Synthesis and biological evaluation of new MET inhibitors with 1,6-naphthyridinone scaffold

Article abstract of DOI:10.1016/j.ejmech.2019.111803

A potent and novel MET inhibitor, 5-((4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)amino)-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-ones (8), was designed and synthesized via a scaffold-hopping strategy of a 2,7-naphthyridinone MET kinase inhibito

Full text of DOI:10.1016/j.ejmech.2019.111803

Journal Pre-proof
Synthesis and biological evaluation of new MET inhibitors with 1,6-naphthyridinone
scaffold
Ming-Shu Wang, Lin-Sheng Zhuo, Fan-Peng Yang, Wen-Jie Wang, Wei Huang,
Guang-Fu Yang
PII:
S0223-5234(19)30955-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.111803
EJMECH 111803
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 August 2019
Revised Date: 2 October 2019
Accepted Date: 19 October 2019
Please cite this article as: M.-S. Wang, L.-S. Zhuo, F.-P. Yang, W.-J. Wang, W. Huang, G.-F. Yang,
Synthesis and biological evaluation of new MET inhibitors with 1,6-naphthyridinone scaffold, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111803.
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Graphical Abstract
Synthesis and Biological Evaluation of New MET Inhibitors
with 1,6-Naphthyridinone Scaffold
Ming-Shu Wang1,† Lin-Sheng Zhuo1,†, Fan-Peng Yang1,†, Wen-Jie Wang1, Wei Huang1*,
and Guang-Fu Yang1,*

1
2
Synthesis and Biological Evaluation of New MET Inhibitors with
1,6-Naphthyridinone Scaffold
3
4
Ming-Shu Wang1,† Lin-Sheng Zhuo1,†, Fan-Peng Yang1,†, Wen-Jie Wang1, Wei Huang1*, and
Guang-Fu Yang1,*
5
6
7
1Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, International Joint
Research Center for Intelligent Biosensor Technology and Health, College of Chemistry, Central China
Normal University, Wuhan 430079, P.R. China
8
9
* Corresponding authors. E-mail address: weihuangwuhan@126..com (W. Huang),
gfyang@mail.ccnu.edu.cn (G. Yang).
10
11 † M.W., L.Z. and F.Y. contributed equally to this work.
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
ABSTRACT:
A
potent
and
novel
MET
inhibitor,
5-((4-((2-amino-3-chloropyridin-4-yl)oxy)-3-fluorophenyl)amino)-3-(4-fluorophenyl)-1,6-na
phthyridin-4(1H)-ones (8), was designed and synthesized via a scaffold-hopping strategy of a
2,7-naphthyridinone MET kinase inhibitor 7. Lead compound 8 had good potency (IC50 of
9.8 nM), but unfavorable pharmacokinetic profiles (F = 12%, CL = 5.0 L/h/kg). Systematic
structural optimization of compound 8 resulted in 9g (MET, IC50 = of 9.8 nM) with a
comparable MET potency to that of compound 2 and a favorable pharmacokinetic profile (F
= 63%, CL = 0.12 L/h/kg). Further study of the derivatization of N(1) amine group of 9g led
to the discovery of 23a with good MET potency (IC50 of 7.1 nM), promising VEGFR-2
selectivity (3226-fold), and a markedly drug-likeness improvement (F = 57.7%, CL = 0.02
L/h/kg). The excellent VEGFR-2 selectivity and favorable drug-likeness of 23g suggest that
the 1,6-naphthyridine moiety could be used as a new scaffold for kinase inhibitor discovery.
KEYWORDS: 1,6-Naphthyridone, MET kinase inhibitor, Pharmacokinetic profiles,
VEGFR-2 selectivity
1

1
2
3
4
5
6
7
8
9
1. Introduction
The heterodimeric transmembrane protein MET is a receptor tyrosine kinase (RTK)
known as a hepatocyte growth factor receptor (HGFR). 1-4 Upon activation by hepatocyte
growth factor receptor, MET demonstrates pivotal roles in many fundamental cellular
processes including survival, proliferation, differentiation, migration, invasion, and
motility.5-7 Dysregulation of MET/HGF signaling has been reported to induce proliferation,
invasion, metastasis, tumor angiogenesis, and drug resistance of cancer cells across a broad
spectrum of tumors. 8-10 Thus, MET has attracted considerable attention as a potential target
for cancer treatment.
10
11
12
Figure 1. Examples of multi-targeted MET inhibitors.
13
14
15
16
17
18
19
20
21
22
To date, several MET-targeting agents have been used to disrupt HGF/MET signaling
including HGF and MET antibodies and small-molecule kinase inhibitors.6,11-13
Antibody-based therapies primarily prevent ligand-mediated activation of the receptor, while
small-molecule inhibitors can prevent ligand-dependent activation as well as
ligand-independent activation; thus, a small-molecule MET inhibitor could benefit a different,
and potentially larger, cancer patient population.14,15 The first small molecule MET inhibitor
is cabozantinib (1), which was approved by the FDA for the treatment of progressive
metastatic medullary thyroid cancer (2012).16 Since then, many derivatives of cabozantinib
have been reported including compound 2 (BMS-777607), which exhibits desirable
drug-likeness and modest selectivity to VEGFR-217.
2

1
2
3
Figure 2. Examples of 2,7-naphthyridine and 1,6-naphthyridine.
4
5
Naphthyridines are common in various natural products and are pharmacologically
important18. Compounds containing a 2,7-naphthyridine scaffold are a novel class of
bispyridine structures in many natural alkaloids and synthetic products (Figure 2, compounds
3, 4).19,20 These products usually possess variant biological activities such as antibiotic,
cell-protecting, and anticancer activity. This makes them attractive pharmaceutically active
fragments for further drug discovery.21
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
The 1,6-naphthyridine is an isomer of 2,7-naphthyridine, and its derivatives can inhibit
multiple kinases including Lck, IGF-1R, Abl, FGFR, MET, and ALK (Figure 2, compound
5).22 Furthermore, 1,6-naphthyridines were designed as FGFR-1 and VEGFR-2 kinase
inhibitors with positions 2- and 7- substituted by an amino and 3- substituted by aryl groups.
This suggests that various substitutions on this active scaffold could confer diverse biological
activities (Figure 2, compound 6).23-25
In our previous research, knowledge of the binding mode of BMS-777607 in MET
kinases led to the introduction of 2,7-naphthyridinone fragments into block C based on the
“scaffold hopping strategy.” The resulting early candidate 7 showed excellent potency.26
Here, in order to further explore SARs and optimize the drug-likeness of naphthyridine-based
derivatives, compound 7 was selected as a starting point for this research.
3

1
2
Figure 3. Design of 1,6-naphthyridine derivatives based on a scaffold-hopping strategy.
3
4
According to our previous studies, compound 7 displayed comparable MET enzymatic
activity as 2 due to the conservation of the key H-bond interactions between the carbonyl
group in block C with residues Asp1222 and the amino group in block A with the Met1160.26
Here, these two key H-bond interactions were reversed, and the 1,6-naphthyridine derivative
8 was designed based on the scaffold-hopping strategy of heteroatom migration to further
optimize block C. Furthermore, inspired by the discovery of VEGFR-2/MET inhibitor
altiretinib,27-29 detailed structure activity relationship (SAR) studies of block A and block C
were carried out to optimize the drug-likeness.
5
6
7
8
9
10
11
12
13
14
15
16
2. Chemistry
The most attractive route to 5-substituted 1,6-naphthyridines 8 was via an intermediate
with a displaceable 5-halo group for direct nucleophilic substitution reactions with the
appropriate amines. With this consideration in mind, we designed an efficient synthetic
strategy illustrated in Scheme 1. The aldol reaction of 10 with ethyl formate followed by
condensation with 2-chloropyridin-4-amine 12 and cyclization yielded the key intermediate
4

1
2
3
4
5
6
7
8
9
chlorinated 1,6-naphthyridine 14. The intermediate 15 was smoothly obtained using a method
modified from the literature.17 Due to the presence of the active N(1) amine group in
intermediate 14, the palladium-catalyzed Buchwald coupling reaction failed to afford
compound 16. Thus, we decided to explore more applicable reaction conditions to assemble
the chlorinated 1,6-naphthyridine 14 and aromatic amine 15 because protection and
deprotection of the amine group would result in a cumbersome synthetic route. Eventually,
we determined that the reaction proceeded smoothly in a PTSA acid-isopropanol system. The
precipitated compound 16 was easily purified and had excellent yields. Finally, Hoffman
degradation delivered 8 in high yield.
10
11
12
13
Scheme 1. Reagents and conditions: i) NaH, ethyl formate, 0°C; ii) HCl, EtOH, reflux; iii) Ph2O, 250°C;
(iv) PTSA, isopropanol, 80°C; and (v) PhI(OAc)2, EtOAc/CH3CN/H2O.
14
15
16
17
18
19
20
21
The synthesis of target compounds 9 and 23 included a route that started from
N-acylated-4-(4-amino-2-fluorophenoxy)pyridin-2-amine (21) as the other key intermediate
(Scheme 2). Briefly, commercially available 2-chloropyridin-4-ol (17) was reacted with
1,2-difluoro-4-nitrobenzene
(18)
under
base
conditions
to
yield
2-chloro-4-(2-fluoro-4-nitrophenoxy)pyridine (19). This was further reacted with different
types of acid amides under Pd-catalyzed conditions to produce intermediate 20. Intermediate
20 was then reduced using iron powder and catalytic amounts of ammonium chloride in
ethanol to obtain key intermediate 21. Another intermediate 22 for the synthesis of
5

1
2
3
compounds 23 was obtained by the subsequent substitution reaction of 14 with different
halides or substituted oxirane in the presence of K2CO3. Then, target compounds 9 and 23
were prepared similar to 16.
4
5
6
7
Scheme 2. Reaction conditions and reagents: i) K2CO3, DMF, r.t.; (ii) NH2COR, dppf, Pd2(dba)3,
Cs2CO3, 1,4-dioxane, 85°C; (iii) Fe, NH4Cl, EtOH, H2O, reflux; (iv) K2CO3, DMF, r.t.; and (v) PTSA,
isopropanol, 80°C.
8
9
3. Results and Discussion
We initially studied if the 1,6-naphthyridine moiety can serve as a key pharmacophoric
10
11
12
13
14
15
16
17
18
19
group to demonstrate MET inhibition. Therefore, the 2-aminopyrimidine core and the
terminal phenyl ring were reserved based on the chemical structure of compound 7 in the
newly designed compound 8. A generally utilized 2-fluoro-1,4-substituted phenyl linker was
also adopted from the newly reported inhibitors. To our delight, 8 (IC50 = 9.8 nM) had similar
MET potency as 7 (IC50 = 9.9 nM) and 2 (IC50 = 7.6 nM). This indicated that the scaffold
hopping of heteroatom migration was an effective strategy and led to a new scaffold for
further optimization.
Next, molecular docking was performed to further verify the design strategy and provide
rational direction for optimization. The docking result demonstrated that 8 interacted with
protein almost exactly as in the solved cocrystal structure of 2 (Figure 4). The
6

1
2
2-aminopyrimidine core anchors the inhibitor to the hinge region via two hydrogen bonds
with Met-1160, whereas the central phenyl ring interacts by π-stacking with Phe-1223; the
carbonyl group and the N(1) amine group of 1,6-naphthyridinone fragment interact by
H-bonding with Asp1222 and Glu1227, respectively. This implicated that
1,6-naphthyridinone provides a new hydrogen bonding pattern to better fit the subpocket of
MET versus 2,7-naphthyridinone; the terminal phenyl ring occupies a allosteric hydrophobic
pocket. The docking results revealed that, in addition to the key pharmacophoric group
1,6-naphthyridinone fragment, the dual hydrogen bond system of 2-aminopyrimidine core is
essential for maintaining activity. More importantly, the newly introduced functionalizable
N(1) amine group of 1,6-naphthyridin-4(1H)-one moiety may provide more optimization
options than 2,7-naphthyridin-2(1H)-one.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
Figure 4. (A) Cocrystal structure of 2 bound to the inactive conformation of MET kinase domain; 17 (B)
Our reported binding mode of 7 with MET;26 (C) The proposed binding mode of 8 with MET; (D) The
binding mode overlay of 8 (purple), 7 (white), and 2 (light blue) with MET. The navy blue dashed lines
represent H-bonds between residues with 8, 7, and 2.
7

1
2
With the binding conformations of 8 in hand and encouraged by the discovery of
VEGFR-2/MET inhibitor altiretinib, a SAR study of block A was first performed (Table 1).
A series of acylation modifications of 2-aminopyrimidine core were performed with the
dual hydrogen bond system reserved in order to optimize its drug-likeness. Overall,
substitution on the 2-aminopyrimidine core was well-tolerated and maintained inhibitory
activity. Compound 9a bearing an acetyl group displayed an IC50 value of 21.4 nM, which
is approximately 2.1-fold less potent than that of compound 8. The potency of compound
9b (MET, IC50 = 4.7 nM) with a propionyl group is twice as high perhaps due to the sterics
of the propionyl group. When either the butyryl group or pentanoyl group with a long alkyl
chain was employed, compound 9c (MET, IC50 = 14.8 nM) and 9d (MET, IC50 = 14.7 nM)
only displayed a slight loss (1.5-fold) in enzyme activity, which might benefit from the
flexibility of the alkyl chain. Meanwhile, incorporation of an isobutyryl group (9e) resulted
in a 1.8-fold loss of potency (MET, IC50 = 17.7 nM). MET potency was slightly impaired
by about 2.9-fold as the sterically bulky pivaloyl group (9f) was employed further. The
VEGFR-2 selectivity was increased by more than 106.7-fold (MET, IC50 = 28.1 nM;
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
VEGFR-2, IC50 >3000 nM).
Table 1. Activity of compounds 9a-l against MET/VEGFR-2 kinasea and metabolic stability in human
18
microsomes.
19
20
8

IC50, nMa
VEGFR-2
Met Stab
(T1/2, min)
Compds.
R1
MET
9.8
8
-
68
38.1
189.8
9a
9b
9c
9d
9e
9f
Me
21.4
4.7
-
Et
20.2
163.0
n-Pr
n-Bu
i-Pr
t-Bu
14.8
14.7
17.7
28.1
19.4
-
-
-
-
26.9
230.1
>3000
9g
9h
8.1
8.8
792.5
-
24.0
61.2
9i
9j
9.8
9.9
>3000
58.9
80.4
434.0
248.9
HO
9k
11.8
288.9
9l
35.0
7.6
>3000
138.7
-
-
2 (BMS-777606)
a
1
2
In vitro kinase assays were performed with the indicated purified recombinant MET or VEGFR-2
kinase domains (nM).
3
4
Steric effects might be the main cause of MET potency loss, and steric effects were
more crucial for maintaining VEGFR-2 activity. Furthermore, the cyclopropanecarbonyl
group (9g), cyclobutanecarbonyl group (9h), and cyclohexanecarbonyl group (9i) were also
employed. Compound 9g (MET, IC50 = 8.1 nM) and compound 9i (MET, IC50 = 9.8 nM)
showed comparable MET activity to 8, while 9h (MET, IC50 = 24.1 nM) showed a 2.4-fold
loss in MET potency. More interestingly, 9g displayed excellent VEGFR-2 inhibitory
activity (IC50 = 8.8 nM), and 9i with a sterically bulky substituent displayed good
selectivity against VEGFR-2 (>306-fold). This underscores the importance of steric effects
in maintaining VEGFR-2 activity. In addition to the alkyl acyl group, we also explored the
5
6
7
8
9
10
11
12
13
2-hydroxyacetyl
group
(9j),
2-hydroxypropanoyl
group
(9k),
and
2-hydroxy-2-methylpropanoyl group (9l). Similar steric effects were also the main factor
9

1
2
3
4
for MET potency and VEGFR-2 selectivity. Compound 9j (MET, IC50 = 9.9 nM) and 9k
(MET, IC50 = 11.8 nM) showed comparable MET activity to compound 8. Compound 9l
(MET, IC50 = 35 nM) showed a 3.5-fold loss in MET potency, while compound 9l also
showed good VEGFR-2 selectivity (>306-fold) because of the sterically bulky substituent.
5
6
Figure
5. (A) The proposed binding mode of 9i (yellow) and 9g (pink) with MET; (B) Surface
7
8
representations of 9i binding to Met; (C) The proposed binding mode of 9i (yellow) and 9g (pink) with
VEGFR-2; (D) Surface representations of 9i binding to VEGFR-2
9
10
11
12
13
14
In order to better understand the SAR of block A and the selectivity between MET and
VEGFR-2 of 9i, molecular docking was further performed. As illustrated in Figure 5,
compound 9g and 9i were both docked into the binding site of MET kinase (PDB: 3F82)
and VEGFR-2 kinase (PDB: 3U6J). For MET kinase, the cyclopropanecarbonyl group (9g)
and cyclohexanecarbonyl group (9i) all formed well hydrophobic interactions with residues
TRY1159 and LSY1161 (Figure 5A, 5B). As for VEGFR-2 kinase, sterically smaller
10

1
2
3
4
5
cyclopropanecarbonyl group of 9g could be well tolerated, however, the replacement of
cyclopropanecarbonyl group by sterically bulky cyclohexanecarbonyl group introduced
steric clash with residue LYS1161, which should be responsible for the impaired VEGFR-2
potency of 9i (Figure 5C, 5D). Taken together, substitutions on the 2-aminopyrimidine
core were more tolerated for MET potency than VEGFR-2. This can lead to the design of
6
7
selective MET inhibitors against VEGFR-2.
Next, selected potent compounds with good MET inhibitory activity were incubated with
8
9
human liver microsomes to initially evaluate metabolic stability (Table 1). Table 1 shows that
compound 9b (bearing propionyl group) had a moderate half-life (T1/2 = 163.0 min)
comparable to that of 8 (T1/2 = 189.8 min). Compound 9g with a cyclopropanecarbonyl group
was very stable in human liver microsomes (T1/2 = 792.5 min), and compound 9i with a
cyclohexanecarbonyl group exhibited high clearance rates (T1/2 = 80.4 min).
10
11
12
13
14
Table 2. Activity of compounds 23a-d against MET/VEGFR-2 kinasea and metabolic stability in
human microsomes.
15
IC50, nMa
VEGFR-2
Met Stab
(T1/2, min)
Compds.
R2
MET
7.1
23a
23b
Me
Et
22,910
1380
882.6
-
15.6
23c
23d
33.8
10,450
-
25.9
7.6
>30,000
138.7
-
-
2
a
16
17
In vitro kinase assays were performed with the indicated purified recombinant MET or VEGFR-2
kinase domains (nM).
11

1
2
Thus, the potential impact of R2 substituent on the MET inhibition was investigated with
9g as the lead compound (Table 2). The introduction of a methyl group to the N(1)-position
(23a) led to a slight increase (1.3-fold) of MET potency (MET, IC50 = 7.1 nM). More
importantly, the methylation of the NH group resulted in a significant increase in VEGFR-2
selectivity by 3226-fold (VEGFR-2, IC50 = 22910 nM). The ethyl group was then employed,
and 23b displayed a 1.2-fold MET potency loss and an 88-fold improvement in VEGFR-2
selectivity.
3
4
5
6
7
8
9
To investigate the optimal substituent on the N(1)-position, compound 23c (bearing a
hydroxyethyl group) and compound 23d (bearing a propan-2-ol group) were further
synthesized. While these compounds had a potency loss against MET (3.4-fold and 2.7-fold),
they still maintain high VEGFR-2 selectivity. These results indicate that sterically suitable
methyl group might be optimal, and fine-regulation of newly introduced functionalizable N(1)
amine group of 1,6-naphthyridin-4(1H)-one moiety compared to 2,7-naphthyridin-2(1H)-one
could also lead to selectivity improvement. Even more amazing is that 23a was slightly more
10
11
12
13
14
15
16
stable (T1/2 = 882.6 min, Table 2) than 9g in human liver microsomes
.
17
18
Figure 6. Preliminary results of kinase profile of 23a (Inhibitory rate in 1 µM)
Then，the broad spectrum kinase profile of 23a is represented in the form of heat map in
Figure 6 using inhibitory rate in 1 µM for fifty other kinases. Like most class II c-Met
19
20
12

1
2
3
4
5
6
7
inhibitors, 23a also showed high inhibitory effects against AXL, FGR, FLT3, cMer, TRKA,
TRKB and TYRO3 (inhibitory rate > 80% in 1 µM). And the inhibitory activity of 23a
against important kinases was further assayed using the homogeneous time resolved
fluorescence (HTRF) method. As shown in Table 3, In contrast to its high potency against
MET (IC50 = 7.1 nM), 23a can also strongly inhibit AXL (IC50 = 8.0 nM) and Mer (IC50 =
4.2 nM) at low nanomolar level. Moreover, 23a demonstrated moderate selectivity against
FLT4 and RON (83.2 fold and 48.7 fold, respectively), and good selectivity against other
8
9
forty two kinases (inhibitory rate < 80% in 1 µM).
Table 3. Kinase selectivity profile of 23a
kinase
AXL
IC50, nM
8.2
kinase
TRKA
IC50, nM
32.9
FLT4
cMER
RON
591.0
4.2
TRKB
58.8
TEK(Tie2)
TYRO3(RSE)
26.3
346.0
41.1
10
11
12
13
14
15
16
Antiproliferative activities of all the synthesized compounds against NIH-H460
(human lung cancer), HT-29 (human colorectal cancer) and MKN-45 (human gastric cancer)
cell lines were also evaluated in vitro using Cabozantinib as a positive control. As shown in
Table 4, most of the new compounds and Cabozantinib displayed moderate inhibitory
activity against the tested cancer cell lines. Compounds 8, 9b and 9e exerted good and
broad-spectrum antiproliferative activity against the tested cancer cell lines, especially for
compound 9e (inhibitory rate of 83.4-95.4% in 10 µM).
17
18
Table 4. Antiproliferative activity of synthesized compounds against NIH-H460, HT-29 and MKN-45
cell lines.
Inhibitory rate (% in 10 µM)a
Compds.
NIH-H460
79.1%
HT-29
56.2%
36.8%
68.7%
MKN-45
85.7%
8
9a
9b
58.2%
77.1%
69.7%
92.1%
13

9c
49.7%
71.8%
84.6%
56.7%
54.6%
43.6%
72.1%
53.6%
56.0%
54.3%
15.6%
11.0%
52.1%
67.4%
54.8%
23.5%
-42.2%
83.4%
-45.8%
25.5%
46.0%
8.7%
64.6%
62.7%
95.4%
62.5%
81.8%
52.8%
55.2%
63.0%
67.3%
57.8%
30.9%
31.9%
41.8%
44.3%
49.1%
9d
9e
9f
9g
9h
9i
9j
18.5%
47.8%
-2.9%
24.8%
15.8%
3.5%
9k
9l
23a
23b
23c
23d
-2.6%
Cabozantinib
29.0%
1
2
aData are presented as the means ± SDs of three independent experiments.
Following the in vitro experiments, in vivo PK properties of three compounds (8, 9g,
23a) were determined in rat. As shown in Table 5, the chemical structure of blocks A and C
had a significant influence on the PK properties after oral administration and intravenous
injection in rats. The newly designed 1,6-naphthyridine derivatives 8 had a worse oral
exposure and oral bioavailability in the rat than the reported 2,7-naphthyridine derivatives.
However, 9g, with the 2-aminopyridine core of block A acylated by the
cyclopropanecarbonyl group, afforded a lower total clearance CL (0.12 L/h/kg) after oral
dose of 5 mg/kg leading to a 12.8-fold higher plasma AUC0-∞ (42.2 h*µg/mL) and a
1.25-fold longer half-life T1/2(6.4 h) compared to 7. A favorable oral bioavailability (F =
63%) was also observed leading to the favorable overall PK profiles of 9g. More
encouragingly, when the methyl group was introduced into the N(1)-position of 9g, a
particularly lower total clearance CL (0.02 L/h/kg; 7-fold lower to that of 9g) of 23a was
3
4
5
6
7
8
9
10
11
12
13
14
observed after an oral dose of 5 mg/kg. This led to a markedly higher plasma AUC0-∞ (232.6
14

1
2
3
4
h*µg/mL; 69-fold higher to that of 7) and a much longer half-life T1/2 (20.6 h; 4-fold longer
to that of 7). Overall, compound 23a displayed the best PK profiles. That means that
fine-regulation of N(1)-position could lead to improvements in drug-likeness.
Table 5. In vivo PK profiles of selected compounds in rata,b.
7c
8
9g
23a
2.5 mg/kga 10 mg/kga 2.5 mg/kga 10 mg/kga
1 mg/kga
(i.v.)
5 mg/kga
(i.g.)
1 mg/kga 5 mg/kga
N.O.
(i.v.)
3.2
-
(p.o.)
(i.v.)
(i.g.)
(i.v.)
25.4
-
(i.g.)
20.6
8.7
t1/2 (h)
tmax (h)
5.1
0.5
1.0
6.8
-
6.4
7.3
2.7
42.2
1.1
0.1
11.4
63
1.2
0.03
16.4
4.5
2.7
Cmax (µg/mL)
-
1.6
0.6
1.9
13.5
0.7
0.08
8.5
-
6.5
7.8
3.1
6.7
2.2
80.6
232.6
AUC0-∞ (h*µg/mL)
Vz (L/kg)
3.6
0.8
-
-
-
0.4
0.6
0.3
-
7.5
5.0
3.0
12
0.4
0.01
10.8
-
0.6
0.02
32.4
58
CL (L/h/kg)
MRT (h)
-
-
54
F (%)
5
6
7
8
a Vehicle: 70% PEG400-30% water. Cmax, maximum concentration; Tmax, time of maximum concentration;
T1/2, half-lif; AUC0-∞, area under the plasma concentration time curve; CL, clearance; VZ, volume of
distribution; and F, oral bioavailability. b Data reported as the average of three animals. cOur previously
reported lead compound26.
9
4. Conclusions
10
11
12
13
14
15
16
17
18
19
20
Our previous research of 2,7-naphthyridinone-based class II MET kinase inhibitors was
combined with a scaffold-hopping strategy of heteroatom migration to make compound 8
with a 1,6-naphthyridin-4(1H)-one moiety. This served as the lead compound with good
potency but poor pharmacokinetic profiles (F = 12%, CL = 5.0 L/h/kg after an oral dose of
10 mg/kg). The detailed SAR studies of block A resulted in the discovery of the new
1,6-naphthyridone-based MET kinase inhibitor 9g (MET, IC50 of 9.8 nM) with a comparable
MET potency to that of compound 2 and favorable pharmacokinetic profiles (F = 63%, CL =
0.1 L/h/kg, AUC0-∞ = 42.2 h*µg/ml, T1/2 = 6.4 h after oral dose of 5 mg/kg). Additional work
led to the functionalizable N(1) amine group and 23a (MET, IC50 of 7.1 nM). More
importantly, by tuning the N(1)-position, 23a displayed an amazing VEGFR-2 selectivity
improvement (3226-fold) as well as an improvement in drug-likeness (F = 58%, CL = 0.02
15

1
2
3
4
L/h/kg, AUC0-∞ = 232.6 h*µg/ml, T1/2 = 20.6 h after oral dose of 5 mg/kg). Excellent
VEGFR-2 selectivity and the favorable drug-likeness of 23a have shown that the
1,6-naphthyridine moiety could be used as a new scaffold in kinase inhibitor discovery.
Further studies are underway.
5
6
5. Experimental
5.1. General Methods
7
Unless otherwise noted, all chemical reagents were commercially available and treated
with standard methods. Silica gel column chromatography (CC). silica gel (200-400 Mesh;
Qingdao Makall Group Co., Ltd; Qingdao; China). Solvents were dried in a routine way
and redistilled. All reactions involving air- or moisture-sensitive reagents were performed
under a nitrogen or argon atmosphere. Melting points of compounds were measured on a
Melt-Temp II apparatus and uncorrected. 1H NMR spectra (400 MHz ) and 13C NMR (100
MHz) spectra were recorded on a Bruker BioSpin AG (Ultrashield Plus AV 400)
spectrometer as deuterochloroform (CDCl3) or dimethyl sulfoxide-d6 (DMSO-d6) solutions
using tetramethylsilane (TMS) as an internal standard (δ = 0) unless noted otherwise. MS
spectra were obtained on an Agilent technologies 6120 quadrupole LC/MS (ESI).
High-resolution mass spectra (HR-MS) were obtained on an Agilent 6224 TOF LC/MS
(USA) All reactions were monitored using thin-layer chromatography (TLC) on silica gel
plates. Yields were of purified compounds and were not optimized.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
5.2. General procedures for the synthesis of intermediates 11, 13, 14
5.2.1. (Z)-ethyl 2-(4-fluorophenyl)-3-hydroxyacrylate (11). A dried flask was charged with
sodium hydride (12 mmol), ethyl 2-(4-fluorophenyl)acetate 10 (10 mmol), ethyl format (12
mmol) and anhydrous DMF (50 mL) under nitrogen. The mixture was heated to 100 °C and
stirred for 3 h. Ice-cold water was added, and the mixture was extracted with
dichloromethane. The organic layer was dried and concentrated. The residue was purified
by chromatography (Hexane/EA = 5:1) to yield 11 as a yellow liquid (90%). 1H NMR (600
MHz, DMSO-d6) δ 11.03 (s, 1H), 7.87 (s, 1H), 7.32 (t, J = 8.0, 5.4 Hz, 2H), 7.12 (t, J = 9.0
Hz, 2H), 4.11 (q, J = 7.2 Hz, 2H), 1.21 (t, J = 7.2 Hz, 3H). MS (ESI): 210.1 (M)+.
16

1
2
5.2.2. (Z)-ethyl 3-((2-chloropyridin-4-yl)amino)-2-(4-fluorophenyl)acrylate (13).
A
solution of 11 (5 mmol), 2-chloropyridin-4-amine (12, 5 mmol) and HCl (20 mmol%) in
3
ethanol (30 mL) was heated to 75 °C under nitrogen for 2 h. The mixture was filtered, and
1
4
the solid was washed with ice-cold ethanol to yield 13 as a yellow solid (83%). H NMR
5
(400 M, DMSO-d6) δ 8.17 (d, J = 5.6 Hz, 1H), 8.05 (d, J = 13.2 Hz, 1H), 7.41-7.45 (m,
1H), 7.06-7.11 (m, 2H), 7.02 (d, J = 9.6 Hz, 1H), 6.84 (s, 2H), 6.72 (d, J = 5.6 Hz, 1H),
6.54 (d, J = 13.2 Hz, 1H), 4.25 (q, J = 7.2 Hz, 1H), 1.29 (t, J = 7.2 Hz, 1H); MS (ESI):
320.1 (M)+.
6
7
8
9
5.2.3. 5-chloro-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one (14). A solution of 13 (3
mmol) in diphenyl oxide (20 mL) was heated to 230 °C under nitrogen for 2 h. The mixture
was cooled to room temperature and the resulted solid was washed with petroleum
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1
(30-60°C) to afford 14 as yellow solid (11%). H-NMR (400 M, DMSO-d6) δ 12.40 (bs,
1H), 8.31 (d, J = 5.6 Hz, 1H), 8.16 (s, 1H), 7.66 (d, J = 5.6 Hz, 2H), 7.47 (d, J = 5.6 Hz,
2H), 7.23 (d, J = 6.8 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) δ173.7, 162.5, 160.9,
150.6, 148.1, 147.6, 138.1, 131.1, 124.4, 117.9, 115.1, 113.0. MS (ESI): 274.0 (M)+.
5.3. General procedures for the synthesis of intermediates 15, 19, 20a-l
5.3.1. 4-(4-amino-2-fluorophenoxy)-3-chloropicolinamide (15). The intermediate 15 was
smoothly obtained using a modified method from previous published work.17 1H NMR (400
MHz, CD3OD) δ 8.29 (d, 1H, J = 5.6 Hz), 7.00 (t, 1H, J = 8.8 Hz), 6.79 (d, 1H, J = 5.6
Hz), 6.63-6.55 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 166.6, 160.8, 154.1, 153.9,
149.0, 148.7, 128.5, 123.7, 115.9, 110.1, 110.0, 101.3; HRMS (ESI): calcd for
C12H9N3O2ClF (M+H+): 282.0440, Found: 282.0447.
5.3.2. 2-chloro-4-(2-fluoro-4-nitrophenoxy)pyridine (19). A dried flask was charged with
2-chloropyridin-4-ol 17 (38.6 mmol), 1,2-difluoro-4-nitrobenzene 18 (38.6 mmol), K2CO3
(46.3 mmol) and DMF (200 ml). The mixture was stirred at room temperature until the
completion of the reaction as monitored by TLC analysis, ice-cold water (600 ml) was
added, and the mixture was stirred for 5h. Then filtered, the filter cake was washed with
1
water and dried under infrared light to yield the desired product 19 (80%). H NMR (600
MHz, DMSO-d6) δ 8.44 (d, J = 10.2 Hz, 1H), 8.40 (d, J = 4.2 Hz, 1H), 8.21 (d, J = 9.0 Hz,
1H), 7.68 (t, J = 7.8 Hz, 1H), 7.33 (s, 1H), 7.20 (s, 1H). MS (ESI): 268.1 (M)+ .
17

1
2
5.3.3. N-(4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl)acetamide (20a). A solution of 19 (3.7
mmol), acetamide (4.4 mmol), dppf (0.372 mmol), Pd2(dba)3 (0.186 mmol) in 1,4-dioxane
(25 mL) was heated to 80 °C and stirred under nitrogen until the completion of the reaction
as monitored by TLC analysis. The mixture was filtered, and the filtrate was concentrate
and purified by chromatography (Hexane/EA = 20:1) to yield 20a as a pink solid (91%). 1H
NMR (600 MHz, DMSO-d6) δ 10.70 (s, 1H), 8.42 (d, J = 10.2 Hz, 1H), 8.28 (d, J = 5.4 Hz,
1H), 8.19 (d, J = 8.4 Hz, 1H), 7.78 (s, 1H), 7.61 (t, J = 8.4 Hz, 1H), 6.83 (s, 1H), 2.06 (s,
3H). MS (ESI): 291.1 (M)+ .
3
4
5
6
7
8
9
5.3.4. N-(4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl)propionamide (20b). Prepared according
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
to
the
procedure
for
the
preparation
of
20a,
from
2-chloro-4-(2-fluoro-4-nitrophenoxy)pyridine 19 and propionamide to yield 20b as yellow
1
solid (85%), H NMR (600 MHz, DMSO-d6) δ 10.70 (s, 1H), 8.46 (d, J = 10.2 Hz, 1H),
8.30 (d, J = 5.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.79 (s, 1H), 7.61 (t, J = 8.4 Hz, 1H),
6.83 (s, 1H), 2.44 (q, J = 7.2 Hz, 2H), 1.10 (t, J = 7.2 Hz, 3H). MS (ESI): 305.1 (M)+ .
5.3.5. N-(4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl)butyramide (20c). Prepared according to
the
procedure
for
the
preparation
of
20a,
from
2-chloro-4-(2-fluoro-4-nitrophenoxy)pyridine 19 and butyramide to yield 20c as white
1
solid (92%), H NMR (400 MHz, DMSO-d6) δ 10.70 (s, 1H), 8.46 (d, J = 10.6 Hz, 1H),
8.31 (d, J = 5.6 Hz, 1H), 8.22 (d, J = 8.8 Hz, 1H), 7.83 (d, J = 2.0 Hz, 1H), 7.64 (t, J = 8.4
Hz, 1H), 6.87 (d, J = 5.6 Hz, 1H), 2.36 (t, J = 7.2 Hz, 2H), 1.57 (m, J = 7.2 Hz, 2H), 0.89
(t, J = 7.6 Hz, 3H). MS (ESI): 319.1 (M)+ .
5.3.6. N-(4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl)pentanamide (20d). Prepared according
to
the
procedure
for
the
preparation
of
20a,
from
2-chloro-4-(2-fluoro-4-nitrophenoxy)pyridine 19 and pentanamide to yield 20d as white
1
solid (92%), H NMR (400 MHz, DMSO-d6) δ 10.65 (s, 1H), 8.43 (dd, J = 10.4, 2.8 Hz,
1H), 8.29 (d, J = 5.6 Hz, 1H), 8.26 – 8.14 (m, 1H), 7.80 (d, J = 2.4 Hz, 1H), 7.62 (t, J = 8.8
Hz, 1H), 6.85 (dd, J = 6.0, 2.4 Hz, 1H), 2.37 (t, J = 7.6 Hz, 2H), 1.52 (q, J = 7.6 Hz, 2H),
1.38 – 1.16 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H). MS (ESI): 333.1 (M)+ .
5.3.7. N-(4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl)isobutyramide (20e). Prepared according
to
the
procedure
for
the
preparation
of
20a,
from
18

1
2
2-chloro-4-(2-fluoro-4-nitrophenoxy)pyridine 19 and isobutyramide to yield 20e as white
1
solid (91%), H NMR (600 MHz, DMSO-d6) δ 10.65 (s, 1H), 8.43 (d, J = 10.2 Hz, 1H),
3
8.29 (d, J = 5.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.80 (s, 1H), 7.62 (t, J = 8.4 Hz, 1H),
6.86 (dd, J = 5.4, 1.8 Hz, 1H), 2.73 (dt, J = 13.2, 6.6 Hz, 1H), 1.04 (s, 6H). MS (ESI):
319.1 (M)+ .
4
5
6
5.3.8. N-(4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl)pivalamide (20f). Prepared according to
7
the
procedure
for
the
preparation
of
20a,
from
8
2-chloro-4-(2-fluoro-4-nitrophenoxy)pyridine 19 and pivalamide to yield 20e as white solid
(87%), 1H NMR (600 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.43 (d, J = 10.2 Hz, 1H), 8.30 (d,
J = 5.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.80 (s, 1H), 7.62 (t, J = 8.4 Hz, 1H), 6.86 (dd, J
= 5.4, 1.8 Hz, 1H), 1.19 (s, 9H). MS (ESI): 333.1 (M)+ .
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
5.3.9.
N-(4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl)cyclopropanecarboxamide
(20g).
Prepared according to the procedure for the preparation of 20a, from
2-chloro-4-(2-fluoro-4-nitrophenoxy)pyridine 19 and cyclopropanecarboxamide to yield
20g as white solid (91%), 1H NMR (600 MHz, DMSO-d6) δ 10.60 (s, 1H), 8.41 (d, J = 9.0
Hz, 1H), 8.28 (d, J = 5.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.83 (s, 1H), 7.62 (t, J = 8.4 Hz,
1H), 6.84 (d, J = 3.6 Hz, 1H), 1.96 (s, 1H), 0.96 – 0.81 (m, 4H). MS (ESI): 317.1 (M)+ .
5.3.10. N-(4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl)cyclobutanecarboxamide (20 h).
Prepared according to the procedure for the preparation of 20a, from
2-chloro-4-(2-fluoro-4-nitrophenoxy)pyridine 19 and cyclobutanecarboxamide to yield 20h
as Light yellow solid (89%), 1H NMR (600 MHz, DMSO-d6) δ 10.45 (s, 1H), 8.41 (d, J =
9.0 Hz, 1H), 8.28 (d, J = 5.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.83 (s, 1H), 7.62 (t, J = 8.4
Hz, 1H), 6.84 (d, J = 3.6 Hz, 1H), 3.40 - 3.30 (m, 1H), 2.23 - 2.13 (m, 2H), 2.08 (d, J = 9.0
Hz, 2H), 1.97 -1.85 (m, 1H), 1.78 (s, 1H). MS (ESI): 331.1 (M)+ .
5.3.11.
N-(4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl)cyclohexanecarboxamide
(20i).
Prepared according to the procedure for the preparation of 20a, from
2-chloro-4-(2-fluoro-4-nitrophenoxy)pyridine 19 and cyclohexanecarboxamide to yield 20i
1
as yellow solid (88%), H NMR (600 MHz, DMSO-d6) δ 10.61 (s, 1H), 8.45 (d, J = 10.2
Hz, 1H), 8.29 (d, J = 5.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.80 (s, 1H), 7.62 (t, J = 8.4 Hz,
19

1
2
1H), 6.86 (t, J = 6.0 Hz, 1H), 2.48 (s, 1H), 1.93 – 1.45 (m, 5H), 1.45 – 0.96 (m, 5H). MS
(ESI): 359.1 (M)+ .
3
5.3.12. N-(4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl)-2-hydroxyacetamide (20j). Prepared
4
according
to
the
procedure
for
the
preparation
of
20a,
from
5
2-chloro-4-(2-fluoro-4-nitrophenoxy)pyridine 19 and 2-hydroxyacetamide to yield 20j as
yellow solid (59%), 1H NMR (400 MHz, DMSO-d6) δ 9.91 (s, 1H), 8.42 (dd, J = 10.4, 2.4
Hz, 1H), 8.28 (d, J = 5.6 Hz, 1H), 8.18 (d, J = 8.8, 2.4, 1.2 Hz, 1H), 7.76 (d, J = 2.4 Hz,
1H), 7.62 (t, J = 8.8 Hz 1H), 6.87 (dd, J = 5.6, 2.4 Hz, 1H), 5.65 (t, J = 6.0 Hz, 1H), 4.01
(d, J = 6.0 Hz, 2H). MS (ESI): 307.1 (M)+ .
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5.3.13.
N-(4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl)-2-hydroxypropanamide
(20k).
Prepared according to the procedure for the preparation of 20a, from
2-chloro-4-(2-fluoro-4-nitrophenoxy)pyridine 19 and 2-hydroxypropanamide to yield 20j
as yellow solid (70%),1H NMR (600 MHz, DMSO-d6) δ 9.84 (s, 1H), 8.45 (d, J = 10.2 Hz,
1H), 8.31 (s, 1H), 8.21 (d, J = 9.6 Hz, 1H), 7.78 (s, 1H), 7.65 (t, J = 6.0 Hz, 1H), 6.91 (s,
1H), 5.86 (s, 1H), 4.20 (s, 1H), 1.27 (s, 3H). MS (ESI): 321.1 (M)+ .
5.3.14.
N-(4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl)-2-hydroxy-2-methylpropanamide
(20l). Prepared according to the procedure for the preparation of 20a, from
2-chloro-4-(2-fluoro-4-nitrophenoxy)pyridine 19 and 2-hydroxy-2-methylpropanamide to
yield 20l as yellow solid (83%), 1H NMR (600 MHz, DMSO-d6) δ 9.62 (s, 1H), 8.54 (d, J =
10.2 Hz, 1H), 8.40 (d, J = 5.4 Hz, 1H), 8.30 (d, J = 9.6 Hz, 1H), 7.84 (s, 1H), 7.75 (t, J =
8.4 Hz, 1H), 7.01 (d, J = 5.4 Hz, 1H), 6.13 (s, 1H), 1.41 (s, 6H). MS (ESI): 335.1 (M)+ .
5.4. General procedures for the synthesis of intermediates 21a-l
5.4.1 N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)acetamide (21a). To the mixture of
N-(4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl)acetamide (20a, 2.06 mmol), NH4Cl (6.18
mmol), ethanol (18 mL) and H2O (2 ml) added iron powder (6.18 mmol) slowly. Upon the
completion of addition, the reaction mixture was heated at reflux for 1 h, and then filtered
immediately. Subsequently, the filtrate was concentrated and anhydrous ether (5.0 mL) was
added to the residue, then stirred for 0.5 h and filtrated to give the compound 21a as a
yellow solid (81%). 1H NMR (600 MHz, DMSO-d6) δ 10.48 (s, 1H), 8.13 (s, 1H), 7.60 (s,
13
1H), 6.95 (s, 1H), 6.79 – 6.15 (m, 3H), 5.44 (s, 2H), 2.02 (s, 3H). C NMR (150 MHz,
20

1
2
DMSO-d6) δ 170.1, 164.0, 154.5, 154.0, 152.3, 150.5, 145.0, 123.4, 121.9, 114.2, 108.5,
101.3, 24.3. MS (ESI): 261.1 (M)+ .
3
5.4.2. N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)propionamide (21b).
Prepared
4
according to the procedure for the preparation of 21a, from 20b to yield 21a as a brown
1
5
solid (85%), H NMR (600 MHz, DMSO-d6) δ 10.42 (s, 1H), 8.11 (s, 1H), 7.61 (s, 1H),
13
6
6.88 (s, 1H), 6.74 – 6.23 (m, 3H), 5.44 (s, 2H), 2.33 (s, 2H), 0.99 (s, 3H). C NMR (150
7
MHz, DMSO-d6) δ 173.4, 166.7, 155.6, 154.3, 149.7, 148.8, 129.5, 124.3, 110.5, 107.3,
101.9, 99.5, 29.7, 9.7. MS (ESI): 276.1 (M+H+).
8
9
5.4.3. N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)butyramide (21c). Prepared according
to the procedure for the preparation of 21a, from 20c to yield 21c as a white solid (88%),
1H NMR (400 MHz, DMSO-d6) δ 10.47 (s, 1H), 8.16 (d, J = 5.6 Hz, 1H), 7.66 (d, J = 2.0
Hz, 1H), 6.99 (t, J = 8.8 Hz, 1H), 6.64 (dd, J = 5.6, 2.4 Hz, 1H), 6.55 (dd, J = 13.2, 2.4 Hz,
1H), 6.46 (dd, J = 8.8, 1.6 Hz, 1H), 5.52 (s, 2H), 2.34 (t, J = 7.2 Hz, 2H), 1.57 (h, J = 7.6
Hz, 2H), 0.89 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 172.6, 166.7, 155.5,
153.9, 149.7, 148.9, 129.5, 124.3, 110.5, 107.3, 101.9, 99.6, 38.4, 18.6, 14.0. MS (ESI):
289.1 (M)+ .
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5.4.4.
N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)pentanamide
(21d).
Prepared
according to the procedure for the preparation of 21a, from 20d to yield 21d as a white
solid (80%),1H NMR (400 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.14 (d, J = 5.6 Hz, 1H), 7.64
(s, 1H), 6.97 (t, J = 9.2 Hz, 1H), 6.62 (dd, J = 5.6, 2.4 Hz, 1H), 6.50 (dd, J = 12.4, 2.0 Hz,
1H), 6.43 (d, J = 8.4 Hz, 1H), 5.46 (s, 2H), 2.34 (t, J = 7.2 Hz, 2H), 1.63 – 1.42 (m, 2H),
13
1.27 (q, J = 7.2 Hz, 2H), 1.01 – 0.73 (m, 3H). C NMR (150 MHz, DMSO-d6) δ 172.8,
166.7, 155.6, 154.3, 149.7, 129.5, 129.4, 124.3, 110.5, 107.3, 101.9, 99.6, 36.2, 27.3, 22.1,
14.10. MS (ESI): 303.1 (M)+ .
5.4.5. N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)isobutyramide (21e). Prepared
according to the procedure for the preparation of 21a, from 20e to yield 21e as a yellow
1
solid (81%), H NMR (600 MHz, DMSO-d6) δ 10.44 (s, 1H), 8.14 (s, 1H), 7.63 (s, 1H),
6.96 (s, 1H), 6.64 (s, 1H), 6.50 (d, J = 12.0 Hz, 1H), 6.41 (s, 1H), 5.46 (s, 2H), 2.70 (s, 1H),
13
1.03 (s, 6H). C NMR (150 MHz, DMSO-d6) δ 176.7, 166.7, 155.6, 154.4, 149.6, 148.8,
129.5, 124.4, 110.5, 107.4, 101.9, 99.5, 34.9, 19.7. MS (ESI): 289.1 (M)+ .
21

1
2
5.4.6. N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)pivalamide (21f). Prepared according
to the procedure for the preparation of 21a, from 20f to yield 21f as a white solid (89%), 1H
NMR (600 MHz, DMSO-d6) δ 9.82 (s, 1H), 8.16 (d, J = 5.4 Hz, 1H), 7.64 (s, 1H), 6.97 (t, J
= 9.0 Hz, 1H), 6.67 (s, 1H), 6.52 (d, J = 13.2 Hz, 1H), 6.43 (d, J = 8.4 Hz, 1H), 5.47 (s,
2H), 1.19 (s, 9H). 13C NMR (150 MHz, DMSO-d6) δ 177.7, 166.7, 155.6, 154.5, 149.4,
148.8, 129.5, 124.4, 110.5, 107.6, 101.9, 100.0, 27.2, 27.2. MS (ESI): 303.2 (M)+ .
3
4
5
6
7
5.4.7.
N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)cyclopropanecarboxamide
(21g).
8
Prepared according to the procedure for the preparation of 21a, from 20g to yield 21g as a
white solid (89%), 1H NMR (600 MHz, DMSO—d6) δ 10.79 (s, 1H), 8.15 (d, J = 5. 4 Hz,
1H), 7.59 (s, 1H), 6.95 (t, J = 9.0 Hz, 1H), 6.67-6.61 (m, 1H), 6.49 (dd, J = 13.2, 2.4 Hz,
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
13
1H), 6.40 (d, J = 8.4 Hz, 1H), 5.44 (s, 2H), 2. 03-1.88 (m, 1H), 0. 76 (br, 4H). C NMR
(150 MHz, DMSO-d6) δ 173.2, 166.7, 155.6, 154.3, 149.7, 148.8, 129.5, 124.3, 110.4,
107.4, 101.9, 99.5, 14.6, 8.1. MS (ESI): 287.1 (M)+ .
5.4.8.
N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)cyclobutanecarboxamide
(21h).
Prepared according to the procedure for the preparation of 21a, from 20h to yield 21h as a
yellow solid (80%), 1H NMR (600 MHz, DMSO-d6) δ 10.37 (s, 1H), 8.15 (s, 1H), 7.67 (s,
1H), 6.99 (s, 1H), 6.80 – 6.06 (m, 3H), 5.51 (s, 2H), 3.38 (s, 1H), 2.17 (s, 2H), 2.06 (s, 2H),
13
1.90 (s, 1H), 1.77 (s, 1H). C NMR (150 MHz, DMSO-d6) δ 174.3, 166.7, 155.6, 154.4,
149.7, 148.9, 124.3, 110.5, 107.3, 101.9, 99.6, 39.6, 24.8, 18.0. MS (ESI): 301.1 (M)+ .
5.4.9.
N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)cyclohexanecarboxamide
(21i).
Prepared according to the procedure for the preparation of 21a, from 20i to yield 21i as a
1
white solid (80%), H NMR (400 MHz, DMSO-d6) δ 10.35 (s, 1H), 8.10 (d, J = 5.6 Hz,
1H), 7.59 (d, J = 2.0 Hz, 1H), 6.94 (t, J = 8.8 Hz, 1H), 6.61 (dd, J = 5.6, 2.4 Hz, 1H), 6.48
(dd, J =13.2, 2.4 Hz 1H), 6.39 (dd, J = 8.8, 2.4 Hz, 1H), 5.46 (s, 2H), 2.44 (s, 1H), 1.71 –
1.67 (m, 5H), 1.43 – 1.05 (m, 5H). 13C NMR (150 MHz, DMSO-d6) δ 175.8, 166.7, 155.6,
154.4, 149.6, 148.9, 129.5, 124.3, 110.5, 107.3, 101.9, 99.5, 44.7, 29.4, 25.8, 25.6. MS
(ESI): 329.2 (M)+ .
5.4.10. N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)-2-hydroxyacetamide (21j). Prepared
according to the procedure for the preparation of 21a, from 20j to yield 21j as a light
1
brown solid (82%), H NMR (400 MHz, DMSO-d6) δ 9.68 (s, 1H), 8.13 (d, J = 5.6 Hz,
22

1
2
1H), 7.59 (s, 1H), 6.95 (t, J = 9.2 Hz, 1H), 6.64 (dd, J = 5.6, 2.4 Hz, 1H), 6.49 (dd, J =
13.2, 2.4 Hz, 1H), 6.40 (dd, J = 8.8, 2.0 Hz, 1H), 5.75 (t, J = 6.0 Hz, 1H), 5.47 (s, 2H), 3.97
13
3
(d, J = 6.0 Hz, 2H). C NMR (150 MHz, DMSO-d6) δ 171.9, 166.8, 155.5, 153.2, 150.0,
4
149.0, 129.4, 124.3, 110.5, 107.8, 101.8, 99.4, 61.9. MS (ESI): 277.1 (M)+ .
5
5.4.11.
N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)-2-hydroxypropanamide
(21k).
6
Prepared according to the procedure for the preparation of 21a, from 20k to yield 21k as a
white solid (81%), 1H NMR (600 MHz, DMSO-d6) δ 9.65 (s, 1H), 8.17 (d, J = 4.8 Hz, 1H),
7.62 (s, 1H), 6.98 (t, J = 8.4 Hz, 1H), 6.70 (s, 1H), 6.51 (d, J = 12.6 Hz, 1H), 6.42 (d, J =
8.4 Hz, 1H), 5.86 (d, J = 4.8 Hz, 1H), 5.48 (s, 2H), 4.16 (t, J = 6Hz, 1H), 1.26 (d, J = 6.6
Hz, 3H). 13C NMR (150 MHz, DMSO-d6) δ 174.3, 166.9, 155.5, 153.2, 150.0, 148.9,
129.4, 110.5, 107.9, 103.9, 101.9, 99.2, 67.7, 21.0. MS (ESI): 291.1 (M)+ .
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5.4.12.
N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)-2-hydroxy-2-methylpropanamide
(21l). Prepared according to the procedure for the preparation of 21a, from 20l to yield 21l
1
as a white solid (79%), H NMR (600 MHz, DMSO-d6) δ 9.41 (s, 1H), 8.19 (s, 1H), 7.64
(s, 1H), 6.99 (s, 1H), 6.88 – 6.29 (m, 3H), 5.97 (s, 1H), 5.44 (s, 2H), 1.35 (s, 6H). 13C NMR
(150 MHz, DMSO-d6) δ 176.1, 167.0, 155.5, 153.0, 150.0, 148.9, 129.4, 124.4, 110.5,
108.1, 101.9, 98.8, 72.9, 27.7. MS (ESI): 305.2 (M)+ .
5.5. General procedures for the synthesis of intermediates 22a-d
5.5.1. 5-chloro-3-(4-fluorophenyl)-1-methyl-1,6-naphthyridin-4(1H)-one (22a). A dried
flask was charged with 5-chloro-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one 14 (1.8
mmol), iodomethane (2.18 mmol), K2CO3 (5.3 mmol) and DMF (5 ml). The mixture was
stirred at room temperature for 3h. Then, the reaction mixturewas poured intowater (60.0
mL), the precipitate was collected by filtration and washed with water, and dried under
infrared light to yield the desired product 22a. 1H NMR (600 MHz, DMSO-d6) δ 8.43 (d, J
= 6.0 Hz, 1H), 8.29 (s, 1H), 7.69 (d, J = 6.6 Hz, 1H), 7.67 (d, J = 6.0 Hz, 1H), 7.61 (d, J =
13
6.0 Hz, 1H), 7.25 (t, J = 8.7 Hz, 2H), 3.85 (s, 3H). C-NMR (100 M, DMSO-d6) δ 173.2,
162.6, 161.0, 151.0, 148.5, 148.0, 143.9, 131.0, 124.1, 118.4, 115.0, 111.4, 41.1. MS (ESI):
288.1 (M)+ .
5.5.2. 5-chloro-1-ethyl-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one (22b). Prepared
according
to
the
procedure
for
the
preparation
of
22a,
from
23

1
2
5-chloro-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one 14 and iodoethane to yield 22b as
1
a white solid (86%), H NMR (600 MHz, DMSO-d6) δ 8.42 (d, J = 6.0 Hz, 1H), 8.31 (s,
3
1H), 7.75 – 7.68 (m, 3H), 7.25 (t, J = 8.7 Hz, 2H), 4.33 (q, J = 7.2 Hz, 2H), 1.36 (t, J = 7.2
Hz, 3H). 13C-NMR (100 M, DMSO-d6) δ 173.3, 162.623, 161.030, 151.403, 148.57336,
147.1, 142.8, 131.1, 124.6, 118.7, 115.1, 111.1, 48.4, 14.3. MS (ESI): 302.1 (M)+ .
4
5
6
5.5.3. 5-chloro-3-(4-fluorophenyl)-1-(2-hydroxyethyl)-1,6-naphthyridin-4(1H)-one (22c).
Prepared according to the procedure for the preparation of 22a, from
5-chloro-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one 14 and oxirane to yield 22c as a
7
8
1
9
white solid (46%), H NMR (600 MHz, DMSO-d6) δ 8.39 (d, J = 6.0 Hz, 1H), 8.19 (s, 1H),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
7.74 (d, J = 6.0 Hz, 1H), 7.72 – 7.65 (m, 2H), 7.24 (t, J =8.4 Hz, 2H), 5.01 (s, 1H), 4.38 (s,
13
2H), 3.76 (s, 2H). C-NMR (100 M, DMSO-d6) δ 173.4, 162.6, 161.0, 151.3, 148.3, 147.8,
144.0, 131.0, 123.8, 118.7, 115.2, 111.4, 59.2, 55.2. MS (ESI): 348.1 (M)+
.
5.5.4.
5-chloro-3-(4-fluorophenyl)-1-(2-hydroxy-2-methylpropyl)-1,6-naphthyridin-4(1H)-one
(22d). Prepared according to the procedure for the preparation of 22a, from
5-chloro-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one 14 and 2,2-dimethyloxirane to yield
22d as a white solid (46%), 1H NMR (600 MHz, DMSO-d6) δ 8.41 (d, J = 6.0 Hz, 1H), 8.21
(s, 1H), 7.76 (d, J = 6.0 Hz, 1H), 7.72 – 7.64 (m, 2H), 7.26 (t, J = 8.7 Hz, 2H), 5.09 (s, 1H),
4.38 (s, 2H), 1.21 (s, 6H). 13C-NMR (100 M, DMSO-d6) δ 173.4, 162.6, 161.0, 151.1, 148.9,
147.7, 144.8, 130.9, 123.3, 118.5, 115.1, 112.7, 70.9, 61.1, 27.6. MS (ESI): 346.1 (M)+ ..
5.6. General procedures for the synthesis of targets 8, 9a-o and 23a-d
5.6.1. 5-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenylamino)-3-(4-fluorophenyl)-
1,6-naphthyridin-4(1H)-one (8).
A
solution of 5-chloro-3-(4-fluorophenyl)-1,6-
naphthyridin-4(1H)-one 14 (0.5 mmol), 4-(4-amino-2-fluorophenoxy)-3-chloropicolin-
amide 15 (0.5 mmol) and PTSA (0.5 mmol) in isopropanol (10 mL) was heated to 90 °C
under nitrogen for 2 h. The mixture was filtered, and the solid was washed with ice-cold
ethanol to yield 3-chloro-4-(2-fluoro-4-((3-(4-fluorophenyl)-4-oxo-1,4-dihydro-1,6-
naphthyridin-5-yl)- amino)phenoxy)picolinamide 16 as yellow solid (80%). A solution of
above carboxy amide derivative 16 (1 mmol) and (diacetoxyiodo)benzene (1.1 mmol) in
acetonitrile (10 mL) was stired at 0 °C for 1 h. The resulted solution was concentrated in
24

1
2
vacuum and purified by chromatography (CH2Cl2/MeOH = 20:1) to yield 8 as a yellow
solid (33%), mp 295-296 °C. 1H-NMR (400 M, DMSO-d6) δ 13.14 (s, 1H), 12.41 (bs, 1H),
8.33 (d, J = 10.8 Hz, 1H), 8.16-8.18 (m, 2H), 7.70-7.76 (m, 3H), 7.46 (d, J = 6.8 Hz, 1H),
7.23-7.29 (m, 3H), 6.86 (d, J = 4.8 Hz, 1H), 6.35 (s, 2H), 5.95 (d, J = 4.8 Hz, 1H);
13C-NMR (100 M, DMSO-d6) δ 178.0, 162.2, 160.1, 157.4, 155.6, 154.4, 152.0, 148.4,
147.2, 146.0, 139.0, 138.3, 134.2, 131.0, 130.8, 123.3, 122.7, 116.0, 114.7, 108.1, 106.9,
103.2, 100.1. MS (ESI). 492.1 [M+H]+. HR-MS (EI) m/z calcd for C25H16ClF2N5O2,
491.0961; found 492.1039 [M+H]+.
3
4
5
6
7
8
9
5.6.2.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
N-(4-(2-fluoro-4-((3-(4-fluorophenyl)-4-oxo-1,4-dihydro-1,6-naphthyridin-5-yl)amino)phe
noxy)pyridin-2-yl)acetamide
4-methylbenzenesulfonate
naphthyridin-4(1H)-one
(9a).
14
A
solution
of
5-chloro-3-(4-fluorophenyl)-1,6-
(0.5 mmol),
N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)acetamide 21a (0.5 mmol) and PTSA (0.5
mmol) in isopropanol (10 mL) was heated to 90 °C under nitrogen for 2 h. The mixture was
filtered, and the solid was washed with ice-cold ethanol to yield
N-(4-(2-fluoro-4-((3-(4-fluorophenyl)-4-oxo-1,4-dihydro-1,6-naphthyridin-5-yl)amino)phe
noxy)pyridin-2-yl)acetamide 4-methylbenzenesulfonate (9a) as white solid (61%), m.p.
248-249 °C; 1H NMR (600 MHz, DMSO-d6) δ 13.42 (s, 1H), 13.18 (s, 1H), 11.57 (s, 1H),
8.31 (s, 2H), 8.16 – 7.94 (m, 2H), 7.74 (s, 2H), 7.61 – 7.41 (m, 4H), 7.37 (s, 1H), 7.32 –
7.20 (m, 2H), 7.16 – 7.06 (m, 3H), 7.04 (s, 2H), 2.28 (s, 3H), 2.15 (s, 3H). 13C NMR (150
MHz, DMSO-d6) δ 177.2, 171.6, 168.5, 162.9, 154.8, 154.4, 153.2, 151.4, 147.5, 145.7,
144.4, 139.2, 138.3, 136.1, 131.8, 131.2, 130.5, 128.5, 125.8, 124.9, 124.6, 121.8, 115.3,
115.2, 109.0, 107.1, 104.4, 100.2, 24.4, 21.2. HRMS (ESI): calcd for C27H19F2N5O3
[M+Na]+ 522.1348, found: 522.1349.
5.6.3.
N-(4-(2-fluoro-4-((3-(4-fluorophenyl)-4-oxo-1,4-dihydro-1,6-naphthyridin-5-yl)amino)phe
noxy)pyridin-2-yl)propionamide 4-methylbenzenesulfonate (9b). Prepared according to the
procedure
for
the
preparation
of
9a,
from
and
5-chloro-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one
14
N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)propionamide 21b to yield 9b as a white
25

1
1
2
solid (52%), m.p. 257-258 °C; H NMR (600 MHz, DMSO-d6) δ 13.42 (s, 1H), 13.16 (s,
1H), 11.53 (s, 1H), 8.36 – 8.25 (m, 2H), 8.12 (s, 1H), 8.03 (d, J = 5.4 Hz, 1H), 7.80 – 7.64
(m, 2H), 7.54 (s, 2H), 7.51 – 7.43 (m, 2H), 7.38 (s, 1H), 7.35 – 7.20 (m, 2H), 7.17 – 7.07
(m, 3H), 7.07 – 6.97 (m, 2H), 2.45 (q, J = 7.2 Hz, 2H), 2.29 (s, 3H), 1.07 (t, J = 7.2 Hz,
3
4
13
5
3H). C NMR (150 MHz, DMSO-d6) δ 177.2, 175.3, 168.8, 162.9, 161.3, 154.8, 154.4,
6
153.2, 151.2, 147.6, 145.6, 143.8, 139.2, 138.4, 131.3, 130.5, 128.6, 125.9, 125.0, 124.7,
115.4, 115.3, 109.2, 107.0, 104.4, 100.2, 29.9, 21.2, 9.1. HRMS (ESI): calcd for
C28H21F2N5O3 [M+Na]+ 536.1505, found: 536.1505.
7
8
9
5.6.4.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
N-(4-(2-fluoro-4-((3-(4-fluorophenyl)-4-oxo-1,4-dihydro-1,6-naphthyridin-5-yl)amino)phe
noxy)pyridin-2-yl)butyramide 4-methylbenzenesulfonate (9c). Prepared according to the
procedure
for
the
preparation
of
9a,
from
and
5-chloro-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one
14
N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)butyramide 21c to yield 9c as a white solid
1
(57%), m.p. 251-252 °C; H NMR (600 MHz, DMSO-d6) δ 13.44 (s, 2H), 11.82 (s, 1H),
8.32 (d, J = 4.8 Hz, 2H), 8.09 (s, 1H), 8.02 (d, J = 6.6 Hz, 1H), 7.80 – 7.72 (m, 2H), 7.60 –
7.51 (m, 2H), 7.51 – 7.44 (m, 3H), 7.29 (t, J = 8.4 Hz, 2H), 7.23 (s, 1H), 7.15 – 7.06 (m,
3H), 2.44 (t, J = 6.6 Hz, 2H), 2.29 (s, 3H), 1.62 (q, J = 7.2 Hz, 2H), 0.91 (t, J = 7.2 Hz, 3H).
13C NMR (150 MHz, DMSO-d6) δ 177.2, 174.5, 168.7, 162.9, 161.3, 154.8, 154.4, 153.2,
151.3, 147.6, 145.7, 143.9, 139.2, 138.3, 131.3, 130.5, 128.6, 125.9, 125.0, 124.7, 115.4,
115.3, 109.1, 107.0, 104.4, 100.4, 38.4, 21.2, 18.2, 13.8. HRMS (ESI): calcd for
C29H23F2N5O3 [M+Na]+ 550.1661, found: 550.1662.
5.6.5.
N-(4-(2-fluoro-4-((3-(4-fluorophenyl)-4-oxo-1,4-dihydro-1,6-naphthyridin-5-yl)amino)phe
noxy)pyridin-2-yl)pentanamide 4-methylbenzenesulfonate (9d). Prepared according to the
procedure
for
the
preparation
of
9a,
from
and
5-chloro-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one
14
N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)pentanamide 21d to yield 9d as a white solid
1
(54%), m.p. 250-251 °C; H NMR (600 MHz, DMSO-d6) δ 13.44 (s, 1H), 13.26 (s, 1H),
11.68 (s, 1H), 8.38 – 8.26 (m, 2H), 8.10 (s, 1H), 8.02 (d, J = 6.0 Hz, 1H), 7.77 – 7.72 (m,
26

1
2
2H), 7.63 – 7.51 (m, 2H), 7.48 (d, J = 7.8 Hz, 2H), 7.38 (s, 2H), 7.30 (t, J = 9.0 Hz, 2H),
7.19 (d, J = 5.4 Hz, 1H), 7.12 (d, J = 7.8 Hz, 2H), 7.06 (d, J = 6.6 Hz, 1H), 2.43 (t, J = 4.8
Hz, 2H), 2.34 (, 3H), 1.57 (q, J = 7.2 Hz, 2H), 1.31 (dt, J = 7.2 Hz, 2H), 0.88 (t, J = 7.2 Hz,
3
13
4
3H). C NMR (150 MHz, DMSO-d6) δ 177.2, 174.7, 168.8, 162.9, 161.3, 154.8, 154.3,
5
153.2, 151.1, 147.6, 145.5, 143.7, 139.2, 138.4, 136.2, 131.3, 131.2, 130.5, 128.6, 125.9,
125.1, 124.7, 115.4, 115.3, 109.3, 107.0, 104.4, 100.3, 36.3, 26.8, 22.0, 21.2, 14.1. HRMS
(ESI): calcd for C30H25F2N5O3 [M+Na]+ 564.1817, found: 564.1819.
5.6.6.
6
7
8
9
N-(4-(2-fluoro-4-((3-(4-fluorophenyl)-4-oxo-1,4-dihydro-1,6-naphthyridin-5-yl)amino)phe
noxy)pyridin-2-yl)isobutyramide 4-methylbenzenesulfonate (9e). Prepared according to the
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
procedure
for
the
preparation
of
9a,
from
and
5-chloro-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one
14
N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)isobutyramide 21e to yield 9e as a white
1
solid (54%), m.p. 215-216 °C; H NMR (600 MHz, DMSO-d6) δ 13.43 (s, 1H), 13.19 (s,
1H), 11.55 (s, 1H), 8.35 – 8.28 (m, 2H), 8.10 (s, 1H), 8.02 (d, J = 6.0 Hz, 1H), 7.74 (s, 2H),
7.54 (s, 2H), 7.48 (d, J = 7.8 Hz, 2H), 7.44 (s, 1H), 7.28 (t, J = 8.4 Hz, 2H), 7.17 (s, 1H),
7.12 (d, J = 7.8 Hz, 2H), 7.04 (d, J = 6.6 Hz, 1H), 2.85 – 2.63 (m, 1H), 2.28 (s, 3H), 1.11
(m, 6H). 13C NMR (150 MHz, DMSO-d6) δ 178.5, 177.2, 168.9, 162.9, 161.3, 154.8, 154.3,
153.2, 151.3, 147.6, 145.5, 143.6, 139.2, 138.4, 131.3, 130.4, 128.6, 125.9, 125.1, 124.8,
122.5, 115.4, 115.3, 109.4, 107.0, 104.4, 100.4, 35.5, 21.2, 19.2. HRMS (ESI): calcd for
C29H23F2N5O3 [M+Na]+ 550.1661, found: 550.1660.
5.6.7.
N-(4-(2-fluoro-4-((3-(4-fluorophenyl)-4-oxo-1,4-dihydro-1,6-naphthyridin-5-yl)amino)phe
noxy)pyridin-2-yl)pivalamide 4-methylbenzenesulfonate (9f). Prepared according to the
procedure
for
the
preparation
of
9a,
from
and
5-chloro-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one
14
N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)pivalamide 21f to yield 9f as light yellow
1
solid (51%), m.p. 196-197 °C; H NMR (600 MHz, DMSO-d6) δ 13.40 (s, 1H), 13.02 (s,
1H), 10.97 (s, 1H), 8.38 (d, J = 6.6 Hz, 1H), 8.30 (d, J = 5.4 Hz, 1H), 8.20 (s, 1H), 8.06 (s,
1H), 7.74 (dd, J = 7.8, 6.0 Hz, 2H), 7.56 (s, 3H), 7.48 (d, J = 7.8 Hz, 2H), 7.32 – 7.25 (m,
27

1
2
3H), 7.12 (d, J = 7.8 Hz, 2H), 7.01 (d, J = 5.4 Hz, 1H), 2.35 – 2.13 (m, 3H), 1.25 (s, 9H).
13C NMR (150 MHz, DMSO-d6) δ 180.0, 177.2, 169.5, 162.9, 161.3, 154.8, 154.5, 153.1,
151.2, 147.5, 145.6, 142.4, 141.2, 139.2, 138.3, 136.5, 131.3, 130.5, 128.6, 125.9, 125.1,
124.7, 115.4, 115.3, 109.9, 107.0, 104.4, 100.7, 26.7, 21.2. HRMS (ESI): calcd for
C30H25F2N5O3 [M+H]+ 542.1998, found: 542.1994.
3
4
5
6
5.6.8.
7
N-(4-(2-fluoro-4-((3-(4-fluorophenyl)-4-oxo-1,4-dihydro-1,6-naphthyridin-5-yl)amino)phe
noxy)pyridin-2-yl)cyclopropanecarboxamide 4-methylbenzenesulfonate (9g). Prepared
8
9
according
to
the
procedure
for
the
preparation
of
14
9a,
from
and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5-chloro-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one
N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)cyclopropanecarboxamide 21g to yield 9g as
light yellow solid (46%), m.p. 220-222 °C; 1H NMR (600 MHz, DMSO-d6) δ 13.43 (s, 1H),
13.23 (s, 1H), 11.94 (s, 1H), 8.34 – 8.27 (m, 2H), 8.09 (d, J = 10.8 Hz, 1H), 8.01 (d, J = 5.4
Hz, 1H), 7.78 – 7.71 (m, 2H), 7.57 – 7.51 (m, 2H), 7.48 (d, J = 6.6 Hz, 2H), 7.42 (s, 1H),
7.29 (t, J = 7.8 Hz, 2H), 7.16 – 7.09 (m, 3H), 7.05 (d, J = 5.4 Hz, 1H), 2.28 (s, 3H), 1.96 (s,
1H), 0.96 – 0.81 (m, 4H). 13C NMR (150 MHz, DMSO-d6) δ 177.2, 175.1, 168.7, 162.9,
161.3, 154.8, 154.3, 153.2, 151.1, 147.6, 145.5, 143.9, 139.2, 138.4, 135.9, 131.3, 131.2,
130.4, 128.6, 125.9, 125.1, 124.8, 115.4, 115.3, 109.2, 107.0, 104.4, 100.3, 21.2, 15.2, 9.5.
HRMS (ESI): calcd for C29H21F2N5O3 [M+H]+ 526.1612 , found: 526.1613.
5.6.9.
N-(4-(2-fluoro-4-((3-(4-fluorophenyl)-4-oxo-1,4-dihydro-1,6-naphthyridin-5-yl)amino)phe
noxy)pyridin-2-yl)cyclobutanecarboxamide 4-methylbenzenesulfonate (9h). Prepared
according
to
the
procedure
for
the
preparation
of
14
9a,
from
and
5-chloro-3-(4-fluorophenyl)-1,6-naphthyridin-4(1H)-one
N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)cyclobutanecarboxamide 21h to yield 9h as
light yellow solid (46%), m.p. 235-236 °C; 1H NMR (400 MHz, DMSO-d6) δ 13.68 (s, 1H),
13.41 (s, 1H), 11.61 (s, 1H), 8.34 – 8.26 (m, 2H), 8.17 (s, 1H), 8.08 (d, J = 6.0 Hz, 1H),
7.77 (dd, J = 8.4, 5.6 Hz, 2H), 7.63 (s, 1H), 7.59 – 7.45 (m, 4H), 7.30 (t, J = 8.8 Hz, 2H),
7.21 – 7.11 (m, 5H), 3.47 – 3.36 (m, 1H), 2.31 (s, 3H), 2.28 – 2.20 (m, 2H), 2.20 – 2.09 (m,
13
2H), 2.02 – 1.90 (m, 1H), 1.87 – 1.75 (s, 1H). C NMR (150 MHz, DMSO-d6) δ 177.3,
28