Synthesis and structure-activity relationship of aminopyridines with substituted benzoxazoles as c-Met kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2012.04.083

A series of hydroxybenzoxazole derivatives was synthesized, and their c-Met kinase inhibitory activity was evaluated. Described herein is a potent c-Met inhibitor by structural modification of the parent benzoxazole scaffold, with particular focus on the hydroxyl substituent of the benzoxazole moiety.

Full text of DOI:10.1016/j.bmcl.2012.04.083

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4044–4048
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure–activity relationship of aminopyridines
with substituted benzoxazoles as c-Met kinase inhibitors
Jongkook Lee a,b, Sun-Young Han a,c, Heejung Jung a, Jeon Yang a,d, Jie-Won Choi a,e, Chong Hack Chae a,
Chi Hoon Park a, Sang Un Choi a, Kwangho Lee a, Jae Du Ha a, Chong Ock Lee a, Jae Wook Ryu a,
Hyoung Rae Kim a, Jong Sung Koh a, Sung Yun Cho a,
⇑
a Bio-Organic Science Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600, Republic of Korea
b College of Pharmacy, Kangwon National University, Chuncheon-si, Kangwon-do 200-701, Republic of Korea
c College of Pharmacy and Research Institute of Pharmaceutical Sciences, Gyeongsang National University, 816 Gil 15, Jinju-daero, Jinju, Gyeongnam 660-751, Republic of Korea
d Department of Chemistry, Chungnam National University, Yuseong, Daejeon 305-764, Republic of Korea
e Department of Chemistry, Sogang University, 1 Sinsoo-Dong, Mapo-Koo, Seoul 121-742, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of hydroxybenzoxazole derivatives was synthesized, and their c-Met kinase inhibitory activity
was evaluated. Described herein is a potent c-Met inhibitor by structural modification of the parent benz-
oxazole scaffold, with particular focus on the hydroxyl substituent of the benzoxazole moiety.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 6 December 2011
Revised 10 April 2012
Accepted 17 April 2012
Available online 25 April 2012
Keywords:
Cancer
c-Met kinase
Aminopyridine
Benzoxazole
c-Met (mesenchymal-epithelial transition factor) and its ligand
hepatocyte growth factor (HGF),1 also called scatter factor (SF), are
attractive targets for cancer therapy.2 Dysregulation of c-Met sig-
naling has been strongly implicated in invasive growth and tumor-
igenic transformation. Binding of HGF to the extracellular domain
of c-Met results in the dimerization and phosphorylation of the
tyrosine residues in the intracellular domains.3 Tyrosine phosphor-
ylation of the c-Met juxtamembrane, catalytic, and cytoplasmic do-
mains regulates the internalization, catalytic activity, and docking
of regulatory substrates, which possibly plays a role in oncogenic
signaling cascades such as MAPK, PI3K, and STAT pathways.4 The
multifunctional binding sites of c-Met trigger a broad spectrum
of biological responses such as cell proliferation, migration, and
invasion.5 Blocking of the c-Met signaling pathway reduces tumor
cell growth and invasion.6 Activating mutations, gene rearrange-
ments, bypass mechanisms, and interactions with other receptor
families are major key factors for acquiring resistance to Met
inhibitors.7
structural modification of the parent benzoxazole scaffold, with
particular focus on the hydroxyl substituent of the benzoxazole
moiety. Here, we present the results of our studies on the
structure–activity relationship (SAR) of modified benzoxazoles.
A series of benzoxazole derivatives were synthesized, and their
biological activity was evaluated. Chlorobenzoxazole derivative 1
was synthesized from 3 (or 4)-chloro-2-aminophenol in place of
aminophenol derivatives as the same method previously describe-
d.8a Chlorobenzoxazole derivatives of 1 were reacted with aryl bro-
mide by a palladium-catalyzed cross-coupling reaction to afford 2,
followed by treatment with trifluoroacetic acid (Scheme 1). Vari-
ous alkylamine and alkoxy derivatives were synthesized by the
Pd-catalyzed cross-coupling reaction, followed by treatment with
trifluoroacetic acid.
Hydroxybenzoxazole derivatives of 6 were synthesized by
Pd-catalyzed hydroxylation of the corresponding chlorobenzoxa-
zole derivatives of 1, followed by treatment with trifluoroacetic
acid. Aromatics were introduced by following a previously pub-
lished method,9 which involved Pd-catalyzed heteroarylation of
the corresponding hydroxybenzoxazoles of 4 to afford a C-aryla-
tion product 7 (Scheme 2).
Recently, we have described c-Met inhibitors with a benzoxaz-
ole scaffold, and presented their biological activity.8 As a part of
our research, we identified strongly potent c-Met inhibitors by
Introduction of an amine to the benzoxazole was easily
accomplished by starting with the aminonitrophenol to afford 8.
A series of aminobenzoxazole derivatives of 10 were synthesized
⇑
Corresponding author. Tel.: +82 42 860 7077.
E-mail address: sycho@krict.re.kr (S.Y. Cho).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.083

J. Lee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4044–4048
4045
tBuHN
N
N
NH2
N
NH2
N
XR1
R
Ar
O
O
O
N
N
i, ii
iii, iv
nTFA
nTFA
(R = Cl)
(R = Cl)
N
N
N
N
N
N
(X = N or O)
NBoc
NH
NH
.
2
1
3
(R = Cl, acetyl, aryl, heteroaryl)
Scheme 1. Reagents and conditions: (i) Pd(PPh3)4, Na2CO3, ArB(OH)2, DMF, 120 °C, 65%; (ii) TFA, rt, >95%; (iii) R1NH2 or R1OH, Pd2(dba)3, X-Phos, NaOBut, toluene, 100 °C,
67%; and (iv) TFA, 60 °C, >95%.
Modifications to the pyrazole moiety were accomplished by
cross coupling 16 with suitable pyrazole derivatives. Treatment
of 17 with trifluoroacetic acid afforded 18 (Scheme 5).
tBuHN
N
N
OH
iii
tBuHN
N
N
OH
O
O
Ar
i
The in vitro c-Met kinase inhibitory activity was tested for all
the compounds. As an initial assessment of the SAR focusing on
the phenyl ring of the benzoxazole, select functional groups such
as halogens, amines, and heteroaryl-oxy were introduced into the
phenyl ring; the results are summarized in Table 1. We first exam-
ined the effect of halogen substitution on the benzoxazole phenyl.
Introduction of chlorine or fluorine (1b, 1c) to the R2 or R4 position
of benzoxazole did not significantly increase the inhibitory activity,
compared to the compounds not subjected to substitution (1a).
Substitution of alkylamines (3a, 3b, 3d) at the benzoxazole ring
generally resulted in decreased inhibitory activity compared to the
compounds not subjected to substitution (1a).10 Loss of potency
was observed with acetyl (1d) and 1-hydroxyethane (1e) substitu-
tion at R3; these groups should have been equivalent hydrogen
bond acceptor and donor as the hydroxyl group. A small increase
in inhibitory activity was observed when hetero-aromatic com-
pounds (2a, 3f, 3g), such as pyridines and O-linked pyrimidine
derivatives as hydrogen bond acceptors, were introduced at R3. A
significant increase of inhibitory activity of 2c was observed when
2-pyridine was substituted at R4. A possible explanation for in-
crease was illustrated by pi-stacking interaction of aromatic group
with Y1230 (vide infra).
1
(R = Cl)
N
N
N
N
NBoc
NBoc
ii
4
5
ii
OH
NH2
N
OH
NH2
N
O
N
Ar
nTFA
O
N
nTFA
N
N
N
N
NH
NH
6
7
Scheme 2. Reagents and conditions: (i) Pd2dba3, KOH, H2O, n-Bu3P, 1,4-dioxane,
75%; (ii) TFA, 60 °C, >95%, (iii) ArBr, Pd2dba3, X-Phos, K3PO4, toluene, 100 °C, 13 h,
45%.
A systematic study of the benzoxazole revealed that inhibitory
activity dramatically improved when a hydroxyl group was intro-
duced (Table 2). Introduction of a hydroxyl at the R2 and R3 of
benzoxazole (6a, 6b) increased the potency by 2- to 3-folds; how-
ever, addition of the hydroxyl group at the R4 position decreased
inhibitory activity (6c). Significant improvement in potency was
observed when a hydroxyl substituent and a substituent adjacent
to the hydroxyl group were introduced. These compounds exhib-
ited a nano- to pico-molar range of inhibitory activities. Excellent
results were observed when hydroxyl and methyl groups were
by reduction of 8, followed by treatment with trifluoroacetic acid
(Scheme 3).
Next, we investigated the substituent effects of the amine func-
tional group. Ureas 11 were synthesized by treating 9 with the cor-
responding isocyanates. Sulfonyl isocyanates, sulfonyl chlorides,
benzoyl chlorides, and aryl bromides were used for the synthesis
of amides 12, sulfonamides 13, alkylamines 14, and sulfonylureas
15, respectively (Scheme 4).
NH2
N
tBuHN
N
N
tBuHN
N
N
NH2
nTFA
NH2
ii
NO2
O
N
O
O
i
.
N
N
N
N
N
N
NH
10
Scheme 3. Reagents and conditions: (i) Pd/C, hydrazine hydrate, EtOH, 2 h, rt, 85%; (ii) TFA, 60 °C, >95%.
NBoc
NBoc
8
9

4046
J. Lee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4044–4048
NH2
N
NH2
N
NH
O
NH
O
O
.
N
NH
S
N
NHR6
O
R6
O
.
iii, ii
vi, ii
i, ii
9
O
N
N
.
N
.
N
iv, ii
v, ii
NH
15
NH
11
NH2
N
NH2
N
NH2
N
NHR6
NH
NH
S
O
R6
R6
O
.
N
O
.
O
N
N
O
O
.
N
.
N
N
.
N
N
.
N
NH
12
NH
14
NH
13
Scheme 4. Reagents and conditions: (i) R6NCO, THF, rt, 65%; (ii) TFA, 60 °C, 85%; (iii) R6SO2NCO, THF, rt, 57%; (iv) R6COCl, NEt3, CH2Cl2, 75%; (v) R6SO2Cl, NEt3, CH2Cl2, 55%;
and (vi) ArBr, Pd(PPh3)4, Na2CO3, PhB(OH)2, DMF, 120 °C, 57%.
tBuHN
N
N
NH2
N
R
tBuHN
N
N
R
O
.
R
O
N
O
B
ii
i
O
O
nTFA
+
N
N
Br
16
Scheme 5. Reagents and conditions: (i) Pd(PPh3)2, Na2CO3, 1,4-dioxane, 61% and (ii) TFA, 60 °C, 83%.
N
N
R5
N
N
R5
R5
17
18
introduced at the R2 and R3 positions of benzoxazole (6d and 6e,
ligand configuration was generated with a good root mean square
displacement (RMSD) of 0.5 Å.
respectively). Interestingly, a hydroxyl group at the R3 position of
benzoxazole significantly improved inhibitory activity (6h), in con-
trast to the results for 6g.
Data from the modeling study suggests that aminopyridine
compounds bind c-Met by many different strong interactions
(Fig. 1). The strong interaction of aminopyridine with c-Met is
attributed to the two hydrogen bonds between the aminopyridine
moiety and the hinge residues M1160 and P1158, as well as to the
partial pi-stacking interactions between the benzoxazole moiety
and Y1230. The nitrogen atom in the piperidine ring makes addi-
tional hydrogen bonds with the backbone carbonyl of K1161.
Hydrophobic interactions of the aminopyridine and benzoxazole
moieties with M1211 also possibly contribute to the tight binding.
Introduction of a hydrogen donating groups in the R2 or R3 [–OH
(6b) or –NH2 (10b) in R3, –OH in R2 (6d, 6e, 6f)] position makes
additional strong hydrogen bonds with the carbonyl oxygen of
D1222 and N1209, which greatly enhances the potency. A small
methyl group adjacent to the hydroxyl group does not make any
significant collisions (6d–6h), but bulky alkyl chains in the R2 posi-
tion make unfavorable interactions with the side chains of A1226
and D1222 in the activation loop, and decrease the potency (3b,
3c). Introduction of aromatic groups at the R4 position (2c, 7a,
7b) makes partial pi-stacking interactions with Y1230, but methyl
groups (6h) give better van der Waals interactions with M1211,
D1164, N1209 and Y1230 giving the best potency.
To investigate the SAR of the substituents at the R4 position of
benzoxazole, we introduced 3-pyridinyl (7a), 2-pyrazinyl (7b),
and phenyl (7c) in place of the methyl group on 6h. No significant
improvement in potency was observed. Introduction of an amine
in place of the hydroxyl group exhibited noticeable potency in-
crease (10a, 10b), which were almost equivalent to the activities
of OH group. We next investigated the size and electronic require-
ments for the formation of H-bond of the moieties at the R3 posi-
tion of benzoxazole (11, 13, 14, 15). Introduction of urea (11),
sulfonylurea (15), and sulfonamide (13a, 13b) in place of the hy-
droxyl group at the R3 position of benzoxazole did not improve
potency.
Preliminary SAR studies were focused on the substituents of
pyrazole for the parent compound 1a (Table 3). N-Substitution of
piperidine with methyl (18a) or N-Me-4-piperidinyl (18b) did
not improve potency. For 18f and 18g, loss of potency was ob-
served with N-substitution of piperidine with acetyl, even if the
hydroxyl group was at the R3 position of benzoxazole.
Modeling studies were performed using the Glide software con-
tained in the Maestro 9.1 software with standard-precision (SP)
options.11 The crystal structure of c-Met (PDB code: 3F6612) was
used with a slight modification in the conformation of Y1230 to
accommodate the bulky substituents at the R4 position. The bound
Selected compounds were tested against Flt3 (Fms-like tyrosine
kinase 3) kinase and in an Hs746T cell proliferation assay; the
results are summarized in Table 4. Flt3 receptor tyrosine kiase, a

J. Lee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4044–4048
4047
Table 1
Table 3
SAR of substituents on benzoxazole
SAR of R4 and R6
R2
5
R1
O
R2
R4
4
3
N
R3
R3
N
NH2
R5
NH2
N
6
R4
N
R5
=
)
(
R5
=
7
(
)
O
N
N
2
N
1
N
R6
N
H
R5
Entry R1
R2
R3
R4
R6
c-Met
Entry
R2
R3
R4
c-Met IC50 (lM)
IC50 lM)
1a
1b
1c
1d
1e
2a
2b
2c
2d
3a
3b
3c
3d
H
H
H
H
H
H
F
H
H
H
H
0.080
0.053
0.041
0.8
0.13
0.028
0.21
0.0016
0.077
0.13
0.16
0.54
Cl
Cl
H
H
H
H
H
H
H
18a
18b
18c
H
H
H
H
H
H
Cl
F
F
H
H
H
Me
1.7
0.24
0.09
N-Me-4-piperidinyl
N-Hydroxyethyl-4-
piperidinyl
N-Acetyl-4-piperidinyl
N-(N,N0-Dimethylacetyl)-
4-piperidnyl
N-Acetyl-4-piperidinyl
N-Acetyl-4-piperidinyl
Hydroxyethyl
Me(CO)
Me(OH)CH
3-Pyridinyl
18d
18e
H
H
H
H
Cl
H
H
Cl
0.22
0.027
3-Pyrazoly
H
H
H
H
H
2-Pyridinyl
4-MeO-Ph
Me2CHNH
H
H
H
18f
18g
18h
H
OH Me
H
H
0.051
0.028
0.053
Me OH Me
NHMe
MeCONH
H
H
H
H
H
H
H
H
H
H
Pyridin-
2-yl
Pyridin-
2-yl
Pyridin-
2-yl
CH3CH2CH2NH
0.15
18i
18j
Me
0.014
0.076
N
N-(N,N0-Dimethylacetyl)-
4-piperidnyl
NH
N
3e
3f
H
H
H
H
H
H
0.063
0.034
0.023
N
N
O
N
N
O
3g
Table 2
SAR of hydroxyl or amine substituents on benzoxazole
R1
R2
R3
N
NH2
N
N
R5
=
)
(
R4
O
N
N
H
R5
Compound R1
R2
R3
R4
c-Met IC50
M)
(
l
Figure 1. A proposed structure for the c-Met complexed with 2c (yellow) and 6h
(apple green): Hydrogen bonding between each compound and c-Met is shown in
green dotted lines.
6a
6b
6c
6d
6e
6f
6g
6h
7a
H
H
H
H
OH
H
H
H
OH
H
Me
Me
Et
OH
OH
OH
H
H
OH
H
H
0.022
0.019
0.11
0.027
0.003
0.009
0.18
OH
up to 30% of cases.13 Compound 2c exhibited potent c-Met and Flt3
enzyme inhibition and weak inhibition of Hs746T proliferation; the
Me OH
H
H
H
H
OH
Me
H
H
H
IC50 values ranged from 0.012 to 0.45
inhibitory activities against Flt3 (IC50 = 4 nM), mutant Flt3
(D835Y, IC50 = 0.3 nM), Ron (IC50 = 0.014 M), and Aurora A (IC50
0.09 M). Compound 6h displayed potent c-Met (IC50 = 0.2 nM)
and Flt3 IC50 = 0.034 M) inhibitory activity, with moderate activity
lM. 6b exhibited potent
Me
3-
Pyridinyl
0.0002
0.004
H
l
=
7b
H
H
OH
2-
0.008
l
Pyrazinyl
l
7c
10a
10b
11
H
H
H
H
H
H
H
H
H
H
NH2
H
OH
H
NH2
PhNH(CO)NH
H
MeSO2NH
4-F-PhSO2NH
4-F-PhNH
4-Me-
Ph
H
H
H
H
H
H
H
H
0.048
0.056
0.014
0.13
in the Hs746T proliferation assay (IC50 = 0.035
lM). A series of
H
Table 4
12
NHCOMe
0.54
SAR for Flt3 and Hs746T assay15
13a
13b
14
H
H
H
H
0.088
0.067
0.41
Compound
c-MetIC50
(l
M)
FLT3 IC50
(
lM)
Hs746T IC50 (lM)
2c
6b
6d
6f
6h
7b
7c
0.0016
0.019
0.027
0.009
0.0002
0.008
0.048
0.014
0.0006
0.004
0.002
0.004
0.034
0.035
0.014
0.0036
0.24
0.45
15
0.045
PhSO2NH(CO)NH
0.012
0.022
0.035
0.18
0.010
3.7
family of PDGFR (platelet-derived growth factor receptor), is consti-
tutively active in many case of acute myeloid leukemia (AML) found
10b

4048
J. Lee et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4044–4048
Ther. 2011, 10, 518; (c) Cepero, V.; Sierra, J. R.; Corso, S.; Hhiso, E.; Casorzo, L.;
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benzoxazole compounds exhibited a low level of oral exposure and
bioavailability (6b, F = 17%, data not shown). To overcome the lack
of pharmacokinetic properties presumably attributed to intrinsic
phenolic properties, we tried to introduce some of bio-isotere14
group instead of hydroxyl group of benzoxazoles, such as pyrazole
(2b), H-bond donor (1e) or acceptor groups (1d, 2c), and other func-
tional groups (13a, 13b, 15). Introduction of functional groups other
than hydroxyl group at R2 and R3 positions could not increase the c-
Met kinase inhibitory activities.
8. (a) Cho, S. Y.; Han, S. Y.; Ha, J. D.; Ryu, J. W.; Lee, J. O.; Jung, H.; Kang, N. S.; Kim,
H. R.; Koh, J. S.; Lee, J. Bioorg. Med. Chem. Lett. 2010, 20, 4223; (b) Han, S.-Y.; Lee,
J. O.; Ahn, S.-H.; Lee, M.-O.; Kang, S.-Y.; Cha, H.-J.; Cho, S. Y.; Ha, J. D.; Ryu, J. W.;
Jung, H. J.; Kim, H. R.; Koh, J. S.; Lee, J. Invest. New Drugs 2012, 30, 518.
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1740.
In summary, we have identified a series of benzoxazoles with a
substituted hydroxyl group as potent c-Met kinase inhibitors. A
series of hydroxyl-substituted benzoxazoles exhibited potent
c-Met and Flt3 dual kinase inhibitory activities, with excellent
inhibition of Hs746T proliferation. Further studies for improving
the pharmacokinetic properties of benzoxazoles in order to in-
crease oral bioavailability and anti-tumor activity are underway
in our research group.
10. Amines (aniline, N-methyl aniline, pyrrolidine, isopropyamine) were
introduced at R1 position of benzoxazole, and the inhibitory activities were
decreased. (data not shown).
11. Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.; Klicic, J. J.; Mainz, D. T.
J. Med. Chem. 2004, 47, 1739.
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Acknowledgments
This work was supported by the Korea Research Institute of
Chemical Technology and the Basic Science Research Program from
NRF (2011-0010374, S.-Y.H.) funded by the government of Korea
(MEST).
15. c-Met and Flt3 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). Flt3 kinase assay was performed as described above
using recombinant Flt3 protein in kinase reaction buffer (250 mM HEPES (pH
7.0), 0.5 mM orthovanadate, 0.05% BSA, 0.1% NaN3, 5 mM MgCl2, 1 mM MnCl2,
1 mM DTT). Reference compounds were included in each assay for plate
uniformity (crizotinib for c-Met and lestaurtinib for Flt3) and select
compounds were subjected to repeat experiments.
References and notes
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