T. Nguyen et al. / European Journal of Medicinal Chemistry 46 (2011) 2917e2929
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6.10. Synthesis of 8-benzyl-8-aza-bicyclo[3.2.1]octan-3-amine (42)
[23]
incubation in 1 M arginineeHCl, 1 mM oxidized glutathione, pH 8.0
at 37 ꢀC overnight. The refolded rPrP was dialyzed against water
and concentrated by centrifugal membrane (Amicon Ultra-15,
MWCO 3000, Millipore). To remove N-terminal histidine tag, rPrP
A solution of ethyl 3-oxo-8-azabicylo[3.2.1]octane-8-carboxylic
acid (2.3 mmol, 0.46 g) in ethanol (1 ml) was mixed with KOH
(0.353 g) in water (5 mL) and heated at 100 ꢀC for 3 h. After cooling
down to room temperature, the solution was diluted with 20 mL of
dichloromethane. The organic layer was dried and concentrated in
vacuo to give crude 8-aza-bicyclo[3.2.1]octan-3-one (39). 1H NMR
(0.2 mg/mL) was digested with 0.3 m
g/mL trypsin at 16 ꢀC for 16 h in
25 mM TriseHCl, pH 7.5. The N-terminal histidine tag-truncated
rPrP was purified by anion-exchange chromatography (UNO-Q,
1 mL, Bio-Rad), buffer was changed to 10 mM NaOAc (pH6.0) with
Amicon Ultra-15, and stored at ꢂ80 ꢀC. Protein concentration was
determined by UV measurement and found to be equivalent to
0.79 mg/ml.
(300 MHz, CDCl3)
(m, 1H), 2.36 (m, 2H), 2.56 (m, 2H), 3.87 (m, 2H). 13C NMR (75 MHz,
CDCl3) 30.0 (2C), 50.8 (2C), 54.9 (2C), 209.7. 39 was reacted with
benzyl chloride as in Section 6.6 to give 8-benzyl-8-aza-bicyclo
[3.2.1]octan-3-one (40). 1H NMR (300 MHz, CDCl3)
1.63 (m, 2H),
2.16 (m, 4H), 2.72 (m, 2H), 3.50 (m, 2H), 3.76 (m, 2H), 7.34 (m, 5H).
13C NMR (75 MHz, CDCl3)
27.7 (2C), 48.1 (2C), 55.1, 58.6 (2C),127.2,
d
1.69 (m, 2H), 1.90 (dd, J1 ¼ 4.1, J2 ¼ 8.8, 2H), 2.31
d
6.14. Evaluation of binding affinity by surface plasmon resonance
d
The assay was performed on a BIAacore 3000 platform (BIAcore,
Uppsala, Sweden) following reported methods with modifications
[27,28]. The sensor chip CM5 and amine coupling kit were
purchased from GE Healthcare Bio-sciences AB (Uppsala, Sweden).
The running buffer was prepared by filtering and degassing
a phosphate buffer 1ꢃ, pH 7.4 containing 2.5% DMSO. Recombinant
human prion protein (amino acids 121e231; hPrP121e231,
molecular weight 12,544.97 Da) was dissolved in 10 mM sodium
d
128.4 (2C), 128.5 (2C), 138.9, 210.1. MS(APCI) m/z [Mþ] 215.3. To
obtain the oxime 41, hydroxylamine hydrochloride (1.7 mmol,
0.117 g) was stirred in methanol (7 mL) at 0 ꢀC. The slurry was
treated with Na2CO3 (0.09 g) and stirred for 5 min after which 40
(1.36 mmol, 0.29 g) in methanol (1 mL) was added. After stirring for
5 h at room temperature, methanol was removed in vacuo, the
residue was treated with dichloromethane and brine, the organic
fractions were combined, dried (anhydrous Na2SO4), and concen-
trated to give 0.2 g of the oxime 41 as a white solid (Yield 65%). 1H
acetate buffer, pH 4.5 (10 mg/ml) and immobilized on the CM5 chip
to reach a density of ca 3000 response units (RU) using amine
coupling. A reference surface was prepared by treating a blank flow
cell in the same way as the flow cell containing the immobilized
peptide. Stock solutions of test compounds (2 mM) were prepared
NMR (300 MHz, CDCl3)
d
1.52 (t, J ¼ 9.6, 1H),1.63 (t, J ¼ 9.6,1H), 2.04
(m, 2H), 2.14 (d, J ¼ 14.7, 1H), 2.26 (dd, J1 ¼ 2.7, J2 ¼ 12.6), 2.62 (d,
J ¼ 14.7, 1H), 2.99 (d, J ¼ 15.6, 1H), 3.36 (b, 2H), 3.66 (s, 2H),
in DMSO and diluted to 50
Each analytical cycle consisted of running buffer for 60 s (stabili-
zation phase), a sample injection of 50 M in running buffer for
120 s (association phase) and running buffer for 150 s (dissociation
phase). Flow rates were kept at 30 L/min. Surface regeneration of
the chip was carried out by injecting 10 mM NaOH (30 s, flow rate of
30 L/min). After regeneration, the surface was allowed to stabilize
mM with phosphate buffer 1ꢃ, pH 7.4.
7.25e7.42 (m, 5H). 13C NMR (75 MHz, CDCl3)
d 26.6, 27.5, 31.3, 37.1,
55.5, 57.8, 58.4, 127.0, 128.2 (2C),128.7 (2C), 138.9, 156.3. MS(ESI) m/
z [M þ Hþ] 231.1. The oxime 41 was reduced with Adam’s catalyst as
described in Section 6.6 to give the title compound 42 as a yellow
oil (76%) which was not characterized and reacted immediately to
give 19 (Section 6.3).
m
m
m
for ca 60 s before the next injection. The binding response curve
was obtained by subtracting the background signal (from the
reference flow cell) from that of the sample flow cell in order to
correct for non-specific binding and bulk effects due to the analyte.
The binding affinity was taken as the maximum response at the end
of the association phase as seen from the binding response curve.
Binding was expressed as % RUmax which is defined as the % theo-
retical maximum binding response units (RU) and determined by
the following equation assuming a 1:1 stoichiometry:
6.11. 3,9-Dichloro-5,6,7,8-tetrahydroacridine (43) [22]
To a mixture of 2-amino-4-chlorobenzoic acid (5 mmol, 0.858 g)
and cyclohexanone (5 mmol, 0.52 mL) was carefully added 5 mL of
POCl3 at 0 ꢀC. The mixture was heated under reflux for 2 h, then
cooled to room temperature, and concentrated to give a slurry. The
residue was diluted with dichloromethane, neutralized with
aqueous K2CO3, and washed with brine. The organic layer was dried
over anhydrous Na2SO4 and concentrated in vacuo to furnish 1.12 g
of 43 as a reddish brown solid (Yield 88%). 1H NMR (300 MHz,
h
i
%RUmax
¼
RUcompound=RUimmobilized protein
CDCl3)
d
1.89 (m, 4H), 2.88 (t, J ¼ 5.3, 2H), 3.06 (9t, J ¼ 5.3, 2H), 7.34
h
i
(dd, J1 ¼ 1.9, J2 ¼ 8.9, 1H), 7.89 (m, 2H). 13C NMR (75 MHz, CDCl3)
ꢃ MWprotein=MWcompound ꢃ 100%
d
22.1, 22.2, 27.1, 33.4, 123.3, 124.7, 126.8, 127.1, 128.9, 135.1, 141.4,
145.9, 160.5. MS(APCI) m/z [Mþ] 252.7.
6.15. Evaluation of permeability by the PAMPA-BBB assay
6.12. Evaluation of cell-based anti-prion activity
The method described by Di et al. [29] was followed with
modifications. Porcine polar brain lipid (PBL) (catalog no. 141101)
was purchased from Avanti Polar Lipids, Inc. (Alabaster, Alabama,
USA). Dodecane was obtained from SigmaeAldrich. The acceptor
plate was a 96-well filter plate (MultiscreenTM-IP, catalog no.
The anti-prion activity of test compounds was determined on
ScN2a, N167 and F3 cell models as described by Nguyen Thi et al.
[19].
6.13. Preparation of human prion protein 121e231 ( hPrP121e231
)
MAIPN4510, PVDF membrane, pore size is 0.45 mm) from Millipore
(Bredford, USA) and the donor plate was a 96-well plate (catalog no.
MATRNPS50, Millipore, Billerica, MA, USA). The 96-well UV plate
(catalog no. 3535) was purchased from Corning Inc. (NY, USA).
Compounds were dissolved in DMSO at 10 mM, and diluted in
Recombinant PrP (rPrP, human PrP 121-231aa with an N-
terminal histidine tag and Factor Xa digestion sequence) was
bacterially expressed as inclusion bodies by the Escherichia coli
protein expression pET system. The inclusion bodies were solubi-
lized in 8 M urea, 25 mM TriseHCl, 300 mM NaCl, pH 7.5 and then
purified by metal chelating chromatography (HiTrap-Ni, GE Health
Science). The purified rPrP was refolded by rapid dilution and
phosphate buffer solution 1ꢃ, pH 7.4e50
on their aqueous solubilities. An aliquot (300
solution was transferred to the donor well. The filter membrane
was coated with 5 L of 20 mg/mL PBL in dodecane and the
mM or 30
mM depending
mL) of the diluted
m