Design, synthesis and evaluation of highly selective pyridone-based class II MET inhibitors

Article abstract of DOI:10.1016/j.bmcl.2014.05.097

The high incidence of MET oncogene activation in human malignancies has prompted researchers to develop MET inhibitors. As part of our efforts to developing effective and safe therapeutic agents against MET-dependent tumors, a pyridone-based class II MET inhibitor, namely, 1-(4-((2-amino-3-iodopyridin-4- yl)-oxy)-3-fluorophenyl)-N-(4-fluorobenzyl)-4-methoxy-6-oxo-1, 6-dihydropyridine-3-carboxamide (3s), was identified. Knowledge of the binding mode of class II MET inhibitors led to the design of new inhibitors that utilize 2-pyridone to conformationally restrain key pharmacophoric groups within the molecule. Integrated molecular docking and SAR studies resulted in the discovery of a novel class of pyridone MET inhibitors with high potency (IC₅₀ of 0.005 μM) and efficient selectivity (>5000 fold) to VEGFR-2, c-Kit and RET kinases.

Full text of DOI:10.1016/j.bmcl.2014.05.097

Accepted Manuscript
Design, Synthesis and Evaluation of Highly Selective Pyridone-based Class II
MET Inhibitors
Nengfang She, Linsheng Zhuo, Wen Jiang, Xiaolei Zhu, Jia Wang, Zhihui Ming,
Xinge Zhao, Xin Cong, Wei Huang
PII:
S0960-894X(14)00606-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.05.097
BMCL 21711
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 April 2014
14 May 2014
29 May 2014
Please cite this article as: She, N., Zhuo, L., Jiang, W., Zhu, X., Wang, J., Ming, Z., Zhao, X., Cong, X., Huang,
W., Design, Synthesis and Evaluation of Highly Selective Pyridone-based Class II MET Inhibitors, Bioorganic &
Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.05.097
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Design, Synthesis and Evaluation of Highly Selective
Pyridone-based Class II MET Inhibitors
Nengfang Shea, Linsheng Zhuoa, Wen Jianga, Xiaolei Zhua, Jia Wangb, Zhihui Mingb,
Xinge Zhaob, Xin Congb and Wei Huanga,c,*
aKey Laboratory of Pesticide and Chemical Biology, Ministry of Education, College of Chemistry,
Central China Normal University, Wuhan 430079, P.R.China
bJiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, Jiangsu Simcere
Pharmaceutical Co. Ltd, Nanjing 210042, P.R.China
cState Key Laboratory of Agricultural Microbiology, College of Plant Science and Technology,
Huazhong Agricultural University, Wuhan 430070, China
Abstract The high incidence of MET oncogene activation in human malignancies has
prompted researchers to develop MET inhibitors. As part of our efforts to developing
effective and safe therapeutic agents against MET-dependent tumors,
a
pyridone-based class II MET inhibitor, namely,
1-(4-((2-amino-3-iodopyridin-4-yl)-oxy)-3-fluorophenyl)-N-(4-fluorobenzyl)-4-metho
xy-6-oxo-1,6-dihydropyridine-3-carboxamide (3s), was identified. Knowledge of the
binding mode of class II MET inhibitors led to the design of new inhibitors that utilize
2-pyridone to conformationally restrain key pharmacophoric groups within the
molecule. Integrated molecular docking and SAR studies resulted in the discovery of
a novel class of pyridone MET inhibitors with high potency (IC50 of 0.005 µM) and
efficient selectivity (> 5000 fold) to VEGFR-2, c-Kit and RET kinases.
Keywords: MET; Kinase inhibitor; Selectivity; Pyridone
Footnotes
*Address correspondence to this author at the College of Chemistry, Central China Normal
University, No 152, Luoyu Road, Hongshan District, Wuhan 430079, P.R. China; Tel:
+86-27-67867958; E-mail: weihuangwuhan@126.com

MET tyrosine kinase is the only known high-affinity receptor of hepatocyte
growth factor (HGF), also known as scatter factor.1 MET is an attractive target of
therapeutic agents used in cancer treatment2 because MET regulates cell proliferation,
migration, invasion and survival; these processes are integral to cancer progression
and metastasis.
MET inhibitors can be categorized into two classes based on their chemical
structures or binding modes.3 Class I inhibitors bind in a U-shaped conformation to
the ATP-binding site at the entrance of a kinase pocket, wrap around Met1211 and
bind to a hinge block; as a result, specifically inhibit MET kinases, such as crizotinib.
Class II inhibitors bind to a region of MET that extends from the ATP binding site to
Ile1145 near the C-c-spiral block. Compoud cabozantinib (1), which is an example of
a class II MET inhibitor, effectively inhibits the kinase activity of MET, VEGFR-2,
c-kit and RET.4 Studies have suggested that class II inhibitors may be more effective
than class I inhibitors against the mutations that disrupt the binding site of class I
because their binding interactions extend beyond the entrance of the active site of
MET.5
EtO
N
O
a
b
F
NH O
F
O
Block D
F
H
H
Block B
O
Cl
N
N
2
O
O
H2N
N
(BMS-777607)
Block C
O
O
F
H
N
N
1
F
N
n
Block A
(cabozantinib)
O
O
O
Cl
3
H2N
N
Figure 1. Representative MET inhibitors (1, 2) and designed compounds (3)
However, class II MET inhibitors are correlated with many off-target effects of
other protein kinases. Although these agents have been generally well characterized,
clinical use has shown that these agents elicit unexpected and serious toxic effects in
various organs. Moreover, many side effects of class II MET inhibitors occur because
these inhibitors exhibit potent but non-specific obstruction of vascular endothelial
growth factor function.3b Hence, novel MET inhibitors with improved selectivity
profiles, particularly to VEGFR-2, and minimal side effects should be developed.

The molecular structure of the class II MET inhibitors can be divided into four
blocks known as A, B, C and D (Figure 1).3 Analyzing the crystal structure of
foretinib (PDB code, 3LQ8) and MET kinase complex, we found that the chain
structure of the block C assumed a pseudocyclic conformation. Therefore, various
mono-ring structures, including pyrazole,6 imidazole7 and pyridine8-10 can be
successfully used to replace the amide linker of blocks B and C via cyclization
strategy a, also known as 2 (BMS-777607).8 In the crystal structure complex of 2 and
MET kinase (PDB code, 3F82), block C also adopted a nearly co-planar conformation
with maximum deviation from planarity of 0.063 Å in the nitrogen atom of
3-carboxamide group. We proposed that a 2-pyridone ring could be used to modify
block C based on the cyclization of the amide linker of blocks B and C (strategy b).
The planarity of block C could be conserved to maintain the molecular conformation.
The carbonyl groups of 2-pyridone ring and carboxyamide may function as potential
H-bond acceptors to retain interactions with key residues in the binding pocket.
Liu et al.5,6 designed a pyrazolone-based MET inhibitor called AMG-458 highly
selective (2420 fold) tor VEGFR-2. However, AMG-458 binds covalently to rat and
human liver microsomes in the absence of NADPH and forms thioether adducts in
vitro and in vivo after aryloxy-quinolines (blocks A and B) are introduced.3a,11
However, 2 (BMS-777607) exhibits efficient druggability and modest selectivity
(46 fold) to VEGFR-2. Therefore, we postulated that block C could be modified and
blocks A and B could be reserved in 2 (BMS-777607); such processes may be
effective strategies that could be used to discover highly selective class II MET
inhibitors with efficient druggability (Figure 1). Hence, we disclosed the
design and discovery of a novel class of pyridone MET inhibitors 3 with good
potency and high selectivity.

O
O
CO2Me
Method A
7
O
R1
O
O
R1
O
F
F
O
R1
CO2H
H
N
H
N
ii), iii)
F
N
F
N
F
NH2
F
N
n
n
F
NH2
Cl
O
O
O
O
HO
v)
iv)
5
X
X
X
X
X
H2N
i)
H2N
H2N
H2N
N
N
N
6
N
8
N
H2N
N
9
3a-g and 3o-t
O
O
O
O
MeO
OMe
10
4
O
O O
R1 = H, OMe, OEt, -OCH2CH=CH2, -O(CH2)2OCH3
vi), iii)
Scheme 1. Reagent and conditions: i) t-BuOK, DMF, 50oC for 6 h, 76-82%; ii) DMAP, Molecular
sieves, MeOH, 60oC for 2 h, 81-85%; iii) NaOH, MeOH, H2O, 50oC for 2 h, 86-92%; iv) BtOH,
EDCI, DMAP, DCM, 25oC for 12 h, 54-85%; v) PhI(OAc)2, EA, DCM, H2O, 0oC for 2 h, 65-83%;
vi) EtOH, 70oC for 2 h, 86-90%.
Considering the order at which different blocks were introduced, we selected two
kinds of synthetic methods to synthesize the target compound 3. Method A was
utilized when blocks A and B exhibited a benzoxy-pyridine structure. The key
carboxylic acid fragment (8) was prepared by subjecting chlorinated pyridine (4) to
nucleophilic substitution with 4-aminophenol (5), cyclization with 712 or 10,13 and
hydrolysis. Compounds 3a–g and 3o–t were synthesized on the basis of Hofmann
degradation (Scheme 1) by performing the condensation of carboxylic acid 8 and
diverse amine.
Method B
R1
O
R1
O
7, i)
iii)
F
NH2
F
N
F
N
CO2H
CO2Me
HO
HO
HO
12
10, ii)
11
5
iv)
Cl
R1
H
N
R2
R3
F
O
Y
R1
H
N
F
O
F
N
N
14
F
HO
N
O
O
O
R2
R3
Y
v)
13
3h-n and 3u-w
N
Scheme 2. Reagent and conditions: i) DMAP, Molecular sieves, MeOH, 60oC for 2h, 81%; ii)
EtOH, 70oC for 2h, 85%; iii) NaOH, MeOH, H2O, 50oC for 2h, 88-95%; iv) BtOH, EDCI,
DMAP, DCM, 25oC for 12h, 62-78%; v) t-BuOK, DMF, 50oC for 1-12h, 65-87%.
Method B was chosen to optimize the structure of block A. The key phenol
fragment (13) was prepared by subjecting 4-amino-2-fluorophenol (5) to cyclization
with 7 or 10, condensation with 4-fluorobenzylamine and hydrolysis. The target
compounds 3h–n and 3u–w were obtained after 13 was subjected to nucleophilic
substitution with chlorinated heterocycle 14 (Scheme 2).

Table 1 MET and VEGFR-2 inhibitory activity of compounds 3a–f
IC50 (µM)
Compds
n
MET
> 25
> 25
> 25
> 25
> 25
0.85
VEGFR-2
0
1
0
1
0
1
-
3a
3b
3c
3d
3e
3f
-
-
-
-
>25
The ability of the synthesized compounds prepared in this study was examined to
inhibit MET and VEGFR-2 activities by using an enzyme assay with a recombinant
kinase domain.15 In Table 1, 1-phenyl-2-pyridone-3-carboxamide derivatives 3a and 3b
and 1-phenyl-2-pyridone-4-carboxamide derivatives 3c and 3d did not inhibit MET
even at the highest concentration of 25 µM. The resulting compound 3f showed a
moderate inhibition of MET (IC50 = 0.85 µM) when the carboxamide group was moved
to 5-position of the 2-pyridone ring and the CH2 group was introduced between the
carboxamide group and block D. This characteristic was inconsistent with the reported
regulation of five atoms in which blocks B and D are usually connected by six chemical
bonds.16 3f did not inhibit VEGFR-2 even at the highest concentration of 25 µM. As a
result, 1-phenyl-2-pyridone-5-carboxamide was used as a potential scaffold to
discover highly selective MET inhibitors.
Table 2 MET inhibitory activity of compounds 3g–w
Compds
block A
A-2
R1
H
H
H
IC50 (µM)
0.39
3g
3h
3i
A-3
0.46
A-4
0.28

A-5
A-7
A-8
A-9
A-10
A-1
A-1
A-1
A-1
A-2
A-2
A-4
A-5
A-6
H
0.43
1.23
0.21
0.57
0.24
0.045
0.55
0.28
0.34
0.005
0.l5
3j
3k
3l
H
H
H
H
3m
3n
3o
3p
3q
3r
3s
OMe
OEt
-OCH2CH=CH2
-O(CH2)2OCH3
OMe
-O(CH2)2OCH3
OMe
3t
0.099
0.21
0.12
0.008
3u
3v
3w
OMe
OMe
1 (cabozantinib)
The initial SAR study of block A was performed. Chlorine atom (3f) was replaced
with an iodine atom (3g) or an alkynyl group (3h, 3i and 3j) in the 3-position; as a result,
a slight increase in MET inhibitory activity (ranging from 1.8 fold increase to 3.0 fold
increase) was observed. The bio-isostere of the pyridine ring with thieno[3,2-b]pyridine
(3l), thieno[3,2-d]pyrimidine (3m) and quinazoline (3n) also resulted in a 1.5 fold to 4.0
fold improvement compared with compound 3f; the substitution of
pyrrolo[2,3-b]pyridine (3k) resulted in a slight decrease in potency.
Figure 2. The bindind mode of 3f (A) and 3o (B) with MET. The blue line respresents a hydrogen
bond. For clarity, only important residues were shown with stick model.
The binding mode of 3f was investigated using an AutoDock 4.2 program to
guide the follow-up structure optimization. Before molecular docking, we
systematically analyzed the PDB structures of MET kinase. The result indicated that

the Glu1227 and Lys1110 residues showed different conformations in different PDB
structures (data not shown). Furthermore, these residues could be roughly grouped
into two conformations. In one of the conformations, a hydrogen bond was formed
between Glu1227 and Lys1110 (PDB code, 3LQ8). In the other conformation,
Glu1227 acquired an opposite conformation and the H-bond was disrupted (PDB code,
3L82). Accordingly, the IC50 values of the ligands in 3LQ8 and 3L82 were 0.5 and 3.9
nM, respectively. In Figure 2A, compound 3f took on a similar binding mode with
class II inhibitors and formed H-bonds with Lys1110, Phe1223 and Met1160.
However, Glu1227 did not form H-bond with Lys1110. To improve the potency, we
proposed that a methoxy group could be introduced to the 4-position of 2-pyridone in
block C; this introduction could regulate the conformations of Glu1227 and Lys1110,
function as a potential H-bond acceptor, and increase the potency of this inhibitor. The
binding mode of 3o is shown in Figure 2B. We observed that Glu1227 indeed formed
an H-bond with Lys1110. The binding energy obtained from the AutoDock was -10.23
kcal/mol and -10.98 kcal/mol for 3f and 3o, respectively. We were intrigued by this
possibility and wondered about the resulting activity of 3o.
In actuality, 3o displayed more potent MET inhibitory activity than 3f (19 fold)
when a methoxy group was introduced to the 4-position of 2-pyridone. However, the
introduction of a group more bulky than a methoxy group decreased the inhibitory
activity at different degrees (3p, 3q and 3r). Furthermore, the SAR study of block A
was performed; 3s (IC50 of 0.005 µM) was substituted with an iodine atom in the
3-position of the pyridine ring and exhibited a 9 fold increase in potency compared with
3o and comparable potency to 1 (cabozantinib, IC50 of 0.008 µM). Bulky groups were
introduced to the 3-position of the pyridine ring (3u, 3v and 3w), resulting in the loss of
potency to some extent (2 fold to 42 fold).
Table 3 Kinase selectivity of compounds 3o and 3s
Enzyme, IC50, µM
Compds
MET
0.045
0.005
0.008
VEGFR-2
>25
c-Kit
>25
RET
>25
3o
3s
>25
>25
>25
0.007
0.004
0.016
1 (cabozantinib)

The inhibitory activity of the most potent compounds 3o and 3s against VEGFR-2,
c-Kit and RET kinase were also assayed using the homogeneous time resolved
fluorescence (HTRF) method.14 3o and 3s demonstrated extraordinary selectivity
against VEGFR-2, c-Kit and RET kinase (> 555 fold and > 5000 fold), while compound
1 (cabozantinib) displayed comparable activity against MET, VEGFR-2, c-Kit and RET
kinase without any selectivity.4
Figure 3. The overlay of the binding model of compounds 3o (A) and 1 (B) with MET and
VEGFR-2 kinases, respectively. The yellow and green stick represent MET complex. The magenta
and lightpink stick represent VEGFR-2 complex. The first number of residue represents in MET
kinase and the second number refers to in VEGFR-2 kinase.
The molecular docking method was also used to investigate the selectivity of
compounds 3o and 1. In Figure 3A, 3o formed H-bonds with Lys1110, Asp1222 and
Met1160 in MET kinase and with Asp1046 and Cys919 in VEGFR-2 kinase. 3o
formed an H-bond with Lys1110 in MET kinase and displayed no interactions with
the corresponding Lys868 residue in VEGFR-2 kinase. Compound 1 (cabozantinib)
formed H-bonds with Lys1110, Asp1222 and Met1160 in MET kinase and formed
H-bonds with Asp1046, Glu885 and Cys919 in VEGFR-2 kinase. Therefore, an
important H-bond between 3o and Lys868 was absent in the 3o/VEGFR-2 kinase
complex and this finding is a key factor affecting the selectivity of 3o. For 1, another
H-bond with Glu885 in VEGFR-2 kinase (Glu1227 in MET kinase) could compensate
for the missing of H-bond with Lys868 (Lys1110 in MET kinase), resulting in low
selectivity between MET and VEGFR-2 kinase.
The effects of selected pyridone compounds on cell proliferation were then
preliminarily evaluated in both MKN-45 and HUVEC cells. 3o and 3s elicited

excellent inhibitory effects on the proliferation of HUVEC cells with an IC50 of
2.41µM to11.1 µM; by comparison, lower potency was observed in MKN-45 cells
(IC50 > 25 µM).
We described the design, synthesis and biological screening of a series of
conformationally constrained analogues of compound 1 (cabozantinib). These novel
compounds contained a structure that replaces the acylthiourea moiety with a novel
2-pyridone-5-carboxamide. On the basis of molecular docking results, we found that
the structural modification of block C formed compound 3o that inhibited MET
activity with an IC50 of 0.045 µM. Further structural optimization of block A yielded
compound 3s that potently inhibited MET (IC50 of 0.005 µM); this result was
comparable to the inhibitory activity of compound 1. Furthermore, 3o and 3s showed
excellent selectivities (> 555 fold and > 5000 fold, respectively) to VEGFR-2, c-Kit,
and RET kinase; by comparison, 1 inhibited the three kinases and MET. Therefore, 1
did not exhibit selectivity. The results of molecular docking clearly indicated that 3o
could form H-bond with Lys1110 in MET kinase but not with VEGFR-2 kinase. In
addition, compounds 3o and 3s effectively inhibited the proliferationof HUVEC cells.
The druggability of 3o and 3s is currently evaluated and these two compounds are
also optimized. Research progress will be reported in due course.

Acknowledgments
This work was supported by the National Natural Science Foundation of China
(No. 21272086). The authors would like to thank Prof. Guangfu Yang of Central
China Normal University for conducting molecular stimulation.
References and Notes
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1
confirmed by 1H NMR and LC/MS. Analytic data of represented compounds 3a: H NMR (300 MHz,
DMSO-d6) δ 11.84 (s, 1H), 8.34 (d, J = 7.4 Hz, 1H), 7.89 (d, J = 6.5 Hz, 1H), 7.77 (d, J = 12.0 Hz, 1H), 7.51
(d, J = 6.0 Hz, 1H), 7.17-7.23 (m, 5H) , 7.06 (t, J = 9.0 Hz, 1H), 6.48 (t, J = 7.2 Hz, 1H), 6.17 (s, 2H), 5.69 (d,
J = 6.0 Hz, 1H). MS (ESI) m/z: 469.0 [M+H]+. HPLC: 95.3%; 3b: 1H NMR (300 MHz, DMSO-d6) δ 9.90 (s,
1H), 8.48 (d, J = 7.2 Hz, 1H), 8.05 (d, J = 6.6 Hz, 1H), 7.83 (d, J = 6.0 Hz, 1H), 7.77 (d, J = 12.0 Hz, 1H),

7.35-7.42 (m, 2H) ,7.11-7.25 (m, 4H), 6.66 (t, J = 7.0 Hz, 1H), 6.47 (s, 2H), 6.02 (d, J = 5.4 Hz, 1H), 4.50 (d,
J = 6.0 Hz, 2H). MS (ESI) m/z: 483.0 [M+H]+. HPLC: 95.9%; 3c: 1H NMR (500 MHz, DMSO-d6) δ 10.52 (s,
1H), 7.80-7.87 (m, 5H), 7.40-7.47 (m, 2H) , 7.20 (t, J = 6.0 Hz, 2H), 7.08 (d, J = 6.0 Hz, 1H), 6.71 (d, J = 7.5
1
Hz, 1H), 6.46 (s, 2H), 6.06 (d, J = 5.5 Hz, 1H). MS (ESI) m/z: 469.0 [M+H]+. HPLC: 96.8%; 3d: H NMR
(300 MHz, DMSO-d6) δ 10.52 (s, 1H), 9.29 (d, J = 6.0 Hz, 1H), 7.67-7.81 (m, 3H), 7.33-7.42 (m, 3H), 7.17 (t,
J = 9.0 Hz, 2H), 6.96 (s, 1H), 6.67 (d, J = 6.0 Hz, 2H), 6.49 (s, 1H), 6.03 (d, J = 6.0 Hz, 1H), 4.44 (d, J = 5.7
Hz, 2H). MS (ESI) m/z: 483.0 [M+H]+. HPLC: 98.0%; 3e: 1H NMR (300 MHz, DMSO-d6) δ 10.13 (s, 1H),
8.53 (d, J = 7.2 Hz, 1H), 8.04 (d, J = 9.6 Hz, 1H), 7.70-7.82 (m, 2H), 7.52-7.68 (m, 2H), 7.48-7.52 (m, 2H),
7.16 (t, J = 9.0 Hz, 2H), 6.63 (d, J = 10.2 Hz, 1H), 6.51 (s, 2H), 6.06 (d, J = 6.0 Hz, 1H). MS (ESI) m/z: 469.1
[M+H]+. HPLC: 95.0%; 3f: 1H NMR (500 MHz, DMSO-d6) δ 8.83 (t, J = 6.0 Hz, 1H), 8.35 (s, 1H), 7.95 (d, J
= 10.0 Hz, 1H), 7.73-7.83 (m, 2H), 7.42 (d, J = 8.5Hz, 2H), 7.36 (d, J = 8.5Hz, 2H), 7.14-7.19 (m, 2H), 6.55
(d, J = 10.0 Hz, 1H), 6.25 (s, 2H), 6.04 (d, J = 5.5 Hz, 1H), 4.42 (d, J = 6.0 Hz, 2H). MS (ESI) m/z: 482.7
[M+H]+. HPLC: 96.2%; 3g: 1H NMR (300 MHz, DMSO-d6) δ 8.85 (t, J = 5.7 Hz, 1H), 8.35 (s, 1H), 7.95 (d,
J = 9.9 Hz, 1H), 7.80 (d, J = 6.0 Hz, 1H), 7.75 (d, J = 10.8 Hz, 1H), 7.32 -7.42 (m, 4H), 7.15 (t, J = 5.5 Hz, J
= 6.0 Hz, 2H) , 6.55 (d, J = 9.6 Hz, 1H), 6.25 (s, 2H), 5.93 (d, J = 5.5 Hz, 1H), 4.42 (d, J = 5.7 Hz, 2H). MS
(ESI) m/z: 575.0 [M+H]+. HPLC: 95.6%; 3h: 1H NMR (500 MHz, DMSO-d6) δ 8.83 (t, J = 6.0 Hz, 1H), 8.34
(s, 1H), 7.96 (d, J = 9.5 Hz, 1H), 7.86 (d, J = 6.0 Hz, 1H), 7.73 (d, J = 11.3 Hz, 1H), 7.42-7.47 (m, 2H),
7.33-7.36 (m, 2H), 7.16 (t, J = 9.5 Hz, 2H), 6.56 (d, J = 9.5 Hz, 1H), 6.25 (s, 2H), 5.93 (d, J = 5.5 Hz, 1H),
4.59 (s, 1H), 4.42 (d, J = 6.0 Hz, 2H). MS (ESI) m/z: 473.1 [M+H]+. HPLC: 96.4%; 3i: 1H NMR (500 MHz,
DMSO-d6) δ 8.83 (t, J = 5.6 Hz, 1H), 8.34 (s, 1H), 7.96 (d, J = 9.5 Hz, 1H), 7.83 (d, J = 6.0 Hz, 1H), 7.72 (d,
J = 11.3 Hz, 1H), 7.41-7.48 (m, 2H), 7.33-7.36 (m, 2H), 7.14 (t, J = 8.7 Hz, 2H), 6.56 (d, J = 9.6 Hz, 1H),
6.37 (s, 2H), 5.91 (d, J = 5.8 Hz, 1H), 5.24 (t, J = 6.0 Hz, 1H), 4.42 (d, J = 5.8 Hz, 2H), 4.35 (d, J = 5.9 Hz,
2H). MS (ESI) m/z: 503.1 [M+H]+. HPLC: 97.5%; 3j: 1H NMR 500 MHz, DMSO-d6) δ 8.83 (t, J = 5.9 Hz,
1H), 8.33 (s, 1H), 7.96 (d, J = 9.5 Hz, 1H), 7.85 (d, J = 5.8 Hz, 1H), 7.71 (d, J = 11.5 Hz, 1H), 7.38-7.43 (m,
2H), 7.33-7.36 (m, 2H), 7.14 (t, J = 6.7 Hz, 2H), 6.56 (d, J = 9.6 Hz, 1H), 6.28 (s, 2H), 6.01 (d, J = 5.8 Hz,
1H), 4.42 (d, J = 5.8 Hz, 2H), 4.35 (d, J = 5.9 Hz, 2H), 2.18 (s, 6H). MS (ESI) m/z: 530.2 [M+H]+. HPLC:
95.5%; 3k: 1H NMR (500 MHz, DMSO-d6) δ 8.84 (t, J = 5.9 Hz, 1H), 8.38 (s, 1H), 8.13 (d, J = 5.4 Hz, 1H),
7.97 (d, J = 9.6 Hz, 1H), 7.76 (d, J = 11.2 Hz, 1H), 7.51 (t, J = 8.7 Hz, 1H), 7.41-7.45 (m, 2H), 7.33-7.37 (m,
2H), 7.15 (t, J = 6.7 Hz, 2H), 6.57 (d, J = 9.6 Hz, 1H), 6.48 (d, J = 5.3 Hz, 1H), 6.30-6.31 (m, 1H), 6.01 (d, J
= 5.8 Hz, 1H), 4.42 (d, J = 5.8 Hz, 2H). MS (ESI) m/z: 473.1 [M+H]+. HPLC: 95.4%; 3l: 1H NMR (300 MHz,
DMSO-d6) δ 8.87 (t, J = 6.0 Hz, 1H), 8.58 (d, J = 5.3 Hz, 1H), 8.38 (s, 1H), 8.21 (d, J = 5.4 Hz, 1H), 7.97 (d,
J = 9.6 Hz, 1H), 7.82 (d, J = 11.3 Hz, 1H), 7.63-7.71 (m, 2H), 7.33-7.52 (m, 3H), 7.15 (t, J = 9.0 Hz, 2H),
6.72 (d, J = 5.3 Hz, 1H), 6.58 (d, J = 9.6 Hz, 1H), 4.43 (d, J = 5.7 Hz, 2H). MS (ESI) m/z: 491.1 [M+H]+.
HPLC: 95.1%; 3m: 1H NMR (300 MHz, DMSO-d6) δ 8.76-8.85 (m, 2H), 8.58 (d, J = 5.1 Hz, 1H), 8.42 (s,
1H), 7.99 (d, J = 9.6 Hz, 1H), 7.71-7.78 (m, 2H), 7.51 (d, J = 8.7 Hz, 1H), 7.31-7.39 (m, 3H), 7.02-7.18 (m,
2H), 6.59 (d, J = 9.6 Hz, 1H), 4.41 (d, J = 5.7 Hz, 2H). MS (ESI) m/z: 490.1 [M+H]+. HPLC: 98.3%; 3n: 1H
NMR (300 MHz, DMSO-d6) δ 8.86 (t, J = 6.0 Hz, 1H), 8.60 (s, 1H), 8.41 (s, 1H), 7.97 (d, J = 9.6 Hz, 1H),
7.76 (d, J = 11.3 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 7.60 (s, 1H), 7.41-7.59 (m, 2H), 7.33-7.38 (m, 2H), 7.17 (t,
J = 9.0 Hz, 2H), 6.57 (d, J = 9.6 Hz, 1H), 4.42 (d, J = 6.0 Hz, 2H), 4.00 (s, 6H). MS (ESI) m/z: 545.2 [M+H]+.
HPLC: 99.4%; 3o: 1H NMR (500 MHz, DMSO-d6) δ 8.51 (t, J = 6.2 Hz, 1H), 8.11 (s, 1H), 7.80 (d, J = 5.7
Hz, 1H), 7.65 (d, J = 5.6 Hz, 1H), 7.33-7.44 (m, 4H), 7.15 (t, J = 6.6 Hz, 2H), 6.44 (s, 2H), 6.07 (d, J = 5.5 Hz,
1H), 6.02 (s, 1H), 4.44 (d, J = 6.1 Hz, 2H), 3.28 (s, 3H). MS (ESI) m/z: 513.1 [M+H]+. HPLC: 95.6%; 3p: 1H
NMR (500 MHz, DMSO-d6) δ 8.31 (t, J = 6.02 Hz, 1H), 8.09 (s, 1H), 7.80 (d, J = 5.5 Hz, 1H), 7.65 (d, J =
5.6 Hz, 1H), 7.33-7.44 (m, 4H), 7.16 (t, J = 6.6 Hz, 2H), 6.45 (s, 2H), 6.06 (d, J = 5.5 Hz, 1H), 6.00 (s, 1H),

4.45 (d, J = 6.1 Hz, 2H), 4.20 (d, J =7.0 Hz, 2H), 1.35 (t, J =7.0 Hz, 3H). MS (ESI) m/z: 527.0 [M+H]+.
HPLC: 95.2%; 3q: 1H NMR (300 MHz, DMSO-d6) δ 8.45 (t, J = 6.0 Hz, 1H), 8.10 (s, 1H), 7.80 (d, J = 6.0
Hz, 1H), 7.65 (d, J = 6.0 Hz, 1H), 7.33-7.43 (m, 4H), 7.11-7.17 (m, 2H), 6.50 (s, 2H), 6.00-6.09 (m, 3H),
5.29-5.44 (m, 2H), 4.74 (d, J = 5.4 Hz, 2H), 4.44 (d, J =7.0 Hz, 2H). MS (ESI) m/z: 539.0 [M+H]+. HPLC:
96.1%; 3r: 1H NMR (500 MHz, DMSO-d6) δ 8.40 (s, 1H), 7.99 (t, J = 5.0 Hz, 1H), 7.82 (d, J = 6.0 Hz, 1H),
7.20-7.46 (m, 5H), 7.03 (t, J = 6.5 Hz, 2H), 6.18 (d, J = 6.0 Hz, 1H), 5.96 (s, 1H), 5.51 (s, 2H), 4.58 (d, J =
5.5 Hz, 2H), 4.19 (t, J = 4.5 Hz, 2H), 3.66 (t, J = 4.5 Hz, 2H), 3.18 (s, 3H). MS (ESI) m/z: 557.0 [M+H]+.
1
HPLC: 95.1%; 3s: H NMR (500 MHz, DMSO-d6) δ 8.51 (t, J = 6.0 Hz, 1H), 8.10 (s, 1H), 7.79 (d, J = 5.5
Hz, 1H), 7.65 (d, J = 11.5 Hz, 1H), 7.33-7.41 (m, 4H), 7.15 (t, J = 7.0 Hz, 2H), 6.25 (s, 2H), 6.02 (s, 1H), 5.94
(d, J = 5.5 Hz, 1H), 4.44 (d, J = 6.5 Hz, 2H), 3.92 (s, 3H). MS (ESI) m/z: 605.0 [M+H]+. HPLC: 96.5%; 3t:
1H NMR (500 MHz, DMSO-d6) δ 8.39 (s, 1H), 7.95 (t, J = 5.0 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.20-7.35
(m, 5H), 7.04 (t, J = 6.0 Hz, 2H), 6.02 (d, J = 6.0 Hz, 1H), 5.96 (s, 1H), 5.40 (s, 2H), 4.58 (d, J = 5.5 Hz, 2H),
4.19 (t, J = 4.5 Hz, 2H), 3.67 (t, J = 4.5 Hz, 2H), 3.18 (s, 3H). MS (ESI) m/z: 649.0 [M+H]+. HPLC: 95.8%;
3u: H NMR (500 MHz, DMSO-d6) δ 8.51 (t, J = 6.0 Hz, 1H), 8.11 (s, 1H), 7.82 (d, J = 10.0 Hz, 1H), 7.64 (d,
J = 11.0 Hz, 1H), 7.42 (d, J = 9.5 Hz, 1H), 7.33-7.36 (m, 3H), 7.15 (t, J = 9.5 Hz, 2H), 6.36 (s, 2H), 6.02 (s,
1H), 5.94 (d, J = 6.0 Hz, 1H), 5.25 (t, J = 6.0 Hz, 1H), 4.44 (d, J = 6.0 Hz, 2H), 4.35 (d, J = 6.0 Hz, 2H), 3.93
(s, 3H). MS (ESI) m/z: 533.0 [M+H]+. HPLC: 99.0%; 3v: 1H NMR (500 MHz, DMSO-d6) δ 8.51 (t, J = 6.0
Hz, 1H), 8.09 (s, 1H), 7.84 (d, J = 5.5 Hz, 1H), 7.63 (d, J = 11.0 Hz, 1H), 7.33-7.39 (m, 4H), 7.14 (t, J = 6.0
Hz, 2H), 6.25 (s, 2H), 6.01-6.06 (m, 2H), 4.44 (d, J = 5.8 Hz, 2H), 4.07 (d, J = 5.5 Hz, 2H), 3.92 (s, 3H), 2.18
1
(s, 6H). MS (ESI) m/z: 560.0 [M+H]+. HPLC: 97.1%; 3w: H NMR (500 MHz, DMSO-d6) δ 12.2 (s, 1H),
8.53 (s, 1H), 8.29 (d, J = 5.5 Hz, 1H), 8.11-8.16 (m, 2H), 7.75 (d, J = 11.0 Hz, 1H), 7.62 (t, J = 8.5 Hz, 1H),
7.30-7.43 (m, 5H), 7.09-7.16 (m, 4H), 6.56 (d, J = 5.0 Hz, 1H), 6.04 (s, 1H), 4.46 (d, J = 5.0 Hz, 2H), 3.94 (s,
3H). MS (ESI) m/z: 572.2 [M+H]+. HPLC: 95.2%.

Figure 1. Representative MET inhibitors (1, 2) and designed compounds (3)

Figure 2. The bindind mode of 3f (A) and 3o (B) with MET. The blue line respresents a hydrogen
bond. For clarity, only important residues were shown with stick model.

Figure 3. The overlay of the binding model of compounds 3o (A) and 1 (B) with MET and
VEGFR-2 kinases, respectively. The yellow and green stick represent MET complex. The magenta
and lightpink stick represent VEGFR-2 complex. The first number of residue represents in MET
kinase and the second number refers to in VEGFR-2 kinase.

O
O
CO2Me
Method A
7
O
R1
O
O
R1
O
F
F
O
R1
CO2H
H
N
H
N
ii), iii)
F
N
F
N
F
NH2
F
N
n
n
F
NH2
Cl
O
O
O
O
HO
v)
iv)
5
X
X
X
X
X
H2N
i)
H2N
H2N
H2N
N
4
N
N
N
8
N
H2N
N
9
3a-g and 3o-t
O
O
O
O
MeO
OMe
10
6
O
O O
R1 = H, OMe, OEt, -OCH2CH=CH2, -O(CH2)2OCH3
vi), iii)
Scheme 1. Reagent and conditions: i) t-BuOK, DMF, 50oC for 6h, 76-82%; ii) DMAP, Molecular
sieves, MeOH, 60oC for 2h, 81-85%; iii) NaOH, MeOH, H2O, 50oC for 2h, 86-92%; iv) BtOH,
EDCI, DMAP, DCM, 25oC for 12h, 54-85%; v) PhI(OAc)2, EA, DCM, H2O, 0oC for 2h, 65-83%;
vi) EtOH, 70oC for 2h, 86-90%.

Scheme 2. Reagent and conditions: i) DMAP, Molecular sieves, MeOH, 60oC for 2h, 81% ii)
EtOH, 70oC for 2h, 85%; iii) NaOH, MeOH, H2O, 50oC for 2h, 88-95%; iv) BtOH, EDCI,
DMAP, DCM, 25oC for 12h, 62-78%; v) t-BuOK, DMF, 50oC for 1-12h, 65-87%.

Table 1 MET and VEGFR-2 inhibitory activity of compounds 3a–f
IC50 (µM)
Compds
n
MET
> 25
> 25
> 25
> 25
> 25
0.85
VEGFR-2
0
1
0
1
0
1
-
3a
3b
3c
3d
3e
3f
-
-
-
-
>25

Table 2 MET inhibitory activity of compounds 3g–w
Compds
3g
block A
A-2
A-3
A-4
A-5
A-7
A-8
A-9
A-10
A-1
A-1
A-1
A-1
A-2
A-2
A-4
A-5
A-6
R1
IC50 (µM)
0.39
H
H
0.46
3h
3i
H
0.28
H
0.43
3j
H
1.23
3k
3l
H
0.21
H
H
0.57
3m
3n
3o
0.24
OMe
0.045
0.55
OEt
3p
3q
3r
-OCH2CH=CH2
-O(CH2)2OCH3
OMe
0.28
0.34
0.005
0.l5
3s
-O(CH2)2OCH3
OMe
3t
0.099
0.21
3u
3v
OMe
OMe
0.12
3w
0.008
1 (cabozantinib)

Table 3 Kinase selectivity of compounds 3o and 3s
Enzyme, IC50, µM
N.O.
MET
0.045
0.005
0.008
VEGFR-2
>25
c-Kit
>25
RET
>25
3o
3s
>25
>25
>25
0.007
0.004
0.016
1 (cabozantinib)

Graphical Abstract
Design, Synthesis and Evaluation of Highly Selective
Pyridone-based Class II MET Inhibitors
Nengfang Shea, Linsheng Zhuoa, Wen Jianga, Xiaolei Zhua, Jia Wangb, Zhihui Mingb,
Xinge Zhaob, Xin Congb and Wei Huanga,c,*
aKey Laboratory of Pesticide and Chemical Biology, Ministry of Education, College of Chemistry,
Central China Normal University, Wuhan, 430079, P.R.China
bJiangsu Key Laboratory of Molecular Targeted Antitumor Drug Research, Jiangsu Simcere
Pharmaceutical Co. Ltd, Nanjing 210042, P.R.China
cState Key Laboratory of Agricultural Microbiology, College of Plant Science and Technology,
Huazhong Agricultural University, Wuhan 430070, China
OMe
O
F
O
F
H
H
H
H
N
N
N
F
N
N
F
N
Docking
& SAR
Cyclization
O
O
O
O
F
O
O
O
N
Cl
H2N
O
O
I
3f
3s
N
Cabozantinib
H2N
N
Novel & selective lead scaffold
(IC50 of 850 nM)
Highly selective MET inhibitor (IC50 of 5 nM)
> 5000 fold over VEGFR-2, c-Kit & RET
non-selective MET inhibitor
A novel series of class II MET inhibitors has been designed that utilize 2-pyridone to
conformationally restrain key pharmacophoric groups within the molecule
via a scaffold-hopping strategy. Integrated molecular docking and SAR studies
resulted in the discovery of a novel class of pyridone MET inhibitor with high
potency (IC50 of 0.005 µM) and efficient selectivity (> 5000 fold) to VEGFR-2, c-Kit
and RET kinase
.