2-Amino-aryl-7-aryl-benzoxazoles as potent, selective and orally available JAK2 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.01.069

A series of novel benzoxazole derivatives has been designed and shown to exhibit attractive JAK2 inhibitory profiles in biochemical and cellular assays, capable of delivering compounds with favorable PK properties in rats. Synthesis and structure-activity relationship data are also provided.

Full text of DOI:10.1016/j.bmcl.2010.01.069

Bioorganic & Medicinal Chemistry Letters 20 (2010) 1724–1727
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
2-Amino-aryl-7-aryl-benzoxazoles as potent, selective and orally available
JAK2 inhibitors
*
Marc Gerspacher , Pascal Furet, Carole Pissot-Soldermann, Christoph Gaul, Philipp Holzer,
Eric Vangrevelinghe, Marc Lang, Dirk Erdmann, Thomas Radimerski, Catherine H. Regnier, Patrick Chene,
Alain De Pover, Francesco Hofmann, Fabienne Baffert, Thomas Buhl, Reiner Aichholz, Francesca Blasco,
Ralf Endres, Jörg Trappe, Peter Drueckes
Novartis Institutes for BioMedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel benzoxazole derivatives has been designed and shown to exhibit attractive JAK2 inhib-
itory profiles in biochemical and cellular assays, capable of delivering compounds with favorable PK
properties in rats. Synthesis and structure–activity relationship data are also provided.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 18 December 2009
Revised 6 January 2010
Accepted 7 January 2010
Available online 21 January 2010
Keywords:
Janus kinase
JAK1
JAK2
JAK3
Tyk2
Kinase inhibitors
JAK2 inhibitor
The four Janus kinases (JAK1, JAK2, JAK3, TYK2) are intracellular
protein tyrosine kinases that have important roles in the mediation
of cytokine receptor signaling.1 The discovery of an acquired acti-
vating point mutation in the pseudokinase domain of JAK2 in a
large proportion of patients suffering from chronic myeloprolifera-
tive neoplasms (cMPNs) has drawn a lot of attention to this kinase
during recent years. For the first time there is a molecular under-
standing of the underlying disease mechanism and, equally of
importance, the mutated JAK2 kinase represents a druggable target
for therapeutic intervention. The JAK2 valine 617 to phenylalanine
mutation (JAK2V617F) is found in nearly every patient with polycy-
themia vera (PV) and in approximately every second patient
suffering from essential thrombocythemia (ET) and myelofibrosis
(PMF).2 Patients typically present an increased hematocrit (PV), in-
creased platelet counts (ET), splenomegaly (PV, PMF), and gradual
loss of bone marrow function (PMF). It is hoped that targeting
JAK2V617F with small molecule inhibitors will alleviate these symp-
toms.3 Here, we report the discovery of benzoxazole derivatives as
potent, selective as well as orally available JAK2 inhibitors.
Based on the structure of a pyrrolopyrimidine derivative 14
which was identified as an inhibitor of JAK2 in a focused screen,
application of several cycles of scaffold morphing, and lead optimi-
zation led to the discovery of a series of novel 2-amino-aryl-7-aryl-
benzoxazoles, exhibiting surprisingly favorable JAK2 inhibitory
and selectivity profiles (Fig. 1). A derivatisation program aiming
at variations of the X and Y aryl/heteroaryl residues quickly
showed that a variety of residues is tolerated for potency in JAK2
inhibition (Table 1). This was considered an advantage as it opened
up the possibility to simultaneously optimize potency, selectivity
and pharmacokinetic properties. Initially, the trimethoxy-phenyl
residue at position 2 was kept constant to study the influence of
N
N
HN
X
N
N
O
HN
O
O
Y
N
2 - 17
O
X = aryl
Y = aryl, heteroaryl
1
* Corresponding author. Tel.: +41 61 696 76 36; fax: +41 61 696 62 46.
E-mail address: marc.gerspacher@novartis.com (M. Gerspacher).
Figure 1. Discovery of benzoxazoles.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.01.069

M. Gerspacher et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1724–1727
1725
Table 1
Inhibition of JAK family kinases5 and inhibition of cell proliferation in SET-2V617F cells6 for compounds 2–17
N
O
H
N
2
X
7
Y
Compd
X
Y
JAK2 inhibition JAK1 inhibition JAK3 inhibition TYK2 inhibition IC50 Inhibition of SET-2V617F
IC50
(l
M)
IC50
(lM)
IC50
(
lM)
(l
M)
proliferation (cellular assay),
GI50 M)
(
l
O
O
N
2
3
4
0.11
0.76
0.45
0.64
3.4
—
O
O
O
O
O
O
0.015
0.44
0.71
5.3
>10
>10
—
—
O
O
>10
Cl
O
O
5
0.84
8.9
>10
>10
—
OMe
N
O
O
6
7
8
0.008
0.15
0.21
0.68
0.67
2.0
1.4
0.50
—
O
O
O
3.6
NHSO2Me
O
O
O
O
0.005
0.047
0.13
0.15
0.13
NHSO2Me
O
O
9
10
11
12
0.008
<0.003
0.014
0.039
0.0275
0.4
0.11
0.063
0.31
0.73
0.16
0.15
0.42
—
O
O
O
O
F
N
O
O
O
0.022
0.082
0.18
—
O
N
O
O
O
0.30
0.62
N
O
N
O
O
N
O
(continued on next page)

1726
M. Gerspacher et al. / Bioorg. Med. Chem. Lett. 20 (2010) 1724–1727
Table 1 (continued)
Compd
X
Y
JAK2 inhibition JAK1 inhibition JAK3 inhibition TYK2 inhibition IC50 Inhibition of SET-2V617F
IC50
(l
M)
IC50
(lM)
IC50
(l
M)
(lM)
proliferation (cellular assay),
GI50
(lM)
N
N
13
0.008
0.005
0.005
0.008
0.005
0.13
—
—
0.49
O
O
O
O
F
N
O
F
14
15
16
17
0.14
0.16
0.19
0.17
0.22
0.20
0.12
0.3
0.20
0.26
0.20
0.41
N
N
O
O
H
N
F
F
0.048
0.067
0.035
N
N
N
O
N
O
H
N
F
F
0.097
O
N
O
various aryl moieties at position 7. A simple phenyl residue (3)
leads to a fairly potent but insoluble and very lipophilic JAK2 inhib-
itor, whereas addition of meta-chloro (4) or para-methoxy groups
(5) leads to a decrease in activity. In order to make compounds
more soluble, polar residues were introduced that are best toler-
ated at the para position as the comparison of compounds 7 and
8 illustrates (additional examples: 6, 9–11). Compound 8 also
shows very good inhibition of SET-2V617F cell proliferation.
Replacement of the trimethoxy-phenyl group with benzamide or
piperazine-phenyl residues also leads to potent and cellularly ac-
tive compounds that can be combined with a variety of 7-aryl moi-
eties (compounds 12–17). It is noteworthy that the introduction of
a methyl group ortho to the carboxamide residue leads to an in-
crease in potency (12 compared to 13). The introduction of 1 (or
2) fluorine atoms to the 7-aryl residue leads to better JAK2 inhibi-
tory activity (9 vs 10). While in general there is relatively favorable
selectivity of the compounds for JAK2 versus the other JAKs, there
is less of an influence of the substitution pattern on the selectivity
factors per se. Apparently, a substitution pattern that leads to an
increase in JAK2 inhibition also leads to increased inhibition of
the other JAKs.
followed by a reaction of the latter with bis(pinacolato)-diboron
to produce the boronic acid derivative 20. The aniline building
block 22 was prepared in a three steps reaction sequence starting
from commercially available 2-methyl-4-nitro benzoic acid (21) as
outlined in the Scheme. The nitro group of 2-bromo-6-nitro-phenol
(23, commercially available) was reduced and the resulting
hydroxyl-aniline derivative 24 was reacted with potassium ethyl
xanthogenate, followed by methylation of the thiol residue to
produce 7-bromo-2-methylsulfanyl-benzoxazole (25). Activation
of the sulfur leaving group of 25 with mCPBA, followed by reaction
with 22 led to 4-(7-bromo-benzoxazol-2-ylamino)-2,N-dimethyl-
benzamide (26). Suzuki coupling of 26 with 20 led to the final
product 4-[7-(3,5-difluoro-4-morpholin-4-yl-methyl-phenyl)-ben-
zoxazol-2-ylamino]-2,N-dimethyl-benzamide (15).
Table 2
Pharmacokinetic properties of 15 in ratsa
Parameter
Mean SD
15
2.3 0.4
3.2 1.7
2391 562
2435 695
3.5 1.9
CL (mL minÀ1 kgÀ1
VSS (L/kg)
)
4
t1/2 term (h)
AUC, iv d.n. (nmol h LÀ1
)
)
AUC, po d.n. (nmol h LÀ1
Tmax (h)
The preparation of the benzoxazole derivatives is relatively
straightforward, convergent and allows introduction of the various
substitution patterns in the last two steps. The synthesis is exem-
plified with the preparation of 15 as shown in Scheme 1. Starting
from commercially available 5-bromo-2-bromomethyl-1,3-di-
fluoro-benzene (18), the morpholine derivative 19 was prepared
Cmax d.n. (nM)
BAV (%)
242 33
102 29
d.n.: dose normalized to 1 mg/kg.
a
Mean of four animals.

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.