732
C. Labrière et al. / Bioorg. Med. Chem. 24 (2016) 721–734
(eluent: CH2Cl2/EtOH, 97/3). The product was triturated with
dichloromethane and ethanol to afford 25 as a yellow powder
(630 mg, 89%). TLC: Rf = 0.20 (CH2Cl2/EtOH, 96/4). Mp 225 °C. IR
stirred at room temperature for 30 min, then ketone 28 (180 mg,
0.5 mmol, 1.0 equiv) was added and the stirring was continued at
room temperature for 12 h. The reaction was quenched with satu-
rated ammonium chloride aqueous solution and extracted with
EtOAc. The combined organic layers were washed with water,
brine, dried over MgSO4, filtered on silica and evaporated under
vacuum to afford 14a as a beige solid (100 mg, 71%). IR mmax
mmax (cmꢁ1): 1579 ( N–H). 1H NMR (d6-
mC@O), 2223 (mCN), 3188 (m
DMSO, 300 MHz) d (ppm): 3.81 (3H, s), 6.93 (1H, dd, J = 8.8 Hz,
J = 2.5 Hz, H60), 7.46 (1H, d, J = 8.8 Hz), 7.64 (1H, dd, J = 8.1 Hz,
J = 4.9 Hz), 7.71 (1H, d, J = 2.5 Hz), 8.27 (1H, s), 8.44 (1H, s), 8.32
(1H, d, J = 8.1 Hz), 8.74 (1H, dd, J = 4.9 Hz, J = 1.6 Hz), 9.08 (1H, d,
J = 2.3 Hz), 12.24 (1H, s). 13C NMR (d6-DMSO, 75.5 MHz) d (ppm):
55.3, 103.2, 113.3, 113.4, 113.6, 117.2, 124.0, 126.9, 128.6, 131.5,
136.0, 136.5, 148.7, 151.4, 152.2, 155.9, 180.7; ESI-MS: m/z 304.1
([M+H]+), 326.1 ([M+Na]+), 358.1 ([M+Na+MeOH]+), HRESI-MS:
m/z 326.0916 (calcd for C18H13N3O2Na, 326.0905).
(cmꢁ1): 2201 ( CN). Mp °C. 1H NMR (d6-DMSO, 500 MHz) d
m
(ppm): 3.81 (3H, s), 4.22 (2H, s), 6.11 (1H, s), 6.84 (1H, dd,
J = 8.9 Hz, J = 2.1 Hz), 7.30 (1H, dd, J = 7.6 Hz, J = 4.6 Hz), 7.32 (1H,
m), 7.34 (1H, d, J = 8.9 Hz), 7.68 (1H, d, J = 7.6 Hz), 7.96 (1H, s),
8.39 (1H, d, J = 4.6 Hz), 8.60 (1H, s), 11.71 (1H, s). 13C NMR (d6-
DMSO, 75.5 MHz) d (ppm): 37.2, 56.4, 90.8, 103.3, 113.4, 113.6,
114.0, 120.6, 124.5, 125.6, 131.1, 133.2, 135.3, 136.2, 148.6,
150.2, 155.9, 156.1; ESI-MS: m/z 290.1 ([M+H]+), 312.1 ([M
+Na]+). HRESI-MS: m/z 312.1115 (calcd for C18H15N3ONa+
312.1113).
4.1.2.13. 1-(5-Methoxy-1H-indol-3-yl)-2-pyridin-3-yl-ethanone
(27).
A
mixture of 3-pyridylacetic (2.36 g, 13.6 mmol,
1.0 equiv) in acetic anhydride (12 mL) was heated in a sealed tube
at 85 °C for one hour, then 5-methoxyindole (2 g, 13.6 mmol,
1.0 equiv) was added. The reaction mixture was heated at 85 °C
for 20 min then at 105 °C for 30 min. After cooling to room temper-
ature the mixture was quenched with water and EtOAc. The mix-
4.1.2.16. (2Z,4E)-2-(5-Methoxy-1H-indol-3-yl)-5-(pyridin-3-yl)
penta-2,4-dienenitrile (31).
To a solution of sodium metha-
nolate (66 mg, 1.2 mmol, 1.2 equiv) in anhydrous ethanol (15 mL)
were added, under an argon atmosphere, (5-methoxy-1H-indol-
3-yl)-acetonitrile 7a (190 mg, 1.0 mmol, 1.0 equiv) and, after
30 min stirring, (E)-3-(pyridin-3-yl)acrylaldehyde 30 (150 mg,
1.1 mmol, 1.1 equiv). The reaction apparatus was protected from
light and the mixture heated at 40 °C for 8 h. The reaction was
allowed to cool to room temperature and then, the solvent was
removed under reduced pressure and the crude taken up in ethyl
acetate. The organic layer was washed with water and brine, dried
over MgSO4 and evaporated. The residue was purified by silica gel
flash-column chromatography (eluent: CH2Cl2/EtOH, 99/1 to 98/2)
to afford 31 as an orange powder (90 mg, 29%). TLC: Rf = 0.30 (CH2-
ture was basified to pH
7 with saturated aqueous Na2CO3
solution. The mixture was extracted with EtOAc. The combined
organic layers were washed with brine, dried (MgSO4) and evapo-
rated. The residue was purified by silica gel flash-column chro-
matography (eluent: CH2Cl2/EtOH, 96/4 to 94/6). The residue was
triturated with ethanol and diethyl ether to afford 27 as a beige
powder (1.74 g, 48%). IR mmax (cmꢁ1): 3130 (
mN–H), 1621 (mC@O).
Mp 210 °C. 1H NMR (d6-DMSO, 500 MHz) d (ppm): 3.75 (3H, s),
4.21 (2H, s), 6.85 (1H, dd, J = 8.7 Hz, J = 2.4 Hz), 7.34 (1H, dd,
J = 7.9 Hz, J = 4.7 Hz), 7.38 (1H, d, J = 8.7 Hz), 7.67 (1H, d,
J = 2.4 Hz), 7.73 (1H, d, J = 7.9 Hz), 8.44 (1H, dd, J = 4.7 Hz,
J = 1.5 Hz), 8.51 (1H, s), 8.55 (1H, d, J = 1.5 Hz), 11.95 (1H, s).13
C
Cl2/EtOH, 96/4). Mp 223 °C. IR mmax (cmꢁ1): 2212 (
m
CN). 1H NMR
NMR (d6-DMSO, 75.5 MHz) d (ppm): 42.4, 55.2, 102.9, 112.8,
115.7, 123.2, 126.3, 131.5, 132.1, 134.8, 137.0, 147.4, 150.4,
155.5, 191.7; ESI-MS: m/z 267.0 ([M+H]+). HRESI-MS: m/z
(d6-DMSO, 500 MHz) (ppm): 3.85 (3H, s), 6.90 (1H, dd,
d
J = 8.9 Hz, J = 2.1 Hz), 7.26 (1H, d, J = 15.3 Hz), 7.37 (1H, dd,
J = 15.3 Hz, J = 11.0 Hz), 7.40 (1H, d, J = 8.9 Hz), 7.44 (1H, dd,
J = 7.9 Hz, J = 4.9 Hz), 7.47 (1H, d, J = 2.1 Hz), 7.64 (1H, d,
J = 11.0 Hz), 7.75 (1H, s), 8.08 (1H, d, J = 7.9 Hz), 8.51 (1H, d,
J = 4.9 Hz), 8.73 (1H, s), 11.67 (1H, s). 13C NMR (d6-DMSO,
75.5 MHz) d (ppm): 56.1, 102.6, 108.9, 110.2, 112.8, 113.7, 117.7,
124.5, 127.6, 128.0, 132.5, 132.8, 133.4, 134.2, 135.7, 149.2,
149.7, 155.1; ESI-MS: m/z 302.1 ([M+H]+), 324.1 ([M+Na]+).
HRESI-MS: m/z 302.1296 (calcd for C19H16N3O, 302.1293).
267.1132 (calcd for
16H14N2O2, 0.2 H2O%: C, 71.20; H, 5.38; N, 10.38. Found: C,
71.08; H, 5.45; N, 10.31.
C
16H15N2O+2 267.1134). Anal. Calcd for
C
4.1.2.14. tert-Butyl 5-methoxy-3-[2-(pyridin-3-yl)acetyl]-1H-
indol-1-carboxylate (28). To solution of 27 (1.00 g,
a
3.8 mmol, 1.0 equiv) and (Boc)2O (1.2 mL, 5.6 mmol, 1.5 equiv) in
CH2Cl2 was added DMAP (17 mg, 0.14 mmol, 0.04 equiv). The reac-
tion mixture was stirred at room temperature for one hour The sol-
vent was removed under reduced pressure. The mixture was
extracted with ethyl acetate. The combined organic extracts were
washed with brine and water, dried over MgSO4, filtered and con-
centrated in vacuo. The residue was purified by silica gel flash-col-
umn chromatography (eluent: EtOAc/EtOH, 100/0 to 90/10). The
residue was triturated with diethyl ether to afford 28 as a white
powder (1.28 g, 93%). IR mmax (cmꢁ1): 1731 (mC@O carbamate), 1665
4.2. Biology
4.2.1. General
Competent BL21(DE3)pLysS cells were obtained from NOVA-
GEN. Chromatographic columns are from GE Healthcare. PIPES,
ATP, lysozyme, DNAse, EGTA, PMSF and kanamycin were obtained
from Sigma-Aldrich. IPTG was purchased from Melford.
(m
C@O). 1H NMR (d6-DMSO, 500 MHz) d (ppm): 1.69 (9H, s), 3.79
4.2.2. Measurements of the inhibition of basal and MT-
stimulated MKLP-2 ATPase activities
(3H, s), 4.42 (2H, s), 7.03 (1H, dd, J = 8.9 Hz, J = 2.1 Hz), 7.37 (1H,
dd, J = 7.3 Hz, J = 4.7 Hz), 7.72 (1H, m), 7.73 (1H, d, J = 2.1 Hz),
7.99 (1H, d, J = 8.9 Hz), 8.47 (1H, d, J = 4.7 Hz), 8.53 (1H, s), 8.77
(1H, s). 13C NMR (d6-DMSO, 75.5 MHz) d (ppm): 28.1, 43.1, 55.8,
85.9, 104.5, 114.8, 116.1, 119.1, 123.8, 128.5, 129.9, 131.6, 134.6,
137.9, 148.2, 149.0, 151.2, 157.1, 193.8; ESI-MS: m/z 367.2 ([M
+H]+), 389.1 ([M+Na]+), 755.3 ([2M+Na]+). HRESI-MS: m/z
367.1667 (calcd for C21H23N2O+4: m/z = 367.1658).
MKLP-256-505 (N-terminal residues 56 to 505 of MKLP-2 cover-
ing the entire motor domain) was expressed and purified as previ-
ously described.16 Steady-state basal and MT-stimulated ATPase
rates were measured using the pyruvate kinase/lactate dehydroge-
nase-enzyme linked assay.30 The amounts of MKLP-256-505 were
optimized at 4
assays. Assays were performed in the presence of increasing inhi-
bitor concentrations (0 M to 100 M). All compounds were mea-
lM for basal and 40 nM for MT-stimulated activity
l
l
4.1.2.15. (Z)-3-(5-Methoxy-1H-indol-3-yl)-4-(pyridin-3-yl)-but-
sured at least in triplicate. Paprotrain served as a positive control.
Kinetics measurements were performed using a 96-well Sunrise
photometer (TECAN, Maennesdorf, Switzerland). The data were
analyzed using Kaleidagraph 4.0 (Synergy Software).
2-enenitrile (29).
phonate (156 L, 1.5 mmol, 3.0 equiv) in THF (4 mL) was added
NaH (44 mg, 80%, 1.5 mmol, 3.0 equiv). The reaction mixture was
To a solution of diethyl(cyanomethyl)phos-
l