Synthesis and chemiluminescent properties of 6,8-diaryl-2-methylimidazo[1, 2-a]pyrazin-3(7H)-ones: Systematic investigation of substituent effect at para-position of phenyl group at 8-position

Article abstract of DOI:10.1016/j.jphotochem.2014.07.016

6,8-Diphenylimidazopyrazinone derivatives having a substituent R (R = CF₃, H, and OMe) at para position of the 8-phenyl group were synthesized and their chemiluminescent properties were investigated. The chemiluminescence maxima (CL_max

Full text of DOI:10.1016/j.jphotochem.2014.07.016

Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 12–25
Contents lists available at ScienceDirect
Journal of Photochemistry and Photobiology A:
Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Synthesis and chemiluminescent properties of
6,8-diaryl-2-methylimidazo[1,2-a]pyrazin-3(7H)-ones: Systematic
investigation of substituent effect at para-position of phenyl group at
8-position
Ryota Saitoa,b,∗, Takashi Hiranoc, Shojiro Makic, Haruki Niwac
a Department of Chemistry, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
b Research Center for Materials with Integrated Properties, Toho University, Chiba, Japan
c Department of Engineering Science, Graduate School of Informatics and Engineering, The University of Electro-Communications, 1-5-1 Chofugaoka, Chofu,
Tokyo 182-8585, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 June 2014
Received in revised form 14 July 2014
Accepted 18 July 2014
Available online 28 July 2014
6,8-Diphenylimidazopyrazinone derivatives having a substituent R (R = CF3, H, and OMe) at para position
of the 8-phenyl group were synthesized and their chemiluminescent properties were investigated. The
chemiluminescence maxima (CLmax) of these compounds were observed to be in the range of 513–553 nm
with a bathochromic shift that increased with the electron-withdrawing character of R, contrary to the
previously observed substituent effect at the 6-postion. The chemiluminescence efficiencies (ꢀCL) of these
imidazopyrazinones were improved by the introduction of a p-substituted phenyl group at the 8-position.
The quantitative investigation of the three quantum efficiencies (ꢀR, ꢀS, and ꢀFL) whose product gives us
ꢀCL revealed that the ꢀCL gains made were largely because of the increase in the values of the fluorescence
quantum yields of the corresponding light emitters (ꢀFL). The yields of the singlet-excited emitters (ꢀS)
during the chemiluminescent reaction were found to be very small (0.015–0.019), suggesting that one
cannot construct an efficient imidazopyrazinone–chemiluminescence system that is comparable to the
aequorin bioluminescence system only by using the electronic effects of substituents.
Keywords:
Imidazo[1,2-a]pyrazin-3(7H)-one
Chemiluminescence
Substituent effect
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
chemiluminescent reaction in aprotic solvents such as dimethyl
sulfoxide (DMSO) under air in the absence of catalyzing proteins
3,7-Dihydroimidazo[1,2-a]pyrazine-3-ones are widely dis-
tributed as luminescent substrates in the bioluminescent systems
of various luminous marine organisms such as a photopro-
tein, aequorin, isolated from the jellyfish Aequorea victoria [1].
During the bioluminescent reaction of aequorin, coelenterazine
(1) that is located at the reaction center of aequorin is oxi-
dized to form coelenteramide (2) in its singlet-excited state.
This reaction has high efficiency, one of the characteristics of
aequorin bioluminescence [2,3]. Coelenterazine also undergoes a
[4]. Various studies have focused on the mechanisms of bio- and
chemiluminescence, and it has been predicted that the bio- and
chemiluminescent reactions of coelenterazine follow the same
molecular mechanism. The widely accepted mechanism for lumi-
nescent oxidation is shown in Scheme 1 [5]. The most important
process in this mechanism is the decomposition of the dioxe-
tanone intermediate I that results in the chemical generation of
the singlet-excited state of the light emitter II [6–8]. Although
the molecular mechanisms of bio- and chemiluminescence are
identical, there is a considerable difference in their luminescent
efficiencies. The chemiluminescence efficiency of coelenterazine
(0.002 in DMSO) [4] is much lower than the bioluminescence
efficiency of aequorin (0.2) [2]. Many studies have attempted
to elucidate this difference in the mechanisms of bio- and
chemiluminescence.
∗
Corresponding author at: Toho University, Department of Chemistry, 2-2-1
Miyama, Funabashi, Chiba 2748510 Japan. Tel.: +81474721926; fax: +81474721926.
E-mail address: saito@chem.sci.toho-u.ac.jp (R. Saito).
http://dx.doi.org/10.1016/j.jphotochem.2014.07.016
1010-6030/© 2014 Elsevier B.V. All rights reserved.

R. Saito et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 12–25
13
Scheme 1. Plausible reaction mechanism during the chemi- and bio-luminescent reactions of imidazopyrazinones.
the variation in the ꢀS values was so small that one cannot judge
the relevance of the CIEEL or the CTIL mechanism using only these
results. Therefore, these mechanisms are still controversial and the
accumulation of related data is required.
We have also reported the chemiluminescence of some
imidazopyrazinones that have a conjugated system at the 8-
position of imidazopyrazinone nucleus in aqueous media [17,18].
These compounds mostly exhibited red-shifted chemilumines-
cence (ꢁmax: 500–530 nm) in basic buffer solutions, indicating
that the chemiluminescent properties of imidazopyrazinones are
strongly susceptible to the electronic substituent effect at the
8-position. However, since then, no attempt has been made to
investigate systematically the substituent effect on chemilumines-
cent properties including chemiluminescence quantum yields.
As part of our ongoing efforts to understand and regu-
late the chemiluminescence of imidazopyrazinones using sub-
stituent effects, we synthesized imidazopyrazinones having
para-substituted phenyl groups on their 8-position, 5a–c, in
which the electronic property of the substituents was systemat-
ically varied from electron-withdrawing (e.g., trifluoromethyl) to
electron-donating (e.g., methoxy group). Herein, we report the
effect of substituent variation on the chemiluminescent properties
of 5a–c. In addition, the syntheses and fluorescence of acetami-
dopyrazines, 6a–c, which are the corresponding products obtained
as a result of the oxidative chemiluminescence reactions of 5a–c,
have also been reported for a detailed understanding of the sub-
stituent effect on chemiluminescence.
The efficient generation of the singlet-excited emitter can
be attributed to a chemically initiated electron exchange lumi-
nescence (CIEEL) mechanism [9] or a charge transfer-induced
luminescence (CTIL) [8,10,11] mechanism. Scheme 1 illustrates the
application of these mechanisms to the imidazopyrazinone bio- or
chemi-luminescence sequence.
According to these hypotheses, the electron transfer from
the amidopyrazine moiety to the dioxetanone ring promotes the
decomposition of the dioxetanone intermediate, generating the
singlet-excited amide anion with high efficiency [12]. In the case
of the bioluminescent reaction of coelenterazine (1), the electron-
donating hydroxyl group, which probably dissociates to form the
more strongly electron-donating phenoxide in aequorin [13], is
considered to be essential for the effective formation of the singlet-
excited coelenteramide [14]. In order to understand the mechanism
of bio- and chemi-luminescence of imidazopyrazinones and, in par-
ticular, to clarify whether the electron-donating substituent plays
an important role in efficient generation of the singlet-excited
emitter, we have been investigating systematically the substituent
effects on the chemiluminescence of the coelenterazine analogues
(3) [7,15] and on the fluorescence of the coelenteramide analogues
(4) [16]. It was found that the efficiencies of the chemical gener-
ation of the singlet-excited light emitters (ꢀS: 0.008–0.015) were
quite small despite the large variation in the Hammett substituent
constant of R from −0.83 (for NMe2) to 0.54 (for CF3). Although
the substituent effect was apparently observed on the ꢀS values,

14
R. Saito et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 12–25
2. Experimental
2.1. General
intensities were recorded on a TD-4000 lumiphotometer (Labo-
science Co., Tokyo, Japan). The condition for this experiment is as
follows: a 15-␮l methanolic solution of 5a–c (4.0 ␮M) was placed
in the lumiphotometer, and the chemiluminescence reaction was
initiated by the injection of 300 ␮l of dehydrated DMSO.
All melting points were measured on a YAMATO Model MP-
21 or a LABORATORY DEVICES Mel-Temp II apparatuses in open
capillaries and are uncorrected. 1H-NMR spectra were recorded
on a JNM-GX270 spectrophotometer (JEOL Ltd., Japan) or a JNM-
LA400 spectrophotometer (JEOL Ltd., Japan). Chemical shifts (ı)
are reported in ppm using tetramethylsilane or an undeuterated
solvent as internal standards in the deuterated solvent used. Cou-
pling constants (J) are given in Hz. Chemical shift multiplicities
are reported as s = singlet, d = doublet, t = triplet, and m = multiplet.
Infrared (IR) spectra were obtained using the spectrophotometers
IR-810 or FT/IR-470plus (JASCO Co., Ltd., Japan). High and low reso-
lution electron impact ionization (EI) mass spectra were measured
on a HITACHI Model M-80B mass spectrometer with a HITACHI
M-0101 data system. The ionization energy and the accelerating
voltage were 70 eV and 3 kV, respectively. Combustion analyses
were performed on a PERKIN ELMER Series II CHNS/O Analyzer
2400. All conventional chemicals used in the present study are
commercially available, and were used as received.
2.5. Uv–vis absorption and fluorescence spectrometry
Spectral grade solvents were used for the measurement of
UV–vis absorption and fluorescence spectra. UV–vis spectra were
recorded on a Model 320 (Hitachi Co., Ltd., Japan) or a V-550 spec-
trophotometer (JASCO Co., Ltd., Japan). Fluorescence spectra were
measured with a Model F-4010 (Hitachi Co., Ltd., Japan) or a Model
F-777 fluorescence spectrophotometer (JASCO Co., Ltd., Japan) and
corrected according to the manufacturer’s instructions (excitation
bandpass: 5 nm; emission bandpass: 5 nm; response: 2.0 s; scan
speed: 60 nm min−1). A solution of compound 4 or 6 for fluo-
rescence measurement was prepared by mixing a stock solution
(100 ␮l) of 4 or 6 in DMSO (20 ␮M) and DMSO containing 0.5 vol%
of 10%-methanolic tetrabutylammonium hydroxide (2.0 ml) within
a 1-cm2 quartz cuvette at 25 ◦C. The reproducibility of each mea-
surement was definitively verified.
2.2. Computational details
2.6. Fluorescence quantum yields [7,16]
Semi-empirical calculations for ground and singlet-excited
states geometries were carried out with the PM7 [19] method
of MOPAC 2012 [20]. Solvent’s dielectric constant was taken into
account for these calculations by applying the conductor-like
screening (COSMO) model [21]. Density functional theory (DFT)
calculations [22], which include time-dependent DFT (TDDFT)
method [23], were performed with the GAMESS program (ver-
sion 11) [24,25] using Becke’s three-parameter function combined
with Lee, Yang and Parr’s correlation function (B3LYP) [26]. The
DFT calculations for geometry optimizations were performed with
the 6-31G(d) (for neutral compounds) or the 6-31+G(d) [27] (for
anionic compounds) basis set, and the TDDFT calculations were
carried out with the 6-31+G(d) basis set. Analysis of the MOPAC
and GAMESS log-files as well as molecular graphics were car-
ried out using the WinmostarTM program [28] and Chem3D Pro
(ver. 12.0). Semi-empirical quantum calculations of the UV/vis
absorption spectra were achieved by the intermediate neglect
of differential overlap/screened (INDO/S) approximation method
[29] using the WinmostarTM with the following parameters: basis
function = SDP, repulsion integral formula = “3” (Nishimoto-Mataga
equation), charge = 0, nuclear repulsion formula = “2” (Za*Zb*␥ab),
PKappa (Kappa value for p electron) = 0.585, DKappa (Kappa value
for d electron) = 0.3, CI = 20, and number of excited states = 1. All cal-
culations were conducted on a PC carrying an Intel CoreTM i3 3220
processor (3.30 GHz) and 8-GB RAM.
The fluorescence quantum yields at 25 ◦C were measured rela-
tive to quinine bisulfate in 0.1 M sulfuric acid and calculated on the
basis of the following equation.
ꢁ
ꢂ
ꢀ
Ifsam ꢁsf am dꢁsf am
n2ref
ODref
ꢀfsam = ꢀfref
ꢁ
ꢂ
ꢀ
2
ODsam
nsam
Ifref ꢁrfef dꢁrfef
where nref and nsam are the refractive indices of the solvents, ODref
ref
sam
and ODsam (≤0.02) are the optical densities, ꢀf (=0.52) and ꢀf
are the quantum yields, and the integrals denote the (computed)
area of the corrected fluorescence spectra, each parameter for the
standard (ref) and sample (sam) solutions, respectively. The repro-
ducibility of each measurement was definitively verified.
2.7. Determination of ꢀR values
The ꢀR values were determined by comparing the HPLC chro-
matogram of the luminescence-spent product with that of the
corresponding synthesized acetamidopyrazine 6a–e. The HPLC
analyses were performed on an Inertsil ODS column (4.6 × 250 mm;
GL Sciences, Inc., Japan) with an acetonitrile/water solvent system
as a mobile phase.
2.8. Synthetic procedures
2.3. Chemiluminescence spectra
2.8.1. Representative procedure for the Suzuki coupling reactions.
A typical example: Synthesis of 2-amino-5-bromo-3-(4-
trifluoromethylphenyl)pyrazine (9a) and 2-amino-3,5-bis(4-
trifluoromethylphenyl)pyrazine (10a)
Chemiluminescence spectra of the imidazopyrazinones 5a–c
were measured on a PMA-10 photonic multichannel analyzer
equipped with an image intensifier (Hamamatsu Photonics K. K.).
Chemiluminescence measurement was achieved by mixing a stock
solution (100 ␮l) of 5a–c in methanol (1.0 mM) and DMSO (2.0 ml)
within a 1-cm2 quartz cuvette under aerobic condition at 25 ◦C. The
reproducibility of each measurement was definitively verified.
To a dioxane solution (6.0 ml) of 2-amino-3,5-dibromopyra-
zine
7 (304 mg, 1.20 mmol) was successively added tetrakis
(triphenylphosphine)palladium (112 mg, 9.6 × 10−5 mol, 8 mol%),
4-trifluoromethylphenylboronic acid 8a (230 mg, 1.21 mmol), and
1 ml of 2-mol dm−3 sodium carbonate under argon atmosphere.
The mixture was then refluxed for 4 h under argon atmosphere.
After cooling to room temperature, water (10 ml) was added, and
organic materials were extracted using chloroform (15 ml × 3). The
combined organic layer was then dried over sodium sulfate. The
removal of the solvent afforded brown solid, which was purified by
2.4. Chemiluminescence quantum yields
The chemiluminescence quantum yields of 5a–c at 25 ◦C were
determined by comparing their luminescence intensities to that
of luminol as a standard (ꢀCL = 0.028) [30]. The luminescence

R. Saito et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 12–25
15
preparative thin-layered chromatography on Merck silica gel (par-
ticle size, 5–40 ␮m; 200 × 200 × 1.75 mm, 5 plates) with a mixed
solvent system (ethylacetate:hexane = 8:3) as a mobile phase to
give 9a (293 mg, 77%) in the form of a pale yellow solid and 10a
(17 mg, 4%) in the form of a pale yellow solid. 9a: mp152–154 ◦C
(ethanol–hexane); ıH (400 MHz, CDCl3) 4.78 (br. s, 2H), 7.77 (d,
J = 8.3, 2H), 7.88 (d, J = 8.3, 2H), 8.13 (s, 1H); ꢂmax (KBr)/cm−1 3415,
3311, 3185, 1642, 1616, 1449, 1406, 1320, 852; m/z (positive-EI)
(relative intensity) 319 (93), 317 (M+, 100), 211 (61), 184 (47).
Anal. Calcd for C11H7BrF3N3: C, 41.53; H, 2.22; N, 13.21. Found:
C, 41.94; H, 2.06; N, 12.89. 10a: mp 194–196 ◦C (ethanol–hexane);
ıH (400 MHz, CDCl3) 4.94 (br. s, 2H), 7.70 (d, J = 8.0, 2H), 7.80 (d,
J = 8.0, 2H), 7.96 (d, J = 8.0, 2H), 8.07 (d, J = 8.0, 2H), 8.55 (s, 1H); ꢂmax
(KBr)/cm−1 3482, 3288, 3152, 1630 (sh), 1618, 1536, 1464, 1425,
1408, 1329, 852; m/z (positive-EI) (relative intensity) 383 (100, M+),
382 (41), 356 (15), 314 (10). Anal. Calcd for C18H11F6N3: C, 56.40;
H, 2.89; N, 10.96. Found: C, 56.54; H, 2.72; N, 10.86.
a yellowish brown solid of 14. Yield: 1.16 g (88%); mp 133 ◦C (deco-
mop); ıH (270 MHz, CDCl3) 5.06 (br. s, 2H), 7.34–7.48 (m, 3H),
7.84–7.89 (m, 2H), 8.41 (s, 1H); m/z (positive-EI) (relative intensity)
251 (91, [M + 2]+), 249 (100, M+), 170 (17), 143 (20), 116 (62).
2.8.6. 2-Amino-5-phenyl-3-(4-trifluoromethylphenyl)pyrazine
(11a)
Method
A (from 9a): In this method 11a was prepared
from 7 (137 mg, 4.30 × 10−4 mol) with tetrakis(triphenylphos-
phine)palladium (25 mg, 2.1 × 10−5 mol), 4-trifluoromethyl-
phenylboronic acid 8a (57.3 mg, 4.70 × 10−4 mol), and 0.5 ml
of 2-mol dm−3 sodium carbonate using a method similar to
that for preparing 9a. Yield: 111 mg (82%). Method B (from
14): 11a was prepared from 14 (251 mg, 1.00 mmol) with
tetrakis(triphenylphosphine)palladium (43 mg, 3.7 × 10−5 mol),
4-trifluoromethylphenylboronic acid 8a (285 mg, 1.5 mmol), and
0.9 ml of 2-mol dm−3 sodium carbonate i using a method similar
to that for preparing 9a, except refluxing for 9 h. The product
was isolated by column chromatography on silica gel (particle
size, 63–200 ␮m; 100 g) with a mixed solvent system (ethylac-
etate:chloroform = 4:1) as eluents. Yield: 301 mg (95%). Compound
11a was obtained as tan needles (from ethanol); mp 190–190 ◦C;
ıH (270 MHz, CDCl3) 4.78 (br. s, 2H), 7.35–7.49 (m, 3H), 7.79 (d,
J = 8.6, 2H), 7.95–8.00 (m, 4H), 8.51 (s, 1H); ꢂmax (KBr)/cm−1 3405,
3305, 3172, 1641, 1614, 1530, 1465, 1406, 1322, 849, 762, 695;
m/z (positive-EI) (relative intensity) 315 (100, M+), 288 (23), 102
(68). HRMS (positive-EI) Calcd for C17H12F3N3: 315.0983. Found:
315.1038.
2.8.2. 2-Amino-5-bromo-3-(4-methoxyphenyl)pyrazine (9c)
Prepared from 7 (304 mg, 1.20 mmol) with tetrakis(triphenyl-
phosphine)palladium (112 mg, 9.6 × 10−5 mol, 8 mol%), 4-
methoxyphenylboronic acid 8c (182 mg,1.20 mmol), and 1.5 ml
of 2-mol dm−3 sodium carbonate using a method similar to
that for preparing 9a: yield, 171 mg (51%); mp 143–145 ◦C
(ethanol–hexane); ıH (400 MHz, CDCl3) 3.87 (s, 3H), 4.76 (br. s,
2H), 7.01 (d, J = 9.0, 2H), 7.69 (d, J = 9.0, 2H), 8.03 (s, 1H); ꢂmax
(KBr)/cm−1 3470, 3292, 3146, 2827, 1631, 1609, 1508, 1440, 1391,
1251, 839. Anal. Calcd for C11H10BrN3O: C, 47.16; H, 3.60; N, 15.00.
Found: C, 47.28; H, 3.46; N, 14.85.
2.8.7. 2-Amino-3,5-diphenylpyrazine (11b)
2.8.3. 2-Amino-3,5-bis(4-methoxyphenyl)pyrazine (10c)
This compound was obtained as a by-product in the synthesis of
5c: yield, 44 mg (12%). Identification of the product was achieved
by comparing its spectral features with that of previously reported
data [31].
Prepared from
7
(401 mg, 1.59 mmol) with tetrakis
(triphenylphosphine)palladium (72 mg, 6.2 × 10−5 mol), phenyl-
boronic acid 8b (491 mg, 4.03 mmol), and 8.0 ml of 2-mol dm−3
sodium carbonate using a method similar to that for preparing
9a, except refluxing for 5 h in 1,2-dimethoxyehane. The product
was isolate by column chromatography on silica gel (particle
size, 63–200 ␮m; 70 g), eluted with a mixed solvent system
(first ethylacetate:chloroform:hexane = 1:1:1, and then ethylac-
etate:chloroform = 1:1) to give 11b as pale yellow solids. Yield:
360 mg (91%); mp 134–135 ◦C; ıH (270 MHz, CDCl3) 4.84 (br. s, 2H),
7.32–7.56 (m, 6H), 7.80–7.84 (m, 2H), 7.95–7.99 (m, 2H), 8.45 (s,
1H); ꢂmax (KBr)/cm−1 3461, 3282, 3144, 1625, 1536, 1459, 1436,
746, 688; m/z (positive-EI) (relative intensity) 247 (100, M+), 220
(17), 116 (12), 102 (22), 89 (10). HRMS (positive-EI) Calcd for
2.8.4. 2-Amino-5-phenylpyrazine (13)
Prepared from 12 (122 mg, 7.01 × 10−4 mol) with tetrakis
(triphenylphosphine)palladium
(33.5 mg,
2.90 × 10−5 mol),
phenylboronic acid 8b (116 mg, 9.53 × 10−4 mol), and 1.0 ml
of 2-mol dm−3 sodium carbonate using a method similar to that
for preparing 9a, except refluxing for 12 h. The product was
isolate by column chromatography on silica gel (particle size,
63–200 ␮m; 30 g), eluting with a mixed solvent system (ethy-
lacetate:chloroform = 1:1), followed by recrystallization from
ethanol–hexane to give 13 as tan needles: yield, 89.2 mg (74%);
mp: 146–147 ◦C; ıH (400 MHz, CDCl3) 4.61 (br. s, 2H), 7.35–7.49
(m, 3H), 7.87–7.90 (m, 2H), 8.80 (d, J = 1.4, 1H), 8.46 (d, J = 1.4, 1H);
ꢂmax (KBr)/cm−1 3340, 3174, 3020, 1653, 1588, 1536, 1480, 1389,
751, 694 (␥CH). Anal. Calcd for C10H9N3: C, 70.16; H, 5.30; N, 24.54.
Found: C, 70.32; H, 5.19; N, 24.63.
C16H13N3: 247.1108. Found: 247.1085.
2.8.8. 2-Amino-5-phenyl-3-(4-methoxyphenyl)pyrazine (11c)
Method
A
(from 9c): In this method 11c was prepared
from 9c (114 mg, 4.07 × 10−4 mol) with tetrakis(triphenyl-
phosphine)palladium (25 mg, 2.1 × 10−5 mol), 4-methoxyphenyl-
boronic acid 8c (57 mg, 4.7 × 10−4 mol), and 0.5 ml of 2-mol dm−3
sodium carbonate in using a method similar to that for prepar-
2.8.5. 2-Amino-3-bromo-5-phenylpyrazine (14)
ing 9a. Yield: 109 mg (96%). Method B (from 14): In this
To a solution of aminopyrazine 13 (902 mg, 5.27 mmol) in chlo-
roform (40 ml) was added tetrabutylammonium tribromide (3.11 g,
6.44 mmol) and pyridine (7.0 ml) under argon atmosphere, and
the mixture was then stirred for 3 h with heating on an oil bath
(55–58 ◦C) under argon. After cooling to room temperature water
was added to the reaction mixture, and then the mixture was
stirred for another 20 min at room temperature. Organic materials
were then extracted with chloroform (50 ml × 3), and the combined
organic layer was dried over sodium carbonate. The removal of the
solvent afforded black mass, which was purified by column chro-
matography on silica gel (particle size, 63–200 ␮m; 530 g), eluting
with a mixed solvent system (ethylacetate:chloroform = 1:1) to give
method 11c was prepared from 14 (50 mg, 0.2 mmol) with
tetrakis(triphenylphosphine)palladium (21 mg, 1.8 × 10−5 mol),
4-methyoxhyphenylboronic acid 8c (40 mg, 0.26 mmol), and 0.3 ml
of 2-mol dm−3 sodium carbonate using a method similar to that
for preparing 9a, except refluxing for 13 h in 1,2-dimethoxyethane.
Yield: 52 mg (94%). Compound 11c was obtained as tan needles
(from ethanol); mp 138–139 ◦C; ıH (270 MHz, CDCl3) 4.78 (br. s,
2H), 7.05 (d, J = 8.9, 2H), 7.32–7.47 (m, 3H), 7.79 (d, J = 8.9, 2H),
7.94–7.99 (m, 4H), 8.42 (s, 1H); ꢂmax (KBr)/cm−1 3449, 3305,
3170, 1636, 1608, 1533, 1510, 1461, 1245, 839, 766, 695; m/z
(positive-EI) (relative intensity) 277 (100, M+). HRMS (positive-EI)
Calcd for C17H15N3O: 277.1215. Found: 277.1188.

16
R. Saito et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 12–25
2.8.9. 3,7-Dihydro-2-methyl-6-phenyl-8-(4-trifluoromethyl-
phenyl)imidazo[1,2-a]pyrazin-3-one (5a)
was added dropwise acetyl chloride (80 ␮l, 1.1 mmol) at 0 ◦C in a
period of 2 min, and the mixture was stirred for 30 min. Saturated
aqueous sodium bicarbonate (2.0 ml) was added to the reaction
mixture, and then organic materials were extracted with chlo-
roform (10 ml × 3). The combined organic layer was dried over
magnesium sulfate, and then the solvent was removed under
reduced pressure to afford crude product, which was purified by
recrystallization from methanol to give the pure product as color-
less needles (41 mg, 58%). mp 183–184 ◦C; ıH (270 MHz, DMSO-d6)
1.93 (s, 3H), 7.41–7.59 (m, 6H), 7.83 (dd, J = 1.6, 7.9, 2H), 8.19 (dd,
J = 1.6, 7.9, 2H), 9.05 (s, 1H), 10.40 (s, 1H); ꢂmax (KBr)/cm−1 3430,
3250, 1670, 1540, 1504, 1492, 1419, 749, 693; m/z (positive-EI) (rel-
ative intensity) 289 (14, M+), 247 (61), 116 (37), 102 (39), 89 (47), 43
(100). HRMS (positive-EI) Calcd for C18H15N3O: 289.1215. Found:
289.1219.
To
a degassed ethanol solution (2.5 ml) of 11a (81 mg,
0.26 mmol) was added pyruvic aldehyde (40 wt% solution in
water, 175 mg, 0.97 mmol) and conc. HCl (100 ␮l). The mixture
was heated at 80 ◦C in a sealed-vessel under argon atmosphere
for 3.5 h. After cooling to room temperature, the solvent was
removed under reduced pressure. Diethyl ether (1.0 ml) and con-
centrated hydrogen chloride (2 drops) were successively added to
the residue and the mixture was cooled to −20 ◦C to form yel-
low precipitate, which was collected by suction filtration. After
drying in vacuo, the hydrochloride salt of 5a was obtained as
pale yellow solids. Yield: 71 mg (68%); mp 153 ◦C (decomp);
ıH (400 MHz, CD3OD) 2.55 (s, 3H), 7.49–7.59 (m, 3H), 8.00 (d,
J = 8.1, 2H), 8.15–8.18 (m, 2H), 8.30 (d, J = 8.1, 2H), 8.79 (s, 1H);
ꢂmax (KBr)/cm−1 3403, 3067, 2931, 1658, 1572, 1532, 1493,
1323, 848, 770, 695. Anal. Calcd for C20H14F3N3O·HCl: C, 61.98;
H, 3.77; N, 10.84. Found: C, 62.09; H, 3.60; N, 10.79. The
hydrochloride-free 5a was readily obtained by passing through
a silica-gel short column with 10:1 chloroform/methanol as elu-
ents. HRMS (positive-EI) Calcd for C20H14F3N3O:369.1089. Found:
369.1011.
2.8.14. 2-Acetamino-5-phenyl-3-(4-methoxyphenyl)pyrazine
(6c)
Prepared from 11c (101 mg, 0.36 mmol) using a method similar
to that for preparing 6b. Yield: 62 mg (53%), colorless needles (from
methanol); mp 193 ◦C; ıH (270 MHz, DMSO-d6) 1.96 (s, 3H), 3.83 (s,
3H), 7.04 (d, J = 8.9, 2H), 7.47–7.59 (m, 3H), 7.82 (d, J = 8.9, 2H), 8.18
(dd, J = 1.6, 7.9, 2H), 8.99 (s, 1H), 10.34 (s, 1H); ꢂmax (KBr)/cm−1
3450, 3240, 1671, 1510, 1372, 1254, 1178; m/z (positive-EI) (rela-
tive intensity) 319 (45, M+), 277 (61), 116 (63), 102 (64), 89 (42), 43
(83). HRMS (positive-EI) Calcd for C19H17N3O2: 319.1312. Found:
319.1280.
2.8.10. 3,7-Dihydro-2-methyl-6,8-diphenylimidazo[1,2-a]
pyrazin-3-one (5b)
Prepared from 11b (110 mg, 0.44 mmol) using a method sim-
ilar to that for preparing 5a. The Product, 5b hydrochloride, was
obtained as yellow needles (71 mg, 49%). mp 128 ◦C (decomp);
ıH (400 MHz, CD3OD) 2.55 (s, 3H), 7.48–7.58 (m, 3H), 7.67–7.73
(m, 3H), 8.06–8.10 (m, 2H), 8.14–8.16 (m, 2H), 8.75 (s, 1H); ꢂmax
(KBr)/cm−1 3401, 3061, 2924, 1658, 1572, 1528, 1492, 773, 694.
Anal. Calcd for C19H15N3O ·HCl·0.75H2O: C, 64.96; H, 5.02; N, 11.96.
Found: C, 65.24; H, 4.95; N, 11.44. HRMS (positive-EI) Calcd for
2.8.15. 3,7-Dihydro-2-methyl-8-(2,6-dimethylphenyl)-6-
phenylimidazo[1,2-a]pyrazin-3-one (16)
To a mixture of 7 (211 mg, 0.84 mmol), 2,6-dimethylphenyl-
boronic acid (126 mg, 0.84 mmol) and triphenylphosphine
(36 mg, 0.14 mmol) in dioxane (3.0 ml) were successively
added bis(triphenylphosphine)palladium(II) dichloride (36 mg,
50.9 ␮mol) and aqueous sodium bicarbonate (2 M, 1.0 ml). The
mixture was then refluxed for 16 h under argon atmosphere.
After cooling to room temperature, water (40 ml) was added to
the reaction mixture and organic materials were extracted with
chloroform (50 ml × 3). The combined organic layer was dried
over magnesium sulfate, and the solvent was evaporated to give
brownish amorphous, which was roughly purified by column
chromatography on silica gel to give orange glassy solid that
C19H15N3O: 301.215. Found: 301.1263.
2.8.11. 3,7-Dihydro-8-(4-methoxyphenyl)-2-methyl-6-
phenylimidazo[1,2-a]pyrazin-3-one (5c)
Prepared from 11c (90 mg, 0.32 mmol) using a method simi-
lar to that for preparing 5a. The Product, 5c hydrochloride, was
obtained as yellow needles (63 mg, 54%). mp 129 ◦C (decomp);
ıH (400 MHz, CD3OD) 2.55 (s, 3H), 7.23 (d, J = 8.8, 2H), 7.50–7.59
(m, 3H), 8.09–8.12 (m, 2H), 8.11 (d, J = 8.8, 2H), 8.64 (s, 1H);
ꢂmax (KBr)/cm−1 3406, 3071, 2935, 1660, 1607, 1534, 1493,
1026, 887, 766, 693. Anal. Calcd for C20H17N3O2·HCl·2.1H2O: C,
59.22; H, 5.52; N, 10.36. Found: C, 58.95; H, 5.02; N, 10.34.
HRMS (positive-EI) Calcd for C20H17N3O2: 331.1321. Found:
331.1364.
contained
2-amino-5-bromo-3-(2,6-dimethylphenyl)pyrazine.
This material was directly reacted with phenylboronic acid (92 mg,
0.75 mmol) without further purification using a method similar
to that for preparing 11a, yielding 15 (95 mg, 41% from 7) as pale
yellow solid; ıH (400 MHz, CDCl3) 2.13 (s, 6H), 4.46 (br, s, 2H), 7.17
(d, J = 7.3, 2H), 7.26 (t, J = 7.3, 1H), 7.33–7.37 (m, 1H), 7.42–7.45 (m,
2H), 7.91–7.93 (m, 2H), 8.48 (s, 1H). Then, 15 was reacted with
pyruvic aldehyde using a method similar to that for preparing 5a
hydrochloride. The obtained 16 hydrochloride was purified by
column chromatography on silica gel with chloroform/methanol
(10/1) as eluents to afford hydrochloride-free 16. Yield: 69 mg (70%
from 15), orange solid; ıH (400 MHz, CDCl3) 2.29 (s, 6H), 2.45 (s,
3H), 7.15 (s, 1H), 7.18 (d, J = 7.6, 2H), 7.31 (t, J = 7.8, 1H), 7.44–7.54
(m, 6H); ꢂmax (KBr)/cm−1 3430, 3034, 2918, 1631, 1558, 1505,
1469, 768, 694, 674. Anal. Calcd for C20H17N3O2·HCl·2.1H2O: C,
59.22; H, 5.52; N, 10.36. Found: C, 58.95; H, 5.02; N, 10.34.
2.8.12. 2-Acetamino-5-phenyl-3-(4-trifluoromethylphenyl)
pyrazine (6a)
A mixture of 11a (50 mg, 0.16 mmol) and acetic anhydride
(1.5 ml, 16 mmol) was stirred at 70 ◦C for 1d. After cooling to room
temperature the resulting colorless precipitate was collected by
suction filtration. The collected material was washed with water,
and then purified by recrystallization from methanol to afford the
product as colorless needles (14 mg, 24%). mp 218 ◦C; ıH (270 MHz,
DMSO-d6) 1.95 (s, 3H), 7.48–7.60 (m, 3H), 7.85 (d, J = 8.2, 2H), 8.03
(d, J = 8.2, 2H), 8.19–8.22 (m, 2H), 9.13 (s, 1H), 10.63 (s, 1H); ꢂmax
(KBr)/cm−1 3430, 3220, 1670, 1328, 852; m/z (positive-EI) (relative
intensity) 357 (39, M+), 315 (100), 116 (58), 102 (56), 77 (37), 69
(78). HRMS (positive-EI) Calcd for C19H14F3N3O: 357.1089. Found:
357.1131.
3. Results and discussion
3.1. Syntheses and electronic properties of imidazopyrazinones
5a–c
2.8.13. 2-Acetamino-3,5-diphenylpyrazine (6b)
To a solution of 11b (60 mg, 0.24 mmol) and anhydrous pyri-
dine (0.70 ml, 8.6 mmol) in anhydrous dichloromethane (2.0 ml)
The preparation of the imidazopyrazinones was achieved in
accordance with Scheme 2. The starting material, 2-amino-3,

R. Saito et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 12–25
17
Scheme 2. Synthesis pathways for 2-amino-3,5-diarylpyrazines 11a–c.
Scheme 3. Alternative methods for preparing 2-amino-3,5-diarylpyrazines 11a and 11c.
5-dibromopyrazine (7), was prepared by the bromination of com-
mercially available 2-aminopyrazine with tetrabutylammonium
tribromide, a useful substituent for bromine [32], according to
the method described in literature [33]. Next, the monoary-
lated aminopyrazines 9a and 9c were obtained in good yield by
a regioselective palladium-catalyzed cross-coupling reaction of
dibromopyrazine 7 with an equimolar of arylboronates 8a and 8c,
respectively. A small amount of the corresponding 3,5-diarylated
products (10a) [18] was also obtained. The C-3 selectivity was
also observed in a Sonogashira coupling reaction [34] using the
Scheme 4. Synthesis of imidazopyrazinones 5a–c and amidopyrazines 6a–c.

18
R. Saito et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 12–25
Fig. 1. Absorption spectra of the imidazopyrazinones 5a–c (100 ␮M) in
Britton–Robinson buffer (pH 7.0).
same substrate. This regioselectivity may be caused by the vicinal
contribution of the 2-amino group during the catalytic cycle. The
resulting bromopyrazines 9a and 9c were then further reacted with
phenylboronic acid 8b under Suzuki-coupling conditions to afford
the diarylaminopyrazines 11a and 11c, respectively. Thus, two
different aryl substituents were introduced at 3- and 5-positions
of 2-aminopyrazine by successive Suzuki coupling reactions.
Diarylaminopyrazines 11a and 11c were also obtained from 2-
amino-5-bromopyrazine 12 in three steps, as shown in Scheme 3.
The spectroscopic features of the diarylaminopyrazines obtained
by both the synthetic routes were identical, confirming the
regioselective introduction of the different aryl groups. 2-Amino-
3,5-diphenylpyrazine 11b was directly synthesized by the Suzuki
cross-coupling reaction of 2-amino-3,5-dibromopyrazine with two
equivalents 8b in good yield. Thus, the present study has provided
additional examples of compounds obtained as a result of the intro-
duction of different conjugate systems at the 3- and 5-positions
of 2-aminopyrazine. The obtained 3,5-diarylaminopyrazines 11a–c
were then condensed with pyruvic aldehyde under acid-catalyzed
conditions [7,34] to afford the intended imidazopyrazinones
5a–c as hydrochloride salts in moderate yields (68% for 5a,
49% for 5b, and 53% for 5c) (Scheme 4). 2-Acetamidopyrazines
6a–c, the corresponding products of the oxidative chemilumi-
nescent reactions of 5a–c, were prepared by the acetylation
of the 3,5-diarylaminopyrazine 11a–c (Scheme 4) [16]. The
structure of the products was verified using 1H-NMR, IR, and
high-resolution mass spectrometries, coupled with combustion
analysis.
In order to understand the electronic effects of the phenyl group
at the 8-position and the substituent R on the central imidazopy-
razinone conjugate system, the electronic absorption spectra of
the synthesized imidazopyrazinones 5a–c were measured, and the
results are shown in Fig. 1. Each compound exhibits the low-
est transition at approximately 435 nm, which is almost equal
to that of 3,7-dihydro-2-methyl-6-phenylimidazo[1,2-a]pyrazin-
3-one, the Cypridina luciferin analogue (CLA) [35], having no
substituent at the 8-position. In addition, the lowest transition
energy is found to be independent of the electronic effect of
the substituent even in a polar medium such as water. These
results imply that the phenyl ring at the 8-position is twisted
in such a manner that there is no interaction between the ␲-
electrons of the central imidazopyrazinone and those of the phenyl
group at the 8-position. The X-ray crystallographic analysis of
5a–c would provide the evidence for this, but it has not been
Fig. 2. Optimized geometries of 5a–c calculated by the PM7-COSMO and the
B3LYP/6-31G(d) methods.
completed because single crystals of these compounds suitable
for the analysis have been hardly obtained. Therefore, the opti-
mized geometry of 5a–c was estimated using both semi-empirical
and density functional theory (DFT) [22,24] calculations. For semi-
empirical approach, PM7 method [19] with the conductor-like
screening model (COSMO) [21] was adopted, and DFT calcula-
tions were performed at the B3LYP/6-31G(d) level [26]. The results
are shown in Fig. 2. In the optimized geometries of 5a–c calcu-
lated by the PM7-COSMO method, the dihedral angle ꢃ between
pyrazine ring and the phenyl group at 8-position were 53.2◦,
50.2◦, and 46.1◦, respectively, whereas those in the geometries
obtained by DFT calculations were 29.1◦, 30.0◦, and 28.4◦, respec-
tively. Thus, DFT calculations gave more planar geometries than the
PM7-COSMO method, and this does not correspond to the experi-
mental results. The absorption spectra of 5a–c were also calculated
at different levels of theory and basis sets, which include a semi-
empirical intermediate neglect of differential overlap/screened
(INDO/S) approximation method [29], and the time-dependent DFT
(TDDFT) method [23] at the B3LYP level with 6-31+G(d) basis set
[27]. The above geometries, calculated by both the PM7-COSMO
and the DFT methods, were used for these calculations. The results
are summarized in Fig. 3 compiled with experimental data. Obvi-
ously, the INDO/S calculations using the geometries obtained by
the PM7-COSMO provides absorption bands exhibiting good agree-
ment with the experimental data. Thus, for the present system,
the semi-empirical calculations are better approaches to obtain
optimized geometries and absorption spectra than the DFT meth-
ods.

R. Saito et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 12–25
19
Fig. 3. Calculated absorption spectra (red sticks) of 5a–c at different levels of theory: (a) 5a (optimized by PM7-COSMO); INDO/S, (b) 5a (PM7-COSMO); TD-B3LYP/6-31+G(d),
(c) 5a (B3LYP/6-31G(d)); INDO/S, (d) 5a (B3LYP/6-31G(d)); TD-B3LYP/6-31+G(d), (e) 5b (PM7-COSMO); INDO/S, (f) 5b (PM7-COSMO); TD-B3LYP/6-31+G(d), (g) 5b (B3LYP/6-
31G(d)); INDO/S, (h) 5b (B3LYP/6-31G(d)); TD-B3LYP/6-31+G(d), (i) 5c (PM7-COSMO); INDO/S, (j) 5c (PM7-COSMO); TD-B3LYP/6-31+G(d), (k) 5c (B3LYP/6-31G(d)); INDO/S,
(l) 5c (B3LYP/6-31G(d)); TD-B3LYP/6-31+G(d). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

20
R. Saito et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 12–25
Table 1
Chemiluminescence properties of imidazopyrazinones 5a–c and 3a–c in aerated
DMSO at 25 ◦C.
a
CLmax (nm)
Quantum yield
Compound (R)
c
d
e
ꢀCLb/10−2
ꢀR
ꢀFL
ꢀS
5a (CF3)
5b (H)
553
525
513
454
462
473
0.52
0.53
0.45
0.06
0.15
0.18
0.79
0.88
0.78
0.78
0.93
0.95
0.34
0.41
0.38
0.05
0.18
0.21
0.019
0.015
0.015
0.015
0.008
0.008
5c (OMe)
3a (CF3)f
3b (H)f
3c (OMe)f
a
Emission maxima of the chemiluminescence spectra.
Chemiluminescence quantum yields.
Chemical yields of the amidopyrazine products by chemiluminescent reactions.
Fluorescence quantum yields of the corresponding light emitters.
b
c
d
e
Quantum efficiency of chemical generation of the singlet-excited states of the
light emitters.
f
Values taken from Ref. [7].
Fig. 5. Plots of the chemiluminescence energies (in eV) for 5a–c measured in aerated
DMSO against Hammett’s substituent constant (ꢄp values) of substituent R.
imidazopyrazinone chemiluminescence is freely controllable sim-
ply using the substituent effects.
These CLmax values corresponded well with the fluores-
cence maxima (FLmax) of the corresponding amide anion of
6a-c measured in DMSO containing a methanolic solution of
tetrabutylammonium hydroxide (Table 2), indicating that each
imidazopyrazinone formed the singlet-excited state of the cor-
responding amide anion (II) in accordance with the proposed
mechanism (Scheme 1). Therefore, the effect of the substituent on
the CLmax corresponds to that on the FLmax of the amide anion.
In order to better understand the effect of substituents on the
electronic structures of the light emitters, amide anion forms of
6a–c, the electronic absorption spectra of 6a–c in DMSO containing
methanolic tetrabutylammonium hydroxide as a base were mea-
sured. For comparison, the absorption spectra of coelenteramide
analogues 4a–c, which are the corresponding 3-benzyl derivatives,
were also measured under the same condition. The results are com-
piled in Table 2, along with the fluorescence data and Stokes shift
values.
3.2. Substituent effect on chemiluminescence properties
3.2.1. Chemiluminescence maxima
The measurement of chemiluminescence of the synthesized
imidazopyrazinones was achieved by mixing a methanolic stock
solution of 5a–c (1.0 mM) and DMSO under aerobic conditions [7].
The observed properties are summarized in Table 1. The chemi-
luminescence maxima (CLmax) of 5a–c were observed at 553,
525, and 513 nm, respectively, as shown in Fig. 4. It has already
been found that the introduction of a conjugating system at the
8-position of the imidazopyrazinone skeleton results in largely
red-shifted chemiluminescence in DMSO [36]. As expected, the
synthesized imidazopyrazinones produced light that was green,
yellow, or yellowish green in DMSO, whereas 8-benzyl ana-
logues 3 emitted blue light (ꢁmax: 454–479 nm) [7] under the
same chemiluminescence reaction conditions. It is notable that
a linear correlation was observed between the luminescence
energy (in eV) for 5a–c and Hammett’s substituent constant (ꢄp)
of the substituent R (Fig. 5). This indicates that the color of
As can be seen in Table 2, all the absorption maxima of 6a–c−
were observed at wavelengths that were shorter than the wave-
lengths at which those of the 3-benzyl derivatives 4a–c− were
observed. In addition, whereas the lowest transition energy was
not affected strongly by the substituent, the fluorescence max-
ima were affected strongly. The electrostatic repulsion between the
anion of the amide group and the phenyl group at the 3-position
may have increased the transition energy and could be one of
the causes of the hypsochromic absorption of 6− as compared to
4−. This substituent-insensitive absorption clearly indicates that
the observed transition does not have a charge transfer character,
and therefore, the dipole change in 6− with the substituent in the
ground state is negligible. From these results, it can be inferred
that the dihedral angle between the pyrazine ring and the phenyl
group at the 3-position would be so large that they might not be
able to effectively conjugate with each other. This consideration is
supplementarily supported by a semi-empirical molecular orbital
calculation. Fig. 6-a shows the optimized geometry of 6b− in DMSO
in the ground state (S0) calculated by the PM7-COSMO method,
in which the dihedral angle between the phenyl at the 3-position
and the central pyrazine ring (ꢃ) is 63.4◦. On the other hand, the
largely red-shifted CLmax, namely, the FLmax of 6−, strongly suggests
that the phenyl group at the 3-position of the emitter conjugates
effectively with the pyrazine ring. According to the PM7-COSMO
calculation, the dihedral angle ꢃ of 6b− in the singlet-excited state
(S1) in DMSO is estimated to be 25.7◦, as shown in Fig. 6-b, more
Fig. 4. Chemiluminescence spectra of 5a–c and 16 in aerated DMSO at 25 ◦C.

R. Saito et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 12–25
21
Table 2
Absorption and fluorescence data of 6a–c and 4a–c [16] in DMSO containing 0.5 vol% of 10%-methanolic tetrabutylammonium hydroxide at 25 ◦C.
Structure
Compound
R
Absorption maximum/nm
Fluorescence
maximum (nm)
Stokes shift (103 cm−1
)
(ε/10−4 cm−1 mol−1 l)
6a–
CF3
345 (2.00)
553
10.90
6b–
6c–
H
OMe
338 (1.77)
340 (1.74)
523
511
10.47
9.84
4a–
CF3
377 (1.91), 330 (sh. 1.59)
450a
4.30
4b–
4c–
H
OMe
364 (sh. 1.49), 314 (1.74)
369 (sh. 1.48), 308 (2.02)
462a
466a
5.73
5.64
a
Values taken from Ref. [7].
Fig. 6. Optimized geometry of the acetamidopyrazine anion 6b− (a) in the ground state (S0) and (b) in the singlet excited state (S1), calculated by the PM7-COSMO method.
planar than that in the S0 state. The dihedral angle ϕ between the
5-phenyl and pyrazine ring in the S1 state was also calculated to
be more planar (24.1◦) than that in the S0 state (40.6◦), showing
that the three aromatic rings in 6b− overlap sufficiently in the S1
state to extend the ␲-conjugation system. An attempt to get the
optimized geometry of 6b− in the S1 by TDDFT method was also
made. However, the calculation did not converge even after a week
of CPU time.
to the fully relaxed state (S1,RX) accompanied by the twisting
motion of the phenyl group especially at 3-position to extend the
␲-conjugation system affording the largely red-shifted fluores-
cence.
Generally, a molecule in the excited state is more polar than
that in the ground state [37,38]. 2-Acetamido-5-phenylpyrazines
have dipole moments in the direction of the pyrazine ring from
the phenyl group at the 5-position because of the electron-
attracting ability of the pyrazine ring. Therefore, the repulsion
between the dipole and the anion in the excited state should
be greater than that in the ground state, and it is quite likely
that 6− adopts a more planar geometry in the lowest singlet-
excited state in order to decrease the repulsion and to stabilize
the anion by enabling charge delocalization through the extended
conjugating system at the 3-position. Based on these consider-
ations, the photophysical behavior of 6− can be represented as
shown in Fig. 7, where S0,t and S0,p denote the ground state of
the twisted and the planar conformers, respectively, c is the veloc-
ity of light, and ꢂabs and ꢂfl are the wavenumbers corresponding
to the absorption and emission maxima, respectively. After exci-
Fig. 7. Schematic explanation for the photophysical process of acetamidopyrazine
6− in DMSO containing 0.5 vol% of 10%-methanolic tetrabutylammonium hydride.
tation, 6− may get relaxed from the Franc–Condon state (S1,FC
)

22
R. Saito et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 12–25
Fig. 8. Schematic illustration of a plausible reaction dynamics during the chemiluminescent reaction of 5.
The same dynamics may be involved in the chemiluminescent
where ꢂabs and ꢂfl are the wavenumbers corresponding to the
absorption and emission maxima, respectively; is the dif-
reaction mechanism. At the initial stage of the chemilumines-
cent reaction, 5a–c adopt twisted geometries at the 8-position. On
proceeding with the reactions, singlet-excited amide anions are
produced, which are likely to take up more planar conformations
that are responsible for the largely red-shifted luminescence, as
illustrated in Fig. 8.
–
g
e
ference in dipole moment between the excited and the ground
states; and ε, n, and a are the static bulk dielectric constant, optical
refractive index of the solvent, and radius of the spherical cavity
containing the dipole, respectively. Assuming the spherical radius
a is constant, under the same conditions (i.e., in the same solvent
and at the same temperature), the Stokes shift becomes a function
To confirm this consideration, imidazopyrazinone 16, which
has two ortho-methyl groups on the 8-phenyl group to restrict
the rotation of the 8-phenyl and take a twisted geometry at this
position, was synthesized, as shown in Scheme 5, and its chemilu-
minescence was measured under the same conditions as those for
5a–c. The obtained chemiluminescence spectrum was depicted in
Fig. 4 together with those for 5a–c. The CLmax for 16 was observed
at 491 nm, which is still longer wavelength than that for 3b but
shorter than that for the corresponding analogue 5b. This indi-
cates that 2,6-dimethylphenyl moiety in the light emitter during
the chemiluminescent reaction of 16 partly conjugates with the
central pyrazine ring to shift its CLmax about 30 nm longer than that
for 3b but not to such an extent that cause the largely red-shifted
one as 5b. These facts led us to conclude that the chemiluminescent
reaction mechanism of the present system involves the rotation of
the substituted phenyl group at the 8-position (5-position for the
amide anion).
Next, we discuss the effect of the substituent R on CLmax. As
shown in Fig. 4, both CLmax and FLmax exhibited a strong depen-
dence on the substituent R. Interestingly, the observed CLmax
exhibited a bathochromic shift when R was changed from the
electron-donating methoxy to the electron-withdrawing triflu-
oromethyl group, whereas the opposite substituent effect was
observed in the case of the phenyl group at the 6-position of 3
wherein an electron-donating substituent R caused CLmax to exhibit
a bathochromic shift [15]. As mentioned above, in the case of 6−,
shifts in CLmax result in corresponding shifts in FLmax, namely, the
Stokes shifts. Generally, a Stokes shift in a fluorescence spectrum is
essentially related to a change in the molecular dipole on excitation
[38], which can be described well by the Lippert–Mataga equation
[39]:
of
and e. In the present system, the changes in the ground
g
state dipoles among 6a–c− as a result of the substituents are small,
as can be seen from the substituent-insensitive absorption behav-
ior (Table 2), and therefore, the large, substituent-sensitive Stokes
shifts observed are considered to be caused predominantly by the
molecular dipole changes in the excited states according to Eq. (1).
That is, the difference in the peak shift among 6a–c− reflects the
difference in the dipole moments in the excited states. In general,
the reorganization of the solvent molecules around a solute pro-
ceeds upon excitation according to the newly formed dipole vector
and thereby stabilize the solute in the singlet-excited state [38].
The degree of this stabilization depends on the magnitude of the
newly formed dipole moment. In general, the greater the polarity of
a molecule in the singlet-excited state, the greater the stabilization
of the excited state energy in a polar solvent such as DMSO. This
results in the large Stokes shift [38]. In the case of 2-acetamido-
5-phenylpyrazines, the dipole moments are directed toward the
pyrazine ring from the phenyl group at the 5-position. In the case
of 4, an electron-donating substituent on the phenyl group at the 5-
position increases the molecular dipole moment of the light emitter
in the singlet-excited state [16], causing red-shifted chemilumines-
cence. In the present system, the larger Stokes shift observed for the
trifluoromethyl analog (6a−) as compared to that observed for the
methoxy analog (6c−) apparently indicates that the former has a
larger dipole moment than the latter in the singlet-excited state.
Fig. 9 displays this dipole change schematically. Assuming, for the
sake of convenience, a coordinate axis defined as shown in Fig. 9-
a, an electron-withdrawing substituent Y on the phenyl group at
the 3-position synergistically increases the magnitude of the x-axis
component of the molecular dipole moment (Fig. 9-b). Thus, it is
evident that CLmax (i.e., FLmax of the corresponding amide anions)
of imidazo[1,2-a]pyrazin-3(7H)-ones shifts to a longer wavelength
with an increase in the dipole moments of the corresponding
ꢃ
ꢄ
ꢅ
ꢆ
2
−
e
g
2
hc
ε − 1
n2 − 1
vabs − vfl
=
−
(1)
a3
2n2 + 1
2ε + 1
Scheme 5. Synthesis of imidazopyrazinone 16. Reagents and conditions: (i) 2,6-
dimethylphenylboronic acid (1 eq.), PdCl2(PPh3)2, PPh3, Na2CO3, aqueous dioxane,
reflux, 16 h; (ii) phenylboronic acid (1 eq.), Pd(PPh3)4, Na2CO3, aqueous dioxane,
reflux, 19 h; (iii) pyruvic aldehyde, HCl, EtOH, 90 ◦C, 4 h.
Fig. 9. Schematic explanation of dipole interactions in the acetamidopyrazine anion,
.
6−

R. Saito et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 12–25
23
emitter in the singlet-excited states as a result of the sub-
stituents, and the seemingly opposite substituent effect on the
CLmax observed in 3 and 5 is now explained.
addition, only a small change with substituents was observed. In
order to understand this substituent-insensitive ꢀS, the electronic
properties of the corresponding dioxetane intermediates 17a–c
during the chemiluminescent reaction of 5a–c were calculated by
the DFT method at the B3LYP level with a 6-31+G(d) basis set. For
comparison, the same calculations for 18a–c, the model structures
of dioxetane intermediates during the chemiluminescent reaction
of 3, were conducted. The calculated total Mulliken charge densities
of the amide nitrogen (qN) and the atoms that constitute the diox-
etanone moiety (qDox) are summarized in Table 3. The qN values
of 17 as well as 18 also scarcely varied with substituent changing
from electron-withdrawing trifluoromethyl (Hammett substituent
constant ꢄp = 0.54) [40] to electron-donating methoxy (ꢄp = –0.27)
[40] groups. The qDox values also exhibited a slight change with sub-
stituents. Thus, it is conjectured that the small charge changes on
the amide nitrogen and on the dioxetane moiety with substituents
are in accordance with the small substituent effect on ꢀS.
Teranishi and Goto have reported similar weak substituent
effects on the ꢀS values for the chemiluminescence of 5-
(5-aryl-2-pyrazinylamino)-1,2,4-trioxane derivatives 19, which
chemiluminesced in the presence of a base via the dioxetane inter-
mediate 20. The observed ꢀS values varied from 0.0026 to 0.0067
[41]. They attributed these observations to the fact that the negative
charge on the nitrogen atom gives it an electron-donating abil-
ity that is sufficiently strong to readily decompose the dioxetane
by itself without the need for any support from electron-donating
substituents. This consideration is applicable to our system.
3.2.2. Chemiluminescence quantum yields
The chemiluminescence quantum yield (ꢀCL
) is generally
defined as the product of three efficiencies, i.e., the fraction of
reacting molecules that follow the correct chemical pathway (ꢀR),
quantum yield of the singlet-excited molecule formed (ꢀS), and
fluorescence quantum yield of the light emitter (ꢀFL) [37]:
ꢀCL = ꢀRꢀSꢀFL
(2)
The values of ꢀCL, ꢀR, and ꢀFL for the present system were
experimentally obtained (see Section 2), and the ꢀS values were
calculated using these data and Eq. (2). The results are compiled
in Table 1, accompanied by those for the 8-benzyl analogues 3a–c
obtained under the same conditions [7]. All compounds underwent
the chemiluminescent reaction with good ꢀR values. It should be
emphasized that the ꢀCL values of 5a–c were 3–8 times larger than
those of 3a–c, and thus, the 8-arylated compounds 5a–c are found
to be highly effective chemiluminescent substrates relative to the
8-alkyl derivatives 3a–c. With respect to the ꢀFL values of 5a–c, they
are obviously larger than those of 3a–c. This result clearly indicates
that the observed enhancement in the ꢀCL values for 5a–c reflects
the improved ꢀFL values of the light emitters.
The ꢀS values observed for 5, which are the focus of our
attention, proved to be almost equal to those observed for 3. In
Thus, our observations do not validate the conventional expla-
nation that an electron-donating substituent on a phenyl group
bound to the central pyrazine ring enhances the ꢀS. However, this
does not deny the applicability of the CIEEL or CTIL mechanism
because the nitrogen anion serves as a strong electron donor. In this
regard, many studies have focused on the chemiluminescence of
dioxetanes, whose ꢀS values exhibited a strong dependency on the
electronic character of the substituents. For instance, 1,2-dioxetane
21, which has two p-substituted phenyl groups directly linked to
the central dioxetane ring, exhibited a drastic enhancement in its
ꢀS value when R was a strongly electron-donating dimethylamino
group [42]. Thus, the CIEEL or CTIL mechanism can still be applicable
for explaining the effective decomposition of dioxetanes to gener-
ate singlet-excited light emitters but not for the high ꢀS value in the
aequorin bioluminescence. Previously, Matsumoto et al. demon-
strated that in the case of the chemiluminescence of 1,1-linked
dioxetane derivatives 22, it is essential to determine the conforma-
tion of the electron-donating phenolate anion moiety relative to the
dioxetane ring in order to determine the luminescence efficiency
[43]. Yamaguchi et al. also demonstrated that efficiently lumi-
nescent dioxetanes take appropriate conformations for efficient
singlet-excited energy productions during their thermal decom-
position reactions [11]. Taking these facts and our present results
into consideration, a possible explanation for the high ꢀS value
in the aequorin bioluminescence is that the conformation of the
dioxetanone intermediate is strictly regulated by the surround-
ing apoprotein, apoaequorin, in such a manner that it is favorable
Table 3
DFT calculations of the electronic properties of the dioxetane intermediate 17 and
18 having substituent R (CF3, H, or OMe) at the B3LYP/6-31+G(d) level.
Structure
Compound
R
qN
qDox
17a
17b
17c
CF3
H
−0.352
−0.362
−0.356
−0.138
−0.135
−0.142
OMe
18a
18b
18c
CF3
H
−0.352
−0.368
−0.368
−0.255
−0.267
−0.272
OMe

24
R. Saito et al. / Journal of Photochemistry and Photobiology A: Chemistry 293 (2014) 12–25
for generating the singlet-excited light emitter, whereas in the
absence of the protein, the corresponding dioxetane intermediate
may take up an arbitrary conformation that predominantly afford
the light emitter not in the singlet-excited state but in the ground
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We have presented the syntheses and the chemiluminescence
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these imidazopyrazinones were definitely improved by the intro-
duction of a p-substituted phenyl group at the 8-position, but
this improvement was not caused a change in the value of ꢀS
but by that in the value of ꢀFL. In contrast to the substituent-
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those absolute values remained small. Therefore, it seems rea-
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imidazopyrazinone-chemiluminescence systems comparable to
aequorin-bioluminescence systems only by changing the electronic
character of substituents. Although the regulation of ꢀS by chang-
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was proved that the chemiluminescence maximum, CLmax, was
controllable using the substituent effect. In proportion to the
electron-attracting ability of the substituent at the para-position
of the 8-phenyl group, CLmax was shifted to longer wavelength.
Interestingly, the direction of this spectral shift with the electronic
property of the substituent is opposite to that observed for coelen-
terazine analogues 3 whose CLmax was shifted to longer wavelength
with increasing the electron-donating ability of the substituent at
the para-position of the 6-phenyl group. These findings provide a
new principle as a guide for designing imidazopyrazinones capable
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Acknowledgements
Proceedings of the 13th International Symposium on Bioluminescence
Chemiluminescence. Progress and Perspectives, World Scientific, Singapore,
2005, pp. 335–338.
&
The authors gratefully acknowledge support for this research by
a Grant-in-Aid for Scientific Research C (No 25410179 for R.S. and
No. 22550031 for T.H.) from the Japan Society for the Promotion
of Science (JSPS). Financial support to R.S. from MEXT-Supported
Program for the Strategic Research Foundation at Private Univer-
sities (2012–2016) is also gratefully appreciated. We would like
to thank Mr. Grant White for his kind assistance in correcting this
manuscript.
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