Synthesis and structure-activity relationships of quinolinone and quinoline-based P2X7 receptor antagonists and their anti-sphere formation activities in glioblastoma cells

Article abstract of DOI:10.1016/j.ejmech.2018.03.023

Screening a compound library of quinolinone derivatives identified compound 11a as a new P2X7 receptor antagonist. To optimize its activity, we assessed structure-activity relationships (SAR) at three different positions, R₁, R₂ and R₃, of the quinolinone scaffold. SAR analysis suggested that a carboxylic acid ethyl ester group at the R₁ position, an adamantyl carboxamide group at R₂ and a 4-methoxy substitution at the R₃ position are the best substituents for the antagonism of P2X7R activity. However, because most of the quinolinone derivatives showed low inhibitory effects in an IL-1β ELISA assay, the core structure was further modified to a quinoline skeleton with chloride or substituted phenyl groups. The optimized antagonists with the quinoline scaffold included 2-chloro-5-adamantyl-quinoline derivative (16c) and 2-(4-hydroxymethylphenyl)-5-adamantyl-quinoline derivative (17k), with IC₅₀ values of 4 and 3 nM, respectively. In contrast to the quinolinone derivatives, the antagonistic effects of the quinoline compounds (16c and 17k) were paralleled by their ability to inhibit the release of the pro-inflammatory cytokine, IL-1β from LPS/IFN-γ/BzATP-stimulated THP-1 cells (IC₅₀ of 7 and 12 nM, respectively). In addition, potent P2X7R antagonists significantly inhibited the sphere size of TS15-88 glioblastoma cells.

Full text of DOI:10.1016/j.ejmech.2018.03.023

Accepted Manuscript
Synthesis and structure-activity relationships of quinolinone and quinoline-based
P2X7 receptor antagonists and their anti-sphere formation activities at glioblastoma
cells
Seung-Hwa Kwak, Seungheon Shin, Ji-Hyun Lee, Jin-Kyoung Shim, Minjeong Kim,
So-Deok Lee, Aram Lee, Jinsu Bae, Jin-Hee Park, Aliaa Abdelrahman, Christa E.
Müller, Steve K. Cho, Seok-Gu Kang, Myung Ae Bae, Jung Yoon Yang, Hyojin Ko,
Yong-Chul Kim
PII:
S0223-5234(18)30266-6
10.1016/j.ejmech.2018.03.023
EJMECH 10288
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 September 2017
Revised Date: 28 February 2018
Accepted Date: 8 March 2018
Please cite this article as: S.-H. Kwak, S. Shin, J.-H. Lee, J.-K. Shim, M. Kim, S.-D. Lee, A. Lee, J.
Bae, J.-H. Park, A. Abdelrahman, C.E. Müller, S.K. Cho, S.-G. Kang, M.A. Bae, J.Y. Yang, H. Ko,
Y.-C. Kim, Synthesis and structure-activity relationships of quinolinone and quinoline-based P2X7
receptor antagonists and their anti-sphere formation activities at glioblastoma cells, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.023.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Synthesis and Structure-Activity Relationships of Quinolinone Leave this area blank for abstract info.
and Quinoline-based P2X7 Receptor Antagonists and
their Anti-sphere Formation Activities at Glioblastoma Cells
Seung-Hwa Kwaka,#, Seungheon Shin , Ji-Hyun Lee , Jin-Kyoung Shim , Minjeong Kim , So-Deok Lee , Aram Lee , Jinsu Bae , Jin-Hee Park , Aliaa
b
c
c
a,†
a
a
b
a,^
d
d
a,b
c
e
e
a*
a,b*
Abdelrahman , Christa E. Müller , Steve K. Cho , Seok-Gu Kang , Myung Ae Bae , Jung Yoon Yang , Hyojin Ko and Yong-Chul Kim
a
b
School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea. Department of BioMedical
c
Science and Engineering (BMSE), Gwangju Institute of Science and Technology (GIST), Gwangju 500-712, Republic of Korea. Department of
d
Neurosurgery, Brain Tumor Center, Severance Hospital, Yonsei University College of Medicine, Seoul 03722, Republic of Korea. PharmaCenter Bonn,
e
Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany. Bio & Drug Discovery
Division, Korea Research Institute of Chemical Technology (KRICT), Daejeon 34114, Republic of Korea

ACCEPTED MANUSCRIPT
Synthesis and Structure-Activity Relationships of Quinolinone
and Quinoline-based P2X7 Receptor Antagonists and their Anti-
sphere Formation Activities in Glioblastoma Cells
a,
b
c
c
a,†
#
Seung-Hwa Kwak , Seungheon Shin , Ji-Hyun Lee , Jin-Kyoung Shim , Minjeong Kim ,
a
a
b
a,^
d
So-Deok Lee , Aram Lee , Jinsu Bae , Jin-Hee Park , Aliaa Abdelrahman , Christa E.
d
a,b
c
e
e
a*
Müller , Steve K. Cho , Seok-Gu Kang , Myung Ae Bae , Jung Yoon Yang , Hyojin Ko
a,b*
and Yong-Chul Kim
a
School of Life Sciences, Gwangju Institute of Science and Technology (GIST), Gwangju 500-712,
Republic of Korea.
b
Department of BioMedical Science and Engineering (BMSE), Gwangju Institute of Science and
Technology (GIST), Gwangju 500-712, Republic of Korea.
c
Department of Neurosurgery, Brain Tumor Center, Severance Hospital, Yonsei University College of
Medicine, Seoul 03722, Republic of Korea.
d
PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An
der Immenburg 4, D-53121 Bonn, Germany.
e
Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology (KRICT),
Daejeon 34114, Republic of Korea
*
Corresponding Author: Prof. Hyojin Ko and Prof. Yong-Chul Kim. Tel: +82-62-715-2502; Fax:
82-62-715-2484. Email address: kohyojin@gist.ac.kr (H. J. Ko); yongchul@gist.ac.kr (Y.- C.
+
Kim).
#
Present address: Department of Chemistry, Duke University, Durham, North Carolina 27708,
United States.
†
Present address: COSMAX, #701, Pangyo inno valley E, 255, Pangyo-ro, Bundang-gu, Seongnam-si,
Gyeonggi-do, Republic of Korea.
^
Present address: New Drug Development Center (NDDC), Daegu-Gyeongbuk, Medical Innovation

ACCEPTED MANUSCRIPT
Foundation (DGMIF), 80 Cheombok-ro, Dong-gu, Daegu 41061, Republic of Korea.

ACCEPTED MANUSCRIPT
ABSTRACT
Screening a compound library of quinolinone derivatives identified compound 11a as a new
P2X7 receptor antagonist. To optimize its activity, we assessed structure-activity relationships
(
SAR) at three different positions, R , R and R , of the quinolinone scaffold. SAR analysis
1 2 3
suggested that a carboxylic acid ethyl ester group at the R position, an adamantyl
1
carboxamide group at R and a 4-methoxy substitution at the R position are the best
2
3
substituents for the antagonism of P2X7R activity. However, since most of the quinolinone
derivatives showed low inhibitory effects in an IL-1β ELISA assay, the core structure was
further modified to a quinoline skeleton with chloride or substituted phenyl groups. The
optimized antagonists with the quinoline scaffold included 2-chloro-5-adamantyl-quinoline
derivative (16c) and 2-(4-hydroxymethylphenyl)-5-adamantyl-quinoline derivative (17k),
with IC values of 4 and 3 nM, respectively. In contrast to the quinolinone derivatives, the
5
0
antagonistic effects of the quinoline compounds (16c and 17k) were paralleled by their ability
to inhibit the release of the pro-inflammatory cytokine, IL-1β, from LPS/IFN-γ/BzATP-
stimulated THP-1 cells (IC50 of 7 and 12 nM, respectively). In addition, potent P2X7R
antagonists significantly inhibited the sphere size of TS15-88 glioblastoma cells.
Keywords: P2X7 receptor, antagonist, IL-1β, EtBr, inflammation, quinoline, quinolinone,
anti-glioma

ACCEPTED MANUSCRIPT
1
. Introduction
Purinergic receptors are plasma membrane proteins that can be classified into three different
families: P1, P2X and P2Y. Broadly, P1 (adenosine) and P2Y receptors are G protein-coupled
receptors (GPCR), and the P2X receptors are ligand-gated ion channels that are activated by
1
-4
extracellular ATP molecules.
In the case of P2X receptors (P2XR), seven different subtypes, P2X1‒P2X7, have been
identified in the immune and nervous systems. Among the P2XR subtypes, P2X7R has drawn
particular interest in terms of its distinctive molecular structure, such as a longer C-terminus
with 100-200 amino acids and pathological functions related to inflammation. In the tertiary
structure of the ion channel, P2X7R is composed of homotrimeric subunits, and the receptor
is mainly expressed in hematopoietic cells, including mast cells, lymphocytes, erythrocytes,
5
and macrophages. The P2X7Rs also play important pathophysiological functions in the
human monocyte cell line THP-1, epidermal Langerhans cells, fibroblasts, and cells in the
central nervous system (CNS) such as microglia and Schwann cells, which suggests that the
receptor is involved in diseases such as chronic inflammation, neurodegeneration, and
6
chronic pain.
P2X7R is activated not only by the endogenous ligand ATP but also by other agonists such as
3
,7,8
BzATP, ADP, UTP and AMP.
As a unique function of the receptor, the activation of
P2X7R results in the formation of permeable pores and the induction of signaling cascades
2
+
+
+
downstream of the receptor. Upon the activation of P2X7R, cations such as Ca , Na and K
can be permeable through the ion channel or pore. This process leads to the activation of
related inflammatory signals, including phospholiphase A2, phospholiphase D, mitogen-
activated protein kinase (MAPK), and nuclear factor kappa-light-chain-enhancer of activated
9
B cells (NF-κB). More importantly, P2X7R activation ultimately stimulates the release of

ACCEPTED MANUSCRIPT
10,11
interleukin-1β (IL-1β),
which is a master cytokine for the mediation of inflammatory
1
2
signals, along with cell proliferation, differentiation and apoptosis. In addition, P2X7R
triggers several other signaling cascades, which ultimately lead to macrophage fusion,
1
3,14
superoxide production in microglia and lymphoid cells.
Thus, therapeutic interventions
targeting P2X7R have been explored as a novel approach for the prevention or treatment of
inflammatory disorders such as arthritis, chronic inflammatory pain, neuropathic pain, and
1
5-18
neurodegenerative diseases.
In addition, P2X7R is over-expressed in various cancer cells,
in which it has been reported to show important functions, such as improved survival,
1
9,20
invasiveness, and metastasis, in cancer microenviroments.
Recently, P2X7R was reported
2
1
to be involved in the activation of microglia located near glioma cells. The inhibition of
P2X7R function showed a decrease in the number of glioma cells, which can induce
glioblastoma. Glioblastoma is the most common type of brain tumor and a major cause of
2
2
their high morbidity.
According to the above diverse pathophysiological functions of the P2X7 receptor, several
P2X7R antagonists have been discovered. For example, KN-62 (1, Figure 1), previously
2
+
known as a type II Ca /calmodulin-dependent kinase inhibitor, was developed as an early-
23
generation P2X7R antagonist. The antagonists developed by the pharmaceutical industry
for the purpose of new drug discovery are compounds 2‒5 in Figure 1. AZD9056 (2) is an
orally bioavailable P2X7R antagonist with an IC value of 10-13 nM (IL-1β ELISA assay),
5
0
targeting rheumatoid arthritis (RA), chronic obstructive pulmonary disease (COPD) and
Crohn’s disease. However, compound 2 failed in phase 2 clinical trials because it did not
2
4
show appreciable efficacy compared with placebo. Another potent and highly selective
P2X7R antagonist, CE-224535 (3), also entered clinical trials for treating patients with RA
2
5
that was inadequately controlled by methotrexate, but it failed to show significant efficacy.
A-438079 (4) and GSK314181A (5) were reported to have anti-nociceptive effects in

ACCEPTED MANUSCRIPT
2
6
27,28
neuropathic and inflammatory pain
and are currently in the discovery stage.
2
9
We have reported diverse classes of P2X7R antagonists, including dichloropyridine (6), and
3
0
2
,5-dioxoimidazolidine derivatives (7). Recently, we reported potent imidazole-based
3
1
P2X7R antagonists (8) with significant anti-migration and invasion activities against
metastatic breast cancers.
3
2
To discover new chemical entities as P2X7R antagonists, a quinolinone library that was
previously reported by our group was screened to identify a hit compound (11a, Table 1) as a
weak P2X7R antagonist. The quinolinone scaffold has been studied extensively to develop
3
3-36
agents for treating immune disorders.
Moreover, we recently reported the optimization of
quinolinone derivatives for their inhibitory effects on IL-2 secretion and their functional
37
mechanism of immunomodulation. In this report, we describe the development of new
P2X7R antagonists by structure-activity relationships (SAR) and the optimization of the
quinolinone-based hit compound, 11a. In addition, the functional effects of the representative
compound, including the inhibition of IL-1β release, which is closely related to immune
disorders, and anti-sphere formation activities against glioblastoma cells, in which P2X7
receptors are overexpressed and function as activation of microglia located near glioblastoma
2
1, 22
cells, were examined.

ACCEPTED MANUSCRIPT
2
. Results and discussion
2
.1. Chemistry
The general synthetic procedures for the quinolinone and quinoline analogs are described in
Schemes 1 and 2, and a full list of synthesized analogs is provided in Tables 1‒5.
3
8
The starting materials, 5-nitroquinolin-2(1H)-one (9a) and methyl 5-nitro-2-oxo-1,2-
3
2
dihydroquinoline-3-carboxylate (9b), were prepared following the previously reported
3
2,38
procedure.
5-Nitro groups of 9a‒b were first reduced to the corresponding amines with
SnCl , and the amide moieties of the quinolinone skeletons were selectively reacted with
2
appropriate benzyl chlorides or benzyl bromides to afford a series of compounds, 10a‒l. The
amino group of compounds 10a‒l was subjected to acylation reactions with appropriate acid
chlorides to afford 11a‒i, 12a‒j and 13a. In this step, coupling reaction conditions with
carboxylic acid moiety (R CO H) failed to yield the desired products. For the synthesis of
2
2
1
3b‒g, the methyl ester of 11i was hydrolyzed under basic conditions using 10% KOH in
MeOH to a carboxylic acid analogue, 13b, which was subsequently subjected to coupling
reactions with several alkyl amines, such as methylamine and dimethylamine, to produce 13f
and 13g and with various alcohols to afford 13c‒e (Scheme 1).
The synthetic scheme for quinoline derivatives from the quinolinone structure is described
in Scheme 2. The quinolinone amide groups of 9a and 9b were first converted into
chloroquinoline moieties by reaction with POCl (14a and 14b). Then, the 5-nitro groups of
3
1
4a and 14b were reduced to the amine with SnCl to afford compounds 15a or 15b, which
2
were subjected to acylation reactions with appropriate acid chloride groups to produce 16a‒e.
Next, for the introduction of diverse groups at the R position, appropriate boronic acid
4
derivatives were reacted with 16c under Suzuki-Miyaura cross-coupling conditions to yield

ACCEPTED MANUSCRIPT
1
7a‒g and 17i. Compound 17h was obtained from the reduction of 17g by using SnCl , and
2
1
7i was further transformed into the corresponding ester analogue 17j by methylation using
CH I and was then subsequently reduced with LAH to afford 17k.
3
2
2
.2. Biology
.2.1. Structure-Activity Relationships (SAR) To investigate the SAR of the quinolinone and
quinoline derivatives, the P2X7R antagonistic activities of all of the synthesized compounds
were evaluated using an ethidium bromide (EtBr) uptake assay in human HEK293 cells
stably expressing hP2X7 receptors. KN-62 (1) and AZD9056 (2) were used as positive
controls, for which the IC values (158 and 2 nM, respectively) were found to be similar to
50
2
3,24
the reported data.
As an initial SAR analysis, aryl moieties (11a‒c), alkyl moieties (11d‒g) and adamantyl
moieties (11h and 11i) with different carbon chain lengths were explored at the R position
2
because compound 10b with no substituent at 5-amino group showed a complete loss of
activity compared to the hit compound 11a (Table 1). In the analysis of the effects of carbon
chain lengths between phenyl and carbonyl groups of compounds 11a‒c, a longer carbon
chain showed a profile of increased antagonistic effects. For cycloalkyl moieties (11e‒g), the
tendency of antagonistic activities related with the carbon chain lengths was opposite
compared with aromatic moieties (11a‒c). Thus, a decreased carbon chain length afforded a
sharp increase in the antagonistic effects, as shown in the case of 11d‒g, with 15% and 20%
inhibition at 1 µM (11d and 11e, No. of carbon in chain: 2), 875 nM (11f, No. of carbon in
chain: 1) and 810 nM (11g, No. of carbon in chain: 0). This profile of P2X7R antagonistic
activities was also observed in the polycycloalkyl derivatives, 11h and 11i, which showed
potent antagonistic effects with IC values of 861 and 120 nM, respectively. Based on this
5
0

ACCEPTED MANUSCRIPT
result, we concluded that intact bulky cycloalkyl carboxamide groups substituted at the 5-
position of the quinolinone structure is important for P2X7R antagonism.
Next, we investigated the SAR of R substituents by modifications of the benzyl substituents
3
to 3-methoxy (12a), halide (12c‒e), alkyl (12f‒g) and electron-withdrawing groups such as
nitro (12h) and cyano (12i), respectively, while maintaining the R position as adamantyl
2
carboxamide (Table 2). Comparing 11i and 12a, changing the substituted position of the 4-
methoxy group to the meta position resulted in a significantly decreased antagonistic effect.
Thus, all other substituents were introduced at the para position of the benzyl moiety. The
activity profile of the series of compounds was as follows: 4-ethyl (12g), 4-methyl (12f) < 4-
trifluoro-methoxy (12b) < 4-fluoro (12c), 4-bromo (12e), 4-nitro (12h), 4-chloro (12d) < 4-
methoxy (11i). Among the derivatives (11i, 12a−j), the halide-substituted compounds (12c−e)
and nitro (12h) showed IC values of 720−880 nM. The simple alkyl substituted derivatives
50
with 4-methyl (12g) or 4-ethyl (12f) groups decreased the P2X7R antagonistic activity
dramatically compared with other halide, nitro and alkoxy substituted analogs including the
most active compound, 11i with 4-methoxy group in Table 2. Basis on the results shown in
Tables 1 and 2, adamantyl carboxamide at R and 4-methoxybenzyl group at R exhibited
2
3
best outcome and were fixed for the further analysis of the SAR of R substituents.
1
Thus, the methyl ester group at the R position was modified to hydrogen, carboxylic acid,
1
various alkyl esters (13c−e) and carboxamides (13f and 13g) to investigate the possible
effects of hydrogen bond acceptors or donors in this position. P2X7R antagonistic activity
was lost upon the removal (13a) or hydrolysis (13b) of the methyl ester group of 11i. The
optimum alkyl moiety of the ester group was determined to be the two carbon ethyl moiety
(
13c), which displayed two-fold increased activity, with an IC value of 62 nM. Surprisingly,
50
carboxamide-substituted compounds 13f and 13g showed large decrease of the antagonistic
effect, suggesting that the property of stronger hydrogen bonding acceptor of amide nitrogen

ACCEPTED MANUSCRIPT
39
through the carbonyl oxygen, than ester might be involved in the interaction with hP2X7R.
However, the quinolinone derivatives showed low inhibitory effects against BzATP-induced
IL-1β release in LPS/IFNc-differentiated human THP-1 cells (Supporting Information, Table
S3). To overcome the functional defects and improve the antagonistic activity, we tried to
1
2 40
attempt to change substituent position from N to C . The results for the activities of
quinolines are shown in Tables 4 and 5. At first, the effects of substituents at the R and R3
2
positions were re-explored with the 2-chloro-quinoline skeleton by employing the similar
moieties investigated for the quinolinone derivatives in Table 1 (Table 4). Unlike the activity
profile of quinolinone derivatives, no substitutions at the R position (13a vs 16d) and the
1
cycloalkyl acetyl group at R position (11f, h and 11i vs 16c, e and 16d, respectively) were
2
favorable for the 2-chloro-quinoline compounds. Therefore, the mode of antagonistic activity
is speculated to be different between the skeletons. Among the quinoline derivatives listed in
Table 4, compound 16c, which possesses an adamantyl acetamide group at the 5 position of
2
-chloro-quinoline, displayed single digit nanomolar antagonistic activity (IC = 4 nM).
50
To explore the effects of substituents at R of the quinoline skeleton, various substituted
4
phenyl groups (17a−k) were introduced by Suzuki coupling reactions with compound 16c
Table 5). The different mode of action of quinoline analogs was re-confirmed by a
(
comparison of compounds 11i and 12j (IC50 value of 120 nM and no activity at 1 µM,
respectively) vs compounds 17a and 17b (IC50 values of 447 and 18 nM, respectively).
Interestingly, demethylated derivative 17c had two-fold increased activity, with high
positional preference at the para position of the phenyl group compared with 17d and 17e.
Therefore, further substitutions were tried with polar moieties having hydrogen donors and
acceptors, including halide, amine, nitro, acid, ester and alcohol at the 4-position of R phenyl
4
group, resulting in 3 ~ 123 nM of IC values. The activity profile of the series of compounds
5
0

ACCEPTED MANUSCRIPT
was as follows: no substitution (17a) < 4-chloro (17f) < 4-nitro (17g) < 4-methoxy (17b) < 4-
amine (17h), 4-hydroxy (17c), 4-carboxylic acid (17i), 4-methyl ester (17j) < 4-
hydroxymethyl (17k). Among the quinoline derivatives, compounds 16c and 17k showed
potent antagonistic effects, with IC50 values of 4 and 3 nM, respectively. In addition, a
4
1
parallel MTT assay of active quinoline derivatives to evaluate their cytotoxicity resulted in
more than 90% survival at 10 µM of the compounds (Table S4 and Table S5).
2
.2.2. Functional Inhibitory Activities of IL-1β Release and Subtype Selectivity Among the
quinolone derivatives, 16c, 16e, 17a−c and 17f−k were further evaluated for their functional
activity based on P2X7R-related signaling in immune cells. The inhibitory activity of the
antagonists on the IL-1β release triggered by the activation of P2X7R was measured in
differentiated THP-1 cells. The trends of inhibitory activities of quinoline-based analogues in
the IL-1β ELISA assay displayed a similar profile to the P2X7R antagonism in the EtBr
uptake assay. Thus, the potent P2X7R antagonists with single digit nanomolar IC values,
5
0
1
6c, 17c and 17h−k showed IC values of 7, 6, 12−33 nM in the suppression of IL-1β
50
release.
To investigate P2XR subtype selectivity, the selected potent P2X7R antagonists 16c and
7b shown in Table 5 were evaluated at other P2XR subtypes. The antagonistic effects of the
1
2
+
compounds were measured by their inhibition of Ca flux in 1321N1 astrocytoma cells
stably expressing the human P2X1−4 receptors. All compounds showed weak or negligible
antagonistic activities at P2X1, 2 and 4 receptors and 17b displayed an IC value of 3.0 µM
5
0
for P2X3R (Table 6), indicating that this class of P2X7R antagonists has a good subtype
selectivity profile.
2
.2.3. Effects of P2X7R Antagonist on TS15-88 cells The potent P2X7R antagonists were

ACCEPTED MANUSCRIPT
assessed for their anti-cancer effects using in-vitro assays of TS15-88 glioblastoma cells.
TS15-88 neurosphere cells were incubated with a 0 or 50 µM concentration of each of the
seven potent antagonists for 24 h and 72 h to measure cell viability. In figure 2, an inhibitory
effect on TS15-88 cell viability was observed within 24 h for all agents, except compounds
1
7b and 17i. After 72 h of treatment, the seven antagonists showed an inhibitory effect on
TS15-88 cells and showed higher effects than 24 h of treatment, except for 17i. The IC50
values of selected compounds AZD9056, 16c, 17c, and 17j were measured as 8.92, 40.0, 25.6,
and 35.6 µM, respectively. As shown in figures 3 and 4, inhibitory effects on neurosphere
formation were also observed for the seven antagonists. Except for 17i, the antagonists
showed over 50 percent critical inhibitory effects on sphere formation after 5 days of 50 µM
drug treatment. In mRNA expression studies of TS15-88 cells after drug treatment, genes that
represent functions of maintaining stemness within the cells were changed. As shown in
figure 5, POU5F1 expression was significantly reduced by 41% after 3 days of 16c treatment.
SOX2 expression did not show any changes. Interestingly, MYC and CD133 expression was
increased by 41% and 30%, respectively, although the total surface area of the TS15-88
neurospheres was significantly decreased by 50 µM 16c (Figures 3 and 4).
2
.2.4. Blood-Brain Barrier (BBB) Permeability of P2X7R Antagonist on zebrafish models.
We have investigated in vivo BBB (blood-brain barrier) permeability of compound 16c using
4
2
the adult zebrafish models since targeting glioblastoma requires BBB penetration of drugs.
Zebrafish models have been applied for in vivo pharmacological studies because of its
features such as small size, high fecundity and rapid growth. An adult male zebrafish was
tested for the possibility of BBB penetration for the most active compound, 16c. The
concentration of 16c at 0.5 h in the blood was measured as 235 ng/mL and 425 ng/mL in the

ACCEPTED MANUSCRIPT
brain tissue, respectively (Figure 6 and Table 7), indicating that 16c may have the property of
significant BBB penetration.
2
.2.5. Blood-Brain Barrier (BBB) Permeability test on Parallel Artificial Membrane
Permeability Assay (PAMPA).
To investigate the BBB permeability of test compounds (17c, 17i, 17k and 17h), we used
PAMPA methods. The solubility at three concentrations before PAMPA proceeded at 50 µM,
the highest concentration at which no precipitation of the compound occurs (Table 8-1).
2
.2.6. Physicochemical Properties
As fundamental parameters of physicochemical properties, the water solubility and cell
permeability of compound 16c, 17c, 17h and 17k showed medium level of kinetic solubility,
and permeability (Table 9-2). The equilibrium solubility of 16c resulted in more than 30 times
less solubility than that of AZD9056. Unfortunately, the in-vitro metabolic stability of the
compounds in liver microsomal fraction turned out to be very poor (Table 9-1). The lack of
metabolic stability of some representative compounds needs to be improved for the
optimization of oral bioavailability in the future.

ACCEPTED MANUSCRIPT
3
. Conclusions
Screening a quinolinone-based compound library led to the identification of 11a as a new
lead compound for P2X7R antagonism. To optimize P2X7R antagonistic potency,
modifications at R , R and R of the quinolinone structure were performed. SAR analysis of
1
2
3
the various derivatives suggested that a carboxylate ethyl ester group at R , adamantyl
1
carboxamide at R and 4-methoxy substitution at R gave the best substituents for the
2
3
antagonism of P2X7R activity. Since the quinolinones showed low functional inhibitory
effects in an IL-1β ELISA assay, further modification of the core structure was examined.
The resulting optimized quinoline-based antagonists, 16c and 17k, showed single digit
nanomolar IC values in both EtBr uptake and IL-1β ELISA assays. Furthermore, the most
5
0
potent representative antagonist, 16c, displayed appreciable functional activities in reducing
the sphere size of TS15-88 glioblastoma cells. These results suggest that the quinoline-based
P2X7R antagonists may have the potential to treat glioblastoma, which is currently
categorized as highly unmet medical needs.

ACCEPTED MANUSCRIPT
4
. Experimental section
4
.1. Chemistry
All the reagents and solvents were purchased from Sigma-Aldrich and TCI and used without
1
further purification. H NMR spectra were determined with a JEOL JNM-ECX 400P
1
3
spectrometer at 400 MHz, and C NMR spectra were recorded using a FT-NMR
spectrometer at 125 MHz (Korea Basic Science Institute, Gwangju); spectra were taken in
CDCl , DMSO-d or MeOH-d . Unless otherwise noted, chemical shifts are expressed as δ
3
6
4
units downfield from internal tetramethylsilane or relative parts per million (ppm) from
CDCl (7.26 ppm), DMSO (2.50 ppm), MeOH (3.31 ppm) and coupling constants (J) are in
3
Hertz (Hz). Data are reported as follows: chemical shift, multiplicity (s, singlet; d, doublet; t,
triplet; m, multiplet), coupling constants and integration. Mass spectroscopy was carried out
on electrospray, and high-resolution mass spectra (m/z) were recorded on FAB and ESI.
High-resolution mass analysis was performed at Korea Basic Science Institute (Daegu) and
National Development Institute of Korean Medicine (Jangheung-Gun).
The purity of all final compounds was determined by analytical HPLC. The determination of
purity was conducted on Agilent 1100 series HPLC system using an Agilent ZORBAX
Extend C18 analytical column (250 × 4.6 mm, 5 mm, 80 Å) in linear gradient solvent systems
-1
(
solvent system was H O:CH CN = 80:20 over 10 min at a flow of 1 mL min ). Peaks were
2 3
detected at 254 nm.
5
-Nitroquinolin-2(1H)-one (9a) See reference 38.
Methyl 5-nitro-2-oxo-1,2-dihydroquinoline-3-carboxylate (9b) See reference 32.

ACCEPTED MANUSCRIPT
General procedure A: For preparation of 10a‒l, 15a‒b and 17h
37
Appropriate benzyl substituted compounds, 14a‒b or 17g (1.0 equiv) was dissolved in
ethanol and SnCl (3.0 equiv) was added. After the reaction mixture was stirred for 4 h at 80
2
o
C in argon gas environment, it was concentrated and partitioned between saturate brine and
ethyl acetate. The organic layer was dried over sodium sulfate, filtered and evaporated under
the vacuum. The resulting residue was purified by silica gel column chromatography to
afford 10a‒l, 15a‒b and 17h.
5
-Amino-1-(4-methoxybenzyl)quinolin-2(1H)-one (10a) Following the general procedure
A, reaction of 1-(4-methoxybenzyl)-5-nitroquinolin-2(1H)-one (Supporting Information)
(
80.0 mg, 0.25 mmol) with SnCl (169.0 mg, 0.75 mmol) in EtOH affords 10a (62.0 mg).
2
1
Yield 89%; H NMR (400 MHz, CDCl ) δ 7.61 (1H, d, J = 8.0 Hz), 7.31 (1H, t, J = 8.0 Hz),
3
7
5
.03 (2H, d, J = 8.0 Hz), 6.83‒6.81 (2H, m), 6.73 (2H, d, J = 8.0 Hz), 6.48 (1H, d, J = 8.0 Hz),
+
.46 (2H, s), 4.11 (2H, s), 3.76 (3H, s). MS (ESI) m/z: 281.96 ([M+H] ).
Methyl 5-amino-1-(4-methoxybenzyl)-2-oxo-1,2-dihydroquinoline-3-carboxylate (10b)
See reference 32.
Methyl 5-amino-1-(3-methoxybenzyl)-2-oxo-1,2-dihydroquinoline-3-carboxylate (10c)
Following the general procedure A, reaction of methyl 1-(3-methoxybenzyl)-5-nitro-2-oxo-
3
7
1
,2-dihydroquinoline-3-carboxylate (80.0 mg, 0.21 mmol) with SnCl (147.0 mg, 0.65
2

ACCEPTED MANUSCRIPT
1
mmol) in EtOH affords 10c (153.0 mg). Yield 70%; H NMR (400 MHz, CDCl ) δ 8.69 (1H,
3
s), 7.81 (1H, dd, J = 4.0 Hz, 4.0 Hz), 7.62 (1H, t, J = 8.0 Hz), 7.53 (1H, d, J = 8.0 Hz), 7.22‒
7
.16 (2H, m), 7.00 (2H, t, J = 8.0 Hz), 5.51 (2H, s), 4.38 (2H, s), 3.97 (3H, s), 3.75 (3H, s).
+
MS (ESI) m/z: 338.87 ([M+H] ).
Methyl
5-amino-2-oxo-1-(4-(trifluoromethoxy)benzyl)-1,2-dihydroquinoline-3-
carboxylate (10d) Following the general procedure A, reaction of methyl 5-nitro-2-oxo-1-
3
7
(
4-(trifluoromethoxy)benzyl)-1,2-dihydroquinoline-3-carboxylate (80.0 mg, 0.18 mmol)
1
with SnCl (128.0 mg, 0.56 mmol) in EtOH affords 10d (72.0 mg). Yield 100%; H NMR
2
(
400 MHz, CDCl ) δ 8.70 (1H, s), 7.62 (1H, d, J = 8.0 Hz), 7.36‒7.27 (4H, m), 6.53‒6.46
3
-
(
2H, m), 5.50 (2H, s), 4.45 (2H, s), 3.98 (3H, s). MS (ESI) m/z: 391.10 ([M-H] ).
Methyl 5-amino-1-(4-fluorobenzyl)-2-oxo-1,2-dihydroquinoline-3-carboxylate (10e)
Following the general procedure A, reaction of methyl 1-(4-fluorobenzyl)-5-nitro-2-oxo-
3
7
1
,2-dihydroquinoline-3-carboxylate (80.0 mg, 0.22 mmol) with SnCl (152.0 mg, 0.67
2
1
mmol) in EtOH affords 10e (199.0 mg). Yield 91%; H NMR (400 MHz, CDCl ) δ 8.70 (1H,
3
s), 7.60 (1H, t, J = 8.0 Hz), 7.51 (1H, d, J = 8.0 Hz), 7.42 (1H, d, J = 8.0 Hz), 7.10 (2H, dd, J
=
4.0 Hz, 8.0 Hz), 6.97 (2H, t, J = 8.0 Hz), 5.50 (2H, s), 4.43 (2H, s), 3.91 (3H, s). MS (ESI)
-
m/z: 325.23 ([M-H] ).
Methyl 5-amino-1-(4-chlorobenzyl)-2-oxo-1,2-dihydroquinoline-3-carboxylate (10f)
Following the general procedure A, reaction of methyl 1-(4-chlorobenzyl)-5-nitro-2-oxo-
3
7
1
,2-dihydroquinoline-3-carboxylate (80.0 mg, 0.21 mmol) with SnCl (145.0 mg, 0.64
2
1
mmol) in EtOH affords 10f (55.0 mg). Yield 76%; H NMR (400 MHz, CDCl ) δ 8.74 (1H,
3

ACCEPTED MANUSCRIPT
s), 7.66 (1H, d, J = 8.0 Hz), 7.63 (1H, t, J = 8.0 Hz), 7.50 (1H, d, J = 8.0 Hz), 7.30 (2H, d, J =
8
.0 Hz), 7.17 (2H, d, J = 8.0 Hz), 5.52 (2H, s), 4.40 (2H, s), 3.91(3H, s). MS (ESI) m/z:
-
3
41.23 ([M-H] ).
Methyl 5-amino-1-(4-bromobenzyl)-2-oxo-1,2-dihydroquinoline-3-carboxylate (10g)
Following the general procedure A, reaction of methyl 1-(4-bromobenzyl)-5-nitro-2-oxo-
3
7
1
,2-dihydroquinoline-3-carboxylate (80.0 mg, 0.19 mmol) with SnCl (130.0 mg, 0.57
2
1
mmol) in EtOH affords 10g (57.0 mg). Yield 77%; H NMR (400 MHz, CDCl ) δ 8.63 (1H,
3
s), 7.84‒7.81 (1H, m), 7.60 (1H, t, J = 8.0 Hz), 7.48‒7.41 (3H, m), 7.10 (2H, d, J = 8.0 Hz),
+
5
.50 (2H, s), 4.43 (2H, s), 3.84 (3H, s). MS (ESI) m/z: 387.73 ([M+H] ).
Methyl 5-amino-1-(4-methylbenzyl)-2-oxo-1,2-dihydroquinoline-3-carboxylate (10h)
Following the general procedure A, reaction of methyl 1-(4-methylbenzyl)-5-nitro-2-oxo-
3
7
1
,2-dihydroquinoline-3-carboxylate (80.0 mg, 0.22 mmol) with SnCl (153.0 mg, 0.68
2
1
mmol) in EtOH affords 10h (71.0 mg). Yield 100%; H NMR (400 MHz, CDCl ) δ 8.62 (1H,
3
s), 7.73‒7.70 (1H, m), 7.58‒7.48 (2H, m), 7.20‒7.00 (4H, m), 5.50 (2H, s), 4.40 (2H, s), 3.94
-
(
3H, s), 2.31 (3H, s). MS (ESI) m/z: 321.03 ([M-H] ).
Methyl
5-amino-1-(4-ethylbenzyl)-2-oxo-1,2-dihydroquinoline-3-carboxylate
(10i)
Following the general procedure A, reaction of methyl 1-(4-ethylbenzyl)-5-nitro-2-oxo-1,2-
3
7
dihydroquinoline-3-carboxylate (80.0 mg, 0.21 mmol) with SnCl (147.0 mg, 0.65 mmol)
2
1
in EtOH affords 10i (63.0 mg). Yield 89%; H NMR (400 MHz, CDCl ) δ 8.68 (1H, s), 7.80‒
3
7
.70 (1H, m), 7.53‒7.46 (2H, m), 7.10‒7.00 (4H, m), 5.49 (2H, s), 4.37 (2H, s), 3.97 (3H, s),
+
2
.62‒2.49 (2H, m), 1.20 (3H, t, J = 8.0 Hz). MS (ESI) m/z: 336.92 ([M+H] ).

ACCEPTED MANUSCRIPT
Methyl
5-amino-1-(4-nitrobenzyl)-2-oxo-1,2-dihydroquinoline-3-carboxylate
(10j)
Following the general procedure A, reaction of methyl 5-nitro-1-(4-nitrobenzyl)-2-oxo-1,2-
3
7
dihydroquinoline-3-carboxylate (80.0 mg, 0.20 mmol) with SnCl (137.0 mg, 0.61 mmol)
2
1
in EtOH affords 10j (68.0 mg). Yield 96%; H NMR (400 MHz, CDCl ) δ 8.70 (1H, d, J =
3
8
8
.0 Hz), 8.15 (2H, d, J = 12.0 Hz), 7.41‒7.32 (2H, m), 7.31‒7.27 (1H, m), 6.51 (1H, d, J =
.0 Hz), 6.49‒6.41 (1H, m), 5.59 (2H, s), 4.45 (2H, s), 3.98 (3H, s). MS (ESI) m/z: 351.76
-
(
[M-H] ).
Methyl 5-amino-1-(4-cyanobenzyl)-2-oxo-1,2-dihydroquinoline-3-carboxylate (10k)
Following the general procedure A, reaction of methyl 1-(4-cyanobenzyl)-5-nitro-2-oxo-
3
7
1
,2-dihydroquinoline-3-carboxylate (80.0 mg, 0.21 mmol) with SnCl (149.0 mg, 0.66
2
1
mmol) in EtOH affords 10k (58.0 mg). Yield 78%; H NMR (400 MHz, CDCl ) δ 8.74 (1H,
3
s), 7.60 (1H, d, J = 8.0 Hz), 7.33‒7.27 (4H, m), 6.53‒6.46 (2H, m), 5.56 (2H, s), 4.45 (2H, s),
+
3
.98 (3H, s). MS (ESI) m/z: 334.84 ([M+H] ).
Methyl 5-amino-1-benzyl-2-oxo-1,2-dihydroquinoline-3-carboxylate (10l) Following the
general procedure A, reaction of methyl 1-benzyl-5-nitro-2-oxo-1,2-dihydroquinoline-3-
3
7
carboxylate (80.0 mg, 0.23 mmol) with SnCl (155.0 mg, 0.69 mmol) in EtOH affords 10l
2
1
(
62.0 mg). Yield 87%; H NMR (400 MHz, CDCl ) 8.79 (1H, s), 7.61 (1H, d, J = 8.0 Hz),
3
7
.34–7.32 (2H, m), 7.22 (3H, t, J = 8.0 Hz), 6.54 (2H, d, J = 8.0 Hz), 5.52 (2H, s), 4.43 (2H,
+
s), 3.99 (3H, s). MS (ESI) m/z: 309.10 ([M+H] ).

ACCEPTED MANUSCRIPT
2
-Chloroquinolin-5-amine (15a) Following the general procedure A, reaction of 14a (75.0
mg, 0.36 mmol) with SnCl (243.0 mg, 1.08 mmol) in EtOH affords 15a (72.0 mg). Yield
2
1
1
00%; H NMR (400 MHz, CDCl ) δ 8.07 (1H, d, J = 8.0 Hz), 7.48 (1H, t, J = 8.0 Hz), 7.42
3
(1H, d, J = 8.0 Hz), 7.27 (1H, d, J = 8.0 Hz), 6.78 (1H, dd, J = 8.0 Hz, 8.0 Hz). MS (ESI) m/z:
-
1
77.54 ([M-H] ).
Methyl 5-amino-2-chloroquinoline-3-carboxylate (15b) Following the general procedure A,
reaction of 14b (80.0 mg, 0.30 mmol) with SnCl (1.0 mg, 0.90 mmol) in EtOH affords 15b
2
1
(
72.0 mg). Yield 100 %; H NMR (400 MHz, CDCl ) δ 8.75 (1H, s), 7.61 (1H, t, J = 8.0 Hz),
3
7
.45 (1H, d, J = 8.0 Hz), 6.83 (1H, dd, J = 8.0 Hz, 8.0 Hz), 3.99 (3H, s). MS (ESI) m/z:
+
2
37.30 ([M+H] ).
2
-(Adamantan-1-yl)-N-(2-(4-aminophenyl)quinolin-5-yl)acetamide (17h) Following the
general procedure A, reaction of 17g (10.0 mg, 0.02 mmol) with SnCl (15.0 mg, 0.06 mmol)
2
in EtOH affords 17h (3.0 mg). Yield 30%; ¹H NMR (400 MHz, CDCl ) δ 8.15 (1H, d, J = 8.0
3
Hz), 8.01 (2H, d, J = 8.0 Hz), 7.95 (1H, d, J = 8.0 Hz), 7.81 (1H, d, J = 8.0 Hz), 7.49‒7.40
(
2H, m), 7.34 (1H, s), 6.79 (2H, d, J = 8.0 Hz), 3.87 (2H, s), 2.26 (2H, s), 2.18 (1H, s), 2.04
1H, s), 1.79‒1.63 (13H, m). MS (ESI) m/z: 412.19 ([M+H]⁺). Purity 96%
(
General procedure B: For preparation of 11a‒i, 12a‒j, 13a and 16a‒e
Compounds 10a‒l, 15a or 15b (1.0 equiv), DIPEA (2.0 equiv) and appropriate carboxylic
acid chloride derivative (1.2 equiv) were stirred in DCM for 15 min at room temperature.

ACCEPTED MANUSCRIPT
After the reaction mixture was partitioned between saturated aqueous NaHCO solution and
3
ethyl acetate. The organic layer was dried over sodium sulfate, filtered and evaporated under
vacuum. The resulting residue was purified by silica gel column chromatography to afford
11a‒i, 12a‒j, 13a and 16a‒e.
Methyl 1-(4-methoxybenzyl)-2-oxo-5-(3-phenylpropanamido)-1,2-dihydroquinoline-3-
carboxylate (11a) Following the general procedure B, acylation reaction of 10b (10.0 mg,
0
3
7
.02 mmol) and hydrocinnamoyl chloride (0.005 mL, 0.03 mmol) affords 11a (4.9 mg). Yield
1
5%; H NMR (400 MHz, CDCl ) δ 8.22 (1H, s), 7.56 (1H, s), 7.36 (1H, t, J = 8.0 Hz), 7.21‒
3
.15 (5H, m), 6.90 (1H, d, J = 8.0 Hz), 6.84‒6.77 (4H, m), 6.58 (1H, d, J = 8.0 Hz), 5.48 (2H,
+
s), 3.96 (3H, s), 3.75 (3H, s), 3.18‒3.01 (4H, m). MS (ESI) m/z: 471.13 ([M+H] ). Purity
9
6%.
Methyl
1-(4-methoxybenzyl)-2-oxo-5-(2-phenylacetamido)-1,2-dihydroquinoline-3-
carboxylate (11b) Following the general procedure B, acylation reaction of 10b (10.0 mg,
0
3
7
.02 mmol) and phenylacetyl chloride (0.004 mL, 0.03 mmol) affords 11b (4.0 mg). Yield
1
0%; H NMR (400 MHz, CDCl ) δ 8.19 (1H, s), 7.57‒7.36 (6H, m), 7.32‒7.27 (2H, m),
3
.24‒7.20 (1H, m), 7.12 (2H, d, J = 8.0 Hz), 6.78 (2H, d, J = 8.0 Hz), 5.45 (2H, s), 3.95 (3H,
-
s), 3.76 (3H, s), 2.16‒2.14 (2H, m). MS (ESI) m/z: 455.39 ([M-H] ). Purity 99%
Methyl
5-benzamido-1-(4-methoxybenzyl)-2-oxo-1,2-dihydroquinoline-3-carboxylate
(11c) Following the general procedure B, acylation reaction of 10b (10.0 mg, 0.02 mmol) and

ACCEPTED MANUSCRIPT
1
benzoyl chloride (0.004 mL, 0.03 mmol) affords 11c (5.0 mg). Yield 39%; H NMR (400
MHz, CDCl ) δ 8.62 (1H, s), 8.24 (1H, s), 7.97 (2H, d, J = 8.0 Hz), 7.64‒7.58 (2H, m), 7.57‒
3
7
.49 (3H, m), 7.24‒7.20 (1H, m), 7.16 (2H, d, J = 8.0 Hz), 6.82 (2H, d, J = 8.0 Hz), 5.46 (2H,
-
s), 3.91 (3H, s), 3.73 (3H, s). MS (ESI) m/z: 441.39 ([M-H] ). Purity 96%
Methyl
5-(3-cyclopentylpropanamido)-1-(4-methoxybenzyl)-2-oxo-1,2-
dihydroquinoline-3-carboxylate (11d) Following the general procedure B, acylation
reaction of 10b (10.0 mg, 0.02 mmol) and cyclopentanepropionyl chloride (0.004 mL, 0.03
1
mmol) affords 11d (5.0 mg). Yield 37%; H NMR (400 MHz, MeOH-d ) 8.25 (1H, s), 7.78
4
(1H, s), 7.47 (1H, t, J = 8.0 Hz), 7.15 (2H, d, J = 8.0 Hz), 7.04 (1H, d, J = 8.0 Hz), 6.84 (1H,
d, J = 8.0 Hz), 6.78 (2H, d, J = 8.0 Hz), 5.53 (2H, s), 3.93 (3H, s), 3.74 (3H, s), 3.18‒3.01
+
(
4H, m), 2.23‒2.18 (9H, m). MS (ESI) m/z: 463.12 ([M+H] ). Purity 95%
Methyl 5-(3-cyclohexylpropanamido)-1-(4-methoxybenzyl)-2-oxo-1,2-dihydroquinoline-
3
-carboxylate (11e) Following the general procedure B, acylation reaction of 10b (10.0 mg,
0
.02 mmol) and 3-cyclohexylpropanoyl chloride (6.0 mg, 0.03 mmol) affords 11e (5.0 mg).
1
Yield 36%; H NMR (400 MHz, CDCl ) δ 8.62 (1H, s), 7.70 (1H, s), 7.52‒7.48 (2H, m), 7.15
3
(3H, d, J = 8.0 Hz), 6.81 (2H, d, J = 8.0 Hz), 5.49 (2H, s), 4.46‒4.41 (2H, m), 3.75 (3H, s),
+
2
9
.52 (2H, m), 2.33‒2.29 (7H, m), 1.54‒1.50 (7H, m). MS (ESI) m/z: 476.79 ([M+H] ). Purity
5%

ACCEPTED MANUSCRIPT
Methyl 5-(2-cyclohexylacetamido)-1-(4-methoxybenzyl)-2-oxo-1,2-dihydroquinoline-3-
carboxylate (11f) Following the general procedure B, acylation reaction of 10b (10.0 mg,
0
.02 mmol) and cyclohexylacetyl chloride (0.004 mL, 0.03 mmol) affords 11f (5.0 mg).
1
Yield 37%; H NMR (400 MHz, CDCl ) δ 8.62 (1H, s), 7.55‒7.49 (2H, m), 7.40 (1H, s), 7.17
3
(3H, d, J = 8.0 Hz), 6.82 (2H, d, J = 8.0 Hz), 5.52 (2H, s), 3.97 (3H, s), 3.76 (3H, s), 2.40‒
2
.30 (2H, m), 2.08‒1.98 (3H, m), 1.91‒1.84 (3H, m), 1.76‒1.69 (2H, m), 1.65‒1.60 (3H, m).
-
MS (ESI) m/z: 461.48 ([M-H] ). Purity 95%
Methyl 5-(cyclohexanecarboxamido)-1-(4-methoxybenzyl)-2-oxo-1,2-dihydroquinoline-
3
-carboxylate (11g) Following the general procedure B, acylation reaction of 10b (5.0 mg,
0
.01 mmol) and cyclohexanecarbonyl chloride (0.002 mL, 0.02 mmol) affords 11g (1.0 mg).
1
Yield 17%; H NMR (400 MHz, CDCl ) δ 8.57 (1H, s), 7.51 (2H, d, J = 8.0 Hz), 7.38 (1H, s),
3
7
2
4
.16 (3H, d, J = 8.0 Hz), 6.81 (2H, d, J = 8.0 Hz), 5.49 (2H, s), 3.98 (3H, s), 3.74 (3H, s),
.08‒1.98 (3H, m), 1.91‒1.84 (3H, m), 1.76‒1.69 (2H, m), 1.65‒1.60 (3H, m). MS (ESI) m/z:
+
48.91 ([M+H] ). Purity 99%
Methyl
5-(2-(adamantan-1-yl)acetamido)-1-(4-methoxybenzyl)-2-oxo-1,2-
dihydroquinoline-3-carboxylate (11h) Following the general procedure B, acylation
reaction of 10b (10.0 mg, 0.02 mmol) and 1-adamantane acetyl chloride (0.006 mL, 0.03
1
mmol) affords 11h (5.0 mg). Yield 33%; H NMR (400 MHz, CDCl ) δ 8.64 (1H, s), 7.52‒
3
7
.49 (2H, m), 7.35 (1H, s), 7.16 (3H, d, J = 8.0 Hz), 6.82 (2H, d, J = 8.0 H), 5.52 (2H, s),
3
.96 (3H, s), 3.76 (3H, s), 2.23‒2.20 (2H, m), 2.16 (1H, s), 2.02 (1H, s), 1.79‒1.63 (13H, m).
-
MS (ESI) m/z: 513.45 ([M-H] ). Purity 95%

ACCEPTED MANUSCRIPT
Methyl
5-(adamantane-1-carboxamido)-1-(4-methoxybenzyl)-2-oxo-1,2-
dihydroquinoline-3-carboxylate (11i) Following the general procedure B, acylation reaction
of 10b (10.0 mg, 0.02 mmol) and adamantane-1-carbonyl chloride (6.0 mg, 0.03 mmol)
1
affords 11i (4.0 mg). Yield 27%; H NMR (400 MHz, CDCl ) δ 8.55 (1H, s), 7.58 (1H, s),
3
7
.51 (1H, t, J = 8.0 H), 7.45 (1H, d, J = 8.0 Hz), 7.16 (3H, t, J = 8.0 Hz), 6.81 (2H, d, J = 8.0
Hz), 5.47 (2H, s), 3.98 (3H, s), 3.76 (3H, s), 2.18 (1H, s), 2.04 (1H, s), 1.79‒1.63 (13H, m).
1
3
C NMR (125 MHz, CDCl ) 206.86, 176.98, 165.88, 158.79, 158.62, 141.97, 138.81, 135.82,
3
1
4
33.34, 128.08, 127.90, 127.87, 121.51, 119.03, 114.22, 113.84, 112.84, 55.24, 52.88, 46.19,
+
1.80, 39.35, 38.65, 36.31, 30.93, 28.11, 27.81. MS (ESI) m/z: 501.47 ([M+H] ). HRMS
+
(
FAB) calcd for C H N O [M] 500.2311, found 500.2310. Purity 97%
3
0
32
2
5
Methyl
5-(adamantane-2-carboxamido)-1-(3-methoxybenzyl)-2-oxo-1,2-
dihydroquinoline-3-carboxylate (12a) Following the general procedure B, acylation
reaction of 10c (10.0 mg, 0.02 mmol) and adamantane-1-carbonyl chloride (6.0 mg, 0.03
1
mmol) affords 12a (4.0 mg). Yield 27%; H NMR (400 MHz, CDCl ) δ 8.57 (1H, s), 7.58
3
(1H, s), 7.53‒7.43 (2H, m), 7.20 (1H, t, J = 8.0 Hz), 7.13 (1H, d, J = 8.0 Hz), 6.77‒6.74 (3H,
m), 5.55 (2H, s), 3.99 (3H, s), 3.75 (3H, s), 2.18 (1H, s), 2.04 (1H, s), 1.79‒1.63 (13H, m).
+
MS (ESI) m/z: 500.94 ([M+H] ). Purity 97%
Methyl
5-(adamantane-2-carboxamido)-2-oxo-1-(4-(trifluoromethoxy)benzyl)-1,2-
dihydroquinoline-3-carboxylate (12b) Following the general procedure B, acylation

ACCEPTED MANUSCRIPT
reaction of 10d (10.0 mg, 0.02 mmol) and adamantane-1-carbonyl chloride (6.0 mg, 0.03
1
mmol) affords 12b (4.0 mg). Yield 28%; H NMR (400 MHz, CDCl ) δ 8.68 (1H, s), 7.57‒
3
7
5
5
.51 (2H, m), 7.42 (1H, s), 7.24‒7.19 (2H, m), 7.14 (2H, d, J = 8.0 Hz), 7.10‒7.07 (1H, m),
.54 (2H, s), 3.95 (3H, s), 2.18 (1H, s), 2.04 (1H, s), 1.79‒1.63 (13H, m). MS (ESI) m/z:
-
53.51 ([M-H] ). Purity 97%
Methyl 5-(adamantane-2-carboxamido)-1-(4-fluorobenzyl)-2-oxo-1,2-dihydroquinoline-
3
-carboxylate (12c) Following the general procedure B, acylation reaction of 10e (10.0 mg,
0
.03 mmol) and adamantane-1-carbonyl chloride (6.0 mg, 0.03 mmol) affords 12c (5.0 mg).
1
Yield 34%; H NMR (400 MHz, CDCl ) δ 8.53 (1H, s), 7.55 (1H, s), 7.53‒7.46 (2H, m), 7.44
3
(1H, t, J = 8.0 Hz), 7.21‒7.17 (2H, m), 6.97 (2H, t, J = 8.0 Hz), 5.52 (2H, s), 3.98 (3H, s),
+
2
.18 (1H, s), 2.04 (1H, s), 1.79‒1.63 (13H, m). MS (ESI) m/z: 488.92 ([M+H] ). Purity 95%
Methyl 5-(adamantane-2-carboxamido)-1-(4-chlorobenzyl)-2-oxo-1,2-dihydroquinoline-
3
-carboxylate (12d) Following the general procedure B, acylation reaction of 10f (10.0 mg,
0
.02 mmol) and adamantane-1-carbonyl chloride (6.0 mg, 0.03 mmol) affords 12d (5.0 mg).
1
Yield 34%; H NMR (400 MHz, CDCl ) δ 8.53 (1H, s), 7.55 (1H, s), 7.53‒7.28 (3H, m),
3
7
.14‒7.00 (3H, m), 6.97 (1H, t, J = 8.0 Hz), 5.53 (2H, s), 3.91 (3H, s), 2.18 (1H, s), 2.04 (1H,
+
s), 1.79‒1.63 (13H, m). MS (ESI) m/z: 505.92 ([M+H] ). Purity 96%
Methyl 5-(adamantane-2-carboxamido)-1-(4-bromobenzyl)-2-oxo-1,2-dihydroquinoline-
3
-carboxylate (12e) Following the general procedure B, acylation reaction of 10g (10.0 mg,

ACCEPTED MANUSCRIPT
0
.02 mmol) and adamantane-1-carbonyl chloride (6.0 mg, 0.03 mmol) affords 12e (5.0 mg).
1
Yield 36%; H NMR (400 MHz, CDCl ) δ 8.51 (1H, s), 7.55 (1H, s), 7.53‒7.41 (2H, m),
3
7
.31‒7.28 (2H, m), 7.11‒7.00 (2H, m), 6.97‒6.94 (1H, m), 5.52 (2H, s), 3.98 (3H, s), 2.18
+
(
1H, s), 2.04 (1H, s), 1.79‒1.63 (13H, m). MS (ESI) m/z: 549.12 ([M+H] ). Purity 96%
Methyl 5-(adamantane-2-carboxamido)-1-(4-methylbenzyl)-2-oxo-1,2-dihydroquinoline-
3
-carboxylate (12f) Following the general procedure B, acylation reaction of 10h (10.0 mg,
0
.03 mmol) and adamantane-1-carbonyl chloride (7.0 mg, 0.03 mmol) affords 12f (5.0 mg).
1
Yield 34%; H NMR (400 MHz, CDCl ) δ 8.56 (1H, s), 7.55 (1H, s), 7.53‒7.43 (3H, m),
3
7
.23‒7.09 (3H, m), 6.97 (1H, t, J = 8.0 Hz), 5.52 (2H, s), 3.95 (3H, s), 2.42 (3H, s), 2.18 (1H,
-
s), 2.04 (1H, s), 1.79‒1.63 (13H, m). MS (ESI) m/z: 483.92 ([M-H] ). Purity 99%
Methyl 5-(adamantane-2-carboxamido)-1-(4-ethylbenzyl)-2-oxo-1,2-dihydroquinoline-3-
carboxylate (12g) Following the general procedure B, acylation reaction of 10i (10.0 mg,
0
.02 mmol) and adamantane-1-carbonyl chloride (6.0 mg, 0.03 mmol) affords 12g (4.0 mg).
1
Yield 27%; H NMR (400 MHz, DMSO-d ) 8.56 (1H, s), 7.55 (1H, s), 7.51 (1H, t, J = 8.0
6
Hz), 7.28 (1H, d, J = 8.0 Hz), 7.13‒7.06 (5H, m), 5.47 (2H, s), 3.80 (3H, s), 2.52‒2.49 (2H,
+
m), 2.03‒1.84 (9H, m), 1.74‒1.63 (9H, m). MS (ESI) m/z: 498.93 ([M+H] ). Purity 96%
Methyl 5-(adamantane-2-carboxamido)-1-(4-nitrobenzyl)-2-oxo-1,2-dihydroquinoline-3-
carboxylate (12h) Following the general procedure B, acylation reaction of 10j (10.0 mg,
0
.02 mmol) and adamantane-1-carbonyl chloride (6.0 mg, 0.03 mmol) affords 12h (4.0 mg).

ACCEPTED MANUSCRIPT
1
Yield 27%; H NMR (400 MHz, CDCl ) δ 8.60 (1H, s), 8.16 (2H, d, J = 8.0 Hz), 7.60 (1H, s),
3
7
.54‒7.46 (2H, m), 7.36 (2H, d, J = 8.0 Hz), 7.00‒6.96 (1H, m), 5.65 (2H, s), 3.98 (3H, s),
-
2
.18 (1H, s), 2.04 (1H, s), 1.79‒1.63 (13H, m). MS (ESI) m/z: 513.84 ([M-H] ). Purity 95%
Methyl 5-(adamantane-1-carboxamido)-1-(4-cyanobenzyl)-2-oxo-1,2-dihydroquinoline-
3
-carboxylate (12i) Following the general procedure B, acylation reaction of 10k (10.0 mg,
0
.03 mmol) and adamantane-1-carbonyl chloride (7.0 mg, 0.03 mmol) affords 12i (4.0 mg).
1
Yield 26%; H NMR (400 MHz, CDCl ) δ 8.59 (1H, s), 7.59 (2H, d, J = 8.0 Hz), 7.55‒7.43
3
(2H, m), 7.30 (2H, d, J = 8.0 Hz), 7.00 (1H, d, J = 8.0 Hz), 5.62 (2H, s), 3.99 (3H, s), 2.18
+
(
1H, s), 2.04 (1H, s), 1.79‒1.63 (13H, m). MS (ESI) m/z: 495.90 ([M+H] ). Purity 95%
Methyl
5-(adamantane-1-carboxamido)-1-benzyl-2-oxo-1,2-dihydroquinoline-3-
carboxylate (12j) Following the general procedure B, acylation reaction of 10l (10.0 mg,
0
.03 mmol) and adamantane-1-carbonyl chloride (7.0 mg, 0.03 mmol) affords 12j (3.0 mg).
1
Yield 22%; H NMR (400 MHz, CDCl ) δ 8.57 (1H, s), 7.60 (2H, d, J = 8.0 Hz), 7.55‒7.43
3
.
(
2H, m), 7.30‒7.03 (4H, m), 5.60 (2H, s), 3.97 (3H, s), 2.18 (1H, s), 2 00 (1H, s), 1.79‒1.60
-
(
13H, m). MS (ESI) m/z: 469.21 ([M-H] ). Purity 97%
N-(1-(4-Methoxybenzyl)-2-oxo-1,2-dihydroquinolin-5-yl)adamantane-2-carboxamide
(13a) Following the general procedure B, acylation reaction of 10a (10.0 mg, 0.03 mmol) and
1
adamantane-1-carbonyl chloride (8.0 mg, 0.04 mmol) affords 13a (7.0 mg). Yield 45%; H
NMR (400 MHz, CDCl ) δ 7.73 (1H, d, J = 8.0 Hz), 7.47 (1H, s), 7.42‒7.36 (2H, m), 7.20‒
3

ACCEPTED MANUSCRIPT
7
1
.09 (3H, m), 6.86‒6.74 (3H, m), 5.50 (2H, s), 3.76 (3H, s), 2.18 (1H, s), 2.04 (1H, s), 1.79‒
+
.63 (13H, m). MS (ESI) m/z: 442.91 ([M+H] ). Purity 99%
Methyl
5-(2-(adamantan-1-yl)acetamido)-2-chloroquinoline-3-carboxylate
(16a)
Following the general procedure B, acylation of 15b (5.0 mg, 0.02 mmol) and 1-
adamantaneacetylchloride (0.003 mL, 0.02 mmol) in DCM affords 16a (4.0 mg). Yield 48%;
1
H NMR (400 MHz, CDCl ) δ 8.62 (1H, s), 7.93‒7.77 (3H, m), 7.67 (1H, s), 4.01 (3H, s),
3
+
2
.30 (2H, s), 2.18 (1H, s), 2.04 (1H, s), 1.79‒1.63 (13H, m). MS (ESI) m/z: 413.52 ([M+H] ).
Purity 94%
Methyl
5-(adamantane-1-carboxamido)-2-chloroquinoline-3-carboxylate
(16b)
Following the general procedure B, acylation of 15b (5.0 mg, 0.02 mmol) and adamantane-1-
1
carbonyl chloride (3.0 mg, 0.02 mmol) in DCM affords 16b (4.0 mg). Yield 48%; H NMR
(
400 MHz, CDCl ) δ 8.62 (1H, s), 7.93‒7.73 (3H, m), 7.65 (1H, s), 4.04 (3H, s), 2.18 (1H, s),
3
+
2
.04 (1H, s), 1.79‒1.63 (13H, m). MS (ESI) m/z: 399.01 ([M+H] ). Purity 97%
2
-(Adamantan-1-yl)-N-(2-chloroquinolin-5-yl)acetamide (16c) Following the general
procedure B, acylation of 15a (50.0 mg, 0.28 mmol) and 1-adamantaneacetylchloride (0.06
1
mL, 0.33 mmol) in DCM affords 16c (37.0 mg). Yield 37%; H NMR (400 MHz, CDCl ) δ
3
8
.14 (1H, d, J = 8.0 Hz), 7.91 (1H, d, J = 8.0 Hz), 7.83 (1H, d, J = 8.0 Hz), 7.73 (1H, t, J =
.0 Hz), 7.43 (1H, d, J = 8.0 Hz), 7.28 (1H, s), 2.24 (2H, s), 2.02 (2H, s), 1.79‒1.63 (13H, m).
8
1
3
C NMR (125 MHz, CDCl ) δ 144.35, 129.42, 128.21, 126.61, 122.68, 118.47, 118.28,
3