Novel 7-formyl-naphthyridyl-ureas derivatives as potential selective FGFR4 inhibitors: Design, synthesis, and biological activity studies

Article abstract of DOI:10.1016/j.bmc.2019.04.018

Total twenty-five 7-formyl-naphthyridyl-urea derivatives were designed, synthesized and evaluated for their inhibition of FGFR4 kinase and antitumor activity. The pharmacological data indicated that most of the tested compounds showed high selectivity towards FGFR4 kinase and could significantly inhibit FGFR4 and the tumor cells lines with the high expression of FGFR4. In particular, compounds 6f, 6g, 6h, 6l, 6m and 6s showed a good performance in pharmacokinetic tests. When tested in mice, the representative compound 6f was found to have good pharmacokinetic parameters, low toxicity, and better tumor inhibiting activity in vivo.

Full text of DOI:10.1016/j.bmc.2019.04.018

Accepted Manuscript
Novel 7-Formyl-naphthyridyl-ureas Derivatives as Potential Selective FGFR4
Inhibitors: Design, Synthesis, and Biological Activity Studies
Chang'an Sun, Lei Fang, Xiaobing Zhang, Peng Gao, Shaohua Gou
PII:
S0968-0896(19)30427-4
https://doi.org/10.1016/j.bmc.2019.04.018
BMC 14872
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
14 March 2019
8 April 2019
10 April 2019
Please cite this article as: Sun, C., Fang, L., Zhang, X., Gao, P., Gou, S., Novel 7-Formyl-naphthyridyl-ureas
Derivatives as Potential Selective FGFR4 Inhibitors: Design, Synthesis, and Biological Activity Studies, Bioorganic
&
Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.04.018
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Novel 7-Formyl-naphthyridyl-ureas Derivatives as Potential Selective FGFR4 Inhibitors: Design,
Synthesis, and Biological Activity Studies
a,c
a,b,∗
a,c
c
a,∗
Chang’an Sun , Lei Fang , Xiaobing Zhang , Peng Gao , Shaohua Gou
a
Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research and School of Chemistry and Chemical Engineering, Southeast
University, Nanjing 211189, China
b
State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, Guilin 541004,
China
c
Jiangsu Hansoh Pharmaceutical Group CO., LTD., Lianyungang 222000, China
*
Corresponding authors: sgou@seu.edu.cn (S. Gou); lei.fang@seu.edu.cn (L.Fang)
Key words: FGFR4; selectivity; antitumor; pharmacokinetic profile
Abstract Total twenty-five 7-formyl-naphthyridyl-urea derivatives were designed, synthesized and evaluated
for their inhibition of FGFR4 kinase and antitumor activity. The pharmacological data indicated that most of the
tested compounds showed high selectivity towards FGFR4 kinase and could significantly inhibit FGFR4 and the
tumor cells lines with the high expression of FGFR4. In particular, compounds 6f, 6g, 6h, 6l, 6m and 6s showed a
good performance in pharmacokinetic tests. When tested in mice, the representative compound 6f was found to have
good pharmacokinetic parameters, low toxicity, and better tumor inhibiting activity in vivo.
1
. Introduction
The biological action of fibroblast growth factors (FGFs) is mediated by specific cell surface receptors belonging
to the Receptor Protein Tyrosine Kinase (RPTK) family. These proteins consist of an extracellular ligand binding
domain, a single transmembrane domain and an intracellular tyrosine kinase domain which undergoes
phosphorylation upon binding of FGF. Four fibroblast growth factor receptors (FGFRs) have been identified to date:
FGFR1, FGFR2, FGFR3, and FGFR4. FGFR signaling is involved in the control of multiple developmental and
physiological processes, as well as being implicated as a driver in certain forms of cancers such as primary breast
cancer,[1] gastric cancer,[2] uterine cervical cancer,[3] colorectal cancer,[4] prostate cancer,[5] pancreatic cancer,[6]
hepatocarcinoma,[7] and non-small-cell lung cancer[8] et al.
A number of pan-FGFR inhibitors, which target the ATP binding site within the kinase domain of the receptor,
have been described and are characterized as being either: biased towards FGFR 1-3 inhibition, with >10-fold lower
1

FGFR4 potency; or equipotent versus all four family members.[9,10] However, for all of these agents the robust
inhibition of FGFR4 often leads to extensive inhibition of the other three isoforms, and as a result will be associated
with the side effects resulting from FGFR 1-3 inhibition.[11] Therefore, the opportunity to identify selective
inhibitors of FGFR4 was investigated with the anticipation that it would provide better tolerated treatments, and
enable more robust inhibition of the target to be explored.
To explore this possibility, scientists have conducted a series of studies in the past years and a number of
inhibitors have reported. PD166866 with a structure of quinazoline [12] and compound 1 with a structural of
imidazolopyridine are reported to achieve FGFR4 selectivity.[13] Furthermore, the P-loop cysteine in the FGFR
family has been targeted for pan-FGFR inhibition in an irreversible-covalent manner with the acrylamide containing
inhibitors FIN-1[14] and PRN1371[15], and in a reversible-covalent manner with the structurally-related cyano-
acrylamides, as exemplified by 2.[16] In 2015, Hagel, et. al reported the first selective FGFR4 inhibitor BLU9931 [17]
via adding an acrylamide moiety to provide a selectivity of less than 10 nM activity against FGFR4 and larger than
00-fold over FGFR1. They also indicated that the structure-based design of this molecule involved the covalent
,
1
targeting of a unique Cysteine 552 (Cys552) residue in the hinge region of the receptor. Based on the structure of
BLU9931, several similar compounds such as BLU554[18], H2B6527[19], 3[20], 4[21], 5[22] were reported (Figure 1).
Figure 1. Structures of representative FGFR inhibitors.
Recently, Novartis report a new compound FGF401, a highly-selective inhibitor of FGFR4 which is currently
undergoing clinical evaluation for the treatment of FGFR4 and β-klotho positive solid tumors.[23-25] Further research
2

indicated that 7-formylpyridine ureas (7-FPUs) improved potency and physicochemical properties compared to 7-
formylquinoline amide, and revealed that the reversible-covalent mechanism of inhibition the 7-FPUs exhibited
slowed binding kinetics. Moreover, the aldehyde, as the putative electrophile, was demonstrated to be a key
structural element for activity. The cyano group in the 5-position was also of particular interest based upon the low
lipophilicity.[26] In an ongoing effort to discover novel FGFR4 inhibitors and build diversification of chemical
library, we continued to develop structurally relevant compounds 6 with diverse substituent on naphthyridine and
pyridine. Herein, we reported the synthesis and the biological characterizations of a new class of 7-FPUs derivatives
with convenient raw materials availability and industrial manufacture (Figure 2).
Figure 2. Core modifications lead to the 7-FPUs derivatives
2
. Results and discussion
Chemistry The designed inhibitors were readily prepared by using a condensation reaction as the key step
(
Scheme 1).27 Briefly, commercially available 6-amino-4-fluoronicotinonitrile 7 was treated with different
2
substituted H-R giving the corresponding intermediates 8 through a nucleophilic substitution reaction. Different 6-
position substituted 7-(dimethoxymethyl)-1,2,3,4-tetrahydro-1,8-naphthyridine compounds 9 were treated with
diphenyl carbonate under the presence of LiHMDS to afford the phenoxycarbonyl protected products 10. After a
nucleophilic substitution reaction of compounds 8 and 10, compounds 11 were reacted with HCl to produce the
designed molecules 6a-6y.
3

Scheme 1 Synthetic Route of Optically Active Compounds 6: Reagents and conditions: a) compound 7, H-R2,
o
DIPEA, DMF, 60 C; b) compound 9, diphenyl carbonate, THF, LiHMDS, rt; c) compound 8, 10, THF, LiHMDS,
2
rt; d) compound 11, HCl, THF/H O = 11/4, rt, 2h.
Kinase and cell inhibition activity The obtained compounds were examined for their ability to inhibit FGFR 1
and 4 using an enzyme assay, and furthermore a cellular assay was performed to determine the inhibition activity of
7
-FPUs derivatives against Hep 3B and HuH-7 with the high expression of FGFR4 as well as SK-HEP-1 cells
without FGFR4 gene amplification[28,29] (Table 1-3). All of the compounds showed significant kinase inhibition
activity against FGFR4 while little activity against FGFR1. Some compounds showed the FGFR4 selectivity >1000
vs FGFR1. The reason is probably that, similar to the previous study of FGF401, the aldehyde was demonstrated to
be a key structural element for activity. Besides, the 7-formyl-naphthyridyl-ureas scaffold was also involved the
covalent targeting of a unique Cysteine 552 (Cys552) residue in the hinge region of the receptor. In the cellular
assay the target compounds showed significant inhibition to the cell lines with the high expression of FGFR4
including cell lines Hep 3B and HuH-7. In contrast, the compounds showed little activity against cell line SK-HEP-1
without FGFR4 gene amplification. So it is clear that the 7-FPUs derivatives had high selectivity to FGFR4 kinase.
1
For initial structure-activity relationship exploration, a series of compounds with a ring substituent of R and an
2
open chain substituent of R were studied. Compound 6a was served as a benchmark to analyze both enzymatic and
cellular potency contributions from different substitution patterns. Similar to the positive control FGF401,
compound 6a showed significant kinase inhibition to FGFR4 with IC50 of 1.6 nM and showed significant inhibition
2
to both cell lines Hep 3B (8.9 nM) and HuH-7 (16.3 nM). As for the R substituent, it was found that changing the
NH group in 6a to S atom in 6b or O atom in 6c led to a little loss of potency; further introduction of an α-methyl
into 6c afforded 6d, which demonstrated an enhancement of cellular potency compared to 6c. Increasing the ring
1
size of R from 2-oxo-morpholin to the 7-membered analogues 2-oxo-oxazepan (e.g. 6e) retained a similar level of
4

2
activity as compared with 6d. Interestingly, when R-1-methoxy-N-methylpropan-2-amine was introduced into R
group, the resulting compound 6f was more potent than the other compounds with IC50 of 1.5 nM against FGFR4
and IC50 of 1.1 nM and 2.5 nM against Hep 3B and HuH-7 respectively. Furthermore, reducing the ring size to the 5-
membered analogues, the resulting compounds 6g and 6h demonstrated a further enhancement of kinase inhibition
activity and cellular potency as compared with 6a. However, less active was observed in compound 6i case which
2
containing S-1-methoxy-N-methylpropan-2-amine as R group.
1
2
Table 1 Activity of 7-FPUs derivatives with a ring substituent of R and an open chain substituent of R
Structure
R2
Enzyme IC50 ± SEM (nM)
Cell IC50 ± SEM（nM）
Entry Compounds
R1
FGFR1
FGFR4
Hep 3B
HuH-7
SK-HEP-1
1
FGF401
>10000
1.9±0.3
8.8±0.8
16.3±0.5
NDa
2
3
4
5
6a
6b
6c
6d
>10000
>10000
>10000
>10000
1.6±0.2
4.6±0.1
4.5±0.03
1.3±0.1
8.9±0.7
17.3±0.7
>10000b
>10000
>10000
>10000
20.8±2.4 32.2±0.8
27.7±1.8 51.8±2.5
2.9±0.3
2.2±0.3
7.8±0.5
4.6±0.1
6
7
6e
6f
>10000
>10000
2.1±0.1
1.5±0.2
>10000
>10000
1.1±0.1
2.5±0.2
8
9
6g
>10000
1.2±0.1
1.9±0.05
2.3±0.2
8.3±0.3
9.0±0.1
>10000
6h
6i
>10000
>10000
1.7±0.02
5.0±0.1
>10000
>10000
1
0
13.6±0.05 40.5±0.6
a
“
ND” means “not determined”. b “>10000” means that the corresponding compound did not show any
activity at the concentration over 10000 nM.
In order to further investigate the influence of R substituent on the activity of the target compounds, compounds
2
1
2
6
j-6r with a ring substituent of R and a ß-ring substituent of R were designed and prepared. Investigation suggested
that the aryl substitution in compounds 6j and 6k showed a significant loss of kinase activity and cell line inhibition
because the aryl substituents had a planar structure while other substituents had a three-dimensional structure. The S-
tetrahydrofuran-3-thiol and R- tetrahydrofuran-3-thiol substituted compounds 6l and 6m exhibited a moderate
FGFR4 inhibitory potency while compound with the substitution of (1R,2R)-2-methoxycyclopentanamine (6n) or
5

(
3S,4S)-3,4-dimethoxytetrahydro-2H-pyran (6o) showed excellent activity in both enzyme and cellular tests,
2
suggesting that the R group size had a great influence on the inhibitory potency. When the tetrahydrofuran-3-amine
2
1
group was introduced to the R , increasing the ring size of R to the 7-membered analogues or reducing the ring size
to the 5-membered analogues resulted in molecules 6p, 6q and 6r which all demonstrated excellent inhibitory
activity of kinase activity and cell line inhibition, respectively.
1
2
Table 2 Activity of 7-FPUs derivatives with a ring substituent of R and an ß-ring substituent of R
Structure
R1
Enzyme IC50 ± SEM (nM)
Cell IC50 ± SEM（nM）
Entry Compounds
R2
FGFR1
FGFR4
Hep 3B
HuH-7
SK-HEP-1
1
2
6j
>10000
71±1
46±4
69±2
>10000
>10000
6k
>10000
38±0.03
69±2.7 20.7±0.01
3
4
6l
>10000
>10000
5.3±0.02
7.5±0.2
14.5±0.3 36.1±0.2
22.7±0.2 50.1±0.3
>10000
>10000
6m
5
6
6n
6o
>10000
>10000
1.0±0.01
2.7±0.04
1.0±0.1
2.7±0.04
>10000
>10000
10.9±1.7 40.8±0.2
3.7±0.01 7.2±0.02
7
8
9
6p
6q
6r
>10000
>10000
>10000
1.4±0.2
1.3±0.1
1.0±0.1
>10000
>10000
>10000
2.3±0.1
1.8±0.2
4.9±0.01
5.4±0.3
1
2
Finally, systematic structural optimization of 7-FPUs derivatives of both ring substituents in R and R revealed
that the substituents dramatically impacted FGFR4 inhibition. As summarized in Table 3, the compound 6s with the
1
2
structure of 2-oxo-morpholin of R and (S)-3-methoxypyrrolidine of R showed a good enzymatic potency against
FGFR4 with IC50 of 3.5 nM and inhibition of Hep 3B cell line (24.5 nM) and HuH-7 cell line (24.7 nM). The
2
relatively low potency of 6t and 6u indicated that the larger substitution of R such as (S)-2-
(
methoxymethyl)pyrrolidine or N,N-dimethylpyrrolidin-3-amine were less tolerable. Our investigation also
6

2
suggested that R-isomer in 2’-position of the R substitution showed a better activity than the S-isomer by comparing
the compounds 6v vs 6w and 6x vs 6y.
1
2
Table 3 Activity of 7-FPUs derivatives with a ring substituent in R and a ring substituent in R
Structure
R1 R2
Enzyme IC50 ± SEM (nM)
Cell IC50 ± SEM（nM）
Entry compounds
FGFR1
FGFR4
Hep 3B
HuH-7
SK-HEP-1
1
2
3
6s
6t
>10000
3.5±0.01
24.9±1.8 27.5±0.1
>10000
>10000
>10000
>10000
>10000
9.1±0.3
9.4±0.7
22.9±3.5 65.3±2.9
93.8±11.5 297±0.6
6u
4
5
6
7
6v
6w
6x
6y
>10000
>10000
>10000
>10000
9.5±0.01
4.7±0.3
4.6±0.3
11.1±0.2
38.7±3.4 29.6±0.9
9.9±0.01 10.2±0.3
13.3±3.2 11.8±0.8
39.3±1.0 34.6±1.3
>10000
>10000
>10000
>10000
Pharmacokinetic study With the excellent in vitro enzyme and cell potencies, the pharmacokinetic properties of
f, 6g, 6h, 6l, 6m and 6s were also evaluated in vivo, and the results were summarized in Table 4. Selected
6
compounds were administrated to male Sprague-Dawley (SD) rats: orally by gavage at a 5 mg/kg dose. The result
suggested that designed 7-formylpyridine urea derivatives showed a good performance in the pharmacokinetic
study. Compounds 6f, 6g, 6h and 6s demonstrated higher plasma concentration than compounds 6l and 6m,
however, longer residence time was observed by 6l and 6m.
7

Table 4 Pharmacokinetic profile of various 7-formyl-naphthyridyl-ureas derivatives
Compound
Dose (mg/kg)
Cmax (ng/mL)
6f
6g
6h
5mg/kg
3287 2335 1640
6l
6m
6s
3377
3762
3356
AUC (ng/mL*h) 9464 12280 11327 1954 6780 14659
t
1/2 (h)
1.27
2.48
1.1
3.2
1.0
2.9
0.79
3.5
0.96
3.65
1.2
2.6
MRT (h)
Kinases Screening Due to the excellent biological activity and good performance in the pharmacokinetic study,
-formylpyridine urea derivatives 6f was further evaluated for the inhibitory activities against other 36 kinase in
7
vitro (Table 5). It was found that compound 6f showed excellent inhibition against FGFR4 kinase (IC50 1.5 nM,
Table 1 entry 7). In contrast, compound 6f showed little activity against the other 36 species of kinase. It is clear that
the 7-formylpyridine urea derivative, 6f, inhibited FGFR4 kinase activity with high selectivity and specificity.
Table 5 Activity of 6f against different kinases
Kinase Name IC50(nM) Kinase Name IC50(nM)
KDR
ROS
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
MAPKAPK3 >10000
MET
RET
ZAP70
TIE2
FAK
TRK-A
AXL
RON
ALK
CKIT
IGF1R
FLT1
FLT4
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
PDGFRa
PDGFRb
EGFR
MUSK
JAK2
JAK3
EphA1
EphB1
HER4
INSR
HER2
SYK
LTK
FLT3
TYK2
JAK1
MAPKAPK2 >10000
FGFR1
FGFR2
FGFR3
>10000
>10000
>10000
In vivo antitumor activity Two different human tumor cell lines (HuH7 and Hep 3B) were used to study the in
vivo efficacy of compound 6f and positive control FGF401 in tumor xenograft nude mouse model (Figures 3 and
4
). At all of the test dosages (10, 30, and 100 mg/kg), 6f significantly inhibited the tumor (HuH7) growth in nude
mice and the inhibitory rates were 28.1%, 62.7%, and 70.8%, respectively. In contrast, the inhibition rate of FGF401
8

(
30 mg/kg) was 52.5%, which is lower than 6f at the same dosage. For the inhibition of Hep 3B tumor growth,
compound 6f showed even better activity. The inhibition rates of 6f (3, 10, and 30 mg/kg) reached 69.8%, 126.5%,
and 164.6%, respectively, while FGF401 (10 mg/kg) showed the worse inhibitory effect (85.9%) at the same
dosage. The results clearly indicated that 6f had a better inhibiting activity than positive control FGF401 in a dose-
dependent manner.
Figure 3. Efficacy of 6f and FGF401 on resistant human hepatocarcinoma HuH7 tumor xenograft in nude mouse
(
n=5).
Figure 4. Efficacy of 6f and FGF401 on resistant human hepatocarcinoma Hep 3B tumor xenograft in nude mouse (n=5).
9

Toxicity studies in mice Long-term toxicity studies were performed in male and female SD mice to determine
potential adverse effects and maximum tolerated dose (MTD) for 6f. Both male and female SD mice were treated for
2
8 days with compound 6f at 50, 150, or 450 (mg/kg)/day and then rehabilitate for another 28 days. The body
weights of all treated mice remained normal during the study when compared to the vehicle-treated controls. In the
50 mg/kg dose group, the test substance-related death was observed, but no toxicity was observed in the male
animals. Under the conditions of this experiment, no significant toxicity dose (NOAEL) was found for the dosage of
50 mg/kg in male animals; no significant toxicity dose (NOAEL) was found for the dosage of 150 mg/kg in female
4
4
animals. Acute toxicity studies were performed in male and female SD mice for once with compound 6f at 200, 600
or 2000 (mg/kg). No biologically significant changes in clinical pathology were observed. The NOAEL in mice
acute toxicity studies was estimated to be 2000 (mg/kg)/day.
3
. Conclusion
A series of 7-formylpyridine urea derivatives 6 were designed and synthesized. The synthesized compounds were
evaluated for their potential as selective FGFR4 Inhibitors. It was found that most of the target compounds showed
potent inhibitory activity to FGFR4 kinase and FGFR4 high-expressing tumor cells, and possessed high selectivity
towards FGFR4 over other kinases. In particular, compounds 6f, 6g, 6h, 6l, 6m and 6s showed a good performance
in pharmacokinetic tests. Moreover, the representative compound 6f also showed a better antitumor activity than
FGF401 in a dose-dependent manner in vivo, and an excellent and a higher level of NOAEL in mice toxicity.
Altogether, the target compounds showed promising exploration values as selective FGFR4 inhibitors for the
treatment of tumor.[30] Further studies on structural optimization and biological activities about these derivatives are
still underway in our laboratory and will be reported in the future.
4
. Experimental section
Chemistry All chemicals were obtained from commercial purchase and solvents were purified and dried by
standard procedures. Flash chromatography (FC): silica gel (SiO
2
; 40 mm, 200~300 mesh). Melting points were
uncorrected and were determined using a capillary apparatus (RDCSY-I). H NMR and 13C NMR Spectra: Bruker
AVANCE-400 Digital NMR Spectrometer, ESI-MS: Thermo Finnigan LCQ advantage MAX. Elemental analysis
was performed on a Vario EL III apparatus.
1
N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((3-oxomorpholino)methyl)-3,4-dihydro-1,8-
naphthyridine-1(2H)-carboxamide (6a).
1
0

Typical procedure:
A mixture of 6-amino-4-fluoronicotinonitrile 7 (4.11 g, 30 mmol), 2-methoxyethanamine (4.5 g, 60 mmol), and
o
DIPEA (1.16 g, 90 mmol) in DMF (120 mL) was stirred at 60 C until the reaction was completed by TLC. After
quenched with water, the reaction solution was extracted with CH Cl
2
2
(3×100 mL), washed with sequentially with
satd NaHCO (aq) and brine and then dried over anhydrous Na SO . Filtration, evaporation, and chromatography on
3
2
4
silica gel afforded the product 8 (3.84g, 67%). MS m/z (ESI): 193.1 [M+H]+
A solution of 4-((2-(dimethoxymethyl)-5,6,7,8-tetrahydro-1,8-naphthyridin-3-yl)methyl)morpholin-3-one 9
o
(
642mg, 2 mmol) and dimethyl carbonate (643mg, 3 mmol) in THF was cooled down to -78 C under N
2
atmosphere,
then a solution of LiHMDS in THF (4mL, 4mmol) was dropped, and warmed to rt, stirring until the reaction was
completed by TLC. The reaction mixture was quenched with an aqueous solution of saturated NH Cl (100 mL) and
extracted with EA (2×50 mL).The combined organic layer was washed with brine and then dried over anhydrous
Na SO . Filtration, evaporation, and chromatography on silica gel afforded the product 10 (400mg, 45%). MS m/z
4
2
4
(
ESI): 442.1 [M+H]+
A solution of 6-amino-4-((2-methoxyethyl)amino)nicotinonitrile 8 (20 mg, 0.09 mmol) and phenyl 7-
(
dimethoxymethyl)-6-((3-oxomorpholino)methyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxylate 10 (38 mg,
o
0
.09 mmol) in THF (2 mL) was cooled down to -78 C under N
2
atmosphere, then a solution of LiHMDS in THF
(
0.2 mL, 0.2mmol) was dropped, and warmed to rt, stirring until the reaction was completed by TLC. The reaction
mixture was quenched with an aqueous solution of saturated NH Cl (20 mL) and extracted with EA (2×30 mL).The
combined organic layer was washed with brine and then dried over anhydrous Na SO . Filtration, evaporation, and
4
2
4
chromatography on silica gel afforded the product 11 (23 mg, 46%). MS m/z (ESI): 580.2 [M+H]+
To the solution of N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-(dimethoxymethyl)-6-((3-
oxomorpholino)methyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide 11 (23 mg, 0.04mmol) in THF/water
(
2/1, 1.5 mL) was added concentrated hydrochloric acid (0.15 mL, 1.8 mmol), the combined solution was stirred for
hour. The reaction mixture was quenched with an aqueous solution of saturated NaHCO (20 mL) and extracted
2
3
2 4
with EA (2×30 mL).The combined organic layer was washed with brine and then dried over anhydrous Na SO .
1
Filtration, evaporation, and chromatography on silica gel afforded the product 6a (15 mg, 70%). HNMR (400MHz,
3
CDCl , ppm) δ13.57 (s, 1 H), 10.24 (s, 1 H), 8.18 (s, 1 H), 7.67 (s, 1 H), 7.58 (s, 1 H), 5.34 (s, 1 H), 5.11 (s, 2 H),
4
.25 (s, 2 H), 4.07-4.10 (t, J=5.60 Hz, 2 H), 3.87-3.90 (m, 2 H), 3.63-3.65 (t, J=5.20 Hz, 2 H), 3.49-3.51 (t, J=5.20
1
1

13
Hz, 2 H), 3.42 (s, 3 H), 3.40-3.42 (m, 2 H), 2.92-2.95 (m, 2 H), 2.05-2.06 (m, 2 H). CNMR (100MHz, CDCl , ppm)
3
δ 193.6, 167.5, 155.7, 155.1, 152.6, 152.3, 151.1, 144.2, 140.7, 128.2, 128.0, 116.5, 93.2, 89.9, 69.9, 68.3, 63.9,
+
5
4
9.0, 46.9, 43.9, 43.8, 42.5, 28.4, 20.9; HRMS calcd for [M+1] Chemical Formula: C24
H
28
N
7
5
O Exact Mass:
93.2146 found 494.2130. HPLC purity: 99.1%.
N-(5-cyano-4-((2-methoxyethyl)thio)pyridin-2-yl)-7-formyl-6-((3-oxomorpholino)methyl)-3,4-dihydro-1,8-
naphthyridine-1(2H)-carboxamide (6b).
1
HNMR (400MHz, CDCl , ppm) δ13.86 (s, 1 H), 10.23 (s, 1 H), 8.35 (s, 1 H), 8.27 (s, 1 H), 7.69 (s, 1 H), 5.11
3
(
s, 2 H), 4.26 (s, 2 H), 4.08-4.11 (m, 2 H), 3.88-3.91 (m, t, J=5.20 Hz, 2 H), 3.75-3.78 (t, J=6.00 Hz, 2 H), 3.47 (s, 3
13
H), 3.41-3.43 (m, 2 H), 3.32-3.36 (m, 2 H), 2.94-2.97 (m, 2 H), 2.06-2.08 (m, 2 H). CNMR (100MHz, CDCl ,
3
ppm) δ193.3, 167.5, 155.3, 154.7, 152.6, 152.3, 150.1, 144.1, 140.8, 128.3, 128.2, 115.6, 107.9, 102.4, 69.8, 68.3,
+
6
5
3.9, 59.9, 47.0, 44.0, 43.9, 31.2, 28.4, 20.8; HRMS calcd for [M+1] Chemical Formula: C24
H
27
N
6
O SExact Mass:
5
11.1764 found 511.1741. HPLC purity: 98.1%.
N-(5-cyano-4-(2-methoxyethoxy)pyridin-2-yl)-7-formyl-6-((3-oxomorpholino)methyl)-3,4-dihydro-1,8-
naphthyridine-1(2H)-carboxamide (6c)
1
HNMR (400MHz, CDCl , ppm) δ13.86 (s, 1 H), 10.24 (s, 1 H), 8.36 (s, 1 H), 7.97 (s, 1 H), 7.68 (s, 1 H), 5.11
3
(
s, 2 H), 4.34-4.35 (m, 2 H), 4.26 (s, 2 H), 4.09 (m, 2 H), 3.89 (m, 2 H), 3.83 (m, 2 H), 3.47 (s, 3 H), 3.42 (m, 2 H),
1
3
2
1
.95 (m, 2 H), 2.06 (m, 2 H). CNMR (100MHz, CDCl , ppm) δ 193.3, 167.5, 167.4, 157.1, 153.2, 152.6, 151.0,
3
44.1, 140.8, 128.3, 128.2, 114.9, 96.5, 95.2, 70.2, 69.0, 68.3, 64.0, 59.6, 44.0, 43.9, 29.7, 22.7, 20.8; HRMS calcd
+
for [M+1] Chemical Formula: C24
H
27
N
6
O Exact Mass: 495.1992 found 495.1970. HPLC purity: 98.1%.
6
(
R)-N-(5-cyano-4-((1-methoxypropan-2-yl)oxy)pyridin-2-yl)-7-formyl-6-((3-oxomorpholino)methyl)-3,4-
dihydro-1,8-naphthyridine-1(2H)-carboxamide (6d)
1
HNMR (400MHz, CDCl , ppm) δ13.81 (s, 1 H), 10.24 (s, 1 H), 8.35 (s, 1 H), 8.00 (s, 1 H), 7.68 (s, 1 H), 5.11
3
(
(
(
s, 2 H), 4.86-4.87 (m, 1 H), 4.26 (s, 2 H), 4.08-4.11 (t, J=5.60 Hz, 2 H), 3.88-3.91 (t, J=5.20 Hz, 2 H), 3.56-3.67
13
m, 2 H), 3.43 (s, 3 H), 3.41-3.42 (m, 2 H), 2.93-2.96 (m, 2 H), 2.04-2.07 (m, 2 H), 1.41-1.43 (m, 3 H). CNMR
100MHz, CDCl
3
, ppm) δ 193.4, 167.5, 167.0, 157.0, 153.4, 152.6, 151.1, 144.1, 140.8, 128.3, 128.2, 115.1, 97.2,
+
9
5.7, 68.3, 63.9, 59.6, 46.9, 43.9, 43.8, 31.9, 29.7, 28.4, 22.7, 20.8; HRMS calcd for [M+1] Chemical Formula:
C
25
H
29
N
6
O
6
Exact Mass: 509.2149 found 509.2128. HPLC purity: 97.8%.
R)-N-(5-cyano-4-((1-methoxypropan-2-yl)oxy)pyridin-2-yl)-7-formyl-6-((2-oxo-1,3-oxazepan-3-yl)methyl)-
,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (6e)
(
3
1
2

1
HNMR (400MHz, CDCl
s, 2 H), 4.86-4.88 (m, 1 H), 4.14-4.16 (m, 2 H), 4.08-4.10 (m, 2 H), 3.56-3.69 (m, 2 H), 3.43 (s, 3 H), 3.30-3.33 (m,
H), 2.93-2.96 (m, 2 H), 2.04-2.06 (m, 2 H), 1.87-1.91 (m, 2 H), 1.73-1.74 (m, 2 H), 1.41-1.42 (d, J=6.00Hz, 3 H).
3
, ppm) δ13.83 (s, 1 H), 10.24 (s, 1 H), 8.35 (s, 1 H), 8.01 (s, 1 H), 7.73 (s, 1 H), 4.95
(
2
13
3
CNMR (100MHz, CDCl , ppm) δ 193.3, 167.1, 160.9, 156.9, 153.2, 152.6, 150.9, 143.9, 140.4, 129.2, 128.1,
+
1
15.1, 97.2, 95.7, 75.3, 75.2, 70.5, 59.6, 49.6, 48.7, 43.8, 28.8, 28.4, 26.0, 20.9, 16.3; HRMS calcd for [M+1]
Chemical Formula: C26 Exact Mass: 523.2305 found 523.2293. HPLC purity: 98.8%.
R)-N-(5-cyano-4-((1-methoxypropan-2-yl)amino)pyridin-2-yl)-7-formyl-6-((2-oxo-1,3-oxazepan-3-yl)methyl)-
H
31
N
6
O
6
(
3
,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (6f)
1
HNMR (400MHz, DMSO-d6, ppm) δ13.50 (s, 1 H), 10.06 (s, 1 H), 8.27 (s, 1 H), 7.71 (s, 1 H), 7.56 (s, 1 H),
6
3
.61-6.62 (d, 1 H), 4.79 (s, 2 H), 4.08-4.10 (m, 2 H), 3.97-3.99 (m, 2 H), 3.83-3.88 (m, 1 H), 3.47-3.51 (m, 1 H),
.38-3.41 (m, 1 H), 3.32 (s, 3 H), 3.26-3.28 (m, 2 H), 2.96 (t, 2 H), 1.93-1.95 (m, 2 H), 1.76-1.79 (m, 2 H), 1.60-1.62
1
3
(
m, 2 H), 1.20-1.21 (d, 3 H). CNMR (100MHz, DMSO-d6, ppm) δ192.6, 160.3, 155.8, 155.4, 154.3, 152.5, 150.7,
1
43.7, 139.4, 129.8, 129.1, 117.5, 93.2, 88.9, 75.1, 70.2, 58.8, 49.3, 49.1, 47.9, 43.9, 28.9, 28.0, 26.0, 20.8, 17.3;
+
HRMS calcd for [M+K] Chemical Formula: C26
H
31
N
7
5
O K Exact Mass: 560.2024 found 560.1992. HPLC
purity ：9 9.9%.
(
R)-N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((3-methoxy-2-oxopyrrolidin-1-
yl)methyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (6g)
1
HNMR (400MHz, CDCl , ppm) δ13.57 (s, 1 H), 10.23 (s, 1 H), 8.18 (s, 1 H), 7.63 (s, 1 H), 7.58 (s, 1 H), 5.34
3
(
s, 1 H), 4.88-4.99 (dd, J=15.20 Hz, 28.00 Hz, 2 H), 4.06-4.09 (m, 2 H), 3.98-4.01 (m, 1 H), 3.63-3.65 (m, 2 H),
3
1
1
.59 (s, 3 H), 3.47-3.51 (m, 2 H), 3.41 (s, 3 H), 3.39-3.40 (m, 1 H), 3.29 (m, 1 H), 2.90-2.93 (m, 2 H), 2.39 (m, 1 H),
1
3
.85-2.04 (m, 3 H). CNMR (100MHz, CDCl , ppm) δ193.4, 173.0, 155.7, 155.2, 152.6, 152.4, 151.1, 144.1, 140.9,
3
28.2, 127.8, 116.3, 93.2, 89.9, 77.9, 69.9, 59.0, 58.1, 44.3, 43.8, 42.5, 40.7, 28.4, 26.2, 20.9; HRMS calcd for
+
[
M+1] Chemical Formula: C25
H
30
N
7
5
O Exact Mass: 508.2308 found 508.2286. HPLC purity: 95.5%.
(
S)-N-(5-cyano-4-((2-methoxyethyl)amino)pyridin-2-yl)-7-formyl-6-((3-methoxy-2-oxopyrrolidin-1-
yl)methyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (6h)
1
HNMR (400MHz, CDCl , ppm) δ13.57 (s, 1 H), 10.23 (s, 1 H), 8.18 (s, 1 H), 7.63 (s, 1 H), 7.58 (s, 1 H), 5.34
3
(
(
s, 1 H), 4.87-4.99 (dd, J=15.20 Hz, 28.00 Hz, 2 H), 3.98-4.09 (m, 3 H), 3.63-3.65 (m, 2 H), 3.58 (s, 3 H), 3.50-3.51
m, 2 H), 3.48 (s, 3 H), 3.37-3.47 (m, 1 H), 3.27-3.29 (m, 1 H), 2.90-2.93 (m, 2 H), 2.36 (m, 1 H), 2.01-2.05 (m, 2
13
H), 1.94-1.98 (m, 1 H). CNMR (100MHz, CDCl , ppm) δ 193.4, 173.0, 155.7, 154.6, 152.6, 152.4, 151.1, 144.1,
3
1
3

1
40.1, 128.2, 127.8, 116.3, 93.2, 89.9, 77.9, 69.9, 59.0, 58.1, 44.3, 43.8, 42.5, 40.7, 28.3, 26.2, 20.9; HRMS calcd
+
for [M+1] Chemical Formula: C25
H
30
N
7
O Exact Mass: 508.2308 found 508.2285. HPLC purity: 97.5%.
5
N-(5-cyano-4-(((S)-1-methoxypropan-2-yl)amino)pyridin-2-yl)-7-formyl-6-(((S)-3-methoxy-2-oxopyrrolidin-1-
yl)methyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (6i)
1
HNMR (400MHz, CDCl , ppm) δ13.54 (s, 1 H), 10.23 (s, 1 H), 8.17 (s, 1 H), 7.63 (s, 1 H), 7.61 (s, 1 H), 5.18-
3
5
3
.20 (d, J=7.6Hz, 1 H), 4.88-4.99 (dd, J=15.60 Hz, 28.00 Hz, 2 H), 4.06-4.09 (m, 2 H), 3.98-4.00 (m, 2 H), 3.58 (s,
H), 3.38-3.49 (m, 6 H), 3.29 (m, 1 H), 2.90-2.93 (m, 2 H), 2.40 (m, 1 H), 1.92-2.06 (m, 3 H), 1.31-1.32 (d, J=6.40
1
3
Hz, 3 H). CNMR (100MHz, CDCl
3
, ppm) δ193.4, 173.0, 155.6, 155.0, 152.6, 152.4, 151.1, 144.0, 140.9, 128.2,
+
1
27.7, 116.5, 93.4, 89.9, 77.9, 75.1, 59.3, 58.1, 48.0, 44.3, 43.8, 40.8, 28.4, 26.2, 20.9, 17.3; HRMS calcd for [M+1]
Chemical Formula: C26 Exact Mass: 522.2465 found 522.2443. HPLC purity: 97.1%.
H
32
N
7
O
5
N-(5-cyano-4-(2-methoxyphenoxy)pyridin-2-yl)-7-formyl-6-((3-oxomorpholino)methyl)-3,4-dihydro-1,8-
naphthyridine-1(2H)-carboxamide (6j)
1
HNMR (400MHz, CDCl , ppm) δ13.78 (s, 1 H), 10.23 (s, 1 H), 8.45 (s, 1 H), 7.65 (s, 1 H), 7.52 (s, 1 H), 7.28-
3
7
2
.32 (m, 1 H), 7.17-7.19 (m, 1 H), 7.02-7.08 (m, 2 H), 5.10 (s, 2 H), 4.24 (s, 2 H), 3.97-3.99 (m, 2 H), 3.87-3.90 (m,
13
H), 3.80 (s, 3 H), 3.39-3.42 (m, 2 H), 2.88-2.91 (m, 2 H), 1.97-2.04 (m, 2 H). CNMR (100MHz, CDCl , ppm)
3
δ193.3, 167.7, 167.5, 156.8, 153.0, 152.2, 151.3, 151.0, 144.1, 141.3, 140.8, 128.2, 128.1, 127.7, 122.6, 121.5,
+
1
14.7, 113.5, 98.5, 95.0, 68.3, 64.0, 56.0, 46.9, 43.9, 43.7, 28.4, 20.8; HRMS calcd for [M+1] Chemical Formula:
C
28
H
27
N
6
6
O Exact Mass: 543.1992 found 543.1974. HPLC purity: 99.0%.
N-(5-cyano-4-((4-methoxypyridin-3-yl)amino)pyridin-2-yl)-7-formyl-6-((3-oxomorpholino)methyl)-3,4-
dihydro-1,8-naphthyridine-1(2H)-carboxamide (6k)
1
HNMR (400MHz, CDCl , ppm) δ13.63 (s, 1 H), 10.22 (s, 1 H), 8.65 (s, 1 H), 8.41-8.42 (d, J=5.20 Hz, 1 H),
3
8
4
.32 (s, 1 H), 7.92 (s, 1 H), 7.65 (s, 1 H), 6.94-6.95 (d, J=5.60 Hz, 1 H), 6.61 (s, 1 H), 5.10 (s, 2 H), 4.25 (s, 2 H),
.00-4.03 (m, 2 H), 3.96 (s, 3 H), 3.87-3.90 (m, 2 H), 3.39-3.42 (m, 2 H), 2.90-2.93 (m, 2 H), 1.99-2.02 (m, 2 H).
13
CNMR (100MHz, CDCl
3
, ppm) δ193.5, 167.5, 158.2, 156.2, 153.3, 153.0, 152.3, 151.1, 148.2, 144.2, 144.1, 140.6,
+
1
28.3, 127.9, 124.4, 116.1, 106.9, 95.6, 91.4, 68.3, 63.9, 55.9, 46.9, 43.9, 43.7, 28.4, 20.8; HRMS calcd for [M+1]
Chemical Formula: C27 Exact Mass: 543.2104 found 543.2082. HPLC purity: 100.0%.
S)-N-(5-cyano-4-((tetrahydrofuran-3-yl)thio)pyridin-2-yl)-7-formyl-6-((3-oxomorpholino)methyl)-3,4-
dihydro-1,8-naphthyridine-1(2H)-carboxamide (6l)
H
27
N
8
O
5
(
1
4

1
HNMR (400MHz, CDCl
3
, ppm) δ13.89 (s, 1 H), 10.23 (s, 1 H), 8.37 (s, 1 H), 8.28 (s, 1 H), 7.69 (s, 1 H), 5.11
(
s, 2 H), 4.35-4.40 (m, 1 H), 4.26 (s, 2 H), 4.00-4.11 (m, 3 H), 3.88-3.99 (m, 4 H), 3.79-3.82 (m, 1 H), 3.41-3.43 (m,
1
3
2
1
4
5
H), 2.94-2.97 (m, 2 H), 2.57-2.60 (m, 1 H), 2.03-2.09 (m, 3 H). CNMR (100MHz, CDCl , ppm) δ193.2, 167.5,
3
54.9, 154.7, 152.6, 152.4, 151.0, 144.1, 140.1, 128.4, 128.3, 115.5, 108.6, 102.1,73.1, 68.3, 67.6, 63.9, 47.0, 44.0,
+
2.3, 32.9, 29.7, 28.4, 20.8; HRMS calcd for [M+1] Chemical Formula: C25
H
27
N
6
O SExact Mass: 523.1764 found
5
23.1743. HPLC purity: 97.6%.
(
R)-N-(5-cyano-4-((tetrahydrofuran-3-yl)thio)pyridin-2-yl)-7-formyl-6-((3-oxomorpholino)methyl)-3,4-
dihydro-1,8-naphthyridine-1(2H)-carboxamide (6m)
1
HNMR (400MHz, CDCl , ppm) δ13.91 (s, 1 H), 10.24 (s, 1 H), 8.37 (s, 1 H), 8.29 (s, 1 H), 7.69 (s, 1 H), 5.11
3
(
s, 2 H), 4.36-4.40 (m, 1 H), 4.26 (s, 2 H), 3.94-4.11 (m, 3 H), 3.88-3.91 (m, 4 H), 3.79-3.82 (m, 1 H), 3.41-3.44 (m,
1
3
2
1
4
5
H), 2.94-2.97 (m, 2 H), 2.59-2.60 (m, 1 H), 2.03-2.08 (m, 3 H). CNMR (100MHz, CDCl , ppm) δ193.2, 167.5,
3
55.0, 154.7, 152.6, 152.2, 151.0, 144.1, 140.1, 128.4, 128.3, 115.4, 108.6, 102.1,73.1, 68.3, 67.6, 63.9, 47.0, 44.0,
+
2.3, 32.9, 29.7, 28.4, 20.8; HRMS calcd for [M+1] Chemical Formula: C25
H
27
N
6
O SExact Mass: 523.1764 found
5
23.1749. HPLC purity: 97.4%.
N-(5-cyano-4-(((1R,2R)-2-methoxycyclopentyl)amino)pyridin-2-yl)-7-formyl-6-((3-oxomorpholino)methyl)-
3
,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (6n)
1
HNMR (400MHz, CDCl , ppm) δ13.54 (s, 1 H), 10.23 (s, 1 H), 8.17 (s, 1 H), 7.74 (s, 1 H), 7.66 (s, 1 H), 5.11
3
(
s, 2 H), 4.87-4.89 (d, J=6.00 Hz, 1 H), 4.25 (s, 2 H), 4.08-4.11 (m, 2 H), 3.87-3.93 (m, 3 H), 3.68-3.69 (m, 1 H),
1
3
3
.42 (s, 3 H), 3.39 (m, 2 H), 2.91-2.95 (m, 2 H), 2.32 (m, 1 H), 1.75-2.05 (m, 6 H), 1.52-1.54 (m, 1 H). CNMR
(
100MHz, CDCl
3
, ppm) δ193.6, 167.5, 155.8, 155.3, 152.5, 151.2, 144.2, 140.6, 128.2, 127.9, 116.6, 94.0, 89.7,
+
8
6.9, 77.2, 68.3, 63.9, 59.2, 57.2, 46.9, 43.9, 43.7, 31.0, 29.8, 28.5, 21.5, 20.9; HRMS calcd for [M+1] Chemical
Formula: C27
H
32
N
7
5
O Exact Mass: 534.2465 found 534.2446. HPLC purity: 97.4%.
N-(5-cyano-4-(((3S,4S)-4-methoxytetrahydro-2H-pyran-3-yl)oxy)pyridin-2-yl)-7-formyl-6-((3-
oxomorpholino)methyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (6o)
1
HNMR (400MHz, CDCl , ppm) δ13.82 (s, 1 H), 10.24 (s, 1 H), 8.36 (s, 1 H), 8.07 (s, 1 H), 7.69 (s, 1 H), 5.11
3
(
s, 2 H), 4.53-4.54 (m, 1 H), 4.26 (s, 2 H), 4.07-4.12 (m, 3 H), 3.88-3.96 (m, 3 H), 3.61 (m, 1 H), 3.58-3.59 (m, 2H),
.55 (s, 3 H), 3.40-3.54 (m, 2 H), 2.93-2.96 (m, 2 H), 2.19-2.22 (m, 1 H), 2.04-2.07 (m, 2 H), 1.71-1.74 (m, 1 H).
3
13
CNMR (100MHz, CDCl , ppm) δ193.3, 167.5, 166.9, 157.0, 153.1, 152.5, 151.0, 144.1, 140.8, 128.3, 128.2, 114.8,
3
1
5

+
9
7.3, 95.7, 78.2, 77.2, 68.3, 66.4, 65.3, 63.9, 57.7, 46.9, 43.9, 43.8, 29.6, 28.4, 20.8; HRMS calcd for [M+1]
Chemical Formula: C27 Exact Mass: 551.2254 found 551.2237. HPLC purity: 98.8%.
S)-N-(5-cyano-4-((tetrahydrofuran-3-yl)amino)pyridin-2-yl)-7-formyl-6-((2-oxo-1,3-oxazepan-3-yl)methyl)-
H
31
N
6
O
7
(
3
,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (6p)
1
HNMR (400MHz, CDCl , ppm) δ13.64 (s, 1 H), 10.24 (s, 1 H), 8.20 (s, 1 H), 7.72 (s, 1 H), 7.62 (s, 1 H), 5.07-
3
5
3
.08 (d, J=6.40 Hz, 1 H), 4.95 (s, 2 H), 4.33 (m, 1 H), 4.01-4.16 (m, 6 H), 3.87-3.90 (m, 1 H), 3.78-3.81 (m, 1 H),
13
.32 (m, 2 H), 2.92-2.96 (m, 2 H), 2.45 (m, 1 H), 1.88-2.06 (m, 5 H), 1.72-1.74 (m, 2 H). CNMR (100MHz, CDCl ,
3
ppm) δ193.5, 160.1, 155.6, 154.8, 152.7, 152.5, 150.9, 144.0, 140.3, 129.1, 128.0, 116.2, 93.7, 90.0, 70.4, 67.0, 53.3,
+
4
9.6, 43.8, 33.2, 31.9, 29.3, 28.8, 26.0, 22.7, 20.9; HRMS calcd for [M+1] Chemical Formula: C26
H
30
N
7
O Exact
5
Mass: 520.2308 found 520.2291. HPLC purity: 97.0%.
R)-N-(5-cyano-4-((tetrahydrofuran-3-yl)amino)pyridin-2-yl)-7-formyl-6-((2-oxo-1,3-oxazepan-3-yl)methyl)-
(
3
,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (6q)
1
HNMR (400MHz, CDCl , ppm) δ13.57 (s, 1 H), 10.17 (s, 1 H), 8.13 (s, 1 H), 7.65 (s, 1 H), 7.55 (s, 1 H), 5.01-
3
5
2
1
3
5
.02 (d, J=6.40 Hz, 1 H), 4.88 (s, 2 H), 4.25 (m, 1 H), 3.94-4.09 (m, 6 H), 3.71-3.82 (m, 2 H), 3.23-3.25 (m, 2 H),
13
.85-2.89 (m, 2 H), 2.37 (m, 1 H), 1.81-1.99 (m, 5 H), 1.65-1.67 (m, 2 H). CNMR (100MHz, CDCl , ppm) δ193.5,
3
60.9, 155.6, 154.8, 152.7, 152.5, 150.9, 144.0, 140.3, 129.1, 128.0, 116.2, 93.7, 90.0, 70.4, 67.0, 53.3, 49.6, 43.8,
+
3.2, 31.9, 29.3, 28.8, 26.0, 22.7, 20.9; HRMS calcd for [M+1] Chemical Formula: C26
H
30
N
7
5
O Exact Mass:
20.2308 found 520.2287. HPLC purity: 96.9%.
N-(5-cyano-4-(((R)-tetrahydrofuran-3-yl)amino)pyridin-2-yl)-7-formyl-6-(((R)-3-methoxy-2-oxopyrrolidin-1-
yl)methyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (6r)
1
HNMR (400MHz, CDCl , ppm) δ13.55 (s, 1 H), 10.16 (s, 1 H), 8.12 (s, 1 H), 7.56 (s, 1 H), 7.54 (s, 1 H), 5.01-
3
5
.02 (d, J=6.40 Hz, 1 H), 4.80-4.92 (dd, J=15.60 Hz, 30.00 Hz, 2 H), 4.06 (m, 1 H), 3.90-4.02 (m, 5 H), 3.80-3.82
(
m, 1 H), 3.71-3.74 (m, 1 H), 3.51 (s, 3 H), 3.32-3.33 (m, 1 H), 3.20-3.22 (m, 1 H), 2.84-2.87 (m, 2 H), 2.33 (m, 2
1
3
H), 1.86-1.98 (m, 4 H). CNMR (100MHz, CDCl
3
, ppm) δ193.3, 173.0, 155.6, 154.8, 152.6, 152.4, 151.1, 144.0,
1
40.9, 128.2, 127.9, 116.2, 93.7, 90.0, 77.9, 73.1, 67.0, 58.1, 53.3, 44.3, 43.8, 40.8, 33.2, 28.3, 26.2, 20.9; HRMS
+
calcd for [M+1] Chemical Formula: C26
S)-N-(5-cyano-4-(3-methoxypyrrolidin-1-yl)pyridin-2-yl)-7-formyl-6-((3-oxomorpholino)methyl)-3,4-
dihydro-1,8-naphthyridine-1(2H)-carboxamide (6s)
H
30
N
7
O Exact Mass: 520.2308 found 520.2281. HPLC purity: 96.7%.
5
(
1
6

1
HNMR (400MHz, CDCl
3
, ppm) δ13.48 (s, 1 H), 10.24 (s, 1 H), 8.20 (s, 1 H), 7.67 (s, 1 H), 7.48 (s, 1 H), 5.11
(
s, 2 H), 4.25 (s, 2 H), 4.06-4.09 (m, 3 H), 3.79-3.89 (m, 6 H), 3.39-3.42 (m, 2 H), 3.37 (s, 3 H), 2.91-2.95 (m, 2 H),
1
3
2
1
2
9
.20-2.22 (m, 1 H), 2.05-2.07 (m, 3 H). CNMR (100MHz, CDCl , ppm) δ193.6, 167.5, 155.2, 154.5, 154.2, 152.6,
3
51.1, 144.2, 140.7, 128.1, 127.9, 119.9, 96.2, 88.3, 79.1, 68.3, 63.9, 56.6, 54.4, 47.4, 46.9, 43.8, 43.7, 30.4, 28.4,
+
0.9; HRMS calcd for [M+1] Chemical Formula: C26
H
30
N
7
O Exact Mass: 520.2308 found 520.2290. HPLC purity:
5
7.3%.
(
S)-N-(5-cyano-4-(2-(methoxymethyl)pyrrolidin-1-yl)pyridin-2-yl)-7-formyl-6-((3-oxomorpholino)methyl)-3,4-
dihydro-1,8-naphthyridine-1(2H)-carboxamide (6t)
1
HNMR (400MHz, CDCl , ppm) δ 13.46 (s, 1 H), 10.24 (s, 1 H), 8.21 (s, 1 H), 7.66 (s, 1 H), 7.60 (s, 1 H),
3
5
1
.11(s, 2 H), 4.56 (m, 1 H), 4.25 (s, 2 H), 4.07-4.10 (m, 2 H), 3.87-3.90 (m, 3 H), 3.56-3.65 (m, 2 H), 3.45-3.49 (m,
13
H), 3.40-3.42 (m, 2 H), 3.38 (s, 3 H), 2.91-2.95 (m, 2 H), 1.97-2.14 (m, 6 H). CNMR (100MHz, CDCl , ppm)
3
δ193.6, 167.5, 155.8, 154.7, 153.6, 152.6, 151.2, 144.2, 140.7, 128.1, 127.9, 120.2, 96.8, 88.6, 72.3, 68.3, 63.9,
+
5
9.4,58.4, 50.3, 46.9, 43.8, 43.7, 28.5, 28.4, 23.2, 20.9; HRMS calcd for [M+1] Chemical Formula: C27
H
31
N
7
O
5
Exact Mass: 534.2465 found 534.2447. HPLC purity: 98.0%.
N-(5-cyano-4-(3-(dimethylamino)pyrrolidin-1-yl)pyridin-2-yl)-7-formyl-6-((3-oxomorpholino)methyl)-3,4-
dihydro-1,8-naphthyridine-1(2H)-carboxamide (6u)
1
HNMR (400MHz, CDCl , ppm) δ13.50 (s, 1 H), 10.23 (s, 1 H), 8.20 (s, 1 H), 7.66 (s, 1 H), 7.45 (s, 1 H), 5.11
3
(
s, 2 H), 4.25 (s, 2 H), 4.07-4.09 (m, 2 H), 3.87-3.92 (m, 4 H), 3.72-3.74 (m, 1 H), 3.58 (m, 1 H), 3.39-3.42 (m, 2 H),
1
3
2
1
4
5
.86-2.98 (m, 3 H), 2.35 (s, 6 H), 2.22 (m, 1 H), 2.03 (m, 3 H). CNMR (100MHz, CDCl , ppm) δ193.6, 167.5,
3
55.6, 154.8, 153.9, 152.7, 151.2, 144.2, 140.7, 128.2, 127.9, 120.0, 95.8, 88.1, 68.3, 65.1, 63.9, 53.3, 48.6, 46.9,
+
4.2, 43.8, 43.7, 29.8, 29.7, 28.5, 20.9; HRMS calcd for [M+1] Chemical Formula: C27
H
33
N
8
4
O Exact Mass:
33.2625 found 533.2603. HPLC purity: 98.5%.
(
S)-N-(5-cyano-4-(3-methoxypyrrolidin-1-yl)pyridin-2-yl)-7-formyl-6-((2-oxooxazolidin-3-yl)methyl)-3,4-
dihydro-1,8-naphthyridine-1(2H)-carboxamide (6v)
1
HNMR (400MHz, CDCl , ppm) δ13.47 (s, 1 H), 10.23 (s, 1 H), 8.20 (s, 1 H), 7.75 (s, 1 H), 7.48 (s, 1 H), 4.88
3
(
s, 2 H), 4.31-4.35 (t, J=8.00 Hz, 2 H), 4.06-4.10 (m, 3 H), 3.78-3.82 (m, 4 H), 3.57-3.61 (m, 2 H), 3.37 (s, 3 H),
1
3
2
1
.93-2.96 (m, 2 H), 2.20 (m, 1 H), 2.01-2.06 (m, 3 H). CNMR (100MHz, CDCl
3
, ppm) δ193.5, 158.9, 155.2, 154.5,
54.2, 152.6, 151.4,144.1, 141.5, 128.2, 127.3, 119.9, 96.2, 88.3, 79.1, 62.2, 56.6, 54.4, 47.4, 45.0, 43.8, 42.2, 30.4,
1
7

+
2
8.4, 20.9; HRMS calcd for [M+1] Chemical Formula: C25
H
28
N
7
5
O Exact Mass: 506.2152 found 506.2131. HPLC
purity: 95.9%.
R)-N-(5-cyano-4-(3-methoxypyrrolidin-1-yl)pyridin-2-yl)-7-formyl-6-((2-oxooxazolidin-3-yl)methyl)-3,4-
(
dihydro-1,8-naphthyridine-1(2H)-carboxamide (6w)
1
HNMR (400MHz, CDCl , ppm) δ13.47 (s, 1 H), 10.24 (s, 1 H), 8.20 (s, 1 H), 7.75 (s, 1 H), 7.49 (s, 1 H), 4.88
3
(
s, 2 H), 4.31-4.35 (t, J=8.00 Hz, 2 H), 4.07-4.09 (m, 3 H), 3.79-3.82 (m, 4 H), 3.57-3.61 (m, 2 H), 3.37 (s, 3 H),
1
3
2
1
2
.93-2.96 (m, 2 H), 2.22 (m, 1 H), 2.02-2.07 (m, 3 H). CNMR (100MHz, CDCl , ppm) δ193.5, 158.9, 155.2, 154.5,
3
54.2, 152.6, 151.4,144.1, 141.5, 128.2, 127.3, 119.9, 96.2, 88.3, 79.1, 62.2, 56.6, 54.4, 47.4, 45.0, 43.8, 42.2, 30.4,
+
8.4, 20.9; HRMS calcd for [M+1] Chemical Formula: C25
H
28
N
7
O Exact Mass: 506.2152 found 506.2132. HPLC
5
purity: 96.5%.
N-(5-cyano-4-((R)-3-methoxypyrrolidin-1-yl)pyridin-2-yl)-7-formyl-6-(((S)-4-methyl-2-oxooxazolidin-3-
yl)methyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (6x)
1
HNMR (400MHz, CDCl , ppm) δ13.48 (s, 1 H), 10.24 (s, 1 H), 8.20 (s, 1 H), 7.76 (s, 1 H), 7.48 (s, 1 H), 5.03-
3
5
3
.07 (d, J=16.40 Hz, 1 H), 4.76-4.81 (d, J=16.40 Hz, 1 H), 4.39-4.44 (t, J=8.00 Hz, 2 H), 4.06-4.09 (m, 3 H), 3.78-
.93 (m, 6 H), 3.37 (s, 3 H), 2.92-2.95 (m, 2 H), 2.20-2.24 (m, 1 H), 2.02-2.05 (m, 3 H), 1.28-1.29(d, J=4.00 Hz, 3
1
3
H). CNMR (100MHz, CDCl
3
, ppm) δ193.5, 158.8, 155.2, 154.5, 154.2, 152.6, 151.2, 143.6, 140.8, 128.4, 128.1,
+
1
19.9, 96.1, 88.3, 79.1, 69.3, 56.6, 54.4, 51.8, 47.4, 43.8, 39.7, 30.4, 28.4, 20.9, 18.5; HRMS calcd for [M+1]
Chemical Formula: C26 Exact Mass: 520.2308 found 520.2292. HPLC purity: 100.0%.
H
30
N
7
O
5
N-(5-cyano-4-((S)-3-methoxypyrrolidin-1-yl)pyridin-2-yl)-7-formyl-6-(((S)-4-methyl-2-oxooxazolidin-3-
yl)methyl)-3,4-dihydro-1,8-naphthyridine-1(2H)-carboxamide (6y)
1
HNMR (400MHz, CDCl , ppm) δ13.48 (s, 1 H), 10.24 (s, 1 H), 8.20 (s, 1 H), 7.76 (s, 1 H), 7.48 (s, 1 H), 5.03-
3
5
3
.07 (d, J=16.00 Hz, 1 H), 4.76-4.81 (d, J=16.00 Hz, 1 H), 4.40-4.44 (t, J=8.00 Hz, 2 H), 4.06-4.09 (m, 3 H), 3.79-
.93 (m, 6 H), 3.37 (s, 3 H), 2.92-2.95 (m, 2 H), 2.21-2.24 (m, 1 H), 2.02-2.05 (m, 3 H), 1.29-1.30 (d, J=4.00 Hz, 3
1
3
H). CNMR (100MHz, CDCl
3
, ppm) δ193.5, 158.8, 155.2, 154.5, 154.2, 152.6, 151.2, 143.6, 140.8, 128.4, 128.1,
+
1
19.9, 96.1, 88.3, 79.1, 69.3, 56.6, 54.4, 51.8, 47.4, 43.8, 39.7, 30.4, 28.4, 20.9, 18.5; HRMS calcd for [M+1]
Chemical Formula: C26 Exact Mass: 520.2308 found 520.2292. HPLC purity: 99.2%.
H
30
N
7
O
5
In-vitro biochemical kinase assays All assays were performed in 384 well microtiter plates. Each assay
contained 8-point serial dilutions for 40 test compounds, as well as four 8-point serial dilutions of staurosporine as
reference compound, plus 16 high and 16 low controls.
1
8

Liquid handling and incubation steps were done on an innovadyne Nanodrop express equipped with a robotic
arm (Thermo CatX, Caliper Twister II) and an incubator (Liconic STX40, Thermo Cytomat 2C450). The assay
plates were prepared by addition of 50 nl per well of compound solution in 90% DMSO. The kinase reactions were
started by stepwise addition of 4.5 ul per well of peptide/ATP-solution and 4.5 uL per well of enzyme solution.
o
Kinase reactions were incubated at 30 C for 60 minutes and subsequently terminated by addition of 16 ul per well
of stop solution. Plates with terminated kinase reaction were transferred to the Caliper LC3000 workstations for
reading. Phosphorylated and unphosphorylated peptides were separated using the Caliper microfluidic mobility shift
technology. Samples from terminated kinase reactions were applied to the chip. Analytes are transported through the
chip by constant buffer flow and migration of the substrate peptide is monitored by the fluorescence signal of its
label. Phosphorylated peptide and unphosphorylated peptides are separated in an electric field by their charge/mass
ratio. Kinase activities were calculated from the amounts of formed phosphor-peptide. IC50 values were determined
from percent inhibition values at different compound concentrations by non-linear regression analysis.
In-vitro cellular assays Hep 3B, HuH-7, and SK-HEP-1 cell lines were used for the cell proliferation assays.
In this experiment, the inhibitory effect of the compound on the proliferation of experimental cells was tested by
CellTiter-Glo method, and the IC50 of the compound inhibiting cell proliferation activity was obtained: Inoculate 50-
4
1
00 uL of experimental cell suspension in a 96-well cell culture plate at a density of 1-5×10 cells/mL, and place the
plate in an incubator for 16-24 h (37 °C, 5% CO
2
). Then add a gradient dilution of the solution of the compound to
) in the incubator. Add 50-100uL
be tested to the culture plate, and incubate the plate for 72 h (37 °C, 5% CO
2
CellTiter-Glo reagent to each well, shake at room temperature or let stand for 5-30 min. Determination of the
chemiluminescence signal value of each plate by a microplate reader, then calculate the inhibition rate by the value
of the chemiluminescence signal and the IC50 of the compound is obtained by curve fitting according to the
inhibition rates of different concentrations.
Pharmacokinetic Assay Rates, half male and half female for 6~7 weeks, are purchased from Shanghai SLAC
Laboratory Animal CO. LTD; feed the mice under grade SPF. Rats were intragastrically administered, fasted for not
less than 12 hours before administration, free to drink water, and fed for 4 hours after administration. Blood samples
were taken from the posterior ocular veins before administration and 5 min, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0, 10 and 24 h
after administration. Determination of 6f concentration in rat plasma by LC-MS and the pharmacokinetic parameters
of rats were calculated using the non-compartmental model of Phoenix WinNonlin 6.4 software (Pharsight, USA).
1
9

In Vivo Efficacy Study BALB/c nude mice were subcutaneously inoculated with 0.1 ml of experimental cell
7
suspension (7×10 /ml) to establish a subcutaneous xenograft model. When the tumors were grown to an average
3
volume of 200-250 mm , they were randomly divided into 5 groups (each group 5 animals) and administered
according to tumor size and mouse body weight. Tumor volume was measured twice a week, and the weight of
tumor-bearing mice was weighed twice a day, and data were recorded. After 14 days of administration, the tumor
tissue was taken and weighed, and photographed at the same time. The T/C values were calculated according to the
National Cancer Institute (NCI) standardized transplant tumor evaluation method.
Long-term Toxicity Studies in Mice BALB/c small nude mice, half male and half female for 6~7 weeks, are
purchased from Shanghai SLAC Laboratory Animal CO. LTD; feed the mice under grade SPF. Successively dose
the mice by intragastric administration with dosage of 50 mg/kg, 150 mg/kg and 450 mg/kg once a day for 4 weeks;
feed the mice for 4 weeks to recovery after dosage; weigh the mice of the same group at the start of the test, after
administration and after the recovery time. Dissect the mice after bloodletting to die, weigh the corresponding
visceral organs, observe and check the tissue.
Acute Toxicity Studies in Mice BALB/c small nude mice, half male and half female for 6~7 weeks, are
purchased from Shanghai SLAC Laboratory Animal CO. LTD; feed the mice under grade SPF. Dose the mice by
intragastric administration with dosage of 200 mg/kg, 400 mg/kg or 2000 mg/kg by single-dose; observe the mice
for 14 days after administration; weigh the mice of the same group at the start of the test and after the observing time.
Dissect the mice after bloodletting to die, weigh the corresponding visceral organs, observe and check the tissue.
ACKNOWLEDGMENT
This work is supported by Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research and Jiangsu
Hansoh Pharmaceutical Corporation together with a project funded by the Priority Academic Program Development
of Jiangsu Higher Education Institutions. Dr. Fang is thankful to the support of the National Science and
Technology Major Foundation of China, and the State Key Laboratory for Chemistry and Molecular Engineering of
Medicinal Resources (Guangxi Normal University) (CMEMR2017-B05).
2
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Novel 7-Formyl-naphthyridyl-ureas Derivatives as
Potential Selective FGFR4 Inhibitors: Design, Synthesis, and
Biological Activity Studies
a,c
a,b,∗
a,c
c
*
Chang’an Sun , Lei Fang , Xiaobing Zhang , Peng Gao , Shaohua Gou
2
3

2
4