Discovery of aminopyridines substituted with benzoxazole as orally active c-Met kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.05.031

We report the synthesis and biological evaluation of aminopyridines substituted with benzoxazole. The SAR of the aminopyridines was explored to improve the inhibitory activity against c-Met and to decrease hERG affinity. These studies led to the discovery

Full text of DOI:10.1016/j.bmcl.2010.05.031

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4223–4227
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of aminopyridines substituted with benzoxazole as orally active
c-Met kinase inhibitors
Sung Yun Cho, Sun-Young Han, Jae Du Ha, Jae Wook Ryu, Chong Ock Lee, Heejung Jung, Nam Sook Kang,
*
Hyoung Rae Kim, Jong Sung Koh, Jongkook Lee
Bio-Organic Science Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 February 2010
Revised 6 May 2010
Accepted 12 May 2010
Available online 15 May 2010
We report the synthesis and biological evaluation of aminopyridines substituted with benzoxazole. The
SAR of the aminopyridines was explored to improve the inhibitory activity against c-Met and to decrease
hERG affinity. These studies led to the discovery of amide 24 which showed good c-Met inhibitory
potency, low affinity to hERG and favorable pharmacokinetic properties in rats.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
c-Met inhibitor
Aminopyridine
Benzoxazole
hERG
c-Met is a receptor tyrosine kinase and plays important roles in
cell survival, proliferation, migration, and angiogenesis under
physiological conditions. Upon the binding of the hepatocyte
growth factor/scatter factor (HGF/SF), c-Met dimerizes, autop-
hosphorylates and induces activation of a number of downstream
signal transducers such as extracellular signal-regulated kinases
1 and 2 (ERK1/2), signal transducers and activators of transcription
(STAT), phosphoinositide-3-kinase (PI3K), Akt and focal adhesion
kinase (FAK).1–3 c-Met has recently attracted considerable interest
as a therapeutic target based on the discovery that aberrant c-Met
activity leads to the formation of various cancers including lung,
gastric, renal, ovarian, prostate, and liver cancers.2,3 HGF/SF and
epidermal growth factor (EGF) induce synergistic phosphorylation
of c-Met, Akt, and ERK1/2,4 and resistance to an epidermal growth
factor receptor (EGFR) inhibitor (gefitinib) was developed by c-Met
amplification in non-small cell lung cancers.5
Activation of c-Met can be regulated by preventing HGF/SF from
binding to the receptor with antibodies, truncated HGF fragments,
decoy receptors, and ribozyme based treatments, and/or blocking
the active site of the kinase domain with small molecules.2,3 Small
molecule c-Met inhibitors could be used broadly to treat cancer
patients with deregulated c-Met because they inhibit ligand
dependent and independent activation of c-Met. A number of small
molecule inhibitors targeting c-Met have been reported and some
of them reached the clinical trials phase.2 Described herein is a ser-
ies of aminopyridine-based inhibitors of c-Met kinase, which were
substituted with benzoxazole.
The syntheses of 6–11 and 13 are shown in Scheme 1. Nicotinoyl
chloride 1, prepared by a procedure explained in the literature,6 was
coupled with aminophenol 2 to provide benzoxazole 3.7 Substitu-
tion of chloride in benzoxazole 3 with t-butylamine furnished bro-
mopyridine 4.8 Suzuki coupling of bromopyridine 4 with various
boronicacidsorboronateaffordeddiarylsubstitutedpyridine5.9 Re-
moval of the t-butyl group of pyridine 5 by treatment with trifluoro-
acetic acid gave aminopyridines 6–11.10 Piperidine 13 was also
obtained from pyrazole 12 in the same conditions for compounds
6–11. Both the t-butyl and Boc groups of compound 12 were re-
moved by treatment with trifluoroacetic acid simultaneously.
Scheme 2 depicts the syntheses of 16 and 18. Benzoxazole 15,
prepared from commercially available nicotinoyl chloride 14 and
aminophenol 2 like benzoxazole 3, was coupled with boronates
to give pyrazoles 16 and 17. The Boc group of pyrazole 17 was
deprotected to produce piperidine 18 by treatment with hydrogen
chloride solution.
Aminopyridine 19 was obtained by the replacement of chloride
with amines from benzoxazole 3 and converted to 3,5-diaryl-
substituted pyridines 20–22 by Suzuki coupling, as shown in
Scheme 3. Deprotection of the Boc group transformed pyridine
22 to piperidine 23.
The syntheses of compounds 24–33 were described in Scheme
4. Piperidine 13 was coupled with carbonyl chlorides to produce
aminopyridines 24–32. Acetate 32 was hydrolyzed to alcohol 33
by treatment with LiOH.
* Corresponding author. Tel.: +82 42 8607178; fax: +82 42 8607160.
E-mail address: jongkook@krict.re.kr (J. Lee).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.031

4224
S. Y. Cho et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4223–4227
O
Cl
O
Cl
Br
N
H2N
HO
N
Cl
i
N
Cl
+
O
N
N
14
Br
O
N
Br
1
2
3
17
i
iii
N
N
t-BuHN
N
ii
iii
O
N
N
NBoc
iv
O
N
4
Br
15
Br
NH2
N
t-BuHN
N
ii
iv
O
O
N
N
N
N
xHCl
O
N
5
6-11
R
N
R
xHCl
O
N
18
N
N
t-BuHN
N
xHCl
16
NH2
N
O
O
N
NH
N
N
iv
xHCl
12
Scheme 2. Reagents and conditions: (i) Et3N, PPTS, 2-aminophenol (2), xylene, rt,
3 h then reflux 24 h, 46%; (ii) Pd(Ph3P)2Cl2, 1-methylpyrazole-4-boronic acid
pinacolester, dioxane, rt, 30 min then addition of 1 M aqueous Na2CO3, reflux, 3 h,
75%; (iii) Pd(Ph3P)2Cl2, 1-(4-N-Boc-piperidine)pyrazole-4-boronic acid pinacolester,
dioxane, rt, 30 min then addition of 1 M aqueous Na2CO3, reflux, 3 h; (iv) 4 M HCl in
dioxane, MeOH, 0 °C, 3 h, 74% for two steps.
13
N
N
N
N
NBoc
NH
Scheme 1. Reagents and conditions: (i) Et3N, PPTS, xylene, rt, 3 h then reflux 24 h,
45%; (ii) t-BuNH2/DMF (1:1), 70 °C, 72 h, 89%; (iii) Pd(Ph3P)2Cl2, RB(OH)2 or
RB(OR1)2, dioxane, rt, 30 min then addition of 1 M aqueous Na2CO3, reflux, 3 h, 65–
85%; (iv) CF3COOH, 60 °C, overnight then 4 M HCl in dioxane, MeOH, 70–95%.
3
i
To find novel inhibitors targeting ATP-binding sites of c-Met, we
designed several inhibitors based on the X-ray structure of c-Met
complexes with known inhibitors.11–13 Initially aminopyridines
substituted with benzoxazole 6–9 were designed and synthesized
(Table 1). Aminopyridines 7 and 9 showed low micromolar activity
in enzymatic assay in vitro.14 To figure out the structure–activity
relationship (SAR) of aminopyridine 9 we first modified the amino
group in the pyridine ring and found that the amino group was
essential for activity (Table 2). Removal of the amino group de-
creased c-Met inhibitory activity very much. Substitution of one
hydrogen of the amino group with methyl or benzyl group also
abolished the c-Met inhibitory activity. The secondary amino
group may not form bidentate H-bonds with a c-Met hinge residue
due to an intramolecular hydrogen bonding with the N or O of
benzoxazole ring.
Next, we sought to replace the N-methyl group in the pyrazole
ring of aminopyridine 9 with other groups (Table 1). The introduc-
tion of the hydroxyethyl group or pyran in the pyrazole ring (com-
pounds 10 and 11) increased the potency by a few fold. Moreover,
the c-Met inhibitory activity of aminopyridine 9 was improved by
more than one order of magnitude by the substitution of the N-
methyl group with piperidine. To further confirm the importance
of the amino group in the aminopyridines, we prepared piperidine
derivatives 18 and 23. As expected, both compounds 18 and 23
were inactive in a c-Met kinase assay (Table 2).14
RHN
N
N
RHN
N
N
O
ii
O
20 21
,
19
iii
Br
N
N
MeHN
N
N
MeHN
N
N
O
O
iv
xHCl
23
22
N
N
N
N
NBoc
NH
Scheme 3. Reagents and conditions: (i) RNH2/DMF, 70 °C, 72 h, 65–70%; (ii)
Pd(Ph3P)2Cl2, 1-methylpyrazole-4-boronic acid pinacolester, dioxane, rt, 30 min
then addition of 1 M aqueous Na2CO3, reflux, 3 h, 80–95%; (iii) Pd(Ph3P)2Cl2, 1-(4-N-
Boc-piperidine)pyrazole-4-boronic acid pinacolester, dioxane, rt, 30 min then
addition of 1 M aqueous Na2CO3, reflux, 3 h, 91%; (iv) CF3COOH, 60 °C, overnight
then 4 M HCl in dioxane, MeOH, 72%.
Recently some small molecules were withdrawn from the mar-
ket because they induced the prolongation of the QT interval of the
surface electrocardiogram (ECG) by blocking the human ether-a-
go-go-related gene (hERG) potassium channel.15 Blockade of the
hERG channel is a significant hurdle in lead optimization activity
due to its potential cardiac toxicity. Thus, we measured the hERG

S. Y. Cho et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4223–4227
4225
Table 2
SAR of pyridines substituted with benzoxazole
NH2
N
O
N
R1
N
i
13
O
xHCl
N
xHCl
N
N
24-31
R2
ii
N
a
Compound
16
R1
H
R2
c-Met IC50
>10
(lM)
R
O
N
N
N
N
NH2
N
NH2
N
18
H
>10
O
O
N
N
NH
NH
N
N
N
N
N
N
20
21
NHMe
NHBn
>10
>10
iii
32
33
N
N
N
N
N
OH
N
OAc
23
NHMe
>10
O
O
a
For assay conditions, see Ref. 14.
Scheme 4. Reagents and conditions: (i) RCOCl, Et3N, CH2Cl2, 0 °C, 1–4 h then 4 M
HCl in dioxane, MeOH, 70–99%; (ii) AcOCH2COCl, Et3N, CH2Cl2, 0 °C, 3 h, 84%; (iii)
LiOH, THF/H2O (4:1), rt, 5 h then 4 M HCl in dioxane, MeOH, 99%.
suggest that the piperidine group of compound 13 should be
responsible for the high hERG affinity. To improve the inhibitory
potency against c-Met and overcome hERG binding, we modified
the piperidine group of compound 13. N-alkylation of the piperi-
dine group did not increase potency but decreased the hERG affin-
ity 10–30-fold (data not shown). Piperidines substituted with
benzoyl, carbamyl, carbonate, and sulfonyl groups did not reveal
better c-Met inhibitory activity than compound 13 but had less
affinity to hERG (Table 3). Gratifyingly, however, the introduction
of acetyl, substituted acetyl and propanoyl groups improved the
potency and strongly decreased the hERG affinity. Acetamide
2417 showed single digit nanomolar c-Met inhibitory activity and
Table 1
Aminopyridines substituted with benzoxazole: c-Met potency and hERG binding
inhibition
NH2
R
N
O
N
xHCl
no affinity to the hERG channel even at 100 lM.
a
Compound
R
c-Met IC50
M)
hERG inhibition @
Acetamide 24 fits in the ATP-binding site of c-Met very well as
shown in Figure 1. A crystal structure of c-Met complex with
K252a was obtained from the protein data bank (pdb entry;
1R0P).11 Calculations for docking was carried out using LigandFit18
interfaced with Accelrys DiscoveryStudio2.5. The crystal structure
of 1R0P was regenerated very well with À4.58 kcal/mol of binding
energy for K252a. We set the O atom and the H atom in the peptide
backbone of P1158 and M1160 as interaction sites with default
parameters in LigandFit. Acetamide 24 bound c-Met very tightly
(À9.56 kcal/mol) when the same docking parameters were used.
The strong interaction of acetamide 24 with c-Met is attributed
to H-bondings of both the aminopyridine moiety with hinge resi-
dues M1160 and P1158, and the acetyl group with Y1230. Nitrogen
in the piperidine ring may contribute to the tight binding by form-
ing water-bridged H-bonding with Y1230 or N1167. Interestingly,
the binding mode of acetamide 24 is quite different from that of
PF-2341066 which is also a 3,5-disubstituted aminopyridine-based
c-Met inhibitor in clinical trials.9b,13,19 Acetamide 24 interacts with
(
l
10 l
Mb
6
7
8
Ph
4-Pyridyl
3-Pyridyl
>10
5.2
>10
Nc
Nc
Nc
N
N
N
N
9
2.1
1.3
15%
10
Nc
OH
N
N
11
13
0.43
0.08
12%
O
N
N
0.75 l
Md
NH
a
b
c
For assay conditions, see Ref. 14.
For assay conditions, see Ref. 16.
Not tested.
Y1230 by a H-bonding instead of a
2,6-di-chloro-3-fluorophenyl ring of PF-2341066 forms a strong
-stacking interaction with Y1230, a -stacking interaction is not
p-stacking interaction. While
d
p
p
IC50 value.
present between benzoxazole group of acetamide 24 and Y1230.
Preliminary pharmacokinetic studies of acetamide 24 in male
rats demonstrated favorable pharmacokinetic properties (Table 4).
In summary, we have designed and synthesized a series of
aminopyridines substituted with benzoxazole, which targets
affinity of some of the aminopyridines prior to further optimiza-
tion. Piperidine 13 bound to the hERG channel with an IC50 value
of 0.75 l
M, but aminopyridines 9 and 11 did poorly.16 The results

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S. Y. Cho et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4223–4227
Table 3
Table 4
SAR of piperidines
Preliminary pharmacokinetic profile for 24 in rats
Parameter
iva
poa
Cmax
Tmax
t1/2
(
l
g/mL)
0.6
2.7
1.5
2.7
NH2
N
1.0
5.6
1.8
2.4
O
N
AUC (lg h/mL)
CL (L/kg h)
Vss (L/kg)
% F
xHCl
47
a
N
N
Dose, 10 mg/kg (three rats used).
NR
a
Compound
24
R
c-Met IC50
0.003
(lM)
hERG inhibition @10
l
Mb
logical evaluation of the compounds, and the results will be re-
ported in a near future.
Ac
0% @ 100 lM
25
26
0.007
0.140
10%
63%
Acknowledgments
O
Bz
This work was supported by the Korea Research Institute of
Chemical Technology and the National Research Foundation of
Korea (NRF) Grant funded by the government of Korea (MEST)
(R01-2008-000-20205-0).
OMe
27
28
29
0.017
0.060
0.357
0%
O
NMe2
0%
O
O
O
References and notes
NEt2
46%
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OMe
30
31
33
0.058
0.371
0.026
40%
1%
SO2Me
OH
13%
O
a
For assay conditions, see Ref. 14.
For assay conditions, see Ref. 16.
b
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PDB code: 2wgj.
14. c-Met kinase assay: Inhibition of kinase activity against recombinant c-Met
protein was measured using homogeneous time-resolved fluorescence (HTRF)
assays. Recombinant proteins containing c-Met kinase domain were purchased
from Millipore. Optimal enzyme, ATP, and substrate concentrations were
established using HTRF KinEASE kit (Cisbio) according to the manufacturer’s
instructions. Assays are composed of the c-Met enzyme mixed with serially
diluted compounds and peptide substrates in a kinase reaction buffer (250 mM
HEPES (pH 7.0), 0.5 mM orthovanadate, 0.05% BSA, 0.1% NaN3, 5 mM MgCl2,
1 mM DTT). Following the addition of reagents for detection, the Time
Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) signal was
measured using an EnVision multi-label reader (Perkin–Elmer). Dose–
response curves were generated to determine IC50 using Prism version 5.01
(GraphPad).
Figure 1. A proposed structure for c-Met complex with acetamide 24 (yellow). H-
bonding interactions between the compound and c-Met are shown in blue dotted
lines.
15. (a) Jamieson, C.; Moir, E. M.; Rankovic, Z.; Wishart, G. J. Med. Chem. 2006, 49,
5029; (b) Lagrutta, A. A.; Trepakova, E. S.; Salata, J. J. Curr. Top. Med. Chem. 2008,
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Bilodeau, M. T. Curr. Top. Med. Chem. 2008, 8, 1128.
16. hERG binding assay: inhibition profiles were measured in HEK293 cells, which
were stably transfected with a hERG channel construct, by a radioligand
binding assay with [3H]astemizole.
c-Met kinase. Some of the compounds showed good c-Met inhibi-
tory activity, low hERG affinity and good oral bioavailability in rats.
Currently, we are carrying out both in vitro and in vivo pharmaco-
17. Characterization data for acetamide 24: 1H NMR (500 MHz, CD3OD) d 9.06 (d,
J = 2.2 Hz, 1H), 8.39 (d, J = 2.2 Hz, 1H), 8.34 (s, 1H), 8.02 (d, J = 0.6 Hz, 1H), 7.87
(ddd, J = 8.0, 1.2, 0.6 Hz, 1H), 7.78 (ddd, J = 8.0, 0.9, 0.9 Hz, 1H), 7.54 (ddd,

S. Y. Cho et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4223–4227
4227
J = 7.8, 7.8, 1.3 Hz, 1H), 7.49 (ddd, J = 7.7, 7.7, 1.1 Hz, 1H), 4.69–4.63 (m, 1H),
4.57–4.50 (m, 1H), 4.12–4.06 (m, 1H), 3.37–3.31 (m, 1H), 2.87 (ddd, J = 13.0,
18. Venkatachalam, C. M.; Jiang, X.; Oldfield, T.; Waldman, M. J. Mol. Graphics
Modell. 2003, 21, 289.
19. Nishii, H.; Chiba, T.; Morikami, K.; Fukami, T. A.; Sakamoto, H.; Ko, K.; Koyano,
H. Bioorg. Med. Chem. Lett. 2010, 20, 1405.
13.0, 2.6 Hz, 1H), 2.26–2.15 (m, 5H), 2.26–2.15 (m, 5H), 2.10–1.90 (m, 2H); 13
C
NMR (125 MHz, CD3OD) d 171.7, 159.4, 151.7, 151.1, 142.0, 141.9, 137.4, 135.5,
128.2, 127.3, 126.8, 121.5, 120.7, 117.4, 112.1, 111.6, 60.4, 41.7, 33.8, 33.1,
21.2; HRMS (EI) m/z calcd for C22H22N6O2 (M+) 402.1804, found 402.1801.