6488 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Bakshi and Wolfe
mL of CH2Cl2, and the organic layer was washed sequentially
with 1 N HCl (3×), saturated NaHCO3, and finally with brine.
The organic layer was dried over Na2SO4 and evaporated to a
colorless oil. The product was purified by flash chromatography
using hexane/EtOAc 4:1 to yield 80-85% of Weinreb amide 2.
Compound 2 (1 mmol) was then reduced to R-(tert-butoxycar-
bonyl)aminoaldehyde 3 by dissolving in diethyl ether (20 mL)
and slowly adding LiAlH4 (5 mmol). After reduction for 30 min,
the product was hydrolyzed with a solution of KHSO4 (3.5
mmol) in water (10 mL). The product was extracted from the
aqueous layer with ether (3 × 50 mL), and the organic layer
was washed with 1 N HCl, saturated NaHCO3, and brine.
Drying (Na2SO4), filtering, and evaporating provided the
corresponding protected aminoaldehyde (74-93% yield), which
was used without further purification. For alkene 4, 1,1,1,3,3,3-
hexamethyldisilazane (1 mL, 1.1 mmol) was added dropwise
to a 0 °C suspension of potassium hydride (35% dispersion in
oil, 1.1 mmol) in anhydrous THF/DMSO (14 mL/3 mL) under
dry N2. After being stirred at 0 °C for 1 h, the resulting solution
was added via cannula to a 0 °C flask containing methyltriph-
enylphosphonium bromide (1.1 mmol). The mixture was stirred
vigorously for 1 h and then cooled to -78 °C. At -78 °C, a
THF solution of the aldehyde (1 mmol) prepared above was
added via cannula over 20 min. After being stirred at -78 °C
for another 10 min, the mixture was allowed to slowly warm
to room temperature (4 h) and then heated to 40-45 °C for 12
h. The mixture was then cooled to room temperature, and the
reaction was quenched with methanol (200 µL), followed by
aqueous Rochelle salts (10 mL of saturated solution and 100
mL of H2O). The mixture was then extracted with EtOAc (2
× 150 mL). The combined extracts were washed with water
and brine. Drying (Na2SO4) and evaporating provided the
crude product, which was purified by flash chromatography
on silica gel (ether/hexane) to give alkene 4 in 45-50% yield.
white solid. The N-protected bromohydrin (0.3 mmol) was
dissolved in methanol (5 mL), and 1 mL of 0.3 M NaOMe in
MeOH was added. After 1.5 h of stirring, water (10 mL) was
added and the solution was extracted with 10 mL of CH2Cl2.
The organic phase was dried over Na2SO4, filtered, and
evaporated to dryness. Flash chromatography afforded the
pure product 5e in 85% yield with 97% de. [R] +6° (c 0.6, CH3-
OH). 1H NMR (200 MHz, CDCl3): δ 1.39-1.40 (s, 9H), 2.66-
2.72 (m, 1H), 2.8 (dd, 1H), 2.82-3 (m, 3H), 3.72-3.78 (m, 1H),
4.42-4.48 (br, m, 1H), 7.2-7.36 (m, 5H).
Synthesis of (Hydroxyethyl)urea Analogues7 (11). To
a solution of oxirane 5 (1 equiv) in 2-propanol was added 20
equiv of primary amine, and the reaction mixture was refluxed
under dry nitrogen for 12 h. However, for the reaction leading
to analogue 21, methylamine was bubbled into the solution to
saturation and the reaction was carried out in a pressured
vessel. The solution was diluted with ethyl acetate and washed
consecutively with water, aqueous 1 N HCl, and saturated
NaHCO3, and the organic phase was dried over Na2SO4,
filtered, and concentrated to provide the amino alcohol 9. The
isocyanates of P2′ amino acids were made separately by
stirring amino acid ester (HCl salt) in a mixture of CH2Cl2
and NaHCO3 for 20 min followed by addition of phosgene
solution (20% in toluene) into the settled CH2Cl2 layer of the
mixture. After an additional 30 min of stirring, the organic
phase was separated, dried over Na2SO4, filtered, and concen-
trated. The (hydroxyethyl)amine 9 was then coupled with the
isocyanate methyl ester in a minimal amount of CH2Cl2 for
5-6 h. The reaction mixture was concentrated, and the
(hydroxyethyl)urea 10 was purified using flash chromatogra-
phy. For C-terminal extension, the methyl ester functionality
was hydrolyzed with LiOH (0.5 M) in aqueous dioxane for 2
h. After evaporation of dioxane, water and CH2Cl2 were added.
The pH of the aqueous solution was reduced to 2 with aqueous
1 N HCl, and the organic layer was separated and washed
with brine, dried over Na2SO4, and concentrated. The resultant
carboxylic acid was then coupled to the P3′ R-amino alkyl ester
in a minimum amount of DMF in the presence of HATU and
DIPEA for 8 h. The reaction mixture was diluted with CH2Cl2
and washed with aqueous 1 N HCl, saturated NaHCO3, and
finally brine. After concentration, the crude product 11 was
purified by flash chromatography, followed by preparative
HPLC on a C18 column. All final compounds and their
intermediates were characterized and assessed for purity by
1H NMR (200 MHz), mass spectrometry (MALDI-TOF), and
analytical HPLC in two different solvent systems (Supporting
Information).
Synthesis of N-Terminal Extended (Hydroxyethyl)-
urea Analogues (12). tert-Butyloxycarbonyl (Boc) protection
was removed from peptidomimetic 11 by treating with TFA
(trifluoroacetic acid) in CH2Cl2 (3:7) for 30-40 min. The
reaction mixture was diluted with CH2Cl2, and the pH was
increased to 7 with a saturated solution of NaHCO3. The
organic layer was extracted and washed with brine, dried with
Na2SO4, and evaporated to provide the free amine. Coupling
with Boc-protected amino acids was carried out as described
above.
Biological Evaluation. Cell Lines, Compound Treat-
ments, and ELISAs.3 Compounds were tested in CHO cells
stably transfected with the 751 amino acid splice variant of
APP (7w cells). Cells were grown to confluence in Dulbecco’s
modified Eagle’s medium (DMEM) containing 200 µg/mL G418
(Gibco BRL). Stock concentrations of the peptide analogues
in DMSO were added to DMEM to reach the final concentra-
tions with 1% DMSO. Positive controls contained 1% DMSO
alone. After 4 or 24 h, the medium was removed and centri-
fuged at 3000g for 5 min, and the supernatant was stored at
-80 °C until the assays were carried out. Sandwich ELISAs
for Aâ40 and Aâ42 were performed using capture antibodies
2G3 (to Aâ40 residues 33-40) for the Aâ40 species and 21F12
(to Aâ42 residues 33-42) for the Aâ42 species. The reporter
antibody was biotinylated 3D6 (to Aâ residues 1-5) in each
assay. Horseradish peroxidase-avidin binding to the reporter
The resulting olefin (1 mmol) was stirred in CH2Cl2 with
m-chloroperoxybenzoic acid (4 mmol) under a nitrogen atmo-
sphere. When the reaction was complete by TLC analysis, the
mixture was diluted with ether, washed sequentially with ice-
cold 10% Na2SO3, saturated NaHCO3, and brine. Drying and
evaporating provided the white crystalline epoxide 5t in 60%
yield with 99% de {[R] -2°(c 0.6, CH3OH)}. 1H NMR (200 MHz,
CDCl3): δ 1.39-1.42 (s, 9H), 2.56-2.6 (m, 1H), 2.69 (dd, 1H),
2.82-3.14 (m, 3H), 4-4.16 (m, 1H), 4.44-4.52 (br, m, 1H), 7.2-
7.36 (m, 5H)
Synthesis of tert-Butyl [R(S*,S*)]-(-)-(1-Oxiranyl-2-
phenylethyl)carbamate (5e). Synthesis of the erythro ep-
oxide was carried out according to the method described by
Albeck et al.12 with slight modifications. Briefly, a solution of
Boc-protected D-phenylalanine 1 (1 mmol) and N-methylmor-
pholine (1.1 mmol) in dry THF (5 mL) under argon atmosphere
was cooled to -15 °C. Isobutyl chloroformate (1 mmol) was
added, and after 5 min the reaction mixture was quickly
filtered and added to a precooled (-15 °C) ethereal solution of
diazomethane (2 mmol). The reaction mixture was stirred for
2 h at 0 °C, and 1 equiv of 48% aqueous HBr was added. The
reaction was continued for another 15-20 min, and the
resultant bromoketone 7 was purified using flash chromatog-
raphy (elution with 3:1 of ether/hexane). For bromohydrin 8,
a solution of (S)-2-methyl-CBS-oxazaborolidine (1.0 M in
toluene, 0.1 mL, 0.1 mmol) and boranemethyl sulfide (2.0 M
in THF, 0.05 mL, 0.1 mmol) in anhydrous THF (2 mL) was
treated simultaneously with a solution of the R-bromoketone
7 in THF (1 mL) and boranemethyl sulfide (2.0 M in THF,
0.33 mL, 0.66 mmol) at 0 °C under argon over 20 min. The
reaction mixture was allowed to warm to room temperature
and stirred for 1 h. The mixture was then cooled to 0 °C before
1 mL of methanol was added carefully (CAUTION: gas
evolution!!). The reaction mixture was then concentrated in
vacuo (Me2S was trapped and oxidized with household bleach),
and the residue was dissolved in 20 mL of EtOAc. The solution
was washed with 1 N of HCl (3 × 5 mL) and water (2 × 5
mL), dried over Na2SO4, filtered, and concentrated. The
product was purified by column chromatography on silica gel
using hexane/ethyl acetate (90:10) as eluant to give 8e as a
Bakshi, Pancham
