Design, synthesis and pharmacological evaluation of a novel mTOR-targeted anti-EV71 agent

Article abstract of DOI:10.1016/j.ejmech.2019.04.048

Due to the limitations of existing anti-EV71 targets, we have been eager to discover a new anti-EV71 agent based on mTOR (the mammalian target of rapamycin), which is an important target for finding antiviral agents based on host cells. Torin2 is a second

Full text of DOI:10.1016/j.ejmech.2019.04.048

Accepted Manuscript
Design, synthesis and pharmacological evaluation of a novel mTOR-targeted anti-
EV71 agent
Tianlong Hao, Yuexiang Li, Shiyong Fan, Wei Li, Shixu Wang, Song Li, Ruiyuan Cao,
Wu Zhong
PII:
S0223-5234(19)30366-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.04.048
EJMECH 11283
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 February 2019
Revised Date: 3 April 2019
Accepted Date: 17 April 2019
Please cite this article as: T. Hao, Y. Li, S. Fan, W. Li, S. Wang, S. Li, R. Cao, W. Zhong, Design,
synthesis and pharmacological evaluation of a novel mTOR-targeted anti-EV71 agent, European Journal
of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.04.048.
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ACCEPTED MANUSCRIPT
Design, synthesis and pharmacological evaluation of a novel
mTOR-targeted anti-EV71 agent
Tianlong Haoa, Yuexiang Lia, Shiyong Fana, Wei Lia, Shixu Wanga, Song Lia, Ruiyuan Cao*, Wu Zhong*
a National Engineering Research Center for the Emergency Drug, Beijing Institute of Pharmacology and
Toxicology, Beijing 100850, PR China
* Corresponding author
Wu Zhong: National Engineering Research Center for the Emergency Drug, Beijing Institute of
Pharmacology and Toxicology, China; E-mail: zhongwu@bmi.ac.cn
Ruiyuan Cao: National Engineering Research Center for the Emergency Drug, Beijing Institute of
Pharmacology and Toxicology, China; E-mail: 21cc@163.com
we discovered a novel anti-EV71 target—mTOR for the first time, and optimized
the structure of a potent anti-EV71 inhibitor, Torin2, to obtain potent activity and
better water solubility anti-EV71 agents.

ACCEPTED MANUSCRIPT
Design, synthesis and pharmacological evaluation of a novel mTOR-targeted
anti-EV71 agent
Tianlong Haoa, Yuexiang Lia, Shiyong Fana, Wei Lia, Shixu Wanga, Song Lia, Ruiyuan Caoa,*, Wu Zhong a,*
a National Engineering Research Center for the Emergency Drug, Beijing Institute of Pharmacology and Toxicology,
Beijing 100850, PR China
* Corresponding author
Wu Zhong: National Engineering Research Center for the Emergency Drug, Beijing Institute of Pharmacology and
Toxicology, China; E-mail: zhongwu@bmi.ac.cn
Ruiyuan Cao: National Engineering Research Center for the Emergency Drug, Beijing Institute of Pharmacology and
Toxicology, China; E-mail: 21cc@163.com
ABSTRACT
Due to the limitations of existing anti-EV71 targets, we have been eager to discover a new
anti-EV71 agent based on mTOR (the mammalian target of rapamycin), which is an important
target for finding antiviral agents based on host cells. Torin2 is a second-generation ATP
competitive mTOR kinase inhibitor (IC50=0.25 nM). Our research team tested the anti-EV71
activity of Torin2 in vitro for the first time. The result showed that Torin2 had significant
anti-EV71 activity (IC50=0.01 µM). In this study, thirty novel Torin2 derivatives were
synthesized and evaluated for anti-EV71 activity. Among them, 11a, 11b, 11d, 11e and 11m
displayed similar activity to Torin2. 11e displayed the most potent activity, with an IC50 value of
0.027 µM, which was closest to Torin2, and displayed potent mTOR kinase inhibitory activity. A
molecular modeling study showed that 11e interacted with Val2240 and Lys2187 via hydrogen
bonds and had a good match with the receptor. Additionally, a mechanism study showed that
most of the compounds had significant inhibition for the mTOR pathway substrates p70S6K and
Akt. The water solubility test of compounds with potent activity revealed that 11a and 11m were
improved by approximately 5-15-fold compared to Torin2. These data suggest that 11a and 11m
may be potential candidates for anti-EV71 treatment.
Keywords: mTOR; anti-EV71 Inhibitor.
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1. Introduction
Hand-foot-and-mouth disease (HFMD) is an infectious disease caused by a variety of
enteroviruses (EVs) that can be transmitted by direct contact with the gastrointestinal and
respiratory systems. According to WHO statistics, a wide range of HFMD epidemics have been
reported in various regions of the Western Pacific since 2010 [1, 2].
Enterovirus 71 (EV71) is the main pathogen that causes HFMD. At present, there are no
effective antiviral drugs and preventive vaccines against EV71, and most of them are still in the
preclinical research stage. The idea of EV71 antiviral therapy is mainly divided into two aspects.
On the one hand, it directly targets the EV71 infection process, and the targets are mainly EV71
RNA-dependent RNA polymerase, VP1, and nonstructural proteins (2A, 2B, 2C, 3A, 3C and
3D). There are also some agents that block the replication process of the virus by cutting off the
EV71 replication-related signaling pathways, including small molecule agents, natural products,
and nucleotide therapy, but most of them lack a rigorous clinical trial basis, and the mechanism
and effect need to be further clarified. Additionally, the long-term effects of certain drugs may
also trigger viral resistance mutations [3-11].
Due to the limitations of existing anti-EV71 virus targets, it is of particular importance to find
new targets for high-efficiency and low-toxicity against EV71. The mammalian target of
rapamycin (mTOR) is a highly conserved serine/threonine protein kinase that belongs to the
phosphoinositide 3-kinase (PI3K)-related protein kinase (PIKK) family [12, 13]. mTOR is a key
element in the PI3K/Akt/mTOR signaling pathway that controls cell growth in response to
energy, nutrients, growth factors and other environmental cues [14]. It exists in two functionally
distinct protein complexes, mTORC1 and mTORC2. The mTORC1 complex is composed of
Raptor, LST8, PRAS40 and Deptor, and is responsible for regulated protein synthesis through
phosphorylation of S6K1 and 4EBP1. The mTORC2 complex consists of Rictor, LST8, SIN1,
Deptor, and Protor, and regulates cell proliferation and survival through phosphorylation of
Akt/PKB. After the virus attaches and enters the host cell, it activates the PI3K/Akt/mTOR
signaling pathway, upregulates the phosphorylation level of downstream functional factors S6K1
and 4EBP1 of mTOR, reduces the host cell apoptosis caused by a viral infection, and makes the
virus continue to infect the cell [15]. A manuscript describing the mechanism of action studies is
in preparation and will be published elsewhere.
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In recent years, studies to inhibit viruses by inhibiting mTOR of host cells have been widely
reported [16-28]. We collected the existing mTOR inhibitors in the preclinical research stage and
tested their activities against the EV71 in vitro [29-41]. Our experimental data showed that
Torin2, which is a mTORC1 and mTORC2 dual inhibitor [42], exhibits potent activity against
EV71 with an IC50 of 0.01 µM. Although Torin2 showed high activity and suitable
bioavailability (F=51%), its water solubility is poor. We measured its solubility in water as 1.29
µg/ml which is almost insoluble according to Ch.P. (Pharmacopoeia of The People's Republic of
China). Moreover, as water solubility is an important physicochemical property affecting the
absorption of oral drugs, we considered to improve the water solubility of Torin2 through
structural modification while maintaining high activity. To overcome these deficiencies, we
initiated a medicinal chemistry campaign that resulted in the identification of a highly potent
anti-EV71 agent targeting mTOR.
2. Results and Discussion
2.1. Chemistry
Based on the early studies [42, 43] and the analysis of Torin2 and mTOR protein
cocrystallization, we optimized the structure of Torin2 and obtained the preliminary structural
formula (1’) of the designed compounds. The heterocycle A and B was an essential part of the
activity, and it needed to contain an electron-rich structure to form a hydrogen bond with the
amino acid residue; a quinoline ring was selected as the core structure. Ring C was mainly used
to maintain its pharmacokinetic properties, and a 3-trifluoromethylphenyl was selected. Part D
filled the hydrophobic pocket region of the acceptor, primarily a five or six-membered aromatic
heterocyclic ring or a flexible long-chain substitution. Therefore, we decided to retain the
quinoline ring structure of Part A and B and the 3-trifluoromethylphenyl structure of Part C. We
redesigned Part D and Part E and investigated its effect on the structure-activity relationship. We
designed series 1, 2 and 3 compounds and investigated the effects of the lactam structure, the
oxazolone structure and the ketene side chain structure of Part E on the activity. For Part D, we
chose aromatic rings, aromatic heterocycles, spiro rings, and chain substituents to investigate the
effect of different conjugated systems and different spatially oriented substitutions on activity.
Figure 1
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6-bromo-4-((3-(trifluoromethyl)phenyl)amino)quinoline-3-carbaldehyde (2) was synthesized
according to a method previously reported in the literature [42]. The Horner-Wadsworth
-Emmons olefination reaction of 2 with ethyl 2-(diethoxyphosphoryl) acetate in the presence of
potassium carbonate provided the intermediate 3 [44]. The Suzuki coupling reaction of
intermediate 3 with different boronic esters in the presence of Pd(PPh3)4 and potassium carbonate
provided the compounds 4a-4b [42]. Intermediate 3 reacted with different substituted aromatic
amines via a coupling reaction to afford compounds 4c-4d [45]. (Scheme 1).
Scheme 1
The oxidation reaction of 2 with MCPBA in the presence of potassium carbonate provided the
intermediate 5, which was treated with NaOH (1N) to give intermediate 6. Then, intermediate 6
reacted with N,N'-Carbonyldiimidazole via a condensation reaction to afford intermediate 7. The
Suzuki coupling reaction of intermediate 7 with different boronic esters in the presence of
Pd(PPh3)4 and potassium carbonate provided the compounds 8a-8c [46]. Intermediate 7 reacted
with different enamides or olefins via a coupling reaction to afford compounds 8d-8f [47].
(Scheme 2).
Scheme 2
Compound 2 reacted with 1-(triphenylphosphoranylidene)propan-2-one via a coupling
reaction to afford intermediate 9. The Suzuki coupling reaction of intermediate 9 with different
boronic esters in the presence of Pd(PPh3)4 and potassium carbonate provided the compounds
10a-10e [48]. (Scheme 3).
Scheme 3
Finally, considering the excellent anti-EV71 activity of compound 4b, a series of analogues,
11a-11o, were synthesized with different substitutions at the R4, R5 and R6-position via different
substituted pyridyl derivatives, such as alkyl, methoxy, fluoro, alkylamide, arylamide-substituted
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pyridine, other ring-substituted pyridine and quinolone to improve activity and pharmacokinetic
properties [42-44]. (Scheme 4).
Scheme 4
2.2. Biology
2.2.1. Anti-EV71 Activities and the SARs
Starting from the lead-compound Torin2, 4a-4d, 8a-8f, and 10a-10e were synthesized, and
their anti-EV71 activities were evaluated in RD cell lines. The results listed in Table 1 indicated
that compared to the compounds of series 2 and series 3, the series 1 compounds generally have
certain antiviral activities, of which compound 4b displayed closer anti-EV71 activity to Torin2.
Table 1
To search for potential compounds that display better activity and pharmacokinetic properties,
the reactions of intermediate 3 with different substituted pyridine derivatives were performed to
obtain compounds 11a-11o. In this series of analogues, we mainly investigated the effects of the
location, size, electronegativity of different substitutions on pyridyl, and obtained some obvious
SARs. The results listed in Table 2 indicated that the position and size of the substitution on
pyridine moiety was crucial for anti-EV71 activity.
Compound 11a, which contained -F at the C-2 (R4) position of the pyridine moiety displayed
some higher anti-EV71 activity compared to compound 11b, which contains -F at the C-6 (R6)
position. Compound 11c, which contained -OCH3 at the C-2 (R4) position, lost activity (IC50
>
200 µM). Moving the -OCH3 group from the C-2 position to the C-6 position significantly
increased the anti-EV71 activity (Compound 11d: IC50 values of 0.04 µM). Therefore, when the
substitution was an electron-donating group and located at the C-6 position, it displayed better
activity.
On the other hand, as shown in compound 11e-11o, when the C-6 (R6) position was
substituted by a N derivative, the anti-EV71 activity of the compound can be maintained.
Compound 11e, 11f and 11h were all substituted by a linear amide group. Among them, the
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propionamide-substituted compound 11e displayed the best activity (IC50 values of 0.027 µM)
close to the reference compound Torin2, but the activity gradually decreased with extension of
the length of the substitution. When a linear substitution (compound 11f) is replaced by a
branched substitution (compound 11g), the activity significantly decreased. The activity of
compound 11i, which was obtained by replacing the terminal substitution with a phenyl group,
was lower than that of compound 11e. Therefore, the SARs in the amide substituted compound
was that the chain substituted compounds were superior to the aromatic ring substituted
compounds. The length of the chain substitution had a significant influence on the activity, the
shorter the substitution length was, the better the activity.
The compound 11k substituted with an aryloxyamide displayed moderate activity, and when
the substitution was replaced by a benzyloxyamide (compound 11l), the activity was greatly
lower. Similarly, when replaced with a t-butoxyamide group, the activity of compound 11j
decreased.
If R6 was a heterocyclic substitution, the compound 11m, having a piperazine substitution,
displayed moderate activity, and if the piperazine of the compound 11m was replaced by a
morphine (compound 11n) or a pyrrolidine (compound 11o), the activity would be significantly
reduced. Therefore, the SARs of the cyclic substituted compound was that the activity of the
six-membered ring-substituted compound was superior to that of the five-membered
ring-substituted compound, and the activity of the double-N-ring-substituted compound was
superior to that of the single-N-ring-substituted compound. The SARs of all the synthesized
compounds are summarized in Figure 2.
Table 2
Figure 2
2.2.2. In vitro mTOR Kinase Inhibitory Activity
To elucidate whether the synthesized compounds target the mTOR kinase, the in vitro mTOR
kinase inhibitory activities of 8a, 10a and 11e, which were the representative compounds of the
respective series, were evaluated in regard to their relatively better anti-EV71 activities among
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all the tested compounds. The inhibitory concentrations that inhibited the mTOR kinase by 50%
(IC50) of compounds 8a, 10a, and 11e were 256.80, 7766, and 29.24 nM, respectively. Figure 3.
Figure 3
2.2.3. Molecular Modeling Study
Compound 11e displayed the best anti-EV71 activity and effective mTOR kinase inhibition in
the initial screening, and we then selected it as the optimized compound for the following
studies. To explore the binding modes of target compounds with the ATP-binding site of mTOR,
molecular docking simulation studies were carried out by using the Libdock module of the
Discovery Studio 2.5. When docking compound 11e, Figure 4A shows that compound 11e could
be docked in the ATP-binding site in a similar conformation to that of the reference compound
Torin2 in the crystal structure (PDB ID code: 4JSV). Since numerous studies have suggested that
the interaction to the hinge region is crucial to mTOR inhibitory activity, the interaction with
Val2240 is essential to many kinds of mTOR inhibitors [49-56]. Compound 11e and Torin2
could overlap in the position of the ring structure. The nitrogen atom on the quinoline group and
the oxygen atom on the carbonyl group of 11e and Torin2, respectively, bond to Val2240 and
Lys2187 via a hydrogen bond. Moreover, the structure of quinoline in 11e and Torin2 is fixed on
the central hydrophobic region of the ATP-binding site with a π-π conjugation effect with
Trp2239. To elucidate the SARs that were concluded from the anti-EV71 activities above,
molecular docking was also performed to study the binding mode of some representative
compounds. Comparing the docking results of 11e and 8a (the representative compounds of
series 2), compound 8a lost the hydrogen bond interaction with the Lys2187 (Figure 4B), which
played a vital role in binding with the ATP-binding site. Similarly, In comparison with 11e, the
decrease in the anti-EV71 activity of 10a might be due to the loss of the hydrogen bond with
Lys2187 (Figure 4C). Moreover, the structure of 10a was reversed, and the interaction between
the ketene side chain and the hydrophobic cavity was decreased compared to 11e. In comparison
with 11e, compounds 11d, 11h and 11m formed the same hydrogen bonds with Val2240 and
Lys2187 in the ATP-binding site. This might explain why their activity was close to 11e.
However, due to the increase in the size of the long chain substitution of the compound 11h
(Figure 4E) and the piperazine substitution of the compound 11m (Figure 4F), it was more
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easily exposed to the solvent environment. On the other hand, compared with 11e, we found the
activity decline of the -OCH3 substituted 11d (Figure 4D) because the -OCH3 substitution was
not enough to occupy the hydrophobic cavity. Therefore, the size of the substitution was critical
for activity.
Figure 4
2.2.4. Cell-based Enzyme Inhibition Assay
As reported in the references, ATP competitive inhibitors of mTOR inhibit both mTORC1 and
mTORC2 complexes and have a greater inhibitory function against mTORC2 than mTORC1
single inhibitors [57-59]. We performed a mTORC1 and mTORC2 cell-based assay to evaluate
inhibitory activity and the molecular mechanism of the synthesized compounds in RD cells.
mTORC1 activates the activity of P70S6K1 by phosphorylating the Thr389 site of P70S6K1,
mTORC2 activates Akt by phosphorylating the Ser473 site of Akt. Therefore, the degree of
phosphorylation of the Thr389 site of P70S6K1 and the Ser473 site of Akt in the test cells can be
detected to reflect the inhibition of intracellular mTORC1 and mTORC2 by the compound. In
this study, both p70 and Akt phosphorylation levels were evaluated in the mTORC1 and
mTORC2 pathways of the RD cell line with the treatment of 20 µM of the compounds in the
medium containing 167 nM of insulin for 2 h. Meanwhile, there were decreasing expression
levels of p70 phosphorylation due to the Rapamycin (a mTORC1 single inhibitor) treatment
under the same assay condition. The results are shown in Figure 5. Based on these observations,
we concluded that compounds 4a, 4b, 4c, 4d, 11a, 11b, 11d, 11e, 11f, 11g, 11h, 11i, 11j, 11k,
11l, 11m, 11n, and 11o could downregulate p70 phosphorylation expression levels. Compounds
4b, 4c, 4d, 11a, 11b, 11d, 11e, 11f, 11g, 11h, 11i, 11j, 11k, 11l, 11n, and 11o significantly
decreased the expression levels of phosphorylated Akt. The assay results showed that most series
3 compounds had dual inhibition of the mTORC1 and mTORC2 pathways. However,
compounds 8a and 8d of series 2 and compound 10a of series 3 can inhibit neither mTORC1 nor
mTORC2, which was consistent with the lower mTOR kinase inhibitory activity of compounds
8a and 10a.
Figure 5
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2.2.4. Absorption Properties and Water Solubility
As the poor absorption properties of Torin2 are the main factors that restrict its druggability,
the absorption properties of compounds 11a, 11b, 11d, 11e, and 11m, which displayed potent
activity, attracted our attention and were predicted using software ADMET Predictor version 8.5
(Simulations Plus Inc., Lancaster, CA, USA) [60]. The computer simulation results listed in
Table 3 revealed that the absorption properties of compounds 11a, 11b, 11d, 11e and 11m were
all in a reasonable range, which suggested that 11a, 11b, 11d, 11e and 11m were excellent
candidates for potential anti-EV71 agents.
Table 3
As the poor absorption of Torin2 may be due to the limited water solubility [42]. We measured
the water solubility of 11a, 11b, 11d, 11e and 11m. The results are listed in Table 4. The water
solubility of 11b, 11d and 11e were worse than Torin2. However, compared to Torin2, the water
solubility of 11a was improved by approximately 5-fold, of which 11m was improved by
approximately 15-fold, which suggested that 11a and 11m may display better absorption
properties than Torin2 for potential anti-EV71 agents and have the potential to become
candidates with good druggability.
Table 4
3. Conclusions
Due to the limitations of existing anti-EV71 targets, it is especially urgent to find new safe and
efficient targets. The past few years witnessed the rapid development of the novel mTOR
inhibitors as antiviral agents. Torin2 analogues are one of the most representative classes
displaying mTOR kinase inhibition. On the basis of our previous work, we focused the study on
the Torin2 derivatives derived from different core structures, including six-membered ring
compounds (series 1), five-membered ring compounds (series 2) and open-loop compounds
(series 3). The comprehensive and detailed SARs we acquired might provide some information
for the design and synthesis of new anti-EV71 agents. In the present work, we evaluated the
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inhibitory activities of the newly synthesized 30 compounds toward EV71 in RD cell lines.
Notably, compound 11e displayed the most potent in vitro anti-EV71 activity. To elucidate the
SARs, we conducted molecular docking studies on representative compounds. The docking
results displayed that compound 11e showed the best anti-EV71 activity and effective mTOR
kinase inhibition in the initial screening and interacted with Val2240 and Lys2187 via the
hydrogen bond, with a good match with the receptor. We therefore conclude that the necessary
hydrogen bonding and appropriate substitution size promoted the activity. To evaluate the
molecular mechanism of the compounds of the examples in RD cells, we performed mTORC1
and mTORC2 cell-based assays. Most compounds had dual inhibition of the mTORC1 and
mTORC2 pathways. As water solubility is an important physicochemical property affecting the
absorption of oral drugs, and water solubility is the main factor hindering Torin2’s druggability,
we predicted the absorption properties and measured the water solubility of synthesized
compounds with potent activity. The results revealed that the absorption properties of the
compounds were all in a reasonable range, and compared to Torin2, the water solubility of 11a
and 11m, which displayed similar anti-EV71 activity to Torin2, was improved by approximately
5-15-fold. All together, we developed anti-EV71 agents based on the mTOR target for the first
time, and found some potential candidates with better water solubility and potent activity.
Research on these compounds is ongoing, and further efforts are in process to find excellent
candidates for potential anti-EV71 agents.
4. Experiment Section
4.1. Chemistry
1
All reagents and solvents were used as received from commercial sources. H-NMR and
13C-NMR spectra were recorded at 400 MHz and 100 MHz on a JNM-ECA-400 instrument
(JEOL Ltd., Tokyo, Japan) in DMSO-D6. Chemical shifts are expressed in δ (ppm), with
tetramethylsilane (TMS) functioning as the internal reference. Coupling constants (J) were
expressed in Hz. High-resolution mass spectra were obtained using a TOF G6230A LC/MS
(Agilent Technologies, New York, NY, USA) with an ESI source. Melting points were
determined using an RY-1 apparatus (Yutong Company, Shanghai, China). Reagents and
1
13
solvents were commercially available without further purification. The H-NMR, C-NMR and
HRMS spectra of the compounds in this article can be found in Supporting Information.
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General Procedure for the Preparation of 4a-4d, 8a-8f, 10a-10e and 11a-11o.
9-bromo-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one (3) Intermediate
2 was synthesized according to a method previously reported in the literature [42]. To a solution
of compound 2 (3.95 g, 10 mmol), K2CO3 (4.15 g, 30 mmol) and ethyl 2-(diethoxyphosphoryl)
acetate (6.73 g, 30 mmol) in dry EtOH was added. The resulting mixture was heated to 100°C
for 12 h before cooling to room temperature. Upon removal of the solvents under a vacuum, the
residue was diluted with water followed by extraction with EtOAc. Purification of the residue by
ISCO (hexanes/EtOAc 5:1) provided compound 3, a yellow solid. (2.87 g, 68.6% yield). 1H
NMR (400 MHz, DMSO-D6) δ(ppm): 9.19 (s, 1H), 8.34 (d, J = 9.5 Hz, 1H), 8.07 (dd, J = 9.3,
8.8 Hz, 2H), 7.95 (dd, J = 17.2, 8.4 Hz, 2H), 7.86 – 7.79 (m, 2H), 6.98 (d, J = 9.5 Hz, 1H), 6.58
13
(d, J = 2.0 Hz, 1H). C NMR (101 MHz, DMSO-D6) δ(ppm): 162.92, 152.05, 148.04, 148.02,
141.58, 141.26, 140.64, 134.03, 133.15, 132.87, 132.02, 131.70, 131.37, 127.70, 126.83, 122.86,
119.21, 118.84, 114.21. HR-MS (ESI) m/z: calcd. for C19H10BrF3N2O [M + H]+: 418.9929,
found: 419.0001. Mp 192–195 °C.
9-(2-oxoindolin-5-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one (4a)
To a solution of compound 3 (4.18 g, 10 mmol) in 1,4-dioxane at room temperature, we
subsequently added Pd(PPh3)4 (1.16 g, 1 mmol), K2CO3 (2.76 g, 20 mmol), and (2-oxoindolin-5
-yl)boronic acid (2.12 g, 12 mmol). After degassing, the resulting mixture was heated to 80 °C
for 4 h before cooling to room temperature. The solution was extracted with EtOAc. The organic
layer was washed with water and brine, dried (MgSO4), filtered, and evaporated to dryness as a
yellow solid. (3.89 g, 82.6% yield) 1H NMR (400 MHz, DMSO-D6) δ(ppm): 10.53 (s, 1H), 9.15
(s, 1H), 8.34 (d, J = 9.5 Hz, 1H), 8.13 (dd, J = 25.1, 8.2 Hz, 3H), 8.05 – 7.87 (m, 2H), 7.83 (d, J
= 7.9 Hz, 1H), 7.15 (d, J = 7.7 Hz, 1H), 6.98 (dd, J = 22.7, 5.5 Hz, 2H), 6.64 – 6.47 (m, 2H),
13
3.51 (s, 2H). C NMR (101 MHz, DMSO-D6) δ(ppm): 177.18, 163.10, 151.41, 148.69, 144.94,
142.53, 141.91, 140.81, 139.01, 137.60, 133.90, 132.15, 131.99, 131.69, 131.42, 129.27, 127.01,
126.31, 125.01, 123.10, 122.90, 122.28, 120.36, 117.81, 114.09, 107.60, 36.04. HR-MS (ESI)
m/z: calcd. for C27H16F3N3O2 [M + H]+: 472.1195, found: 472.1268. Mp 281–282 °C.
9-(6-(dimethylamino)pyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(
1H)-one (4b) The title compound was obtained similarly to 4a. The boronic acid was replaced
1
with (6-(dimethylamino)pyridin-3-yl)boronic acid. (yellow solid, yield: 82.4%). H NMR (400
MHz, DMSO-D6) δ(ppm): 9.10 (s, 1H), 8.32 (d, J = 9.5 Hz, 1H), 8.13 (s, 1H), 8.06 (dd, J = 8.1,
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3.5 Hz, 2H), 8.00 (d, J = 2.4 Hz, 1H), 7.96 (dd, J = 8.7, 1.8 Hz, 1H), 7.90 (t, J = 7.9 Hz, 1H),
7.80 (d, J = 8.0 Hz, 1H), 7.06 (dd, J = 8.9, 2.6 Hz, 1H), 6.93 (t, J = 5.6 Hz, 2H), 6.57 (d, J = 8.9
Hz, 1H), 3.05 (s, 6H). 13C NMR (101 MHz, DMSO-D6) δ(ppm): 163.12, 158.83, 150.85, 148.29,
146.25, 142.17, 141.98, 140.82, 135.40, 135.28, 133.89, 132.03, 131.59, 131.39, 131.26, 128.31,
127.14, 126.64, 122.30, 122.18, 121.11, 117.99, 114.08, 105.82, 38.10. HR-MS (ESI) m/z: calcd.
for C26H19F3N4O [M + H]+: 461.1511, found: 461.1585. Mp 220–222 °C.
General procedure for the preparation of 9-((3-aminophenyl)amino)-1-(3-(trifluoromethyl)
phenyl)benzo[h][1,6]naphthyridin-2(1H)-one(4c) To a solution of compound 3 (4.18 g, 10
mmol) in 1,4-dioxane at room temperature, we subsequently added Pd(dba)3 (0.23 g, 0.25 mmol),
CsCO3 (4.88 g, 15 mmol), 1,3-phenylenediamineand (1.62 g, 15 mmol) and 4,5-Bis
(diphenylphosphino)-9,9-dimethylxanthene (0.14 g, 0.25 mmol). The resulting mixture was
heated to 100 °C for 2 h under a N2 atmosphere. The solution was extracted with EtOAc. The
organic layer was washed with water and brine, dried (MgSO4), filtered, and evaporated to
1
dryness as a yellow solid. (3.52 g, 78.8% yield). H NMR (400 MHz, DMSO-D6) δ(ppm): 8.85
(s, 1H), 8.22 (d, J = 9.5 Hz, 1H), 7.94 (s, 1H), 7.86 (d, J = 9.0 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H),
7.63 (dd, J = 14.4, 6.4 Hz, 2H), 7.53 (d, J = 7.9 Hz, 1H), 7.29 (dd, J = 9.0, 2.4 Hz, 1H), 6.89 –
6.80 (m, 2H), 6.43 (d, J = 2.3 Hz, 1H), 6.21 (dd, J = 7.9, 1.3 Hz, 1H), 6.07 (t, J = 2.0 Hz, 1H),
13
5.87 – 5.75 (m, 1H), 5.15 (s, 2H). C NMR (101 MHz, DMSO-D6) δ(ppm): 163.08, 149.72,
147.18, 144.29, 142.66, 142.42, 140.96, 140.81, 140.76, 133.62, 131.57, 131.43, 130.78, 130.47,
130.08, 125.39, 123.80, 122.80, 121.75, 118.98, 114.25, 108.69, 107.17, 105.64, 105.16. HR-MS
(ESI) m/z: calcd. for C25H17F3N4O [M + H]+: 447.1354, found: 447.1427. Mp 217–218 °C.
9-((6-aminopyridin-2-yl)amino)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1
H)-one (4d) The title compound was obtained similarly to 4c. 1,3-phenylenediamineand was
replaced with pyridine-2,6-diamine. (yellow solid, yield: 81.3%). 1H NMR (400 MHz,
DMSO-D6) δ(ppm): 8.91 (s, 1H), 8.50 (s, 1H), 8.25 (d, J = 9.5 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H),
7.87 (d, J = 9.0 Hz, 1H), 7.66 (dt, J = 13.4, 7.8 Hz, 3H), 7.52 (dd, J = 9.0, 2.3 Hz, 1H), 7.22 –
13
7.11 (m, 2H), 6.86 (d, J = 9.4 Hz, 1H), 5.91 (d, J = 7.9 Hz, 1H), 5.71 (t, J = 14.5 Hz, 3H). C
NMR (101 MHz, DMSO-D6) δ(ppm): 163.02, 148.31, 141.68, 141.18, 140.71, 134.17, 131.40,
130.91, 130.87, 130.58, 125.83, 125.80, 125.66, 125.63, 125.51, 125.11, 122.80, 122.05, 120.74,
118.47, 114.24, 114.22, 100.00, 97.26. HR-MS (ESI) m/z: calcd. for C24H16F3N5O [M + H]+:
448.1307, found: 448.1380. Mp 259–261 °C.
12

ACCEPTED MANUSCRIPT
6-bromo-4-((3-(trifluoromethyl)phenyl)amino)quinolin-3-yl formate (5) To a solution of
compound 2 (3.95 g, 10 mmol) in DCM at room temperature, we added 3-chloroperoxybenzoic
acid (3.45 g, 20 mmol). The resulting mixture was stirred at room temperature for 1 h. Upon
removal of the solvents, the residue was subjected to column purification (DCM/MeOH 10:1) to
1
furnish the desired compound 5 as a yellow solid. (3.25 g, 79.2% yield). H NMR (400 MHz,
DMSO-D6)δ(ppm): 9.07 (s, 1H), 8.85 (s, 1H), 8.03 – 7.87 (m, 3H), 7.79 – 7.75 (m, 1H), 7.70
13
(ddd, J = 4.5, 2.1, 1.0 Hz, 1H), 7.60 – 7.52 (m, 2H), 7.40 (dd, J = 23.0, 6.1 Hz, 1H). C NMR
(101 MHz, DMSO-D6) δ(ppm): 166.63, 163.07, 148.84, 146.18, 142.35, 141.86, 133.26, 132.09,
131.44, 131.20, 130.20, 129.37, 128.46, 125.23, 124.19, 123.54, 122.24. HR-MS (ESI) m/z:
calcd. for C17H10BrF3N2O2 [M + H]+: 410.9878, found: 410.9950. Mp 176–179 °C.
6-bromo-4-((3-(trifluoromethyl)phenyl)amino)quinolin-3-ol (6) A solution of compound 5
(4.10 g, 10 mmol) in NaOH (1 N) at room temperature was heated to 50 °C for 4 h. After the
mixture was cooled to room temperature, a solution of AcOH (1 N) was added to neutralize the
solution followed by dilution with water and extraction with EtOAc. After the organic layer was
dried with Na2SO4, the solvents were removed and the residue was purified by ISCO
1
(hexanes/EtOAc 5:1) to furnish compound 6 as a yellow solid. (3.27 g, 85.6% yield). H NMR
(400 MHz, DMSO-D6) δ(ppm): 10.47 (s, 1H), 8.68 (d, J = 22.9 Hz, 2H), 8.25 – 8.14 (m, 1H),
7.93 – 7.85 (m, 1H), 7.67 (dd, J = 8.9, 2.2 Hz, 1H), 7.37 (t, J = 7.9 Hz, 1H), 7.07 (d, J = 7.7 Hz,
1H), 6.98 (s, 1H), 6.92 – 6.83 (m, 1H). 13C NMR (101 MHz, DMSO-D6) δ(ppm):145.27, 144.95,
144.83, 142.65, 131.99, 130.00, 129.71, 127.31, 126.45, 126.30, 124.90, 123.60, 120.17, 119.17,
115.00, 111.79. HR-MS (ESI) m/z: calcd. for C16H10BrF3N2O [M + H]+: 382.9929, found:
383.0001. Mp 184–186 °C.
8-bromo-1-(3-(trifluoromethyl)phenyl)oxazolo[5,4-c]quinolin-2(1H)-one (7) To a solution of
compound 6 (3.82 g, 10 mmol) in dry THF at room temperature, we added
N,N'-Carbonyldiimidazole (2.43 g, 15 mmol). The resulting mixture was stirred at room
temperature for 4 h. Upon removal of the solvents, the residue was subjected to column
purification (DCM/MeOH 10:1) to furnish the desired compound 7 as a yellow solid. (3.10 g,
1
75.9% yield). H NMR (400 MHz, DMSO-D6)δ(ppm): 9.18 (s, 1H), 8.31 (d, J = 1.9 Hz, 1H),
8.15 (ddd, J = 8.6, 1.7, 0.6 Hz, 2H), 8.07 – 7.99 (m, 2H), 7.83 (dd, J = 9.1, 2.2 Hz, 1H), 7.65 (s,
1H). 13C NMR (101 MHz, DMSO-D6) δ(ppm): 153.07, 144.44, 137.15, 135.69, 135.17, 134.88,
13

ACCEPTED MANUSCRIPT
133.47, 133.16, 132.17, 132.05, 131.92, 131.07, 127.84, 126.17, 122.60, 120.55, 116.46. HR-MS
(ESI) m/z: calcd. for C17H8BrF3N2O2 [M + H]+: 408.9721, found: 408.9793. Mp 193–195 °C.
8-(6-methylpyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)oxazolo[5,4-c]quinolin-2(1H)-one (8a)
To a solution of compound 7 (4.08 g, 10 mol) in 1,4-dioxane at room temperature, we
subsequently added Pd(PPh3)4 (1.16 g, 1 mmol), K2CO3 (2.76 g, 20 mmol), and
(6-methylpyridin-3-yl)boronic acid (1.64 g, 12 mmol). After degassing, the resulting mixture
was heated to 80 °C for 4 h before cooling to room temperature. The solution was extracted with
EtOAc. The organic layer was washed with water and brine, dried (MgSO4), filtered, and
evaporated to dryness. As a yellow solid (3.59 g, 85.3% yield). 1H-NMR (400 MHz, DMSO-D6)
δ (ppm): 9.36 (s, 1H), 8.82 (d, J = 2.0 Hz, 1H), 8.42 – 8.35 (m, 2H), 8.28 (ddd, J = 15.9, 8.6, 4.5
Hz, 3H), 8.12 (d, J = 8.0 Hz, 1H), 8.03 (t, J = 7.9 Hz, 1H), 7.93 (d, J = 8.4 Hz, 1H), 7.29 (d, J =
1.8 Hz, 1H), 2.77 (s, 3H). 13C NMR (101 MHz, DMSO-D6) δ (ppm): 154.29, 153.07, 143.94,
142.35, 140.01, 137.09, 135.01, 134.77, 133.92, 133.47, 133.21, 132.16, 131.37, 131.04, 130.01,
129.06, 128.13, 127.94, 126.13, 125.40, 119.51, 115.11, 19.81. HR-MS (ESI) m/z: calcd. for
C23H14F3N3O2 [M + H]+: 422.1038, found: 422.1111. Mp 299–300 °C.
8-(5-methoxypyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)oxazolo[5,4-c]quinolin-2(1H)-one
(8b) The title compound was obtained similarly to 8a. The boronic acid was replaced with
(5-methoxypyridin-3-yl)boronic acid. (yellow solid, yield: 83.1%). 1H-NMR (400 MHz,
DMSO-D6) δ(ppm): 9.17 (s, 1H), 8.38 (s, 1H), 8.28 (d, J = 2.7 Hz, 1H), 8.22 (d, J = 8.9 Hz, 2H),
8.18 – 8.11 (m, 3H), 8.01 (t, J = 7.9 Hz, 1H), 7.32 – 7.29 (m, 1H), 7.15 (d, J = 1.8 Hz, 1H), 3.84
(s, 3H). 13C NMR (101 MHz, DMSO-D6) δ (ppm): 156.10, 153.20, 145.50, 139.82, 138.08,
137.03, 135.46, 135.28, 134.94, 133.72, 132.94, 132.12, 131.75, 131.42, 131.10, 128.13, 127.75,
126.40, 125.41, 118.26, 118.16, 115.49, 56.08. HR-MS (ESI) m/z: calcd. for C23H14F3N3O3 [M +
H]+: 438.0987, found: 438.1060. Mp 257–258 °C.
8-(quinolin-3-yl)-1-(3-(trifluoromethyl)phenyl)oxazolo[5,4-c]quinolin-2(1H)-one (8c) The title
compound was obtained similarly to 8a. The boronic acid was replaced with quinolin-3
1
-ylboronic acid. (gray solid, yield: 82.2%). H-NMR (400 MHz, DMSO-D6) δ (ppm): 9.19 (s,
1H), 8.85 (d, J = 2.4 Hz, 1H), 8.44 – 8.40 (m, 2H), 8.30 – 8.23 (m, 3H), 8.19 (d, J = 8.0 Hz, 1H),
8.08 – 8.02 (m, 2H), 7.96 (d, J = 7.3 Hz, 1H), 7.83 – 7.78 (m, 1H), 7.71 – 7.67 (m, 1H), 7.29 (s,
1H).13C-NMR (101 MHz, DMSO-D6) δ (ppm): 153.19, 148.99, 147.48, 145.45, 137.09, 135.53,
135.29, 134.94, 133.77, 132.96, 132.21, 131.94, 131.84, 131.41, 131.09, 130.70, 129.23, 128.77,
14

ACCEPTED MANUSCRIPT
128.14, 128.03, 127.87, 127.80, 126.37, 125.47, 118.32, 115.62. HR-MS (ESI) m/z: calcd. for
C26H14F3N3O2 [M + H]+: 458.1038, found: 458.1111. Mp 242–244 °C.
(E)-1-(3-(2-oxo-1-(3-(trifluoromethyl)phenyl)-1,2-dihydrooxazolo[5,4-c]quinolin-8-yl)allyl)ur
ea (8d) To a solution of compound 7 (4.08 g, 10 mmol) in DMF at room temperature, we added
Pd(OAc)2 (0.45 g, 2 mmol), 1-allylurea (2.00 g, 20 mmol), Tri(o-tolyl)phosphine (1.22 g, 4
mmol) and Et3N(10.12 g, 100 mmol). The resulting mixture was heated to 100 °C for 2 h under
N2 atmosphere. The residue was diluted with water followed by extraction with EtOAc. The
organic layer was washed with water and brine, dried (MgSO4), filtered, and evaporated to
1
dryness, yellow solid (3.64g, 84.9% yield). H NMR (400 MHz, DMSO-D6) δ(ppm): 9.06 (s,
1H), 8.30 (s, 1H), 8.17 – 8.09 (m, 2H), 8.05 – 7.96 (m, 2H), 7.86 (d, J = 9.1 Hz, 1H), 6.78 (s,
1H), 6.25 – 6.11 (m, 3H), 5.51 (s, 2H), 3.70 (t, J = 5.4 Hz, 2H). 13C NMR (101 MHz, DMSO-D6)
δ(ppm): 158.96, 153.22, 145.27, 136.96, 135.64, 135.22, 133.80, 133.40, 132.65, 132.21, 131.99,
131.07, 130.65, 128.48, 127.67, 126.85, 126.22, 117.52, 115.55, 115.17, 113.65. HR-MS (ESI)
m/z: calcd. for C21H15F3N4O3 [M + H]+: 429.1096, found: 429.1169. Mp 158–160 °C.
(E)-4-(2-oxo-1-(3-(trifluoromethyl)phenyl)-1,2-dihydrooxazolo[5,4-c]quinolin-8-yl)but-3-enen
itrile (8e) The title compound was obtained similarly to 8d. 1-allylurea was replaced with
1
acrylonitrile. (yellow solid, yield: 82.7%). H NMR (400 MHz, DMSO-D6)δ(ppm): 9.16 (d, J =
3.0 Hz, 1H), 8.25 (s, 1H), 8.12 (t, J = 8.1 Hz, 3H), 8.05 (dd, J = 9.1, 1.7 Hz, 1H), 8.01 – 7.95 (m,
13
1H), 7.51 (d, J = 16.6 Hz, 1H), 7.09 (s, 1H), 6.42 (d, J = 16.6 Hz, 1H). C NMR (101 MHz,
DMSO-D6) δ(ppm): 153.18, 149.82, 148.24, 146.64, 137.23, 135.43, 134.83, 133.26, 133.03,
132.43, 132.08, 131.74, 128.30, 127.76, 125.87, 125.50, 122.53, 118.88, 115.22, 99.21. HR-MS
(ESI) m/z: calcd. for C20H10F3N3O2 [M + H]+: 382.0725, found: 382.0798. Mp 235–237 °C.
methyl(E)-3-(2-oxo-1-(3-(trifluoromethyl)phenyl)-1,2-dihydrooxazolo[5,4-c]quinolin-8-yl)acr
ylate (8f) The title compound was obtained similarly to 8d. 1-allylurea was replaced with methyl
acrylate. (yellow solid, yield: 82.3%). 1H NMR (400 MHz, DMSO-D6) δ(ppm): 9.17 (d, J = 11.8
Hz, 1H), 8.31 (s, 1H), 8.15 (d, J = 8.0 Hz, 2H), 8.11 (s, 2H), 8.01 (t, J = 7.9 Hz, 1H), 7.37 (d, J =
16.0 Hz, 1H), 7.08 (s, 1H), 6.50 (d, J = 16.0 Hz, 1H), 3.71 (s, 3H). 13C NMR (101 MHz,
DMSO-D6) δ(ppm): 166.73, 153.17, 146.48, 143.43, 137.17, 135.26, 135.06, 133.39, 133.14,
132.67, 132.22, 132.14, 132.08, 131.63, 129.21, 127.75, 127.13, 121.89, 120.20, 115.33, 52.25.
HR-MS (ESI) m/z: calcd. for C21H13F3N2O4 [M + H]+: 415.0827, found: 415.0900. Mp 243–245
°C.
15

ACCEPTED MANUSCRIPT
(E)-4-(6-bromo-4-((3-(trifluoromethyl)phenyl)amino)quinolin-3-yl)but-3-en-2-one (9) To a
solution
of
compound
2
(3.95
g,
10
mmol)
in
DMSO,
then
1-(triphenylphosphoranylidene)propan-2-one (3.18 g, 10 mmol) was added. The resulting
mixture was heated to 120 °C for 4 h before cooling to room temperature. The residue was
diluted with water followed by extraction with EtOAc. Purification of the residue by ISCO
1
(hexanes/EtOAc 5:1) provided compound 9 as a yellow solid. (3.79 g, 87.3% yield). H NMR
(400 MHz, DMSO-D6) δ(ppm): 9.46 (s, 1H), 9.13 (s, 1H), 8.47 (d, J = 1.9 Hz, 1H), 7.94 (dt, J =
8.9, 5.4 Hz, 2H), 7.45 (t, J = 8.3 Hz, 1H), 7.35 (d, J = 16.5 Hz, 1H), 7.23 (d, J = 7.7 Hz, 1H),
7.12 (d, J = 5.7 Hz, 2H), 6.89 (d, J = 16.5 Hz, 1H), 1.98 (s, 3H). 13C NMR (101 MHz,
DMSO-D6) δ(ppm): 198.15, 151.28, 148.24, 145.30, 144.10, 138.46, 134.00, 132.24, 132.07,
131.98, 130.76, 129.35, 129.24, 128.30, 126.43, 124.78, 121.43, 120.32, 118.81, 27.19. HR-MS
(ESI) m/z: calcd. for C20H14BrF3N2O [M + H]+: 435.0242, found: 435.0313. Mp 115–119 °C.
(E)-4-(6-(6-aminopyridin-3-yl)-4-((3-(trifluoromethyl)phenyl)amino)quinolin-3-yl)
but-3
-en-2-one(10a) To a solution of compound 9 (4.34 g, 10 mmol) in 1,4-dioxane at room
temperature, Pd(PPh3)4 (1.16 g, 1 mmol), K2CO3 (2.76 g, 20 mmol), and (4-aminophenyl)
boronic acid (1.64 g, 12 mmol) were subsequently added. After degassing, the resulting mixture
was heated to 80 °C for 4 h before cooling to room temperature. The solution was extracted with
EtOAc. The organic layer was washed with water and brine, dried (MgSO4), filtered, and
evaporated to dryness as a yellow solid (3.69 g, 82.6% yield). 1H-NMR (400 MHz, DMSO-D6) δ
(ppm): 9.52 (s, 1H), 9.12 (s, 1H), 8.43 (d, J = 1.6 Hz, 1H), 8.10 (d, J = 8.7 Hz, 1H), 8.02 (dd, J =
8.7, 1.8 Hz, 1H), 7.48 (d, J = 4.1 Hz, 1H), 7.45 (d, J = 4.4 Hz, 1H), 7.24 (d, J = 7.8 Hz, 1H), 7.19
– 7.14 (m, 3H), 6.98 (d, J = 1.7 Hz, 1H), 6.94 – 6.88 (m, 2H), 6.65 (dd, J = 8.0, 1.4 Hz, 1H), 5.24
13
(s, 2H), 2.03 (s, 3H). C NMR (101 MHz, DMSO-D6) δ(ppm): 198.13, 150.31, 149.74, 148.90,
145.80, 145.10, 140.66, 139.75, 138.83, 130.73, 130.46, 130.12, 130.04, 127.79, 125.98, 123.54,
121.33, 118.50, 117.20, 115.32, 114.16, 113.82, 112.95, 27.17. HR-MS (ESI) m/z: calcd. for
C26H20F3N3O [M + H]+: 448.1558, found: 448.1632. Mp 183–185 °C.
(E)-4-(6-(3-aminophenyl)-4-((3-(trifluoromethyl)phenyl)amino)quinolin-3-yl)but-3-en-2-one(1
0b) The title compound was obtained similarly to 10a. The boronic acid was replaced with
1
(3-aminophenyl)boronic acid. (yellow solid, yield: 84.7%). H-NMR (400 MHz, DMSO-D6)
δ(ppm): 9.49 (s, 1H), 9.10 (s, 1H), 8.39 (s, 1H), 8.08 (d, J = 8.7 Hz, 1H), 8.00 (dd, J = 8.7, 1.5
Hz, 1H), 7.45 (dd, J = 15.8, 7.0 Hz, 2H), 7.22 (d, J = 7.7 Hz, 1H), 7.13 (dd, J = 7.9, 4.5 Hz, 3H),
16

ACCEPTED MANUSCRIPT
6.90 (dd, J = 24.7, 8.4 Hz, 3H), 6.61 (d, J = 6.7 Hz, 1H), 5.22 (s, 2H), 2.01 (s, 3H). 13C NMR
(101 MHz, DMSO-D6) δ(ppm): 198.13, 150.33, 149.75, 148.88, 145.79, 145.10, 140.64, 139.75,
138.80, 130.76, 130.46, 130.14, 130.04, 128.69, 127.82, 125.98, 123.54, 123.27, 121.31, 118.52,
117.21, 115.28, 114.14, 113.81, 112.92, 27.21. HR-MS (ESI) m/z: calcd. for C26H20F3N3O [M +
H]+: 448.1558, found: 448.1630. Mp 184–186 °C.
(E)-4-(6-(5-methoxypyridin-3-yl)-4-((3-(trifluoromethyl)phenyl)amino)quinolin-3-yl)but-3-en-
2-one (10c) The title compound was obtained similarly to 10a. The boronic acid was replaced
1
with (5-methoxypyridin-3-yl)boronic acid. (green solid, yield: 88.5%). H-NMR (400 MHz,
DMSO-D6) δ(ppm): 9.51 (s, 1H), 9.12 (s, 1H), 8.58 (dd, J = 25.3, 1.4 Hz, 2H), 8.35 (d, J = 2.7
Hz, 1H), 8.20 (dd, J = 8.7, 1.7 Hz, 1H), 8.12 (d, J = 8.7 Hz, 1H), 7.74 – 7.65 (m, 1H), 7.53 –
7.38 (m, 2H), 7.26 (d, J = 7.7 Hz, 1H), 7.18 (d, J = 6.9 Hz, 2H), 6.89 (d, J = 16.4 Hz, 1H), 3.92
13
(s, 3H), 2.01 (s, 3H). C NMR (101 MHz, DMSO-D6) δ(ppm): 198.13, 156.14, 151.01, 149.16,
145.48, 145.31, 140.70, 138.79, 137.25, 136.10, 135.29, 130.75, 130.44, 130.06, 127.88, 123.23,
123.16, 122.46, 122.03, 119.22, 118.13, 117.66, 114.50, 114.47, 56.25, 27.17. HR-MS (ESI)
m/z: calcd. for C26H20F3N3O2 [M + H]+: 464.1508, found: 464.1580. Mp 164–167 °C.
(E)-4-(4'-((3-(trifluoromethyl)phenyl)amino)-[3,6'-biquinolin]-3'-yl)but-3-en-2-one (10d) The
title compound was obtained similarly to 10a. The boronic acid was replaced with
1
quinolin-3-ylboronic acid. (yellow solid, yield: 90.9%). H-NMR (400 MHz, DMSO-D6)
δ(ppm): 9.57 (s, 1H), 9.38 (d, J = 2.1 Hz, 1H), 9.13 (s, 1H), 8.75 (d, J = 7.3 Hz, 2H), 8.34 (d, J =
8.6 Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 8.13 – 8.02 (m, 2H), 7.81 (t, J = 7.7 Hz, 1H), 7.68 (t, J =
7.5 Hz, 1H), 7.54 – 7.35 (m, 2H), 7.28 (d, J = 7.8 Hz, 1H), 7.22 (d, J = 1.9 Hz, 2H), 6.89 (d, J =
16.4 Hz, 1H), 2.00 (s, 3H). 13C NMR (101 MHz, DMSO-D6) δ(ppm): 198.12, 151.01, 150.08,
149.07, 147.47, 145.48, 145.40, 138.91, 135.49, 133.87, 132.53, 130.92, 130.78, 130.39, 130.14,
129.26, 128.95, 128.08, 127.85, 127.77, 125.96, 123.35, 123.25, 122.47, 122.14, 118.04, 117.71,
114.58, 27.12. HR-MS (ESI) m/z: calcd. for C29H20F3N3O [M + H]+: 484.1558, found: 484.1631.
Mp 173–176 °C.
(E)-4-(6-(6-fluoropyridin-3-yl)-4-((3-(trifluoromethyl)phenyl)amino)quinolin-3-yl)but-3-en-2-
one (10e) The title compound was obtained similarly to 10a. The boronic acid was replaced with
(6-fluoropyridin-3-yl)boronic acid. (yellow solid, yield: 89.3%). 1H-NMR (400 MHz,
DMSO-D6) δ(ppm): 10.22 (s, 1H), 9.08 (s, 1H), 8.87 – 8.71 (m, 2H), 8.51 (td, J = 8.3, 2.7 Hz,
1H), 8.24 (dd, J = 8.8, 1.8 Hz, 1H), 8.13 (d, J = 8.7 Hz, 1H), 7.54 (t, J = 7.8 Hz, 1H), 7.45 – 7.26
17

ACCEPTED MANUSCRIPT
(m, 4H), 7.20 (d, J = 16.4 Hz, 1H), 6.79 (d, J = 16.4 Hz, 1H), 1.92 (d, J = 3.9 Hz, 3H). 13C NMR
(101 MHz, DMSO-D6) δ(ppm): 197.97, 162.22, 149.07, 147.65, 146.57, 146.42, 144.28, 141.27,
141.19, 138.71, 134.70, 133.55, 130.74, 128.03, 125.85, 123.76, 123.14, 122.56, 122.46, 119.10,
116.46, 116.28, 110.51, 110.14, 26.83. HR-MS (ESI) m/z: calcd. for C25H17F4N3O [M + H]+:
452.1308, found: 452.1381. Mp 133–134 °C.
9-(2-fluoropyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one
(11a) The title compound was obtained similarly to 4a. The boronic acid was replaced with
(2-fluoropyridin-3-yl)boronic acid. (white solid, yield: 89.9%). 1H NMR (400 MHz, DMSO-D6)
δ(ppm): 9.22 (s, 1H), 8.36 (d, J = 9.5 Hz, 1H), 8.29 – 8.22 (m, 1H), 8.16 (d, J = 8.7 Hz, 1H),
8.09 (s, 1H), 7.99 – 7.89 (m, 2H), 7.89 – 7.77 (m, 2H), 7.53 – 7.45 (m, 1H), 7.44 – 7.35 (m, 1H),
6.98 (dd, J = 9.5, 3.8 Hz, 1H), 6.89 (d, J = 1.7 Hz, 1H). 13C NMR (101 MHz, DMSO-D6)
δ(ppm): 163.07, 160.88, 158.52, 152.23, 148.89, 147.72, 147.57, 142.59, 141.67, 141.39, 140.79,
133.83, 132.02, 131.21, 130.99, 130.65, 126.98, 126.59, 125.99, 122.94, 122.48, 122.26, 117.64,
114.15. HR-MS (ESI) m/z: calcd. for C24H13F4N3O [M + H]+: 436.0995, found: 436.1068. Mp
158–161 °C.
9-(6-fluoropyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one
(11b) The title compound was obtained similarly to 4a. The boronic acid was replaced with
(6-fluoropyridin-3-yl)boronic acid. (white solid, yield: 87.6%). 1H NMR (400 MHz, DMSO-D6)
δ(ppm): 9.19 (s, 1H), 8.36 (d, J = 9.5 Hz, 1H), 8.15 (d, J = 8.7 Hz, 2H), 8.09 – 8.00 (m, 2H),
7.97 – 7.86 (m, 2H), 7.84 (d, J = 8.0 Hz, 1H), 7.69 (td, J = 8.2, 2.7 Hz, 1H), 7.23 (dd, J = 8.5, 2.8
Hz, 1H), 6.96 (dd, J = 14.4, 5.6 Hz, 2H). 13C NMR (101 MHz, DMSO-D6) δ(ppm): 163.05,
151.97, 148.96, 145.77, 145.61, 142.48, 141.89, 140.80, 140.60, 140.52, 134.08, 133.70, 133.13,
132.14, 131.75, 129.11, 127.14, 126.70, 123.55, 122.47, 117.79, 114.19, 110.36, 109.99. HR-MS
(ESI) m/z: calcd. for C24H13F4N3O [M + H]+: 436.0995, found: 436.1068. Mp 264–266 °C.
9-(2-methoxypyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one
(11c) The title compound was obtained similarly to 4a. The boronic acid was replaced with
(2-methoxypyridin-3-yl)boronic acid. (white solid, yield: 88.2%). 1H NMR (400 MHz,
DMSO-D6) δ(ppm): 9.18 (s, 1H), 8.35 (d, J = 9.5 Hz, 1H), 8.17 (dd, J = 4.8, 2.1 Hz, 1H), 8.11 –
8.05 (m, 2H), 7.96 – 7.88 (m, 2H), 7.87 – 7.76 (m, 2H), 7.05 – 6.92 (m, 3H), 6.80 (d, J = 1.7 Hz,
1H), 3.81 (s, 3H). 13C NMR (101 MHz, DMSO-D6) δ(ppm): 163.10, 160.34, 151.69, 148.64,
147.02, 142.48, 141.86, 140.83, 138.76, 134.06, 133.95, 131.99, 131.42, 131.11, 130.30, 126.99,
18

ACCEPTED MANUSCRIPT
126.49, 125.75, 123.11, 122.82, 122.24, 117.62, 117.51, 113.99, 53.90. HR-MS (ESI) m/z: calcd.
for C25H16F3N3O2 [M + H]+: 448.1195, found: 448.1268. Mp 272–275 °C.
9-(6-methoxypyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-one
(11d) The title compound was obtained similarly to 4a. The boronic acid was replaced with
(6-methoxypyridin-3-yl)boronic acid. (white solid, yield: 87.4%). 1H NMR (400 MHz,
DMSO-D6) δ(ppm): 9.16 (s, 1H), 8.34 (d, J = 9.5 Hz, 1H), 8.16 – 8.08 (m, 2H), 8.07 – 7.96 (m,
3H), 7.89 (t, J = 7.9 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.28 (dd, J = 8.7, 2.6 Hz, 1H), 6.94 (dd, J
= 10.5, 5.6 Hz, 2H), 6.80 (dd, J = 8.6, 0.4 Hz, 1H), 3.88 (s, 3H). 13C NMR (101 MHz,
DMSO-D6) δ(ppm): 163.76, 163.08, 151.50, 148.67, 145.35, 142.37, 141.95, 140.81, 137.55,
134.32, 133.93, 132.12, 131.57, 131.28, 128.83, 128.61, 127.20, 126.68, 125.58, 122.70, 122.34,
117.88, 114.13, 110.92, 53.90. HR-MS (ESI) m/z: calcd. for C25H16F3N3O2 [M + H]+: 448.1195,
found: 448.1267. Mp 211–216 °C.
N-(5-(2-oxo-1-(3-(trifluoromethyl)phenyl)-1,2-dihydrobenzo[h][1,6]naphthyridin-9-yl)pyridin
e-2-yl) propionamide (11e) The title compound was obtained similarly to 4a. The boronic acid
1
was replaced with (6-propionamidopyridin-3-yl)boronic acid. (white solid, yield: 90.9%). H
NMR (400 MHz, DMSO-D6) δ(ppm): 10.64 (s, 1H), 9.18 (s, 1H), 8.35 (d, J = 9.5 Hz, 1H), 8.07
(ddd, J = 16.3, 11.5, 5.5 Hz, 6H), 7.96 – 7.83 (m, 2H), 7.49 (dd, J = 8.7, 2.5 Hz, 1H), 6.99 (dd, J
= 22.2, 5.5 Hz, 2H), 2.43 (q, J = 7.5 Hz, 2H), 1.08 (t, J = 7.5 Hz, 3H). 13C NMR (101 MHz,
DMSO-D6) δ(ppm): 173.64, 163.08, 152.34, 151.49, 148.56, 145.94, 142.49, 141.94, 140.82,
136.41, 134.14, 132.06, 131.63, 131.52, 131.31, 130.07, 128.94, 128.78, 127.07, 126.56, 125.58,
122.44, 117.89, 114.14, 113.41, 29.80, 9.89. HR-MS (ESI) m/z: calcd. for C27H19F3N4O2 [M +
H]+: 489.1460, found: 489.1533. Mp 249–250 °C.
N-(5-(2-oxo-1-(3-(trifluoromethyl)phenyl)-1,2-dihydrobenzo[h][1,6]naphthyridin-9-yl)pyridin
-2-yl)butyramide (11f) The title compound was obtained similarly to 4a. The boronic acid was
replaced with (6-butyramidopyridin-3-yl)boronic acid. (white solid, yield: 81.9%). 1H NMR (400
MHz, DMSO-D6) δ(ppm): 10.65 (s, 1H), 9.16 (s, 1H), 8.34 (d, J = 9.5 Hz, 1H), 8.12 (d, J = 8.8
Hz, 3H), 8.08 – 7.97 (m, 3H), 7.96 – 7.82 (m, 2H), 7.49 (dd, J = 8.7, 2.5 Hz, 1H), 6.99 (dd, J =
13
24.4, 5.5 Hz, 2H), 2.40 (t, J = 7.3 Hz, 2H), 1.69 – 1.55 (m, 2H), 0.92 (t, J = 7.4 Hz, 3H). C
NMR (101 MHz, DMSO-D6) δ(ppm): 172.82, 163.08, 152.30, 151.60, 148.78, 145.94, 142.37,
141.96, 140.82, 136.40, 134.08, 132.05, 131.68, 131.30, 130.13, 128.87, 127.05, 126.54, 125.58,
19

ACCEPTED MANUSCRIPT
122.86, 122.41, 122.35, 117.88, 114.14, 113.46, 38.43, 18.86, 14.13. HR-MS (ESI) m/z: calcd.
for C28H21F3N4O2 [M + H]+: 503.1617, found: 503.1689. Mp 279–280 °C.
N-(5-(2-oxo-1-(3-(trifluoromethyl)phenyl)-1,2-dihydrobenzo[h][1,6]naphthyridin-9-yl)pyridin
-2-yl)isobutyramide (11g) The title compound was obtained similarly to 4a. The boronic acid
1
was replaced with (6-isobutyramidopyridin-3-yl)boronic acid. (white solid, yield: 82.8%). H
NMR (400 MHz, DMSO-D6) δ(ppm): 10.64 (s, 1H), 9.17 (s, 1H), 8.35 (d, J = 9.4 Hz, 1H), 8.17
– 8.09 (m, 3H), 8.09 – 8.00 (m, 3H), 7.95 – 7.84 (m, 2H), 7.48 (dd, J = 8.7, 2.5 Hz, 1H), 7.03 (d,
J = 1.5 Hz, 1H), 7.00 – 6.93 (m, 1H), 2.79 (dt, J = 13.6, 6.8 Hz, 1H), 1.11 (d, J = 6.8 Hz, 6H).
13C NMR (101 MHz, DMSO-D6) δ(ppm): 176.90, 163.09, 152.43, 151.62, 148.79, 145.94,
142.39, 141.97, 140.83, 136.38, 134.17, 134.07, 132.06, 131.70, 131.63, 131.30, 130.14, 128.88,
127.08, 126.56, 122.40, 120.17, 117.90, 114.14, 113.53, 34.97, 19.91. HR-MS (ESI) m/z: calcd.
for C28H21F3N4O2 [M + H]+: 503.1617, found: 503.1689. Mp 213–215 °C.
N-(5-(2-oxo-1-(3-(trifluoromethyl)phenyl)-1,2-dihydrobenzo[h][1,6]naphthyridin-9-yl)pyridin
-2-yl)pentanamide (11h) The title compound was obtained similarly to 4a. The boronic acid was
1
replaced with (6-pentanamidopyridin-3-yl)boronic acid. (white solid, yield: 86.3%). H NMR
(400 MHz, DMSO-D6) δ(ppm): 10.64 (s, 1H), 9.17 (s, 1H), 8.35 (d, J = 9.5 Hz, 1H), 8.12 (dd, J
= 8.5, 6.3 Hz, 3H), 8.08 – 7.97 (m, 3H), 7.96 – 7.83 (m, 2H), 7.49 (dd, J = 8.7, 2.5 Hz, 1H), 7.03
(d, J = 1.6 Hz, 1H), 6.96 (d, J = 9.4 Hz, 1H), 2.42 (t, J = 7.4 Hz, 2H), 1.63 – 1.51 (m, 2H), 1.32
(dq, J = 14.6, 7.3 Hz, 2H), 0.97 – 0.85 (m, 3H). 13C NMR (101 MHz, DMSO-D6) δ(ppm):
172.97, 163.08, 152.31, 151.62, 148.79, 145.95, 142.39, 141.97, 140.83, 136.41, 134.15, 134.10,
132.06, 131.70, 131.62, 131.30, 130.14, 128.90, 127.06, 126.58, 122.43, 122.36, 117.90, 114.15,
113.46, 36.25, 27.56, 22.30, 14.29. HR-MS (ESI) m/z: calcd. for C29H23F3N4O2 [M + H]+:
517.1773, found: 517.1846. Mp 274–277 °C.
N-(5-(2-oxo-1-(3-(trifluoromethyl)phenyl)-1,2-dihydrobenzo[h][1,6]naphthyridin-9-yl)pyridin
-2-yl)-2-phenylacetamide (11i) The title compound was obtained similarly to 4a. The boronic
acid was replaced with (6-(2-phenylacetamido)pyridin-3-yl)boronic acid. (white solid, yield:
1
83.7%). H NMR (400 MHz, DMSO-D6) δ(ppm): 10.94 (s, 1H), 9.17 (s, 1H), 8.34 (d, J = 9.5
Hz, 1H), 8.18 – 7.99 (m, 6H), 7.94 – 7.82 (m, 2H), 7.49 (dd, J = 8.7, 2.5 Hz, 1H), 7.43 – 7.29 (m,
4H), 7.30 – 7.22 (m, 1H), 7.02 (d, J = 1.7 Hz, 1H), 6.96 (d, J = 9.4 Hz, 1H), 3.76 (s, 2H).13C
NMR (101 MHz, DMSO-D6) δ(ppm): 170.78, 163.08, 152.21, 151.64, 148.77, 146.02, 142.41,
141.96, 140.83, 136.50, 136.23, 134.15, 134.01, 132.07, 131.68, 131.30, 130.41, 129.77, 128.86,
20

ACCEPTED MANUSCRIPT
127.16, 127.08, 126.55, 125.58, 122.87, 122.49, 122.37, 117.90, 114.14, 113.47, 43.44. HR-MS
(ESI) m/z: calcd. for C32H21F3N4O2 [M + H]+: 551.1617, found: 551.1689. Mp 143–145 °C.
Tert-butyl(5-(2-oxo-1-(3-(trifluoromethyl)phenyl)-1,2-dihydrobenzo[h][1,6]naphthyridin-9-yl)
pyridin-2-yl)carbamate (11j) The title compound was obtained similarly to 4a. The boronic acid
was replaced with (6-((tert-butoxycarbonyl)amino)pyridin-3-yl)boronic acid. (white solid, yield:
1
85.6%). H NMR (400 MHz, DMSO-D6) δ(ppm): 10.02 (s, 1H), 9.16 (s, 1H), 8.35 (d, J = 9.5
Hz, 1H), 8.12 (dd, J = 11.2, 2.3 Hz, 3H), 8.04 (dt, J = 8.8, 4.4 Hz, 2H), 7.91 (t, J = 7.8 Hz, 1H),
7.82 (dd, J = 17.8, 8.4 Hz, 2H), 7.33 (dd, J = 8.8, 2.5 Hz, 1H), 6.98 (dd, J = 14.7, 5.6 Hz, 2H),
13
1.50 (s, 9H). C NMR (101 MHz, DMSO-D6) δ(ppm): 163.08, 153.22, 152.68, 151.55, 148.76,
146.11, 142.37, 141.94, 140.83, 136.10, 134.19, 134.10, 132.07, 131.81, 131.65, 131.28, 129.32,
128.81, 127.09, 126.60, 122.89, 122.41, 117.91, 114.14, 112.28, 80.30, 28.54. HR-MS (ESI)
m/z: calcd. for C29H23F3N4O3 [M + H]+: 533.1722, found: 533.1795. Mp 234–236 °C.
Phenyl(5-(2-oxo-1-(3-(trifluoromethyl)phenyl)-1,2-dihydrobenzo[h][1,6]naphthyridin-9-yl)pyr
idin-2-yl)carbamate (11k) The title compound was obtained similarly to 4a. The boronic acid
was replaced with (6-((phenoxycarbonyl)amino)pyridin-3-yl)boronic acid. (white solid, yield:
83.3%). 1H NMR (400 MHz, DMSO-D6) δ(ppm): 10.98 (s, 1H), 9.19 (d, J = 10.9 Hz, 1H), 8.35
(dt, J = 11.1, 5.6 Hz, 1H), 8.22 – 8.10 (m, 3H), 8.08 – 8.02 (m, 1H), 7.90 (ddd, J = 14.7, 13.6,
7.7 Hz, 3H), 7.47 (ddd, J = 11.5, 9.8, 5.8 Hz, 3H), 7.38 – 7.21 (m, 4H), 7.03 (d, J = 1.9 Hz, 1H),
6.97 (dd, J = 9.4, 1.3 Hz, 1H). 13C NMR (101 MHz, DMSO-D6) δ(ppm): 163.08, 152.54, 152.07,
151.66, 150.82, 150.60, 150.53, 148.83, 147.05, 146.22, 142.41, 141.96, 140.83, 137.51, 136.51,
134.10, 132.10, 131.70, 130.30, 130.01, 128.87, 127.13, 126.24, 122.57, 121.87, 117.91, 115.72,
114.16, 112.60. HR-MS (ESI) m/z: calcd. for C31H19F3N4O3 [M + H]+: 553.1409, found:
553.1482. Mp 150–152 °C.
Benzyl(5-(2-oxo-1-(3-(trifluoromethyl)phenyl)-1,2-dihydrobenzo[h][1,6]naphthyridin-9-yl)pyr
idin-2-yl)carbamate (11l) The title compound was obtained similarly to 4a. The boronic acid was
replaced with (6-(((benzyloxy)carbonyl)amino)pyridin-3-yl)boronic acid. (yellow solid, yield:
82.7%). 1H NMR (400 MHz, DMSO-D6) δ(ppm): 10.49 (s, 1H), 9.19 (d, J = 14.3 Hz, 1H), 8.35
(d, J = 9.5 Hz, 1H), 8.27 – 7.94 (m, 5H), 7.94 – 7.73 (m, 3H), 7.61 – 7.19 (m, 6H), 7.08 – 6.88
(m, 2H), 5.21 (s, 2H). 13C NMR (101 MHz, DMSO-D6) δ(ppm): 163.08, 153.99, 152.38, 151.60,
148.79, 148.72, 146.14, 142.39, 141.95, 140.82, 137.01, 136.37, 134.10, 132.08, 131.67, 131.28,
129.74, 128.99, 128.84, 128.55, 128.38, 128.00, 127.11, 126.59, 122.54, 122.36, 117.91, 114.15,
21

ACCEPTED MANUSCRIPT
112.32, 66.44. HR-MS (ESI) m/z: calcd. for C32H21F3N4O3 [M + H]+: 567.1566, found: 567.1639.
Mp 150–153 °C.
9-(6-(4-methylpiperazin-1-yl)pyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthy
ridin-2(1H)-one (11m) The title compound was obtained similarly to 4a. The boronic acid was
replaced with (6-(4-methylpiperazin-1-yl)pyridin-3-yl)boronic acid. (yellow solid, yield: 85.1%).
1H NMR (400 MHz, DMSO-D6) δ(ppm): 9.12 (s, 1H), 8.33 (d, J = 9.5 Hz, 1H), 8.12 (s, 1H),
8.09 – 8.02 (m, 2H), 8.02 – 7.94 (m, 2H), 7.90 (t, J = 7.9 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.11
(dd, J = 8.9, 2.6 Hz, 1H), 6.94 (dd, J = 5.6, 3.8 Hz, 2H), 6.78 (d, J = 8.9 Hz, 1H), 3.56 – 3.46 (m,
4H), 2.45 – 2.31 (m, 4H), 2.23 (s, 3H).13C NMR (101 MHz, DMSO-D6) δ(ppm): 163.12, 158.84,
151.02, 148.40, 146.18, 142.22, 141.98, 140.83, 135.75, 134.98, 133.97, 132.04, 131.61, 131.46,
131.28, 128.41, 127.09, 126.64, 123.77, 122.22, 121.39, 117.98, 114.10, 107.06, 54.84, 46.35,
44.95. HR-MS (ESI) m/z: calcd. for C29H24F3N5O [M + H]+: 516.1933, found: 516.2006. Mp
161–162 °C.
9-(6-morpholinopyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(1H)-
one (11n) The title compound was obtained similarly to 4a. The boronic acid was replaced with
1
(6-morpholinopyridin-3-yl)boronic acid. (yellow solid, yield: 84.2%). H NMR (400 MHz,
DMSO-D6) δ(ppm): 9.12 (s, 1H), 8.33 (d, J = 9.5 Hz, 1H), 8.13 (s, 1H), 8.06 (dd, J = 8.2, 5.0 Hz,
2H), 8.03 – 7.95 (m, 2H), 7.90 (t, J = 7.9 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H), 7.15 (dd, J = 8.9, 2.6
Hz, 1H), 6.94 (dd, J = 5.6, 3.9 Hz, 2H), 6.79 (d, J = 8.9 Hz, 1H), 3.81 – 3.65 (m, 4H), 3.57 –
3.42 (m, 4H). 13C NMR (101 MHz, DMSO-D6) δ(ppm): 163.11, 158.98, 151.06, 148.42, 146.13,
142.23, 141.98, 140.83, 135.80, 134.89, 133.95, 132.05, 131.94, 131.46, 131.29, 128.44, 127.08,
126.61, 124.29, 122.23, 121.48, 117.97, 114.10, 107.05, 66.42, 45.45. HR-MS (ESI) m/z: calcd.
for C28H21F3N4O2 [M + H]+: 503.1617, found: 503.1689. Mp 286–287 °C.
9-(6-(pyrrolidin-1-yl)pyridin-3-yl)-1-(3-(trifluoromethyl)phenyl)benzo[h][1,6]naphthyridin-2(
1H)-one (11o) The title compound was obtained similarly to 4a. The boronic acid was replaced
1
with (6-(pyrrolidin-1-yl)pyridin-3-yl)boronic acid. (yellow solid, yield: 83.3%). H NMR (400
MHz, DMSO-D6) δ(ppm): 9.10 (s, 1H), 8.32 (d, J = 9.5 Hz, 1H), 8.13 (s, 1H), 8.10 – 7.98 (m,
3H), 7.98 – 7.85 (m, 2H), 7.79 (d, J = 8.0 Hz, 1H), 7.02 (dd, J = 8.8, 2.6 Hz, 1H), 6.92 (dd, J =
10.8, 5.6 Hz, 2H), 6.36 (d, J = 8.8 Hz, 1H), 3.40 (t, J = 6.5 Hz, 4H), 1.95 (t, J = 6.6 Hz, 4H). 13C
NMR (101 MHz, DMSO-D6) δ(ppm): 163.12, 156.77, 150.79, 148.24, 146.66, 142.15, 141.98,
140.83, 135.49, 135.13, 133.86, 132.03, 131.57, 131.37, 131.24, 128.29, 127.15, 126.64, 122.16,
22

ACCEPTED MANUSCRIPT
122.07, 120.98, 118.01, 114.08, 106.46, 46.96, 25.51. HR-MS (ESI) m/z: calcd. for
C28H21F3N4O [M + H]+: 487.1667, found: 487.1741. Mp 252–254 °C.
4.2. Biological Evaluations
4.2.1. In vitro Antiviral Assay
The test compound and the positive compound were dissolved in DMSO to 100 mM
depending on the mass and molecular weight of the test compound and positive compound. The
test compound was diluted to a concentration of 800 µM using a cell maintenance solution and
then diluted by a dilution factor of 3 times for a total of 10 concentrations. The cell maintenance
medium (DMEM medium with 2% FBS) was used to serially dilute the positive drug Torin2 to
the same dilution as the compound to be tested. The diluted compound was then added to a
white-walled clear bottom 96-well plate at 50 µl per well. An equal volume of cell maintenance
medium was added to both the cell control group and the virus control group. The EV71 virus
strains were removed from -80 °C and equilibrated to room temperature. The virus was diluted to
100 TCID50 using the maintenance medium and was then added to the above 96-well plate cells
at 50 µl per well. The cell control group was added with an equal volume of maintenance
medium. The RD cells were seeded at a concentration of 1ⅹ105 / ml in a 96-well plate with a
white-walled clear bottom, 100 µl per well, and finally a volume of 200 µl per well, and the final
concentration of the drug was 0.25 times that of the pretreatment concentration. The plates were
cultured at 37 °C for 4 days. The buffer and substrate of the CellTiter-Glo® Chemiluminescent
Cell Viability Assay reagent (Promega) were mixed in the dark to prepare a working solution,
and cell viability assay were performed according to the manufacturer’s protocol. The IC50 (50%
inhibitory concentration) was calculated using software Origin 8.0.
4.2.2. mTOR Enzyme Assay
The mTOR kinase activities of all the compounds were determined using a LanthaScreen
Kinase Activity Assay (Invitrogen) following the manufacturer’s instructions, with compound
Torin 2 as a positive control. Briefly, the mTOR enzyme (0.5 µg/mL, Invitrogen), ATP (3 µM,
Sigma), GFP-4E-BP1 peptide (0.4 µM, Invitrogen) and test compounds were diluted in kinase
buffer (50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MnCl2, 2 mM DTT and 0.01% Tween-20).
The reactions were performed in black 384-well proxiplates (PerkinElmer) at room temperature
for 1 h and stopped by adding EDTA to 10 mM. Tb-p4E-BP1 (pThr46) antibody (Invitrogen)
was then added to each well to a final concentration of 2 nM, and the mixture was incubated at
23

ACCEPTED MANUSCRIPT
room temperature for 30 min. The intensity of the light emission was measured with an Envision
2104 reader (PerkinElmer) in TR-FRET mode (excitation at 340 nm and emission at 495 nm/520
nm). All of the compounds were tested in duplicate, and the results were expressed as IC50.
4.2.3. Western Blot Analysis
Cell lysates were prepared in loading buffer (Beyotime) and heated at 100 °C for 10 min, and
proteins were then separated by gel electrophoresis using the NuPAGE Novex 4−12% Bis-Tris
gel system. Proteins were then transferred onto a PVDF membrane using the Bio-Rad
transmembrane system. The PVDF membranes were then probed with a rabbit anti-p70 S6
antibody (CST, 2708), a rabbit anti-phospho-p70 S6 antibody (CST, 9234), a rabbit anti-Akt
antibody (CST, 4691), a rabbit anti- phosphor-Akt antibody (CST, 4058), or a rabbit anti-Actin
antibody (CST, 4970) and visualized using the corresponding IRDye anti-rabbit antibody
(LI-COR, 926-32211). The membrane was then scanned using the LI-COR Odyssey.
4.2.4. Molecular Modeling
The molecular docking process was as follows: first, crystal structures of mTOR were
downloaded from the RCSB Protein Data Bank (http://www.rcsb.org). The selected protein Data
Bank (PDB) ID was mTOR (4JSV, resolution 3.5 Å). Next, the water molecules in the crystal
were cleared, and hydrogen atoms and electric charges were added. Subsequently, molecule
structures of ligands were drawn by ChemBioDraw 12.0 (PerkinElmer) and introduced into
protein crystal cells. Finally, the molecular docking was carried out by using the LIB-DOCK
module of the Discovery Studio 2.5 package. After the docking process was finished, the best
conformation (Figure 4) was selected and the hydrogen bonds were displayed according to the
docking result.
Acknowledgments
We are grateful for the financial supports of the National Science and Technology Major
Projects for "Major New Drugs Innovation and Development" (2018ZX09711003) of China.
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