M. Gerspacher et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1724–1727
1727
In summary, compounds from a series of novel benzoxazole
derivatives display attractive JAK2 inhibitory profiles in biochemi-
cal and cellular assays. In addition, a selected example (15) was
demonstrated to exhibit favorable PK properties in rats.
Br
Br
O
O
N
a
B
b
F
F
F
F
References and notes
N
F
Br
18
F
O
19
1. (a) Harpur, A. G.; Andres, A. C.; Ziemiecki, A., et al Oncogene 1992, 7, 1347; (b)
Saharinen, P.; Vihinen, M.; Silvennoinen, O. Mol. Biol. Cell. 2003, 14, 1448; (c)
Yamoka, K.; Saharinen, P.; Pesu, M., et al Genome Biol. 2004, 5, 253.
2. (a) James, C.; Ugo, V.; Le Couedic, J.-P., et al Nature 2005, 434, 1144; (b) Levine, R.
L.; Wadleigh, M.; Cools, J., et al Cancer Cell 2005, 7, 673; (c) Valentino, L.; Pierre J.
Biochem. Pharmacol. 2006, 71, 713.
20
NH2
O
N+O
O
c, d, e
3. (a) Vainchenker, W.; Constaninescu, S. N. Am. Soc. Hematol. Educ. Program 2005,
195; (b) Tefferi, A.; Barosi, G.; Mesa, R. A., et al Blood 2006, 108, 1497.
4. (a) Choi, H.-S.; Wang, Z.; Richmond, W.; He, X.; Yang, K.; Jiang, T.; Sim, T.;
Karanewsky, D.; Gu, X.-J.; Zhou, V.; Liu, Y.; Ohmori, O.; Caldwell, J.; Gray, N.; He,
Y. Bioorg. Med. Chem. Lett. 2006, 16, 2173; (b) Choi, H.-S.; Wang, Z.; Richmond,
W.; He, X.; Yang, K.; Jiang, T.; Karanewsky, D.; Gu, X.-J.; Zhou, V.; Liu, Y.; Che, J.;
Lee, C. C.; Caldwell, J.; Kanazawa, T.; Umemura, I.; Matsuura, N.; Ohmori, O.;
Honda, T.; Gray, N.; He, Y. Bioorg. Med. Chem. Lett. 2006, 16, 2689.
5. Detailed description of the enzyme assays: Gerspacher, M.; Furet, P.;
Vangrevelinghe, E. PCT Int. Appl. WO2008031594, 2008; Chem. Abstr. 2008,
148, 379605.
O
NH
22
O
OH
Br
21
OH
O
OH
N+
f
H2N
Br g, h
N
O
O
S
6. Human megakaryoblastic SET-2 cells (number ACC608, DSMZ, Braunschweig,
Germany) were cultured in standard RPMI medium supplemented with 10% of
Br
23
24
25
fetal calf serum (FCS), 2 mM
L-glutamine and 1% (v/v) penicillin/streptomycin.
To determine the anti-proliferative activity of candidate JAK2 inhibitors, SET-2
cells were incubated for 72 h with an eight point concentration range of
compound and cell proliferation relative to DMSO vehicle control treated cells
was measured using the colorimetric WST-1 (catalogue number 1644807, Roche
Diagnostics GmbH, Penzberg, Germany) cell viability readout. Of each triplicate
treatment the mean was calculated and these data were plotted in XLfit 4 (XLfit
four curve fitting software for Microsoft Excel, ID Business Solutions Ltd,
Guildford, Surrey, United Kingdom) to determine the respective GI50 values.
7. Pharmacokinetic properties were determined in conscious, fed, permanently
cannulated female rats. For po administration by gavage, aq solution of 15 in
citrate buffer pH 3 was used as a vehicle (2.5 mL/kg), whereas for the iv route
15 was administered into the femoral vein as a solution in NMP (30%) in PEG
N
N
O
H
H
22
20
N
N
O
j
i
Br
O
O
F
F
NH
NH
N
26
15
O
200 (0.5 mL/kg). Blood samples (approx. 70
femoral artery for 48 h after iv administration and for 24 h after oral dosing.
Iv and po administration was in the same animals with 48 h washout
period between administrations (cross-over design). After CH3CN precipitation
of blood samples (50 L), dried residues were re-dissolved in methanol/water,
lL) were collected from the
Scheme 1. Preparation of 15. Reagents and conditions: (a) morpholine, Et3N, DMF;
(b) bis-(pinacolato)-diboron, Pd(dppf)Cl2, CH2Cl2, KOAc, DMA, 80 °C; (c) (COCl)2,
DMF (cat.), CH2Cl2; (d) MeNH2 (2 M in THF), CH2Cl2; (e) H2, Pd/C, MeOH, THF; (f) Ra-
Ni, MeOH, THF; (g) potassium ethyl xanthogenate, EtOH; (h) MeI, K2CO3, DMF; (i)
mCPBA, CH2Cl2, rf; (j) Pd(PPh3)4, K3PO4, 1,2-dimethoxyethane, 100 °C.
a
l
separated by HPLC on a C18 reversed-phase HPLC column, followed by MS/
MS analysis on a triple quadrupole mass analyzer (Finnigan TSQ Quantum).
The compound was detected as a fragment of its protonated quasi-molecular
ion [M+H]+. A structurally closely related compound was used as analytical
internal standard. Quantification of blood levels of the parent compound was
based on a seven-level calibration curve (in triplicate) using blank rat blood
samples spiked with stock solutions of external and internal standards.
Based on its most attractive broader overall in vitro and physi-
cochemical profile, 15 was selected for pharmacokinetic property
assessment.7 As shown in Table 2, 15 has a low clearance and
low volume of distribution at steady state (VSS) and a terminal
t1/2 of 3.2 h after iv administration to rats. After po administration,
15 exhibits a high Cmax, a moderately late Tmax and a very high oral
bioavailability. Profiling of 15 in a variety of animal models is still
ongoing and will be published in due course.
Pharmacokinetic parameters were estimated using
a non-compartmental
approach. AUCs iv and po were calculated using the trapezoidal rule, then
extrapolated to infinity using the terminal half-life calculated by log-linear
regression from the last three (measurable) blood levels after iv
administration.