Functionalization at C2, C3, and C4 of quinolines: Discovery of water-soluble betaine dyes of C3 quinolinium derivatives with solvatochromic and pH-sensitive properties

Article abstract of DOI:10.1016/j.dyepig.2020.108341

During functionalization at C2, C3, and C4 of quinoline, we came across a one-step metal free synthesis of C3 quinolinium derivatives of mesomeric betaine dyes. Some mesomeric betaine dyes had solvatochromic properties, and were sensitive to pH and solvates in the water. Certain betaine dyes had different properties in acidic and basic environments; the reversible interconversion between yellow (neutral or basic condition) and colorless (acidic condition) forms of the dye occurs by pH change. The changes in UV absorbance intensities of the dyes in the presence of cyclodextrins indicated the inclusion complex formation between cyclodextrins and dyes. The betaine dyes were partially dissolved in water due to the presence of positive and negative charges in the molecule. These betaine dyes did not exhibit cytotoxic activity, hence may be used in biological research.

Full text of DOI:10.1016/j.dyepig.2020.108341

Journal Pre-proof
Functionalization at C2, C3, and C4 of quinolines: Discovery of water-soluble betaine
dyes of C3 quinolinium derivatives with solvatochromic and pH-sensitive properties
Sasithorn Sangher, Chatchai Kesornpun, Thammarat Aree, Chulabhorn Mahidol,
Somsak Ruchirawat, Prasat Kittakoop
PII:
S0143-7208(19)31550-5
DOI:
https://doi.org/10.1016/j.dyepig.2020.108341
DYPI 108341
Reference:
To appear in: Dyes and Pigments
Received Date: 5 July 2019
Revised Date: 25 February 2020
Accepted Date: 6 March 2020
Please cite this article as: Sangher S, Kesornpun C, Aree T, Mahidol C, Ruchirawat S, Kittakoop P,
Functionalization at C2, C3, and C4 of quinolines: Discovery of water-soluble betaine dyes of C3
quinolinium derivatives with solvatochromic and pH-sensitive properties, Dyes and Pigments (2020), doi:
https://doi.org/10.1016/j.dyepig.2020.108341.
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Graphical Abstract
Functionalization at C2, C3, and C4 of quinolines: Discovery of water-soluble betaine
dyes of C3 quinolinium derivatives with solvatochromic and pH-sensitive properties
Sasithorn Sangher, Chatchai Kesornpun, Thammarat Aree, Chulabhorn Mahidol, Somsak
Ruchirawat, Prasat Kittakoop*

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Functionalization at C2, C3, and C4 of quinolines: Discovery of water-soluble
betaine dyes of C3 quinolinium derivatives with solvatochromic and pH-
sensitive properties
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Sasithorn Sangher a†,Chatchai Kesornpun a, Thammarat Aree b, Chulabhorn Mahidol a,c,
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Somsak Ruchirawat a,c,d, Prasat Kittakoop a,c,d,
*
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a Chulabhorn Research Institute, Kamphaeng Phet 6 Road, Laksi , Bangkok 10210, Thailand
b Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330,
Thailand
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c Chulabhorn Graduate Institute, Chemical Biology Program, Chulabhorn Royal Academy,
Kamphaeng Phet 6 Road, Laksi, Bangkok 10210, Thailand
d Center of Excellence on Environmental Health and Toxicology (EHT), CHE, Ministry of
Education, Thailand
† Deceased 24 March 2017
*Corresponding author
E-mail address: prasat@cri.or.th (P. Kittakoop)

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ABSTRACT
During functionalization at C2, C3, and C4 of quinoline, we came across a one-step metal
free synthesis of C3 quinolinium derivatives of mesomeric betaine dyes. Some mesomeric
betaine dyes had solvatochromic properties, and were sensitive to pH and solvates in the
water. Certain betaine dyes had different properties in acidic and basic environments; the
reversible interconversion between yellow (neutral or basic condition) and colorless (acidic
condition) forms of the dye occurs by pH change. The changes in UV absorbance intensities
of the dyes in the presence of cyclodextrins indicated the inclusion complex formation
between cyclodextrins and dyes. The betaine dyes were partially dissolved in water due to the
presence of positive and negative charges in the molecule. These betaine dyes did not exhibit
cytotoxic activity, hence may be used in biological research.
Keywords: betaine dyes; quinolinium derivatives; quinoline derivatives; solvatochromism;
green chemistry; water-soluble dyes; pH-sensitive dye

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1. Introduction
Recently, we are interested in the synthesis of quinoline derivatives for medicinal
chemistry research using quinolinium salts. The quinoline derivative, TMC207 (Bedaquiline),
which was approved as an anti-tuberculosis drug in 2012, has prompted us to prepare
quinoline derivatives [1]. Recently, quinoline derivatives are of interest for their applications
as sensors and materials [2, 3]. N-Methylquinolinium derivatives have a high potential for
photonic applications [4] and are used in a photocatalytic degradation of phenolic pollutants
[5]. During functionalization at C2, C3, and C4 of quinoline, we discovered a one-step metal
free synthesis of C3 quinolinium derivatives of mesomeric betaine dyes. We employed
chemistry of quinolinium salts (1) for the preparation of quinoline derivatives.
Chemistry of quinolinium salts (1) has long been established, for example, for the
synthesis of 2-4 (Figure 1) [6-8]. Quinophthalone (5) (Figure 1) or quinoline yellow SS, a
greenish yellow dye, is a commercially available C2 quinoline derivative, and it has been
used for many decades as a coloring agent for many materials, for example, paper, wools,
paints, and polyester fibers. However, quinophthalone (5) is cytotoxic, and this limits
biocompatibility [9, 10]. Recently, quinophthalone (5) was found to show uncommon N-to-O
excited state intramolecular proton transfer between photobases [11]. Reichardt's dye is
pyridinium N phenolate betaine dye with various applications, for example, as indicators of
polarity of solvents, ionic liquids, and solid surfaces [12].
While using the chemistry of quinolinium salts (1) for C2, C3 and C4
functionalization of quinoline, we found that treating a cyclic diketone (6) with quinolinium
salts (1) simply gave products 7, 8, and 9 under basic conditions (Figure 1). Among the
products obtained, C3 quinolinium derivatives (8) are betaine dyes with interesting
properties. Moreover, the carbon-carbon bond formation at C3 of quinolinium salts (1) is
unprecedented in the chemistry of quinolinium salt. Normally, the carbon-carbon bond

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formation of pyridiniums and quinoliniums gives the functionalization at 2- and 4-positions
of an arene using organometallic reagents [13, 14], but not at C3 position; the present work
demostrates a simple carbon-carbon bond formation at C3 of quinolinium salt. Certain
mesomeric betaine dyes (8) had solvatochromic properties, and they were sensitive to pH and
water contents. The betaine dyes (8) were partially dissolved in water because they had both
positive and negative charges in their molecule, and the dye 8 did not exhibit cytotoxic
activity; therefore they may be applied for cell-based assays. We report herein a one-step
metal free synthesis of C3 quinolinium derivatives of mesomeric betaine dyes, as well as
certain propeties and cytotoxicity.
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Figure 1. Previous investigation of chemistry of quinolinium salts, structure of
quinophthalone (5), and present work: A, C2 functionalization of quinoline derivative (7) and
B, C3 and C4 functionalization of quinoline derivatives (8 and 9).
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2. Experimental
2.1. General remarks
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Melting points were measured using Stuart SMP30 melting point apparatus without
correction. The NMR spectra were recorded either on a Bruker Avance 300 or 400 MHz
spectrometers. Chemical shifts (δ) were reported in ppm relative to TMS or residual solvent
signals (TMS, 0.00 ppm; acetone-d6, 2.05 ppm for 1H NMR and 29.8 ppm for 13C NMR;
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CDCl3, 7.26 ppm for 1H NMR and 77.0 ppm for 13C NMR; DMSO-d6, 2.50 ppm of 1H NMR
and 39.5 ppm for 13C NMR; CD3OD; 3.31 ppm for 1H NMR and 49.0 ppm for 13C NMR).
The following abbreviations are used to indicate the multiplicity in 1H NMR spectra: s,
singlet; br s, broad singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m, multiplet. IR
spectra were obtained from a PerkinElmer Spectrum ONE spectrophotometer using Universal
Attenuated Total Reflectance (UATR) technique. UV-Vis spectra were obtained from a
Shimadzu UV-1700 PharmaSpect UV-Vis spectrophotometer. The UV-Vis spectra of
aqueous solution of 8de and 8dg were recorded on Agilent Cary 100 UV-Vis
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spectrophotometer. The pH values of aqueous solutions of 8de and 8dg were monitored by
MettlerToledo SevenCompact pH meter. HRMS data were obtained from a Bruker
MicroTOF mass spectrometer. Column chromatography was packed either with Silica gel 60
(Merck, 0.063–0.200 mm) or with Sephadex LH-20 (GE Health Care Bio-Sciences AB).
MPLC was carried out on Büchi C-605, C-620, C-660 separation modules coupled with
Büchi UV-monitor C-630 with reversed-phase C18 MPLC column (26x460 mm). HPLC was
carried out using Waters e2695 and Waters 1525 separation modules coupled with Waters
2998 photodiode array detector. Analytical HPLC column was Waters Sunfire reversed-phase
C18 (4.6x250 mm, 5 µm), and preparative HPLC column was Waters Sunfire reversed-phase
C18 (19x250 mm, 5 µm). Chemicals and reagents (with the purity > 95%) were obtained

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from commercial sources e.g. Merck, Sigma-Aldrich, and TCI, using without purification,
unless otherwise indicated.
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2.2. General procedures for the preparation of 1-methylquinolinium iodide salt (1)
Iodomethane (3 eq.) was added to a septum-sealed reaction flask containing 1.0 g of
an individual quinoline, then acetone (10 mL) was added to a reaction flask. The reaction was
kept stirring for 24-40 h at room temperature. Completion of reaction was indicated by a
precipitation of 1-methylquinolinium iodide salt (1). A yellow precipitate of 1 was collected
by filtration over a filter paper, and washed with MeOH in order to remove quinoline and
MeI. The salt 1 was obtained with 90-95% yield, and it was used without further purification.
Synthesis of the salts 1a, 1b, 1d, and 1e were previously reported [15-18]. 1c was obtained as
yellow amorphous solid, 1H NMR (400 MHz, DMSO-d6):
δ 9.67 (d, J = 5.6 Hz, 1H), 9.52 (d,
J = 8.9 Hz, 1H), 8.91 (d, J = 9.0, Hz, 1H), 8.78 (d, J = 7.7 Hz, 1H), 8.43 (dd, J = 9.0, 8.9 Hz,
1H), 8.37 (dd, J = 7.7, 5.6 Hz, 1H), 4.71 (s, 3H);13C NMR (75 MHz, DMSO-d6):
δ
151.7,
146.2, 142.3, 138.4, 133.8, 127.0, 125.4, 124.4, 121.6, 46.6; ESI-TOF MS (m/z): [M]+calcd.
for C10H9N2O2, 189.0665; found, 189.0658.
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2.3. General procedures for the reaction of 1-methylquinolinuim iodide salt (1) and 1,3-
dicarbonyl compounds (6)
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1-Methylquinolinium iodide salt (1, 200 mg), 1,3-dicarbonyl compounds (6, 1.0-1.2
eq.), and K2CO3 (1.0-1.2 eq.) were sequentially added to a reaction flask. A mixture (2.0 mL)
of EtOH:water (1:1, v/v) was added to the reaction flask. The reaction mixture was kept
stirring overnight (18-24 h) at room temperature. The reaction was terminated either by the
addition of water or by filtration (depending on the reaction). The reaction mixture was
evaporated to dryness and separated by chromatographic techniques, or subjected to reversed-
phase C18 MPLC.

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2.3.1. 2-(5-Bromo-1-methylquinolin-2(1H)-ylidene)-5,5-dimethylcyclohexane-1,3-dione (7ac)
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1a (203.7 mg, 0.576 mmol), dimedone (6c) (82.4 mg, 0.582 mmol), and K2CO3 (86.9 mg,
0.622 mmol) were sequentially added to a reaction flask. A mixture (2.0 mL) of EtOH:water
(1:1, v/v) was added to the reaction flask, and the mixture was kept stirring at room
temperature for 18 h. Then, cold water was added to the reaction mixture; precipitates were
filtered through a filter paper and washed with water, giving the precipitates and water
soluble parts. The precipitates were further separated by Sephadex-LH20 column
chromatography (SepCC, 1.5x120 cm, eluted with DCM:MeOH, 50:50, v/v), to yield 11.3
mg of 7ac. The water soluble part was evaporated to dryness, and the dried material was
extracted with DCM, to yield 76.9 mg of 7ac. The total amount of 7ac was 88.2 mg (0.242
mmol, 42% yield). 7ac was obtained as yellow rod crystal; mp.: decomposed at 150 ºC 1H
NMR (300 MHz, CDCl3): δ 8.52 (d, J = 9.5 Hz, 1H), 8.17 (d, J = 9.4 Hz, 1H), 7.93 (d, J =
9.1, Hz, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.66 (dd, J = 8.7, 7.9 Hz, 1H), 4.04 (s, 3H), 2.45 (s,
4H), 1.16 (s, 3H), 1.14 (s, 3H);13C NMR (75 MHz, CDCl3):
δ
192.4 (2C), 161.6, 140.6,
136.8, 131.9, 130.9, 129.9, 125.6, 123.6, 117.7, 110.3, 51.5 (2C), 43.0, 30.9, 28.6 (2C); IR
(UATR): νmax 3446, 2953, 2865, 1625, 1562, 1546, 1502, 1466, 1420, 1402, 1368, 1301,
1226, 1178, 1140, 1082, 1031, 951, 890, 828, 793, 754, 731, 694 cm-1; UV-VIS (EtOH): λmax
(log ε) 428 (4.22), 270 (4.39), 250 (4.40), 233 (4.57) nm; ESI-TOF MS (m/z): [M+H]+calcd.
for C18H19BrNO2, 360.0594; found, 360.0608.
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2.3.2. 5,5-Dimethyl-2-(1-methyl-5-nitroquinolin-2(1H)-ylidene)cyclohexane-1,3-dione (7cc)
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1c (212.0 mg, 0.664 mmol), 6c (96.4 mg, 0.681 mmol), and K2CO3 (89.1 mg, 0.638 mmol)
were sequentially added to a reaction flask. A mixture (2.0 mL) of EtOH:water (1:1, v/v) was
added to the reaction flask; it was stirred at room temperature for 18 h. To the reaction
mixture was added cold water, giving precipitates. The precipitates were filtered through a
filter paper, and washed with water. The precipitates were extracted with DCM, and the

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DCM extract was further separated by preparative thin layer chromatography (PTLC) (20x20
cm; developed with 25% MeOH:EtOAc), to yield 47.6 mg of 7cc (0.1445 mmol, 21% yield).
7cc was obtained as yellow amorphous solid; 1H NMR (300 MHz, CDCl3):
δ
8.71 (d, J = 9.8
Hz, 1H), 8.31 (d, J = 9.8 Hz, 1H), 8.27 (dd, J = 7.9, 0.8 Hz, 1H), 8.23 (d, J = 8.9 Hz, 1H),
7.92 (dd, J = 8.7, 8.0 Hz, 1H), 4.03 (s, 3H), 2.47 (s, 4H), 1.15 (s, 3H), 1.14 (s, 3H); 13C NMR
(75 MHz, CDCl3): δ 192.8 (2C), 161.4, 146.3, 140.2, 131.9, 131.5, 130.3, 123.5, 123.2,
119.4, 110.9, 51.7 (2C), 44.0, 30.8, 28.5 (2C); IR (UATR): νmax 3421, 3083, 2954, 2867,
1630, 1552, 1529, 1507, 1466, 1409, 1363, 1331, 1267, 1231, 1175, 1140, 1108, 1076, 1033,
1014, 959, 931, 888, 834, 807, 755, 731, 699, 622 cm-1; UV-VIS (EtOH): λmax (log ε) 445
(4.02), 302 (3.74), 268 (4.19), 233 (4.20), 218 (4.25) nm; ESI-TOF MS (m/z): [M+H]+ calcd.
for C18H19N2O4, 327.1339; found 327.1349.
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2.3.3. 2-(5-Bromo-1-methylquinolin-2(1H)-ylidene)cyclohexane-1,3-dione (7ad)
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1a (202.5 mg, 0.579 mmol), 1,3-cyclohexandione (6d) (67.8 mg, 0.599 mmol), and K2CO3
(89.7 mg, 0.643 mmol) were sequentially added to a reaction flask. To the reaction mixture
was added 2.0 mL of a mixture of EtOH:water (1:1, v/v), and the reaction mixture was kept
stirring at room temperature for 18 h. Then, water was added to the reaction mixture, to give
precipitates. Collection of precipitates was by filtration through a filter paper. Precipitates
were washed with water, then extracted with DCM, to yield 76.0 mg of 7ad (0.236 mmol,
41%). 7ad was obtained as an orange amorphous solid; 1H NMR (300 MHz, CDCl3):
δ 8.53
(d, J = 9.4 Hz, 1H), 8.18 (d, J = 9.4 Hz, 1H), 7.94 (d, J = 8.8 Hz, 1H), 7.91 (d, J = 7.9 Hz,
1H), 7.67 (dd, J = 8.7, 7.9 Hz, 1H), 4.05 (s, 3H), 2.60-2.50 (m, 4H), 2.10-2.00 (m, 2H); 13C
NMR (75 MHz, CDCl3): δ 193.1 (2C), 161.9, 140.6, 136.7, 131.9, 130.9, 130.0, 125.6, 123.6,
117.8, 111.4, 42.8, 37.8 (2C), 20.4; IR (UATR): νmax 3605, 3073, 2369, 2131, 1975, 1624,
1564, 1503, 1385, 1181, 1082, 985, 830, 808 cm-1; UV-Vis (EtOH): λmax (log ε) 428 (4.43),

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300 (4.09), 268 (4.61), 250 (4.65), 233 (4.78) nm; ESI-TOF MS (m/z): [M+H]+ calcd. for
C16H15BrNO2, 322.0281; found, 322.0293.
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2.3.4. 2-(5-Chloro-1-methylquinolin-2(1H)-ylidene)cyclohexane-1,3-dione (7bd)
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1b (202.3 mg, 0.662 mmol), 6d (67.8 mg, 0.668 mmol), and K2CO3 (95.3 mg, 0.683 mmol)
were sequentially added to a reaction flask. A mixture (2.0 mL) of EtOH:water (1:1, v/v) was
added to the reaction flask, and the mixture was further kept stirring at room temperature for
18 h. Then, water was added to the reaction mixture, giving precipitates. The precipitates
were filtered through a filter paper, and washed with water. The precipitates were extracted
with DCM, to yield 42.6 mg of 7bd (0.161 mmol, 24% yield). 7bd was obtained as a yellow
amorphous solid; 1H NMR (300 MHz, CDCl3):
δ 8.55 (d, J = 9.4 Hz, 1H), 8.18 (d, J = 9.4
Hz, 1H), 7.89 (d, J = 7.9 Hz, 1H), 7.77-7.68 (m, 2H), 4.06 (s, 3H), 2.60-2.54 (m, 4H), 2.10-
1.99 (m, 2H); 13C NMR (75 MHz, CDCl3):
δ
193.1 (2C), 161.9, 140.5, 134.1, 133.3, 131.6,
129.8, 127.3, 124.4, 117.1, 111.3, 42.7, 37.7 (2C), 20.3; IR (UATR): νmax 3024, 2949, 2826,
1987, 1676, 1622, 1600, 1586, 1565, 1550, 1505, 1472, 1445, 1424, 1406, 1370, 1324, 1308,
1292, 1253, 1231, 1214, 1179, 1171, 1130, 1093, 1075, 984, 922, 889, 857, 835, 799, 779,
755, 707, 666 cm-1; UV-Vis (EtOH): λmax (log ε) 428 (4.21), 300 (3.86), 268 (4.37), 248
(4.40), 231 (4.56) nm; ESI-TOF MS (m/z): [M+H]+ calcd. for C16H15ClNO2, 288.0786; found
288.0776.
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2.3.5. 2-(5-Nitro-1-methylquinolin-2(1H)-ylidene)cyclohexane-1,3-dione (7cd)
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1c (205.6 mg, 0.650 mmol), 6d (75.5 mg, 0.667 mmol), and K2CO3 (95.0 mg, 0.680 mmol)
were sequentially added to a reaction flask. A mixture (2.0 mL) of EtOH:water (1:1, v/v) was
added to the reaction flask; the mixture was kept stirring at room temperature for 18 h. Then,
the reaction mixture was subjected to SepCC (1.5x120 cm, eluted with DCM:MeOH, 50:50,
v/v), giving fractions A1-A10. The fractions A4-A10 were combined and further separated by

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silica gel column chromatography (SGCC, 2x20 cm, eluted with 20% MeOH:EtOAc), to
yield 50.6 mg 7cd (0.170 mmol, 26% yield). 7cd was obtained as a yellow amorphous solid;
1H NMR (300 MHz, CDCl3):
δ
8.72 (d, J = 9.7 Hz, 1H), 8.33 (d, J = 9.7 Hz, 1H), 8.28 (d, J =
7.9 Hz, 1H), 8.25 (d, J = 8.9 Hz, 1H), 7.93 (dd, J = 8.6, 8.1 Hz, 1 H), 4.05 (s, 3H), 2.59 (dd, J
= 6.6, 6.2 Hz, 4H), 2.10-2.00 (m, 2H); 13C NMR (75 MHz, CDCl3):
δ
193.4 (2C), 161.7,
146.3, 140.2, 132.1, 131.5, 130.3, 123.5, 123.2, 119.4, 112.0, 43.8, 38.0 (2C), 20.1; IR
(UATR): νmax 3432, 3083, 2943, 2865, 1628, 1552, 1507, 1467, 1425, 1410, 1363, 1323,
1227, 1174, 1128, 1012, 984, 910, 861, 811, 757, 732 cm-1; UV-Vis (EtOH): λmax (log ε) 444
(4.07), 303 (3.87), 266 (4.30), 217 (4.43) nm; ESI-TOF MS (m/z): [M+H]+ calcd. for
C16H15N2O4, 299.1026; found, 299.1037.
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2.3.6. 2-(5-Bromo-1-methylquinolin-2(1H)-ylidene)cyclopentane-1,3-dione (7ae)
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1a (205.2 mg, 0.586 mmol), 1,3-cyclopentanedione (6e) (58.4 mg, 0.589 mmol), and K2CO3
(84.8 mg, 0.608 mmol) were sequentially added to a reaction flask. A mixture (2.0 mL) of
EtOH:water (1:1, v/v) was added to the reaction flask, and the mixture was stirred at room
temperature for 18 h. To the reaction mixture was added water, giving precipitates. The
precipitates were filtered through a filter paper and washed with water. The precipitates were
extracted with DCM, giving 92.6 mg of the material, which was further separated by SepCC
(1.5x120 cm, eluted with DCM:MeOH, 50:50, v/v), giving fraction B1-B19. Fractions B8-
B19 were combined and separated by SGCC (1.5x20 cm, eluted with 25% MeOH:EtOAc), to
yield 42.6 mg of 7ae (0.116 mmol, 24% yield). 7ae was obtained as a yellow amorphous
solid; 1H NMR (400 MHz, CDCl3):
δ 8.56 (d, J = 9.5 Hz, 1H), 8.46 (d, J = 9.5 Hz, 1H), 7.91
(d, J = 8.8 Hz, 1H), 7.88 (d, J = 7.8 Hz, 1H), 7.68 (dd, J = 8.3, 8.2 Hz, 1 H), 4.12 (s, 3H),
2.65 (s, 4H); 13C NMR (100 MHz, CDCl3):
δ
200.6 (2C), 156.4, 141.0, 137.9, 132.3, 130.6,
126.1, 125.2, 123.8, 117.6, 109.1, 43.5, 34.1 (2C); IR (ATR): νmax 2921, 1668, 1601, 1559,
1511, 1481, 1430, 1413, 1397, 1314, 1275, 1242, 1213, 1182, 1081, 1065, 982, 959, 829,

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789, 747, 728, 700, 667 cm-1; UV-Vis (EtOH): λmax (log ε) 416 (4.25), 327 (3.66), 297 (3.93),
246 (3.37), 230 (4.47) nm; ESI-TOF MS (m/z): [M+H]+ calcd. for C15H13BrNO2, 318.0130;
found 318.0126.
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2.3.7. 2-(5-Chloro-1-methylquinolin-2(1H)-ylidene)cyclopentane-1,3-dione (7be)
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256
1b (201.5 mg, 0.586 mmol), 6e (66.7 mg, 0.673 mmol), and K2CO3 (93.4 mg, 0.673 mmol)
were sequentially added to a reaction flask. To the reaction mixture was added 2.0 mL of a
mixture of EtOH:water (1:1, v/v), and the mixture was stirred at room temperature for 18 h.
Then, water was added to the reaction mixture, to give precipitates, and the precipitates were
filtered through a filter paper and washed with water. The precipitates were extracted with
DCM, giving 167.0 mg of the extract, which was further separated by SepCC (1.5 x120 cm,
eluted with DCM:MeOH, 50:50, v/v), giving fractions C1-C23. Fractions C4-C22 were
combined and further separated by SGCC (1.5x20 cm, eluted with 25% MeOH:EtOAc), to
yield 42.3 mg of 7be (0.154 mmol, 23% yield). 7be was obtained as a yellow amorphous
solid; 1H NMR (300 MHz, CDCl3):
δ 8.58 (d, J = 9.5 Hz, 1H), 8.46 (d, J = 9.5 Hz, 1H), 7.86
(d, J = 8.5 Hz, 1H), 7.75 (dd, J = 8.5, 7.7 Hz, 1H), 7.69 (dd, J = 7.7, 1.1 Hz, 1H), 4.12 (s,
3H), 2.64 (s, 4H); 13C NMR (75 MHz, CDCl3):
δ
200.6 (2C), 156.5, 140.9, 135.1, 133.4,
132.0, 127.0, 125.9, 124.0, 116.8, 109.1, 43.5, 34.1 (2C); IR (UATR): νmax 2922, 1670, 1603,
1562, 1511, 1482, 1430, 1430, 1412, 1396, 1349, 1313, 1274, 1213, 1181, 1121, 1092, 1067,
982, 961, 829, 794, 747, 728, 700, 666 cm-1; UV-Vis (EtOH): λmax (log ε) 416 (4.33), 326
(3.65), 296 (3.96), 246 (4.36), 228 (4.48) nm; ESI-TOF MS (m/z): [M+H]+ calcd for
C15H13ClNO2, 274.0629, found 274.0635.
257
2.3.8. 2-(5-Nitro-1-methylquinolin-2(1H)-ylidene)cyclopentane-1,3-dione (7ce)
258
259
1c (203.0 mg, 0.642 mmol), 6e (64.9 mg, 0.642 mmol), and K2CO3 (104.2 mg, 0.746 mmol)
were sequentially added to a reaction flask. A mixture (2.0 mL) of EtOH:water (1:1, v/v) was

12
260
261
262
263
264
265
266
267
268
269
270
271
272
added to the reaction flask; this mixture was stirred at room temperature for 18 h. Then, the
reaction mixture was evaporated to dryness, then extracted with DCM, giving the DCM
extract, which was further separated by SepCC (1.5x120 cm, eluted with DCM:MeOH,
50:50, v/v), giving fractions D1-D16. Fractions D14 and D15 were combined to give 7ce
(46.6 mg, 0.164 mmol, 25 % yield). 7ce was obtained as a yellow amorphous solid; 1H NMR
(400 MHz, CDCl3):
δ 8.80 (d, J = 9.7 Hz, 1H), 8.62 (d, J = 9.7 Hz, 1H), 8.28 (d, J = 7.8 Hz,
1H), 8.23 (d, J = 8.8 Hz, 1H), 7.95 (dd, J = 8.4, 8.2 Hz, 1H), 4.14 (s, 3H), 2.69 (s, 4H); 13C
NMR (100 MHz, CDCl3): δ 200.5 (2C), 156.1, 146.5, 140.5, 133.1, 130.9, 128.1, 123.3,
123.0, 119.2, 109.5, 44.3, 34.1 (2C); IR (UATR): νmax 3092, 2354, 1621, 1607, 1558, 1513,
1474, 1434, 1420, 1398, 1361, 1318, 1277, 1219, 1190, 1166, 1076, 1006, 967, 886, 834,
801, 770, 750, 729, 680, 666 cm-1; UV-Vis (EtOH): λmax (log ε) 430 (4.27), 302 (3.97), 234
(4.37), 216 (4.45) nm. ESI-TOF MS (m/z): [M+H]+ calcd for C15H13N2O4, 285.0870; found,
285.0867.
273
2.3.9. 4,4-Dimethyl-1-(1-methylquinolin-1-ium-3-yl)-2,6-dioxocyclohexan-1-ide (8dc)
274
275
276
277
278
279
280
281
282
283
284
1d (201.5 mg, 0.558 mmol), 6c (95.4 mg, 0.674 mmol), and K2CO3 (93.3 mg, 0.668 mmol)
were sequentially added to a reaction flask. To the reaction mixture was added 2 mL of a
mixture of EtOH:water (1:1, v/v); the mixture was kept stirring at room temperature for 18 h.
Then, the mixture was directly subjected to MPLC (reversed-phase C18, 26x460 mm,
gradient elution of 0-95% MeOH:water), to yield 28.8 mg of 8dc, (0.101 mmol, 18% yield).
8dc was obtained as a yellow amorphous solid; 1H NMR (300 MHz, CDCl3):
δ 10.11 (d, J =
1.5 Hz, 1H), 9.72 (br s, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.87 (d, J = 8.7 Hz, 1H), 7.80 (td, J =
8.0, 1.2 Hz, 1H), 7.79 (td, J = 7.9, 0.9 Hz, 1H), 4.43 (s, 3H), 2.40 (s, 4H), 1.11 (s, 6H); 13C
NMR (75 MHz, CDCl3): δ 192.1 (2C), 151.2, 142.3, 135.4, 133.5, 131.5, 130.0, 129.9, 128.4,
116.3, 105.0, 51.5 (2C), 44.8, 30.6, 28.5 (2C); IR (UATR): νmax 3405, 3039, 2952, 2921,
2864, 1602, 1563, 1502, 1431, 1393, 1326, 1267, 1141, 1120, 1045, 969, 895, 878, 764, 742,

13
673, 699 cm-1; UV-Vis (EtOH): λmax (log ε) 437 (3.52), 284 (4.51), 231 (4.49) nm; ESI-TOF
285
286
MS (m/z): [M+H]+ calcd. for C18H20NO2, 282.1489; found, 282.1492.
287
2.3.10. 1-(1-Methylquinolin-1-ium-3-yl)-2,6-dioxocyclohexan-1-ide (8dd)
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
1d (205.5 mg, 0.581 mmol), 6d (81.2 mg, 0.702 mmol), and K2CO3 (97.8 mg, 0.700 mmol)
were sequentially added to a reaction flask. A mixture (2.0 mL) of EtOH:water (1:1, v/v) was
added to the reaction flask, and the mixture was stirred at room temperature for 18 h. The
reaction mixture was subjected to MPLC separation (reversed-phase C18, 26x460 mm,
gradient elution of 10-95% MeOH:water). Fractions from MPLC containing of 8dd were
further separated by HPLC (Waters Sunfire C18 reversed-phase, 19x250 mm, gradient
elution of 20-30% MeOH:water over 40 min, 10 mL/min); the product 8dd was eluted with
the retention time of 22.6 min (18.8 mg, 0.08 mmol, 13% yield). 8dd was obtained as orange
amorphous solid; 1H NMR (300 MHz, CDCl3):
δ 9.98 (d, J = 1.4 Hz, 1H), 9.67 (s, 1H), 8.05
(d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.84 (td, J = 8.8, 1.3 Hz, 1H), 7.75-7.69 (m,
1H), 4.45 (s, 3H), 2.52 (t, J = 6.4 Hz, 4H), 1.97 (quin, J = 6.6 Hz, 2H);13C NMR (75 MHz,
CDCl3): δ 192.8 (2C), 151.6, 143.2, 135.4, 133.7, 131.8, 130.2, 129.9, 128.5, 116.3, 106.6,
44.8, 37.5 (2C), 20.6; IR (UATR): νmax 3394, 3058, 2936, 2865, 1601, 1562, 1501, 1433,
1391, 1349, 1328, 1294, 1244, 1183, 1131, 1073, 1038, 986, 906, 860, 765, 733, 700 cm-1;
UV-Vis (EtOH): λmax (log ε) 437 (3.52), 282 (4.44), 231 (4.46) nm; ESI-TOF MS (m/z):
[M+H]+ calcd. for C16H16NO2, 254.1176; found, 254.1169.
304
2.3.11. 1-(1-Methylquinolin-1-ium-3-yl)-2,5-dioxocyclopentan-1-ide (8de)
305
306
307
308
1d (200.4 mg, 0.573 mmol), 6e (67.7 mg, 0.683 mmol), and K2CO3 (93.8 mg, 0.672 mmol)
were sequentially added to a reaction flask. To the reaction mixture was added 2.0 mL of a
mixture of EtOH:water (1:1, v/v, 2.0 mL), and the mixture was stirred at room temperature
for 18 h. The reaction mixture was separated by MPLC (reversed-phase C18, 26x460 mm,

14
309
310
311
312
313
gradient elution of 5-95% MeOH:water), to yield 68.7 mg of 8de, (0.287 mmol, 50% yield).
8de was obtained as yellow rod crystal; mp: decomposed at 280-290 ºC; 1H NMR (300 MHz,
CDCl3): δ 10.87 (d, J = 1.5 Hz, 1H), 10.20 (s, 1H), 8.14 (d, J = 7.5 Hz, 1H), 7.93 (d, J = 8.8
Hz, 1H), 7.87 (ddd, J = 8.7, 6.8, 1.7 Hz, 1H), 7.75 (td, J = 7.0, 1.0 Hz, 1H), 4.53 (s, 3H), 2.52
(s, 4H); 13C NMR (75 MHz, CDCl3):
130.5, 128.9, 116.4, 102.6, 45.1, 33.6 (2C); 1H NMR (400 MHz, CD3OD-d4):
δ
202.9 (2C), 146.3, 135.4, 134.0, 133.9, 131.5, 130.7,
314
315
316
317
318
319
320
321
δ 10.30 (d, J =
1.4 Hz, 1H), 9.80 (s, 1H), 8.29 (d, J = 8.9 Hz, 1H), 8.16 (dd, J = 8.2, 1.1 Hz, 1H), 7.99 (ddd,
J = 8.8, 7.1, 1.5 Hz, 1H), 7.84 (ddd, J = 8.0, 7.2, 0.7 Hz, 1H), 4.61 (s, 3H), 2.47 (s, 4H); 13C
NMR (100 MHz, CD3OD-d4): δ 204.5 (2C), 148.1, 138.2, 136.7, 133.7, 132.8, 131.4, 130.9,
130.4, 119.1, 105.2, 46.1, 33.8 (2C); IR (UATR): νmax 3043, 2917, 1599, 1553, 1470, 1447,
1413, 1341, 1288, 1256, 1138, 992, 926, 755 cm-1; UV-Vis (EtOH): λmax (log ε) 434 (3.93),
324 (4.31), 299 (4.30), 271 (4.59), 229 (4.57) nm; ESI-TOF MS (m/z): [M+H]+ calcd. for
C15H14NO2, 240.1019; found, 240.1017.
322
2.3.12. 2,2-Dimethyl-5-(1-methylquinolin-1-ium-3-yl)-4,6-dioxo-1,3-dioxan-5-ide (8df)
323
324
325
326
327
328
329
330
331
332
1d (201.5 mg, 0.570 mmol), 2,2-dimethyl-1,3-dioxane-4,6-dione (6f) (102.2 mg, 0.695
mmol), and K2CO3 (96.0 mg, 0.688 mmol) were sequentially added to a reaction flask. A
mixture (2.0 mL) of EtOH:water (1:1, v/v) was added to the reaction flask, and the mixture
was kept stirring at room temperature for 18 h. The reaction mixture was subjected to MPLC
separation (reversed-phase C18, 26x460 mm, gradient elution of 10-95% MeOH:water), to
yield 59.2 mg of 8df (0.208 mmol, 36% yield). 8df was obtained as orange block crystal; mp:
decomposed at 201-204 ºC; 1H NMR (300 MHz, CD3OD-d4):
δ 9.95 (d, J = 1.6 Hz, 1H),
9.51 (d, J = 1.5 Hz, 1H), 8.24 (dd, J = 8.9, 0.6 Hz, 1H), 8.11 (ddd, J = 8.2, 1.4, 0.5 Hz, 1H),
7.97 (ddd, J = 8.8, 7.1, 1.5 Hz, 1H), 7.82 (ddd, J = 8.1, 7.1, 1.0 Hz, 1H), 4.59 (s, 3H), 1.70 (s,
6H); 13C NMR (75 MHz, CD3OD-d4):
δ 167.9 (2C), 151.0, 141.5, 135.8, 135.2, 133.5, 131.0,

15
333
334
335
336
337
130.7, 130.3, 118.9, 102.8, 76.3, 46.0, 26.1 (2C); IR (UATR): νmax 3465, 3078, 2993, 2940,
1671, 1607, 1516, 1487, 1459, 1431, 1300, 1288, 1261, 1205, 1184, 1163, 1145, 1120, 1076,
1046, 988, 964, 934, 908, 880, 854, 831, 780, 764, 740, 699 cm-1; UV-Vis (EtOH): λmax (log
ε) 436 (2.85), 322 (3.27), 276 (3.57), 228 (3.64) nm; ESI-TOF MS (m/z): [M+H]+ calcd. for
C16H16NO4, 286.1074; found, 286.1076.
338
2.3.13. 2-(1Methylquinolin-1-ium-3-yl)-1,3-dioxo-2,3-dihydro-1H-inden-2-ide (8dg)
339
340
341
342
343
344
345
346
347
348
349
350
351
352
1d (202.0 mg, 0.571 mmol), 1,3-indandione (6g) (104.3 mg, 0.692 mmol), and K2CO3 (95.2
mg, 0.682 mmol) were added to a reaction flask, respectively. A mixture of EtOH:water (1:1,
v/v, 2.0 mL) was added to the reaction flask, and the reaction mixture was kept stirring at
room temperature for 18 h. The reaction mixture was subjected to MPLC separation
(reversed-phase C18, 26x460 mm, gradient elution of 10-95% MeOH:water), to yield 46.4
mg of 8dg (0.161 mmol, 28 % yield). 8dg was obtained as red amorphous solid; 1H NMR
(300 MHz, DMSO-d6):
δ 10.46 (d, J = 1.4 Hz, 1H), 9.75 (s, 1H), 8.26 (d, J = 8.7 Hz, 1H),
8.20 (d, J = 8.1 Hz, 1H), 7.86 (ddd, J = 8.3, 7.3, 1.1 Hz, 1H), 7.79 (dd, J = 7.9, 7.1 Hz, 1H),
7.41 (dd, J = 5.1, 3.1 Hz, 2H), 7.34 (dd, J = 5.2, 3.0 Hz, 2H), 4.57 (s, 3H); 13C NMR (100
MHz, DMSO-d6): δ 189.3 (2C), 145.6, 139.7 (2C), 133.7, 132.9, 131.2, 130.8 (2C), 130.6,
130.0, 129.0, 128.9, 118.6, 118.3 (2C), 97.1, 45.7; IR (UATR): νmax 3046, 1599, 1557, 1516,
1491, 1467, 1438, 1408, 1373, 1348, 1284, 1194, 1141, 1096, 957, 916, 890, 758, 719 cm-1;
UV-Vis (MeOH): λmax (log ε) 462 (3.80), 360 (4.37), 284 (4.33), 246 (4.09), 224 (4.36) nm;
ESI-TOF MS (m/z): [M+H]+ calcd. for C19H14NO2, 288.1019; found, 288.1027.
353
2.3.14. 2-(1-Methylquinolin-1-ium-3-yl) acetate (8dfM)
354
355
356
8df (6.0 mg, 0.021 mmol) was dissolved in 6.0 mL of 1.0 M HCl; this mixture was stirred at
room temperature for 48 h. The mixture was then neutralized with 1.0 M NaOH to pH 7, and
it was evaporated to dryness. The dry material was extracted with MeOH, to yield 3.5 mg of

16
357
358
359
360
361
362
363
8dfM (0.017 mmol, 80% yield). 8dfM was obtained as pale pinkish amorphous solid, 1H
NMR (300 MHz, CD3OD):
δ 9.34 (s, 1H), 8.98 (S, 1H), 8.46 (d, J = 8.9 Hz, 1H), 8.34 (d, J =
8.2 Hz, 1H), 8.22 (m, 1H), 8.01 (dd, J = 7.7, 7.6 Hz, 1H), 4.68 (s, 3H), 3.86 (br s, 2H); 13C
NMR (75 MHz, CD3OD):
δ 176.2, 152.8, 147.5, 138.6, 136.0, 134.5, 131.2, 131.1, 130.9,
119.4, 46.0, 42.1; IR (UATR): νmax 3382, 2925, 2854, 1591, 1529, 1373, 1237, 1118, 1038,
867, 775, 688 cm-1; UV-Vis (EtOH): λmax (log ε) 322 (3.56), 238 (4.25), 202 (4.14) nm; ESI-
TOF MS (m/z): [M+Na]+ calcd. For C12H11NO2Na, 224.0687; found, 224.0683.
364
2.3.15. 5,5-Dimethyl-2-(1-methylquinolin-4(1H)-ylidene)cyclohexane-1,3-dione (9ec)
365
366
367
368
369
370
371
372
373
374
375
376
377
1e (200.7 mg, 0.501 mmol), 6c (143.8 mg, 1.016 mmol), and K2CO3 (92.7 mg, 0.664 mmol)
were sequentially added to the reaction flask. A mixture of EtOH:water (1:1, v/v, 2.0 mL)
was added to the reaction flask, and the mixture was stirred at room temperature for 18 h. The
reaction mixture was separated by MPLC (reversed-phase C18, 26x460 mm, gradient elution
of 0-95% MeOH:water), to yield 126.3 mg of 9ec (0.449 mmol, 89% yield). 9ec was obtained
as yellow amorphous solid; 1H NMR (300 MHz, CDCl3):
δ 8.22 (d, J = 8.5, 1.3 Hz, 1H), 8.16
(d, J = 6.7 Hz, 1H), 8.01 (d, J = 6.7 Hz, 1H), 7.85 (ddd, J = 8.7, 7.0, 1.6 Hz, 1H), 7.68 (d, J =
8.7 Hz, 1H), 7.59 (ddd, J = 8.5, 7.0, 1.1 Hz, 1H), 4.14 (s, 3H), 2.47 (s, 4H), 1.19 (s, 6H); 13C
NMR (75 MHz, CDCl3): δ 192.1 (2C), 162.4, 142.9, 138.4, 134.0, 132.7, 128.5, 126.1, 123.4,
115.7, 111.5, 51.7 (2C), 43.2, 30.9, 28.8 (2C); IR (UATR): νmax 3393, 2954, 2921, 2865,
1616, 1503, 1397, 1357, 1245, 1227, 1171, 1142, 1117, 1074, 1037, 890, 842, 804, 761, 732
cm-1; UV-Vis (EtOH): λmax (log ε) 454 (4.10), 318 (4.00), 279 (4.69), 234 (4.81), 202 nm:
ESI-TOF MS (m/z): [M+H]+ calcd. for C18H20NO2, 282.1489; found, 282.1479.
378
2.3.16. 2-(1-Methylquinolin-4(1H)-ylidene)cyclohexane-1,3-dione (9ed)
379
380
1e (299.9 mg, 88% purity, 0.667 mmol), 6d (169.4 mg, 1.465 mmol), and K2CO3 (110.8 mg,
0.794 mmol) were sequentially added in a reaction flask. To the reaction mixture was added

17
381
382
383
384
385
386
387
388
389
390
391
2.0 mL of a mixture of EtOH:water (1:1, v/v); this mixture was stirred at room temperature
for 18 h. The reaction mixture was separated by MPLC (reversed-phase C18, 26x460 mm,
gradient elution of 0-95% MeOH:water), to yield 71.4 mg of 9ed (0.282 mmol, 42% yield).
9ed was obtained as yellow amorphous solid; 1H NMR (300 MHz, CDCl3):
8.6, 1.0 Hz, 1H), 8.18 (d, J = 6.7 Hz, 1H), 8.09 (d, J = 6.7 Hz, 1H), 7.87 (ddd, J = 8.7, 7.0,
δ 8.26 (dd, J =
1.6 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.61 (ddd, J = 8.4, 7.0, 1.2 Hz, 1H), 4.20 (s, 3H), 2.59
(dd, J = 6.4, 6.3 Hz, 4H), 2.13-2.03 (m, 2H); 13C NMR (75 MHz, CDCl3);
δ
193.0 (2C),
162.9, 142.8, 138.4, 134.1, 132.8, 128.6, 126.1, 123.5, 115.7, 112.7, 43.2, 38.0 (2C), 20.7; IR
(UATR): νmax 3022, 2924, 2850, 2359, 1613, 1514, 1401, 1359, 1328, 1183, 984, 855, 763
cm-1; UV-Vis (EtOH) λmax (log ε) 454 (3.36), 318 (3.27), 278 (3.95), 234 (4.07) nm; ESI-
TOF MS (m/z): [M+H]+ calcd. for C16H16NO2, 254.1176; found, 254.1167.
392
2.3.17. 2-(1-Methylquinolin-4(1H)-ylidene)cyclopentane-1,3-dione (9ee)
393
394
395
396
397
398
399
400
401
402
403
404
1e (301.4 mg, 88% purity, 0.671 mmol), 6e (135.4 mg, 1.339 mmol), and K2CO3 (113.7 mg,
0.814 mmol) were sequentially added to a reaction flask. A mixture (2.0 mL) of EtOH:water
(1:1, v/v) was added to the reaction flask, and the mixture was kept stirring at room
temperature for 18 h. The reaction mixture was subjected to MPLC separation (reversed
phase C18, 26x460 mm, gradient elution of 0-95% MeOH:water), to yield 29.5 mg of 9ee (
0.117 mmol, 17% yield). 9ee was obtained as yellow rod crystal; mp: decomposed at 182-185
ºC; 1H NMR (300 MHz, CDCl3):
δ 8.62 (ddd, J = 8.5, 1.4, 0.4 Hz, 1H), 8.35 (d, J = 6.9 Hz,
1H), 8.08 (d, J = 6.9 Hz, 1H), 7.89 (ddd, J = 8.7, 7.0, 1.6 Hz, 1H), 7.70 (dd, J = 8.6. 0.3 Hz,
1H), 7.64 (ddd, J = 8.4, 7.0, 1.2 Hz, 1H), 4.19 (s, 3H), 2.63 (s, 4H); 13C NMR (75 MHz,
CDCl3): δ 201.5 (2C), 156.3, 142.9, 138.8, 134.2, 132.9, 125.5, 125.3, 117.4, 115.3, 111.8,
43.0, 33.8 (2C); IR (UATR): νmax 3027, 2914, 1583, 1546, 1488, 1433, 1419, 1355, 1325,
1239, 1166, 986, 842, 783, 759 cm-1; UV-Vis (EtOH): λmax (log ε) 434 (3.36), 321 (2.91), 256

18
(3.53), 233 (3.63); ESI-TOF MS (m/z): [M+Na]+ calcd. for C15H13NO2Na, 262.0838; found,
405
406
262.0833.
407
2.4. Single crystal X-ray analysis of compounds 7ac, 8de, 8df and 9ee
408
409
X-ray diffraction data of 8de crystallized in monoclinic P21/c and 8df in triclinic were
collected at 296(2) K using a Bruker X8 APEXII KAPPA CCD diffractometer (MoKα
410
411
412
413
414
415
416
417
418
419
420
421
radiation,
Pna21 were collected at 296(2) K using a Bruker X8 PROSPECTOR KAPPA CCD
diffractometer (CuKα radiation, = 1.54178 Å). Data processing including integration,
λ = 0.71073 Å), whereas data of 7ac in monoclinic P21/n and 9ee in orthorhombic
λ
reduction together with multi-scan absorption correction, and merging were carried out with
standard softwares implemented in the APEX2 program suite [19]. The four structures were
solved by intrinsic phasing method with SHELXTL XT [20] and refined anisotropically by
full matrix least-squares on F2 with SHELXTL XLMP [21]. Absolute configuration of 9ee
was established by anomalous dispersion with copper radiation [Flack parameters (x) [22]
close to zero, 0.28(18)]. Crystal data of compounds 7ac, 8de, 8df and 9ee (CIFs) have been
deposited with the Cambridge Crystallographic Data Centre (CCDC 1864613–1864616).
They can be obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/cif. Details
of crystal data and refinement parameters are given in Supplementary material, Table S1.
422
2.5. Assay for cytotoxicity
423
424
425
426
427
428
Cytotoxic activity of compounds toward MRC-5 normal cell line (Normal human embryonic
lung cell line) and HepG2 (Human hepatocellular liver carcinoma cell line), HuCCA-1
(Human lung cholangiocarcinoma cancer cell line), and A549 (Human lung adenocarcinoma
cell line) cancer cells was assessed with the MTT assay [23]. Cells were grown under 5%
CO2 with 95% humidity, and cell culture medium was added to containing the test
compound, which was prepared in the culture medium to give a final concentration of 50

19
429
430
431
432
433
434
435
µg/mL. After incubation of cells with the test compound for 48h, cell viability was
determined using MTT assay. Briefly, the MTT reagent (100 mL) (0.5 mg/mL in serum-free
cell culture medium) was added to each well, and the microtiter plates were further incubated
for 2.5-4 h at 37 °C under 5% CO2 with 95% humidity. DMSO (100 mL) was added to the
medium in order to dissolve the purple formazan, and the absorbance at 550 nm was
measured using a Spectra-Max Plus 384 microplate reader with a reference wavelength of
650 nm.
436
437
438
3. Results and discussion
3.1 Synthesis
439
440
441
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450
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452
We found that quinolinium salts with electron withdrawing groups substituted at a
position 5, e.g., 5-bromo-, 5-chloro-, and 5-nitro quinolinium salts (1a-1c) had reactivity
toward 1,3-dicarbonyl compounds (Figure 2). At room temperature, treating a salt 1a with
dicarbonyls 6a and 6b in the presence of base, K2CO3, gave polycyclic compounds
(unpublished data). However, treating salts 1a-1c with cyclic diketones (6c, 6d, and 6e) gave
C2-functionalized quinoline derivatives 7 with yields of 19%-42% (Figure 2). The structure
of 7ac was confirmed by X-ray analysis (ORTEP plot in Supplementary material, Fig. S1).
C2-Functionalized quinoline derivatives with structures similar to 7ac and 7ad were
previously prepared by the reactions of 2-chloro- and 2-methylthio-1-methylquinolinium
iodides or quinoline N-oxides with 1,3-dicarbonyl compounds [24-26]. Previous methods for
the synthesis of compound 7 required more steps or using strong conditions, for example,
involving sequential insertions of oxygen and sulfur atoms at C2 of quinoline substrate [24]
and refluxing with acetic anhydride or using sodium hydride [25, 26]. The present method
provides a simple preparation of C2 quinoline derivatives.

20
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454
Figure 2. Structures of quinolinium salts (1a-1e) and dicarbonyl compounds (6) used in this
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work and syntheses of C2 quinoline derivatives (7).
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463
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467
Next we tried to prepare C3- and C4-substituted quinoline derivatives by coupling 3-
bromo- and 4-bromoquinolinium salts with diketones. However, the 4-iodoquinolinium salt
(1e) (Figure 3) was obtained instead of 4-bromoquinolinium salt, when treating 4-
bromoquinoline with MeI. The exchange of one halogen atom with another halogen atom for
1e is possibly similar to the Finkelstein reaction; complete halogen exchange reactions were
previously observed among cholro, bromo, and iodo compounds [27]. Treating salts 1d and
1e with cyclic diketones gave products 8 and 9 with yields between 13% and 89% (Figure 3).
Structures of 8de (ORTEP plot and bond distances of 8de, Figure 3) and 9ee (Supplementary
material, Fig. S2) were confirmed by X-ray analysis. Compounds with similar structures to
that of C4-functionalized quinolines (9ec, 9ed, and 9ee) were previously prepared from
substrates, 4-chloro- or 4-tosyloxyquinolines, 1-alkyl-2-alkylthioquinolinium iodides, and N-

21
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469
470
471
phenacylphenanthrolinium bromides [24, 28]. However, previous reports for the preparation
of compound 9 involving sequential insertions of oxygen and sulfur atoms at C2 of quinoline
substrate [24] or requiring the presence of oxygen or halogen at C2 of quinoline substrate
[28].
472
Br
EtOH/H2O (1:1), K2CO3
Diketones (6)
+
N
Compound 8
I
rt, 18-24 h
1d
O
O
O
O
O
O
N
N
N
8dd (13%)
8dc (18%)
8de (50%)
ORTEP plot of 8de
I
Bond distance of 8de
EtOH/H2O (1:1), K2CO3
rt, 18-24 h
Diketones (6)
+
N
Compound 9
I
1e
O
O
O
O
O
O
N
N
N
9ee (17%)
9ed (42%)
9ec (89%)
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474
Figure 3. Synthesis of C3- and C4-substituted quinoline derivatives, ORTEP plot and bond
distances (in Å) of 8de.
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477
478
The formation of mesomeric betaines 8dc, 8dd, and 8de is unprecedented. Recently,
479
Schaefer III computationally proposed that cyclopentadiene and cyclooctatetraene could be

22
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potential compounds for the preparation of zwitterions [29]. Accordingly, cyclooctatetraene,
1,3-cycloheptanedione, and Meldrum’s acid were used as substrates. Only Meldrum’s acid
gave 8df with yield of 36% (Figure 4). The structure of 8df was confirmed by X-Ray analysis
(ORTEP plot in Supplementary material, Fig. S3). Treating a salt 1d with 1,3-indandione
gave 28% yield of 8dg (Figure 4). Previously, 8dg was prepared by 6 step-synthesis, and at
least 2 steps used Pd metal for Sonogashira-Hagihara coupling [30]. The present work
provides a one-step and metal-free method for the preparation of such mesomeric betaine.
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Figure 4. Synthesis of 8df, 8dg and 8dfM.
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3.2 Solvatochromic properties of the betaine dyes
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502
Among the compounds synthesized, betaines 8dc, 8dd, 8de, 8df, and 8dg exhibited
interesting properties, and these dyes were studied for their solvatochromism and cytotoxic
properties. The betaine dye 8de had different properties in acidic and basic environments; it
is paritally dissolved in H2O or in basic solution (1.0 M NaOH), giving yellow color, but it is
well dissolved in acidic solution (solubility > 1.0 mg/mL), giving colorless solution. As
shown in Figure 5a, lanes 1 and 4, the dye 8de was partially dissolved in H2O or in 1.0 M
NaOH, giving a yellow solution, while it was completely dissolved in 1.0 M HCl
(concentration 1.0 mg/mL), but giving colorless solution (Figure 5a, lane 2). The dye 8de was
also completely dissolved in a mixture of HCl solution (1.0 M):EtOH (1:1, v/v) or NaOH
solution (1.0 M):EtOH (1:1, v/v), giving pale yellow and yellow solutions (Figure 5a, lanes 3
and 5), respectively. Interestingly, a color of a solution of 8de in 1.0 M HCl was changed
from colorless to yellow after neutralization with 1.0 M NaOH (Figure 5b, lane 2); this

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506
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511
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518
yellow solution was evaporated to dryness, giving a yellow solid of 8de (revealed by 1H
NMR spectrum). This experiment indicated that 8de was transformed into a colorless
compound under acidic condition, which could be converted back to its original structure
(yellow) by neutralization. Upon these data, it is concluded that the reversible interconversion
between yellow (neutral or basic condition) and colorless (acidic condition) forms of the dye
8de occurs by pH change. Such reversible interconversion was also observed for 8dc and
8dd, but not for 8dg. The electron delocalization in an aromatic ring of the1,3-indandione
moiety in 8dg might significantly prevent such reversible interconversion. Wavelength of
maximum absorbance (λmax) and molar extinction coefficient of compounds 8dc, 8dd, 8de,
and 8dg in MeOH, MeCN, and CHCl3 are summarized in Table 1. These dyes are not
fluorescent. Intersetingly, the zwitterion 8df took at least 30 h to become colorless in 1.0 M
HCl, but it was not converted back to yellow after neutralization. Detailed analysis (see
Supplementary material) revealed that 8df was transformed to the zwitterion 8dfM (Figure
4), suggesting that these mesomeric betaines may be usefull for the preparation of C3
substituted-quinoline zwitterion via a chemical degration; a positive iminium ion is stabilized
by the newly generated carbanion after degradation of these mesomeric betaines.
519

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520
521
Figure 5. a, 8de in: H2O (lane 1), 1.0 M HCl (lane 2), 1.0 M HCl:EtOH (1:1, v/v) (lane 3),
1.0 M NaOH (lane 4), and 1.0 M NaOH:EtOH (1:1, v/v) (lane 5). b, Neutralization of an
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523
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527
acidic solution of 8de with 1.0 M NaOH leads to color change from colorless to yellow (lane
2). c, Solutions of 8de in H2O, acetone:H2O (50:50, v/v), MeOH, MeCN, acetone, DMSO,
CHCl3, THF, and EtOAc (lanes 1-9). d, Addition of H2O to solutions of 8de in H2O,
acetone:H2O (50:50, v/v), MeOH, MeCN, acetone, DMSO, CHCl3, THF, and EtOAc (lanes
1-9).
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530
531
532
533
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535

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Table 1. Wavelength of maximum absorbance (λmax) and molar extinction coefficient of
compounds 8dc, 8dd, 8de, and 8dg in MeOH, MeCN, and CHCl3
Compound
8dc
λmax, Molar extinction coefficient at visible region
MeOH
MeCN
CHCl3
λmax 429 nm, Log
ε
3.45
λmax 474 nm, Log
ε
ε
ε
ε
3.57 λmax 477 nm, Log
ε
3.45
3.40
8dd
λmax 426.5 nm, Log
ε
3.38 λmax 426 nm, Log
3.49 λmax 464.5 nm, Log
ε
8de
λmax 426 nm, Log
ε
ε
3.60
3.87
λmax 468 nm, Log
λmax 484 nm, Log
3.69 λmax 477 nm, Log
ε
ε
3.63
3.78
8dg
λmax 426 nm, Log
3.87 λmax 506 nm, Log
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539
540
Reversible interconversion between yellow and colorless forms of 8de was monitored
by UV-Vis spectrum over pH ranges 1.0-12.7; when pH of the solution increased, the
absorbances at wavelengths 227 nm, 270 nm, and 400 nm increased, while those at 250 nm
and 358 nm decreased (Figure 6). The pKa value of compound 8de was 2.5 (Supplementary
material, Fig. S4). Note that color changes of 8de solution were clearly observed at pH ranges
of 1.5 (colorless) to 4.0 (yellow). Accordingly, five cycles of pH adjustment using HCl and
NaOH solutions were performed, starting from pH 4.0 to pH 1.5 (yellow to colorless), then
from pH 1.5 back to pH 4.0 (colorless to yellow) (Figure 7), confirming the interconversion
of yellow and colorless forms of 8de. The yellow material obatined after five cycles of pH
changes had identical 1H NMR spectrum to that of 8de, indicating that the dye 8de is stable
in both acidic and basic conditions. Structure elucidation of the colorless form of 8de was
conducted in an NMR tube, using trifluoroacetic acid (TFA) as an acid (see Supplementary
material). The upfield shift of 13C NMR resonance of the two ketone carbonyls was observed
from δC 204.5 for yellow zwitterion (before the addition of TFA) to δC 199.6 for colorless
form (after the addition of TFA), supporting the presence of a double bond of an enol form
(Figure 7). However, the 13C NMR resonance exhibited only one line for the carbonyl signal
at δC 199.6, therefore, the posibility for the presence of the keto structure of the colorless
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551
552
553
554
555
556

26
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form could not be ruled out. Note that, when pH of the dye 8dg solution increased, the
absorbances at wavelengths 283 nm, 348 nm, and 435 nm increased, while those at 240 nm
and 263 nm decreased (Supplementary material, Fig. S5). However, unlike the dye 8de, the
color of 8dg solutions remained unchanged under various pH conditions.
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564
Figure 6. UV-Vis spectrum (0.01 mg/mL in water) of 8de over pH ranges of 1.0-12.7, and a
plot of pH and absorbance at 400 nm.
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567

27
568
O
O
Acid
2
Base
O
OH
N
N
Cl
H
8de
Enol form
Yellow
Colorless
569
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572
Figure 7. UV-Vis spectrum over pH ranges 1.5-4.0 (0.01mg/mL in water) and the proposed
structure of the colorless form of 8de.
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577
The dye 8de had different colors, i.e., yellow, orange, and red, when dissolved in
organic solvents; its color depended on solvents. Accordingly, the solvatochromism of 8de
was preliminarily investigated. Solutions of 8de in H2O, acetone:H2O (50:50, v/v), and
MeOH were yellow (Figure 5c, lanes 1-3), while those in MeCN (lane 4), acetone (lane 5),

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584
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591
DMSO (lane 6), CHCl3 (lane 7), THF (lane8), and EtOAc (lane 9) were orange-red. UV-Vis
spectrum of 8de in these solvents is shown in Figure 8; λmax values at the visible region in
water (400 nm), acetone:H2O (50:50) (420 nm), MeOH (426 nm), MeCN (468 nm), DMSO
(469 nm), CHCl3 (477 nm), acetone (479 nm), THF (490 nm), and EtOAc (490 nm) are
displayed in Figure 8. The λmax value of 8de in water (400 nm) is 90 nm different from that of
THF and EtOAc. Addition of H2O to the solutions of MeCN (lane 4), acetone (lane 5),
DMSO (lane 6), THF (lane 8), and EtOAc (lane 9) led to a color change from orange-red to
yellow. However, red solution of 8de in CHCl3 remained unchanged (Figure 5d, lane 7,
bottom layer). After the addition H2O, solutions of 8de in CHCl3 and EtOAc had two layers;
both H2O layers were yellow (Figure 5d, lane 7, top layer; lane 9, bottom layer). This
experiment demonstrated that the present betaine dye can be dissolved in both polar (water)
and non-polar solvents (CHCl3 and EtOAc) since it was in both layers of the solvents.
Moreover, the dye 8de was sensive to water contents; red shift of ca 40 nm was observed at
the visible region of 8de in EtOH (compared to that in H2O, Figure 9).
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593