Prion Inhibition by 3,5-Dicarbonitrile-Based Compounds
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 1 71
Hz, 1H), 3.60 (m, 2H), 3.48 (m, 2H), 3.34 (q, J ) 7.2 Hz, 4H),
1.35 (t, J ) 7.2 Hz, 6H); LCMS (ESI) m/z 342 (MH+); HRMS m/z
calculated for C17H20N5OS (MH+) 342.1389, found 342.1375; purity
method A, 99%, method B, 100%.
2-Amino-6-(2-(diisopropylamino)ethylthio)-4-furan-2-ylpyri-
dine-3,5-dicarbonitrile (20). 1H NMR (CD3OD/CD3CO2D) δ 7.88
(d, J ) 1.2 Hz, 1H), 7.51 (d, J ) 3.6 Hz, 1H), 6.75 (dd, J ) 2.0,
3.6 Hz, 1 H), 3.83 (quintet, J ) 6.8 Hz, 2H), 3.61 (m, 2H), 3.44
(m, 2H), 1.42 (d, J ) 6.8 Hz, 12H); LCMS (ESI) m/z 370 (MH+);
HRMS m/z calculated for C19H24N5OS (MH+) 370.1702, found
370.1701; purity method A, 95%, method B, 100%.
(bs, 1H), 7.18 (d, J ) 3.2 Hz, 1H), 6.96 (d, J ) 8.4 Hz, 1H), 6.86
(dd, J ) 1.6, 3.6 Hz, 1H), 4.30 (d, J ) 4.8 Hz, 4H); LCMS (ESI)
m/z 419 (MH+); HRMS m/z calculated for C21H14N4O4NaS (MNa+)
441.0633, found 441.0631; purity method A, 74%, method B, 95%.
Duplex Cellular Toxicity and Antiprion Assay. ScN2a cells
were maintained in filter-sterilized (0.2 mm) MEM, supplemented
with FBS and GlutaMax. On day 1, media was aspirated from a
confluent 100 mm plate of ScN2a cells and cells were detached by
addition of 0.05% trypsin-EDTA (1 mL). MEM media was added
(approximately 10 mL), and the cell concentration was determined
using Packed Cell Volume tubes (TPP). The ScN2a cell concentra-
tion was adjusted to 4 × 105 cells mL-1 with MEM media. A 96-
well, tissue-culture-treated, clear-bottom, black plate (Greiner Bio-
One) wetted with MEM media (90 µL) was incubated at 37 °C
prior to use. To this plate, 100 µL of the ScN2a cell suspension
was transferred, and the cells were allowed to settle for 2 h prior
to addition of the test compound. Compound libraries were prepared
in 100% DMSO at the required concentrations in a 96-well format
and then diluted 1/20 with sterile PBS prior to use. Compounds (10
µL) were transferred to the 96-well cell culture plate, and the plates
were incubated at 37 °C. Final DMSO concentrations did not exceed
0.25% (v/v). Media were aspirated on day 5, and cells were washed
twice with PBS (200 µL). Calcein-AM (100 µL, 2.5 µg mL-1
solution in PBS) was added, and the plates were incubated at
37 °C for 25 min. Fluorescent emission intensity was quantified
using a fluorescence plate reader, ex/em ) 492 nm/525 nm.
The calcein-AM solution was aspirated, 50 µL of lysis buffer
(10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.5% sodium deoxy-
cholate; 0.5% Nonidet P-40) was added, and then plates were
shaken for 5 min at room temperature. Benzonase nuclease (25
µL, 7.5 U/mL in 50 mM Tris-HCl, pH 8.0; 20 mM NaCl; 2 mM
MgCl2) was added, and plates were incubated at 37 °C for 15 min
or until the DNA pellet was no longer visible. Proteinase K (25
µL, 25 µg mL-1 solution in lysis buffer) was added, and the plates
were incubated at 37 °C for 1 h. Proteolysis was inhibited by the
addition of PMSF (10 µL, 20 mM solution in ethanol), with a 10
min incubation at room temperature. PK-digested PrPSc was
precipitated by the addition of PTA (110 µL, 7.5% solution in 64.75
mM MgCl2, pH 7.4). Plates were incubated overnight at 37 °C and
then centrifuged at 2200 rpm for 60 min using a Beckman-Coulter
GS-6R centrifuge. The supernatant was aspirated. The PTA-
precipitated protein pellet was denatured with 6 M guanidine
thiocyanate (55 µL) in coating buffer (55 µL, 0.1 M sodium
bicarbonate, pH 8.6) for 5 min at room temperature with shaking.
The suspension (100 µL) was transferred to Immunlon II ELISA
plates (Fisher Scientific), sealed, and incubated overnight at 4 °C.
ELISA plates were washed twice with TBST (20 mM Tris-HCl,
137 mM NaCl, 0.05% Tween-20, pH 7.5), blocked with 200 µL of
3% BSA as a solution in TBS (20 mM TrisHCl, 137 mM NaCl,
pH 7.5), sealed, and incubated at 37 °C. After 1 h, the ELISA plates
were washed twice with TBST and incubated with 100 µL of anti-
PrP antibody D18 (1 µg mL-1) as a solution in 1% BSA/TBS.
ELISA plates were sealed and incubated at 37 °C for 2 h, then
washed seven times with TBST. Goat antihuman IgG Fab AP
conjugate (100 µL) diluted 1:2500 with 1% BSA/TBS was added
to the plates, sealed, and incubated at 37 °C for 1 h. Plates were
washed seven times with TBST. Plates were developed with
p-nitrophenyl phosphate (100 µL, 1 mg mL-1) as a solution in
alkaline phosphatase buffer (1 M diethanolamine, 0.5 mM
MgCl2‚6H20, pH 9.8). Absorbance at 405 nm was measured using
a microplate reader. Dose-response curves and EC50 values were
computed using SigmaPlot.
2-(2-(Pyrrolidin-1-yl)ethylthio)-6-amino-4-(furan-2-yl)pyridine-
1
3,5-dicarbonitrile (21). H NMR (CD3OD/CD3CO2D) δ 7.88 (s,
1H), 7.50 (d, J ) 3.6 Hz, 1H), 6.75 (dd, J ) 1.6, 3.6 Hz, 1H), 3.58
(m, 6H), 3.48 (m, 2H), 2.12 (m, 4H); LCMS (ESI) m/z 340 (MH+);
HRMS m/z calculated for C17H18N5OS (MH+) 340.1232, found
340.1244; purity method A, 97%, method B, 100%.
2-(2-(Piperidin-1-yl)ethylthio)-6-amino-4-(furan-2-yl)pyridine-
1
3,5-dicarbonitrile (22). H NMR (CD3OD/CD3CO2D) δ 7.88 (d,
J ) 2.0 Hz, 1 H), 7.50 (d, J ) 3.6 Hz, 1H), 6.75 (dd, J ) 1.6, 3.6
Hz, 1H), 3.65 (m, 2H), 3.59 (m, 2H), 3.45 (m, 2H), 3.04 (m, 2H),
1.95 (m, 2H), 1.83 (m, 3H), 1.55 (m, 1H); LCMS (ESI) m/z 354
(MH+); HRMS m/z calculated for C18H20N5OS (MH+) 354.1389,
found 354.1381; purity method A, 95%, method B, 100%.
2-(3-(Piperidin-1-yl)propylthio)-6-amino-4-(furan-2-yl)pyri-
dine-3,5-dicarbonitrile (23). 1H NMR (DMSO-d6) δ 8.10 (d, J )
1.2 Hz, 1H), 7.39 (d, J ) 3.6 Hz, 1H), 6.83 (dd, J ) 1.6, 3.6 Hz,
1H), 3.43 (m, 2H), 3.23 (t, J ) 7.2 Hz, 2H), 3.14 (m, 2H), 2.85
(m, 2H), 2.04 (m, 2H), 1.80 (m, 2H), 1.62 (m, 3H), 1.35 (m, 1H);
LCMS (ESI) m/z 368 (MH+); HRMS m/z calculated for C19H22N5-
OS (MH+) 368.1545, found 368.1537; purity method A, 93%,
method B, 100%.
2-(2-Morpholinoethylthio)-6-amino-4-(furan-2-yl)pyridine-
1
3,5-dicarbonitrile (24). H NMR (CD3OD/CD3CO2D) δ 7.88 (d,
J ) 1.2 Hz, 1H), 7.50 (d, J ) 3.2 Hz, 1H), 6.75 (dd, J ) 2.0, 3.6
Hz, 1H), 3.92 (m, 4H), 3.59 (m, 2H), 3.46 (m, 2H), 3.35 (m, 4H);
LCMS (ESI) m/z 356 (MH+); HRMS m/z calculated for C17H17N5O2-
NaS (MNa+) 378.1001, found 378.1002; purity method A, 87%,
method B, 100%.
2-(2-(Dibutylamino)ethylthio)-6-amino-4-(furan-2-yl)pyridine-
1
3,5-dicarbonitrile (25). H NMR (CD3OD/CD3CO2D) δ 7.88 (s,
1H), 7.50 (d, J ) 3.6 Hz, 1H), 6.75 (dd, J ) 1.6, 3.6 Hz, 1H), 3.61
(m, 2H), 3.52 (m, 2H), 3.25 (m, 4H), 1.72 (m, 4H), 1.45 (m, 4H),
1.01 (t, J ) 7.2 Hz, 6H); LCMS (ESI) m/z 398 (MH+); HRMS m/z
calculated for C21H28N5OS (MH+) 398.2015, found 398.2011; purity
method A, 95%, method B, 100%.
2-(2-(N-Benzyl-N-methylamino)ethylthio)-6-amino-4-(furan-
2-yl)pyridine-3,5-dicarbonitrile (26). 1H NMR (CD3OD/CD3-
CO2D) δ 7.89 (d, J ) 1.6 Hz, 1H), 7.50 (d, J ) 3.6 Hz, 1H), 7.46
(m, 5H), 6.76 (dd, J ) 1.6, 3.6 Hz, 1H), 4.41 (s, 2H), 3.60 (m,
2H), 3.51 (m, 2H), 2.97 (s, 3H); LCMS (ESI) m/z 390 (MH+);
HRMS m/z calculated for C21H20N5OS (MH+) 390.1389, found
390.1385; purity method A, 89%, method B, 100%.
General Procedure B. Synthesis of Thienopyridine Com-
pounds. Thienopyridines 6 were prepared according to the proce-
dure of Dyachenko et al. Briefly, pyridine compounds 5 were treated
with 10% aqueous KOH (1 equiv) in DMF (0.4 mL) for 5 h at the
room temperature. The product was precipitated by the addition of
H2O (0.4 mL) and collected by filtration, washed with ice-cold H2O
(5 mL), ice-cold ethanol (5 mL), and hexanes (5 mL) and dried
under high vacuum. Compounds were obtained in 60-80% yields.
3,6-Diamino-4-furan-2-yl-2-(2-nitrobenzoyl)thieno[2,3-b]py-
1
ridine-5-carbonitrile (28). H NMR (DMSO-d6) δ 8.17 (d, J )
Duplex Solubility and PAMPA Assay. Compound stocks at
10 mM were serially diluted over a concentration range of 500-
3.13 µM in DMSO (final volume 200 µL) in a UV-compatible,
96-well plate (Greiner Bio-One), in triplicate. Absorbance at 320
nm was quantified using a SpectraMax microplate reader and a
standard curve was derived such that r2 > 0.85. Standard curves
for control compounds were determined at an appropriate absorption
maxima. A membrane-embedded solubility plate (Millipore) was
wetted with previously filtered donor buffer (275 µL, 50 mM
8.0 Hz, 1H), 8.12 (bs, 1H), 7.90 (t, J ) 7.2 Hz, 1H), 7.78 (d, J )
8.0 Hz, 1H), 7.75 (bs, 1H), 7.70 (d, J ) 7.2 Hz, 1H), 7.22 (bs,1H),
6.88 (dd, J ) 1.6, 3.6 Hz, 1H); LCMS (ESI) m/z 406 (MH+);
HRMS m/z calculated for C19H12N5O4S (MH+) 406.0610, found
406.0602; purity method A, 69%, method B, 95%.
3,6-Diamino-4-furan-2-yl-2-(2,3-dihydrobenzo[b][1,4]dioxan-
2-oyl)thieno[2,3-b]pyridine-5-carbonitrile (29). 1H NMR (DMSO-
d6) δ 8.10 (s, 1H), 7.66 (bs, 1H), 7.24 (d, J ) 8.4 Hz, 1H), 7.19