells.28
hydroxyethylene 1 afforded a C/E ) 100. The net effect was a
much more potent cellular inhibitor of Aâ production than had
been observed in the statine and the HE classes of BACE
inhibitors with comparable enzyme potency.
To address the effect of nitrogen insertion on the P1′ SAR,
the diastereomeric D-Ala analog 3b was prepared. This com-
pound exhibited less than 4-fold loss in enzyme inhibitory
activity compared to 3a (Table 1). While studying the P1′ SAR,
we continued improving the N-terminal isophthalate and found
that methylation of the 3-position of the aromatic ring (3c)
resulted in a slight improvement of activity. In this new
N-terminal variant, dimethylation of the P1′ methylene resulted
in only a 3-fold decrease of activity (3d).
This relatively flat P1′ SAR suggested that the C-terminal
amide may no longer be optimal, which could be attributed to
the mainframe shift caused by the nitrogen insertion in the
peptidic backbone. We, therefore, became interested in exploring
the possibility of replacing the peptidic C-terminus to lower
the number of amide bonds and, thus, further enhance the
pharmacological profile of this series. We performed a library
scan of commercially available primary amines and discovered
that the C-terminal amide could be replaced with a benzyl amine.
The resulting inhibitor 6a exhibited submicromolar BACE
enzyme activity, which corresponds to only a slight attenuation
(2-fold) from amino acid HEA 3c (Table 2). In addition,
compound 6a demonstrated a cell-to-enzyme ratio (C/E) of 0.3,
an improvement from that found in the amino acid HEA
Chemistry
The synthesis of compounds 3c and 6b was accomplished
with a well-known three-step procedure (Scheme 1) starting
from the requisite erythro R-amino epoxide 4, which was
prepared according to literature procedures.20 Epoxide opening
in the presence of silica gel21 or alumina17,22 afforded the Boc-
protected amino alcohol 5a, which was then deprotected under
acidic conditions and underwent amide coupling to provide 3c.
The sequence to form the benzyl HEA 6b was similar, with
the only difference being the epoxide opening, which could be
accomplished by heating excess amine in the presence of
epoxide 4 in isopropanol. Other HEA analogs 2, 3b-d, and
6a,c were prepared according to the same three-step procedure
using the appropriate epoxides,20,23 N- and C-terminus precur-
sors.
Binding Mode of HEA Inhibitor to BACE. Crystallization
of human BACE in the presence of 6b afforded the opportunity
to use X-ray crystallography to characterize the binding mode
of this compound within the BACE enzyme active site.
Compound 6b sits at the active site between the catalytic

778 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Maillard et al.
between the protonated HEA nitrogen and the Asp 32 carbox-
ylate side chain (interatomic distance 2.7 Å) and the Gly 34
main chain carbonyl oxygen (interatomic distance 3.0 Å).
These hydrogen bonding interactions are complemented by
four key hydrophobic interactions within various subsites. The
N-terminal dipropyl groups of the dipropylamide are locked into
a configuration that projects one of the propyl groups into the
S3 subsite. The presence of the second propyl group helps to
maintain the amide group out-of-plane with respect to the phenyl
plane of the isophthalate. In fact, one hydrogen on the first
carbon is within van der Waals contact of one of the fluorines
present on the di-fluoro phenylalanine (di-F Phe, 3.7 Å). The
amide carbonyl makes a 68° out-of-plane twist with respect to
the isophthalate group. This twist diminishes the conjugation
of this system, which would be very costly without steric
constraint provided by the tertiary amide propyl groups interact-
ing with the S3 subsite. Aryl esters or ketones would pay 4-5
kcal/mol to achieve the 68° out-of-plane twist observed for the
dipropyl amide isophthalate groups. A second set of hydrophobic
interactions are made by the isophthalate itself with Thr 230,
Gln 73, and Arg 235. The isophthalate moiety of 6b is
sandwiched between Thr230 and Gln73 due in part to the close
interaction of the Gln73 side chain.
Figure 2. X-ray crystal structure of compound 6b cocrystallized with
human BACE (inhibitor shown with yellow carbon atoms and yellow
protein backbone) overlaid with the HE inhibitor OM00-3.24 The
molecular surface shown is derived from the BACE cocrystal structure
with 6b and is colored by electrostatic potential (red, negatively charged;
blue, positively charged).
Hydrophobic interactions in S1 provide a key component of
the affinity of 6b. This pocket is lined with a number of
hydrophobic and aromatic side chains (Leu 30, Gln73, Tyr71,
Phe 108, Trp115, and Leu118) that create a pocket specifically
designed for a Phe-like group on the inhibitor. Gln 73 packs
very closely with the isophthalate group, creating a sandwiching
interaction. A fourth hydrophobic interaction occurs in S2′ with
the C-terminal meta-iodo benzyl group. The meta-substitution
on the C-terminal aromatic group positions the iodine directly
into the center of the S2′ subsite. The C-terminal aromatic group
also completes a series of stacking interactions between aromatic
groups from the protein (Trp 115, Phe 108, Tyr 71, and Tyr
198) and aromatic groups from the inhibitor (diF Phe and
C-terminal benzyl).27 These interactions might explain the
increased activity of 6b compared to 3c. Additionally, an usually
close contact between the aryl hydrogen of the aromatic group
and the main chain carbonyl of Gly 34 (3.2 Å) is observed.
This proton is positioned similarly to the C-terminal amide N-H
of OM00-3.
Figure 3. Interactions between compound 6b and human BACE.
Hydrogen bonds are shown with dotted lines. Close interactions detailed
in the text are shown with double arrows. Measured distances are
indicated along the dashed lines.
aspartates (residues 32 and 228) and the flap (residues 66-
76). The inhibitor makes seven key hydrogen bonds with the
protein. Interatomic distances consistent with two hydrogen
bonds are observed between the N-terminal amide carbonyl
oxygen of 6b and the Thr 232 main chain nitrogen and its side
chain hydroxyl (both distances 2.9 Å). Distances and orientations
of the flap main chain nitrogen indicate that one hydrogen bond
exists between the second amide carbonyl oxygen of 6b and
the Gln 73 main chain nitrogen (2.9 Å). Another hydrogen bond
exists between the second amide nitrogen and the main chain
carbonyl oxygen of Gly 230 (interatomic distance of 3.1 Å).
As noted above, the erythro configuration of the hydroxy-
ethylamine TSI is preferred. The overlay of the crystal structures
of BACE in complex with inhibitor OM00-324 reveals the
differences between the HE and HEA insert binding modes to
the catalytic aspartates (Figures 2 and 3). The nitrogen insertion
in 6b caused a shift in the position of the isostere hydroxyl
group away from its common central position in OM00-3
between the aspartate residues.25,26 In contrast to other aspartyl
protease inhibitors that mimic the tightly bound water molecule
shared between the two aspartates, this HEA hydroxyl interacts
with only one of the aspartate residuessAsp 228 (interatomic
distance 2.6 Å). Two additional hydrogen bonds are observed
Conclusion
Inhibitors of BACE have the potential to become efficacious
agents for the treatment of Alzheimer’s disease. In this article,
we reported the development of a new series of hydroxyethyl
secondary amine-based BACE inhibitors displaying nanomolar
potencies in the enzymatic assay. In the initial series containing
a peptidic C-terminus, the most active analog 3c exhibited a
strong preference for the (R) stereochemistry at the transition
state hydroxyl and an excellent C/E ratio. The analysis of the
crystal structures of HEA 6b revealed that the HEA insert
created a new trajectory for the peptidic C-terminus compared
to HE analogs, which permitted the replacement of the C-
terminus amide bond by a phenyl ring. Optimization of the
phenyl ring by reintroduction of a P2′ substituent in the meta-
position led to inhibitors 6b,c, low nanomolar inhibitors in both
enzymatic and the 293 cell-based assays. Compounds 6b,c are
examples of very potent BACE inhibitors with a nonpeptidic
C-terminii that extends into the S2′ subsite. These compounds
represent a major step toward our goal of identifying a candidate
with the required pharmacological properties for CNS activity.

Peptidomimetic Inhibitors of Human â-Secretase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 779
(1S,2R)-N-[1-(3,5-Difluoro-benzyl)-2-hydroxy-3-(S)-(1-isobu-
tylcarbamoyl-ethylamino)-propyl]-5-methyl-N′,N′-dipropyliso-
phthalamide (3c). Compound 5a (271 mg, 0.61 mmol) was
dissolved in 20 mL of 15% trifluoroacetic acid in dichloromethane
at 0 °C. After 3 h, the reaction mixture was concentrated in vacuo.
The crude amine salt was used without further purification. The
crude amine trifluoroacetate salt was dissolved in dry dichlo-
romethane (20 mL) and cooled to 0 °C. Triethylamine (0.5 mL,
3.5 mmol) and 5-methyl-N,N-dipropyl-isophthalamic acid (176 mg,
0.67 mmol) were added, followed by hydroxybenzotriazole (HOBt,
165 mg, 1.22 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcar-
bodiimide hydrochloride (EDC, 233 mg, 1.22 mmol). This mixture
was stirred at 0 °C for 1 h then allowed to warm to room
temperature overnight. The mixture was then partitioned between
10% citric acid (10 mL) and EtOAc (10 mL), and the aqueous layer
was extracted with EtOAc (3 × 10 mL). The combined organic
layers were washed (NaHCO3, NaCl), dried (Na2SO4), filtered, and
concentrated under reduced pressure. Flash chromatography gave
a white foam as product (282 mg, 79%): Rf ) 0.3 (10% MeOH/
Experimental Section
Chemistry. All reagents were obtained from Aldrich, and all
solvents were obtained from VWR. When anhydrous solvents were
necessary, Aldrich Sure-Seal solvents were used without further
drying. Reaction progress was monitored with analytical thin-layer
chromatography (TLC) plates on 0.25 mm Merck F-254 silica gel
glass plates. Visualization was achieved using phosphomolybdic
acid (PMA), potassium permanganate, or ninhydrin spray reagents
or UV illumination. Flash chromatography was performed on E.
Merck silica gel 60 (230-400 mesh). 1H NMR spectra were
obtained at 300 MHz, respectively, on a Varian or Bruker
spectrometer and are reported in parts per million downfield relative
to tetramethylsilane (TMS). Low-resolution mass spectra were
obtained with a Hewlett-Packard 1100MSD single quad detector
using electron spray ionization. Elemental analyses were obtained
at the University of California at Berkeley and at Desert Analytics,
Tucson, AZ. Compound 1 was prepared as a standard using the
literature procedure.10 Analog 4 was prepared according to literature
procedures20 starting from the commercially available (S)-2-tert-
butoxycarbonylamino-3-(3,5-difluorophenyl)-propionic acid (Syn-
thetech, Inc., Albany, Oregon).
(1S,2R)-[1-(3,5-Difluorobenzyl)-2-hydroxy-3-(S)-(1-isobutyl-
carbamoyl-ethylamino)-propyl]-carbamic Acid tert-Butyl Ester
(5a). Silica gel (Whatman, 230-400 mesh ASTM, 60 A, 1.0 g)
was added to a dichloromethane (5 mL) solution of L-alanine
isobutylamine (104 mg, 0.72 mmol) and (1R,2S)-[2-(3,5-difluo-
rophenyl)-1-oxiranylethyl]-carbamic acid tert-butyl ester (4; 200
mg, 0.67 mmol). The resulting suspension was concentrated under
reduced pressure, and after standing at room temperature for 3 days,
the mixture was purified by flash chromatography (5-95% MeOH/
CH2Cl2) to give a white foam as product (190 mg, 64%): 1H NMR
(300 MHz, CDCl3) δ 6.75 (m, 2H), 6.65 (m, 1H), 4.45 (d, 1H),
3.75 (m, 1H), 3.55 (m, 1H), 3.2-2.9 (m, 4H), 2.95-2.60 (m, 3H),
1.75 (m, 1H), 1.2-1.3 (m, 12H), 0.9 (d, 3H); (M + H)+ ) 444.2.
(1S,2R)-[1-(3,5-Difluorobenzyl)-2-hydroxy-3-(3-iodobenzyl-
amino)propyl]-carbamic Acid tert-Butyl Ester (5b). Compound
5b was prepared in 70% yield from 4 and 3-iodobenzylamine using
1
CH2Cl2); H NMR (300 MHz, CDCl3) δ 7.45-7.2 (m, 2H), 7.1
(m, 1H), 6.80 (m, 2H), 6.65 (m, 1H), 4.35 (m, 1H), 3.70 (m, 1H),
3.40 (m, 2H), 3.10-2.95 (m, 7H), 2.65 (m, 2H), 2.5 (m, 1H), 2.32
(m, 3H), 1.70 (m, 4H), 1.45 (m, 2H), 1.25 (2d, 4H), 0.95 (m, 3H),
0.85 (m, 7H), 0.7 (m, 3H); (M + H)+ ) 589.3. Anal. (C32H46F2N4O4‚
TFA‚1.5H2O) C, H, N.
(1S,2R)-N-[1-(3,5-Difluorobenzyl)-2-hydroxy-3-(1-isobutylcar-
bamoyl-1-methyl-ethylamino)-propyl]-5-methyl-N′,N′-dipropyl-
isophthalamide (3d). Compound 3d was prepared according to
1
the same procedure described for compound 3c. H NMR (300
MHz, CDCl3) δ 7.45-7.2 (m, 2H), 7.15 (s, 1H), 6.80 (m, 2H),
6.65 (m, 1H), 4.35 (m, 1H), 3.70 (m, 1H), 3.40 (m, 2H), 3.10-
2.80 (m, 7H), 2.60 (m, 2H), 2.5 (m, 1H), 2.30 (m, 3H), 1.70 (m,
4H), 1.45 (m, 2H), 1.30 (m, 4H), 0.95 (m, 3H), 0.85 (m, 7H), 0.7
(m, 3H); (M + H)+ ) 603.6. Anal. (C33H48F2N4O4‚TFA‚0.5H2O)
C, H, N.
(1S,2R)-N-[1-(3,5-Difluorobenzyl)-2-hydroxy-3-(benzylamino)-
propyl]-5-methyl-N′,N′-dipropylisophthalamide (6a). Compound
6a was prepared from 5b using the two-step procedure described
below for 6c. The white foam obtained was dissolved in dichlo-
romethane and treated with HCl/Et2O. After stirring overnight, the
precipitate was collected by filtration and dried. 1H NMR (CDCl3)
δ 7.55-7.40 (m, 5H), 7.33 (s, 2H), 6.80 (dd, J ) 8.2, 2.3 Hz, 2H),
6.62 (tt, J ) 9.0, 2.2 Hz, 1H), 4.30-4.10 (m, 3H), 4.00-3.80 (m,
1H), 3.47 (t, J ) 7.7 Hz, 2H), 3.40-3.20 (m, 1H), 3.20-3.00 (m,
4H), 2.82 (dd, J ) 14.4, 11.7 Hz, 1H), 2.41 (s, 3H), 1.80-1.60
(m, 2H), 1.60-1.40 (m, 2H), 1.00 (t, J ) 7.4 Hz, 3H), 0.69 (t, J )
7.4 Hz, 3H); (M + H)+ ) 552.3. Anal. (C32H39F2N3O3‚2H2O‚HCl)
C, H, N.
(1S,2R)-N-[1-(3,5-Difluorobenzyl)-2-hydroxy-3-(3-iodobenzyl-
amino)-propyl]-5-methyl -N′,N′-dipropylisophthalamide (6b).
Compound 6b was prepared from 5b using the two-step procedure
described below for 6c. The white foam obtained was dissolved in
dichloromethane and treated with trifluoroacetic acid. After stirring
overnight, the precipitate was collected by filtration and dried. 1H
NMR (CDCl3) δ 7.80 (s, 1H), 7.75 (d, J ) 8.2 Hz, 1H), 7.50 (s,
2H), 7.40 (d, J ) 8.2 Hz, 1H), 7.15 (m, 2H), 6.80 (dd, J ) 8.2, 2.3
Hz, 2H), 6.60 (tt, J ) 9.0, 2.2 Hz, 1H), 4.15 (m, 2H), 3.95 (m,
1H), 3.75 (m, 1H), 3.50-3.35 (m, 2H), 3.10 (m, 3H), 2.85-2.60
(m, 3H), 2.32 (s, 3H), 1.70 (m, 2H), 1.45 (m, 2H), 0.98 (t, J ) 7.0
Hz, 3H), 0.71 (t, J ) 7.1 Hz, 3H); (M + H)+ ) 678.2. Anal.
(C32H38F2IN3O2‚TFA) C, H, N.
(1S,2R)-N-[1-(3,5-Difluorobenzyl)-2-hydroxy-3-(3-methoxy-
benzylamino)-propyl]-5-methyl-N′,N′-dipropylisophthalamide (6c).
(1S,2R)-[1-(3,5-Difluorobenzyl)-2-hydroxy-3-(3-methoxybenzyl-
amino)propyl]-carbamic acid tert-butyl ester, prepared as compound
5b (255 mg, 0.59 mmol), was dissolved in 2 mL of 50%
trifluoroacetic acid in dichloromethane at rt. After 1 h, the reaction
mixture was concentrated in vacuo. The crude amine salt was used
without further purification. The crude amine trifluoroacetate salt
was dissolved in dry DMF (3 mL) and cooled to 0 °C. Triethylamine
(0.5 mL, 3.6 mmol) and 5-methyl-N,N-dipropyl-isophthalamic acid
1
the procedure detailed above for 5a. H NMR (300 MHz, CDCl3)
δ 7.68 (s, 1H), 7.60 (d, J ) 8.2 Hz, 1H), 7.25 (s, 2H), 6.75 (dd, J
) 8.0, 2.3 Hz, 2H), 6.65 (tt, J ) 9.0, 2.2 Hz, 1H), 3.65 (m, 3H),
3.45 (m, 1H), 2.95 (dd, J ) 12.0, 5.0 Hz, 2H), 2.7-2.8 (m, 3H),
1.38 (s, 9H); (M + H)+ ) 533.1. Anal. (C22H27F2INO3) C, H, N.
(1S,2R)-N-[1-(3,5-Difluorobenzyl)-2-hydroxy-3-(S)-(1-isobu-
tylcarbamoyl-ethylamino)-propyl]-N′,N′-dipropyl-isophthal-
amide (2). Compound 2 was prepared according to the same
procedure described for compound 3c starting from the threo
epoxide.20a 1H NMR (300 MHz, CDCl3) δ 7.7 m (1H), 7.40-7.20
(m, 3H), 6.80 (m, 2H), 6.60 (m, 1H), 4.35 (m, 1H), 3.75 (m, 1H),
3.40 (m, 2H), 3.25 (m, 1H), 3.2-2.95 (m, 6H), 2.75 (m, 2H), 2.55
(m, 1H), 1.70 (m, 4H), 1.50 (m, 2H), 1.30 (m, 4H), 0.95 (m, 3H),
0.85 (m, 7H), 0.7 (m, 3H); (M + H)+ ) 575.4. Anal. (C31H44F2N4O4‚
TFA‚2H2O) C, H, N.
(1S,2S)-N-[1-(3,5-Difluorobenzyl)-2-hydroxy-3-(S)-(1-isobu-
tylcarbamoyl-ethylamino)-propyl]-N′,N′-dipropyl-isophthal-
amide (3a). Compound 3a was prepared according to the same
procedure described for compound 3c. 1H NMR (300 MHz, CDCl3)
δ 7.70 (m, 1H), 7.4 (m, 1H), 7.1 (m, 2H), 6.8 (m, 2H), 6.65 (m,
1H), 4.35 (m, 1H), 3.70 (m, 1H), 3.45 (m, 2H), 3.10-2.95 (m,
6H), 2.65 (m, 2H), 2.45 (m, 1H), 1.70 (m, 2H), 1.50 (m, 1H), 1.25
(2d, J ) 7 Hz, 4H), 0.95 (t, 3H), 0.90 (m, 7H), 0.7 (m, 3H); (M +
H)+ ) 575.6. Anal. (C31H44F2N4O4‚TFA‚H2O) C, H, N.
(1S,2R)-N-[1-(3,5-Difluorobenzyl)-2-hydroxy-3-(R)-(1-isobu-
tylcarbamoyl-ethylamino)-propyl]-N′,N′-dipropyl-isophthal-
amide (3b). Compound 3b was prepared according to the same
procedure described for compound 3c. 1H NMR (300 MHz, CDCl3)
δ 7.80 (m, 1H), 7.7 (m, 1H), 7.3 (m, 2H), 6.8 (m, 2H), 6.58 (m,
1H), 4.30 (m, 1H), 3.70 (m, 1H), 3.40 (m, 2H), 3.10-2.80 (m,
6H), 2.70 (m, 2H), 2.60 (m, 1H), 1.70 (m, 2H), 1.50 (m, 1H), 1.25
(2d, J ) 7 Hz, 4H), 0.95 (t, 3H), 0.90 (m, 7H), 0.7 (m, 3H); (M +
H)+ ) 575.4. Anal. (C31H44F2N4O4‚TFA‚H2O) C, H, N.

780 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4
Maillard et al.
(258 mg, 0.59 mmol) were added, followed by hydroxybenzotri-
azole (HOBt, 157 mg, 1.16 mmol) and 1-(3-dimethylaminopropyl)-
3-ethylcarbodiimide hydrochloride (EDC, 230 mg, 1.2 mmol). This
mixture was stirred at 0 °C for 5 min then allowed to warm to
room temperature overnight. The mixture was then partitioned
between 10% citric acid (10 mL) and EtOAc (10 mL), and the
aqueous layer was extracted with EtOAc (3 × 10 mL). The
combined organic layers were washed (NaHCO3, NaCl), dried (Na2-
SO4), filtered, and concentrated under reduced pressure. Flash
chromatography gave a white foam as product (222 mg, 65%): Rf
Supporting Information Available: Combustion analysis data
of final compounds. This material is available free of charge via
the Internet at http://pubs.acs.org.
References
(1) Selkoe, D. J. Translating cell biology into therapeutic advances in
Alzheimer’s disease. Nature 1999, 399 (6738 Suppl), A23-A31.
(2) Jorge, G.; Blas, F. Amyloidosis and Alzheimer’s disease. AdV. Drug
DeliVery ReV. 2002, 54, 1539-1551.
(3) Citron, M. â-Secretase inhibition for the treatment of Alzheimer’s
diseasespromise and challenge. Trends Pharmacol. Sci. 2004, 25,
92-97. Citron, M. Strategies for disease modification in Alzheimer’s
disease. Nat. ReV. 2004, 5, 677-685. Jorge, G.; Blas, F.; Ghosh, A.;
Hong L.; Tang J. â-Secretase as a therapeutic target for inhibitor
drugs. Curr. Med. Chem. 2002, 9, 1135-1144.
(4) Schenk, D.; Barbour, R.; Dunn, W.; Gordon, G.; Grajeda, H.; Guido,
T.; Hu, K.; Huang, J.; Johnson-Wood, K.; Khan, K.; Kholodenko,
D.; Lee, M.; Liao, Z.; Lieberburg, I.; Motter, R.; Mutter, L.; Soriano,
F.; Shopp, G.; Vasquez, N.; Vandevert, C.; Walker, S.; Wogulis,
M.; Yednock, T.; Games, D.; Seubert, P. Nature 1999, 400, 173-
177.
1
) 0.3 (10% MeOH/CH2Cl2); H NMR (CDCl3) δ 7.45 (s, 1H),
7.34 (s, 1H), 7.30-7.10 (m, 3H), 6.89 (d, J ) 7.2 Hz, 2H), 6.85-
6.70 (m, 1H), 6.80 (dd, J ) 8.2, 2.3 Hz, 2H), 6.62 (tt, J ) 9.0, 2.2
Hz, 1H), 4.40-4.25 (m, 1H), 3.90-3.70 (m, 2H), 3.79 (s, 3H),
3.70-3.55 (m, 1H), 3.55-3.35 (m, 2H), 3.20-2.95 (m, 3H), 2.90
(dd, J ) 14.2, 8.9 Hz, 1H), 2.82-2.68 (m, 2H), 2.32 (s, 3H), 2.11
(br s, 2H), 1.80-1.58 (m, 2H), 1.58-1.40 (m, 2H), 0.98 (t, J )
7.0 Hz, 3H), 0.71 (t, J ) 7.1 Hz, 3H); (M + H)+ ) 582.6. Anal.
(C33H41F2N3O4) C, H, N.
BACE Enzyme and Aâ Cell Assays. â-Cleavage ELISA assays
were carried out in 200 mM sodium acetate, pH 4.8, 0.06% Triton
X-100, with 10 µg/mL MBPAPPC125 (or MBP-C125). MBP-C125
is a fusion protein containing the maltose-binding protein at the
amino terminal end connected to the carboxyl-terminal 125 amino
acids of APP (amyloid precursor protein) at the carboxyl terminal
end. Reaction mixtures were incubated at 37 °C for 1-2 h, and
the quenched reaction mixtures were then loaded onto 96-well plates
coated with a polyclonal antibody raised to MBP. Generated
â-cleaved product was detected using biotinylated Sw192 or
biotinyated Wt192 as specific reporter antibodies and quantitated
against the appropriate MBP-C125 standard. The 293Swe cell
assays were performed according to the protocol previously
described.28
X-Ray Crystallography. A DNA construct of human BACE
was prepared in the E. coli pQE70 expression vector missing 35
amino acids of the pro-segment (sequence started at Arg36 and
continued to Ser342) and containing a C-terminal His6 tag. Details
of the expression, purification, and crystallization will be published
in detail elsewhere. In short, the crystallization conditions were as
follows: 8 mg/mL purified BACE, 16% PEG 3000, and 0.1 M
sodium acetate, pH 4.6. Crystals were grown at 20 °C and grew to
approximately 0.6 × 0.15 mm. Before data collection, the crystals
were cryoprotected in crystallization buffer plus 30% glycerol and
flash frozen in liquid nitrogen crystals of BACE in the presence of
6b diffracted to 1.7 Å resolution at the Advanced Photon Source
(IMCA-CAT beamline 17-ID) with unit cell dimensions of a )
73.1 Å, b ) 105.1 Å, c ) 50.5 Å, R ) γ ) 90°, â ) 94.8° in
space group C2 with one molecule per asymmetric unit. The
complex of BACE with compound 6b was refined using CNX to
an R-factor of 20.6% and a free-R factor of 23.1%. The coordinates
for the structure of the BACE/compound 6b complex have been
deposited in the Protein Data Bank under access code 2IQG.
(5) Esler, W. P.; Wolfe, M. S. A portrait of Alzheimer secretasessNew
features and familiar faces. Science 2001, 293, 1449-1454.
(6) Vassar, R.; Bennett, B. D.; Babu-Khan, S.; Kahn, S.; Mendiaz, E.
A.; Denis, P.; Teplow, D. B.; Ross, S.; Amarante, P.; Loeloff, R.;
Luo, Y.; Fisher, S.; Fuller, J.; Edenson, S.; Lile, J.; Jarosinski, M.
A.; Biere, A. L.; Curran, E.; Burgess, T.; Louis, J.-C.; Collins, F.;
Treanor, J.; Rogers, G.; Citron, M. â-Secretase cleavage of Alzhei-
mer’s amyloid precursor protein by the transmembrane aspartic
protease BACE. Science 1999, 286, 735-741.
(7) Yan, R.; Bienkowski, M. J.; Shuck, M. E.; Miao, H.; Tory, M. C.;
Pauley, A. M.; Brashier, J. R.; Stratman, N. C.; Mathews, W. R.;
Buhl, A. E.; Carter, D. B.; Tomasselli, A. G.; Parodi, L. A.;
Heinrikson, R. L.; Gurney, M. E. Membrane-anchored aspartyl
protease with Alzheimer’s disease â-secretase activity. Nature 1999,
402, 533-537.
(8) Sinha, S.; Anderson, J. P.; Barbour, R.; Basi, G. S.; Caccavello, R.;
Davis, D.; Doan, M.; Dovey, H. F.; Frigon, N.; Hong, J.; Jacobson-
Croak, K.; Jewett, N.; Keim, P.; Knops, J.; Lieberburg, I.; Power,
M.; Tan, H.; Tatsuno, G.; Tung, J.; Schenk, D.; Seubert, P.;
Suomensaari, S. M.; Wang, S.; Walker, D.; Zhao, J.; McConlogue,
L.; John, V. Purification and cloning of amyloid precursor. Nature
1999, 402, 537-540.
(9) Roberds, S. L.; Anderson, J.; Basi, G.; Bienkowski, M. J.; Branstetter,
D. G.; Chen, K. S.; Freedman, S.; Frigon, N. L.; Games, D.; Hu, K.;
Johnson-Wood, K.; Kappenman, K. E.; Kawabe, T.; Kola, I.; Kuehn,
R.; Lee, M.; Liu, W.; Motter, R.; Nichols, N. F.; Power, M.;
Robertson, D. W.; Schenk, D.; Schoor, M.; Shopp, G. M.; Shuck,
M. E.; Sihna, S.; Svensson, K. A.; Tatsuno, G.; Tintrup, H.; Wijsman,
J.; Wright, S.; McConlogue, L. BACE knockout mice are healthy
despite lacking the primary â-secretase activity in the brain: implica-
tions for Alzheimer’s disease therapeutics. Hum. Mol. Genet. 2001,
10, 1317-1324.
(10) (a) Hom, R. K.; Gailunas, A. F.; Mamo, S.; Fang, L. Y.; Tung, J. S.;
Walker, D. E.; Davis, D.; Thorsett, E. D.; Jewett, N. E.; Moon, J.
B.; John, V. Design and synthesis of hydroxyethylene-based pepti-
domimetic inhibitors of human â-secretase. J. Med. Chem. 2004, 47,
158-164. (b) Hom, R. K.; Fang, L. Y.; Mamo, S.; Tung, J. S.; Guinn,
A. C.; Walker, D. E.; Davis, D. L.; Gailunas, A. F.; Thorsett, E. D.;
Sinha, S.; Knops, J. E.; Jewett, N. E.; Anderson, J. P.; John, V. Design
and synthesis of statine-based cell-permeable peptidomimetic inhibi-
tors of human â-secretase. J. Med. Chem. 2003, 46, 1799-1802.
(11) Randolph, J. T.; DeGoey, D. A. Peptidomimetic inhibitors of HIV
protease. Curr. Top. Med. Chem. 2004, 4, 1079-1095. Abdel-
Rahman, H.; Al-karamany, G.; El-Koussi, N. HIV protease inhibitors;
peptidomimetic drugs and future perspective. Curr. Med. Chem. 2002,
9, 1905-1922.
(12) Turner, R.; Koelsch, G.; Hong, L.; Castenheira, P.; Ghosh, A.; Tang,
J. Subsite specificity of memapsin 2 (â-secretase): Implications for
inhibitor design. Biochemisty 2001, 40 (34), 10001-10006.
(13) Roberts, N. A.; Matin, J. A.; Kinchington, D.; Broadhurst, A. V.;
Craig, J. C.; Duncan, I. B.; Galpin, S. A.; Handa, B. K.; Kay, J.;
Kro¨hn, A.; Lambert, R. W.; Merrett, J. H.; Mills, J. S.; Parkes, K. E.
B.; Redshaw, S.; Ritchie, D. L.; Taylor, D. L.; Thomas, G. J.; Machin,
P. J. Rational design of peptide-based HIV proteinase inhibitors.
Science 1990, 248, 358.
Acknowledgment. The authors gratefully acknowledge Don
E. Walker for the HPLC purification of compounds 3a-d and
Andrea Gailunas for the resynthesis of compound 6a. In
addition, the authors would like to acknowledge the use of the
IMCA-CAT beamline 17-ID at the Advanced Photon Source,
which is supported by the companies of the Industrial Macro-
molecular Crystallography Association through a contract with
the Center for Advanced Radiation Sources at the University
of Chicago. Use of the Advanced Photon Source was supported
by the U.S. Department of Energy, Office of Science, Office
of Basic Energy Sciences, under Contract No. W-31-109-Eng-
38.
(14) (a) Dann, J.; Stammers, D.; Harris, C. J.; Arrowsmith, R.; Davies,
D.; Hardy, G.; Morton, J. Human renin: A new class of inhibitors.
Biochem. Biophys. Res. Commun. 1986, 134, 71-77. (b) Ryono, D.;
Free, C.; Neubeck, R; Samaniego, S.; Godfrey, J.; Petrillo, E. Proc.
Am. Pept. Symp., 9th 1985, 739.
Note Added after ASAP Publication. There was an error
in the numbering of the aspartate residues in the third paragraph
of Chemistry in the version published ASAP January 10, 2007;
the corrected version was published ASAP January 18, 2007.

Peptidomimetic Inhibitors of Human â-Secretase
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 4 781
(15) Ajay, B.; Murcko, M. Designing libraries with CNS activity. J. Med.
Chem. 1999, 42, 4942-4951.
DeCrescenzo, G.; Heintz, R.; Reed, K.; Talley, J.; Bryant, M.; Clare,
M.; Houseman, K.; Marr, J.; Mueller, R.; Vazquez, M.; Shieh, H.-
S.; Stallings, W.; Stegeman, R. Discovery of a novel class of potent
HIV-1 protease inhibitors containing the (R)-(hydroxyethyl)urea
isostere. J. Med. Chem. 1993, 36, 288-291. (c) Rotella, D.
Stereoselective synthesis of erythro R-amino epoxides. Terahedron
Lett. 1995, 36, 5453-5456.
(16) (a) March, D. R.; Abbenante, G.; Bergman, D. A.; Brinkworth, R.
I.; Wickramasinghe, W.; Begun, J.; Martin, J. L.; Fairlie, D. P.
Substrate-based cyclic peptidomimetics of Phe-Ile-Val that inhibit
HIV-1 protease using a novel enzyme-binding mode. J. Am. Chem.
Soc. 1996, 118, 3375-3379. (b) Dohnalek, J.; Hasek, J.; Duskova,
J.; Petrokova, H.; Hradilek, M.; Soucek, M.; Konvalinka, J.; Brynda,
J.; Sedlacek, J.; Fabry, M. Hydroxyethylamine isostere of an HIV-1
protease inhibitor prefers its amine to the hydroxy group in binding
to catalytic aspartates. A synchrotron study of HIV-1 protease in
complex with a peptidomimetic inhibitor. J. Med. Chem. 2002, 45,
1432-1438.
(21) Bennett, F.; Patel, N.; Girijavallabhan, V.; Ganguly, A. The synthesis
of novel HIV-protease inhibitors via silica gel assisted addition of
amines to epoxides. Synlett 1993, 703-704.
(22) Posner, G.; Rogers, D. Organic reactions at alumina surfaces. Mild
and selective opening of arene and related oxides by weak oxygen
and nitrogen nucleophiles. J. Am. Chem. Soc. 1977, 99, 8214-8218.
(23) Luly, J.; Dellaria, J.; Plattner, J.; Soderquist, N. A synthesis of
protected aminoalkyl epoxides from R-amino acids. J. Org. Chem.
1987, 52, 1487-1492.
(24) Hong, L.; Turner, R.; Koelsch, G.; Shin, D.; Ghosh, A. K.; Tang, J.
Crystal structure of memapsin 2 (â-secretase) in complex with an
inhibitor OM00-3. Biochemistry 2002, 41, 10963-10967.
(25) Hong, L.; Koelsch, G.; Lin, X.; Wu, S.; Terzyan, S.; Ghosh, A. K.;
Zhang, X. C.; Tang, J. Structure of the protease domain of memapsin
2 (â-secretase) complexed with inhibitor. Science 2000, 290 (5489),
150-153.
(26) Rich, D. Pepstatin-derived inhibitors of aspartic proteinases. A close
look at an apparent transition-state analog inhibitor. J. Med. Chem.
1985, 28, 263-273.
(27) Patel, S.; Vuillard, L.; Cleasby, A.; Murray, W.; Yon, J. Apo and
inhibitor complex structures of BACE (â-secretase). J. Mol. Biol.
2004, 343, 407-416.
(28) Dovey, H.; John, V.; Anderson, J.; Chen, L.; de Saint Andrieu, P.;
Fang, L.; Freedman, S.; Folmer, B.; Goldbach, E.; Holsztynska, E.;
Hu, K.; Johnson-Wood, K.; Kennedy, S.; Kholodenko, D.; Knops,
J.; Latimer, L.; Lee, M.; Liao, Z.; Lieberburg, I.; Motte, R.; Mutter,
L.; Nietz, J.; Quinn, K. Functional γ-secretase inhibitors reduce
â-amyloid peptide levels in brain. J. Neurochem. 2001, 76, 173-181.
(17) Tucker, T. J.; Lumma, W. C., Jr.; Payne, L. S.; Wai, J. M.; De Solms,
S. J.; Giuliani, E. A.; Darke, P. L.; Heimbach, J. C.; Zugay, J. A.;
Schleif, W. A.; Quintero, J. C.; Emini, E. A.; Huff, J. R.; Anderson,
P. A. A series of potent HIV-1 protease inhibitors containing a
hydroxyethyl secondary amine transition state isostere: Synthesis,
enzyme inhibition, and antiviral activity. J. Med. Chem. 1992, 35,
2525-2533.
(18) Stachel, S.; Coburn, C.; Steele, T.; Loutzenhiser, E.; Gregro, A.;
Rajapakse, H.; Lai, M.-T.; Crouthamel, M.-C.; Xu, M.; Tugusheva,
K.; Lineberger, J.; Pietrak, B.; Espeseth, A.; Shi, X.-P.; Chen-Dodson,
E.; Holloway, M.; Munshi, S.; Simon, A.; Kuo, L.; Vacca, J.
Structure-based design of potent and selective cell-permeable inhibi-
tors of human â-secretase (BACE-1). J. Med. Chem. 2004, 47, 6447-
6450.
(19) Yang, W.; Lu, W.; Lu, Y.; Zhong, M.; Sun, J.; Thomas, A.;
Wilkinson, J.; Fucini, R.; Lam, M.; Randal, M.; Shi, X.-P.; Jacobs,
J.; McDowell, R.; Gordon, E.; Ballinger, M. Aminoethylenes: A
tetrahedral intermediate isostere yielding potent inhibitors of the
aspartyl protease BACE-1. J. Med. Chem. 2006, 49, 839-842.
(20) (a) Rich, D.; Sun, C. Q.; Vara Prasad, J.; Pathiasseril, A.; Toth, M.;
Marshall, G.; Clare, M.; Mueller, R.; Houseman, K. Effect of
hydroxyl group configuration in hydroxyethylamine dipeptide iso-
steres on HIV protease inhibition. Evidence for multiple binding
modes. J. Med. Chem. 1991, 34, 1222-1225. (b) Getman, D.;
JM061242Y