Design and Synthesis of Hydroxyethylene-Based Peptidomimetic Inhibitors of Human β-Secretase

Article abstract of DOI:10.1021/jm0304008

The hydroxyethylene (HE) transition state isostere was developed as a scaffold to provide potent, small molecule inhibitors of human β-secretase (BACE). The previous work on the statine series proved critical to the discovery of HE structure-activity rela

Full text of DOI:10.1021/jm0304008

158
J . Med. Chem. 2004, 47, 158-164
Design a n d Syn th esis of Hyd r oxyeth ylen e-Ba sed P ep tid om im etic In h ibitor s of
Hu m a n â-Secr eta se
Roy K. Hom,† Andrea F. Gailunas,† Shumeye Mamo,† Larry Y. Fang,† J ay S. Tung,† Donald E. Walker,†
David Davis,† Eugene D. Thorsett,† Nancy E. J ewett,† J oseph B. Moon,‡ and Varghese J ohn*,†
Elan, 800 Gateway Boulevard, South San Francisco, California 94080, and Pfizer Corporation, 301 Henrietta Street,
Kalamazoo, Michigan 49007
Received August 15, 2003
The hydroxyethylene (HE) transition state isostere was developed as a scaffold to provide potent,
small molecule inhibitors of human â-secretase (BACE). The previous work on the statine series
proved critical to the discovery of HE structure-activity relationships. Compound 20 with the
N-terminal isophthalamide proved to be the most potent HE inhibitor (IC50 ) 30 nM) toward
BACE. Unlike the statine series, we identified HE inhibitors without carboxylic acids on the
C terminus, leading to enhanced cell penetration and making them attractive candidates for
further drug development in Alzheimer’s disease.
In tr od u ction
Two proteases, â- and γ-secretase, are involved in the
sequential proteolysis of membrane-anchored amyloid
precursor protein (APP), which results in the generation
of amyloid â (Aâ) peptide that is thought to be causal
for the pathology and subsequent cognitive decline in
Alzheimer’s disease (AD).1,2 Agents that inhibit Aâ
production via inhibition of these proteolytic pathways
F igu r e 1. Comparison of statine and HE inhibitors.
would be useful in preventing Aâ plaque deposition in
the brain and could be of therapeutic benefit in AD.3
selective inhibitors could be designed, but the high
peptidic nature of the compounds limited their utility
â-Secretase (BACE) has been shown by us and others
to be a membrane-bound aspartyl protease.4-6 The
for drug development. For a variety of reasons, we also
cleavage of APP by BACE occurs in the lumina and is
decided to move away from the statinelike transition
state mimic. Thus, we directed our interest toward other
transition state isosteres, potentially exhibiting more
promising properties such as increased solubility and
enhanced oral bioavailability. Because the HE isostere
contains one less secondary amide linkage than the
statines, we selected this isostere as a starting point
considered to be the rate-limiting step in the processing
of APP to Aâ peptide. BACE cleavage of APP results in
the production of the membrane-bound â-C-terminal
fragment, which is then further cleaved to Aâ by
γ-secretase. BACE knockout homozygote mice show a
complete absence of Aâ production and have no reported
side effects.7 BACE, which is highly expressed in the
for evaluation for favorable pharmacokinetic properties
central nervous system, is thus an attractive therapeutic
(Figure 1).
target for the design of inhibitors of Aâ production. We
Before embarking on small molecule HE peptidomi-
have previously elaborated on the development of pep-
metics, we had already obtained data, which suggested
tidic inhibitors,8 as well as the synthesis of small
that potent HEs could be developed. Replacing the
molecule statine compounds exhibiting BACE mecha-
statine moiety with the HE central core in the hep-
nism specific inhibition.9 In this paper, we describe the
tapeptide series resulted in a 10-fold increase in BACE
evolution of low molecular weight BACE inhibitors
inhibitory activity (Figure 2).8 Tang and co-workers had
containing hydroxyethylene (HE) units as dipeptide
reported potent HE inhibitors of BACE but containing
isosteres.
many amino acid residues and no reported cell activi-
ties.11 However, we needed BACE inhibitors with sub-
Ba ck gr ou n d
stantially fewer amino acid residues and lower molec-
Numerous studies have indicated that the number of
ular weights, such that the molecules would be likely
amide bonds is directly correlated with decreasing
to exhibit cellular permeability as well as oral bioavail-
metabolic stability and oral bioavailability of com-
ability and metabolic stability required for drug candi-
pounds, particularly peptidomimetics, which limits their
dates. We therefore embarked on the following study
use as drug development candidates.10 Our previous
to produce BACE potent, small molecular weight HEs
containing no R-amino acids.
We had noticed that the statine compound 2 was
similar in activity to the analogous HE inhibitor 3. This
suggested that since the prime side of the HE molecule
differed the most from the statines, the structure-
studies on BACE inhibitors demonstrated that potent,
* To whom correspondence should be addressed. Tel: (650)877-7623.
Fax: (650)877-7486. E-mail: varghese.john@elan.com.
† Elan.
‡ Pfizer Corporation.
10.1021/jm0304008 CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/09/2003

Inhibitors of Human â-Secretase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 159
Molecu la r Mod elin g
Additional aid in the design of the HEs was provided
by molecular modeling. Figure 3 shows a molecular
model comparison of HE-based compound 1 to the
corresponding statine inhibitor. Models of inhibitors
bound to the BACE active site were constructed using
the Monte Carlo multiple minimum conformational
searching method as implemented in the BatchMin
program.13 Binding modes found using this method were
generally similar to that of the inhibitor OM99-2,
reported by Tang et al.14 Models of HE- and statine-
based peptidic inhibitors showed nearly identical bind-
ing modes for the N-terminal residues. The HE dipep-
tide isostere, containing the same number of atoms in
the backbone chain as a natural dipeptide, permits
substratelike registration in the enzyme binding site,
with all peptidic amide groups forming hydrogen bonds
to the enzyme. The statine dipeptide isostere backbone,
however, is shorter by one atom, causing a misregis-
tration of the C-terminal residues. In our models, this
led to divergent conformations between the two inhibitor
series at the C terminus and an inability of the statines
to fill the S1′ subsite.
Syn th esis
The HE peptide isosteres were synthesized from
commercially available Boc-3, 5-difluorophenylalanine
(Synthetech), using an amino alcohol-derived chiral
auxiliary.15 An example of the synthetic sequence is
illustrated in Scheme 1 with an ethyl P1′ substituent.
The key intermediates, such as compound 4, were
readily generated by methods previously described.15a
Removal of the Boc group and amide coupling to the
isophthalamide N terminus afforded intermediate 5.
Finally, the lactone 5 was ring-opened using trimethyl-
aluminum to introduce chemical diversity in the inhibi-
tors 6-14.16
F igu r e 2. HE and statine BACE inhibitors.
Resu lts a n d Discu ssion
activity relationships (SARs) of the HEs on the C-
terminal end should diverge from that observed with
the statines. However, the N-terminal SARs would
likely be similar between series. On the basis of this
hypothesis, we decided to embark on the synthesis of a
variety of HEs, with the optimal N terminus discovered
in the statine series, differing at the C terminus and at
the P1′ substituent. The isophthalamide was found to
be an optimal N terminus in the statine series (see
below). This group was discovered through application
of known asparagine replacements to the P2-P3 region
of the molecule.12
The C-terminal SARs of the HE compounds are
summarized in Table 1. Because carboxylic acid func-
tions proved to be vital for potency in the statine series,9
various acid linkers were attached. The two carbon-
linked COOH in inhibitor 6 exhibited a moderate 2800
nM BACE enzyme inhibitory activity. With the ad-
ditional methylene spacer found in compound 7, the
activity increased approximately 10-fold to give a 310
nM inhibitor. Further increasing the length of the
spacer (compounds 8-10) essentially did not alter the
activity of the inhibitor. Restricting the alkyl chain
within a ring (compound 11) led to a 4-fold activity
Sch em e 1a
a
CF3COOH, room temperature. bEDC, HOBt, Et3N, N,N-dipropylisophthalamic acid, 91%, two steps. cAlMe3, RNH2, 1,2-dichloroethane,
reflux.

160 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Hom et al.
F igu r e 3. Modeled structure of HE-containing compound 1 (white) bound to BACE. The analogous statine-based inhibitor is
shown for comparison (orange). Hydrogen bonds to the inhibitor are indicated by red dashes. The two C-terminal residues of both
inhibitors are omitted from the figure. Conformations of the two inhibitors are nearly identical on the N-terminal side of the
transition state isosteres but diverge on the C-terminal side. The valinelike side chain of compound 1 fills the S1′ pocket, but the
positioning of the first statine prime side amide prevents similar branching into S1′.
carboxylic acid interacts via hydrogen bonding to BACE
at the C-terminal end, since the neutral ester was
equally active. Brief attempts at replacing the alkyl
carboxylates with an ether (compound 13) or an aryl
(compound 14) C terminus resulted in loss of activity.
Analogues of compound 11 were then synthesized and
allowed for optimization of the P1′ substituent. Alter-
ations at the P1′ substituent led to a SAR consistent with
that found in the substrate data (Table 2). The smaller
Ala analogue (compound 15) was consistently the most
potent in the BACE inhibition assay. Successively larger
substituents (compounds 16, 18, and 19) eroded activity
in a systematic fashion. The P1′ Gly analogue (inhibitor
17) also demonstrated an approximate 10-fold loss in
activity as compared with 15, perhaps due to a loss of
conformational preference toward the active form. In
contrast to the statines,9 where Val was optimal in P1′,
the most potent HEs contained small P1′ residues.
The lack of a potency difference between the ester and
the acids (Table 1) was striking, especially considering
the large difference observed in ester and acid forms of
statine analogues.9 This suggested that unlike the
statines, the HEs would tolerate nonpolar C termini.
Analogue 20 represents the best example of these,
where the C-terminal isobutyl substituent is comparable
in potency to the carboxylate C-terminal analogue 15,
without the concomitant difficulty in cell permeability
usually exhibited by carboxylic acids (Figure 4).17
Comparison of the most potent HE inhibitor to the
most potent statine inhibitor showed that the HEs were
more cell permeable than the statines (Figure 4).
Although its inherent enzyme potency is poorer than
F igu r e 4. Comparison of HE and statine inhibitors.
increase as compared with the straight chain (inhibitor
10) analogue. The formation of the methyl ester (com-
pound 12) led to little or no loss in activity, in contrast
to the approximately 10-fold loss in activity in the
statine BACE inhibitors.9 These data suggest that the
the best statine analogue, analogue 20 did, in fact,
demonstrate a ratio of cell to enzyme activity of 100 (10
times better than two of the most potent statine
compounds 21 and 22). Compound 20 was the culmina-
tion of improvements over our initial inhibitor 1 as well

Inhibitors of Human â-Secretase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 161
Ta ble 1. C-Terminal SAR of HE BACE Inhibitors
Con clu sion
In our goal to develop Alzheimer’s drug candidates
through BACE inhibition, we investigated the HE
transition state isostere as a scaffold to provide potent,
small molecule inhibitors of BACE. With the N-terminal
isophthalamide similar to those discovered in the statine
series, compound 20 proved to be among the most potent
(IC50 ) 30 nM) toward BACE. Unlike the statine series,
nonpolar C termini were tolerated, resulting in inhibi-
tors that were more cell permeable and contained no
amino acid residues.
Exp er im en ta l Section
Gen er a l. Compounds 1, 2, and 22 have been previously
described.8,9 All reagents were obtained from Aldrich, and all
solvents were obtained from VWR, unless otherwise indicated.
Anhydrous solvents (e.g., tetrahydrofuran (THF), dimethyl
formamide (DMF), and dichloromethane (DCM)) were used as
received.
Reaction progress was monitored with analytical thin-layer
chromatography plates on 0.25 mm Merck F-254 silica gel
glass plates. Visualization was achieved using phosphomolyb-
dic acid, potassium permanganate, or ninhydrin spray re-
agents or UV illumination.
1H and 13C NMR spectra were obtained at 300 and 75 MHz,
respectively, on a Varian or Bruker spectrometer and are
reported in parts per million downfield relative to tetrameth-
ylsilane or to proton resonances resulting from incomplete
deuteration of the NMR solvent (δ scale). Mass spectra were
obtained at the University of California at Berkeley, using
chemical ionization. Elemental analyses were performed at the
University of California at Berkeley or at Desert Analytics,
Tucson, Arizona.
Where noted, compounds were determined to be >95% pure
by high-performance liquid chromatography (HPLC). Purity
of compounds were determined on a Phenomenex Luna C18-
(2), 3 µm column, 4.6 mm i.d. × 30 mm length, with [J ] 20-
70% acetonitrile/water/0.1% trifluoroacetic acid or [K] 50-95%
acetonitrile/water/0.1% trifluoroacetic acid mobile phase, 1.5
mL/min elution at 35 °C at 210, 254, or 280 nm wavelength.
MBP -C125 BACE En zym e Assa y. â-Cleavage ELISA
assays were carried out in 200 mM sodium acetate, pH 4.8,
0.06% Triton X-100, with 10 µg/mL-1 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 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-C26
standard.
a
Concentration necessary to inhibit 50% of enzyme activity in
the MBP-C125 assay;4 average of two or more runs. Standard
deviation of assays is (5%.
Ta ble 2. P1′ SAR of HE BACE Inhibitors
P ep tid e Syn th esis. The peptide inhibitors were generated
in a peptide synthesizer using Boc-protected amino acids for
chain assembly. All chemicals, reagents, and Boc amino acids
were purchased from Applied Biosystems (ABI; Foster City,
CA) with the exception of DCM and DMF, which were from
Burdick and J ackson, and Boc-statine, which was purchased
from Neosystem. The starting resin, Boc-Phe-OCH2-Pam resin,
was also purchased from ABI. All amino acids were coupled
following preactivation to the corresponding 1-hydroxybenzo-
triazole (HOBT) ester using 1.0 equiv of HOBT and 1.0 equiv
of N,N-dicyclohexylcarbodiimide in DMF. The Boc protecting
group on the amino acid R-amine was removed with 50%
trifluoroacetic acid in DCM after each coupling step and prior
to hydrogen fluoride (HF) cleavage. Amino acid side chain
protection was as follows: Glu(Bzl). All other amino acids were
used with no further side chain protection including Boc-
Statine (Bzl, benzyl; CBZ, carbobenzyloxy; Cl-CBZ, chlorocar-
bobenzyloxy; OBzl, O-benzyl). The side chain-protected peptide
resin was deprotected and cleaved from the resin by reacting
compd
X
IC50 (nM)a
11
15
16
17
18
19
Et
Me
Pr
H
i-Bu
Bn
50
26
45
400
310
27 000
a
Concentration necessary to inhibit 50% of enzyme activity in
the MBP-C125 assay;4 average of two or more runs. Standard
deviation of assays is (5%.
as over the Tang inhibitors, all of which were high
molecular weight (>700 amu) compounds containing
natural amino acid residues and subject to low cell
permeability and low metabolic stability.

162 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Hom et al.
with anhydrous HF at 0 °C for 1 h. This generated the fully
deprotected crude peptide as a C-terminal carboxylic acid.
25.4, 24.1, 21.8, 20.6, 12.1, 11.4, 10.9. MH+ (CI): 632.2. HPLC
retention time ) 2.525 [J ], 1.076 [K].
8-[6-(3,5-Diflu or op h en yl)-5-(S)-(3-d ip r op ylca r ba m oyl-
ben zoylam in o)-2-(R)-eth yl-4-(S)-h ydr oxyh exan oylam in o]-
octa n oic Acid (10). Rf ) 0.4 (10% MeOH/CH2Cl2). H NMR
HP LC P u r ifica tion of P ep tid es. Following HF treatment,
the peptide was extracted from the resin in acetic acid and
lyophilized. The crude peptide was then purified using reverse
phase HPLC on a Phenomenex Luna C18(2), 5 µm column 4.6
mm i.d. × 25 cm in length [P] or a Vydac C18, 10 µm column
4.6 mm i.d. × 25 cm in length [V]. The solvent systems used
with these columns were [A] ) 0.1% trifluoroacetic acid/H2O
and [B] ) 0.1% trifluoroacetic acid/acetonitrile as the mobile
phase. The gradient conditions were as follows: (i) 20-50%
[B] in 30 min at 1 mL/min; (ii) 30% [B] hold 5 min, 30-60%
[B] in 30 min at 1 mL/min; and (iii) 35% [B] hold 5 min; 35-
65% [B] in 30 min at 1 mL/min. The purified peptide was
subjected to mass spectrometry and analytical reverse phase
HPLC to confirm its composition and purity.
1
(CDCl3): δ 7.90-7.65 (m, 2 H), 7.55-7.30 (m, 3 H), 6.83 (d, J
) 6.1 Hz, 2 H), 6.61 (tt, J ) 9.0, 2.2 Hz, 1 H), 6.39 (t, J ) 5.5
Hz, 1 H), 5.60 (br s, 1 H), 4.19 (dd, J ) 5.2, 3.0 Hz, 1 H), 3.69
(dt, J ) 9.7, 3.3 Hz, 1 H), 3.60-3.25 (m, 3 H), 3.25-3.00 (m,
3 H), 3.00 (d, J ) 7.4 Hz, 2 H), 2.45-2.25 (m, 1 H), 2.25 (t, J
) 7.2 Hz, 2 H), 1.80-1.20 (m, 19 H), 0.98 (t, J ) 7.2 Hz, 3 H),
0.85 (t, J ) 7.1 Hz, 3 H), 0.72 (t, J ) 7.2 Hz, 3 H). 13C NMR
(CDCl3): δ 177.1, 176.3, 171.4, 167.9, 162.9 (dd, J ) 248, 13
Hz, 2 C), 142.5 (t, J ) 9.1 Hz, 1 C), 136.6, 134.7, 129.1, 128.6,
128.4, 125.7, 112.2 (dd, J ) 16.8, 7.3 Hz, 2 C), 101.8 (t, J ) 25
Hz, 1 C), 69.4, 56.0, 50.9, 46.7, 45.5, 39.1, 38.9, 37.7, 37.4, 34.0,
29.1, 28.6, 28.3, 26.0, 25.2, 24.3, 21.87, 20.6, 12.2, 11.4, 10.9.
MH+ (CI): 660.4. HPLC retention time ) 2.707 [J ], 1.324 [K].
Com p ou n d 3. A 1:1 mixture of diastereomers. Gradient
conditions (P, 1): Rf ) 19.4, 21.0. MNa+(CI) 786.8. HPLC
retention time ) 2.787 [J ], 1.432 [K].
4-{[6-(3,5-Diflu or o-ph en yl)-5-(S)-(3-dipr opylcar bam oyl-
ben zoylam in o)-2-(R)-eth yl-4-(S)-h ydr oxy-h exan oylam in o]-
m eth yl}cycloh exa n eca r boxylic Acid (11). Rf ) 0.3 (10%
N-[2-(S)-(3,5-Diflu or op h en yl)-1-(R)-(4-eth yl-5-oxo-THF -
2-yl)eth yl]-N′,N′-d ip r op ylisop h th a la m id e (5). Rf ) 0.1 (2%
MeOH/CH2Cl2). 1H NMR (CDCl3): δ 7.93-7.79 (m, 2 H), 7.55-
7.40 (m, 3 H), 6.76 (dd, J ) 8.0, 2.0 Hz, 2 H), 6.64 (tt, J ) 9.0,
2.2 Hz, 1 H), 4.64 (q, J ) 8.4 Hz, 1 H), 4.63-4.48 (m, 1 H),
3.70-3.40 (m, 2 H), 3.11 (t, J ) 7.1 Hz, 2 H), 2.96 (dd, J )
13.8, 8.4 Hz, 1 H), 2.87 (dd, J ) 13.8, 7.4 Hz, 1 H), 2.52-2.28
(m, 2 H), 2.12-1.95 (m, 1 H), 1.95-1.40 (m, 8 H), 0.95 (t, J )
7.4 Hz, 6 H), 0.72 (t, J ) 7.0 Hz, 3 H). MH+ (CI): 500.8. Anal.
(C28H34F2N2O4‚0.5H2O) C, H, N.
3-[6-(3,5-Diflu or op h en yl)-5-(S)-(3-d ip r op ylca r ba m oyl-
ben zoylam in o)-2-(R)-eth yl-4-(S)-h ydr oxyh exan oylam in o]-
p r op ion ic Acid (6). Rf ) 0.23 (10% MeOH/CH2Cl2). 1H NMR
(CDCl3): δ 7.85-7.60 (m, 3H), 7.52-7.40 (m, 2H), 7.30-7.10
(m, 2H), 6.83 (app d, J ) 6.5 Hz, 2H), 6.61 (tt, J ) 9.0, 2.2 Hz,
1H), 4.30-4.10 (m, 1H), 3.70-3.22 (m, 5H), 3.22-3.10 (m, 2H),
3.10-2.80 (m, 2H), 2.50-2.20 (m, 3H), 1.80-1.30 (m, 7H), 0.99
(t, J ) 7.3 Hz, 3H), 0.84 (t, J ) 7.3 Hz, 3H), 0.73 (t, J ) 7.3
Hz, 3H). MH+ (CI): 590.3. HPLC retention time ) 2.392 [J ],
0.685 [K].
1
MeOH/CH2Cl2). H NMR (CD3OD): δ 8.16 (d, J ) 9.1 Hz, 0.2
H), 7.95 (t, J ) 5.7 Hz, 0.8 H), 7.90-7.75 (m, 1 H), 7.68 (s, 1
H), 7.60-7.45 (m, 2 H), 6.89 (dd, J ) 8.4, 2.1 Hz, 2 H), 6.72
(tt, J ) 10.3, 2.2 Hz, 1 H), 4.40-4.22 (m, 1 H), 3.64 (dt, J )
9.9, 2.9 Hz, 1 H), 3.47 (t, J ) 7.2 Hz, 2 H) 3.17 (t, J ) 7.0 Hz,
2 H), 3.10-2.80 (m, 4 H), 2.44 (sep, J ) 4.7 Hz, 1 H), 2.30-
2.10 (m, 1 H), 1.96 (d, J ) 11.5 Hz, 2 H), 1.90-1.20 (m, 14 H),
1.10-0.80 (m, 3 H), 0.99 (t, J ) 7.3 Hz, 3 H), 0.89 (t, J ) 7.3
Hz, 3 H), 0.70 (t, J ) 7.3 Hz, 3 H). 13C NMR (CD3OD): δ 179.9,
178.2, 173.1, 169.4, 164.3 (dd, J ) 246.7, 13.0 Hz, 2 C), 144.9
(t, J ) 9.2 Hz, 1 C), 138.5, 136.1, 130.5, 130.0, 129.3, 126.3,
113.1 (dd, J ) 17.0, 7.5 Hz, 2 C), 102.5 (t, J ) 25.8 Hz, 1 C),
71.4, 57.1, 52.2, 48.0, 46.4, 44.5, 38.6, 37.7, 37.5, 31.1, 29.9,
27.4, 22.9, 21.7, 12.4, 11.7, 11.3. MH+ (CI): 658.4. HPLC
retention time ) 2.560 [J ], 0.897 [K].
8-[6-(3,5-Diflu or o-p h en yl)-5-(S)-(3-d ip r op ylca r ba m oyl-
ben zoyla m in o)-2-(R)-et h yl-4-(S)-h yd r oxy-h exa n oyla m i-
i
n o]octa n oic Acid Meth yl Ester (12). Rf ) 0.4 (5% PrOH/
CHCl3). 1H NMR (CDCl3): δ 7.85-7.65 (m, 2 H), 7.55-7.35
(m, 2 H), 6.89 (d, J ) 9.0 Hz, 1 H), 6.80 (dd, J ) 8.1, 2.0 Hz,
2 H), 6.63 (tt, J ) 9.0, 2.3 Hz, 1 H), 5.98 (t, J ) 5.6 Hz, 1 H),
5.19 (s, 1 H), 4.17 (q, J ) 7.6 Hz, 1 H), 3.85-3.70 (m, 1 H),
3.66 (s, 3 H), 3.46 (t, J ) 7.1 Hz, 2 H), 3.40-2.90 (m, 6 H),
2.40-2.20 (m, 1 H), 2.29 (t, J ) 7.5 Hz, 2 H), 1.90-1.15 (m,
18 H), 0.99 (t, J ) 7.0 Hz, 3 H), 0.86 (t, J ) 7.3 Hz, 3 H), 0.74
(t, J ) 6.9 Hz, 3 H). 13C NMR (CDCl3): δ 176.5, 174.3, 170.9,
167.0, 162.9 (dd, J ) 248.2, 12.9 Hz, 2 C), 142.4 (t, J ) 9.1
Hz, 1 C), 137.6, 134.7, 129.3, 127.8, 127.6, 125.1, 112.1 (dd, J
) 16.8, 7.5 Hz, 2 C), 101.9 (t, J ) 25.1 Hz, 1 C), 67.6, 55.6,
51.5, 50.8, 46.5, 46.0, 39.5, 38.3, 36.6, 34.0, 29.3, 28.9, 28.7,
26.5, 24.72, 24.69, 21.9, 20.7, 12.2, 11.4, 11.0. MH+ (CI) 674.4.
HPLC retention time ) 3.052 [J ], 1.815 [K].
4-[6-(3,5-Diflu or op h en yl)-5-(S)-(3-d ip r op ylca r ba m oyl-
ben zoylam in o)-2-(R)-eth yl-4-(S)-h ydr oxyh exan oylam in o]-
bu tyr ic Acid (7). Rf ) 0.075 (5% MeOH/CH2Cl2). 1H NMR
(CDCl3): δ 7.90-7.70 (m, 2 H), 7.55-7.25 (m, 3 H), 7.10-6.75
(m, 1 H), 6.81 (d, J ) 6.3 Hz, 2 H), 6.59 (t, J ) 9.0 Hz, 1 H),
4.80 (br s, 1 H, OH), 4.24 (app d, J ) 6.6 Hz, 1 H), 3.71 (d, J
) 8.6 Hz, 1 H), 3.60-3.40 (m, 2 H), 3.40-3.20 (m, 1 H), 3.13
(t, J ) 7.0 Hz, 2 H), 3.10-2.85 (m, 3 H), 2.45-2.25 (m, 1 H),
2.25-2.10 (m, 2 H), 2.00-1.80 (m, 1 H), 1.90-1.10 (m, 10 H),
0.97 (t, J ) 7.2 Hz, 3 H), 0.82 (t, J ) 7.1 Hz, 3 H), 0.72 (t, J )
7.2 Hz, 3 H). MH+ (CI): 603.7. HPLC retention time ) 2.406
[J ], 0.716 [K].
5-[6-(3,5-Diflu or op h en yl)-5-(S)-(3-d ip r op ylca r ba m oyl-
ben zoylam in o)-2-(R)-eth yl-4-(S)-h ydr oxyh exan oylam in o]-
p en ta n oic Acid (8). 1H NMR (CDCl3): δ 7.90-7.70 (m, 2 H),
7.55-7.30 (m, 3 H), 6.82 (d, J ) 6.1 Hz, 2 H), 6.59 (tt, J ) 9.0,
2.1 Hz, 1 H), 4.90 (br s, 1 H, OH), 4.28 (tt, J ) 12.2, 7.1 Hz, 1
H), 3.80-2.80 (m, 9 H), 2.36 (t, J ) 6.4 Hz, 2 H), 2.30-2.00
(m, 2 H), 2.00-1.20 (m, 12 H), 0.98 (t, J ) 7.3 Hz, 3 H), 0.87
(t, J ) 7.1 Hz, 3 H), 0.73 (t, J ) 7.2 Hz, 3 H). MH+ (CI): 618.3.
HPLC retention time ) 2.460 [J ], 0.751 [K].
6-[6-(3,5-Diflu or op h en yl)-5-(S)-(3-d ip r op ylca r ba m oyl-
ben zoylam in o)-2-(R)-eth yl-4-(S)-h ydr oxyh exan oylam in o]-
h exa n oic Acid (9). Rf ) 0.15 (5% MeOH/CH2Cl2). 1H NMR
(CDCl3): δ 7.90-7.65 (m, 2 H), 7.55-7.30 (m, 2 H), 6.81 (app
d, J ) 6.3 Hz, 2 H), 6.60 (tt, J ) 9.0, 2.2 Hz, 1 H), 6.20-5.90
(m, 1 H), 4.35-4.10 (m, 1 H), 3.80-3.40 (m, 4 H), 3.30-2.80
(m, 5 H), 2.60-2.30 (m, 2 H), 2.20 (t, J ) 6.3 Hz, 2 H), 2.00-
1.80 (m, 1 H), 1.80-1.10 (m, 14 H), 0.98 (t, J ) 7.3 Hz, 3 H),
0.88 (t, J ) 7.2 Hz, 3 H), 0.73 (t, J ) 7.2 Hz, 3 H). 13C NMR
(CDCl3): δ 176.7, 176.2, 171.6, 167.7, 162.8 (dd, J ) 48.0, 12.8
Hz, 1 C), 142.2 (t, J ) 9.2 Hz, 1 C), 136.3, 135.0, 128.9, 128.7,
128.5, 125.8, 112.5-112.0 (m, 1 C), 101.8 (t, J ) 25.2 Hz, 1
C), 69.2, 55.8, 50.9 46.8, 45.6, 38.9, 37.5, 37.3, 33.8, 28.6, 26.0,
(1S,2S,4R)-N-[1-(3,5-Diflu or ob en zyl)-4-(syn ,syn )-(3,5-
d im eth oxycycloh exylca r ba m oyl)-2-h yd r oxyh exyl]-N′,N′-
d ip r op ylisop h th a la m id e (13). Rf ) 0.15 (5% iPrOH/CHCl3).
1H NMR (CDCl3): δ 7.85-7.75 (m, 1 H), 7.71 (s, 1 H), 7.55-
7.40 (m, 2 H), 6.84 (d, J ) 9.0 Hz, 1 H), 6.75 (app d, J ) 6.2
Hz, 1 H), 6.62 (tt, J ) 9.0, 2.2 Hz, 1 H), 6.43 (d, J ) 8.4 Hz, 1
H), 5.00 (d, J ) 3.6 Hz, 1 H), 4.17 (q, J ) 8.2 Hz, 1 H), 4.00-
3.80 (m, 1 H), 3.80-3.60 (m, 1 H), 3.60-3.40 (m, 2 H), 3.40-
3.20 (m, 2 H), 3.34 (s, 6 H), 3.20-3.05 (m, 2 H), 3.05-2.85 (m,
2 H), 2.40-1.95 (m, 4 H), 1.85-1.50 (m, 8 H), 1.50-1.30 (m, 2
H), 1.30-1.10 (m, 2 H), 0.99 (t, J ) 7.0 Hz, 3 H), 0.86 (t, J )
7.3 Hz, 3 H), 0.75 (t, J ) 6.9 Hz, 3 H). MH+ (CI): 660.4. Anal.
(C36H51F2N3O6) C, H, N.
(1S,2S,4R)-N-[4-Ben zylcar bam oyl-1-(3,5-diflu or oben zyl)-
2-h yd r oxy-h exyl]-N′,N′-d ip r op yl-isop h th a la m id e (14). Rf
1
) 0.3 (5% MeOH/CH2Cl2). H NMR (CDCl3): δ 7.75-7.60 (m,
2 H), 7.45-7.12 (m, 7 H), 7.08 (d, J ) 9.0 Hz, 1 H), 6.85-6.65
(m, 3 H), 6.61 (tt, J ) 9.0, 2.2 Hz, 1 H), 5.10 (br s, 1 H), 4.44
(dd, J ) 14.7, 6.1 Hz, 1 H), 4.30-4.10 (m, 1 H), 4.19 (dd, J )
14.7, 5.1 Hz, 1 H), 3.67 (br s, 1 H), 3.37 (t, J ) 7.3 Hz, 2 H),
3.10 (t, J ) 7.3 Hz, 2 H), 2.97 (dd, J ) 13.6, 7.7 Hz, 1 H), 2.88

Inhibitors of Human â-Secretase
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1 163
(dd, J ) 13.6, 7.4 Hz, 1 H), 2.40-2.20 (m, 1 H), 1.80-1.20 (m,
8 H), 0.95 (t, J ) 7.1 Hz, 3 H), 0.83 (t, J ) 7.4 Hz, 3 H), 0.71
(t, J ) 7.1 Hz, 3 H). 13C NMR (CDCl3): δ 176.2, 171.1, 167.0,
162.8 (dd, J ) 248.0, 12.8 Hz, 2 C), 142.4 (t, J ) 9.1 Hz, 1 C),
138.1, 137.1, 134.8, 129.1, 128.8, 128.6, 128.0, 127.7, 127.4,
125.0, 112.1 (dd, J ) 16.6, 7.5 Hz, 2 C), 101.8 (t, J ) 25.1 Hz,
1 C), 68.1, 55.3, 50.8, 46.6, 45.4, 43.5, 38.1, 37.2, 25.1, 21.8,
20.6, 12.1, 11.4, 10.9. MH+ (CI): 608.3. Anal. (C35H43F2N3O4)
C, H, N.
4-(a n ti)-{[6-(3,5-Diflu or o-p h en yl)-5-(S)-(3-d ip r op ylca r -
b a m o y l-b e n zo y la m in o )-4-(S )-h y d r o x y -2-(R )-m e t h y l-
h exan oylam in o]m eth yl}cycloh exan ecar boxylic Acid (15).
1H NMR (CD3OD): δ 8.32 (d, 1H), 7.9-7.6 (m, 3H), 3.5-3.4
(m, 1H), 6.9-6.6 (m, 3H), 4.54 (m, 1H), 3.78 (m, 1H), 3.50 (m,
2H), 3.20 (m, 2H), 3.1-2.9 (m, 4H), 2.6-2.1 (m, 2H), 2.0-1.3
(m, 4H), 1.3 (d, 3H), 0.99 (m, 3H), 0.78 (m, 3H). MNa+ (CI):
666.4. Anal. (C35H47F2N3O6) C, H, N.
4-(a n ti)-{[6-(3,5-Diflu or op h en yl)-5-(S)-(3-d ip r op ylca r -
b a m o y l-b e n zo y la m in o )-4-(S )-h y d r o x y -2-(R )-p r o p y l-
h exan oylam in o]m eth yl}cycloh exan ecar boxylic Acid (16).
1H NMR (CD3OD): δ 8.22 (d, 1H), 8.0-7.6 (m, 4H), 6.9-6.7
(m, 3H), 4.44 (m, 1H), 3.6-2.9 (m, 10H), 2.50 (m, 1H), 2.20
(m, 1H), 2.0-1.3 (m, 22H), 1.0-0.7 (m, 13H). MNa+ (CI):
694.4. Anal. (C37H51F2N3O6‚H2O) C, H, N.
2.45 (m, 1H), 1.80-1.60 (m, 7H), 1.60-1.40 (m, 3H), 1.13 (d,
J ) 7.1 Hz, 3H), 0.98 (t, J ) 7.2 Hz, 3H), 0.85 (dd, J ) 6.7, 2.0
Hz, 6H), 0.73 (t, J ) 7.0 Hz, 3H). 13C NMR (CDCl3): δ 177.2,
170.8, 167.0, 162.9 (dd, J ) 248.3, 12.9 Hz, 2C), 142.3 (t, J )
9.1 Hz, 1C), 137.6, 134.7, 129.4, 128.8, 127.6, 125.1, 112.1 (dd,
J ) 16.7, 7.4 Hz, 2C), 101.9 (t, J ) 25.4 Hz, 1C), 67.6, 55.1,
50.7, 46.8, 46.5, 38.3, 38.2, 38.0, 28.4, 21.9, 20.7, 20.0, 17.0,
11.4, 11.0. MH+ 560.3. Anal. (C31H43F2N3O4) C, H, N.
5-{2-[5-(3,5-Diflu or op h en yl)-4-(3-d ip r op ylca r b a m oyl-
benzoylamino)-3-hydroxypentanoylamino]-3-methylbutyryl-
1
a m in o}cycloh exa n e-1,3-d ica r boxylic Acid (21). H NMR
(CD3OD): δ 8.2 (m, 1H, amide), 7.85 (d, 1H, aryl), 7.75 (s, 1H,
aryl), 7.5 (m, 2H, aryl), 6.9 (d, 2H, aryl), 6.65 (m, 1H, aryl),
4.4 (m, 1H), 4.1 (m, 2H), 3.8 (m, 1H), 3.65 (m, 2H), 3.2 (m,
2H), 3.0 (m, 2H), 2.5 (m, 4H), 2.1 (m, 4H), 1.7 (m, 2H), 1.5 (m,
2H), 1.35 (m, 4H), 1.0 (m, 9H, CH3 ×3), 0.7 (m, 3H, CH3). 13C
NMR (CD3OD): δ 172.0, 168.1, 167.3, 167.2, 163.5, 160.2,
160.1, 157.0, 156.8, 138.8, 132.5, 130.2, 124.6, 124.1, 123.6,
120.6, 107.4, 96.6, 64.8, 54.5, 50.1, 46.2, 46.3, 36.4, 36.3, 35.5,
32.0, 29.3, 29.2, 25.7, 25.6, 16.8, 15.7, 13.6, 12.4, 5.6, 5.2. MH+
(CI): 745.2. Anal. (C38H52F2N4O10‚H2O) C, H, N.
Refer en ces
4-(a n ti)-{[6-(3,5-Diflu or o-p h en yl)-5-(S)-(3-d ip r op ylca r -
ba m oyl-ben zoyla m in o)-4-(S)-h yd r oxy-h exa n oyla m in o]-
(1) Selkoe, D. J . Translating cell biology into therapeutic advances
in Alzheimer’s disease Nature 1999, 399 (Suppl. 6738), A23-
A31.
1
m eth yl}cycloh exa n eca r boxylic Acid (17). H NMR (CD3-
OD): δ 7.98 (t, J ) 5.7 Hz, 1H), 7.84 (dt, J ) 7.1, 1.8 Hz, 1H),
7.70 (s, 1H), 7.60-7.45 (m, 2H), 6.92 (app dd, J ) 8.3, 1.8 Hz,
2H), 6.74 (tt, J ) 9.2, 2.2 Hz, 1H), 4.45-4.30 (m, 1H), 1.00 (t,
J ) 7.4 Hz, 3H), 0.71 (t, J ) 7.3 Hz, 3H). 13C NMR (CD3OD):
δ 180.0, 175.9, 173.1, 169.4, 164.3 (dd, J ) 246.6, 13.1 Hz,
2C), 144.9 (t, J ) 9.3 Hz, 1C), 138.5, 136.1, 130.5, 130.0,
129.3, 126.3, 113.2 (dd, J ) 17.0, 7.5 Hz, 2C), 102.5 (t, J )
25.8 Hz, 1C), 72.9, 56.3, 52.3, 46.4, 44.5, 38.6, 38.1, 33.6, 31.3,
31.0, 29.9, 22.9, 21.7, 11.7, 11.3. MH+ (CI): 686.3. Anal.
(C34H45F2N3O6‚0.5H2O) C, H, N.
4-(a n ti)-{[6-(3,5-Diflu or o-p h en yl)-5-(S)-(3-d ip r op ylca r -
b a m oyl-b e n zoyla m in o)-4-(S )-h yd r oxy-2-(R )-isob u t yl-
h exan oylam in o]m eth yl}cycloh exan ecar boxylic Acid (18).
1H NMR (CD3OD): δ 7.97 (t, J ) 5.7 Hz, 1H), 7.83 (dt, J )
7.1, 1.8 Hz, 1H), 7.70 (s, 1H), 7.70-7.55 (m, 2H), 6.90 (app
dd, J ) 8.3, 1.8 Hz, 2H), 6.74 (tt, J ) 9.2, 2.2 Hz, 1H), 4.40-
4.20 (m, 1H), 3.64 (tt, J ) 9.4, 3.0 Hz, 1H), 3.48 (t, J ) 7.5 Hz,
2H), 3.18 (t, J ) 7.5 Hz, 2H), 3.20-2.85 (m, 4H), 2.63 (sept, J
) 4.8 Hz, 1H), 2.18 (tt, J ) 12.1, 3.4 Hz, 1H), 1.95 (d, J ) 9.0
Hz, 2H), 1.85-1.65 (m, 5H), 1.65-1.25 (m, 9H), 1.20-1.10 (m,
1H), 1.00 (t, J ) 7.4 Hz, 3H), 0.90 (dd, J ) 11.4, 6.4 Hz, 6H),
0.72 (t, J ) 7.3 Hz, 3H). 13C NMR (CD3OD): δ 180.0, 178.7,
173.3, 169.7, 163.8 (dd, J ) 248.0, 12.8 Hz, 2 C), 145.0, 138.6,
130.6, 130.1, 129.4, 126.4, 113.1 (dd, J ) 16.6, 7.5 Hz, 2 C),
102.6 (t, J ) 25.1 Hz, 1 C), 71.1, 56.9, 52.2, 46.5, 44.4, 43.4,
42.7, 38.5, 38.3, 37.5, 31.0, 29.8, 27.2, 23.6, 22.8, 22.4, 21.7,
11.6, 11.2. MH+ (CI): 630.3. Anal. (C38H53F2N3O6) C, H, N.
(2) Sinha, S.; Lieberburg, I. Cellular mechanisms of â-amyloid
production. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11049-11053.
(3) Dovey, H. F.; J ohn, V.; Anderson, J . P.; Chen, L. Z.; De Saint
Andrieu, P.; Fang, L. Y.; Freedman, S. B.; Folmer, B.; Goldbach,
E.; Holsztynska, E. J .; Hu, K. L.; J ohnson-Wood, K. L.; Kennedy,
S. L.; Kholodenko, D.; Knops, J . E.; Latimer, L. H.; Lee, M.; Liao,
Z.; Lieberburg, I. M.; Motter, R. N.; Mutter, L. C.; Nietz, J .;
Quinn, K. P.; Sacchi, K. L.; Seubert, P. A.; Shopp, G. M.;
Thorsett, E. D.; Tung, J . S.; Wu, J .; Yang, S.; Yin, C. T.; Schenk,
D. B.; May, P. C.; Altstiel, L. D.; Bender, M. H.; Boggs, L. N.;
Britton, T. C.; Clemens, J . C.; Czilli, D. L.; Dieckman-McGinty,
D. K.; Droste, J . J .; Fuson, K. S.; Gitter, B. D.; Hyslop, P. A.;
J ohnstone, E. M.; Li, W.-Y.; Little, S. P.; Mabry, T. E.; Miller,
F. D.; Ni, B.; Nissen, J . S.; Porter, W. J .; Potts, B. D.; Reel, J .
K.; Stephenson, D.; Su, Y.; Shipley, L. A.; Whitesitt, C. A.; Yin,
T.; Audia, J . E. Functional gamma-secretase inhibitors reduce
beta-amyloid peptide levels in the brain. J . Neurochem. 2001,
76, 173-181.
(4) Sinha, S.; Anderson, J . P.; Barbour, R.; Basi, G. S.; Caccavello,
R.; Davis, D.; Doan, M.; Dovey, H. F.; Frigon, N.; Hong, J .; Croak,
K.; J ewett, N.; Keim, P.; Knops, J .; Lieberburg, I.; Power, M.;
Tan, H.; Tatsuno, G.; Tung, J .; Schenk, D.; Seubert, P.; Suo-
mensaari, S. M.; Wang, S.; Walker, D.; Zhao, J .; McConologue,
L.; J ohn, V. Purification and cloning of amyloid precursor protein
â-secretase from human brain. Nature 1999, 402, 537-540.
(5) Yan, R.; Bienkowski, M. J .; Shuck, M. E.; Miao, H.; Tory, M. C.;
Pauley, A. M.; Brashier, J . R.; Stratman, N. C.; Mathews, R.
W.; Buhl, A. E.; Carter, D. B.; Tomasselli, A. G.; Parodi, L. A.;
Heinrickson, R. L.; Gurney, M. Membrane-anchored aspartyl
protease with Alzheimer’s disease â-secretase activity. Nature
1999, 402, 533-536.
(6) Vassar, R.; Bennett, B. D.; Babu-Khan, 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 .; J arosinski, M. A.;
Biere, A. L.; Curran, E.; Burgess, T.; Louis, J . C.; Collins, F.;
Treanor, J .; Rogers, G.; Citron, M. â-Secretase cleavage of
Alzhemeier’s amyloid precursor protein by the transmembrane
aspartic protease BACE. Science 1999, 286, 735-741.
(7) (a) Roberds, S. L.; Anderson, J .; Basi, G.; Bienkowski, M. J .;
Branstetter, D. G.; Chen, K. S.; Freedman, S. B.; Frigon, N. L.;
Games, D.; Hu, K.; J ohnson-Wood, K.; Kappenman, K. E.;
Kawabe, T. 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.; Sinha, 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 brain: implications for Alzheimer’s
disease therapeutics. Hum. Mol. Genet. 2001, 10, 1317-1324.
(b) Luo, Y.; Bolon, B.; Kahn, S.; Bennett, B. D.; Babu-Khan, S.;
Denis, P.; Fan, W.; Kha, H.; Zhang, J .; Gong, Y.; Martin, L.;
Louis, J .-C.; Yan, Q.; Richards, W. G.; Citron, M.; Vassar, R.
Mice deficient in BACE1, the Alzheimer’s â-secretase, have
normal phenotype and abolished â-amyloid generation. Nat.
4-(a n t i)-{[2-(R )-Be n zyl-6-(3,5-d iflu or op h e n yl)-5-(S )-
(3-d ip r op ylca r b a m oyl-b e n zoyla m in o)-4-(S )-h yd r oxy-
h exa n oyla m in o]m et h yl}cycloh exa n eca r b oxylic
Acid
(19). 1H NMR (CD3OD): δ 8.20 (d, 1H), 7.7-7.6 (m, 6H), 7.4-
7.2 (m, 6H), 6.9-6.7 (m, 3H), 4.4 (br s, 1H), 3.6 (m, 1H), 3.5-
2.7 (m, 15H), 2.2-1.6 (m, 13H), 1.3-1.2 (m, 4H), 1.00 (m, 3H),
0.70 (m, 5H). 13C NMR (CD3OD): δ 180.1, 177.4, 177.33,
173.30, 169.7, 169.6, 166.25, 166.1, 163.0, 162.8, 145.0, 144.9,
141.0, 138.6, 136.3, 130.3, 130.3, 130.1, 129.5, 129.4, 127.5,
126.4, 113.4, 113.3, 113.2, 113.1, 102.9, 102.6, 102.2, 101.5,
71.3, 57.1, 57.1, 52.2, 48.0, 46.4, 46.3, 44.3, 40.5, 38.3, 37.6,
30.7, 30.6, 29.8, 29.7, 22.8, 21.7,11.0, 11.2. MNa+ (CI): 743.
Anal. (C41H51F2N3O6‚0.5H2O) C, H, N.
N-[1-(3,5-Diflu or oben zyl)-2-h yd r oxy-4-isobu tylca r ba m -
oylp en t yl]-N′,N′-d ip r op ylisop h t h a la m id e (20). 1H NMR
(CDCl3): δ 7.80-7.65 (m, 2H), 7.50-7.35 (m, 2H), 6.87 (d, J
) 9.1 Hz, 1H), 6.81 (app d, J ) 9.1 Hz, 1H), 6.63 (tt, J ) 9.0,
2.2 Hz, 1H), 5.98 (t, J ) 5.9 Hz, 1H), 5.11 (d, J ) 3.7 Hz, 1H),
4.20 (q, J ) 7.9 Hz, 1H), 3.77 (app d, J ) 9.2 Hz, 1H), 3.46 (t,
J ) 7.4 Hz, 2H), 3.25-3.08 (m, 2H), 3.08-2.90 (m, 4H), 2.65-

164 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 1
Hom et al.
Neurosci. 2001, 4, 231-232. (c) Cai, H.; Wang, Y.; McCarthy,
D.; Wen, H.; Borchelt, D. R.; Price, D. L.; Wong, P. C. BACE1 is
the major â-secretase for generation of Aâ peptides by neurons.
Nat. Neurosci. 2001, 4, 233-234.
(14) Hong, L.; Koelsch, G.; Lin, X.; Wu, S.; Terzyan, S.; Ghosh, A.
K.; Zhang, X. C.; Tang, J . Structure of Protease Domain of
Memapsin 2 (â-Secretase) Complexed with Inhibitor. Science
2000, 290, 150-153.
(8) Tung, J . S.; Davis, D. L.; Anderson, J . P.; Walker, D. E.; Mamo,
S.; J ewett, N.; Hom, R. K.; Sinha, S.; Thorsett, E. D.; J ohn, V.
Design of substrate-based inhibitors of human â-secretase. J .
Med. Chem. 2002, 45, 259-262.
(9) Hom, R. K.; Fang, L. Y.; Mamo, S.; Tung, J . S.; Guinn, A. C.;
Knops, J . E.; Walker, D. E.; Davis, D.; Gailunas, A. F.; Thorsett,
E. D.; Sinha, S.; Knops, J . E.; J ewett, N. J .; Anderson, J . P.;
J ohn, V. Design and synthesis of statine-based cell-permeable
peptidomimetic inhibitors of human â-secretase. J . Med. Chem.
2003, 46, 1799-1802.
(10) (a) Greenlee, W. J . Renin inhibitors. Med. Res. Rev. 1990, 10
(2), 173-236. (b) Glenn, M. P.; Fairlie, D. P. Mimetics of the
peptide beta-strand. Mini Rev. Med. Chem. 2002, 2 (5), 433-
445.
(11) Ghosh, A. K.; Bilcer, G.; Harwood, C.; Kawahama, R.; Shin, D.;
Hussain, K. A.; Hong, L.; Loy, J . A.; Nguyen, C.; Koelsch, G.;
Ermolieff, J .; Tang, J . Structure-based design: potent inhibitors
of human brain memapsin 2 (â-secretase). J . Med. Chem. 2001,
44, 2865-2868.
(12) Kaldor, S. W.; Dressman, B. A.; Hammond, M.; Appelt, K.;
Burgess, J . A.; Lubbehusen, P. P.; Muesing, M. A.; Hatch, S.
D.; Wiskerchen, M. A.; Baxter, A. J . Isophthalic acid deriva-
tives: amino acid surrogates for the inhibition of HIV-1 protease.
Bioorg. Med. Chem. Lett. 1995, 5, 721-726.
(13) Chang, G.; Guida, W. C.; Still, W. C. An Internal Coordinate
Monte Carlo Method for Searching Conformational Space. J . Am.
Chem. Soc. 1989, 111, 4379-4386.
(15) (a) Dragovich, P. S.; Prins, T. J .; Zhou, R.; Fuhrman, S. A.;
Patick, A. K.; Matthews, D. A.; Ford, C. E.; Meador, J . W., III;
Ferre, R. A.; Worland, S. T. Structure-based design, synthesis,
and biological evaluation of irreversible human rhinovirus 3C
protease inhibitors. 3. Structure-activity studies of ketometh-
ylene-containing peptidomimetics. J . Med. Chem. 1999, 42,
1203-1212. (b) Askin, D.; Wallace, M. A.; Vacca, J . P.; Reamer,
R. A.; Volante, R. A.; Shinkai, I. Highly diastereoselective
alkylations of chiral amide enolates: new routes to hydroeth-
ylene dipeptide isostere inhibitors of HIV-1 protease. J . Org.
Chem. 1992, 57, 2771-2773. (c) McWilliams, J . C.; Armstrong,
J . D., III; Zheng, N.; Bhupathy, M.; Volante, R. P.; Reider, P. J .
Tandem asymmetric transformations: an asymmetric 1, 2-
migration from a higher order zincate coupled with a stereose-
lective homoaldol reaction. J . Am. Chem. Soc. 1996, 118, 11970-
11971.
(16) (a) Martin, S. F.; Dwyer, M. P.; Lynch, C. L. Application of
AlMe3-mediated amidation reactions to solution phase peptide
synthesis. Tetrahedron Lett. 1998, 39, 1517-1520. (b) Basha,
A.; Lipton, M.; Weinreb, S. M. A mild, general method for
conversion of esters to amides. Tetrahedron Lett. 1977, 4171-
4174.
(17) The detailed protocol for the human embryonic kidney cellular
assay can be found in ref 3.
J M0304008