Characterization of Push-Pull-Type Benzo[X]quinoline Derivatives (X = g or f): Environmentally Responsive Fluorescent Dyes with Multiple Functions

Article abstract of DOI:10.1021/acs.joc.0c01878

Benzo[X]quinoline (X = g or f: BQX) derivatives bearing bis-trifluoromethyl and amine groups have been designed as push-pull-type fluorescent dyes. Through the synthesis of BQX derivatives from 2,7-diaminonaphthalene, linear-type (BQL) and angular-type (BQA) structural isomers were obtained. X-ray structures of single crystals from six given BQX derivatives revealed that the BQL and BQA series adopt planar- and bowl-shaped structures. In the fluorescence spectra, interestingly, the BQL series emitted in the near-infrared region over 700 nm in polar solvents. Based on the visible absorptions and base properties related to the amine moiety, the ammonia responsiveness was investigated using an ion-exchange reaction by the BQX-HCl salt. By exploiting the environmentally responsive fluorescence probe, cell imaging through confocal laser microscopy was conducted using HeLa and 3T3-L1 cells, emitting specific lipid droplets. The results indicate that BQX derivatives have multiple functions and may be applied in materials chemistry and biochemistry.

Full text of DOI:10.1021/acs.joc.0c01878

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Article
Characterization of Push−Pull-Type Benzo[X]quinoline Derivatives
(X = g or f): Environmentally Responsive Fluorescent Dyes with
Multiple Functions
Yasufumi Fuchi,*,§ Tomohiro Umeno,§ Yuichiro Abe, Keita Ikeno, Ryu Yamasaki, Iwao Okamoto,
Kazuteru Usui,* and Satoru Karasawa*
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ABSTRACT: Benzo[X]quinoline (X = g or f: BQX) derivatives bearing bis-
trifluoromethyl and amine groups have been designed as push−pull-type fluorescent
dyes. Through the synthesis of BQX derivatives from 2,7-diaminonaphthalene, linear-
type (BQL) and angular-type (BQA) structural isomers were obtained. X-ray
structures of single crystals from six given BQX derivatives revealed that the BQL and
BQA series adopt planar- and bowl-shaped structures. In the fluorescence spectra,
interestingly, the BQL series emitted in the near-infrared region over 700 nm in polar
solvents. Based on the visible absorptions and base properties related to the amine
moiety, the ammonia responsiveness was investigated using an ion-exchange reaction
by the BQX-HCl salt. By exploiting the environmentally responsive fluorescence
probe, cell imaging through confocal laser microscopy was conducted using HeLa and 3T3-L1 cells, emitting specific lipid droplets.
The results indicate that BQX derivatives have multiple functions and may be applied in materials chemistry and biochemistry.
Scheme 1. Molecular Structures of Fluorescent
Benzoquinoline Derivatives
1. INTRODUCTION
a
Push−pull-type fluorescent dyes possessing electron-donor and
-acceptor groups on π-conjugated rings have applications in
materials chemistry and biochemistry.1 For example, Prodan
and dansyl analogs, which are well-known push−pull-type
fluorescent dyes consisting of a bicyclic naphthalene frame-
work,2 yield electron spectra with solvatochromic behavior
owing to photoinduced intramolecular charge transfer (PICT)
in an excited state.3 Solvent-dependent emission changes
triggered by the PICT indicate the environmentally responsive
characteristics of fluorescent dyes. Therefore, various fluo-
rophores with push−pull-type fluorescent dyes have been used
for applications as environmentally responsive fluorescent
probes. In biochemistry,4 Nile Red, which is composed of a
polycyclic aromatic hydrocarbon, is a fluorescent cellular
bioprobe and exhibits a bathochromic shift in polar solvents.5
In addition to Nile Red, various fluorescent probes have been
used for specific staining of lipid droplets (LDs) by utilizing
their environmentally responsive properties.6,7 LDs are
composed of neutral lipids and are considered intracellular
fat storage organelles.8
a
(A) Previous reported analogues; (B) TFMAQ and benzo[X]-
quinoline derivatives (BQL series (X = g) and BQA series (X = f))
with electron-donor and -acceptor substituents on π-rings.
Additionally, quinoline analogs, such as quinine sulfate,9
have been frequently used as bicyclic fluorophores.10−12
Previous studies have also introduced bis-trifluoromethyl and
amino groups on the quinoline ring, whereby fluorescent
aminoquinoline derivatives (TFMAQ)13 have been developed
as fluorescent dyes (Scheme 1) that respond to various
external stimuli such as solvent polarity,13 heat,13,14 light,15 and
mechanical stimuli.14,16 Moreover, amine-responsive films with
Received: August 4, 2020
Published: September 17, 2020
https://dx.doi.org/10.1021/acs.joc.0c01878
© XXXX American Chemical Society
J. Org. Chem. XXXX, XXX, XXX−XXX
A

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a
Scheme 2. Scheme for the Syntheses of BQL and BQA and Their Derivatives
a
Reagents and conditions: (i) NaHSO3, 28% NH3 aq., sealed tube, 170 °C, 92%; (ii) tosyl chloride, pyridine, rt, 75%; (iii) hexafluoroacetylacetone,
montmorillonite K10, 1,4-dioxane, 60 °C; (iv) conc. H2SO4, 100 °C, 5% for BQL and 32% for BQA; (v) bromobenzene, Pd(OAc)2, t-Bu3P, t-
BuOK, toluene, 100 °C, 31% for BQL-Ph and 38% for BQL-diPh; (vi) bromobenzene, Pd(DPPF)Cl2, DPPF, t-BuOK, 1,4-dioxane, 100 °C, 62%;
(vii) bromobenzene, Pd(OAc)2, t-Bu3P, t-BuOK, toluene, 100 °C, 99%.
absorption color change were developed as an amine sensor.17
In addition to materials chemistry, there have been recent
attempts to develop such materials for use in biochemistry. For
example, specific fluorescence imaging of LDs with a greenish
fluorescent color,18 apoptosis-inducing reagents such as
photosensitizers,19 and thermoresponsive nanomaterials as in
vivo tumor-imaging agents have been reported.20
properties. Finally, based on polarity-responsive fluorescence,
intracellular fluorescence imaging of LDs in live cells was
described for bioapplication.
2. RESULTS AND DISCUSSION
2.1. Synthesis. The scheme for synthesizing the BQX
framework is shown in Scheme 2. According to a previous
report,26 the dihydroxyl group of 2,7-dihydroxynaphthalene
was converted to the diamine group via the Bucherer reaction
in the presence of sodium hydrogen sulfite under an aqueous
ammonia solution into a sealed tube. Considering the low yield
of the linear-type BQL derivative,22−25 to increase the reaction
yield of BQL, one amine group of compound 1 was selectively
protected by a tosyl group, producing compound 2 with a 75%
yield. Direct synthesis from compound 1 to BQL via Combes
quinoline synthesis yielded the unexpected product BQA
(23%). In contrast, BQL and BQA were obtained in 5 and
32% yields through a two-step synthesis from the protected 2
via an imine intermediate by Combes quinoline synthesis.
From the viewpoint of the resonance energy between the
phenanthrene (angular-type) and anthracene (linear-type)
isomers,27 the former was more stable than the latter,
suggesting that the angular-type BQX is predominantly
obtained via the Combes quinoline cyclization. Previous
reports indicate that the linear-type BQX is the main product
when concentrated sulfuric acid is used in the cyclization
step.28 Therefore, BQL synthesis requires further investigation
to obtain a higher yield via kinetic control by considering
factors such as orbital overlaps and functional groups.
Modifications of amine groups by phenyl substitution via
Buchwald−Hartwig cross-coupling were conducted in the
presence of a Pd catalyst with a phosphine ligand. BQL-Ph and
BQL-diPh were synthesized simultaneously in one batch in the
presence of Pd(OAc)2 using t-Bu3P (catalyst A), obtaining 31
and 38% yields, respectively. Using a stepwise phenyl-
The bicyclic framework needs to be expanded by aromatic
rings to prompt the various forms of responsiveness at a lower
energy and to obtain near-infrared spectra using push−pull-
type compounds. In this study, we designed novel tricyclic
benzo[X]quinoline (BQX) derivatives consisting of a ring-
expanded TFMAQ framework. The tricyclic frameworks
introduced with the electron push−pull groups are expected
to show a red shift in the electron spectra. Various additional
advantages, such as a large Stokes shift and an enhancement of
fluorescence intensity compared to those without the push−
pull groups, may be realized. In previous studies related to
benzoquinoline analogs, a representative 7,8-benzoquinoline
(benzo[h]quinoline) composed of the angular-type structural
isomer (Scheme 1A) was investigated for use as a fluorescent
dye.21 However, the examination of the fluorescent dyes using
linear-type BQX derivatives (benzo[g]quinoline22 or benzo-
[g]isoquinoline23−25 in Scheme 1A) was limited because of the
difficulty encountered in their selective synthesis. These BQX
derivatives also emitted in the non-near-infrared region (<550
nm) in the solution. Through the introduction of push−pull-
type substituents on the benzoquinoline ring, long-wavelength
emissions (>650 nm) are expected. This paper reports on the
syntheses, structural analyses, and photophysical properties of
novel fluorescent benzo[g]quinoline (BQL) and benzo[f]-
quinoline (BQA) derivatives, together with their phenyl-
substituted derivatives (-Ph and -diPh). Ammonia responsive-
ness using an ion-exchange reaction of BQX-HCl salt was also
examined, utilizing the strong visible absorptions and base
B
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substituent reaction, BQA-Ph was prepared from BQA in the
presence of Pd(DPPF)Cl2 in 62% yield. Further, BQA-diPh
synthesis was conducted from BQA-Ph using catalyst A in a
quantitative yield. The molecular structures of BQX derivatives
were characterized by nuclear magnetic resonance (NMR)
spectroscopy, infrared (IR) spectroscopy, and electrospray
ionization mass spectrometry (ESI).
2.2. X-ray Crystal Structure Analysis. The crystal
structures of BQX derivatives were characterized by X-ray
structure analysis. Single crystals suitable for X-ray analysis of
all six BQX derivatives (BQL, -Ph, and -diPh, and BQA, -Ph,
and -diPh) were obtained by recrystallization from various
solvents. The molecular structures and Oak Ridge thermal
ellipsoid plot (ORTEP) drawings with molecular packings are
shown in Figures 1 and S1−S6. The crystallographic
parameters and significant short contacts and angles are
summarized in Tables 1 and S1−S4.
Table 1. Selected Intramolecular Angles and Distances, and
Significant Intermolecular Interactions
angle/deg
Q-
distance/Å
BQX (space
group) and Z′
D−
d
a
b
c
e
Ph
θ1
F−H
A
interactions (Å)
BQL (Fdd2),
Z′ = 1
BQL-Ph (P21/
c), Z′ = 1
BQL-diPh (C2/ 1.5
c), Z′ = 1
BQA (P21)
Z′ = 2
2.4
23.4
2.82
34.7
36.6,
2.41
7.4 π−π (3.40), HB
(3.34)
1.6
2.52
2.52
2.16,
7.4 π−π (3.47), CH−n
(2.60)
7.4 π−π (3.76), F−F
(2.94),
6.3,
6.4
6.3 π−π (3.52, 3.60), HB
41.1
2.19
(3.10, 3.27)
BQA-Ph (P1
̅
)
7.5
8.9
16.8
2.18
6.3 π−π (3.26-3.38),
Z′ = 1
CH−n (2.74)
BQA-diPh (P1
̅
)
33.4
2.18
6.3 π−π (3.56), CH−π
(2.68−2.83)
Z′ = 1
a
Torsion angles between quinoline rings and fused phenyl rings
b
(LQ‑Ph). Dihedral angles between fused edged-phenyl rings in BQX
c
and the amine moiety (LPh‑NR1R2 in Figure 1C). Between F atoms in
CF3 groups and H atoms (C5 atoms for BQL series and C10 atoms
d
for BQA series). Between the centroid in pyridine rings and N atoms
e
in the amine moiety. Significant intermolecular interactions for
packing. HB, hydrogen bond; π−π, π−π interaction; CH−n, CH−n
interaction; F−F, F−F interaction; CH−π, CH−π interaction.
= 23.4°, and 36.6 and 41.1° for BQL and BQA. This suggests
that the amine of BQL contributes to a relatively high sp2
hybrid character.13 The 1.1 Å difference in the D−A distance
and the sp2 hybrid character may be responsible for the
electron spectra in BQX derivatives and the base property
(pKa) at the amine moieties (vide infra). In terms of the
packing of BQL, the H atoms on the amine moiety formed a
long hydrogen bond with N1 (N···N: 3.34 Å) of the
neighboring molecule, forming zigzag chains along the c-axis
(Figure S1). The planar BQL molecule interacted with
neighboring molecules by proximal π−π stacking (3.40 Å),
forming a head-to-head slipped-columnar fusion (Tables 1, S3,
and Figure S1). For BQA, BQA formed zigzag chains with
intermolecular hydrogen bonds at the N9 and N4 atoms of
3.10 and 3.27 Å along the a-axis, respectively. Furthermore,
π−π stacking with head-to-head slipped-columnar fusion (3.52
or 3.60 Å, Figure S4) was also observed with the twisted form.
For the monophenyl-substituted BQL-Ph and BQA-Ph
(Figures S2 and S5), the phenylamine moieties adopted syn
conformations, wherein both derivatives formed a round shape.
In peripheral amine moieties, through the decrease in dihedral
angles (θ1 = 2.82 and 16.8° for BQL-Ph and BQA-Ph), the
planarity within the BQX rings was maintained (LQ‑Ph = 1.6
and 7.5° for BQL-Ph and BQA-Ph). This yielded a higher sp2
hybridization character compared to those of BQL and BQA.
In the packings, the hydrogen bonds based on the secondary
amine group were absent in both BQX-Ph. Instead of
hydrogen bonds, CH−n interactions involving H atoms at
the phenyl rings and the lone pair of N atoms in the pyridine
rings were observed with 2.60 and 2.74 Å for BQL-Ph and
BQA-Ph, respectively. Surprisingly, closed π−π interactions of
3.26 and 3.38 Å with a head-to-tail fusion and the phenyl rings
at the phenylamine moiety were observed in BQA-Ph,
suggesting a strong electron effect in the electron spectra in
the solid state (vide infra).
Figure 1. (A) Structural features of BQL and BQA; (B) top- and
side-views of the crystal structures of BQL and BQA. Reddish broken
lines indicate D−A distances between the centroids of pyridine rings
and amine moieties, which were 7.4 and 6.3 Å for BQL (a) and BQA
(b and c), respectively. Reddish dotted lines represent CH···F
interactions of 2.49 (d), 2.19 (e), and 2.16 Å (f); (C) dihedral angles
θ1 between the fused Ph rings and amine moieties (LPh‑NR1R2).
In the crystal structures of BQL and BQA (Figures 1, S1,
and S4), the molecular twist based on the torsions of the BQX
ring and the donor−acceptor (D−A) distances were
considerably different. The torsion angles between a quinoline
ring and the fused phenyl rings on the BQX rings (LQ‑Ph in
Table 1) were estimated at 2.4 and 6.3−6.4°, forming a
pseudo-planar and a twisted bowl-shaped structure for BQL
and BQA, respectively (Figure 1A,B). This twisted structure of
BQA yielded the upward or downward forms frequently
observed in benzo[f ]quinoline derivatives.29,30 Triggered by
the bowl-shaped structure of BQA, the intramolecular short
contacts between F and H atoms on C10 atoms were below 2.2
Å, which was shorter than the sum of the van der Waals radii
(2.67 Å) (Figure 1B).31 The corresponding distance in BQL
was 2.41 Å (F−H at C5). The different isomers based on the
benzo[X]quinoline framework (X = g or f) exhibited different
D−A distances (between the donor and the acceptor moiety),
which were estimated based on the distance between the
centroid of the pyridine ring and the N atom of the amine
moiety, yielding 7.4 and 6.3 Å for BQL and BQA, respectively
(Figure 1B). The amine moieties adopted a pyramidal form,
and the dihedral angles between the fused phenyl rings and the
plane consisting of LN‑H‑H (Figure 1C) were estimated to be θ1
For the diphenylamine derivatives (BQL- and BQA-diPh in
Figures S3 and S6), the dihedral angles of peripheral tertiary
amine moieties were 34.7 and 33.4° for BQL-diPh and BQA-
C
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diPh, suggesting that the contribution of sp3 hybridizations of
the amine moiety increased, and the donor effect of the phenyl
rings may have diminished via twisting. In BQA-diPh, the
torsion angle within the BQX rings had increased (LQ‑Ph
=
8.9°) compared to those in BQA (6.3 and 6.4°) and BQA-Ph
(7.5°). The twisting of the BQX ring was also enhanced by an
increase in the substitution number of the phenyl ring. Owing
to the bulkiness of phenyl substitutions in the packing, the π−π
interactions had elongated to 3.76 and 3.56 Å, whereby
multiple weak interactions such as CH−F, F−F, and F−H
appeared.
The X-ray analyses provided insights into the differences in
D−A distances, molecular twisting, and molecular packing.
The 1.1 Å difference in the D−A distance between the BQL
and BQA derivatives and the twisting difference of BQX rings
may be the cause of the disparity in the donor effect in the
ultraviolet−visible (UV−vis) spectra and the pKa value,
respectively. The packing differences may affect the differences
in the solid electron spectra (Table 4 and Figure 5).
2.3. Photophysical Properties in Solution. The
absorption and emission spectra of BQL and BQA were
acquired in eight organic solvents: n-hexane (Hex), 1,4-
dioxane (DOX), dibutylether (Bu2O), chloroform (CHCl3),
ethyl acetate (AcOEt), n-butyl alcohol (BuOH), acetonitrile
(MeCN), and methanol (MeOH). The absorption and
emission spectra (excited at 450−480 nm for BQL and
400−420 nm for BQA), together with photographs of each
solution (in Hex, CHCl3, and AcOEt), taken under UV light
(365 nm), are shown in Figures 2, 3A, and S7−S10. The
resulting parameters including absorption or fluorescence
Figure 3. (A) Photographs taken under UV light (365 nm) in n-
hexane, chloroform, and ethyl acetate. Lippert−Mataga plots of (B)
BQL series and (C) BQA series. Circles, squares, and triangles
represent BQX, BQX-Ph, and BQX-diPh, respectively. The solid
lines are the least-squares fittings of each plot. Stokes shifts (Δν) and
fl
abs
Δf values were calculated from ν − ν (cm−1) and (ε − 1)/(2ε +
max
max
1)-(n2 − 1)/(2n2 + 1), respectively. ε and n represent the dielectric
constant and refractive index, respectively.
(ε), absolute fluorescence quantum yields (ϕfl), and
fluorescence lifetimes (τ) are summarized in Tables 2,3, and
S5−S10. The radiative rate constants (kr) and nonradiative
decay constants (knr) were determined based on the given ϕfl
and τ values, respectively.
abs
maxima (λmax or λmaxfl), molar absorption coefficient values
In the absorption of BQL and BQA (Figure 2), broadened
peaks over 400 nm with ε values between 2.0 × 103 and 3.0 ×
103 were observed at the longest absorptions. The λmaxab values
Table 2. Photophysical Values of BQL and BQA Solutions
a
(20 μM)
b
ε × 103
λ
Φfl
τ/ns
knr
ab
max
fl
max
solvent
λ
BQL
Hex
DOX
Bu2O
CHCl3
AcOEt
BuOH
MeCN
MeOH
Hex
446
468
467
452
473
484
469
479
404
417
420
410
423
417
418
424
2.69
3.34
2.99
3.00
3.25
3.09
3.10
2.42
2.19
2.38
2.39
2.29
2.43
2.55
2.41
2.32
537
610
588
607
636
679
673
721
481
569
547
574
589
627
638
657
0.67
0.09
0.16
0.07
34.2
6.41
9.17
3.87
6.54
n.d
0.010
0.14
0.092
0.24
0.03
0.15
<0.01
<0.01
<0.01
0.10
0.09
0.14
0.07
0.05
0.01
<0.01
<0.01
n.d
n.d
c
BQA
4.65
10.6
13.4
4.17
5.25
0.95
n.d.
n.d
0.19
0.086
0.064
0.22
0.18
1.0
DOX
Bu2O
CHCl3
AcOEt
BuOH
MeCN
MeOH
a
ab
fl
The values of λmax and λmax are in nm. n.d., not detected.
Excitation at a maximum of the corresponding excited spectra under
b
Figure 2. Emission (solid line) and absorption (dotted line) spectra
of (A) BQL and (B) BQA in the given eight solvents.
individual conditions. The knr values were calculated from (1 − φ)/τ
c
ab
(ns−1). λmax values were estimated using multipeak fitting.
D
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decay process may be present in the BQA series. The observed
fluorescence quenching in polar solutions (especially in BQL
series) and the positive solvatochromic behavior may be
caused by the stabilization of the PICT state in the excited
state17,32 for typical push−pull-type fluorophores.
To quantitatively elucidate the photophysical properties
according to Lippert−Mataga plots,13,17,33 the Stokes shift
(Δν, cm−1) values were plotted as a function of solvent
polarity (Δf) (Figure 3B,C). From the slope of the linear fitted
lines, the difference in the dipole moment between the ground
and excited states (Δμ) was determined using eq 1
Table 3. Selected Photophysical Data of BQL-Ph, BQL-
diPh, BQA-Ph, and BQA-diPh in various solvents
a
b
c
fl
max
solvents
λambasx/nm
ε
λ
/nm
Φfl
BQL-Ph
Hex
458
471
478
489
475
487
483
485
422
433
440
439
444
445
442
4.34
4.57
4.72
4.98
7.23
7.20
6.44
6.59
2.22
2.08
2.45
2.43
2.75
2.65
2.96
2.82
547
0.63
0.11
0.05
n.d.
0.85
0.28
0.18
n.d.
0.10
0.09
0.07
n.d.
0.13
0.19
0.12
n.d.
CHCl3
AcOEt
MeOH
Hex
CHCl3
AcOEt
MeOH
Hex
CHCl3
AcOEt
MeOH
Hex
CHCl3
AcOEt
MeOH
626
638
731
567
636
644
743
494
587
588
669
510
592
599
677
BQL-diPh
d
2Δμ2
BQA-Ph
Δν =
Δf + constant
hca3
(1)
where h and c are Planck’s constant and the velocity of light,
respectively, and a is the Onsager radius of the molecule. The
radii of BQL and BQA were 4.50 and 4.14 Å, respectively, as
estimated from the density-functional theory (DFT) calcu-
lations.34 In the plots, a positive correlation between Δf and
Δμ was observed in the same manner as for TFMAQ.13 The
estimated Δμ values for BQL and BQA were 8.5 and 9.0
Debye (D), respectively (slightly higher than that of TFMAQ
(8.1 D)),13 indicating that BQA is significantly influenced by
solvent polarity in the excited state. The D−A distances of
BQL and BQA estimated by X-ray analysis were 7.4 and 6.3 Å
(Table 1), respectively, with shorter D−A distances being
expected to produce larger Δμ values. The phenyl-substituted
species, BQL-Ph (a = 4.68 Å), BQL-diPh (a = 4.86 Å), BQA-
Ph (a = 4.59 Å), and BQA-diPh (a = 4.68 Å), yielded Δμ
values of 9.4, 11.2, 10.6, and 11.5 D, respectively. The ΔΔμ
values (ΔBQX‑diPh − ΔBQX‑Ph) were 1.8 and 0.9 for BQL series
and BQA series, respectively. This suggests that the donor
effect of the phenyl ring of the BQA series was limited owing
to the twisting of the BQA rings (Table 1).
The fluorescence decays of BQL and BQA in various
solutions were traced, and τ values were obtained using first-
order decay analysis (Figures S11−16 and Tables 2, S5, S8). In
the nonpolar Hex solution, the τ value of BQL was notably
larger (34.2 ns) than that of BQA (4.65 ns). As the solvent
polarity increased, the τ of BQL decreased, accompanied by a
reduction in ϕfl and an emission red shift. In contrast, the
solvent dependence of τ for BQA was smaller than that for
BQL. The nonradiative decay constants (knr) were determined
based on the given τ and ϕfl of BQL, according to the
relationship (1 − ϕfl)/τ, yielding a strong solvent dependence
(0.010−0.22 ns−1 in Hex, DOX, Bu2O, CHCl3, and AcOEt).
This result suggests that nonradiative decay of BQL is
regulated by the polarity surrounding the fluorophores; this
means that the PICT transition is stabilized by more polar
solvents for fluorescent molecules bearing donor−acceptor
groups. In contrast, the knr of BQA yielded a subtle solvent
dependence (0.064−0.22 ns−1 in Hex, DOX, Bu2O, CHCl3,
and AcOEt), suggesting the presence of another decay process.
One possible route for the decay process in BQA is energy loss
due to the rapid conformational change related to the twisted
BQX rings. This means that the nonplanar twisted-BQA ring
may move up or down triggered by heat; these conformers
would be in equilibrium in the solution under ambient
conditions (Figure 4). To confirm the earlier hypothesis,
variable-temperature (VT) NMR was conducted for BQA in
CD2Cl2 from 20 to −80 °C (Figure S17). Upon lowering the
temperature, a remarkable upfield-shift in the resonance of H10
d
BQA-diPh
442
a
b
The unit is × 103 M−1 cm−1. Excitation at a maximum of the
c
corresponding excited spectrum under individual conditions. n.d.,
not detected. λmax values were estimated by multipeak fitting.
d
ab
of BQL (446−484 nm) were considerably longer than those of
BQA (404−424 nm). These absorptions are attributed to the
S0 → S1 transitions, including a charge-transfer character based
on the highest occupied molecular orbital (HOMO) → lowest
unoccupied molecular orbital (LUMO) transition (vide infra).
ab
With the increase in solvent polarity, λmax showed a red shift
ab
based on positive solvatochromic behavior. The different λmax
between BQL and BQA was a result of the twisted structure of
the BQX rings. BQA, which possessed large torsion angles
ab
within the BQX ring, showed a short λmax owing to limited
conjugation. In the fluorescence spectra, BQL had a longer
wavelength emission (537−721 nm) compared to BQA (481−
657 nm) as for the absorption spectra. As solvent polarity
fl
increased, the λmax of BQL showed a red shift. Interestingly,
near-infrared emissions over 650 nm were observed in polar
solvents such as BuOH, MeCN, and MeOH. In terms of the
solvent dependence of the ϕfl values, BQL had a drastic
decrease from a nonpolar (0.67) to a polar solvent (<0.01);
however, BQA demonstrated only a small dependence in the
nonpolar and medium-polar solvents (0.14 and 0.05).
In phenyl-substituted BQL-Ph and BQL-diPh (Figures S7
and S8; Tables 3, S6 and S7), there was a red shift in the
absorption and emission spectra due to the donor effect of the
phenyl rings. There was also a considerable increase in the ε
values. As for BQL, the emission spectra in the polar solvents
had near-infrared emissions around 700 nm. Through the
substitution of two phenyl rings, the ϕfl values of BQL-diPh
increased to 0.85 in nonpolar n-hexane compared to those of
BQL and BQL-Ph (0.67 and 0.63). This enhancement in ϕfl
resulted from the loss of the H atoms in the amine moiety and
the substituted phenyl ring effect. In contrast, in BQA-Ph and
BQA-diPh (Figures S9 and S10; Tables 3, S9, and S10),
similar red shifts of absorption (with enhancement of ε values)
and emissions were clearly observed. However, the near-
infrared emission over 700 nm was absent even for BQA-diPh.
For the ϕfl of BQA-diPh, a subtle increase in Hex was
observed (0.13) owing to the loss of the NH group, compared
to BQA and BQA-Ph (0.10). These results suggest that, as
opposed to polarity dependence, another essential nonradiative
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resulting absorption and fluorescence spectra of BQX
derivatives in the solid state are shown in Figure S19, and
images under room and UV lighting are shown in Figure 5A.
The photophysical parameters are summarized in Table 4. In
Table 4. Photophysical Parameters of BQX Derivatives in
the Solid State
π−π stacking
a
b
c
Φfl
distance/Å
θ2/deg
ab
max
fl
max
λ
/nm
λ
/nm
Figure 4. Proposed equilibrium between twisted-BQA conformers in
the solution state. Bottom figures are the side-views obtained by X-ray
structures with dotted reddish lines (CH···F interactions by 2.16 Å).
BQL
552
613
0.01
0.06
0.01
0.06
0.03
0.26
3.40
3.46
3.70
3.52, 3.60
3.27
45.4
46.9
55.4
BQL-Ph
BQL-diPh
BQA
BQA-Ph
BQA-diPh
510
543
423
440
459
585
625
545
575
542
and a subtle upfield-shift for H6 were observed. Based on X-ray
analysis, the H10 atom in the twisted-BQA interacted with the
F atom to form a CH−F interaction. In the solution state at 23
°C, the validity of the CH−F interaction should diminish
owing to the rapid rotation of the CF3 groups, causing the
upfield-shift of the proton.35 However, the corresponding
proton was observed at 7.90 ppm at 23 °C from 7.82 ppm at
−80 °C, exhibiting a downfield-shift with increasing temper-
ature. This discrepancy between the chemical shifts can be
explained as the resonance effect of the BQA ring becoming
dominant with increasing temperature; i.e., at lower temper-
atures, the conformational change in BQA became slow to
adopt the twisted form, reducing the contribution of the
resonance effect. At that time, the corresponding proton
showed an upfield-shift. In contrast, with increasing temper-
ature, the BQA ring can quickly move up and down, enhancing
the contribution of the resonance effect and thus resulting in
the downfield-shift of the proton. For BQL (Figure S18),
although the CH−F interaction was negligible in the X-ray
structure (Table 1), a gentle upfield-shift of the corresponding
H5 proton was observed with decreasing temperature. The
result suggests that the dominant contribution to the chemical
shift is not the CH−F interaction but the alteration of the
resonance effect in the BQX ring. Moreover, these results
suggest that the conformational change between the twisted
conformers is responsible for the fluorescence quenching based
on a nonradiative decay process.
3.56
a
Diffuse-reflectance method with conversion using a Kubelka−Munk
b
c
function using diluted KBr samples. See Table 1. π−π stacking
angles in Figure 5B.
ab
contrast to the solution samples, the λmax values of the BQL
series were strongly dependent on the crystal structures,13−15
as opposed to the number of phenyl rings. This yielded the
ab
λmax values with the order BQL > BQL-diPh > BQL-Ph. In
the X-ray crystal structure, BQL crystallized with the highest
density (1.724 g/cm3 in Table S1) and with the shortest π−π
stacking distance between BQX rings (Table 4), suggesting
ab
that BQL exhibited the longest λmax and the second longest
fl
λmax among the BQL series. However, owing to the strong
intermolecular hydrogen bond and the close π−π stacking, a
considerably small ϕfl was obtained (<0.01) based on
aggregation-induced quenching.36 Despite BQL-diPh carrying
two phenyl rings, there was limited red shift of λmaxab and λmax
.
fl
The π−π stacking angles (θ2 in Figure 5B) among the head-to-
tail columnar packings were estimated to be 45.4, 46.9, and
55.4° for BQL, BQL-Ph, and BQL-diPh, respectively (Figure
5B). The largest θ2, 55.4°, in BQL-diPh was larger than 54.7°,
indicating the behavior at the borderline between J and H-
aggregation.37 This means that BQL and BQL-Ph crystallized
with J-aggregation (<54°), whereas BQL-diPh crystallized with
H-aggregation (>54°), resulting in a blue shift of absorption
accompanied by weak emission. Accordingly, the solid BQL-
diPh absorbed and emitted with a limited red shift, together
with a small ϕfl value. In contrast, the λmaxab of the BQA series
was dependent on the number of phenyl rings. However, the
λmax and ϕfl values were dependent on the crystal packings.
fl
The monophenyl BQA-Ph emitted with the longest wave-
length and the smallest ϕfl values because of its shortest π−π
stacking distance (3.27 Å) among the BQA series. Diphenyl
BQA-diPh emitted the shortest wavelength and the highest ϕfl
value because of its missing hydrogen bond, long π−π stacking
distance (3.50 Å), and the largest angle (LQ‑Ph = 8.9° and θ1 =
33.4° in Table 1), which were related to the twisted BQX ring
and the amine moiety.
Figure 5. (A) Images of crystalline samples taken under room and UV
lighting (365 nm); (B) angles (θ2) between π−π stackings of the
BQL series with head-to-head slipped-columnar fusion.
2.5. Computational Chemistry. DFT calculations were
performed to support the electronic states of BQX derivatives
using theoretical calculations. The initial geometries of
molecules obtained by X-ray analysis were optimized at the
B3LYP/6-31G** level. Tamm−Dancoff (TD)-DFT, incorpo-
rating the TD approximation calculations of the molecules for
UV−vis spectra, was conducted at the B3LYP/6-311G** level
of theory. In the HOMO and the LUMO of BQL (Figure 6A),
the orbitals were distributed over all of the atoms of the BQX
2.4. Photophysical Properties in the Solid State. In
solution conditions, the BQX derivatives exhibited visible
absorption and fluorescence color with moderate ε and ϕfl
values. In addition, the bicyclic TFMAQ derivatives emitted in
the visible region with moderate ϕfl even in the solid state.13−15
As such, investigations of photophysical properties in the solid
state for tricyclic BQX derivatives were conducted. The
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Figure 6. Left panels in (A, B): graphical representations of the highest occupied and the lowest unoccupied Kohn−Sham orbital (isovalue = 0.02)
of (A) BQL and (B) BQA. Right panels indicate UV−vis spectra (n-hexane: black solid lines) together with the calculated spectra (reddish broken
lines) by TD-DFT for (A) BQL and (B) BQA. (C) Electron density difference between the ground and the first excited state (Δρ(r) = ρES(r) −
ρ
GS(r)) with Sr and t indices. Bluish and greenish surfaces represent positive and negative regions, respectively (isocontour value = 0.01). (D)
Correlation plot between the transition dipole moments (μ) and the given ε values at the longest wavelength of all BQL derivatives (R2 = 0.997).
ring, with the exception of the CF3 groups in the HOMO.
However, the orbitals tended to be distributed at the amine
moiety (donor) in the HOMO and at the quinoline ring
(acceptor) in the LUMO. For BQA (Figure 6B), there was a
clear deviation of the molecular orbital (MO) distributions
between the HOMO and LUMO. In the HOMO, the MOs
were almost completely distributed around the amine moiety,
while an absence of MO around the quinoline ring with CF3
groups was observed. In the LUMO, the MOs tended to be
distributed around the pyridine ring, as opposed to the amine
moiety. These results suggest that BQA readily undergoes
charge separation between the HOMO → LUMO transition
compared to BQL. With an increase in the number of phenyl
rings, there was a larger deviation of the MO distribution
between the electron-donor and -acceptor groups (Figure
6A,B). For quantitative analysis, the numbers of contributions
of a hole and an electron contained in the individual MOs were
revealed. In the HOMO and LUMO, the contributions of
BQL (Figures S20−S22) and BQA (Figures S23−S25) were
estimated at 96.3 and 96.5% based on the hole and at 95.0 and
96.0% based on the electron, respectively. This indicates that
the charge separation occurs mainly between the HOMO →
LUMO transition in both derivatives. To evaluate the
relationship between the amplitude of charge separation and
the structures of the BQX derivatives, using the Multiwfn
program,38 the Sr and t indices38,39 were estimated to be 0.644
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a.u. and 0.688 Å for BQL and 0.573 a.u. and 1.573 Å for BQA,
respectively (Figures 6, S26, S27, and Table S12). The lower
Sr40 and higher t indices41 mean a larger charge separation;
BQA causes a larger charge separation compared to BQL. This
result agrees with the Δμ values (8.5 and 9.6 D for BQL and
BQA in Figure 3A, respectively). This indicates that the
contribution of the charge separation in the excited state for
BQA is larger than that for BQL. Moreover, the results
correlate with the D−A distance from X-ray analysis. The
distance between the centroid in the fused-pyridine ring and
the amine moiety was considered the D−A distance, yielding
7.4 and 6.3 Å for BQL and BQA, respectively (Table 1). These
results were supported by the qct index42 using the program
Gaussian (Figures S26, S27, and Table S12). For the phenyl-
substituted BQX, there was a similar change across all indices
(Sr, t, and qct) as the number of phenyl rings increased. This
suggests that the charge separation was accelerated by phenyl
substitution (Figure 6C and Table S12).
TD-DFT calculations were performed (Figure 6 in the right
panels) to reproduce the UV−vis absorptions (Figure 2). The
longest broadened absorptions at 456 and 404 nm (n-hexane)
for BQL and BQA were assigned as the S0 → S1 transition (f =
0.074 and 0.053 for BQL and BQA, respectively) based on the
HOMO → LUMO transition. The broadened shapes were also
reproduced. In addition to the longest absorptions, all other
absorptions were assigned (Figure 6 and Table S11). The
calculated transition dipole moment (μ) linearly correlated
with the ε values of the BQX derivatives (Figure 6D and Table
S12).
2.6. Exploiting as an Ammonia Sensor. The amine
moieties in the BQX framework possess roles as an electric
donor effect and as an acidic response. Utilizing the strong
visible absorptions (ε = 2.0−3.0 × 103) based on the D−A
effect and acid−base reactivity, the ammonia responsiveness of
solid BQX-HCl salts (Figure 7A) was investigated. The base
properties of BQL and BQA were assessed to estimate their
pKa values in HCl solutions (<pH 2) and McIlvaine buffer
solutions43 (>pH 2) containing citric acid and disodium
phosphate (Figure S27). The pKa values were 1.8 and 2.2 for
the protonated-BQL and BQA, respectively, indicating that
BQA possesses a stronger base property than BQL and
TFMAQ (pKa = 0.9 for the protonated TFMAQ). The
different basicities between protonated-BQL and -BQA are a
result of the length of the π-conjugation system and the
reduction in the electron-withdrawing effect by the CF3 group;
BQA consists of a shorter π-conjugation system owing to its
Figure 7. (A) Schematic of an ammonia sensor taking advantage of
the acid−base reaction between BQX and HCl and the ion-exchange
reaction between BQX-HCl and NH3; (B, C): solid-state spectral
changes and photographs before and after exposures to HCl gas and
NH3 gas for (B) BQL and (C) BQA diluted by KBr (2 wt %). In the
spectra, reddish, bluish, and black solid lines represent BQX after
exposure to HCl and the resulting BQX-HCl after exposure to NH3
gas. In the photographs, top (B or C-1), middle (B or C-2), and
bottom (B or C-3) panels represent BQX after exposure to HCl gas
for 10 min and the resulting BQX-HCl salt after exposure to NH3 gas
for 5 s.
color change accompanied by a large difference in absorption
before and after the exposure processes indicate that BQX-
HCl salts may behave as ammonia sensors. Additionally, BQX-
HCl salts are capable of being exploited as an alternate Kaiser
test,44 which is frequently used in peptide syntheses.45
ab
twisted bowl-shaped structure, producing a λmax that is
2.7. Fluorescence Imaging in Live Cells. As per the
observed fluorescence properties, the BQX derivatives
fluoresce with moderate quantum yields in nonpolar environ-
ments, and quenching occurs in polar environments such as
aqueous solutions. This molecular property enables specific
detection of nonpolar environments with a high signal-to-noise
ratio (SNR) in biological systems. Based on the greenish
fluorescence of LDs obtained using TFMAQ analogs,13 the BQ
derivatives were applied as a reddish fluorescent staining
reagent for LDs in live cells (Figure 8A). Cultured HeLa cells
were stained with BQL or BQA and costained with LipidTOX
Deep Red (Life Technologies),46 an established probe for
neutral lipids43 (Figure 8C,D). Cells were incubated with 10
μM BQL or BQA in a culture medium, and fluorescence
imagery was obtained using confocal laser microscopy without
washout (extinction/emission (Ex/Em) = 488/595 nm for
BQL, Ex/Em = 403/525 nm for BQA, Ex/Em = 637/700 nm
shorter than that for BQL (Table 2). Moreover, its MOs are
more localized at the amine moiety in the HOMO, compared
to that of BQL (Figure 6A,B). Under a closed HCl atmosphere
prepared using 33% HCl solution, the color of the exposed
BQX changed gradually, exhibiting a pale-brownish solid from
a reddish solid for BQL (Figure 7B) and a colorless solid from
a yellowish solid for BQA (Figure 7C). These color changes
were confirmed by the absorption spectra, and the
corresponding round maxima at 500 and 450 nm decreased
to yield the corresponding HCl salts through an acid−base
reaction. The production of HCl salts was also confirmed by
IR spectra (Figure S28). Surprisingly, the HCl salts rapidly
reacted with NH3 gas prepared using 28% NH3 solution, such
that the color change was reversed. At this time, via the ion-
exchange reactions, the production of NH4Cl salt was observed
in the IR spectra (Figure S28). This quick responsiveness and
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addition, the intracellular spectrum of BQA (Figure S31B)
yielded an emission maximum at approximately 550 nm,
indicating that its polarity is between those of chloroform and
ethyl acetate, consistent with the BQL results.
The cytotoxicities of BQL and BQA were subsequently
investigated to determine whether the compounds are
appropriate for live-cell imaging (Figure S32). The cytotoxicity
toward HeLa cells incubated with each compound for 2 h at 37
°C was evaluated using the MTS reagent (3-(4,5-dimethylth-
iazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium), which is a water-soluble tetrazolium salt.
After incubation with high concentrations (100 μM) of BQL,
the MTS assay demonstrated cell viability of over 70%,
showing that BQL has low cytotoxicity. In contrast, a cell
viability of less than 30% was obtained after incubation with
100 μM of BQA, indicating its high cell-growth inhibiting
activity for HeLa cells. The difference in cytotoxicity between
BQL and BQA may be attributed to the structure of the BQ
unit fused to the TFMAQ framework (linear or angular).
Pyrido carbazole derivatives19 fused to TFMAQ with angular-
type structures have previously been shown to possess high
cytotoxicity. Furthermore, the photostabilities of BQL and
BQA in methanol solution (20 μM) were examined under
irradiation by light generated with a high-pressure mercury
lamp. The resulting values were compared with the photo-
stability of Nile Red, a fluorescent probe commonly utilized for
LDs (Figure S33). The photostabilities were evaluated on the
basis of the degradation of corresponding absorption peaks.
The decay of Nile Red was the most rapid, whereas BQL
showed a more moderate decay than BQA, indicating that
photostability had the order BQL> BQA> Nile Red.
Considering the emission color, cytotoxicity, and photo-
stability, BQL is more suitable for fluorescence imaging of
LDs in live cells than BQA. Fluorescence imaging of LDs was
then conducted in differentiated 3T3-L1 adipocytes49 stained
with the three BQL derivatives synthesized in this study
(Figure S34). The differentiated cells were incubated with 5
μM of each compound in the culture medium, and the
fluorescence images were tracked by confocal laser microscopy
without washout (Ex/Em = 488/595 nm). In BQL-stained
images (Figure S34A), specific fluorescence signals in differ-
entiated 3T3-L1 cells were observed, revealing spherical
clusters of LDs. In contrast, signals were absent in the images
obtained with BQL-Ph and BQL-diPh (Figures S34B,C). It is
possible that BQL-Ph and BQL-diPh exhibited no fluo-
rescence within the cells because of the high hydrophobicity of
the phenyl groups, preventing a homogenous distribution of
molecules in the aqueous cellular environment. A TFMAQ-8Ar
derivative with hydrophobic tert-butyl groups has previously
been reported to not incorporate well into the LDs in
differentiated 3T3-L1 cells.17 Thus, among the BQ derivatives
developed in this study, BQL is the most suitable probe for
fluorescence imaging of LDs.
Figure 8. (A, B): Schematic of cell fluorescence imaging using BQX.
(A) Cell with an enlarged lipid droplet (LD) before treatment with
BQX; (B) LD fluorescence imaging after incubation with BQX. BQX
incorporated into LD is represented by a strong emission color.
Unincorporated BQX shows a dark color. (C, D) Fluorescence
images and corresponding scatter plots of HeLa cells acquired by
confocal laser microscopy. Fluorescence staining was conducted using
(C) BQL or (D) BQA (10 μM in a culture medium) and LipidTOX
Deep Red costain. R values (Pearson’s correlation coefficient) were
(A) 0.92 and (B) 0.83 for each scatter plot. Scale bars are 50 μm.
for LipidTOX Deep Red). In the fluorescence imageries for
BQL, BQA, and LipidTOX, a granular collection of fluorescent
regions was observed in the cells. The fluorescence imageries
obtained after treatment with BQL, BQA, and LipidTOX were
analyzed using scatter diagrams and Pearson’s correlation
coefficient47 to assess the similarities between the distributions
of BQX and LipidTOX. The R values for BQL and BQA were
0.92 and 0.83, respectively. This indicates the colocalization of
BQXs with LipidTOX in LDs in living HeLa cells. Therefore,
BQX may be used as red-colored probes for fluorescence
imaging of LDs in living cells by exploiting their specific
fluorescence in hydrophobic environments. Notably, BQL is
capable of detecting LDs at longer fluorescence wavelengths
(lower energies) than the TFMAQ-8Ar series18 (Ex/Em =
403/525 nm) that has previously been reported. To date,
several probes with red-colored fluorescence for LD imaging
have been reported.6,7 In comparison, BQL has a relatively
large Stokes shift and significantly higher SNR between
nonpolar (e.g., ϕfl = 0.67 in Hex) and polar (e.g., ϕ < 0.01
in MeOH) environments. From the confocal laser microscopy
fluorescence imaging of HeLa cells, fluorescence imagery and
emission spectra of BQL and BQA within cells and the
intracellular matrix were obtained at various wavelengths
(Figures S29−S31). As shown by the images acquired with
BQL, potent fluorescence signals excited at 488 nm were
collected from 560 to 660 nm, resulting in intracellular
emission with a peak at 610 nm (Figure S31A). This suggests
that the polarity of the LDs of HeLa cells is lower than that of
the AcOEt solution and higher than that of the CHCl3 solution
(Table 2). The polarity of the LDs of MCF7 cells has
previously been reported48 to be between those of AcOEt and
n-butyl acetate, consistent with these results for HeLa cells. In
3. CONCLUSIONS
Push−pull-type tricyclic benzo[X]quinoline (X = g and f)
derivatives bearing donor and acceptor groups were newly
prepared as conformational isomers consisting of linear-type
(BQL) and angular-type (BQA) structural isomers. To
enhance the electron-donor effect, the phenyl group was
introduced into the amine groups; a total of six derivatives
were prepared as environmentally responsive fluorescent dyes.
In X-ray analyses, the BQL series adopted pseudo-planar
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layer was acidified by 10% HCl aq. The separated aqueous layer was
neutralized by NaOH aq. and extracted with diethylether (100 mL).
The organic layer was dried over MgSO4, filtered, and evaporated in
vacuo. The resulting residue was used for next reactions without
further purification as compound 2 (234 mg, 75%). Melting point:
156−158 °C; IR (KBr): 3406, 3339, 1636, 1516, 1346, 1320, 1159,
and 1091 cm−1; 1H NMR (600 MHz, CDCl3) δ: 7.67 (d, J = 8.3 Hz,
2H), 7.54 (dd, J = 6.8, 8.4 Hz, 2H), 7.27 (d, J = 1.8 Hz, 1H), 7.18 (d,
J = 8.2 Hz, 2H), 6.92 (dd, J = 2.2, 8.6 Hz, 1H), 6.86 (dd, J = 2.2, 8.6
Hz, 1H), 6.83 (d, J = 1.9 Hz, 1H), 6.70 (br s, 1H), 3.87 (br s, 2H),
2.34 (s, 3H); 13C{1H}NMR (150 MHz, CDCl3) δ: 144.9, 143.8,
136.1, 135.2, 134.5, 129.6 (2C), 129.1, 129.0, 127.2 (2C), 125.5,
117.7, 117.4, 116.3, 108.0, 21.5; HRMS (ESI) m/z [M + H]+ calcd
for C17H17N2O2S 313.1011, found: 313.1007.
structures, whereas the BQA series exhibited twisting of the
benzoquinoline ring with torsion angles of 6.3−8.9°. In the
crystal packings, the BQL series crystallized with head-to-head
slipped-columnar fusion. In the BQA series, the three
individual derivatives crystallized with different fusions. In
the electron spectra, the BQL series emitted relatively strongly
in nonpolar solutions and in the near-infrared region over 700
nm for polar solutions. This near-infrared emission is the
longest wavelength among the benzoquinoline derivatives
previously reported.22−25 In contrast, the BQA series emitted
weakly compared to the BQL series owing to the vibrations
attributed to the upward and downward twisted-rings. In the
Lippert−Mataga plots, the twisted-BQA series possessed
higher Δμ values compared to those of the BQL series,
indicating that the charge separation in the excited state was
larger than that in the BQL series. In the solid spectra,
absorption and emission were considerably controlled by the
crystal packings. In particular, the π−π stacking distance
significantly impacted the ϕfl values; the highest and lowest ϕfl
were from BQA-diPh, which possesses the longest π−π
stacking distance, and BQA-Ph, which possesses the shortest
π−π stacking distance, respectively. The ammonia responsive-
ness was investigated using BQX-HCl salt by utilizing the
relatively strong visible color and base properties. It resulted in
a rapid responsiveness toward NH3 gas by an ion-exchange
reaction. The LDs in living cells were stained with reddish
fluorescence without a washout by utilizing the environ-
mentally responsive fluorescence properties of BQL. The
results indicate that BQX derivatives have applications in
materials chemistry and biochemistry. To enhance their
multifunctionality, molecular designs are under investigation
involving changing substituents and the π conjugation system.
4.2.3. 2,4-Bis(trifluoromethyl)benzo[g]quinolin-8-amine (BQL)
and 1,3-Bis(trifluoromethyl)benzo[f]quinolin-9-amine (BQA). Hex-
afluoroacetylacetone (1.4 mL, 9.7 mmol) and montmorillonite K10
(1.13 g) were added to a solution of compound 2 (755 mg, 2.42
mmol) in dry 1,4-dioxane (12 mL) under a N2 atmosphere. The
reaction mixture was stirred at 60 °C for 10 h under a N2 atmosphere
in an oil bath. The mixture was cooled to r.t. and filtered through
celite, and the resulting filtrate was evaporated in vacuo. To this
residue was added conc. H2SO4 (2.4 mL), and the resulting mixture
was heated at 100 °C for 5 h in an oil bath. Following neutralization
by NaOH aq., the mixture was extracted with AcOEt three times. The
organic layer was dried over Na2SO4, filtered, and evaporated in vacuo.
The residue was purified by silica gel column chromatography (n-
hexane/AcOEt = 20/1) to afford BQL (38.0 mg, 5%) as a red solid
and BQA (252 mg, 32%) as a yellow solid. Spectral data of BQL;
melting point: 228−235 °C; IR (KBr): 3464, 3351, 1636, 1277, 1197,
1
and 1135 cm−1; H NMR (600 MHz, CDCl3) δ: 8.59 (s, 2H), 7.97
(d, J = 9.0 Hz, 1H), 7.82 (s, 1H), 7.17 (dd, J = 2.2, 8.9 Hz, 1H), 7.14
(d, J = 2.0 Hz, 1H), 4.22 (br s, 2H); 13C{1H}NMR (150 MHz,
2
2
CDCl3) δ: 147.4 (q, JCF = 35.6 Hz), 145.7, 144.5, 136.7 (q, JCF
=
1
32.1 Hz), 136.6, 130.4, 129.4, 125.8, 123.7, 123.2 (q, JCF = 273.4
1
Hz), 122.7, 121.1 (q, JCF = 274.0 Hz), 118.1, 111.8, 105.2;
19F{1H}NMR (282 MHz, CDCl3) δ: −62.8, −68.4; HRMS (ESI) m/
z [M + H]+ calcd for C15H9F6N2 331.0669, found: 331.0707; anal.
calcd for C15H8F6N2: C, 54.56; H, 2.44; N, 8.48. Found: C, 54.78; H,
2.60; N, 8.34.
4. EXPERIMENTAL SECTION
1
4.1. General Information. H, 13C, and 19F NMR spectra were
measured by a Bruker Biospin AVANCE II 600 or III 300
spectrometer using CDCl3 as the solvent. VT-NMR spectra were
recorded by JEOL JNM-ECZ400S in CD2Cl2. Infrared spectra for
tableting samples with KBr were recorded using a JASCO FT-IR
spectrometer. High-resolution electrospray ionization mass spectra
were recorded using a JEOL JMS-T 100LP spectrometer. UV−Vis
spectra in solutions were recorded using JASCO V760 spectrometers.
UV−Vis spectra in solid states were recorded using JASCO V570
spectrometers equipped with a calibrated integrating sphere system.
The fluorescence spectra and quantum yields of solutions and solids
were recorded on a JASCO FP-8500 spectrofluorimeter equipped
with a calibrated integrating sphere system. The fluorescence decay
plots were recorded by HORIBA FluoroCube.
Spectral data of BQA; melting point: 132−138 °C, IR (KBr): 3457,
1
3399, 1635, 1540, 1276, 1188, and 1137 cm−1, H NMR (300 MHz,
CDCl3) δ: 8.13 (s, 1H), 8.00−7.97 (m, 2H), 7.84 (d, J = 9.0 Hz, 1H),
7.79 (d, J = 8.5 Hz, 1H), 7.16 (dd, J = 2.1, 8.5 Hz, 1H), 4.13 (br, 2H);
2
13C{1H}NMR (150 MHz, CDCl3) δ: 150.9, 146.3 (q, JCF = 35.6
2
Hz), 145.9, 135.3 (q, JCF = 32.6 Hz), 133.6, 130.2, 128.6, 126.9,
1
1
124.2, 123.9 (q, JCF = 273.5 Hz), 123.1, 121.2 (q, JCF = 273.3 Hz),
3
3
118.6, 114.8 (q, JCF = 7.2 Hz), 111.8 (q, JCF = 8.1 Hz); 19F{1H}-
NMR (282 MHz, CDCl3) δ: −58.0, −67.9; HRMS (ESI) m/z [M +
H]+ calcd for C15H9F6N2 331.0669, found: 331.0705; anal. calcd for
C15H8F6N2: C, 54.56; H, 2.44; N, 8.48. Found: C, 54.60; H, 2.47; N,
8.50.
4.2. Synthesis Procedure. 4.2.1. 2,7-Diaminonaphthaline (1).26
A mixture of 2,7-dihydroxynaphthaline (2.00 g, 12.5 mmol) and
NaHSO3 (5.20 g, 50.0 mmol) in 28% ammonia solution (100 mL)
was placed in a pressure reactor. The reaction mixture was sealed and
heated to 170 °C (500 rpm) in a heating block. After being stirred for
9 h, the reaction mixture was cooled to room temperature and basified
with 10% NaOH aq to pH > 12. The resulting precipitate was
collected by filtration and dried in vacuo to afford 1 (1.82 g, 92%) as a
4.2.4. N-Phenyl-2,4-bis(trifluoromethyl)benzo[g]quinolin-8-
amine (BQL-Ph) and N,N-Diphenyl-2,4-bis(trifluoromethyl)benzo-
[g]quinolin-8-amine (BQL-diPh). Pd(OAc)2 (10 mg, 15 mol %), (t-
Bu)3P (8.7 mg, 15 mol %), and t-BuOK (64 mg, 0.6 mmol) were
added to a solution of BQL (95 mg, 0.29 mmol) in dry toluene (3
mL), and this solution was degassed under a nitrogen atmosphere.
The reaction mixture was heated in an oil bath and stirred at 100 °C
for 8 h after bromobenzene (45 μL, 0.43 mmol) was added. The
reaction mixture was cooled to room temperature, diluted with
diethylether, and washed with water. The organic layer was dried over
MgSO4, filtered, and evaporated in vacuo. The resulting residue was
purified by silica gel column chromatography (n-hexane/AcOEt =
200/1) to afford BQL-Ph (36 mg, 31%) and BQL-diPh (53 mg,
38%) as red solids.
1
tan solid. H NMR (400 MHz, CDCl3) δ: 7.50 (d, J = 8.8 Hz, 2H),
6.76 (d, J = 2.0 Hz, 2H), 6.70 (dd, J = 2.4, 8.8 Hz, 2H), 3.77 (br s,
4H).
4.2.2. N-(7-Aminonaphthalen-2-yl)-4-methylbenzenesulfona-
mide (2). A solution of tosyl chloride (270 mg, 1.4 mmol) in
pyridine (10 mL) was added dropwise to a solution of 2,7-
diaminonaphthalene 1 (158 mg, 1.0 mmol) in pyridine (5 mL)
over 1 h at 0 °C. The reaction mixture was warmed to r.t. and stirred
overnight before being quenched with water (100 mL). The resulting
mixture was extracted with diethylether (100 mL), and the organic
Spectral data of BQL-Ph: melting point: 179−185 °C; IR (KBr):
3404, 1636, 1600, 1528, 1442, 1281, 1195, and 1139 cm−1; 1H NMR
(600 MHz, CDCl3) δ: 8.62 (s, 1H), 8.61 (br s, 1H), 8.02 (d, J = 9.1
Hz, 1H), 7.84 (s, 1H), 7.55 (d, J= 2.1 Hz, 1H), 7.44−7.38 (m, 3H),
J
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7.29 (d, J = 7.4 Hz, 2H), 7.13 (t, J = 7.4 Hz, 1H), 6.15 (br s, 1H);
4.3. Recrystallization. Recrystallizations of six BQX derivatives
were performed at rt using the following solvents: BQL (CHCl3),
BQL-Ph (CH3CN), BQL-diPh (CH3CN), BQA (n-hexane/AcOEt),
BQA-Ph (CH2Cl2/ether), and BQA-diPh (CH2Cl2/ether).
4.4. Single-Crystal X-ray Diffraction. X-ray reflection data were
collected on a Rigaku R-AXIS RAPID II diffractometer and
MicorMax-007HF with graphite monochromated Cu Kα radiation
(λ = 1.54187 Å) at 93 1 K. The molecular structures were solved
using direct methods (SHELXL 97).50 The crystallographic data file
(cif.) in this paper has been deposited with the Cambridge
Crystallographic Data Center (CCDC). The given parameters are
summarized in Tables S1, S2, and Figures S44−S49.
4.5. Absorption and Emission Spectra and Fluorescent
Quantum Yield Measurements. The solution samples (20 μM) for
UV−vis absorption and fluorescence spectra were prepared using
eight solvents (n-hexane, 1,4-dioxane, dibutylether, CHCl3, AcOEt, n-
butanol, acetonitrile, and MeOH). The solutions were placed in
quartz cells (1 cm) and were bubbled by a nitrogen gas for 1 min
before the measurements. The fluorescence spectra were acquired by
excitation at the absorption maxima (440−450 nm for BQL
derivatives and 400−450 nm for BQA derivatives) of BQX. The
fluorescence quantum yields were calculated from fluorescence
spectra measured in a calibrated integrating sphere system as absolute
quantum yields.
2
13C{1H}NMR (150 MHz, CDCl3) δ: 147.5 (q, JCF = 35.5 Hz),
2
144.6, 143.0, 141.1, 136.7 (q, JCF = 32.0 Hz), 136.4, 130.3, 130.1,
129.7 (2C), 126.6, 123.50, 123.47, 123.3, 123.1 (q, 1JCF = 273.2 Hz),
1
121.1 (q, JCF = 273.5 Hz), 120.3 (2C), 118.6, 112.1, 106.4;
19F{1H}NMR (282 MHz, CDCl3) δ: −61.9, −68.0; HRMS (ESI) m/
z [M + H]+ calcd for C21H13F6N2 407.0983, found: 407.0980; anal.
calcd for C21H12F6N2: C, 62.08; H, 2.98; N, 6.89. Found: C, 61.87; H,
3.06; N, 6.72.
Spectral data of BQL-diPh: melting point: 171−174 °C; IR (KBr):
1
1634, 1594, 1488, 1430, 1283, 1198, and 1124 cm−1; H NMR (600
MHz, CDCl3) δ: 8.60 (s, 1H), 8.54 (s, 1H), 7.95 (d, J = 9.9 Hz, 1H),
7.84 (s, 1H), 7.49−7.46 (m, 2H), 7.37−7.33 (m, 4H), 7.23−7.20 (m,
4H), 7.18−7.15 (m, 2H); 13C{1H}NMR (150 MHz, CDCl3) δ: 147.4
2
2
(q, JCF = 35.5 Hz), 147.2, 146.7 (2C), 144.4, 136.6 (q, JCF = 32.5
Hz), 136.0, 130.6, 129.6 (4C), 129.5, 127.3, 126.6, 125.5 (4C), 124.5
1
1
(2C), 123.3, 123.1 (q, JCF = 273.3 Hz), 121.0 (q, JCF = 273.7 Hz),
119.2, 115.1, 112.4; 19F{1H}NMR (282 MHz, CDCl3) δ: −61.9,
−68.0; HRMS (ESI) m/z [M + H]+ calcd for C27H17F6N2 483.1296,
found: 483.1289; anal. calcd for C27H16F6N2: C, 67.22; H, 3.34; N,
5.81. Found: C, 67.38; H, 3.24; N, 5.77.
4.2.5. N-Phenyl-1,3-bis(trifluoromethyl)benzo[f ]quinolin-9-
amine (BQA-Ph). Pd(DPPF)Cl2·CH2Cl2 (110 mg, 15 mol %),
DPPF (80 mg, 15 mol %), and t-BuOK (100 mg, 0.9 mmol) were
added to a solution of BQA (290 mg, 0.88 mmol) in dry 1,4-dioxane
(5 mL), and this solution was degassed under a nitrogen atmosphere.
The reaction mixture was heated in an oil bath and stirred at 100 °C
for 10 h after bromobenzene (0.3 mL, 2.9 mmol) was added. The
reaction mixture was cooled to room temperature, diluted with
AcOEt, and washed with water. The organic layer was dried over
Na2SO4, filtered, and evaporated in vacuo. The resulting residue was
purified by silica gel column chromatography (n-hexane/AcOEt =
100/1) to afford BQA-Ph (220 mg, 62%) as an orange solid. Melting
point: 129−132 °C; IR (KBr): 3415, 1620, 1596, 1540, 1498, 1274,
4.6. Fluorescence Lifetime Measurements. The solution
samples (20 μM) as mentioned above were also used for fluorescence
lifetime measurements. The fluorescence decay plots were collected
by a HORIBA FluoroCube using Ludox (Merck) for calibration. The
obtained plots were analyzed by Das Analysis (HORIBA) to calculate
the τ values by a linear fitting model with first-order exponential
decay.
4.7. DFT Calculation.39,42 Geometries of the studied molecules
were optimized at the B3LYP/6-31G** level of theory using the
16A.03 revision of the Gaussian 16 program package. The all-
optimized geometries were further confirmed as minima by carrying
out frequency calculations. No imaginary frequencies were found.
TD-DFT incorporating the Tamm−Dancoff approximation (TDA)
calculations of the studied molecules for UV spectra were performed
at the B3LYP/6-311G** level of theory. Bulk solvent effects (n-
hexane) were evaluated using the integral equation formalism variant
of the polarizable continuum model (IEFPCM). The Dindex (the
hole−electron distance and the charge-transfer length) and charge
density difference (CCD) were derived from Gaussian output files
and were calculated using Multiwfn to describe the ICT character
quantitatively, in which the Iop (9/40 = 4) keyword was used to get
more configuration coefficients.
1
1192, and 1140 cm−1; H NMR (600 MHz, CDCl3) δ: 8.31 (s, 1H),
8.12 (s, 1H), 8.00 (d, J = 8.9 Hz, 1H), 7.89 (d, J = 8.9 Hz, 1H), 7.85
(d, J = 8.6 Hz, 1H), 7.47 (dd, J = 2.1, 8.5 Hz, 1H), 7.40−7.35 (m,
2H), 7.25−7.22 (m, 2H), 7.11−7.07 (m, 1H), 6.10 (s, 1H);
2
13C{1H}NMR (150 MHz, CDCl3) δ: 150.9, 146.5 (q, JCF = 35.7
Hz), 143.4, 141.4, 135.4 (q, 2JCF = 32.6 Hz), 133.4, 130.1, 129.5 (2C),
128.6, 128.1, 125.0, 123.7 (q, 1JCF = 273.5 Hz), 123.4, 121.1 (q, 1JCF
=
3
273.4 Hz), 120.0, 119.7 (2C), 120.0, 114.9 (q, JCF = 7.1 Hz), 113.6
3
(q, JCF = 7.7 Hz); 19F{1H}NMR (282 MHz, CDCl3) δ: −58.1,
−67.5; HRMS (ESI) m/z [M + H]+ calcd for C21H13F6N2 407.0983,
found: 407.0949; anal. calcd for C21H12F6N2: C, 62.08; H, 2.98; N,
6.89. Found: C, 62.05; H, 2.89; N, 7.00.
4.8. pH Titrations of BQL and BQA Solutions. BQL and BQA
were dissolved in 1,4-dioxane. To the BQX solutions were added the
HCl solutions (>pH 2.0) or the McIlvaine buffer solutions (<pH
2.0),43 and 25 μM solutions were prepared with different pH values
(pH: 0.0−7.0). UV−vis spectra were conducted, and the change in
absorptions was plotted as a function of the pH value. By a sigmoidal
fitting, the pKa values of the protonated-BQL and BQA were
estimated to be 1.8 and 2.2, respectively.
4.9. Ammonia Responsiveness. BQL and BQA were diluted by
KBr and mixed using a mortar and pestle for approximately 3 min,
preparing the 2.0 wt % samples. The resulting mixed samples were
exposed by an HCl gas that was prepared using a 33% HCl solution.
After exposure for 10, 30, and 60 min, photos of the individual
samples were taken. The resulting HCl salts after exposure for 60 min
were exposed by an NH3 gas that was prepared using a 28% NH3
solution. After exposure for 5 s, the photos for the resulting samples
were taken.
4.10. Cell Culture and Fluorescence Imaging of Hela Cells.
HeLa cells were purchased from the Japanese Collection of Research
Bioresources (JCRB) Cell Bank. HeLa cells were grown in Dulbecco’s
modified Eagle’s medium (DMEM) including 10% fetal bovine serum
(FBS), 4.5 g/L glucose, 100 units/mL penicillin, and 100 μg/mL
streptomycin (Nacalai tesque) in a humidified atmosphere of 5% CO2
at 37 °C. The cells were grown to confluence in 35 mm glass-based
4.2.6. N,N-Diphenyl-1,3-bis(trifluoromethyl)benzo[f ]quinolin-9-
amine (BQA-diPh). Pd(OAc)2 (2.2 mg, 10 mol %), (t-Bu)3P (4
mg, 20 mol %), and t-BuOK (33 mg, 0.3 mmol) were added to a
solution of BQA (41 mg, 0.1 mmol) in dry toluene (1 mL), and this
solution was degassed under a nitrogen atmosphere. The reaction
mixture was heated in an oil bath and stirred at 100 °C for 3 h after
bromobenzene (20 μL, 0.2 mmol) was added. The reaction mixture
was cooled to room temperature, diluted with diethylether, and
extracted with water. The organic layer was dried over MgSO4,
filtered, and evaporated in vacuo. The resulting residue was purified by
silica gel column chromatography (n-hexane) to afford BQA-diPh (49
mg, 99%) as a yellow solid. Melting point: 146−148 °C; IR (KBr):
1
1615, 1595, 1523, 1496, 1271, 1189, and 1143 cm−1; H NMR (300
MHz, CDCl3) δ: 8.25 (d, J = 1.7 Hz, 1H), 8.04 (s, 1H), 8.02 (d, J =
8.9 Hz, 1H), 7.92 (d, J= 8.9 Hz, 1H), 7.82 (d, J = 8.7 Hz, 1H), 7.58
(dd, J = 2.1, 8.7 Hz, 1H), 7.38−7.30 (m, 4H), 7.22−7.11 (m, 6H);
13C{1H}NMR (150 MHz, CDCl3) δ: 150.7, 147.7, 147.1 (2C), 146.6
2
2
(q, JCF = 35.8 Hz), 135.6 (q, JCF = 32.7 Hz), 133.3, 129.6 (4C),
129.5, 128.8, 128.3, 125.9, 125.4 (4C), 124.3, 124.2 (2C), 123.7,
1
2
123.3 (q, JCF = 273.4 Hz), 121.13 (q, JCF = 273.0 Hz), 121.08 (q,
3JCF = 8.0 Hz), 115.1 (q, JCF = 7.7 Hz); 19F{1H}NMR (282 MHz,
3
CDCl3) δ: −58.9, −67.5; HRMS (ESI) m/z [M + H]+ calcd for
C27H17F6N2 483.1296, found: 483.1288; anal. calcd for C27H16F6N2:
C, 67.22; H, 3.34; N, 5.81. Found: C, 66.93; H, 3.48; N, 5.53.
K
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dishes and treated with 10 μM BQL or BQA and 1000× of HCS
LipidTOX Deep Red (Invitrogen) in DMEM before fluorescence
imaging. The fluorescence signals from the live cells were captured by
confocal laser microscopy (Nikon A1R). Excitation laser wavelengths
were 403 nm for BQA, 488 nm for BQL, and 637 nm for LipidTOX
Deep Red.
Satoru Karasawa − Faculty of Pharmaceutical Sciences, Showa
Pharmaceutical University, Machida 194-8543, Japan;
orcid.org/0000-0002-3107-442X; Email: karasawa@
ac.shoyaku.ac.jp
Authors
4.11. Evaluation of Photostability.18,19 The compounds in
methanol (20 μM, 2 mL) solution were placed into quartz cells with a
stirring bar. These solutions were irradiated on the stirrer by a high-
pressure Hg lamp (1000 W, Ushio) through a collecting lens. The
reactions were followed by absorption spectrometry after irradiation
(1, 2, 5, 10, 30, and 60 min).
Tomohiro Umeno − Faculty of Pharmaceutical Sciences, Showa
Pharmaceutical University, Machida 194-8543, Japan
Yuichiro Abe − Graduate School of Pharmaceutical Sciences,
Kyushu University, Fukuoka 812-8582, Japan; orcid.org/
0000-0003-4283-8509
Keita Ikeno − Faculty of Pharmaceutical Sciences, Showa
Pharmaceutical University, Machida 194-8543, Japan
Ryu Yamasaki − Faculty of Pharmaceutical Sciences, Showa
Pharmaceutical University, Machida 194-8543, Japan;
orcid.org/0000-0002-8976-2962
Iwao Okamoto − Faculty of Pharmaceutical Sciences, Showa
Pharmaceutical University, Machida 194-8543, Japan
4.12. Cell Culture and Differentiation Induction of 3T3-L1
Cells. 3T3-L1 cells were purchased from the JCRB (Japanese
Collection of Research Bioresources) Cell Bank. 3T3-L1 cells were
grown in DMEM including 10% FBS, 1.0 g/L glucose, 100 units/mL
penicillin, and 100 μg/mL streptomycin in a humidified atmosphere
of 5% CO2 at 37 °C. The cells were grown in 35 mm glass-based
dishes and differentiated by adding 3-isobutyl-1-methylxanthine (0.5
mM), insulin (10 μg/mL), and dexamethasone (1 μM) to the culture
media incubated in a CO2 incubator for three days. The media were
replaced with other media including insulin (10 μg/mL), and cells
were incubated in a CO2 incubator for a further three days. The
compounds (BQL, BQL-Ph, and BQL-diPh) in dimethyl sulfoxide
(DMSO) solutions were diluted in culture media (1 μM) before
fluorescence imaging. The bright-field and fluorescence images were
captured by confocal laser microscopy (Nikon, A1R). Excitation laser
wavelength at 488 nm and emission wavelength at 595 nm were used
for fluorescence imaging.
4.13. MTS Assay. HeLa cells were dispensed into a 96-well plate
at 5000 cells/well and incubated at 37 °C overnight. The compounds
BQL and BQA in DMSO were diluted with a culture medium (100
μL/well) at several concentrations (n = 3) in the above-mentioned
wells. The prepared plates were incubated at 37 °C for 2 h, washed
with DMEM twice, and replaced with fresh DMEM containing FBS.
These plates were incubated with CellTiter 96 (Promega, 10 μL/well)
for 3 h at 37 °C before absorbance at 490 nm in each well was
measured by Varioskan Flash (Thermo Scientific). The ratio of
absorbance with nontreated wells was plotted as cell viability rates.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c01878
Author Contributions
§Y.F. and T.U. contributed equally.
Author Contributions
S.K., conceptualization and completion of the original draft
manuscript; I.O., reviewing and editing; Y.F., synthesis of
molecules, spectral characterizations, X-ray analysis, all cell
imaging, and partial writing; T.U., synthesis of molecules,
spectral characterizations, all ammonia-responsiveness tests,
and X-ray analysis; Y.A., conceptualization, synthesis of
molecules, spectral characterizations, and X-ray analysis; K.I.,
partial spectral characterization; K.U., X-ray analysis, all DFT
calculations; R.Y., VT-NMR and reviewing and editing.
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.0c01878.
ACKNOWLEDGMENTS
■
■
sı
This work was supported by a Grant-in-Aid for Scientific
Research (C) (Grant Numbers 19K07016 for S.K. and
17K08212 for Y.F.) from the Japan Society for Promotion
Sciences (JSPS) and a Grant-in-Aid for Young Scientists of
Showa Pharmaceutical University (T.U.). Y.F. also acknowl-
edges support from the Takeda Science Foundation. The
authors thank Dr. K. Hamada and Prof. A. Mizutani of Showa
Pharmaceutical University for valuable advice related to 3T3-
L1 cells. The computations were performed at the Research
Institute for Information Technology, Kyushu University.
X-ray structures and the absorption and fluorescence
spectra of BQX; fluorescence decay curves of BQX;
DFT calculation results of BQX; pH titration of BQL
and BQA; IR spectra change for solid BQL and BQA;
Cartesian coordinates obtained from DFT calculation;
cell imaging using BQL, BQL-diPh, BQA, and BQA-
diPh; intracellular fluorescence spectra of BQL and
BQA; cell viability assays of BQL and BQA; photo-
stability of the MeOH solution of BQL and BQA;
copies of the 1H, 13C, and 19F NMR as well as IR spectra
for compound 2 and BQX; and thermal ellipsoid plots
of crystal structures for BQX (PDF)
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AUTHOR INFORMATION
Corresponding Authors
■
Yasufumi Fuchi − Faculty of Pharmaceutical Sciences, Showa
Pharmaceutical University, Machida 194-8543, Japan;
Email: fuchi@ph.bunri-u.ac.jp
Kazuteru Usui − Faculty of Pharmaceutical Sciences, Showa
Pharmaceutical University, Machida 194-8543, Japan;
orcid.org/0000-0002-2175-5221; Email: usui@
ac.shoyaku.ac.jp
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