Unsymmetric indolylmaleimides: Synthesis, photophysical properties and amyloid detection

Article abstract of DOI:10.1016/j.jphotochem.2014.04.027

Various unsymmetric indolylmaleimides were synthesized. Their photophysical properties and affinities for amyloid fibrils were evaluated. Some unsymmetric indolylmaleimides have large Stokes shifts of more than 120 nm, fluorescence emission maxima wavelen

Full text of DOI:10.1016/j.jphotochem.2014.04.027

Accepted Manuscript
Title: Unsymmetric indolylmaleimides: synthesis,
photophysical properties and amyloid detection
Author: Manabu Nakazono Konen Obayashi Yuji Oshikawa
Kazushi Tani Genki Suenaga Yukio Ando Shinkoh Nanbu
Ryoichi Kuwano
PII:
S1010-6030(14)00201-9
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.jphotochem.2014.04.027
JPC 9669
To appear in:
Journal of Photochemistry and Photobiology A: Chemistry
Received date:
Revised date:
Accepted date:
7-1-2014
13-3-2014
15-4-2014
Please cite this article as: M. Nakazono, K. Obayashi, Y. Oshikawa,
K. Tani, G. Suenaga, Y. Ando, S. Nanbu, R. Kuwano, Unsymmetric
indolylmaleimides: synthesis, photophysical properties and amyloid detection,
Journal of Photochemistry and Photobiology A: Chemistry (2014),
http://dx.doi.org/10.1016/j.jphotochem.2014.04.027
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Graphical abstract
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Some unsymmetric indolylmaleimides have large Stokes shifts of more than 120 nm,
fluorescence maxima emission wavelengths of more than 550 nm and different
emissions under UV irradiation at 365 nm. 3-(1H-Indol-3-yl)-1-methyl-4-phenyl-
1H-pyrrole-2,5-dione detects amyloid fibrils in senile systemic amyloidosis.
CH
3
Different emissions
Amyloid detection
N
O
O
R2
N
R1
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Highlights
●Synthesis of various unsymmetric indolylmaleimides
●Unsymmetric indolylmaleimides having large Stokes shifts and different
fluorescence emissions
●Unsymmetric indolylmaleimide detecting amyloid fibrils in senile systemic
amyloidosis
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Unsymmetric indolylmaleimides: synthesis, photophysical properties
and amyloid detection
a,
b
a
a
Manabu Nakazono *, Konen Obayashi , Yuji Oshikawa , Kazushi Tani ,
c
c
d
e
Genki Suenaga , Yukio Ando , Shinkoh Nanbu , Ryoichi Kuwano
a
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi,
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Higashi-ku, Fukuoka 812-8582, Japan
b
Diagnostic Unit for Amyloidosis, Department of Laboratory Medicine, Kumamoto
University Hospital,1-1-1 Honjo, Kumamoto 860-8556, Japan
c
Department of Neurology, Graduate School of Medical Sciences, Kumamoto
University,1-1-1 Honjo, Kumamoto 860-8556, Japan
d
Faculty of Science and Technology, Sophia University, 7-1 Kioi-Cho, Chiyoda-ku,
Tokyo 102-8554, Japan
e
Department of Chemistry, Graduate School of Sciences, Kyushu University, 6-10-1
Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan
* Corresponding author.
Tel.: +81-92-642-6597
E-mail address: nakazono@phar.kyushu-u.ac.jp
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ABSTRACT
Various unsymmetric indolylmaleimides were synthesized. Their photophysical
properties and affinities for amyloid fibrils were evaluated. Some unsymmetric
indolylmaleimides have large Stokes shifts of more than 120 nm, fluorescence maxima
emission wavelengths of more than 550 nm and different emissions under UV
irradiation at 365 nm. From the results of histopathologic methods using stains, 3-(1H-
Indol-3-yl)-1-methyl-4-phenyl-1H-pyrrole-2,5-dione has high selectivity for amyloid
fibrils in senile systemic amyloidosis.
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Keywords: Fluorescence, Indolylmaleimide, π-conjugation, Amyloid
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1. Introduction
Recently, indolylmaleimide (IM) derivatives have been a focus in the development of
anticancer medicines based on inhibition of protein kinases [1−4]. Furthermore, IM
compounds have been evaluated as luminescent materials for organic light-emitting
diodes [5,6]. Some research groups [7−10], including us [11−14], have studied the
fluorescence (FL) and chemiluminescence properties of symmetric
bisindolylmaleimides. However, the potential use of IM derivatives in clinical
diagnosis has been unexplored. There have been two studies on the FL properties of
unsymmetric IMs [6,7]. A monosubstituted IM compound is obtained from the reaction
of indolylmagnesium bromide and 2,3-dibromo-N-methylmaleimide [15]. The resulting
monosubstituted IM is readily transformed to unsymmetric disubstituted IMs through
Suzuki-Miyaura coupling. The advantage of this synthetic method is that various
substituents, including fluorophores, can be introduced at the C=C bond of the
maleimide moiety. When the indolylmaleimides are modified with a functional group
possessing high affinity for a specific analyte, the utility of the unsymmetric IM
derivatives as FL probes is drastically increased.
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Amyloidosis is a disorder of protein metabolism in which normally soluble
autologous proteins are deposited in tissues as abnormal insoluble fibrils, which cause
structural and functional disruptions [16−21]. Among several histopathologic methods
using stains, Congo red staining is one of the most popular detection methods of
amyloid deposits in tissues [22,23]. However, Congo red-stained histochemical
specimens are not always easily interpreted. In previous studies, styrylbenzene
derivatives exhibited high selectivity for amyloid fibrils in systemic amyloidoses
[24−27]. For example, (trans, trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-
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hydroxy)styrylbenzene (BSB) was developed for in vivo detection of amyloid deposits
in patients with various systemic and localized forms of amyloidosis [25].
Styrylbenzene derivatives such as BSB have π−conjugated structure. In this study, we
designed and synthesized various π−conjugated unsymmetric indolylmaleimides,
cleared their photophysical properties and carefully checked the reactivity of the
compounds with amyloid fibrils.
2. Experimental
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2.1. Chemicals
Ethylmagnesium bromide (tetrahydrofuran solution), trans-2-phenylvinyl boronic
acid, 3-quinine boronic acid, 2,2'-bithiophene-5,5'-diboronic acid bis(pinacol)ester, 4-
formylphenylboronic acid, pyrene-1-boronic acid, 9H-carbazole-2-boronic acid pinacol
ester and 4,4'-biphenyl-diboronic acid were purchased from Aldrich (Milwaukee, USA).
Indole, sodium hydride, phenylboronic acid, palladium(II) acetate,
tetrakis(triphenylphosphine)palladium(0), naphthalene-2-boronic acid, sodium
methoxide, di-tert-butyl dicarbonate, 4-bromomethyl-7-methoxycoumarin and 4-
dimethylaminopyridine were purchased from Wako Chemicals (Osaka, Japan).
Diethyl(4-iodobenzyl)phophonate and rhodamin B were purchased from Tokyo Kasei
Kogyo (Tokyo, Japan). Cesium fluoride was purchased from Nakalai Tesque Inc.
(Kyoto, Japan). 3-Bromo-4-(1H-indol-3-yl)-1-methyl-1H-pyrrole-2,5-dione and tert-
butyl 3-(4-bromo-1-methyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1H-indole-1-
carboxylate were prepared by the reported method [15]. All other chemicals and
solvents were of analytical reagent grade.
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2.2. Apparatus
1
13
The H-NMR (500 MHz) and C-NMR (125.7 MHz) spectra were obtained using a
Varian UNITY plus (USA) spectrometer. The HR ESI-TOF-MS spectra of compounds
1−13 were obtained using a Bruker/micrOTOF II (Karlsruhe, Germany). The FAB MS
spectra were obtained using a JEOL JMS-HX110A (Tokyo, Japan). The absorbance and
fluorescence spectra of compounds 1−13 were obtained using a Jasco V-530
absorptiometer and FP-6500 fluorometer (Tokyo, Japan). The slit widths at the
excitation and emission of the fluorometer were 5 nm. All the FL spectra were
corrected.
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1
1
1
1
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2
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2.3. Synthesis and characterization
All reactions were done under an atmosphere of inert gas.
2.3.1. 3-(1H-indol-3-yl)-1-methyl-4-phenyl-1H-pyrrole-2,5-dione (1)
3-Bromo-4-(1H-indol-3-yl)-1-methyl-1H-pyrrole-2,5-dione (0.12 g, 0.39 mmol),
phenylboronic acid (0.1 g, 0.82 mmol) and K
2 3
CO (0.08 g, 0.58 mmol) were added to
dioxane-H O (28 mL, 6:1). Palladium(II) acetate (0.06 g, 0.27 mmol) was then added
2
to the mixture, and the solution was refluxed for 17 h. Ethyl acetate (200 mL), 1 M
aqHCl (100 mL) and 10% (w/v) aqNaCl (100 mL) were added to the mixture. The
organic layer was dried with anhydrous Na
2
SO
4
. The filtrate was concentrated and
purified by column chromatography (CHCl
3
:CH
3
OH=20:1) to produce compound 1 as a
1
red solid (0.04 g, 33% yield). M.p. 203−205 °C. H NMR (500 MHz, (CD
3
)
2
S=O) 3.03
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(s, 3H, CH
3
), 6.3−6.32 (d, J = 8 Hz, 1H, ArH), 6.65−6.68 (t, J = 8 Hz, 1H, ArH),
7.03−7.06 (t, J = 8 Hz, 1H, ArH), 7.31−7.36 (m, 3H, ArH), 7.39−7.43 (m, 3H, ArH), 8
1
3
3 2
(s, 1H, ArH), 11.9 (s, 1H, indole NH). C NMR (125.7 MHz, (CD ) S=O) 24.0, 104.1,
112.1, 119.7, 121.1, 122.0, 123.8, 128.0, 128.6, 129.5, 130.6, 131.1, 132.0, 136.5, 171.0,
+
171.3. HRMS-ESI (m/z) calculated for C19
325.0952.
H
14
N
2
O
2
[M + Na] 325.0953, found
2.3.2. 3-(1H-indol-3-yl)-1-methyl-4-styryl-1H-pyrrole-2,5-dione (2)
Tert-butyl 3-(4-bromo-1-methyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1H-indole-
1-carboxylate (0.4 g, 1 mmol), trans-2-phenylvinyl boronic acid (0.22 g, 1.49 mmol)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2 3 2
and K CO (0.14 g, 1 mmol) were added to dioxane-H O (30 mL, 4:1).
Tetrakis(triphenylphosphine)palladium (0) (0.12 g, 0.1 mmol) was then added to the
mixture, and the solution was refluxed for 16.5 h. Ethyl acetate (200 mL), 0.5 M aqHCl
(100 mL) and 10% (w/v) aqNaCl (100 mL) were added to the mixture. The organic
layer was dried with anhydrous MgSO
4
. The filtrate was concentrated and purified by
column chromatography (ethyl acetate:hexane=1:2→CHCl
3
:CH
3
OH=20:1) to produce
1
compound 2 as a red solid (0.03 g, 9% yield). M.p. 166−168 °C. H NMR (500 MHz,
3 2 3
(CD ) S=O) 3 (s, 3H, CH ), 7.1−7.13 (t, J = 7 Hz, 1H, ArH), 7.2−7.24 (m, 2H, ArH),
7.29−7.37 (m, 3H, ArH), 7.48−7.53 (m, 3H, ArH), 7.62−7.64 (d, J = 8 Hz, 1H, ArH),
7.79−7.82 (d, J = 16.5 Hz, 1H, CH=CH), 7.88−7.89 (d, J = 2.5 Hz, 1H, ArH), 12 (s, 1H,
1
3
3 2
indole NH). C NMR (125.7 MHz, (CD ) S=O) 23.7, 104.8, 112.5, 118.7, 120.5,
122.4, 125.3, 126.3, 126.6, 128.9, 129.0, 130.3, 131.7, 135.9, 136.6, 136.7, 170.6,
+
171.0. HRMS-ESI (m/z) calculated for C21
351.1106.
H
16
N
2
O
2
[M + Na] 351.1109, found
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2.3.3. 3-(1H-indol-3-yl)-1-methyl-4-(naphthalen-2-yl)-1H-pyrrole-2,5-dione (3)
3-Bromo-4-(1H-indol-3-yl)-1-methyl-1H-pyrrole-2,5-dione (0.12 g, 0.39 mmol),
2 3
naphthalene-2-boronic acid (0.14 g, 0.8 mmol) and K CO (0.08 g, 0.58 mmol) were
added to dioxane-H O (28 mL, 6:1). Palladium(II) acetate (0.06 g, 0.27 mmol) was
2
added to the mixture, and the solution was refluxed for 15 h. Ethyl acetate (200 mL), 1
M aqHCl (100 mL) and 10% (w/v) aqNaCl (100 mL) were added to the mixture. The
organic layer was dried with anhydrous Na
purified by column chromatography (CHCl
red solid (0.1 g, 71% yield). M.p. 230−232 °C. H NMR (500 MHz, (CD
(s, 3H, CH ), 6.35−6.36 (d, J = 8.5 Hz, 1H, ArH), 6.51−6.54 (t, J = 8 Hz, 1H, ArH),
2
SO
4
. The filtrate was concentrated and
3
:CH
3
OH=20:1) to produce compound 3 as a
1
3 2
) S=O) 3.06
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
3
7−7.01 (m, 1H, ArH), 7.4−7.43 (m, 2H, ArH), 7.48−7.55 (m, 2H, ArH), 7.77−7.79 (d, J
= 8.5 Hz, 1H, ArH), 7.83−7.85 (d, J = 8 Hz, 1H, ArH), 7.87−7.88 (d, J = 8 Hz, 1H,
1
3
ArH), 8.02 (s, 1H, ArH), 8.09 (s, 1H, ArH), 11.94 (s, 1H, indole NH). C NMR (125.7
MHz, (CD S=O) 24.0 (CH ), 104.2, 112.1, 119.7, 120.9, 122.0, 124.0, 126.4, 126.6,
3
)
2
3
126.8, 127.3, 127.5, 127.8, 128.1, 128.3, 129.2, 131.1, 132.2, 132.3, 132.5, 136.5,
+
171.0, 171.3. HRMS-ESI (m/z) calculated for C23
375.1109.
H
16
N
2
O
2
[M + Na] 375.1109, found
2.3.4. 3-(1H-indol-3-yl)-1-methyl-4-(quinolin-3-yl)-1H-pyrrole-2,5-dione (4)
Tert-butyl 3-(4-bromo-1-methyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1H-indole-
1-carboxylate (0.6 g, 1.5 mmol), 3-quinoline boronic acid (0.35 g, 2.25 mmol) and
Na
Tetrakis(triphenylphosphine)palladium(0) (0.25 g, 0.22 mmol) was then added to the
mixture, and the solution was refluxed for 20.5 h. Ethyl acetate (300 mL) and H O (200
2 3 2
CO (1.2 g, 15.5 mmol) were added to dioxane-H O (120 mL, 4:1).
2
4
mL) were added to the mixture. The organic layer was dried with anhydrous MgSO .
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The filtrate was concentrated and purified by column chromatography (ethyl
acetate:hexane=4:5) to produce compound 4 as a red solid (0.29 g, 56% yield). M.p.
1
249−251 °C. H NMR (500 MHz, (CD
3
)
2
S=O) 3.08 (s, 3H, CH
3
), 6.37−6.38 (d, J = 8
Hz, 1H, ArH), 6.56−6.59 (m, 1H, ArH), 7.01−7.04 (m, 1H, ArH), 7.45−7.47 (d, J = 8
Hz, 1H, ArH), 7.59−7.63 (m, 1H, ArH), 7.76−7.8 (m, 1H, ArH), 7.95−7.97 (m, 2H,
ArH), 8.08 (s, 1H, ArH), 8.48 (s, 1H, ArH), 8.73 (s, 1H, ArH), 12.04 (s, 1H, indole NH).
1
3
3 2
C NMR (125.7 MHz, (CD ) S=O) 24.1, 104.1, 112.4, 120.1, 120.6, 122.2, 123.7,
124.2, 124.6, 126.9, 127.1, 128.6, 128.7, 130.3, 131.7, 133.1, 136.2, 136.7, 146.6,
+
150.1, 170.7, 171.1. HRMS-ESI (m/z) calculated for C22
found 376.1051.
H
15
N
3
O
2
[M + Na] 376.1062,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2.3.5. 3-([2,2'-Bithiophen]-5-yl)-4-(1H-indol-3-yl)-1-methyl-1H-pyrrole-2,5-dione (5)
Tert-butyl 3-(4-bromo-1-methyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1H-indole-
1-carboxylate (0.21 g, 0.52 mmol) was added to dioxane (40 mL), and then
tetrakis(triphenylphosphine)palladium(0) (0.06 g, 0.052 mmol) was added to the
mixture. The solution was stirred for 10 min. 2,2'-Bithiophene-5,5'-diboronic acid
bis(pinacol)ester (0.13 g, 0.3 mmol) and cesium fluoride (0.47 g, 3 mmol) were added
to the mixture. The mixture was refluxed for 22 h. The solution was then filtered by
celite, and ethyl acetate (200 mL) and H
2
O (200 mL) were added to the filtrate. The
. The filtrate was concentrated and
organic layer was dried with anhydrous MgSO
4
purified by column chromatography (ethyl acetate:hexane=2:5) to produce tert-butyl 3-
(4-([2,2'-bithiophen]-5-yl)-1-methyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1H-
1
indole-1-carboxylate as a red solid (0.05 g, 20% yield). M.p. 114−116 °C. H NMR
(500 MHz, (CD
3 2 3
) S=O) 1.67 (s, 9H, t-Bu), 3.04 (s, 3H, CH ), 7.09−7.11 (m, 2H, ArH),
7.16−7.19 (m, 1H, ArH), 7.24−7.26 (m, 2H, ArH), 7.37−7.40 (m, 2H, ArH), 7.57−7.58
1
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4
5
(d, J = 5 Hz, 1H, ArH), 8.01 (s, 1H, ArH), 8.15−8.17 (d, J = 8 Hz, 1H, ArH). FAB MS
+
(m/z) [M] 490.08.
Tert-butyl 3-(4-([2,2'-bithiophen]-5-yl)-1-methyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-
2 2
3-yl)-1H-indole-1-carboxylate (0.05 g, 0.061 mmol) was added to CH Cl (30 mL).
o
The solution was stirred at 0 C for 15 min, and then trifluoroacetic acid (3 mL) was
added to the solution. The mixture was stirred at 0 °C for 20 min and stirred at an
ambient temperature for 4 h. Ethyl acetate (150 mL), 1 M aqNaHCO
aqK CO (100 mL) were added to the mixture. The organic layer was dried with
anhydrous MgSO . The filtrate was concentrated and purified by column
3
(50 mL) and 1 M
2
3
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
chromatography (ethyl acetate:hexane=2:5) to produce compound 5 as a red solid
1
(0.03 g, 79% yield). M.p. 218−220 °C. H NMR (500 MHz, (CD
3
)
2
S=O) 3.03 (s, 3H,
CH ), 6.87−6.89 (d, J = 8 Hz, 1H, ArH), 6.97−6.99 (t, J = 7 Hz, 1H, ArH), 7.08−7.12
3
(m, 2H, ArH), 7.15−7.19 (m, 1H, ArH), 7.23−7.24 (d, J = 4 Hz, 1H, ArH), 7.34−7.35
(dd, J = 1 Hz, 4 Hz, 1H, ArH), 7.51−7.55 (m, 2H, ArH), 7.91−7.92 (d, J = 3 Hz, 1H,
1
3
3 2
ArH), 11.96 (s, 1H, indole NH). C NMR (125.7 MHz, (CD ) S=O) 24.1, 103.9, 112.5,
120.0, 121.5, 122.1, 123.6, 123.8, 124.8, 127.7, 128.5, 128.6, 129.2, 136.0, 136.6,
+
140.4, 170.7, 170.8. HRMS-ESI (m/z) calculated for C21
413.0394, found 413.0393.
14 2 2 2
H N O S [M + Na]
2.3.6. 3-(1H-indol-3-yl)-1-methyl-4-(4-styrylphenyl)-1H-pyrrole-2,5-dione (6)
3-Bromo-4-(1H-indol-3-yl)-1-methyl-1H-pyrrole-2,5-dione (0.12 g, 0.39 mmol), 4-
2 3
formylphenylboronic acid (0.12 g, 0.8 mmol) and K CO (0.08 g, 0.58 mmol) were
added to dioxane-H O (30 mL, 5:1). Palladium(II) acetate (0.034 g, 0.15 mmol) was
2
then added to the mixture, and the solution was refluxed for 5 h. Ethyl acetate (200
mL), 0.5 M aqHCl (100 mL) and 10% (w/v) aqNaCl (100 mL) were added to the
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mixture. The organic layer was dried with anhydrous Na
2
SO
4
. The filtrate was
concentrated and purified by column chromatography (CHCl
3
:CH OH=20:1) to produce
3
4-(4-(1H-indol-3-yl)-1-methyl-2,5-dioxo-2,5-dihydro-1H-1-pyrrol-3-yl)
1
benzaldehyde as a red solid (0.09 g, 69% yield). H NMR (500 MHz, (CD
3
)
2
S=O) 3.04
(s, 3H, CH ), 6.26−6.28 (d, J = 8 Hz, 1H, ArH), 6.68−6.71 (t, J = 7.5 Hz, 1H, ArH),
3
7.05−7.08 (t, J = 8.5 Hz, 1H, ArH), 7.44−7.46 (d, J = 8 Hz, 1H, ArH), 7.6−7.61 (d, J =
8.5 Hz, 2H, ArH), 7.85−7.87 (d, J = 8 Hz, 2H, ArH), 8.07−8.08 (d, J = 3 Hz, 1H, ArH),
10 (s, 1H, CHO), 12.04 (s, 1H, indole NH).
4-(4-(1H-indol-3-yl)-1-methyl-2,5-dioxo-2,5-dihydro-1H-1-pyrrol-3-yl)benz-
aldehyde (0.14 g, 0.42 mmol) and diethyl benzylphophonate (0.2 ml, 0.96 mmol) were
added to DMF (2 mL). The solution was stirred at 0 °C for 15 min. Sodium methoxide
(28% in methanol solution, 0.1 ml, 0.52 mmol) was added the solution and the mixture
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
was stirred at 90 °C for 18 hr. Ethyl acetate (200 mL) and H
2
O (150 mL) were added to
SO . The filtrate was
the mixture. The organic layer was dried with anhydrous Na
2
4
concentrated and purified by column chromatography (ethyl acetate:hexane=1:1) to
produce compound 6 as a red solid (0.01 g, 6% yield).
1
M.p. 248−250 °C. H NMR (500 MHz, (CD
3
)
2
S=O) 3.04 (s, 3H, CH
3
), 6.44−6.45 (d, J
= 8 Hz, 1H, ArH), 6.7−6.73 (t, J = 7 Hz, 1H, ArH), 7.04−7.07 (t, J = 7 Hz, 1H, ArH),
7.25−7.29 (m, 2H, ArH), 7.31−7.41 (m, 3H, ArH), 7.43−7.45 (m, 3H, ArH), 7.55−7.6
1
3
(m, 4H, ArH), 8 (s, 1H, ArH), 11.9 (s, 1H, indole NH). C NMR (125.7 MHz,
(CD S=O) 24.0, 104.3, 112.2, 119.8, 121.3, 122.1, 123.8, 126.1, 126.6, 127.6, 127.8,
3 2
)
128.1, 128.7, 129.4, 129.5, 129.7, 129.8, 131.1, 131.5, 136.6, 136.8, 137.3, 171.1,
+
171.3. HRMS-ESI (m/z) calculated for C27
427.1420.
H
20
N
2
O
2
[M + Na] 427.1422, found
1
2
Page 12 of 35

1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
2.3.7. 3-(4-(2-([1,1'-Biphenyl]-4-yl)vinyl)phenyl)-4-(1H-indol-3-yl)-1-methyl-1H-
pyrrole-2,5-dione (7)
Diethyl(4-iodobenzyl)phophonate (0.12 ml, 0.52 mmol), phenylboronic acid (0.1 g,
2 3
0.82 mmol), K CO (0.1 g, 0.72 mmol) and palladium acetate(II) (0.03 g, 0.13 mmol)
were added to DMF (3 mL). The solution was stirred at 110 °C for 13 h and cooled to
room temperature. 4-(4-(1H-indol-3-yl)-1-methyl-2,5-dioxo-2,5-dihydro-1H-1-
pyrrol-3-yl)benzaldehyde (0.12 g, 0.36 mmol) and sodium methoxide (28% in methanol
solution, 0.2 mL, 1 mmol) were added the solution and the mixture was stirred at 90 °C
for 3 hr. CHCl
added to the mixture. The organic layer was dried with anhydrous Na
was concentrated and purified by flash column chromatography (CHCl
to produce compound 7 as a red solid (0.05 g, 20% yield). M.p. > 300 °C. H NMR
(500 MHz, (CD S=O) 3.04 (s, 3H, CH ), 6.45−6.46 (d, J = 8 Hz, 1H, ArH), 6.71−6.74
3
(200 mL), 1 M aqHCl (100 mL) and 10% (w/v) aqNaCl (100 mL) were
SO . The filtrate
:CH OH=40:1)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
4
3
3
1
3
)
2
3
(t, J = 7.5 Hz, 1H, ArH), 7.05−7.08 (t, J = 7.5 Hz, 1H, ArH), 7.31−7.37 (m, 3H, ArH),
7.44−7.48 (m, 5H, ArH), 7.58−7.59 (d, J = 8.5 Hz, 2H, ArH), 7.69−7.71 (d, J = 8 Hz,
1
3
6H, ArH), 8.01 (s, 1H, ArH), 11.93 (s, 1H,indole NH). C NMR (125.7 MHz,
(CD S=O) 24.0, 104.3, 112.2, 119.8, 121.2, 122.1, 123.8, 126.1, 126.4, 126.9, 127.1,
127.4, 127.6, 127.9, 128.8, 128.9, 129.7, 129.8, 131.1, 131.5, 136.0, 136.6, 137.3,
3 2
)
+
139.3, 139.5, 171.0, 171.3. HRMS-ESI (m/z) calculated for C33
503.1735, found 503.1726.
24 2 2
H N O [M + Na]
2.3.8. 3-(1H-indol-3-yl)-1-methyl-4-(4-(4-styryl)styryl)phenyl)-1H-pyrrole-2,5-dione
(8)
Diethyl(4-iodobenzyl)phophonate (0.12 mL, 0.52 mmol), trans-2-phenylvinyl-
2 3
boronic acid (0.12 g, 0.81 mmol), K CO (0.1 g, 0.72 mmol) and palladium acetate(II)
1
3
Page 13 of 35

1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
(0.03 g, 0.13 mmol) were added to DMF (2.5 mL). The solution was stirred at 110 °C
for 18 h and returned to room temperature. 4-(4-(1H-indol-3-yl)-1-methyl-2,5-
dioxo-2,5-dihydro-1H-1-pyrrol-3-yl)benzaldehyde (0.1 g, 0.3 mmol) in DMF solution
(2 mL) and sodium methoxide (28% in methanol solution, 0.2 mL, 1 mmol) were added
to the solution and the mixture was stirred at 90 °C for 2.5 hr. CHCl
aqHCl (100 mL) and 10% (w/v) aqNaCl (100 mL) were added to the mixture. The
organic layer was dried with anhydrous Na SO . The filtrate was concentrated and
3
purified by flash column chromatography (CHCl :CH OH=40:1) to produce compound
3
(200 mL), 1 M
2
4
3
1
8 as a red solid (0.01 g, 4% yield). M.p. > 300 °C. HNMR (500 MHz, (CD
3
)
2
S=O)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
3
3.04 (s, 3H, CH ), 6.44−6.46 (d, J = 8.5 Hz, 1H, ArH), 6.71−6.74 (t, J = 7.5 Hz, 1H,
ArH), 7.05−7.08 (t, J = 8 Hz, 1H, ArH), 7.23−7.34 (m, 6H, ArH), 7.36−7.39 (t, J = 8
Hz, 2H, ArH), 7.44−7.45 (d, J = 8.5 Hz, 3H, ArH), 7.56−7.61 (m, 7H, ArH), 8.01 (s, 1H,
1
3
3 2
ArH), 11.93 (s, 1H, indole NH). C NMR (125.7 MHz, (CD ) S=O) 24.0, 104.3, 119.8,
121.3, 122.1, 123.8, 126.1, 126.5, 126.9, 127.0, 127.6, 127.9, 128.5, 128.7, 129.7,
129.8, 131.5, 136.2, 136.6, 137.0, 137.4, 171.1, 171.3. HRMS-ESI (m/z) calculated for
-
C
35
H
26
N
2
O
2
[M - H] 505.1916, found 505.1894.
2.3.9. 3-(1-Ethyl-1H-indol-3-yl)-1-methyl-4-(4-4-(styryl)styryl)phenyl)-1H-pyrrole-
2,5-dione (9)
Compound 8 (0.01 g, 0.02 mmol) was added to DMF (3 mL). The solution was
stirred at 0 °C for 15 min. Sodium hydride (60%, 2 mg, 0.05 mmol) was added to the
solution and the mixture was stirred at room temperature for 20 min and returned to 0
°C. Iodoethane (5 μL, 0.063 mmol) was added to the solution and the mixture was
stirred at room temperature for 1.5 hr. The mixture was concentrated and purified by
3
flash column chromatography (CHCl ) to produce compound 9 as a red solid (0.01 g,
1
4
Page 14 of 35

1
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
94% yield). M.p. > 300 °C. H NMR (500 MHz, (CD
3
)
2
S=O) 1.43−1.46 (t, J = 7 Hz,
3H, CH
3
), 3.04 (s, 3H, CH
3
), 4.32−4.37 (q, J = 7 Hz, 2H, CH ), 6.45−6.47 (d, J = 7.5
2
Hz, 1H, ArH), 6.75−6.77 (t, J = 7.5 Hz, 1H, ArH), 7.1−7.13 (t, J = 6.5 Hz, 1H, ArH),
7.27−7.29 (m, 5H, ArH), 7.36−7.39 (t, J = 7.5 Hz, 2H, ArH), 7.43−7.45 (d, J = 8.5 Hz,
1
3
2H, ArH), 7.54−7.61 (m, 9H, ArH), 8.1 (s, 1H, ArH). C NMR (125.7 MHz,
(CD S=O) 15.2, 24.0, 103.6, 124.4, 126.1, 126.5, 126.9, 127.0, 127.5, 127.9, 128.7,
3 2
)
129.7, 129.8, 133.2, 136.1, 136.6, 137.0, 171.0, 171.3. HRMS-ESI (m/z) calculated for
+
C
37
H
30
N
2
O
2
[M + Na] 557.2205, found 557.2195.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2.3.10. 3-(1H-indol-3-yl)-1-methyl-4-(pyren-1-yl)-1H-pyrrole-2,5-dione (10)
Tert-butyl 3-(4-bromo-1-methyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1H-indole-
1-carboxylate (0.13 g, 0.32 mmol), pyrene-1-boronic acid (0.08 g, 0.32 mmol) and
2 3 2
K CO (0.044 g, 0.32 mmol) were added to dioxane-H O (20 mL, 4:1).
Tetrakis(triphenylphosphine)palladium(0) (0.08 g, 0.07 mmol) was then added to the
mixture, and the solution was refluxed for 16 h. Ethyl acetate (100 mL), 0.5 M aqHCl
(100 mL) and 10% (w/v) aqNaCl (100 mL) were added to the mixture. The organic
4
layer was dried with anhydrous MgSO . The filtrate was concentrated and purified by
column chromatography (ethyl acetate:hexane=1:2) to produce compound 10 as a red
1
solid (0.07 g, 50% yield). M.p. 295−297 °C. H NMR (500 MHz, (CD
3
)
2
S=O) 3.13 (s,
3H, CH
3
), 5.92−5.93 (d, J = 8.5 Hz, 1H, ArH), 6.10−6.14 (m, 1H, ArH), 6.73−6.77 (m,
1H, ArH), 7.24−7.25 (d, J = 8.5 Hz, 1H, ArH), 7.99−8.07 (m, 5H, ArH), 8.20−8.26 (m,
1
3
3H, ArH), 8.30−8.33 (dd, J = 4 Hz, 7.5 Hz, 2H, ArH), 11.84 (s, 1H, indole NH).
C
NMR (125.7 MHz, (CD S=O) 24.2, 105.1, 111.9, 119.6, 120.0, 121.8, 123.6, 123.7,
3 2
)
124.5, 125.2, 125.4, 125.5, 126.3, 126.4, 127.2, 127.3, 127.8, 128.0, 128.4, 129.1,
130.2, 130.6, 131.0, 131.5, 135.2, 136.2, 171.1, 171.5. HRMS-ESI (m/z) calculated for
1
5
Page 15 of 35

+
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
29 18 2 2
C H N O [M + Na] 449.1266, found 449.1257.
2.3.11. Tert-butyl 3-(1-methyl-2,5-dioxo-4-(pyren-1-yl)-2,5-dihydro-1H-pyrrol-3-yl)-
1H-indole-1-carboxylate (11)
Compound 10 (0.05 g, 0.12 mmol), 4-dimethylaminopyridine (0.01 g, 0.08 mmol)
and Di-tert-butyl dicarbonate (0.1 ml, 0.42 mmol) were added to THF (10 mL). The
solution was stirred at 0 °C for 1.5 h. Ethyl acetate (100 mL) and H
added to the mixture. The organic layer was dried with anhydrous Na
was concentrated and purified by column chromatography (ethyl acetate:
2
O (100 mL) were
2
SO . The filtrate
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
hexane=1:2) to produce compound 11 as a red solid (0.05 g, 83% yield). M.p. 173−175
1
°C. H NMR (500 MHz, (CD
3
)
2
S=O) 1.54 (s, 9H, t-butyl), 3.16 (s, 3H, CH
3
), 6.27−6.28
(d, J = 8 Hz, 1H, ArH), 6.4−6.43 (t, J = 8 Hz, 1H, ArH), 6.96−6.99 (t, J = 8.5 Hz, 1H,
ArH), 7.88−7.9 (d, J = 8.5 Hz, 1H, ArH), 8.02−8.04 (d, J = 7.5 Hz, 1H, ArH), 8.06−8.08
(d, J = 7 Hz, 2H, ArH), 8.09−8.11 (d, J = 9.5 Hz, 1H, ArH), 8.2−8.22 (d, J = 9.5 Hz, 1H,
ArH), 8.25−8.27 (d, J = 8.5 Hz, 2H, ArH), 8.31−8.32 (d, J = 8 Hz, 1H, ArH), 8.33−8.34
1
3
3 2
(d, J = 7.5 Hz, 1H, ArH). C NMR (125.7 MHz, (CD ) S=O) 24.4, 27.4, 84.9, 109.6,
114.6, 120.5, 122.4, 123.4, 123.7, 124.5, 124.6, 125.0, 125.7, 125.8, 126.5, 126.7,
127.2, 127.7, 128.1, 128.4, 129.0, 129.5, 130.1, 130.6, 131.5, 133.1, 134.4, 148.3,
+
170.3, 170.9. HRMS-ESI (m/z) calculated for C34
549.1776.
H
26
N
2
O
4
[M + Na] 549.1790, found
2.3.12. 3-(9H-carbazol-2-yl)-4-(1H-indol-3-yl)-1-methyl-1H-pyrrole-2,5-dione (12)
Tert-butyl 3-(4-bromo-1-methyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)-1H-indole-
1-carboxylate (0.51 g, 1.26 mmol), 9H-carbazole-2-boronic acid pinacol ester (0.62 g,
2 3 2
1.9 mmol) and K CO (0.35 g, 2.53 mmol) were added to dioxane-H O (50 mL, 4:1),
1
6
Page 16 of 35

1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
and then tetrakis(triphenylphosphine)palladium(0) (0.16 g, 0.14 mmol) was added to the
mixture. The solution was refluxed for 16 h. Ethyl acetate (200 mL), 0.5 M aqHCl
(100 mL) and 10% (w/v) aqNaCl (100 mL) were added to the mixture. The organic
4
layer was dried with anhydrous MgSO . The filtrate was concentrated and purified by
column chromatography (ethyl acetate:hexane=4:5) to produce compound 12 as a red
1
solid (0.25 g, 51% yield). M.p. 293−295 °C. H NMR (500 MHz, (CD
3
)
2
S=O) 3.06 (s,
3H, CH ), 6.32−6.34 (d, J = 8 Hz, 1H, ArH), 6.51−6.54 (m, 1H, ArH), 6.96−6.99 (m,
3
1H, ArH), 7.13−7.2 (m, 2H, ArH), 7.37−7.44 (m, 3H, ArH), 7.64 (s, 1H, ArH),
7.99−8.02 (m, 2H, ArH), 8.07−8.09 (d, J = 7.5 Hz, 1H, ArH), 11.24 (s, 1H, carbazole
1
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
3 2
NH), 11.88 (s, 1H, indole NH). C NMR (125.7 MHz, (CD ) S=O) 24.0, 104.5, 111.0,
112.1, 112.2, 118.7, 119.6, 120.2, 120.4, 121.2, 121.9, 122.0, 122.6, 124.0, 126.0, 127.5,
129.2, 130.9, 131.2, 136.5, 139.2, 140.3, 171.2, 171.6. HRMS-ESI (m/z) calculated for
+
C
25
H
17
N
3
O
2
[M + Na] 414.1218, found 414.1204.
2.3.13. Tert-butyl 2-(4-(1-(tert-butoxycarbonyl)-1H-indol-3-yl)-1-methyl-2,5-dioxo-
2,5-dihydro-1H-pyrrol-3-yl)-9H-carbazole-9-carboxylate (13)
Compound 12 (0.04 g, 1 mmol), 4-dimethylaminopyridine (0.03 g, 0.25 mmol) and
Di-tert-butyl dicarbonate (0.45 mL, 1.9 mmol) were added to THF (10 mL). The
solution was stirred at 0 °C for 1.5 h. Ethyl acetate (150 mL) and H
added to the mixture. The organic layer was dried with anhydrous Na
was concentrated and purified by column chromatography (ethyl acetate:
2
O (100 mL) were
2
SO . The filtrate
4
hexane=1:2) to produce compound 13 as a red solid (0.06 g, 100% yield). M.p.
1
140−142 °C. H NMR (500 MHz, (CD
3
)
2
S=O) 1.42 (s, 9H, t-butyl), 1.67 (s, 9H, t-
), 6.44−6.46 (d, J =8 Hz, 1H, ArH), 6.75−6.78 (t, J = 7.5 Hz,
1H, ArH), 7.18−7.21 (t, J = 8 Hz, 1H, ArH), 7.39−7.42 (t, J = 7.5 Hz, 1H, ArH),
butyl), 3.09 (s, 3H, CH
3
1
7
Page 17 of 35

1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
7.52−7.55 (t, J = 8 Hz, 1H, ArH), 7.56−7.58 (d, J = 8 Hz, 1H, ArH), 8.07−8.08 (d, J = 8
Hz, 1H, ArH), 8.14 (s, 1H, ArH), 8.16−8.18 (d, J = 8.5 Hz, 2H, ArH), 8.22−8.24 (d, J =
1
3
3 2
8.5 Hz, 1H, ArH), 8.25 (s, 1H, ArH). C NMR (125.7 MHz, (CD ) S=O) 24.2, 26.8,
27.4, 27.6, 31.3, 84.0, 84.9, 109.1, 114.9, 115.8, 117.3, 119.9, 120.5, 121.4, 122.6,
123.5, 124.4, 124.9, 125.9, 126.1, 128.1, 129.1, 129.3, 134.1, 134.8, 136.8, 138.5,
+
148.5, 149.9, 170.2, 170.7. HRMS-ESI (m/z) calculated for C35
614.2267, found 614.2267.
33 3 6
H N O [M + Na]
2.4. Histochemical analysis
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
BSB is π−conjugated molecule and have styrylbenzene moieties. Unsymmetric
indolylmaleimides (compounds 1−4, compounds 6−8), which had aromatic moiety such
as styrylbenzene, were selected to evaluate the selectivity and sensitivity for amyloid
fibrils. To evaluate the selectivity and sensitivity for amyloid fibrils, we used tissue
samples of patients with senile systemic amyloidosis (SSA) caused by deposition of
wild-type transthyretin (WT-TTR) [28]. Each tissue sample was obtained by biopsy or
autopsy from the abdominal wall of patients with senile systemic amyloidosis. Sections
were cut 3 μm thick from paraffin-embedded blocks of tissue and were stained with
alkaline Congo red. Congo red reactivity was confirmed under polarized light. For the
staining of our new compounds, sections were immersed in a solution containing 0.01%
of each compound and 50% ethanol for 30 minutes. Then, the sections were quickly
2 3
differentiated in saturated Li CO and rinsed with 50% ethanol before examination by
fluorescence.
3. Results and discussions
1
8
Page 18 of 35

1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
3.1. Synthesis of unsymmetric maleimides (compounds 1−13)
The expansion of π-conjugation has a major impact on the FL property of FL
compounds [29−31]. Various π-conjugated polymers are designed and synthesized to
produce particularly desirable emissions. In this study, we developed unsymmetric IMs
that exhibit large Stokes shift and FL maxima emission wavelengths of more than 550
nm. To achieve this, various aromatic groups such as naphthyl, carbazolyl and pyrenyl
were introduced to maleimide moiety. Furthermore, unsymmetric IMs having
bithiophene and 4-phenylstylyl moieties were synthesized to achieve a longer shift in
the FL emission maximum wavelength. Unsymmetric IMs were synthesized as shown
in Scheme 1. After indole was metalated with ethylmagnesium bromide, the resulting
indolylmagnesium was coupled with another substrate (halide) to give 3-Bromo-4- (1H-
indol-3-yl)-1-methyl-1H-pyrrole-2,5-dione. The remaining bromo group of 3-Bromo-4-
(1H-indol-3-yl)-1-methyl-1H-pyrrole-2,5-dione was transformed by arylboronic acids
(Scheme 1(a)) through Suzuki-Miyaura coupling. This synthetic method of
unsymmetric IMs is thus facile and promising. A Wittig-Horner reaction was used to
introduce the 4-phenylstylyl moiety (Scheme 1(b)). Table 1 shows the unsymmetric
IMs prepared through the above methods.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
3.2 Absorption and emission studies
3
All compounds 1−13 were dissolved in DMF and CH CN. The absorption and FL
spectra of compounds 1−13 were measured in DMF and CH
3
CN. The absorption
spectra of compounds 1, 2, 3, 5, 8 and 12 in DMF are shown in Fig. 1. The absorption
1
9
Page 19 of 35

1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
maxima wavelengths of most IMs were over 430 nm, and the absorption maximum
wavelength of compound 5 was approximately 500 nm, the largest value among
compounds 1−13. The FL excitation and emission spectra of compounds 1, 2, 3, 5, 8
and 12 in DMF were obtained (Fig. S1). The FL emission spectra of compounds 1, 2, 3,
5, 8 and 12 in DMF are shown in Fig. 2. The FL emission maximum wavelength of
compound 5 in DMF was 597 nm and red FL emission was observed. Apparently, the
introduction of the benzene, naphthalene, styrylbenzene and bithiophene into the
maleimide moiety shifted the FL emission maxima to longer wavelengths. The
expansion of π–conjugation should cause an obvious change in the FL properties of
IMs. The Stokes shifts of compounds 7−13 were more than 100 nm (Table 2). The
introduction of pyrene caused the largest Stokes shift. In DMF, the FL intensity of
compound 13 was the strongest. The FL quantum yield of compound 3 in DMF was
higher than those of other compounds. Compound 10 has a pyrene in the structure.
When compound 10 was excited at 342 nm in DMF, the excimer FL was observed (Fig.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
3
S2). The optical properties of compounds 1−13 in CH CN (Table S1) were similar with
that of compounds 1−13 in DMF. The FL emission of compounds 3, 13, 7, 9 and 5
exhibited different emissions under UV irradiation at 365 nm (Fig. 3).
3.3 Histochemical analysis
Congo red appears orange-red and produces apple-green birefringence under
polarized light (Fig. 4 (A), (B)). Compound 1 showed the highest strength of
fluorescence with amyloid deposited the tissues in a patient with SSA in our compounds
(Fig. 4 (C)). Unsymmetric indolylmaleimide such as compound 1 was useful
compound to detect SSA amyloid fibrils. The fibril in SSA amyloid is derived from
2
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7
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0
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7
8
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0
1
2
3
4
5
normal transthyretin [33]. The FL intensity of compound 1 increased apparently in the
presence of transthyretin fibrils (Fig. S4). A few of aggregation-induced emission (AIE)
phenomena were found. For example, quinolinemalononitrile exhibits extraordinary
2
self-assembly property and strong emission in H O-tetrahydrofuran (THF) solution
[34]. When 1,2-Bis[4-(3-sulfonatopropoxy)phenyl]-1,2-diphenyl-
ethene salt (BSPOTPE) was mixed with performed insulin fibrils, BSPOTPE emitted a
strong green light [35]. The absorption and FL spectra of compound 1 were measured
in H
S5). Compound 1 did not exhibit AIE phenomenon in H
3
intensity of compound 1 in THF was much stronger than that of compound 1 in CH CN
2
O-THF solution, however, the FL emission enhancement were not observed (Fig.
2
O-THF solution. The FL
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
and DMF (Fig. S6). Thus, the FL intensity of compound 1 increased in a hydrophobic
solvent. Compound 1 binds to SSA amyloid, the FL intensity of compound 1 may
increase under highly hydrophobic environment.
4. Conclusions
Various fluorescent unsymmetric indolylmaleimides were synthesized in a facile
manner. The introduction of various aromatic compounds into the maleimide moiety in
unsymmetric indolylmaleimides produced a large Stokes shift and exhibited different
emissions under UV irradiation at 365 nm. We showed that unsymmetric
indolylmaleimides can be used for detecting amyloid fibrils. These results should
provide significant information for the development of useful fluorescent unsymmetric
indolylmaleimides.
Acknowledgements
2
1
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1
The authors thank the Japan Society for the Promotion of Science for their financial
support (Grant No. 23590043). This work was performed under a Cooperative
Research Program of the “Network Joint Research Center for Materials and Devices.”
The authors thank Prof. Toshinobu Masuda and Prof. Sayuri Yokoyama (Daiichi
1
3
University of Pharmacy, Fukuoka, Japan) for C-NMR measurements.
AppendixA. Supplementary data
1
1
Supplementary data associated with this article can be found, in the online version, at
http://dx.doi.org/
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Figure legends
Scheme 1. Synthesis of unsymmetric indolylmaleimides.
Fig. 1. The absorption spectra of compounds 1, 2, 3, 5, 8 and 12 in DMF. The
concentration of compounds 1, 2, 3, 5, 8 and 12 in DMF was 10 μM.
Fig. 2.
The fluorescence emission spectra of compounds 1, 2, 3, 5, 8 and 12 in DMF.
The concentration of compounds 1, 2, 3, 5, 8 and 12 in DMF was 10 μM.
The excitation wavelengths of compounds 1, 2, 3, 5, 8 and 12 were 439,
492, 432, 513, 451 and 454 nm.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
Fig. 3. The emission color of the compounds 3, 13, 7, 9 and 5 in DMF under UV
irradiation at 365 nm.
Fig. 4. Congo red, compound 1, compound 4 and compound 8 staining of blood
vessels in abdominal fat tissue in an 81-year-old male SSA patient. (A)
Congo red staining (B) Congo red staining (polarized light) (C) compound
1 staining (D) compound 4 staining (E) compound 8 staining.
Scale bars = 200 μm.
Table 1. Structure of unsymmetric indolylmaleimides.
Table2. Optical properties of compounds 1−13 in DMF. Absorption maximum
wavelength (λabs), molar absorption coefficients (ε), excitation maximum
wavelength (λex), emission maximum wavelength (λem) and FL quantum
F
yields (Φ ).
a.
b.
The concentration of compounds 1−13 in DMF is 10 μM. Ref. [32].
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Scheme 1
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