Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7815
were carried out on Shimadzu LC-MS 2010 EV system. Chemi-
cal purity for all target compounds was determined by HPLC
with a C18 column (Phenomenex Luna 5 μ C18(2) 100A,
250 mm ꢀ 4.6 mm), detected by ELSD and MS, confirming
g95% purity.
2-(4-Nitrophenyl)imidazo[2,1-b][1,3]benzothiazol-7-ol (3b):
General Procedure A. A mixture of 2-amino-1,3-benzothia-
zol-6-ol (20.0 g, 0.12 mol) and 2-bromo-40-nitroacetophenone
(29.3 g, 0.12 mol) in 600 mL of ethanol was heated to reflux
overnight. The reaction mixture was cooled to 0 °C in an
ice-water bath. Filtration of the resulting precipitate pro-
vided 2-(4-nitrophenyl)imidazo[2,1-b][1,3]benzothiazol-7-ol
(3b) as a yellow solid (17.0 g, 46% yield). 1H NMR (DMSO-
d6) δ 10 (s, 1H), 8.9 (s, 1H), 8.3 (d, J = 8.6 Hz, 2H), 8.1 (d, J =
8.6 Hz, 2H), 7.8 (d, J = 8.7 Hz, 1H), 7.4 (s, 1H), 6.9 (d, J = 8.7
Hz, 1H). LC-MS (ESI) m/z 312 (M þ H)þ.
General Procedure E for Preparation of Hydrochloride Salt.
The free base was dissolved in a mixture of dichloromethane
(20 mL) and methanol (1 mL). A solution of 1.0 M HCl in ethyl
ether (1.1 equiv for all compounds except 7, for which 2.5 equiv
were used) was added dropwise, followed by addition of ethyl
ether. The precipitate was collected by filtration to give N-(5-
tert-butyl-isoxazol-3-yl)-N0-{4-[7-(2-morpholin-4-yl-ethoxy)-
imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea dihydrochloride
(7) (2.441 g, 98%). 1H NMR (DMSO-d6) δ 11.0 (br, 1H), 9.68 (s,
1H), 9.26 (s, 1H), 8.66 (s, 1H), 7.93 (d, J=8.9 Hz, 1H), 7.78 (m,
3H), 7.53 (d, J=8.7 Hz, 2H), 7.26 (dd, J=2.4 and 8.9 Hz, 1H), 6.53
(s, 1H), 4.50 (t, J=4.1 Hz, 2H), 3.97 (m, 2H), 3.81 (t, J=12.1 Hz,
2H), 3.6 (overlapping, 4H), 3.23 (m, 2H), 1.30 (s, 9H). LC-MS
(ESI) m/z 561 (M þ H)þ. Anal. (C29H32N6O4S 2HCl) C, H, N. C:
3
calcd 54.97; found 54.54. H: calcd 5.22; found 5.87. N: calcd 13.26;
found 13.16.
7-(2-Morpholin-4-yl-ethoxy)-2-(4-nitrophenyl)imidazo[2,1-b][1,3]-
benzothiazole (18c): General Procedure B. To a mixture of 2-(4-
nitrophenyl)imidazo[2,1-b][1,3]benzothiazol-7-ol (3b) (4.67 g,
15.0 mmol) in DMF (100 mL) was added potassium carbonate
(5.52 g, 40 mmol) and 4-(2-chloroethyl)morpholine hydrochloride
(3.72 g, 20 mmol). The mixture was heated to 60 °Cfor6hand70°C
for 12 h. The reaction mixture was poured into water (400 mL),
filtered, and washed with water and ethyl ether to give 7-(2-
morpholin-4-yl-ethoxy)-2-(4-nitrophenyl)imidazo[2,1-b][1,3]-
benzothiazole (18c), which was used in the next reaction without
further purification. 1H NMR (DMSO-d6) δ 9.00 (s, 1H), 8.31 (d,
J=8.1 Hz, 2H), 8.09 (d, J=8.1 Hz, 2H), 7.92 (d, J=8.8 Hz, 1H),
7.72 (d, J=1.5Hz, 1H), 7.2(dd, J=8.8 and 1.5Hz, 1H), 4.17 (t, J=
5.4 Hz, 2H), 3.59 (t, J = 4.2 Hz, 4H), 3.34 (overlapping
with solvent, 4H), 2.73 (t, J =5.4 Hz, 2H). LC-MS (ESI) m/z
425 (M þ H)þ.
Kinase Competition Binding. KinomeScan competition bind-
previously.13,15 Kinases were produced displayed on T7 phage
or by expression in HEK-293 cells and tagged with DNA,
binding reactions were performed at room temperature for
one hour, and the fraction of kinase not bound to test compound
determined by capture with an immobilized affinity ligand and
quantitation by quantitative PCR.
MV4-11 Cell Proliferation. MV4-11 cells were cultured in
Iscove’s media with 10% FBS and RPMI complete with 10%
FBS, respectively. For proliferation assays, cells were cultured
overnight in low serum media (0.5% FBS) and then seeded in a
96-well plate at 40000 cells per well. Inhibitors were added to the
cells and incubated at 37 °C for 72 h. Cell viability was measured
using the Cell Titer-Blue cell viability assay from Promega
(Madison, WI).
2-(4-Aminophenyl)-7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b]-
[1,3]benzothiazole (19c): General Procedure C. A mixture of 7-(2-
morpholin-4-yl-ethoxy)-2-(4-nitrophenyl)imidazo[2,1-b][1,3]-
benzothiazole (18c) (15.0 mmol) and ammoniumchloride(2.14g,
40 mmol) in ethanol was heated to reflux, and then iron powder
(8.378 g, 150 mmol) was added. The mixture was heated to reflux
for 3 h and then immediately filtered through celite and washed
with hot ethanol. The filtrate was concentrated to approximately
1/5 of the original volume, neutralized with saturated NaHCO3
solution, and extracted with dichloromethane. The combined
extracts was dried over MgSO4 and concentrated to give 2-(4-
aminophenyl)-7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b][1,3]-
benzothiazole (19c) as an orange solid (4.067 g, 69% for two
steps). 1H NMR (DMSO-d6) δ 8.40 (s, 1H), 7.86 (d, J=8.3 Hz,
1H), 7.70 (s, 1H), 7.51 (d, J=7.6 Hz, 2H), 7.22 (d, J=8.3 Hz,
1H), 6.61 (d, J=7.6 Hz, 2H), 4.34 (t, J=5.6 Hz, 2H), 3.76 (m,
4H), 2.99 (m, 6H). LC-MS (ESI) m/z 395 (M þ H)þ.
Pharmacokinetics. Precatheterized (jugular vein), male Spra-
gue-Dawley rats (230-300 g; Charles River, Hollister, CA) and
female athymic nude mice (20-35 g; Harlan, Livermore, CA)
were acclimated at the vivarium for at least three days following
delivery and prior to entering a study. Rats were fasted over-
night before dosing. Compounds were administered orally (PO)
by gavage at 10 mg/kg in 22% hydroxypropyl-β-cyclodextrin
(HPBCD) or 0.5% methylcellulose. Blood was collected into
K3EDTA tubes for plasma at 15 min, 30 min, 1 h, 2 h, 4 h, 6 h,
and 24 h postdose. Three rats were used per study, however, for
blood collection in mice 150 μL retro-orbital bleeds were taken
semilongitudinally using nine animals, taking 2-3 time-points
per animal for a total of n = 3 plasma concentration-time
curves. Plasma samples, calibration, and quality control stan-
dards (25 μL) were extracted with four volumes of acetonitrile
containing an internal standard and analyzed using LC-MS/MS
analysis (Sciex 4000 Qtrap). Sample separation was achieved on
a Zorbax SBC8 5 μm column (2.1 or 4.6 mm ꢀ 50 mm) using a
0.8 mL/min flow rate over a 3 min gradient from 5 to 95% ACN
containing 0.05% formic acid. The parent compound to frag-
ment mass transitions were monitored. Pharmacokinetic para-
meters were calculated from the normalized LC-MS/MS peak
areas using a noncompartmental model and the linear trape-
zoidal estimation method with WinNonlin (Pharsight v5.2).
Efficacy in Tumor Xenograft Studies. Xenograft studies were
performed at Piedmont Research Center LLC (Morrisville,
NC). MV4-11 human leukemia cells (1 ꢀ 107) that had been
cultured in Iscove’s Modified Dulbecco’s Medium supplemen-
ted with 10% heat-inactivated fetal bovine serum, 100 units/mL
penicillin G, 100 μg/mL streptomycin sulfate, 0.25 μg/mL
amphotericin B, 2 mM glutamine, 0.075% sodium bicarbonate,
and 25 μg/mL gentamicin were harvested during logarithmic
phase growth and resuspended at a concentration of 5 ꢀ 107
cells/mL in 50% Matrigel matrix (BD Biosciences) and 50%
PBS, and implanted subcutaneously into the right flank of
female athymic nude mice (nu/nu, Harlan). Sixteen days later,
mice were sorted into groups, each consisting of 10 mice with
individual tumor sizes of 126-221 mm3 and group mean tumor
N-(5-tert-Butyl-isoxazol-3-yl)-N0-{4-[7-(2-morpholin-4-yl-
ethoxy)imidazo[2,1-b][1,3]benzothiazol-2-yl]phenyl}urea Dihy-
drochloride (7): General Procedure D. A suspension of
2-(4-aminophenyl)-7-(2-morpholin-4-yl-ethoxy)imidazo[2,1-b]-
[1,3]benzothiazole (19c) (4.06 g, 10.3 mmol) and 5-tert-butyl-
isoxazole-3-isocyanate (5) (1.994 g, 12 mmol) in toluene (60 mL)
was heated at 120 °C overnight. The reaction was quenched with
a mixture of dichloromethane and water containing a little
methanol, and the mixture was neutralized with saturated aqu-
eous NaHCO3. The aqueous phase was extracted twice with
dichloromethane, and the combined organic extracts were dried
over MgSO4 and filtered. The filtrate was concentrated to a
volume of about 20 mL and ethyl ether was added, resulting in
the formation of a solid. The precipitate was collected by
filtration, washed with ethyl ether, and dried under vacuum to
give the free base of 7 (2.342 g, 41%). 1H NMR (DMSO-d6) δ 9.6
(br, 1H), 8.9 (br, 1H), 8.61 (s, 1H), 7.86 (d, J=8.9 Hz, 1H), 7.76
(d, J=8.0 Hz, 2H), 7.69 (d, J=1.3 Hz, 1H), 7.51 (d, J=8.0 Hz,
2H), 7.18 (dd, J=1.3 and 8.9 Hz, 1H), 6.52 (s, 1H), 4.16 (t, J=5.7
Hz, 2H), 3.59 (t, J=4.2 Hz, 4H), 3.36 (overlapping, 4H), 2.72 (t,
J=5.7 Hz, 2H), 1.30 (s, 9H).