Discovery and optimization of a series of imidazo[4,5-b]pyrazine derivatives as highly potent and exquisitely selective inhibitors of the mesenchymal–epithelial transition factor (c-Met) protein kinase

Article abstract of DOI:10.1016/j.bmc.2016.07.019

Aberrant c-Met activation has been implicated in multiple tumor oncogenic processes and drug resistance. In this study, a series of imidazo[4,5-b]pyrazine derivatives was designed and synthesized, and their inhibitory activities were evaluated in vitro. Structure–activity relationship (SAR) was investigated systematically and docking analysis was performed to elucidate the binding mode, leading to the identification of the most promising compound 1D-2 which exhibited significant inhibitory effect on both enzymatic (IC₅₀?=?1.45?nM) and cellular (IC₅₀?=?24.7?nM in H1993 cell line) assays, as well as exquisite selectivity and satisfactory metabolic stability in human and rat liver microsomes.

Full text of DOI:10.1016/j.bmc.2016.07.019

Bioorganic & Medicinal Chemistry 24 (2016) 4281–4290
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and optimization of a series of imidazo[4,5-b]pyrazine
derivatives as highly potent and exquisitely selective inhibitors of the
mesenchymal–epithelial transition factor (c-Met) protein kinase
⇑
⇑
Fei Zhao, Jing Zhang, Leduo Zhang, Yu Hao, Chen Shi, Guangxin Xia, Jianxin Yu , Yanjun Liu
Central Research Institute, Shanghai Pharmaceuticals Holding Co., Ltd, Building 5, No. 898 Halei Road, Shanghai 201203, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Aberrant c-Met activation has been implicated in multiple tumor oncogenic processes and drug resis-
tance. In this study, a series of imidazo[4,5-b]pyrazine derivatives was designed and synthesized, and
their inhibitory activities were evaluated in vitro. Structure–activity relationship (SAR) was investigated
systematically and docking analysis was performed to elucidate the binding mode, leading to the iden-
tification of the most promising compound 1D-2 which exhibited significant inhibitory effect on both
enzymatic (IC50 = 1.45 nM) and cellular (IC50 = 24.7 nM in H1993 cell line) assays, as well as exquisite
selectivity and satisfactory metabolic stability in human and rat liver microsomes.
Received 26 May 2016
Revised 9 July 2016
Accepted 11 July 2016
Available online 12 July 2016
Keywords:
c-Met inhibitors
Imidazo[4,5-b]pyrazines
Docking study
Ó 2016 Elsevier Ltd. All rights reserved.
Structure–activity relationship
1. Introduction
To date, several drug candidates acting on c-Met have been
approved. PF-02341066 (Crizotinib, developed by Pfizer Inc)11 is
As a member of the family of receptor tyrosine kinases (RTK),
mesenchymal–epithelial transition factor (c-Met) is a proto-onco-
gene and its endogenous ligand is hepatocyte growth factor
(HGF).1 The HGF/c-Met signaling pathway plays an important role
in a variety of biological activities such as proliferation, survival
and tissue repair, etc. Although these activities are essential in
the development of mammalian cells, they are also implicated in
cancer progression such as proliferation, invasion and metastasis.2
In normal tissues the expression level of MET is very low, but over-
expression has been found in a number of major human malignan-
cies and was considered to be related to high tumors grade and
poor prognosis.3–6 Moreover, it was reported that the resistance
of tumor cells in the course of chemotherapy, radiotherapy and
other RTK based target therapy was associated with aberrant c-
Met signaling and amplification of the Met has been detected in
20% of acquired resistance to epidermal growth factor receptor
(EGFR) inhibitors.7,8
a dual inhibitor of c-Met and anaplastic lymphoma kinase (ALK),
and it was approved on August 26, 2011 for treatment of non-small
cell lung carcinoma (NSCLC). XL184 (Cabozantinib, developed by
Exelixis Inc),12 as a inhibitor of c-Met and VEGFR2, was approved
for treatment of medullary thyroid cancer (MTC) in November
2012. In recent years, a number of different types of small molecule
c-Met inhibitors were reported.13–19 The first generation of c-Met
inhibitors have broad kinase activities, while the second genera-
tion are relatively selective. Although so far none of selective inhi-
bitors has been approved, a number of drug candidates have
entered clinical trials, such as PF4217903,20 JNJ38877605,21
INCB28060,22 etc.
As shown in Figure 1, chemically these reference compounds all
share a relatively conserved structure, including a right ring of
quinoline moiety (P1), a methylene linker, a condensed five-mem-
bered and six-membered bi-heteroaryl ring core and a hydrophilic
aromatic ring side chain at the 6-position (P2). A survey of the liter-
ature on the binding information suggests that the molecule of the
selective c-Met inhibitor makes a turn at the methylene linker and
adopt a bent ‘U-shaped’ conformation binding mode with the inhi-
Since its central role in tumor cell biology, discovery of c-Met
tyrosine kinase inhibitors especially ATP-competitive small mole-
cule inhibitor has recently attracted much attention in targeted
cancer therapies. This research was developed rapidly as a result
of the report of crystal structures of c-Met in 2002 and 2003.9,10
bitor wrapped around Met-1211. There is a
p–p stacking interac-
tion between the fused ring core and the A-loop residue Tyr-
1230, which is important for overall potency. The N-3 nitrogen of
the fused ring is necessary and forms a hydrogen bond with the
N–H of the DFG Asp-1222 residue. The side chain at the 6-position
⇑
Corresponding authors. Tel.: +86 21 61871700; fax: +86 21 50128885.
E-mail addresses: yujx@sphchina.com (J. Yu), liuyj@sphchina.com (Y. Liu).
http://dx.doi.org/10.1016/j.bmc.2016.07.019
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.

4282
F. Zhao et al. / Bioorg. Med. Chem. 24 (2016) 4281–4290
P
1
P
2
Tyr-1159
N
N
N
N
N
PF4217903
N
HO
N
t
Tyr-1230
n
e
c
e
l
v
c
e
l
o
a
b
i
s
Met-1160
N
s
s
o
e
N
n
i
g
N
H
r
W
W
Aryl
docking analysis
W
F
N
W
N
F
N
W
N
JNJ38877605
N
N
3
N
H
N
Asp-1222
N
O
N
HN
bioisosterism
28060
INCB
N
F
N
N
N
N
Aryl
N
N
N
R
N
R= -H, -OH or -CH
3
SAR analysis
N
Aryl
N
N
N
N
Figure 1. Design of molecular scaffolds based on known drug candidates.
was considered to direct toward the solvent-accessible region of
c-Met. Besides, the quinoline is docked into the hinge region and
the nitrogen atom participates in a hydrogen bond with Met-1160
HOAc and POCl3 to give 6-((6-bromo-2-methyl-1H-imidazo
[4,5-b]pyrazin-1-yl)methyl)quinoline (d), followed by Suzuki cou-
pling with 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-
2-yl)-1H-pyrazole to provide compound 1. When c1 reacted with
CDI, 6-bromo-1-(quinolin-6-ylmethyl)-1H-imidazo[4,5-b]pyrazin-
2-ol (e) was obtained, which was subjected to Suzuki coupling
with corresponding boric acid or boric acid ester to produce
compounds 2–4. Preparation of compound 5 was accomplished
in a similar manner to compounds 1–4 using the corresponding
intermediate 6-((6-bromo-1H-imidazo[4,5-b]pyrazin-1-yl)methyl)
quinoline (f1), which was prepared from the reaction of intermedi-
ate c1 with diethoxymethyl acetate.
All the other title compounds (6–27, 1C–10G) were synthesized
by a procedure similar to that described for the synthesis of com-
pound 5 using corresponding boric acid or boric acid ester reacted
with corresponding intermediate f1–f9 which was prepared from a
series of different substituted amines b1–b9 (synthetic procedures
see Supplementary material) as a starting material (Scheme 2).
and there is also a p–p stacking interaction between the aromatic
quinoline ring and benzene ring of Tyr-1159.16,20,23–25 With this
binding information in mind, we hypothesized that the nitrogen
at the 2-position of the fused ring is unnecessary for the binding
and could be replaced. Here, we wish to efficiently identify a series
of potent and selective c-Met inhibitors. To this end, we maintained
the quinoline moiety as well as the 1-methyl-1H-4-pyrazole sub-
stituent at the 6-position of the fused ring which is usually used
in active c-Met inhibitors, while taking imidazo[4,5-b]pyrazine as
the core structure by means of bioisosteric replacement and intro-
ducing different substituents such as methyl group or hydroxyl
group at the 2-position to investigate the influence on the activity
preliminarily. In addition, further optimization of the active scaffold
in order to improve the potency in both enzymatic and cellular
assays was also investigated and the exploration was mainly
focused on the methylene linker and quinoline moiety.
2.2. Biological evaluation
2. Results and discussion
2.1. Chemistry
In the early studies, compounds 1–5 were firstly synthesized
and their ability to inhibit the c-Met kinase and non-small cell lung
cancer cell line H1993 in vitro were measured. As shown in Table 1,
only compound 5 (6-((6-(1-methyl-1H-pyrazol-4-yl)-1H-imidazo
[4,5-b]pyrazin-1-yl)methyl)quinoline) demonstrated moderate
biochemical potency toward c-Met with an IC50 value of 9.9 nM
and possessed inhibitory activity on H1993 cell line with an IC50
Outlined in Scheme 1 is the synthesis of title compounds 1–5. A
reaction of 2-amino-3,5-dibromopyrazine (a) with quinolin-6-
ylmethanamine (b1) in the presence of N,N-diisopropyl ethylamine
(DIPEA) afforded intermediate 6-bromo-N2-(quinolin-6-ylmethyl)
pyrazine-2,3-diamine (c1). Subsequently c1 was treated with
value of 0.617 lM. All the other compounds displayed quite weak

F. Zhao et al. / Bioorg. Med. Chem. 24 (2016) 4281–4290
4283
N
N
NH2
Br
H2N
+
Br
N
a
b1
i
N
N
N
N
N
N
NH2
NH
N
N
N
N
ii
vi
Br
Br
Br
d
c1
N
f1
N
N
iii
iv
iii
N
N
N
N
N
N
N
N
N
N
OH
N
N
N
N
Br
N
N
N
N
e
N
1
5
v
N
N
N
N
N
N
N
N
N
OH
OH
OH
N
N
N
N
N
N
N
N
N
2
3
4
Scheme 1. Reagents and conditions: (i) DIPEA, NMP, 180 °C, 12 h; (ii) HOAc, POCl3, reflux, 4 h; (iii) 1-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Pd
(dppf)2Cl2ÁDCM complex, K2CO3, dioxane, H2O, 110 °C, 4 h; (iv) CDI, dioxane, reflux, overnight; (v) corresponding boric acid or boric acid ester, Pd(dppf)2Cl2ÁDCM complex,
K2CO3, dioxane, H2O, 110 °C, 4 h; (vi) diethoxymethyl acetate, 150 °C, 12 h.
N
N
NH2
R1
Br
NH R1
L
N
N
NH2
Br
L
ii
i
H2N
+
Br
Br
R2
N
R2
c1 c9
N
R3
R3
a
b1 b9
-
-
N
N
N
N
N
N
iii
N
L
R1
R3
N
L
R
R1
R2
f1 f9
N
R2
N
R3
6 27 1C 10G
-
-
,
-
R1, R2=H, F, or –CH3; R3=H or F; L=-CH2- or –CH(CH3)- or –CH(CH2CH3)-
Scheme 2. Reagents and conditions: (i) DIPEA, NMP, 180 °C, overnight; (ii) diethoxymethyl acetate, 150 °C, 12 h; (iii) corresponding boric acid or boric acid ester, Pd
(dppf)2Cl2ÁDCM complex, K2CO3, dioxane, H2O, 110 °C, 4 h.
or almost no inhibitory activity toward c-Met. We can conclude
that the core structure has a great effect on the inhibitory activity,
and the introduction of methyl group or hydroxyl group at the
2-position of imidazole can lead to the loss of c-Met inhibitory
potency. Therefore compound 5 was selected as an attractive
starting point in the next study for chemistry optimization and
structure–activity relationship (SAR) exploration to gain further
insight on the potential of this structural class.
Thus, we synthesized eleven compounds (6–16) varied by dif-
ferent substituents at the 6-position of the imidazo[4,5-b]pyrazine
core ring to investigate their inhibitory activities. As shown in
Table 2, all these compounds demonstrated c-Met inhibitory

4284
F. Zhao et al. / Bioorg. Med. Chem. 24 (2016) 4281–4290
Table 1
SAR of the core
further improvement of potency by enhancing this stacking inter-
action. Some strong electron-withdrawing groups were considered
to introduce to this moiety. It is well known that fluorine atom was
widely used in drug design due to its special characterization and
many anti-cancer drugs contained fluorine atom, such as 5-FU and
Gefitinib, etc. In the next work, fluorine atom was introduced to
different positions of quinoline moiety to alter the electron density
in the ring with the hope of increased potency. Instead of fluorine
atom, methyl group was also investigated. Therefore, other seven
types of compounds (C–I) were designed and synthesized.
As Table 4 shows, many of these compounds exhibited better
activities in both enzymatic and cellular assays. The inhibitory
activities could improve significantly after introduction of a methyl
group on the bridging methylene group as we originally designed.
However, the corresponding compounds of type E and F displayed
quite weak inhibitory activities (activities in the cellular assay
were not tested). It can be inferred that the size of the introduced
group is crucial for the activity. In order to explore the reason,
compound 17 and its analogue which was introduced an ethyl
N
N
N
N
R
1
R
2
N
H1993b IC50
>10
(lM)
a
a
Compd
R1
R2
Enzyme IC50 (nM)
1
–CH3
–OH
640.9
N
N
N
N
2
>1000
>10
3
4
–OH
–OH
>1000
>1000
>10
>10
N
Table 2
5
–H
9.9
0.3
0.617
0.034
N
SAR about the derivatives of type A
N
Control
PF4217903
N
N
N
N
a
IC50 are based on triple runs of experiments.
H1993: human non-small-cell lung cancer cell line with c-Met overexpression.
b
R
N
activities with IC50 values ranging between 12 and 81 nM, which
support our previous molecular design. Besides, this result sug-
gested that the property of the substituent at the 6-position had
a certain degree of influence on the activity. It looks like the intro-
duction of heteroaryl groups can improve the inhibitory activity.
It reported that around the methylene group of inhibitor there
is a hydrophobic pocket with Leu-1157, Lys-1110, and Val-1092
residues and a small hydrophobic group on the methylene group
may fit into the pocket well and form hydrophobic interaction to
potentially improve the potency.23 Taking in consideration the sig-
nificance of the group on the methylene linker, compounds of type
B were synthesized. Meanwhile, the substituents at the 6-position
that taken into consideration were mainly heteroaryl groups, aryl
groups and non aromatic ring based on the results of type A. As
shown in Table 3, all these compounds exhibited relatively potent
inhibition in both enzymatic and cellular assays especially for com-
a
a
Compd
R
Enzyme IC50 (nM)
H1993 IC50
7.909
(lM)
6
66.59
N
N
7
8
9
13.25
15.94
39.96
2.893
2.325
>10
HN
HO
N
N
N
N
pounds 17, 19–21 and 24–26 (IC50 < 1 lM in H1993 cell line). This
10
12.37
4.948
result was encouraging since an unexpectedly significant increase
in cellular activity was obtained by the addition of a methyl group.
For example, compounds 17, 19 and 20 inhibited 3-fold, 5-fold and
15-fold more potently than their corresponding compounds of
unbranched type A, respectively. The influence of substituents at
the 6-position on the activity is consistent with that of type A.
However, great efforts should be made when compared with the
positive control, which is 7-fold more potent in the enzymatic
assay and nearly 6-fold more potent in the cellular assay than
our most potent compound 17.
In an ongoing effort to further improve the potency, chemistry
optimization was carried out as follows. Considering the potent
activities of type B and the possible oxidative metabolism of the
methylene,26,27 the methyl group on the linker was maintained
in the follow-up study, and ethyl group was also investigated.
The selected substituents at the 6-position were mainly heteroaryl
groups and aryl groups. On the other hand, we turned our attention
to the quinoline moiety. It is generally accepted that this structure
H2N
11
12
17.53
17.57
1.159
7.488
N
13
14
15
27.91
81.24
24.59
>10
>10
>10
F3C
F3CO
F
16
33.24
0.33
6.661
0.034
HN
HCl
Control
PF4217903
is significant for potency and c-Met selectivity, and there is a p–p
stacking interaction between the aromatic quinoline ring and
benzene ring of Tyr-1159. We realized that there is still room for
a
IC50 are based on triple runs of experiments.

F. Zhao et al. / Bioorg. Med. Chem. 24 (2016) 4281–4290
4285
Table 3
illustrated with type D. All compounds of this type demonstrated
high potency toward c-Met with IC50 < 5 nM, and several com-
pounds exhibited potent activities in cellular assay at a double-digit
nanomolar level. Compounds 1D, 7D and 8D even showed more
potent activities than positive control. When both 5-position and
7-position were substituted with fluorine atom (type G), the inhibi-
tory activity was further improved since almost all compounds
exhibited much more potent activity in both enzymatic (IC50 < 2 nM
except for compounds 6G and 10G) and cellular assays (IC50 < 85 nM
in H1993 cell line except for compounds 4G, 5G and 10G). But when
5-position or 7-position was substituted with a methyl group (type
H and I), the resulting compounds displayed weak inhibitory activ-
SAR about the derivatives of type B
N
R
N
N
a
a
Compd
R
Enzyme IC50 (nM)
H1993 IC50
0.197
(lM)
17
2.49
N
N
N
N
ities especially in cellular assay (IC50 > 0.5 lM except for com-
pounds 1H and 9H), and it seems compounds of type I exhibited
more weak activities in enzymatic assay than type H. This result is
basically consistent with our prediction. The docking study of
compound 1D revealed that there was an extra weak hydrogen bond
between 7-F and hydrogen atom at the 2-position of core ring,
which is beneficial to the stability of binding conformation. How-
ever, this intramolecular interaction was disappeared when fluorine
atom was substituted for methyl group. For compound 1G, besides
the weak hydrogen bond (displayed as green dashed line) men-
tioned in compound 1D, there were an extra halogen bond between
5-F and N-7 of pyrazine ring (displayed as blue dashed line) and a
Pi–anion interaction between pyrazole ring and Asp-1164 (dis-
played as orange dashed line). All these interactions are beneficial
to the stability (Fig. 3). These docking results were in good agree-
ment with our experimental results.
For the same type, the kind of substituents at the 6-position of
core ring could influence the activities to some degree. The activi-
ties were quite potent when the substituents were heteroaryl
groups, which is in accordance with the previous result. The activ-
ities in cellular assay were reduced or even loss when the sub-
stituent was phenyl group (5C–5G), even though some
compounds demonstrated quite potent c-Met kinase inhibitory
activities. It also can be inferred that the introduction of nitrogen
atom could improve the potency, and compounds with N-
monomethylated amide showed relatively weak activities than
compounds with nonsubstituted amide.
Given their excellent activities in both assays, several com-
pounds of type D and G were selected to further evaluate, including
metabolic stabilities in human and rat liver microsomes (HLM and
RLM), direct inhibition (DI) and time-dependent inhibition (TDI) on
5CYPs in HLM. As shown in Table 5, compounds 6D and 6G were
unstable in both HLM and RLM probably because of the instability
of ester group on benzene ring. Compound 9G was relatively unsta-
ble in RLM, which indicate species difference. The rest compounds
were stable in both HLM and RLM. Compound 9G demonstrated
both DI and TDI on most of the 5CYPs. Almost all of the compounds
(include positive control) displayed DI on CYP2C9 except for com-
pound 1D, and this compound did not show DI or TDI on 5CYPs. It
can be found that there is no obvious connection between the inhi-
bition on 5CYPs and the number of fluorine atoms, but the kind of
substituents at the 6-position has some influences in the inhibition
on 5CYPs especially for the DI. The cell membrane permeability of
compounds 1D, 7D and 8D in Caco-2 cell model was also tested
(see Supplementary material). All the selected compounds dis-
played high permeability with Papp > 10 Â 10À6 cm/s and there
were no efflux transporters involved (efflux ratio <2).
18
38.45
3.285
19
20
11.40
12.71
0.519
0.148
HN
HO
N
N
N
21
22
3.67
5.17
0.614
1.220
N
O
23
24
6.41
3.95
1.109
0.191
O
F
HN
O
F
H2N
25
26
2.74
3.30
0.238
0.956
O
F
N
H
N
27
15.19
0.33
2.661
0.034
HN
HCl
PF4217903
Control
a
IC50 are based on triple runs of experiments.
group on the methylene linker were docked into the active site of
c-Met. As shown in Figure 2, the two compounds adopted the sim-
ilar binding mode with nonphosphorylated c-Met, however, the
binding orientation was changed to some extent after introduction
of a bigger ethyl group. Unlike compound 17, the
p–p stacking
interaction of Tyr-1230 with the pyrazole ring was disappeared
and there was an unfavorable acceptor–acceptor interaction of
Arg-1208 with the N-4 on the pyrazine ring (displayed as red
dashed line) between this analogue and kinase. For now, it looks
that methyl group is the best substituent.
Another conclusion can be inferred is that the introduction of flu-
orine atom on the quinoline moiety can improve the activities in
both enzymatic and cellular assays, but it depends on the substi-
tuted position. The introduction of a fluorine atom at the 8-position
(type C) led to the loss of activity with IC50 > 10 lM in H1993 cell
line. However, a dramatic increase in inhibitory potency in both
Through a comprehensive comparison, compounds 1D and 8D
were selected for further evaluation. It is worth to mention that,
in previous docking study on compound 1D, we found there was
some difference in the binding mode between these two configura-
tions. The weak hydrogen bond between 7-F and hydrogen atom at
the 2-position of core ring which is beneficial to the stability of
binding conformation for compound (S)-1D was disappeared in
assays was achieved after moving fluorine atom to 7-position, as

4286
F. Zhao et al. / Bioorg. Med. Chem. 24 (2016) 4281–4290
Table 4
SAR about the derivatives of types C–I
C: R1=R2=H, R3=F; L=-CH(CH3)-
D: R1=R3=H, R2=F; L=-CH(CH3)-
E: R1=R3=H, R2=F; L=-CH(CH2CH3)-
N
N
N
N
L
R
R1
R3
F
: R1=F, R2=R3=H; L=-CH(CH2CH3)-
G: R1=R2=F, R3=H; L=-CH(CH3)-
H: R1=CH3, R2=R3=H; L=-CH(CH3)-
R2
N
I
: R1=R3=H, R2=CH3; L=-CH(CH3)-
a
a
a
a
Compd
R
Enzyme IC50 (nM)
H1993 IC50 (nM)
Compd
R
Enzyme IC50 (nM)
H1993 IC50 (nM)
b
1C
91.8
>10,000
31.3
5F
81.0
1.8
–
N
N
1D
1E
1.6
10.6
5G
513.9
60.2
b
–
b
O
1F
13.5
–
6D
2.9
O
F
b
1G
1H
1I
1.0
3.2
32.6
35.5
6E
6F
6G
6H
18.1
39.2
4.9
–
–
b
206.1
b
–
29.4
b
5.9
–
b
2C
278.7
3.6
>10,000
186.8
6I
64.5
–
HN
N
2D
b
HN
2E
8.7
–
7D
2.3
32.7
O
F
b
b
2F
2H
37.7
2.7
–
7E
7F
7I
9.4
7.7
31.0
–
–
–
b
599.1
b
HO
3D
3E
4.5
9.3
175.4
N
N
b
H2N
–
8D
1.7
19.5
O
F
b
3F
3G
3H
22.5
1.0
4.2
–
8E
8F
8I
2.5
1.6
11.7
225.8
664.8
–
83.7
726.2
b
4C
190.3
>10,000
171.2
9C
133.8
>10,000
108.8
N
N
4D
4E
4F
4G
4H
4I
3.8
9D
9E
9F
9G
9H
9I
3.4
b
b
14.1
100.0
1.6
4.4
68.3
–
–
17.2
16.4
1.3
4.9
38.1
–
–
b
b
161.6
949.8
46.1
449.9
b
b
–
–
b
5C
155.4
>10,000
570.2
10C
>1000
–
b
5D
5E
4.6
28.1
10G
Control
23.8
0.3
–
b
–
PF4217903
33.6
a
IC50 are based on triple runs of experiments.
The – indicates not tested.
b

F. Zhao et al. / Bioorg. Med. Chem. 24 (2016) 4281–4290
4287
Table 5
Leadlikeness assessment in vitro of compounds
Compd
HLM stabilitya CLint RLM stabilityb CLint DI
l/min/mg l/min/mg
TDI
(l
(l
protein)
protein)
1D
6D
7D
9
73
16
18
125
85
NIc
2C9
NIc
NIc
3A4, 2D6, 3A4
2C9,
2C19
2C9
2C9,
2C19
2C9
8D
1G
14
11
85
63
NIc
NIc
3G
4G
2
14
10
77
NIc
3A4, 2C9, NIc
2C19
6G
9G
290
51
179
275
2C9
2D6, 2C9, 3A4,
NIc
Figure 2. Overlay of compound 17 (green) and its analogue (yellow) bound with
the nonphosphorylated c-Met, displaying the protein in surface representation. The
residues Ile-1084, Ala-1108, Pro-1158, Tyr-1159, Met-1160, Asp-1164, Arg-1208,
Met-1211, Ala-1221, Asp-1222, Ala-1226, Tyr-1230 and Asp-1231 were defined as
the active site.
2C19
2D6,
1A2
NIc
PF4217903
5
8
2C9
a
b
c
When CLint < 50
When CLint < 100
The NI indicates no inhibition on 5CYPs in HLM.
l
l/min/mg protein, compound is stable in HLM.
l
l/min/mg protein, compound is stable in RLM.28
Figure 3. Overlay of compound 1D (green) and compound 1G (yellow) bound with
the nonphosphorylated c-Met, displaying the protein in surface representation.
Figure 4. Overlay of compounds (S)-1D (green) and (R)-1D (yellow) bound with the
nonphosphorylated c-Met.
compound (R)-1D, however, there was an extra halogen bond
between 7-F of compound (R)-1D and Pro-1158 (Fig. 4, displayed
as blue dashed line). This result predicted that these two com-
pounds perhaps exhibited different activities.
was selected, including highly homologous kinase AXL (tyrosine-
protein kinase receptor UFO) and c-Met family member RON
(macrophage-stimulating protein receptor). As expected, this com-
pound is highly selective for c-Met, showing an IC50 of >1000 nM
against other tested tyrosine kinases (Table 7).
Therefore, the enantiomerically pure compounds 1D-1, 1D-2,
8D-1 and 8D-2 were synthesized and their inhibitory activities
were tested. It can be found from Table 6 that compounds with
an (S)-configuration displayed more excellent activities than their
corresponding (R)-configured compounds in enzymatic assays,
which indicated that (S)-configuration was much beneficial to
the binding. Unexpectedly, their activities in the cellular assay var-
ied dramatically since compounds 1D-2 and 8D-2 showed 10-fold
and 28-fold more potent activities than their enantiomers, respec-
tively. And these two compounds all displayed more excellent
activities on H1993 cell line than the positive control. We re-eval-
uated the metabolic stability as well as the inhibition on 5CYPs in
HLM of these two compounds. Compound 1D-2 was stable in both
HLM and RLM. However, compound 8D-2 was unstable in RLM,
which was less stable than their racemic compound 8D. Different
from racemate 1D, compound 1D-2 demonstrated DI on CYP2C9.
Besides DI on CYP2C9, compound 8D-2 also displayed DI on
CYP3A4. These two compounds all did not show TDI on 5CYPs.
Considering the high biochemical efficiency and satisfactory
metabolic stability, compound 1D-2 was selected to further inves-
tigate the kinase selectivity. A panel of 18 human tyrosine kinases
3. Conclusion
In summary, what described here is the identification and
rationally guided optimization process of a series of selective c-
Met tyrosine kinase inhibitors with an imidazo[4,5-b]pyrazine
scaffold by structural modification of the lead compound 5. Many
derivatives displayed potent activities in both enzymatic (at a sin-
gle-digit nanomolar level) and cellular (at a double-digit nanomo-
lar level on H1993 cell line) assays. The SAR was investigated
systematically and particularly focused on the quinoline moiety,
methylene linker and a range of substitutes at 6-position of the
core. According to the data collected in this study, it can be con-
cluded that the introduction of a methyl group on the methylene
linker is beneficial for the activity; while the result is opposite
when ethyl group was introduced instead of methyl group. The
introduction of fluorine atom at quinoline moiety could improve
potency except for 8-position. Molecular docking experiments
were used to provide a rational explanation of the molecular

4288
F. Zhao et al. / Bioorg. Med. Chem. 24 (2016) 4281–4290
Table 6
In vitro evaluation of compounds
a
a
Compd
Configuration
Enzyme IC50
(nM)
H1993 IC50
(nM)
HLM stabilityb CLint
RLM stabilityc CLint
DI
TDI
(
ll/min/mg protein)
(ll/min/mg protein)
d
d
d
d
1D-1
1D-2
8D-1
8D-2
Control
R
S
R
S
1.94
1.45
2.79
1.32
0.33
239.4
24.7
329.1
11.8
–
8
–
–
–
–
49
2C9
NIe
d
d
d
d
–
–
–
23
5
131
8
3A4, 2C9
2C9
NIe
NIe
PF4217903
33.6
a
b
c
IC50 are based on triple runs of experiments.
When CLint < 50 l/min/mg protein, compound is stable in HLM.
When CLint < 100
l/min/mg protein, compound is stable in RLM.28
The – indicates not tested.
The NI indicates no inhibition on 5CYPs in HLM.
l
l
d
e
4.2. Biological evaluation
Table 7
Kinase-selectivity profile of compound 1D-2
4.2.1. In vitro enzymatic assay
Kinase
IC50 (nM)
Kinase
IC50 (nM)
Inhibition of test compound on c-Met kinase activity was deter-
mined by measuring the phosphorylation level of TK substrate-
biotin peptide in a Homogenous Time-Resolved Fluorescence
AXL
RON
>30,000
>30,000
EGFR
EGFR
[T790M]
ErbB4
c-Src
>30,000
>30,000
(HTRF) assay. Into a white 384-well plate was added 2
5Â compound in reaction buffer. Next, 4 L of reaction buffer
(50 mM HEPES pH 7.0, 0.02% NaN3, 0.01% BSA, 0.1 mM Na2VO3,
5 mM MgCl2 and 1 mM DTT, containing 1 M TK substrate-biotin
lL/well of
ALK
Flt-1
>30,000
>30,000
20,618
>30,000
27,606
26,197
>30,000
>30,000
>30,000
>30,000
>30,000
>30,000
>30,000
>30,000
l
VEGFR2
c-Kit
PDGFRa
PDGFRb
RET
ABL
EphA2
EphB2
IGF1R
FGFR1
l
and 1 ng c-Met enzyme) was added to each well. After 5–10 min
preincubation, the kinase reaction was initiated by the addition
of 4 lL of 18 lM ATP in reaction buffer. After 30 min incubation
at room temperature, the enzyme reaction was stopped by
EDTA-containing buffer, which also contained europium-
conjugated anti-phosphoresidue antibody and streptavidin-XL665
(SA-XL665) to allow for detection of the phosphorylated peptide
product. Following 1 h incubation at room temperature fluores-
cence was measured with excitation of 337 nm and dual emission
of 665 and 620 nm on Envision microplate reader (PerkinElmer).
Signal was expressed in terms of HTRF ratio (fluorescence intensity
at 665 nm/fluorescence intensity at 620 nm * 10,000).
mechanism of the differences in activity. The analysis indicated
this promotion of activity is partly owing to the contribution to
maintain the stability of the favorable binding conformation
through intramolecular hydrogen bond or halogen bond. This dis-
covery verified the experimental results and had some inspiration
and reference value for subsequent research. The R/S configuration
had significant influence on the activity, and the compounds with
an (S)-configuration showed more potent activity especially in cel-
lular assay. Among all compounds tested, the most promising com-
pound 1D-2 displayed excellent potency, satisfactory metabolic
stability in both HLM and RLM as well as exquisite kinase selectiv-
ity. All these properties indicated its druggability. Further
optimizations and studies on the in vivo pharmacodynamic and
pharmacokinetic properties are underway in our research group
and will be reported in due course.
4.2.2. In vitro growth inhibition assay
NCI-H1993 cell line was purchased from the American Type Cul-
ture Collection (ATCC, Manassas, VA, USA). Cells were maintained in
RPMI 1640 media and supplemented with 10% fetal bovine serum
(Gibco Life Technologies, Grand Island, NY). NCI-H1993 cells were
seeded at 5000 cells/well in 96-well plates and incubated overnight.
On the next day, the cells were exposed to various concentrations of
compounds and further cultured for 72 h. After chromogenic reac-
tion with Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kuma-
moto, Japan), the OD450 (with reference of OD650) was measured
using a Flexstation 3 reader (Molecular Devices, USA). IC50 values
were calculated using the GraphPad Prism Software. Each experi-
ment was carried out thrice, each time in duplicate.
4. Experimental
4.1. Chemistry
All reagents and solvents employed were purchased from com-
mercial sources and used without further purification. Reactions
were followed by TLC using HSGF 254 silica-gel plates (0.15–
0.2 mm thickness). Silica-gel chromatography was done using the
appropriate size Biotage prepacked silica filled cartridges. Melting
points were determined using a WRR digital apparatus, and were
uncorrected. NMR data were recorded on a Bruker 400 MHz or
500 MHz Digital NMR Spectrometer. Chemical shifts are expressed
as d units using tetramethylsilane as the external standard (in NMR
description, s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
dd, double doublet; and br s, broad singlet). All coupling constants
(J) are reported in Hertz. Mass spectra were measured with an
Agilent quadrupole 6110 spectrometer using an ESI source coupled
to an Agilent 1200 HPLC system.
4.2.3. Metabolic stability in human and rat liver microsomes
This assay utilized a 150
human or rat liver microsomes (0.5 mg/ml) to preincubate with
M test compound for 5 min at 37 °C in 100 mM phosphate buf-
ll incubation system containing
1
l
fer (pH 7.4). The reactions were initiated by adding NADPH
(1 mM). Midazolam was used as positive control. After 0, 5, 10
and 30 min incubations at 37 °C, the reactions were stopped by
adding 300 ll acetonitrile containing tinidazole of 0.1 lg/ml as
internal standard. The samples were vortexed for 10 min, and then
centrifuged at 6000g for 10 min twice at 4 °C and an aliquot of
supernatant was sampled for LC–MS/MS analysis. Calculate the
The preparation of all the title compounds can be found in
Supplementary material because of the limited length of the paper.
CLint
test compounds.
(ll/min/mg protein) by determining the relative quantity of

F. Zhao et al. / Bioorg. Med. Chem. 24 (2016) 4281–4290
4289
4.2.4. Direct inhibition on 5CYPs in HLM
This assay utilized a 100 l incubation system containing
human liver microsomes (0.2 mg/ml), substrate mixture (Midazo-
lam 10 M, Testosterone 100 M, Dextromethophan 10 M,
Diclofenac 20 M, Phenacetin 100 M, (S)-(+)-Mephenytoin
100 M), 10 M test compound, which were preincubated for
5 min at 37 °C in 100 mM phosphate buffer (pH 7.4). The selective
inhibitors of each P450 isoform (Ketoconazole 10 M for CYP3A4,
Quinidine 10 M for CYP2D6, Sulfaphenazole 100 M for CYP2C9,
Naphthoflavone 10 M for CYP1A2, Tranylcypromine 1000
PF4217903 cocrystallized in the binding site of 3ZXZ. And all the
ligands were prepared using Prepare Ligands protocol and Mini-
mize Ligands protocol in Discovery Studio subsequently. To vali-
date the docking method, the PF4217903 was used to carry out
the Ligandfit docking experiment. The docking results were evalu-
ated through comparison of the best docked binding mode with
the experimental one. The RMSD was used to compare differences
between the atomic distances of the docked poses and the real
cocrystallized pose to measure docking reliability. The RMSD of
the heavy atom is only about 0.1432 Å. It indicated that the Ligand-
fit docking method is fit for the cMet-small molecular ligand sys-
tem. The best pose was outputted on the basis of ligandscore 1
and the protein–ligand interactions. All the compounds used in
the docking study are (S)-configuration unless otherwise stated.
l
l
l
l
l
l
l
l
l
l
l
l
lM
for CYP2C19) were used as positive controls. The reactions were
initiated by adding NADPH (1 mM). After 20 min incubations at
37 °C, the reactions were stopped by adding 300
ll acetonitrile
containing tinidazole of 0.1 g/ml as internal standards. The sam-
l
ples were vortexed for 10 min, and then centrifuged at 6000g for
10 min twice at 4 °C and an aliquot of supernatant was sampled
for LC–MS/MS analysis. Calculate the relative activity of the
enzyme by determining the relative quantity of the substrate
metabolites.
Acknowledgement
We thank NeoTrident Co., Ltd for excellent docking assistance.
Supplementary data
4.2.5. Time-dependent inhibition on 5CYPs in HLM
This assay utilized a 200
human liver microsomes (0.2 mg/ml), 10
which were preincubated for 0 min, 5 min, 10 min and 20 min at
l
l incubation system containing
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2016.07.019.
lM test compound,
37 °C after adding NADPH (1 mM) or PBS. Then the system was
References and notes
incubated for 10 min at 37 °C after adding 180
bation mixture containing NADPH (1 mM) and substrate mixture
(Midazolam 5 M, Testosterone 50 M, Dextromethophan 5 M,
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reactions were stopped by adding 600 l acetonitrile containing
tinidazole of 0.1 g/ml as internal standards. The samples were
ll secondary incu-
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