Structural development of a type-1 ryanodine receptor (RyR1) Ca2+-release channel inhibitor guided by endoplasmic reticulum Ca2+ assay

Article abstract of DOI:10.1016/j.ejmech.2019.06.076

Type-1 ryanodine receptor (RyR1) is a calcium-release channel localized on sarcoplasmic reticulum (SR) of the skeletal muscle, and mediates muscle contraction by releasing Ca²⁺ from the SR. Genetic mutations of RyR1 are associated with skeletal muscle diseases such as malignant hyperthermia and central core diseases, in which over-activation of RyR1 causes leakage of Ca²⁺ from the SR. We recently developed an efficient high-throughput screening system based on the measurement of Ca²⁺ in endoplasmic reticulum, and used it to identify oxolinic acid (1) as a novel RyR1 channel inhibitor. Here, we designed and synthesized a series of quinolone derivatives based on 1 as a lead compound. Derivatives bearing a long alkyl chain at the nitrogen atom of the quinolone ring and having a suitable substituent at the 7-position of quinolone exhibited potent RyR1 channel-inhibitory activity. Among the synthesized compounds, 14h showed more potent activity than dantrolene, a known RyR1 inhibitor, and exhibited high RyR1 selectivity over RyR2 and RyR3. These compounds may be promising leads for clinically applicable RyR1 channel inhibitors.

Full text of DOI:10.1016/j.ejmech.2019.06.076

Accepted Manuscript
2+
Structural development of a type-1 ryanodine receptor (RyR1) Ca -release channel
2+
inhibitor guided by endoplasmic reticulum Ca assay
Shuichi Mori, Hiroto Iinuma, Noriaki Manaka, Mari Ishigami-Yuasa, Takashi
Murayama, Yoshiaki Nishijima, Akiko Sakurai, Ryota Arai, Nagomi Kurebayashi,
Takashi Sakurai, Hiroyuki Kagechika
PII:
S0223-5234(19)30603-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.076
EJMECH 11479
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 March 2019
Revised Date: 17 May 2019
Accepted Date: 27 June 2019
Please cite this article as: S. Mori, H. Iinuma, N. Manaka, M. Ishigami-Yuasa, T. Murayama, Y.
Nishijima, A. Sakurai, R. Arai, N. Kurebayashi, T. Sakurai, H. Kagechika, Structural development
2+
of a type-1 ryanodine receptor (RyR1) Ca -release channel inhibitor guided by endoplasmic
2+
reticulum Ca assay, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.06.076.
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ACCEPTED MANUSCRIPT
Graphical abstract

ACCEPTED MANUSCRIPT
Structural development of a type-1 ryanodine receptor (RyR1) Ca2+-release channel inhibitor guided
by endoplasmic reticulum Ca2+ assay
Shuichi Mori, Hiroto Iinuma, Noriaki Manaka, Mari Ishigami-Yuasa, Takashi Murayama,* Yoshiaki
Nishijima, Akiko Sakurai, Ryota Arai, Nagomi Kurebayashi, Takashi Sakurai, and Hiroyuki
Kagechika*
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University (TMDU), Tokyo
101-0062, Japan.
Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of
Medicine, Tokyo 113-8421, Japan.
Abstract
Type-1 ryanodine receptor (RyR1) is a calcium-release channel localized on sarcoplasmic reticulum
(SR) of the skeletal muscle, and mediates muscle contraction by releasing Ca2+ from the SR. Genetic
mutations of RyR1 are associated with skeletal muscle diseases such as malignant hyperthermia and
central core diseases, in which over-activation of RyR1 causes leakage of Ca2+ from the SR. We
recently developed an efficient high-throughput screening system based on the measurement of Ca2+
in endoplasmic reticulum, and used it to identify oxolinic acid (1) as a novel RyR1 channel inhibitor.
Here, we designed and synthesized a series of quinolone derivatives based on 1 as a lead compound.
Derivatives bearing a long alkyl chain at the nitrogen atom of the quinolone ring and having a
suitable substituent at the 7-position of quinolone exhibited potent RyR1 channel-inhibitory activity.
Among the synthesized compounds, 14h showed more potent activity than dantrolene, a known
RyR1 inhibitor, and exhibited high RyR1 selectivity over RyR2 and RyR3. These compounds may
be promising leads for clinically applicable RyR1 channel inhibitors.
Key words: Ryanodine receptor, quinolone, calcium ion channel

ACCEPTED MANUSCRIPT
1. Introduction
Ryanodine receptors (RyRs) are huge calcium-release channels with molecular masses of >2 MDa,
located on the sarcoplasmic reticulum (SR) membrane of skeletal and cardiac muscles. RyR1 and its
isoform RyR2 are expressed in skeletal muscles and cardiac muscles, respectively, and play essential
roles in muscle contraction.1–4 In addition, RyR3 is expressed ubiquitously in various tissues and is
involved in many biological processes, including neurotransmission. In the case of skeletal muscles,
an action potential promotes mechanical coupling of dihydropyridine receptor and RyR1, followed
by Ca2+ release from the SR store though the channel pore of RyR1. The resulting increase of
cytoplasmic Ca2+ level induces skeletal muscle contraction. Genetic mutations of RyR1 are known to
be associated with several muscle diseases, such as malignant hyperthermia (MH) and central core
diseases (CCD),5, 6 in which overactivation of the mutated RyR1 channel causes leakage of Ca2+
from the SR store. In addition, the symptoms of several diseases, such as muscular dystrophy7 and
Alzheimer disease,8 are thought to be caused, at least in part, by over-activation of non-mutated
RyR1 induced by external factors. Therefore, inhibitors of RyR1 channel activity are expected to be
candidates for treating RyR1-related diseases.
Dantrolene (Figure 1) is the only RyR1 channel-targeting drug currently in clinical use; it is
employed to treat MH,9 a lethal disease caused by abnormal Ca2+ release from mutated RyR1
channels, triggered by administration of volatile anesthetics. Dantrolene inhibits the abnormal Ca2+
release by direct interaction with RyR1, ameliorating the symptoms of MH.10, 11 Unfortunately, it is
not applicable to chronic muscle diseases related to RyR1, such as CCD. Although novel RyR1
inhibitors are needed, not much research has been done in this area.12 This is mainly due to lack of
efficient assay method of the RyR1 channel activity that is suitable for high-throughput screening
(HTS) and structural development studies.13
We have recently developed an efficient HTS system for RyR1 inhibitors based on the
measurement of the Ca2+ concentration in the endoplasmic reticulum (ER) ([Ca2+]ER) of HEK293
cells,14–16 and we screened oxolinic acid (1) as an RyR1-specific inhibitor (Figure 1) from a chemical
library of well-characterized drugs (1535 compounds) owned by Tokyo Medical and Dental
University (TMDU).16 Compound 1 not only inhibited mutant RyR1 channels, but also inhibited the
activation of WT RyR1 channel by caffeine, a known RyR channel activator. Thus, it could
potentially treat not only diseases due to mutated RyR1, but also diseases due to over-activation of
WT RyR by other factors or modifications. Compound 1 is a first-generation quinolone antibiotic,
which has been used for the treatment of urinary tract infections17 and is currently using for infection
prevention in aquaculture.18 Its activity is due to the inhibition of bacterial DNA gyrase, a
bacteria-specific enzyme.19 The toxicity of 1 toward humans is low, and indeed, no serious adverse
effects have been reported. Thus, 1 seems to be a promising drug candidate for RyR1-related
diseases. However, the inhibitory potency of 1 (EC50: 0.81 µM) was lower than that of dantrolene

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(EC50: 0.059 µM) by one order of magnitude (Figure 2c). Therefore, in this study, we evaluated the
structure-activity relationship of 1, focusing on structural development to obtain more potent RyR1
channel inhibitors.
Figure 1. Structures of dantrolene and oxolinic acid (1).
2. Results and Discussion
2.1. Molecular design and synthesis
In the previous library screening of RyR1 channel inhibitors, none of 17 quinolone antibiotics,
many of which were new quinolones having a fluorine group and a large substituent such as
piperadine at 6- and 7-position of quinolone, respectively, exhibited RyR1 channel-inhibitory
activity.16 Based on these results, we designed a novel series of quinolone derivatives having a small
substituent at the 6-, 7-, or 8-position of quinolone and different-sized alkyl groups at the 1-nitrogen
atom.
First, quinolones without a substituent on the benzene moiety and with various alkyl groups on the
nitrogen atom were synthesized to investigate the importance of the N-substituent. Alkylation of
1,4-dihydro-4-oxoquinoline-3-carboxylic acid (DOCA) with alkyl halide by using cesium carbonate
as a base gave N,O-dialkylated compounds 2. The ester group of 2 was hydrolyzed in aqueous
sodium hydroxide solution to afford the target carboxylic acids 3 (Scheme 1).
Next, we focused on the effect of substituents on the benzene part of the quinolone ring. A series of
substituted quinolone derivatives 4 – 15 was synthesized (Scheme 2). Substituted anilines were
coupled with diethyl ethoxymethylenemalonate to afford compounds 16 – 27. Intramolecular
cyclization of 16 – 27 by heating in diphenylether gave the corresponding quinolones 28 – 39.
N-Alkylations of 28 – 39 were performed using sodium hydride as a base, since the reactions did not
proceed under the conditions used for the synthesis of 2 (i.e., with cesium carbonate). In some cases,
the N-alkylated products included transesterification products with the alkylating reagents. The ester
groups of 40 – 51 were hydrolyzed in aqueous sodium hydroxide solution to afford 4 – 15 (Scheme
2).

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Scheme1. Synthesis of the quinolone derivatives 3 with various N-substituents.
EtO2C
CO2Et
OEt
O
O
diphenylether
220 °C
EtO2C
4
3
4
3
CO2Et
6
7
OEt
X
X
X
NH2
EtOH, reflux
N
H
N
H
2
2
8
16: X = 4-Me (90%)
17: X = 3-Me (88%)
24: X = 3,4-di-OMe (quant.)
25: X = 2,3-di-OMe (quant.)
28: X = 6-Me (50%)
29: X = 7-Me (50%)
30: X = 6-OMe (51%)
31: X = 7-OMe (40%)
32: X = 8-OMe (55%)
33: X = 7-SMe (52%)
34: X = 7-CF3 (21%)
35: X = 7-OCF3 (45%)
36: X = 6,7-di-OMe (28%)
37: X = 7,8-di-OMe (68%)
18: X = 4-OMe (90%)
19: X = 3-OMe (91%)
20: X = 2-OMe (quant.)
21: X = 3-SMe (quant.)
22: X = 3-CF3 (quant.)
23: X = 3-OCF3 (quant.)
4
6
O
O
26: X =
27: X =
(quant.)
(92%)
38: X =
39: X =
(62%)
(25%)
O
O
3
4
7
6
3
7
O
O
O
O
R-X
6
7
6
7
NaH
n-Propyl: c
n-Octyl:
n-Decyl:
OY
NaOH aq.
MeOH
R =
OH
h
j
X
X
DMF
N
R
N
R
8
8
12h: X = 6,7-di-OMe (39%)
13h: X = 7,8-di-OMe (80%)
40c
40h
41c
41h
42c
42h
43c
43h
44h
45h
48h: X = 6,7-di-OMe, Y = Et (12%)
49h: X = 7,8-di-OMe, Y = R (15%)
4c: X = 6-Me (74%)
4h: X = 6-Me (quant.)
5c: X = 7-Me (60%)
5h: X = 7-Me (70%)
6c: X = 6-OMe (60%)
6h: X = 6-OMe (82%)
7c: X = 7-OMe (60%)
7h: X = 7-OMe (62%)
8h: X = 8-OMe (63%)
: X = 6-Me, Y = R (72%)
: X = 6-Me, Y = R (54%)
: X = 7-Me, Y = R (90%)
: X = 7-Me, Y = R (64%)
: X = 6-OMe, Y = R (81%)
: X = 6-OMe, Y = R (75%)
: X = 7-OMe, Y = Et (77%)
: X = 7-OMe, Y = R (73%)
: X = 8-OMe, Y = Et (45%)
: X = 7-SMe, Y = Et (73%)
6
6
O
O
14h: X =
(quant.)
(quant.)
(36%)
50h: X =
, Y = R (40%)
, Y = Et (40%)
, Y = Et (35%)
O
O
7
6
O
O
7
6
14j
: X =
50j
: X =
O
O
7
6
7
6
9h
: X = 7-SMe (quant.)
46h: X = 7-CF3, Y = Et (13%)
47h: X = 7-OCF3, Y = Et (23%)
10h: X = 7-CF3 (59%)
51h
: X =
15h: X =
11h
: X = 7-OCF3 (91%)
7
7
Scheme 2. Synthesis of the quinolone derivatives 4 – 11 substituted on the benzene ring.

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2.2. RyR1 channel-inhibitory activity
The RyR1 channel-inhibitory activities of the synthesized compounds were evaluated by means of
our [Ca2+]ER assay,16 based on the fact that inhibitors suppress Ca2+ leakage from the ER in HEK293
cells carrying a disease-associated mutation of RyR1, resulting in an increase of [Ca2+]ER due to Ca2+
uptake via the Ca2+ pump (Figure 2a). [Ca2+]ER was measured with R-CEPIA1er, an ER Ca2+-sensing
fluorescent protein,20 and time-lapse fluorescence measurements were performed using a
FlexStation3 fluorometer with 96-well plates. Figure 2b shows typical results of fluorescence
measurements in HEK293 cells expressing RyR1 with the R2163C mutation. Addition of 0.1 – 30
µM 1 gradually increased the [Ca2+]ER signal and reached a plateau in a dose-dependent manner,
indicating that 1 suppressed the Ca2+ leakage via RyR1. Figure 2c shows the dose dependency of
R2163C RyR1 channel-inhibitory activity. The vertical axis of Figure 2c represents the [Ca2+]ER
increase ratio F/F0 which was obtained by normalizing the average fluorescence for the last 100
seconds (F) to that for the first 100 seconds (F0) in the time-lapse fluorescent measurement (Figure
2b). The dose-response curve of 1 was fitted by the Michaelis-Menten equation, and the EC50 value
was determined to be 0.81 µM. The value was one-order higher than that of dantrolene (EC50: 0.059
µM). In contrast, DOCA, a quinolone derivative not alkylated at the 1-nitrogen atom or substituted
at the benzene ring, had no effect on the R-CEPIA1er fluorescence at concentrations up to 30 µM.

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(a)
mutant RyR1
Ca2+ pump
drugs
Ca2+
Ca2+
Ca2+
(b)
(c)
Figure 2. (a) Schematic illustration of the [Ca2+]ER assay principle. (b) Time-lapse R-CEPIA1er
fluorescence measurement with HEK293 cells expressing mutant RyR1. Compound 1 (0.1, 0.3, 1,
and 30 µM) and DMSO was added at 100 seconds (arrow). (c) Dose-dependence of the effect of 1,
dantrolene, and DOCA on [Ca2+]ER in R2163C RyR1 cells. Data are mean ± SD (n = 7). Dotted
line denotes 1 F/F0, which means no inhibitory activity.

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We initially assessed the effects of alkylation at the 1-nitrogen atom of DOCA. Table 1 summarizes
the EC50 values of RyR1 channel-inhibitory activity of 3a – 3m, evaluated by [Ca2+]ER assay. The
N-alkylated DOCAs 3a – 3m showed inhibitory activity, and its potency depended on the length of
the alkyl chain. Specifically, the inhibitory potency increased as the N-alkyl chain of 3 became
longer. Especially, 3h bearing an n-octyl group showed the highest inhibitory activities (EC50: 0.27
µM), being more potent than the lead compound 1 (EC50: 0.81 µM). Further elongation of alkyl
chain (n-nonyl and n-decyl group for 3i and 3j, respectively) resulted in the decrease of inhibitory
activity. In addition, 3k, 3l, and 3m, bearing a cyclopropylmethyl, cyclohexylmethyl, and benzyl
group, respectively, exhibited weak inhibitory activities. The bulkiness of the longer alkyl chains or
these cyclic substituents might interfere with the interaction of the compound with the target site.
Table 1 RyR1 channel-inhibitory activities of 3.
Compound
R
EC50 (µM)
inactive
DOCA
3a
3b
3c
3d
3e
3f
H
Me
>10
Et
>10
n-Pr
3.2 (±0.3)
3.3 (±0.5)
6.7 (±1.0)
5.3 (±0.9)
0.56 (±0.10)
0.27 (±0.05)
0.69 (±0.05)
2.0 (±0.2)
>10
n-Bu
n-Pentyl
n-Hexyl
n-Heptyl
n-Octyl
n-Nonyl
n-Decyl
Cyclopropylmethyl
Cyclohexyllmethyl
Benzyl
3g
3h
3i
3j
3k
3l
5.7 (±1.1)
>10
3m
*EC50 values were obtained by fitting the Michaelis-Menten equation, y = 1 + Amax ([x]/[x] + EC50),
to the dose-response curve.

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Next, we examined the effect of substitution of the quinolone ring at the 6-, 7-, or 8-position (4 –
15). Table 2 summarizes the inhibitory activity of N-n-propyl and N-n-octyl quinolones 4 – 8.
N-n-octyl quinolones (h series) exhibited more potent RyR1 channel-inhibitory activities than the
corresponding N-n-propyl quinolones (c series) for 7-substituted quinolones (5 and 7), while h and c
series exhibited comparable activities for 6-substituted quinolones (4 and 6). Among the N-n-octyl
quinolones, 5h and 7h having a 7-methyl and a 7-methoxy group, respectively, exhibited more
potent inhibitory activity than non-substituted quinolone 3h. However, 4h, 6h, and 8h having a
6-methyl, 6-methoxy, and 8-methoxy group, respectively, exhibited lower inhibitory activity than 3h.
These results indicate that introduction of a 7-substituent on the quinolone ring is favorable for the
inhibitory activity.
Table 2. RyR1 channel-inhibitory activities of 3 – 8.
Compound
X
R
EC50 (µM)
3.2 (±0.3)
3c
4c
5c
6c
7c
3h
4h
5h
6h
7h
8h
H
n-Pr
6-Me
7-Me
6-OMe
7-OMe
H
n-Pr
1.1 (±0.1)
n-Pr
1.1 (±0.1)
n-Pr
4.4 (±0.7)
n-Pr
0.88 (±0.11)
0.27 (±0.05)
1.2 (±0.1)
n-Octyl
n-Octyl
n-Octyl
n-Octyl
n-Octyl
n-Octyl
6-Me
7-Me
6-OMe
7-OMe
8-OMe
0.16 (±0.02)
5.5 (±0.8)
0.076 (±0.011)
7.8 (±1.41)
*EC50 values were derived from dose-response curve fitted by the Michaelis-Menten equation: y = 1
+ Amax ([x]/[x] + EC50).

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Next, we examined the RyR1 channel-inhibitory activities of N-n-octyl quinolones having various
substituents at the 7-position (Table 3). Substitution of the methoxy group of 7h to a methylthio
group (9h) decreased the inhibitory activity. In addition, substitution of the methoxy group to a
trifluoromethyl (10h) or a trifluoromethoxy group (11h) greatly decreased the inhibitory activity.
Furthermore, 12h having 6,7-dimethoxy substitution was inactive, while 13h having 7,8-dimethoxy
substitution exhibited moderate inhibitory activity. On the other hand, compound 14h having the
6,7-methylenedioxo structure, corresponding to the cyclic analog of the inactive 6,7-dimethoxy
compound 12h, exhibited more potent inhibitory activity than 7h. The results suggested that the
orientation of methoxy group at the 7-position was important for the activity. Thus, the orientation
that the 7-methyoxy group directs to the 6-position would be favorable. In the compound 14h, the
7-substituent was fixed in the preferable orientation, while the repulsion between two methoxy
groups in compound 12h caused unfavorable conformation. Compound 15h having 6,7-propano
group exhibited much lower activity than 14h, which indicated the significance of oxygen atoms.
Compound 14j having N-n-decyl group did not show RyR1 channel-inhibitory activity, which
indicated that the n-octyl group is the most suitable for the activity as in the case of N-alkylated
DOCAs (Table 1). Among the synthesized quinolones, 14h was the most potent RyR1 channel
inhibitor, whereas 12h, 14j, and 15h having the similar structural elements to 14h showed little or no
activity, suggesting that structural requirements for potent inhibitory activity are strict.
Figure 3a shows dose dependency of 1, 5h, 7h and 14h for RyR1 channel-inhibitory activity. The
potency was greatly increased by structure development. 14h (EC50: 0.012 µM) was nearly 70-fold
more potent than 1 (EC50: 0.81 µM) and even five-fold more potent than dantrolene (EC50: 0.059
µM) (Figure 2c). We previously reported that 1 does not inhibit RyR2, a cardiac isoform16.
Compounds 5h, 7h and 14h did not affect [Ca2+]ER of HEK293 cells expressing RyR2 (Figure 3b).
On the other hand, these compounds as well as 1 slightly increased [Ca2+]ER of RyR3 cells at 10 µM
(Figure 3c), while the activity was very weak.

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Table 3. RyR1 channel-inhibitory activities of 7-substituted quinolones 7h and 9h – 15h.
Compound
7h
X
R
EC50 (µM)
0.076 (±0.011)
0.41 (±0.091)
> 10
7-OMe
7-SMe
7-CF3
n-Octyl
n-Octyl
n-Octyl
n-Octyl
n-Octyl
n-Octyl
9h
10h
11h
7-OCF3
6,7-(OMe)2
7,8-(OMe)2
7.1 (±1.0)
inactive
12h
13h
1.3 (±0.27)
6
O
14h
14j
n-Octyl
n-Decyl
n-Octyl
0.012 (±0.001)
inactive
O
7
6
O
O
7
15h
9.8 (±0.7)
1
H
Et
0.81 (±0.09)
dantrolene
-
-
0.059 (±0.011)
*EC50 values were derived from dose-response curve fitted by the Michaelis-Menten equation: y = 1
+ Amax ([x]/[x] + EC50).

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(a)
(b)
(c)
Figure 3 (a) Dose-dependent effects of 1, 5h, 7h, and 14h on [Ca2+]ER in R2163C RyR1 cells. (b)
and (c) Effects of 1, 5h, 7h, and 14h at 10 µM on [Ca2+]ER in RyR2 (b) and RyR3 (c) cells.
Data are mean ± SD (n = 6). Dotted line denotes 1 F/F0, which means no inhibitory activity. *P<0.05
compared with DMSO (one-way analysis of variance with Dunnett's test).

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2.3. Ryanodine binding-inhibitory activity
To examine whether the synthesized quinolone derivatives inhibit RyR1 directly, their effects on
the binding of [3H]ryanodine to RyR1 were examined. Ryanodine, a poisonous alkaloid, binds to
RyR1 in the open state.13b, c Namely, the binding of [3H]ryanodine to RyR1 could be a good indicator
of the channel activity. Microsomes isolated from R2163C RyR1 HEK293 cells were used for the
binding assay. The binding of [3H]ryanodine to RyR1 exhibited biphasic Ca2+ dependency with a
peak at about 1 µM Ca2+ (Figure 4a). We tested all the synthesized compounds at 10 µM
concentration and found that only three compounds (5h, 7h and 14h) show inhibitory activity. Upon
addition of 5h, 7h and 14h, the binding of [3H]ryanodine decreased with a slight shift of the Ca2+
dependency to a higher concentration range of Ca2+. The inhibitory effect of 5h, 7h and 14h on
[3H]ryanodine binding was dose-dependent and the IC50 values in the presence of 1 µM Ca2+ were
1.3 µM, 1.6 µM and 0.73 µM, respectively (Figure 4b). These values were more than one-order
higher than EC50 values obtained by [Ca2+]ER assay (see Tables 2 and 3). This apparent discrepancy
in the inhibitory activity between [Ca2+]ER assay and [3H]ryanodine-binding assay may be explained
by the presence of soluble factors that would promote the inhibitory action of quinolone compounds.
Since [3H]ryanodine-binding assay was carried out with isolated microsomes, soluble factors would
have been largely removed during the preparation of microsomes. Another possibility would be that
a conformational change in the quinolone-binding site would occur during the microsome-isolation
process, which decreased the binding affinity. These results suggest that quinolone compounds
directly inhibit the RyR1 channel activity.
We also tested the effects of the synthetic compounds (5h, 7h and 14h) as well as 1 on
[3H]ryanodine binding to RyR2 and RyR3. Consistent with the [Ca2+]ER assay, no effects were
observed on RyR2 (Fig. 4c). The compounds did not inhibit RyR3 (Figure 4d). This appears to be
inconsistent with the results of [Ca2+]ER assay. The inhibitory effect on RyR3 might be too weak to
be detected by [3H]ryanodine binding assay.

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(a)
0.15
0.10
0.05
0.00
DMSO
14h
8
7
6
5
4
3
2
pCa
(b)
0.15
0.15
0.15
0.10
0.05
0.00
7h
5h
14h
0.10
0.05
0.00
0.10
0.05
IC50 = 1.3 ± 0.4 µM
IC50 = 1.6 ± 1.2 µM
IC50 = 0.73 ± 0.19 µM
0.00
0
0
0
0.01
0.01
0.1
1
10
100
0.01
0.1
1
10
100
0.1
1
10
100
Compounds ( M)
Compounds ( M)
Compounds ( M)
(c)
(d)
Figure 4. Effects of 5h, 7h, and 14h on the [3H]ryanodine binding. (a) Ca2+-dependent [3H]ryanodine
binding to R2163C RyR1 in the presence (closed circles) and absence (open circles) of the
compound (10 µM). (b) Dose-dependent [3H]ryanodine-binding inhibition by the compound in the
presence of 1 µM Ca2+. (c) and (d) Effects of 1, 5h, 7h, and 14h at 10 µM on [3H]ryanodine binding
to RyR2 (b) and RyR3 (c). Data are mean ± SD (n = 4)

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3. Conclusion
We designed and synthesized a series of quinolone derivatives as RyR1 channel inhibitor
candidates, based on our previously identified inhibitor, oxolinic acid (1), as the lead compound.
HEK293 cell-based [Ca2+]ER assay revealed that the N-alkyl group of the quinolone structure is
important for the RyR1 channel-inhibitory activity, and introduction of a long alkyl chain, such as an
n-octyl group, is also favorable. In addition, suitable modification of the 7-position of the quinolone
ring increased the inhibitory activity. Notably, 14h exhibited extremely potent activity, much higher
than that of dantrolene. The synthesized quinolone derivatives exhibited high selectivity for RyR1
over RyR2 and RyR3. [3H]Ryanodine-binding assay suggested that the potent RyR1
channel-inhibitory activities of the quinolone derivatives involved direct interaction with RyR1.
Detailed investigation of the mechanism behind the RyR1-inhibitory activities is in progress.
The lead compound 1 is a first-generation quinolone antibiotic, and its toxicity to humans is quite
low.17 Therefore, 1 and its derivatives may be promising candidates for the treatment of
RyR1-related diseases, such as MH and CCD. Although there are several reports for development of
RyR inhibitors based on known drugs or natural products,12 this is the first report on the structural
development of RyR channel inhibitors based on a lead compound obtained from an HTS study. Our
powerful HTS platform should be applicable for further structural development of RyR modulators,
as well as discovery of modulators of RyR2 and RyR3.
4. Experimental
4.1. General
All reagents were purchased from Sigma-Aldrich, Tokyo Kasei, Wako Chemicals, and Kanto
Chemicals and were used without further purification. NMR spectra were recorded on Bruker
AVANCE 400 or Bruker AVANCE 500 spectrometers. Chemical shifts for NMR are reported as parts
1
of per million (ppm) relative to chloroform (7.26 ppm for H NMR and 77.23 ppm for 13C NMR),
DMSO-d6 (2.50 ppm for 1H NMR and 39.51 ppm for 13C NMR), and methanol-d4 (3.31 ppm for 1H
NMR and 49.00 ppm for 13C NMR). Mass spectra were collected on a Bruker Daltonics
microTOF-2focus spectrometer in the positive ion modes. Melting points were obtained on a
Yanagimoto micro melting point apparatus without correction.
4.2. Synthesis
4.2.1. General procedure for synthesis of 2a–k
A mixture of 1,4-Dihydoro-4-oxoquinoline-3-carboxylic acid (200 mg, 1.05 mmol) and Cs2CO3
(1.03 g, 3.17 mmol) in DMF (3 mL) was stirred at room temperature for 10 min. Then, the
corresponding alkyl halide (3.17 mmol) was added and the stirring was continued at 55 °C for 9 h.
After the solvent was removed in vacuo, the residue was extracted with ethyl acetate and water. The

ACCEPTED MANUSCRIPT
organic layer was washed with brine, and dried over sodium sulfate. The solvent was removed under
reduced pressure to give 2.
4.2.1.1. Methyl 1-methyl-4-quinolone-3-carboxylate (2a)
White solid (80% yield): 1H NMR (500 MHz, CDCl3) δ 8.56 (dd, J = 8.5, 1.5 Hz, 1 H), 8.53 (s, 1 H),
7.74 (ddd, J = 8.5, 7.0, 1.5 Hz, 1 H), 7.49 (ddd, J = 8.4, 7.0, 1.1, 1 H), 7.45 (br d, J = 8.0 1 H), 3.95
(s, 1 H), 3.91 (s, 1 H).
4.2.1.2. Ethyl 1-ethyl-4-quinolone-3-carboxylate (2b)
White solid (90% yield): 1H NMR (400 MHz, CDCl3) δ 8.57 (dd, J = 8.2, 1.3 Hz, 1 H), 8.52 (s, 1 H),
7.70 (ddd, J = 8.5, 7.2, 1.8 Hz, 1 H), 7.48–7.43 (m, 2 H), 4.42 (q, J = 7.2 Hz, 2 H), 4.27 (q, J = 7.5
Hz, 2 H), 1.56 (t, J = 7.2 Hz, 3 H), 1.43 (t, J = 7.2 Hz, 3 H).
4.2.1.3. Propyl 1-propyl-4-quinolone-3-carboxylate (2c)
White solid (76% yield): 1H NMR (500 MHz, CDCl3) δ 8.60 (s, 1 H), 8.56 (dd, J = 8.0, 1.5 Hz, 1 H),
7.70 (ddd, J = 8.6, 7.0, 1.6 Hz, 1 H), 7.48–7.44 (m, 2 H), 4.31 (t, J = 6.9 Hz, 2 H), 4.21 (t, J = 7.3 Hz,
2 H), 1.95 (sext, J = 7.5 Hz, 2 H), 1.83 (sext, J = 7.4 Hz, 2 H), 1.05 (t, J = 7.5 Hz, 3 H), 1.04 (t, J =
7.4 Hz, 3 H).
4.2.1.4. Butyl 1-butyl-4-quinolone-3-carboxylate (2d)
White solid (86% yield): 1H NMR (500 MHz, CDCl3) δ 8.53 (dd, J = 7.9, 1.5 Hz, 1 H), 8.44 (br s, 1
H), 7.66 (ddd, J = 8.7, 7.1, 1.6 Hz, 1 H), 7.44–7.41 (m, 2 H), 4.33 (t, J = 6.8 Hz, 2 H), 4.17 (t, J = 6.6
Hz, 2 H), 1.86 (quin, J = 7.4 Hz, 2 H), 1.77 (quin, J = 7.1 Hz, 2 H), 1.50 (quin, J = 7.3 Hz, 2 H), 1.44
(quin, J = 6.9 Hz, 2 H), 1.00–0.95 (m, 6 H).
4.2.1.5. Pentyl 1-pentyl-4-quinolone-3-carboxylate (2e)
1
White solid (quant.): H NMR (500 MHz, CDCl3) δ 8.54 (dd, J = 8.0, 1.7 Hz, 1 H), 8.46 (s, 1 H),
7.67 (ddd, J = 8.6, 8.1, 1.3 Hz, 1 H), 7.44–7.40 (m, 2 H), 4.32 (t, J = 6.9 Hz, 2 H), 4.17 (t, J = 7.4 Hz,
2 H), 1.89 (quin, J = 6.8 Hz, 2 H), 1.80 (quin, J = 7.1 Hz, 2 H), 1.45–1.38 (m, 8 H), 0.95–0.89 (m, 6
H).
4.2.1.6. Hexyl 1-hexyl-4-quinolone-3-carboxylate (2f)
White solid (86% yield): 1H NMR (500 MHz, CDCl3) δ 8.53 (dd, J = 7.9, 1.7 Hz, 1 H), 8.44 (s, 1 H),
7.66 (ddd, J = 8.6, 7.2, 1.6 Hz, 1 H), 7.44–7.39 (m, 2 H), 4.31 (t, J = 7.0 Hz, 2 H), 4.16 (t, J = 7.5 Hz,
2 H), 1.87 (quin, J = 7.4 Hz, 2 H), 1.78 (quin, J = 7.3 Hz, 2 H), 1.45–1.28 (m, 12 H), 0.89–0.87 (m,
6 H).

ACCEPTED MANUSCRIPT
4.2.1.7. Heptyl 1-heptyl-4-quinolone-3-carboxylate (2g)
White solid (98% yield): 1H NMR (500 MHz, CDCl3) δ 8.55 (dd, J = 7.2, 1.7 Hz, 1 H) 8.46 (s, 1 H),
7.68 (ddd, J = 7.9, 7.9, 1.5 Hz, 1 H), 7.46–7.42 (m, 2 H), 4.32 (t, J = 6.9 Hz, 2 H), 4.17 (t, J = 7.5 Hz,
2 H), 1.89 (quin, J = 7.4 Hz, 2 H), 1.80 (quin, J = 7.1 Hz, 2 H), 1.45–1.24 (m, 16 H), 0.92–0.85 (m,
6 H).
4.2.1.8. Octyl 1-octyl-4-quinolone-3-carboxylate (2h)
White solid (98% yield): 1H NMR (500 MHz, CDCl3) δ 8.53 (dd, J = 8.0, 1.3 Hz, 1 H), 8.43 (s, 1 H),
7.66 (ddd, J = 8.5, 7.2 1.5 Hz, 1 H), 7.45–7.38 (m, 2 H), 4.30 (t, J = 7.3 Hz, 2 H), 4.15 (t, J = 7.1 Hz,
2 H), 1.85 (quin, J = 7.2 Hz, 2 H), 1.78 (quin, J = 7.2 Hz, 2 H), 1.43–1.26 (m, 20 H), 0.88–0.85 (m,
6 H).
4.2.1.9. Nonyl 1-nonyl-4-quinolone-3-carboxylate (2i)
White solid (98% yield): 1H NMR (500 MHz, CDCl3) δ 8.57 (dd, J = 8.3, 1.7 Hz, 1 H), 8.48 (s, 1 H),
7.69 (ddd, J = 8.6, 7.2 1.6 Hz, 1 H), 7.47–7.31 (m, 2 H), 4.34 (t, J = 7.0 Hz, 2 H), 4.18 (t, J = 7.5 Hz,
2 H), 1.92 (quin, J = 6.6 Hz, 2 H), 1.81 (quin, J = 6.9 Hz, 2 H), 1.48–1.25 (m, 24 H), 0.90–0.87 (m,
6 H).
4.2.1.10. Decyl 1-decyl-4-quinolone-3-carboxylate (2i)
White solid (98% yield): 1H NMR (400 MHz, CDCl3) δ 8.53 (d, J = 8.1 Hz, 1 H), 8.48 (s, 1 H), 7.66
(ddd, J = 8.5, 7.4 1.0 Hz, 1 H), 7.47–7.42 (m, 2 H), 4.34 (t, J = 7.0 Hz, 2 H), 4.18 (t, J = 7.4 Hz, 2 H),
1.90 (quin, J = 6.6 Hz, 2 H), 1.81 (quin, J = 7.0 Hz, 2 H), 1.49–1.23 (m, 28 H), 0.90–0.87 (m, 6 H).
4.2.1.11. Cyclopropylmethyl 1-(cyclopropylmethyl)-4-quinolone-3-carboxylate (2k)
1
White solid (98% yield): H NMR (500 MHz, CDCl3) δ 8.57 (s, 1 H), 8.56 (br d, J = 7.9 Hz, 1 H),
7.69 (ddd, J = 8.6, 7.1, 1.6 Hz, 1 H), 7.55 (br d, J = 8.5 Hz, 1 H), 7.47–7.42 (m, 2 H), 4.17 (d, J = 8.1
Hz, 2 H), 4.04 (d, J = 7.9 Hz, 2 H), 1.41–1.24 (m, 2 H), 0.76 (ddd, J = 7.3, 5.6, 4.3 Hz, 2 H), 0.61
(ddd, J = 7.3, 5.8, 4.1 Hz, 2 H), 0.49–0.46 (m, 2 H), 0.41–0.37 (m, 2 H).
4.2.1.12. Cyclohexylmethyl 1-(cyclohexylmethyl)-4-quinolone-3-carboxylate (2l)
White solid (98% yield): 1H NMR (500 MHz, CDCl3) δ 8.57 (dd, J = 8.1, 1.6 Hz, 1 H), 8.41 (s, 1 H),
7.68 (ddd, J = 8.6, 7.9, 1.6 Hz, 1 H), 7.44 (ddd, J = 8.4, 8.0, 0.5, 1 H), 7.41 (br d, J = 8.5, 1 H), 4.15
(d, J = 6.7 Hz, 1 H), 3.99 (d, J = 7.3 Hz, 1 H), 1.91–1.69 (m, 11 H), 1.34–1.03 (m, 11 H).
4.2.1.13. Benzyl 1-benzyl-4-quinolone-3-carboxylate (2m)

ACCEPTED MANUSCRIPT
White solid (78% yield): 1H NMR (500 MHz, CDCl3) δ 8.61 (s, 1 H), 8.56 (dd, J = 8.1, 1.5 Hz, 1 H),
7.55 (ddd, J = 7.8, 7.1, 1.7 Hz, 1 H), 7.54–7.51 (m, 2 ), 7.41 (ddd, J = 7.6, 7.3, 1.8 Hz, 1 H),
7.39–7.29 (m, 7 H), 7.16 (br d, J = 8.2 Hz, 2 H), 5.41 (s, 2 H), 5.38 (s, 2 H).
4.2.2. General procedure for synthesis of 16–27
A solution of corresponding aniline (9.32 mmol) and diethyl ethoxymethylenemalonate (9.32
mmol) in ethanol (20 mL) was heated at 90 °C for 18 h. After the solvent was removed under
reduced pressure, the residue was extracted with ethyl acetate and water. The organic layer was
washed with brine, dried over sodium sulfate and concentrated under reduced pressure. The crude
product was purified by open column chromatography (eluent: n-hexane/ethyl acetate = 4 : 1) to give
corresponding compound 16–27.
4.2.2.1. Diethyl 2-[(4-toluidino)methylene]malonate (16)
Pale yellow oil (90% yield): 1H NMR (400 MHz, CDCl3) δ 10.98 (d, J = 13.4 Hz, 1 H), 8.50 (d, J =
13.8 Hz, 1 H), 7.15 (d, J = 8.3 Hz, 2 H), 7.01 (d, J = 8.2 Hz, 2 H), 4.30 (q, J = 7.1 Hz, 2 H), 4.23 (q,
J = 7.1 Hz, 2 H), 2.31 (s, 3 H), 1.37 (t, J = 7.1 Hz, 3 H), 1.32 (t, J = 7.2 Hz, 3 H).
4.2.2.2. Diethyl 2-[(3-toluidino)methylene]malonate (17)
Pale yellow oil (88% yield): 1H NMR (400 MHz, CDCl3) δ 10.96 (d, J = 13.6 Hz, 1 H), 8.51 (d, J =
13.7 Hz, 1 H), 7.23 (t, J = 7.4 Hz, 1 H), 6.97–6.90 (m, 3 H), 4.30 (q, J = 7.2 Hz, 2 H), 4.24 (q, J =
7.2 Hz, 2H), 2.35 (s, 3 H), 1.34 (t, J = 7.2 Hz, 3 H), 1.32 (t, J = 7.2 Hz, 3 H).
4.2.2.3. Diethyl 2-[(4-methoxyphenylamino)methylene]malonate (18)
Pale yellow oil (90% yield): 1H NMR (500 MHz, CDCl3) δ 10.98 (d, J = 13.7 Hz, 1 H), 8.43 (d, J =
13.8 Hz, 1 H), 7.07 (d, J = 8.9 Hz, 2 H), 6.90 (d, J = 8.9 Hz, 2 H), 4.29 (q, J = 7.1 Hz, 2 H), 4.23 (q,
J = 7.1 Hz, 2 H), 3.80 (s, 3 H), 1.37 (t, J = 7.1 Hz, 3 H), 1.31 (t, J = 7.1 Hz, 3 H).
4.2.2.4. Diethyl 2-[(3-methoxyphenylamino)methylene]malonate (19)
Pale yellow oil (91% yield): 1H NMR (400 MHz, CDCl3) δ 10.96 (d, J = 13.5 Hz, 1 H), 8.50 (d, J =
13.5 Hz, 1 H), 7.26 (t, J = 8.1 Hz, 1 H), 6.73–6.64 (m, 3 H), 4.30 (q, J = 7.1 Hz, 2 H), 4.24 (q, J =
7.1 Hz, 2 H), 3.80 (s, 3 H), 1.37 (t, J = 6.4 Hz, 3 H), 1.34 (t, J = 6.2 Hz, 3 H).
4.2.2.5. Diethyl 2-[(2-methoxyphenylamino)methylene]malonate (20)
Pale yellow oil (quant.): 1H NMR (400 MHz, CDCl3) δ 11.10 (d, J = 13.6 Hz, 1 H), 8.55 (d, J = 14.0
Hz, 1 H), 7.23 (br d, J = 8.0 Hz, 1 H), 7.08 (t, J = 7.6 Hz, 1 H), 6.97 (t, J = 7.6 Hz, 1 H), 6.92 (br d, J
= 8.4 Hz, 1 H), 4.32 (q, J = 7.2 Hz, 2 H), 4.24 (q, J = 7.2 Hz, 2 H), 3.92 (s, 3 H), 1.38 (t, J = 7.2 Hz,

ACCEPTED MANUSCRIPT
3 H), 1.32 (t, J = 6.8 Hz, 3 H).
4.2.2.6. Diethyl 2-[[(3-methylthio)phenylamino]methylene]malonate (21)
Pale yellow oil (quant.): 1H NMR (400 MHz, CDCl3) δ 10.97 (d, J = 13.6 Hz, 1 H), 8.49 (d, J = 13.6
Hz, 1 H), 7.27 (t, J = 8.0 Hz, 1 H), 7.01 (dd, J = 7.6, 0.8 Hz, 1 H), 6.97 (t, J = 2.0 Hz, 1 H), 6.90 (dd,
J = 8.0, 2.0 Hz, 1 H), 4.30 (q, J = 7.2 Hz, 2 H), 4.25 (q, J = 6.8 Hz, 2 H), 1.38 (t, J = 7.2 Hz, 3 H),
1.33 (t, J = 7.2 Hz, 3 H).
4.2.2.7. Diethyl 2-[[(3-trifluoromethyl)phenylamino]methylene]malonate (22)
Pale yellow oil (quant.): 1H NMR (500 MHz, CDCl3) δ 11.11 (d, J = 13.2 Hz, 1 H), 8.50 (d, J = 13.4
Hz, 1 H), 7.50 (t, J = 7.9 Hz, 1 H), 7.39 (br d, J = 7.8 Hz, 1 H), 7.36 (s, 1 H), 7.31 (br d, J = 8.0 Hz,
1 H), 4.31 (q, J = 7.1 Hz, 2 H), 4.26 (q, J = 7.1 Hz, 2 H), 1.38 (t, J = 7.1 Hz, 3 H), 1.34 (t, J = 7.2 Hz,
3 H).
4.2.2.8. Diethyl 2-[[(3-trifluoromethyloxy)phenylamino]methylene]malonate (23)
Pale yellow oil (quant.): 1H NMR (400 MHz, CDCl3) δ 11.03 (d, J = 13.4 Hz, 1 H), 8.46 (d, J = 13.4
Hz, 1 H), 7.39 (t, J = 8.1 Hz, 1 H), 7.06 (ddd, J = 8.3, 1.8, 0.8 Hz, 1 H), 7.00 (ddd, J = 8.3, 1.2, 0.8
Hz, 1 H), 6.98 (br s, 1 H), 4.31 (q, J = 7.1 Hz, 2 H), 4.26 (q, J = 7.1 Hz, 2 H), 1.38 (t, J = 7.2 Hz, 3
H), 1.33 (t, J = 7.1 Hz, 3 H).
4.2.2.9. Diethyl 2-[(3,4-dimethoxyphenylamino)methylene]malonate (24)
Pale yellow oil (quant.): 1H NMR (400 MHz, CDCl3) δ 10.99 (d, J = 13.6 Hz, 1 H), 8.43 (d, J = 13.6
Hz, 1 H), 6.84 (d, J = 8.8 Hz, 1 H), 6.70 (dd, J = 8.8, 2.8 Hz, 1 H), 6.64 (d, J = 2.4 Hz, 1 H), 4.29 (q,
J = 6.8 Hz, 2 H), 4.23 (q, J = 7.2 Hz, 2 H), 3.88 (s, 3 H), 3.86 (s, 3 H), 1.37 (t, J = 7.2 Hz, 3 H), 1.31
(t, J = 7.2 Hz, 3 H).
4.2.2.10. Diethyl 2-[(2,3-dimethoxyphenylamino)methylene]malonate (25)
Pale yellow oil (quant.): 1H NMR (400 MHz, CDCl3) δ 11.14 (d, J = 14.0 Hz, 1 H), 8.53 (d, J = 14.0
Hz, 1 H), 7.06 (t, J = 8.0 Hz, 1 H), 6.87 (dd, J = 8.4, 0.8 Hz, 1 H), 6.68 (dd, J = 8.4, 1.2 Hz, 1 H),
4.32 (q, J = 7.2 Hz, 2 H), 4.25 (q, J = 7.2 Hz, 2 H), 3.92 (s, 3 H), 3.88 (s, 3 H), 1.38 (t, J = 7.2 Hz, 3
H), 1.33 (t, J = 7.2 Hz, 3 H).
4.2.2.11. Diethyl [[3,4-(methylenedioxy)phenylamino]methylene]malonate (26)
White solid (quant.): 1H NMR (400 MHz, CDCl3) δ 10.96 (d, J = 13.6 Hz, 1 H), 8.38 (d, J = 13.6 Hz,
1 H), 6.78 (d, J = 8.3 Hz, 1 H), 6.68 (d, J = 2.3 Hz, 1 H), 6.58 (dd, J = 8.32, 2.3 Hz, 1 H), 5.99 (s, 2
H), 4.29 (q, J = 7.1 Hz, 2 H), 4.23 (q, J = 7.1 Hz, 2 H), 1.38 (t, J = 7.1 Hz, 3 H), 1.32 (t, J = 7.1 Hz,

ACCEPTED MANUSCRIPT
3 H).
4.2.2.12. Diethyl [(5-indanylamino)methylene]malonate (27)
1
White solid (92% yield): H NMR (400 MHz, CDCl3) δ 10.98 (d, J = 13.3 Hz, 1 H), 8.50 (d, J =
13.8 Hz, 1 H), 7.19 (d, J = 7.9 Hz, 1 H), 7.02 (s, 1 H), 6.90 (d, J = 7.8 Hz, 1 H), 4.30 (q, J = 7.1 Hz,
2 H), 4.21 (q, J = 7.2 Hz, 2 H), 2.91 (t, J = 7.2 Hz, 2 H), 2.88 (t, J = 7.2 Hz, 2 H), 2.10 (quin, J = 7.2
Hz, 2 H), 1.37 (t, J = 7.1 Hz, 3 H), 1.32 (t, J = 7.1 Hz, 3 H).
4.2.3. General procedure for synthesis of 28–39
A solution of compound 16–27 (7.36 mmol) in diphenylether (50 mL) was heated at 250 °C for 10
h. After cooling, precipitated solid was separated by filtration, washed with diethylether. The residue
was dried under reduced pressure to give corresponding quinolone 28–39.
4.2.3.1. Ethyl 6-methyl-4-quinolone-3-carboxylate (28)
Brown slid (50% yield): 1H NMR (400 MHz, DMSO-d6) δ 12.24 (br s, 1 H), 8.50 (s, 1 H), 7.94 (s, 1
H), 7.52 (s, 2 H), 4.21 (q, J = 7.1 Hz, 2 H), 2.42 (s, 3 H), 1.28 (t, J = 7.1 Hz, 3 H).
4.2.3.2. Ethyl 7-methyl-4-quinolone-3-carboxylate (29)
Brown solid (50% yield): 1H NMR (400 MHz, DMSO-d6) δ 12.19 (br s, 1 H), 8.49 (s, 1 H), 8.04 (d,
J = 8.2 Hz, 1 H), 7.37 (br s, 1 H), 7.23 (dd, J = 8.2, 1.0 Hz, 1 H), 4.20 (q, J = 7.1 Hz, 2 H), 2.43 (s, 3
H), 1.27 (t, J = 7.1 Hz 3 H).
4.2.3.3. Ethyl 6-methoxy-4-quinolone-3-carboxylate (30)
Brown solid (51% yield): 1H NMR (400 MHz, DMSO-d6) δ 12.29 (br s, 1 H), 8.49 (s, 1 H), 7.58 (d,
J = 8.6 Hz, 1 H), 7.57 (d, J = 2.6 Hz, 1 H), 7.34 (dd, J = 9.0, 3.0 Hz, 1 H), 4.21(q, J = 7.1 Hz, 2 H),
3.85 (s, 3 H), 1.28 (t, J = 7.1 Hz, 3 H).
4.2.3.4. Ethyl 7-methoxy-4-quinolone-3-carboxylate (31)
Brown solid (40% yield): 1H NMR (500 MHz, DMSO-d6) δ 12.10 (br s, 1 H), 8.48 (s, 1 H), 8.05 (dd,
J = 7.8, 1.8 Hz, 1 H), 7.01 (s, 1 H), 7.00 (dd, J = 8.6, 1.5 Hz, 1 H), 4.20 (q, J = 7.1 Hz, 2 H), 3.86 (s,
3 H), 1.27 (t, J = 7.1 Hz, 3 H).
4.2.3.5. Ethyl 8-methoxy-4-quinolone-3-carboxylate (32)
Brown solid (55% yield): 1H NMR (400 MHz, DMSO-d6) δ 11.91 (s, 1 H), 8.33 (s, 1 H), 7.69 (dd, J
= 7.2, 1.6 Hz, 1 H), 7.34 (t, J = 8.0 Hz, 1 H), 7.31 (dd, J = 8.0, 2.0 Hz, 1 H), 4.19 (q, J = 7.2 Hz, 1
H), 3.98 (s, 3 H), 1.26 (t, J = 7.2 Hz, 3 H).

ACCEPTED MANUSCRIPT
4.2.3.6. Ethyl 7-(methylthio)-4-quinolone-3-carboxylate (33)
Brown solid (52% yield): 1H NMR (400 MHz, DMSO-d6) δ 12.15 (br s, 1 H), 8.51 (s, 1 H), 8.02 (d,
J = 8.4 Hz, 1 H), 7.35 (d, J = 1.6 Hz, 1 H), 7.26 (dd, J = 8.8, 2.0 Hz, 1 H), 4.20 (q, J = 6.8 Hz, 2 H),
2.56 (s, 3 H), 1.27 (t, J = 6.8 Hz, 3 H).
4.2.3.7. Ethyl 7-(trifluoromethyl)-4-quinolone-3-carboxylate (34)
Brown solid (21% yield): 1H NMR (400 MHz, DMSO-d6) δ 12.45 (br s, 1 H), 8.69 (s, 1 H), 8.33 (d,
J = 8.3 Hz, 1 H), 7.95 (br s, 1 H), 7.64 (d, J = 8.2 Hz, 1 H), 4.21 (q, J = 7.0 Hz, 2 H), 1.28 (t, J = 7.0
Hz, 3 H).
4.2.3.8. Ethyl 7-(trifluoromethyloxy)-4-quinolone-3-carboxylate (35)
Brown solid (45% yield): 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1 H), 8.24 (d, J = 8.9 Hz, 1 H),
7.55 (s, 1 H), 7.33 (d, J = 8.7 Hz, 1 H), 4.21 (q, J = 6.8 Hz, 2 H), 1.28 (t, J = 7.1 Hz, 3 H).
4.2.3.9. Ethyl 6,7-dimethoxy-4-quinolone-3-carboxylate (36)
1
Brown solid (28% yield): H NMR (400 MHz, DMSO-d6) δ 12.10 (s, 1 H), 8.44 (s, 1 H), 7.51 (s, 1
H), 7.05 (s, 1 H), 4.42 (q, J = 7.2 Hz, 2 H), 3.88 (s, 3 H), 3.84 (s, 3H), 1.27 (t, J = 7.2 Hz, 3 H).
4.2.3.10. Ethyl 7,8-dimethoxy-4-quinolone-3-carboxylate (37)
Brown solid (68% yield): 1H NMR (400 MHz, DMSO-d6) δ 11.82 (s, 1 H), 8.33 (s, 1 H), 7.87 (d, J =
8.8 Hz, 1 H), 7.23 (d, J = 9.2 Hz, 1 H), 4.19 (q, J = 7.2 Hz, 2 H), 3.94 (s, 3 H), 3.88 (s, 3 H), 1.26 (t,
J = 7.2 Hz, 3 H).
4.2.3.11. Ethyl 6,7-(methylenedioxy)-4-quinolone-3-carboxylate (38)
Brown solid (62% yield): 1H NMR (400 MHz, DMSO-d6) δ 8.45 (s, 1 H), 7.46 (s, 1 H), 7.02 (s, 1 H),
6.12 (s, 2 H), 4.17 (q, J = 7.0 Hz, 2 H), 1.26 (t, J = 7.1 Hz, 3 H).
4.2.3.12. Ethyl 6,7-(trimethylene)-4-quinolone-3-carboxylate (39)
Brown solid (25% yield): 1H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1 H), 7.97 (s, 1 H), 7.42 (s, 1 H),
4.20 (q, J = 7.1 Hz, 2 H), 2.97 (t, J = 7.6 Hz, 2 H), 2.95 (t, J = 7.2 Hz, 2 H), 2.06 (quin, J = 7.4 Hz, 2
H), 1.27 (t, J = 7.1 Hz, 3 H).
4.2.4. General procedure for synthesis of 40–51
A solution of 28–39 (0.17 mmol) in dry DMF (2 mL) was added to a suspension of sodium hydride
(60%, 0.34 mmol) in dry DMF (3 mL) at 0 °C. After stirring for 10 min at room temperature, a

ACCEPTED MANUSCRIPT
solution of corresponding alkyl halide (0.34 mmol) was added and the mixture was heated at 55 °C
for 9 h. The solvent was removed in vacuo and the residue was extracted with ethyl acetate and water.
The organic layer was washed with brine and dried over sodium sulfate. After evaporation, residue
was purified by silica gel column chromatography (ethyl acetate/hexane 3:1) to give corresponding
compound 40–51. The obtained products were the mixtures of the ethyl esters and transesterification
products with the alkylating reagents. They were used in the next hydrolysis step without further
separation.
4.2.4.1. Propyl 6-methyl-1-propyl-4-quinolone-3-carboxylate (40c)
Orange oil (72% yield): 1H NMR (400 MHz, CDCl3) δ 8.45 (s, 1 H), 8.36 (d, J = 1.0 Hz, 1 H), 7.50
(dd, J = 8.7, 2.1 Hz, 1 H), 7.35 (d, J = 8.6 Hz, 1 H), 4.30 (t, J = 6.8 Hz, 2 H), 4.14 (t, J = 7.3 Hz, 2
H), 2.48 (s, 3 H), 1.94 (sext, J = 6.6 Hz, 2 H), 1.83 (sext, J = 7.2 Hz, 2 H), 1.06 (t, J = 7.5 Hz, 3 H),
1.03 (t, J = 7.4 Hz, 3 H),.
4.2.4.2. Octyl 6-methyl-1-octyl-4-quinolone-3-carboxylate (40h)
Orange oil (54% yield): 1H NMR (400 MHz, CDCl3) δ 8.46 (s, 1 H), 8.36 (d, J = 1.1 Hz, 1 H), 7.50
(dd, J = 8.6, 2.0 Hz, 1 H), 7.35 (d, J = 8.7 Hz, 1 H), 4.33 (t, J = 6.9 Hz, 2 H), 4.16 (t, J = 7.5 Hz, 2
H), 2.49 (s, 3 H), 1.89 (quin, J = 7.4 Hz, 2 H), 1.81 (quin, J = 7.3 Hz, 2 H), 1.48–1.27 (m, 20 H),
0.89 (t, J = 6.9 Hz, 6 H).
4.2.4.3. Propyl 7-methyl-1-propyl-4-quinolone-3-carboxylate (41c)
1
Orange oil (90% yield): H NMR (500 MHz, CDCl3) δ 8.42 (br s, 1 H), 8.41 (d, J = 9.0 Hz, 1 H),
7.23 (d, J = 8.4 Hz, 1 H), 7.18 (s, 1 H), 4.27 (t, J = 6.8 Hz, 2 H), 4.12 (t, J = 7.4 Hz, 2 H), 2.50 (s, 3
H), 1.92 (sext, J = 6.1 Hz, 2 H), 1.80 (sext, J = 7.1 Hz, 2 H),, 1.04–0.99 (m, 6 H).
4.2.4.4. Octyl 7-methyl-1-octyl-4-quinolone-3-carboxylate (41h)
Orange oil (64% yield): 1H NMR (500 MHz, CDCl3) δ 8.43 (br s, 1 H), 8.44 (d, J = 8.4 Hz, 1 H),
7.25 (d, J = 8.4 Hz, 1 H), 7.19 (s, 1 H), 4.32 (t, J = 7.0 Hz, 2 H), 4.13 (t, J = 7.2 Hz, 2 H), 2.52 (s, 3
H), 1.96–1.86 (m, 2 H), 1.80 (quin, J = 7.3 Hz, 2 H), 1.48–1.25 (m, 20 H), 0.88 (t, J = 5.8 Hz 6 H).
4.2.4.5. Propyl 6-methoxy-1-propyl-4-quinolone-3-carboxylate (42c)
Orange oil (81% yield): 1H NMR (500 MHz, CDCl3) δ 8.45 (s, 1 H), 7.98 (d, J = 3.1 Hz, 1 H), 7.40
(d, J = 9.2 Hz, 1 H), 7.29 (dd, J = 9.2, 3.1 Hz, 1 H), 4.30 (t, J = 6.8 Hz, 2 H), 4.16 (t, J = 7.4 Hz, 2
H), 3.92 (s, 3 H), 1.93 (sext, J = 7.4 Hz, 2 H), 1.83 (sext, J = 7.3 Hz, 2 H), 1.06 (t, J = 7.5 Hz, 3 H),
1.02 (t, J = 7.4 Hz, 3 H).

ACCEPTED MANUSCRIPT
4.2.4.6. Octyl 6-methoxy-1-octyl-4-quinolone-3-carboxylate (42h)
Orange oil (75 % yield): 1H NMR (500 MHz, CDCl3) δ 8.42 (s, 1 H), 7.97 (d, J = 3.0 Hz, 1 H), 7.38
(d, J = 9.2 Hz, 1 H), 7.28 (dd, J = 9.2, 3.0 Hz, 1 H), 4.32 (t, J = 7.0 Hz, 2 H), 4.16 (t, J = 7.5 Hz, 2
H), 3.92 (s, 3 H), 1.87 (quin, J = 7.5 Hz, 2 H), 1.83 (quin, J = 7.3 Hz, 2 H), 1.48–1.19 (m, 20 H),
0.88 (t, J = 7.1 Hz, 3 H), 0.87 (t, J = 6.9 Hz, 3 H).
4.2.4.7. Ethyl 7-methoxy-1-propyl-4-quinolone-3-carboxylate (43c)
Orange oil (77% yield): 1H NMR (500 MHz, CDCl3) δ 8.70 (s, 1 H), 8.50 (d, J = 9.0 Hz, 1 H), 7.18
(dd, J = 9.0, 2.2 Hz, 1 H), 6.93 (d, J = 2.2 Hz, 1 H), 4.22 (t, J = 7.4 Hz, 2 H), 3.99 (s, 3 H), 3.73 (q, J
= 7.0 Hz, 2 H), 1.99 (sext, J = 7.4 Hz, 2 H), 1.25 (t, J = 7.1 Hz, 3 H), 1.06 (t, J = 7.4 Hz, 3 H).
4.2.4.8. Octyl 7-methoxy-1-octyl-4-quinolone-3-carboxylate (43h)
Orange oil (73% yield): 1H NMR (500 MHz, CDCl3) δ 8.44 (d, J = 9.0 Hz, 1 H), 8.36 (s, 1 H), 6.98
(dd, J = 9.1, 2.3 Hz, 1 H), 6.76 (d, J = 2.2 Hz, 1 H), 4.29 (t, J = 7.0 Hz, 2 H), 4.08 (t, J = 7.5 Hz, 2
H), 3.91 (s, 3 H), 1.86 (quin, J = 7.4 Hz, 2 H), 1.77 (quin, J = 7.3 Hz, 2 H), 1.45–1.23 (m, 20 H),
0.86 (t, J = 7.1 Hz, 6 H).
4.2.4.9. Ethyl 8-methoxy-1-octyl-4-quinolone-3-carboxylate (44h)
Orange oil (45% yield): 1H NMR (400 MHz, CDCl3) δ 8.36 (s, 1 H), 8.18 (dd, J = 8.4, 1.6 Hz, 1 H),
7.34 (t, J = 8.0 Hz, 1 H), 7.15 (dd, J = 8.0, 1.2 Hz, 1 H), 4.46 (t, J = 7.2 Hz, 2 H), 4.39 (q, J = 7.2 Hz,
2 H), 3.95 (s, 3 H), 1.78 (quin, J = 7.2 Hz, 2 H), 1.40 (t, J = 7.2 Hz, 3 H), 1.31–1.25 (m, 10 H), 0.87
(t, J = 6.8 Hz, 3H).
4.2.4.10. Ethyl 7-(methylthio)-1-octyl-4-quinolone-3-carboxylate (45h)
Orange oil (73% yield): 1H NMR (400 MHz, CDCl3) δ 8.38 (s, 1 H), 8.36 (d, J = 8.0 Hz, 1 H), 7.21
(dd, J = 8.8, 1.6 Hz, 1 H), 7.11 (d, J = 1.6 Hz, 1 H), 4.35 (q, J = 7.2 Hz, 2 H), 4.10 (t, J = 7.2 Hz, 2
H), 2.54 (s, 3 H), 1.84 (quin, J = 7.2 Hz, 2 H), 1.38 (t, J = 7.2 Hz, 3H), 1.35–1.24 (m, 10 H), 0.85 (t,
J = 6.8 Hz, 3H).
4.2.4.11. Ethyl 1-octyl-7-(trifluoromethyl)-4-quinolone-3-carboxylate (46h)
Orange oil (13% yield): 1H NMR (400 MHz, CDCl3) δ 8.66 (d, J = 8.3 Hz, 1 H), 8.53 (s, 1 H), 7.68
(s, 1 H), 7.65 (d, J = 8.5 Hz, 1 H), 4.41 (q, J = 7.1 Hz, 2 H), 4.21 (t, J = 7.4 Hz, 2 H), 1.90 (quin, J =
7.4 Hz, 2 H), 1.42 (t, J = 7.1, 3 H), 1.25–1.39 (m, 10 H), 0.88 (t, J = 6.7 Hz, 3 H).
4.2.4.12. Ethyl 1-octyl-7-(trifluoromethyloxy)-4-quinolone-3-carboxylate (47h)
Orange oil (23% yield): 1H NMR (400 MHz, CDCl3) δ 8.58 (d, J = 8.8 Hz, 1 H), 8.48 (s, 1 H), 7.27

ACCEPTED MANUSCRIPT
(d, J = 8.9 Hz, 1 H), 7.22 (s, 1 H), 4.40 (q, J = 7.1 Hz, 2 H), 4.13 (t, J = 7.4 Hz, 2 H), 1.88 (quin, J =
7.6 Hz, 2 H), 1.42 (t, J = 7.1 Hz, 3 H), 1.40–1.27 (m, 10 H), 0.88 (t, J = 6.0 Hz, 3 H).
4.2.4.13. Ethyl 6,7-dimethoxy-1-octyl-4-quinolone-3-carboxylate (48h)
Yellow solid (12% yield): 1H NMR (400 MHz, methanol-d4) δ 8.91 (s, 1 H), 7.50 (s, 1 H), 7.34 (s, 1
H), 4.44 (q, J = 7.2 Hz, 2 H), 4.24 (t, J = 6.4 Hz, 2 H), 4.02 (s, 3 H), 3.99 (s, 3 H), 1.91 (quin, J = 6.4
Hz, 2 H), 1.57 (quin, J = 6.8 Hz, 2 H), 1.45 (t, J = 7.2 Hz, 3 H), 1.42–1.31 (m, 8 H), 0.90 (t, J = 7.2
Hz, 3H).
4.2.4.14. Octyl 7,8-dimethoxy-1-octyl-4-quinolone-3-carboxylate (49h)
Orange oil (15% yield): 1H NMR (400 MHz, CDCl3) δ 8.37 (s, 1 H), 8.34 (d, J = 8.8 Hz, 1 H), 7.10
(d, J = 9.2 Hz, 1 H), 4.38 (t, J = 7.2 Hz, 2 H), 4.32 (t, J = 6.8 Hz, 2 H), 4.00 (s, 3 H), 3.88 (s, 3 H),
1.79 (quin, J = 6.8 Hz, 2 H), 1.78 (quin, J = 6.8 Hz, 2 H), 1.48–1.24 (m, 20 H), 0.88 (t, J = 6.8 Hz, 3
H), 0.86 (t, J = 7.2 Hz, 3 H).
4.2.4.15. Octyl 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylate (50h)
Pale yellow solid (40% yield): 1H NMR (400 MHz, CDCl3) δ 8.34 (s, 1 H), 7.91 (s, 1 H), 6.82 (s, 1
H), 6.10 (s, 2 H), 4.31 (t, J = 7.0 Hz, 2 H), 4.08 (t, J = 7.4 Hz, 2 H), 1.86 (quin, J = 7.7 Hz, 2 H),
1.79 (quin, J = 7.2 Hz, 2 H), 1.47–1.27 (m, 20 H), 0.88 (t, J = 7.2 Hz, 3 H), 0.87 (t, J = 6.8 Hz, 3 H).
4.2.4.16. Ethyl 1-decyl-6,7-(methylenedioxy)-4-quinolone-3-carboxylate (50j)
Pale orange solid (40% yield): 1H NMR (400 MHz, CDCl3) δ 9.01 (s, 1 H), 7.49 (s, 1 H), 7.41 (s, 1
H), 6.15 (2 H), 4.44 (q, J = 7.2 Hz, 2 H), 4.18 (t, J = 6.4 Hz, 2 H), 1.89 (quin, J = 6.8 Hz, 2 H), 1.44
(t, J = 7.2 Hz, 3 H), 1.34–1.27 (m, 10 H), 0.88 (t, J = 7.2 Hz, 3H).
4.2.4.17. Ethyl 1-octyl-6,7-(trimethylene)-4-quinolone-3-carboxylate (51h)
Pale orange solid (35% yield): 1H NMR (400 MHz, CDCl3) δ 8.41 (s, 1 H), 8.36 (s, 1 H), 7.25 (s, 1
H), 4.40 (q, J = 7.1 Hz, 2 H), 4.14 (t, J = 7.4 Hz, 2 H), 3.04 (t, J = 7.2 Hz, 2 H), 3.02 (t, J = 7.2 Hz, 2
H), 2.17 (quin, J = 7.6 Hz, 2 H), 1.88 (quin, J = 7.5 Hz, 2 H), 1.41 (t, J = 7.1, 3 H), 1.36–1.26 (m, 10
H), 0.87 (t, J = 7.2 Hz, 3 H).
4.2.5. General procedure for synthesis of 3–15
2 M NaOH (3 mL) was added to a solution of compound 2 or 40–51 (130 mg, 0.431 mmol) in
MeOH (5 mL), and the mixture was stirred at room temperature for 8 h. The reaction mixture was
quenched with ice /2 M HCl and stirred for 1 h to form a white precipitate. After the mixture was
filtrated, the precipitate was washed with H2O and recrystallized from each suitable solvent to give

ACCEPTED MANUSCRIPT
desired carboxylic acid 3–15.
4.2.5.1. 1-Methyl-4-quinolone-3-carboxylic acid (3a)
Colorless needle (99% yield) recrystallized from EtOH/AcOEt: mp 291–293 °C; 1H NMR (400 MHz,
CDCl3) δ 14.89 (s, 1 H), 8.78 (s, 1 H), 8.59 (dd, J = 8.8, 2.0 Hz, 1 H), 7.64 (br t, J = 8.0, 1 H), 7.63
13
(br d, J = 8.8), 4.05 (s, 1 H); C NMR (126 MHz, DMSO-d6) δ 177.8, 166.1, 150.1, 140.3, 134.2,
126.4, 125.6, 125.2, 118.2, 107.4, 41.7; HRMS (ESI, m/z) Calcd. for C11H10NO3 [M+H]+: 204.0661.
Found 204.0656.
4.2.5.2. 1-Ethyl-4-quinolone-3-carboxylic acid (3b)
Colorless needle (97% yield) recrystallized from EtOH/AcOEt: mp 249–250 °C; 1H NMR (500 MHz,
DMSO-d6) δ 14.89 (s, 1 H), 9.07 (s, 1 H), 8.40 (dd, J = 8.2, 1.5 Hz, 1 H), 8.05 (br d, J = 8.7, 1 H),
7.98 (ddd, J = 8.7, 7.0, 1.6, 1 H), 7.67 (ddd, J = 8.1, 7.2, 0.9, 1 H), 4.62 (q, J = 7.2, 2 H), 1.43 (t, J =
7.2, 3 H); 13C NMR (126 MHz, DMSO-d6) δ 177.7, 166.0, 149.1, 139.0, 134.2, 126.2, 125.9, 125.5,
118.0, 107.7, 48.9, 14.5; HRMS (ESI, m/z) Calcd. for C12H12NO3 [M+H]+: 218.0817. Found
218.0812.
4.2.5.3. 1-Propyl-4-quinolone-3-carboxylic acid (3c)
Colorless block (91% yield) recrystallized from EtOH/AcOEt: mp 155–157 °C; 1H NMR (500 MHz,
CDCl3) δ 14.97 (s, 1 H), 8.78 (s, 1 H), 8.58 (dd, J = 8.0, 1.4 Hz, 1 H), 7.86 (ddd, J = 8.2, 7.0, 1.5 Hz,
1 H), 7.64–7.59 (m, 2 H), 4.31 (t, J = 7.5 Hz, 2 H), 2.00 (sxt, J = 7.5 Hz, 2 H), 1.07 (t, J = 7.5 Hz, 3
H); 13C NMR (126 MHz, DMSO-d6) δ 177.7, 166.0, 149.5, 139.2, 134.2, 126.3, 125.9, 125.5, 118.2,
107.4, 54.9, 22.0, 10.5; HRMS (ESI, m/z) Calcd. for C13H14NO3 [M+H]+: 232.0974. Found
232.0971.
4.2.5.4. 1-Butyl-4-quinolone-3-carboxylic acid (3d)
Colorless plate (90% yield) recrystallized from n-Hex/AcOEt: mp 173–176 °C; 1H NMR (500 MHz,
CDCl3) δ 14.98 (s, 1 H), 8.77 (s, 1 H), 8.56 (d, J = 8.1 Hz, 1 H), 7.86 (ddd, J = 8.7, 7.1, 1.6 Hz, 1 H),
7.63 (d, J = 8.6 Hz, 1 H), 7.60 (t, J = 7.5 Hz, 1 H), 4.34 (t, J = 7.5 Hz, 2 H), 1.92 (quin, J = 7.6 Hz, 2
H), 1.47 (sext, J = 7.5 Hz, 2 H), 1.02 (t, J = 7.5 Hz, 3 H); 13C NMR (126 MHz, CDCl3) δ 178.5,
167.1, 148.4, 139.3, 134.0, 127.4, 126.6, 126.2, 116.4, 108.7, 54.7, 31.0, 19.9, 13.5; HRMS (ESI,
m/z) Calcd. for C14H16NO3 [M+H]+: 246.1130. Found 246.1125.
4.2.5.5. 1-Pentyl-4-quinolone-3-carboxylic acid (3e)
Colorless plate (80% yield) recrystallized from n-Hex/AcOEt: mp 144–146 °C; 1H NMR (500 MHz,
CDCl3) δ 14.98 (br s, 1 H), 8.77 (s, 1 H), 8.57 (dd, J = 8.1, 1.5, Hz, 1 H), 7.85 (ddd, J = 8.7, 7.1, 1.6

ACCEPTED MANUSCRIPT
Hz, 1 H), 7.62 (d, J = 8.5 Hz, 1 H), 7.60 (ddd, J = 8.1, 7.2, 0.7 Hz, 2 H), 4.32 (t, J = 7.6 Hz, 2 H),
1.95 (quin, J = 7.5 Hz, 2 H), 1.43–1.38 (m, 4 H), 0.93 (t, J = 7.1 Hz, 3 H); 13C NMR (126 MHz,
CDCl3) δ 178.5, 167.1, 148.4, 139.3, 134.0, 127.4, 126.7, 126.2, 116.4, 108.7, 54.9, 28.8, 28.7, 22.2,
13.8; HRMS (ESI, m/z) Calcd. for C15H18NO3 [M+H]+: 260.1287. Found 260.1282.
4.2.5.6. 1-Hexyl-4-quinolone-3-carboxylic acid (3f)
White solid (92% yield) recrystallized from n-Hex/AcOEt: mp 153–155 °C; 1H NMR (500 MHz,
CDCl3) δ 14.98 (br s, 1 H), 8.76 (s, 1 H), 8.58 (dd, J = 8.1, 1.4 Hz, 1 H), 7.86 (ddd, J = 8.7, 7.1, 1.6
Hz, 1 H), 7.62 (d, J = 7.9 Hz, 2 H), 7.60 (t, J = 7.1 Hz, 2 H), 4.32 (t, J = 7.6 Hz, 2 H), 1.94 (quin, J =
13
7.5 Hz, 2 H), 1.45 (quin, J = 7.5 Hz, 2 H), 1.34 (m, 4 H), 0.91 (t, J = 7.1 Hz, 3 H); C NMR (126
MHz, CDCl3) δ 178.5, 167.2, 148.4, 139.3, 134.0, 127.5, 126.7, 126.2, 116.4, 108.7, 54.9, 31.2, 29.0,
26.3, 22.4, 13.9; HRMS (ESI, m/z) Calcd. for C16H20NO3 [M+H]+: 274.1443. Found 274.1442.
4.2.5.7. 1-Heptyl-4-quinolone-3-carboxylic acid (3g)
Colorless plate (99% yield) recrystallized from n-Hex/AcOEt: mp 119–121 °C; 1H NMR (500 MHz,
CDCl3) δ 14.98 (br s, 1 H) 8.77 (s, 1 H), 8.58 (dd, J = 8.1, 1.4 Hz, 1 H), 7.85 (ddd, J = 8.7, 7.1, 1.6
Hz, 1 H), 7.62 (d, J = 9.1 Hz, 2 H), 7.60 (ddd, J = 7.9, 7.2, 0.7 Hz, 2 H), 4.32 (t, J = 7.5 Hz, 2 H),
1.94 (quin, J = 7.5 Hz, 2 H), 1.46–1.27 (m, 8 H), 0.89 (t, J = 6.9 Hz, 3 H); 13C NMR (126 MHz,
CDCl3) δ 178.5, 167.1, 148.4, 139.3, 134.0, 127.5, 126.7, 126.2, 116.4, 108.7, 54.9, 31.5, 29.1, 28.7,
26.6, 22.5, 14.0; HRMS (ESI, m/z) Calcd. for C17H22NO3 [M+H]+: 288.1600. Found. 288.1592.
4.2.5.8. 1-Octyl-4-quinolone-3-carboxylic acid (3h)
1
White solid (88% yield) recrystallized from n-Hex/AcOEt: mp 119–122 °C; H NMR (500 MHz,
CDCl3) δ 14.98 (br s, 1 H), 8.77 (s, 1 H), 8.57 (dd, J = 8.1, 1.5 Hz, 1 H), 7.86 (ddd, J = 8.7, 7.1, 1.6
Hz, 1 H), 7.62 (d, J = 8.6 Hz, 1 H), 7.60 (t, J = 7.6 Hz, 1 H), 4.31 (t, J = 7.6 Hz, 2 H), 1.94 (quin, J =
7.6 Hz, 2 H), 1.45–1.23 (m, 10 H), 0.88 (t, J = 7.5 Hz, 3 H); 13C NMR (126 MHz, CDCl3) δ 178.5,
167.1, 148.4, 139.3, 134.0, 127.4, 126.7, 126.2, 116.4, 108.7, 54.9, 31.6, 29.1, 29.0, 29.0, 26.6, 22.5,
14.0; HRMS (ESI, m/z) Calcd. for C18H24NO3 [M+H]+: 302.1756. Found 302.1749.
4.2.5.9. 1-Nonyl-4-quinolone-3-carboxylic acid (3i)
1
White solid (86% yield) recrystallized from n-Hex/AcOEt: mp 103–104 °C; H NMR (500 MHz,
CDCl3) δ 14.97 (s, 1 H), 8.77 (s, 1 H), 8.58 (dd, J = 8.1, 1.5 Hz, 1 H), 7.86 (ddd, J = 8.7, 7.1, 1.6 Hz,
1 H), 7.63 (d, J = 8.9 Hz, 1 H), 7.59 (t, J = 7.2 Hz, 1 H), 4.32 (t, J = 7.6 Hz, 2 H), 1.94 (quin, J = 7.5
Hz, 2 H), 1.46–1.23 (m, 12 H), 0.88 (t, J = 7.1 Hz, 3 H); 13C NMR (126 MHz, CDCl3) δ 178.5, 167.1,
148.4, 139.3, 134.0, 127.5, 126.7, 126.2, 116.4, 108.7, 54.9, 31.7, 29.3, 29.1, 29.1, 29.1, 26.6, 22.6,
14.1; HRMS (ESI, m/z) Calcd. for C19H26NO3 [M+H]+: 316.1913. Found 316.1912.

ACCEPTED MANUSCRIPT
4.2.5.10. 1-Decyl-4-quinolone-3-carboxylic acid (3j)
1
White solid (82% yield) recrystallized from n-Hex/AcOEt: mp 106–107 °C; H NMR (500 MHz,
CDCl3) δ 14.98 (br s, 1 H), 8.77 (s, 1 H), 8.57 (dd, J = 8.2, 1.5 Hz, 1 H), 7.86 (ddd, J = 8.7, 7.1, 1.7
Hz, 1 H), 7.63 (d, J = 9.3 Hz, 1 H), 7.60 (t, J = 7.5 Hz, 1 H), 4.32 (t, J = 7.6 Hz, 2 H), 1.94 (quin, J =
7.5 Hz, 2 H), 1.46–1.22 (m, 14 H), 0.88 (t, J = 7.5 Hz, 3 H); 13C NMR (126 MHz, CDCl3) δ 178.5,
167.1, 148.4, 139.3, 134.0, 127.5, 126.7, 126.2, 116.4, 108.7, 54.9, 31.8, 29.4, 29.3, 29.2, 29.1, 29.1,
26.6, 22.6, 14.1; HRMS (ESI, m/z) Calcd. for C20H28NO3 [M+H]+: 330.2069. Found 330.2060.
4.2.5.11. 1-(Cyclopropylmethyl)-4-quinolone-3-carboxylic acid (3k)
1
White solid (97% yield) recrystallized from n-Hex/AcOEt: mp 207–209 °C; H NMR (500 MHz,
CDCl3) δ 15.00 (br s, 1 H), 8.86 (s, 1 H), 8.58 (dd, J = 8.1, 1.5 Hz, 1 H), 7.86 (ddd, J = 8.6, 7.1, 1.6
Hz, 1 H), 7.74 (d, J = 8.6 Hz, 1 H), 7.61 (ddd, J = 8.0, 7.2, 0.8 Hz, 1 H), 4.19 (d, J = 7.1 Hz, 2 H),
1.46–1.38 (m, 1 H), 0.81 (m, 2 H), 0.52 (m, 2 H); 13C NMR (126 MHz, CDCl3) δ 178.5, 167.2,
147.8, 139.7, 134.0, 127.4, 126.5, 126.2, 116.4, 108.8, 58.8, 10.1, 4.78; HRMS (ESI, m/z) Calcd. for
C14H14NO3 [M+H]+: 244.0974. Found 244.0967.
4.2.5.12. 1-(Cyclohexylmethyl)-4-quinolone-3-carboxylic acid (3l)
1
White solid (77% yield) recrystallized from n-Hex/AcOEt: mp 220–221 °C; H NMR (500 MHz,
CDCl3) δ 14.98 (br s, 1 H), 8.69 (s, 1 H), 8.58 (dd, J = 8.4, 1.6 Hz, 1 H), 7.85 (ddd, J = 8.6, 7.1, 1.6
Hz, 1 H), 7.62–7.59 (m, 2 H), 4.13 (d, J =7.4 Hz, 2 H), 1.99–1.90 (m, 1 H), 1.80–1.60 (m, 5 H),
1.25–1.06 (m, 5 H); 13C NMR (126 MHz, CDCl3) δ 178.5, 167.1, 148.8, 139.6, 133.9, 127.4, 126.6,
126.2, 116.6, 108.3, 60.9, 37.0, 30.6, 25.9, 25.4; HRMS (ESI, m/z) Calcd. for C17H20NO3 [M+H]+:
286.1443. Found 286.1437.
4.2.5.13. 1-Benzyl-4-quinolone-3-carboxylic acid (3m)
Pale pink solid (50% yield) recrystallized from n-Hex/AcOEt: mp 235–236 °C; 1H NMR (500 MHz,
CDCl3) δ 14.91 (s, 1 H), 8.92 (s, 1 H), 8.58 (dd, J = 8.1, 1.5 Hz, 1 H), 7.72 (ddd, J = 8.6, 7.1, 1.6 Hz,
1 H), 7.55 (ddd, J = 8.3, 7.4, 0.7 Hz, 1 H), 7.53 (d, J = 8.7 Hz, 1 H), 7.39–7.35 (m, 3 H), 7.18 (d, J =
6.5, 2 H), 5.54 (s, 2 H); 13C NMR (126 MHz, CDCl3) δ 178.7, 167.0, 149.2, 139.7, 134.1, 133.3,
129.6, 129.0, 127.3, 126.7, 126.3, 126.1, 117.3, 109.0, 58.2; HRMS (ESI, m/z) Calcd. for C17H14NO3
[M+H]+: 280.0974. Found 280.0970.
4.2.5.14. 6-Methyl-1-propyl-4-quinolone-3-carboxylic acid (4c)
1
White solid (74% yield) recrystallized from n-Hex/AcOEt: mp 231–234 °C; H NMR (500 MHz,
CDCl3) δ 15.11 (br s, 1 H) 8.73 (s, 1 H), 8.36 (d, J = 0.8 Hz, 1 H), 7.67 (dd, J = 8.8, 2.1 Hz, 1 H),

ACCEPTED MANUSCRIPT
7.53 (d, J = 8.7 Hz, 1 H), 4.29 (t, J = 7.4 Hz, 2 H), 2.55 (s, 3 H), 1.99 (sext, J = 7.4 Hz, 2 H), 1.05 (t,
J = 7.4 Hz, 3 H); 13C NMR (126 MHz, CDCl3) δ 178.3, 167.3, 147.9, 137.4, 136.7, 135.4, 126.8,
126.5, 116.3, 108.4, 56.3, 22.4, 21.0, 11.0; HRMS (ESI, m/z) Calcd. for C14H16NO3 [M+H]+:
246.1130. Found 246.1127.
4.2.5.15. 6-Methyl-1-octyl-4-quinolone-3-carboxylic acid (4h)
1
Colorless block (quant.) recrystallized from n-Hex/AcOEt: mp 115–116 °C; H NMR (500 MHz,
CDCl3) δ 15.11 (br s, 1 H), 8.72 (s, 1 H), 8.35 (d, J = 0.9 Hz, 1 H), 7.67 (dd, J = 8.8, 2.1 Hz, 1 H),
7.52 (d, J = 8.8 Hz, 1 H), 4.30 (t, J = 7.5 Hz, 2 H), 2.55 (s, 3 H), 1.92 (quin, J = 7.5 Hz, 2 H),
13
1.44–1.21 (m, 10 H), 0.88 (t, J = 7.0 Hz, 3 H); C NMR (126 MHz, CDCl3) δ178.3, 167.3, 147.8,
137.4, 136.6, 135.4, 126.7, 126.5, 116.3, 108.4, 54.9, 31.6, 29.1, 29.0, 29.0, 26.6, 22.5, 21.0, 14.0;
HRMS (ESI, m/z) Calcd. for C19H26NO3 [M+H]+: 316.1913. Found 316.1906.
4.2.5.16. 7-Methyl-1-propyl-4-quinolone-3-carboxylic acid (5c)
1
White solid (60% yield) recrystallized from n-Hex/AcOEt: mp 228–230 °C; H NMR (500 MHz,
CDCl3) δ 15.09 (br s, 1 H), 8.72 (s, 1 H), 8.45 (d, J = 8.3 Hz, 1 H), 7.42 (d, J = 8.2 Hz, 1 H), 7.37 (br
s, 1 H), 4.27 (t, J = 7.4 Hz, 2 H), 2.61 (s, 3 H), 1.99 (sext, J = 7.4 Hz, 2 H), 1.06 (t, J = 7.4 Hz, 3 H);
13C NMR (126 MHz, CDCl3) δ 178.3, 167.3, 148.3, 145.4, 139.5, 127.9, 127.2, 124.5, 116.0, 108.4,
56.2, 22.5, 22.3, 11.0; HRMS (ESI, m/z) Calcd. for C14H16NO3 [M+H]+: 246.1130. Found 246.1122.
4.2.5.17. 7-Methyl-1-octyl-4-quinolone-3-carboxylic acid (5h)
1
White solid (70% yield) recrystallized from n-Hex/AcOEt: mp 150–152 °C; H NMR (500 MHz,
CDCl3) δ 15.10 (s, 1 H), 8.71 (s, 1 H), 8.44 (d, J = 8.3 Hz, 1 H), 7.41 (br d, J = 8.2 Hz, 1 H), 7.37 (br
s, 1 H), 4.29 (t, J = 7.5 Hz, 2 H), 2.61 (s, 3 H), 1.93 (quin, J = 7.5 Hz, 2 H), 1.45–1.28 (m, 10 H),
13
0.89 (t, J = 6.9 Hz, 3 H); C NMR (126 MHz, CDCl3) δ 178.3, 167.3, 148.2, 145.4, 139.5, 127.9,
127.2, 124.5, 116.0, 108.5, 54.8, 31.6, 29.0, 26.6, 22.6, 22.5, 22.5, 14.0; HRMS (ESI, m/z) Calcd. for
C19H26NO3 [M+H]+: 316.1913. Found 316.1905.
4.2.5.18. 6-Methoxy-1-propyl-4-quinolone-3-carboxylic acid (6c)
1
White solid (60% yield) recrystallized from n-Hex/AcOEt: mp 234–236 °C; H NMR (500 MHz,
CDCl3) δ 15.13 (br s, 1 H), 8.70 (s, 1 H), 7.90 (d, J = 3.0 Hz, 1 H), 7.57 (d, J = 9.4 Hz, 1 H), 7.45
(dd, J = 9.3, 3.0 Hz, 1 H), 4.29 (t, J = 7.4 Hz, 2 H), 3.97 (s, 3 H), 1.98 (sext, J = 7.4 Hz, 2 H), 1.04 (t,
J = 7.4 Hz, 3 H); 13C NMR (126 MHz, CDCl3) δ 177.7, 167.4, 157.9, 146.8, 133.8, 128.1, 124.7,
118.1, 107.9, 106.2, 56.5, 56.0, 22.5, 11.0; HRMS (ESI, m/z) Calcd. for C14H16NO4 [M+H]+:
262.1079. Found 262.1074.

ACCEPTED MANUSCRIPT
4.2.5.19. 6-Methoxy-1-octyl-4-quinolone-3-carboxylic acid (6h)
1
Colorless needle (82% yield) recrystallized from n-Hex/AcOEt: mp 133–134 °C; H NMR (500
MHz, CDCl3) δ 15.13 (s, 1 H), 8.70 (s, 1 H), 7.91 (d, J = 2.9 Hz, 1 H), 7.57 (d, J = 9.4 Hz, 1 H), 7.45
(dd, J = 9.3, 2.7 Hz, 1 H), 4.31 (t, J = 7.4 Hz, 2 H), 3.97 (s, 3 H), 1.93 (quin, J = 7.3 Hz, 2 H),
1.42–1.27 (m, 10 H), 0.88 (t, J = 6.90 Hz, 3 H); 13C NMR (126 MHz, CDCl3) δ177.7, 167.4, 157.9,
146.7, 133.8, 128.1, 124.8, 118.1, 108.0, 106.2, 56.0, 55.1, 31.6 29.2, 29.0, 29.0, 26.6, 22.5, 14.0;
HRMS (ESI, m/z) Calcd. for C19H26NO4 [M+H]+: 332.1862. Found 332.1855.
4.2.5.20. 7-Methoxy-1-propyl-4-quinolone-3-carboxylic acid (7c)
1
White solid (60% yield) recrystallized from n-Hex/AcOEt: mp 221–222 °C; H NMR (500 MHz,
CDCl3) δ 15.15 (br s, 1 H), 8.70 (s, 1 H), 8.50 (d, J = 9.0 Hz, 1 H), 7.18 (dd, J = 9.0, 1.7 Hz, 1 H ),
6.93 (d, J = 1.6 Hz, 1 H), 4.23 (t, J = 7.1 Hz, 2 H), 3.99 (s, 3 H), 1.99 (sext, J = 7.1 Hz, 2 H), 1.06 (t,
J = 7.4 Hz, 3 H); 13C NMR (126 MHz, CDCl3) δ177.9, 167.3, 164.2, 148.6, 141.2, 129.4, 120.5,
114.2, 108.4, 99.9, 56.4, 56.0, 22.1, 11.1; HRMS (ESI, m/z) Calcd. for C14H16NO4 [M+H]+:
262.1079. Found 262.1076.
4.2.5.21. 7-Methoxy-1-octyl-4-quinolone-3-carboxylic acid (7h)
Colorless plate (62% yield) recrystallized from n-Hex/AcOEt: mp 125–126 °C; 1H NMR (500 MHz,
CDCl3) δ 15.17 (br s, 1 H), 8.68 (s, 1 H), 8.47 (d, J = 9.4 Hz, 1 H), 7.17 (dd, J = 9.1, 2.0 Hz, 1 H),
6.93 (d, J = 2.1 Hz, 1 H), 4.24 (t, J = 7.6 Hz, 2 H), 3.99 (s, 3 H), 1.93 (quin, J = 7.4 Hz, 2 H),
1.44–1.27 (m, 10 H), 0.88 (t, J = 7.0 Hz, 3 H); 13C NMR (126 MHz, CDCl3) δ 177.8, 167.3, 164.2,
148.3, 129.3, 120.4, 114.3, 108.3, 99.7, 55.9, 54.8, 31.6, 29.0, 29.0, 28.7, 26.6, 22.5, 14.0; HRMS
(ESI, m/z) Calcd. for C19H26NO4 [M+H]+: 332.1862. Found 332.1861.
4.2.5.22. 8-Methoxy-1-octyl-4-quinolone-3-carboxylic acid (8h)
White solid (63% yield) recrystallized from n-Hex/AcOEt: mp 139–140 °C; 1H NMR (400 MHz,
CDCl3) δ 15.00 (br s, 1 H), 8.62 (s, 1 H), 8.18 (dd, J = 8.0, 1.2 Hz, 1 H), 7.50 (t, J = 8.0 Hz, 1 H),
7.30 (dd, J = 8.8, 1.2 Hz, 1 H), 4.61 (t, J = 7.6 Hz, 2 H), 4.02 (s, 3 H), 1.84 (quin, J = 7.2 Hz, 2 H),
1.32–1.26 (m, 10 H), 0.88 (t, J = 6.8 Hz, 3 H); 13C NMR (126 MHz, CDCl3) δ 178.09, 167.35,
150.62, 150.48, 130.64, 129.18, 126.85, 119.22, 115.71, 108.39, 60.98, 56.73, 31.87, 31.77, 29.34,
29.28, 26.64, 22.78, 14.24; HRMS (ESI, m/z) Calcd. for C19H26NO4 [M+H]+: 332.1856. Found
332.1849.
4.2.5.23. 7-(Methylthio)-1-octyl-4-quinolone-3-carboxylic acid (9h)
White solid (quant.) recrystallized from n-Hex/AcOEt: mp 138–139 °C; 1H NMR (400 MHz, CDCl3)
δ 8.68 (s, 1 H), 8.42 (d, J = 8.8 Hz, 1 H), 7.39 (dd, J = 8.8, 1.6 Hz, 1 H), 7.28 (d, J = 1.2 Hz, 1 H),