Synthesis of boradiazaindacene-imidazopyrazinone conjugate as lipophilic and yellow-chemiluminescent chemosensor for superoxide radical anion

Article abstract of DOI:10.1016/j.tet.2009.11.086

As a chemiluminescent chemosensor that emits yellow light on reacting with a superoxide radical anion (O₂^{{radical dot}-}) and has a lipophilic character, a 6-phenylimidazo[1,2-a]pyrazin-3(7H)-one derivative possessing a boradiazaindac

Full text of DOI:10.1016/j.tet.2009.11.086

Tetrahedron 66 (2010) 583–590
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of boradiazaindacene–imidazopyrazinone conjugate as lipophilic
and yellow-chemiluminescent chemosensor for superoxide radical anion
*
Ryota Saito , Ayako Ohno, Eri Ito
Department of Chemistry, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 October 2009
Received in revised form 18 November 2009
Accepted 19 November 2009
Available online 23 November 2009
As a chemiluminescent chemosensor that emits yellow light on reacting with a superoxide radical anion
(Oꢀ2ꢁ) and has a lipophilic character, a 6-phenylimidazo[1,2-a]pyrazin-3(7H)-one derivative possessing
a boradiazaindacene (BODIPY) at the para position of 6-phenyl (1) was synthesized. The lipophilicity of 1
was investigated by reversed-phase liquid chromatography, and its log Pow value was found to be 3.57.
This value was much higher than that of 2-methyl-6-(4-methoxypheyl)imidazo[1,2-a]pyrazin-3(7H)-one
(MCLA, log Pow¼1.19) and 6-[4-[2-{N0-(5-fluoresceinyl)thioureido}ethoxy]phenyl]-2-methylimidazo[1,2-
a]pyrazin-3(7H)-one (FCLA, log Pow¼ꢁ0.08), and it was comparable to that of benzenoid hydrocarbons.
The Oꢀ2ꢁ-induced chemiluminescence of 1 was investigated using the hypoxanthine/xanthine oxidase
system as the source of Oꢀ2ꢁ, and as a result, yellow emission was observed. The maximum wavelength
was observed at 542 nm, and it was longer than that of FCLA.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
one (FCLA), has been developed to detect Oꢀ2ꢁ at longer wavelength
than MCLA.6 On reacting with Oꢀ2ꢁ at physiological pH conditions,
A superoxide radical anion (O2ꢀꢁ) is a highly reactive free radical
formed by the univalent reduction of oxygen during normal me-
tabolism, and it plays important roles in biological systems. In
mammalian cells, for example, Oꢀ2ꢁ acts as a signaling molecule that
regulates important biological events such as protein phosphory-
lation,1 cell growth,2 and apoptosis.3 Polymorphonuclear leuko-
cytes and other phagocytes also produce Oꢀ2ꢁ as a chemical bomb to
kill infections and ingested bacteria, respectively,4 and to maintain
healthy bodies. However, the increased production or insufficient
extinction of Oꢀ2ꢁ causes cellular or tissue damage, inducing serious
disease states such as ischemia reperfusion injury, Alzheimer’s
disease, and carcinogenesis.4 Thus, the detection of production,
distribution, and quantity of O2ꢀꢁ in cells and tissues is of particular
clinical relevance.
2-Methyl-6-(4-methoxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one
(MCLA) has been widely recognized as the most popular probe used
for this purpose. MCLA can specifically react with Oꢀ2ꢁ immediately
after its production to emit visible light (lmax¼465 nm in buffers at
physiological pH conditions) without any light sources for excitation,
accordingly enabling sensitive and real-time detection of Oꢀ2ꢁ.5 A fluo-
rescein-linking MCLA derivative, 6-[4-[2-{N0-(5-fluoresceinyl)
thioureido}ethoxy]phenyl]-2-methylimidazo[1,2-a]pyrazin-3(7H)-
FCLA emits green light (lmax¼532 nm). This feature makes FCLA
more useful than MCLA, because the blue light emitted by MCLA is
absorbed by some biomaterials.7 FCLA has the additional advantage
of better water solubility over MCLA owing to the hydrophilicity of
the fluorescein moiety, which is charged at the physiological pH
conditions. However, because of the hydrophilic property, fluores-
cein-linking small molecules cannot easily permeate cell mem-
branes. Therefore, the development of MCLA-based lipophilic
chemiluminescent probes with emission at a longer wavelength
than MCLA is still required for the successful detection of Oꢀ2ꢁ inside
cells. For this purpose, we have previously synthesized imidazo
[1,2-a]pyrazin-3(7H)-ones that have two lipophilic phenyl groups at
the 6- and 8-positions of the imidazopyrazinone skeleton. Although
these compounds exhibited O2ꢀꢁ-induced chemiluminescence with
an emission maxima around 500–530 nm in buffer solutions under
basic conditions, their chemiluminescence spectra measured under
neutral conditions showed peaks at around 400 nm.8 In the present
study, as part of our ongoing efforts to develop long-wavelength-
light emitting imidazopyrazinones, we have designed a BODIPY-
attached imidazopyrazinone, 1. BODIPY (4,4-difluoro-4-bora-3a,4a-
diaza-s-indacene) fluorophores are currently of special interest as
substitutes for fluoresceins because of their superior photostability
to fluorescein,9 insensitivity toward solvent polarity and pH,10 and
sufficiently high lipophilicity to permeate cell membranes, as well as
high extinction coefficients and fluorescence quantum yields
approaching unity.11–14
* Corresponding author. Tel.: þ81 47 472 1926.
E-mail address: saito@chem.sci.toho-u.ac.jp (R. Saito).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.11.086

584
R. Saito et al. / Tetrahedron 66 (2010) 583–590
Herein, we describe the synthesis, lipophilicity, and chem-
gave BODIPY–aminopyrazine conjugate 6 (method A). We also
followed an alternative reaction procedure (method B) that in-
volves shorter steps than method A, beginning with the same
starting material 2. In this method, 2 was reacted with 2-amino-5-
iluminescence of the BODIPY-attached imidazopyrazinone that
emits yellow light both in an aprotic organic solvent and in a buffer
with an Oꢀ2ꢁ-generating system at a physiological pH condition.
Me
N
O
HO
O
O
N
Me
N
Me
N
O
O
N
H
COOH
N
N
N
N
H
N
H
F
B
1
H
N
N
F
HN
MeO
O
S
MCLA
FCLA
2. Results and discussion
2.1. Synthesis
bromopyrazine to give 2-amino-5-(4-formylphenyl)pyrazine 7,
which was then successively reacted with pyrrole, chloranil, and
boron trifluoride etherate to give 6. The total yield of 6 from 2 via
method A was 13%, while that via method B was 3%, apparently
indicating that method A is superior to method B even though the
number of steps in method A is more than method B. We
considered that the existence of free amino group in 7 was
unfavorable for the conversion of 7 to 6 and lead to the low yield
via method B. To confirm this, we attempted the same reaction
with N-acetyl derivative of 7 (8) to obtain 9 (in Scheme 1) but
failed because 8 was hardly soluble in conventional organic
solvents including dimethyl sulfoxide (DMSO).
The synthesis of the BODIPY-attached imidazopyrazinone 1 was
achieved as shown in Scheme 1. First, boronic acid 2 was protected
with 2,2-dimethylpropan-1,3-diol to give boronate 3.15 Compound
3 was then reacted with 3-ethyl-2,4-dimethylpyrrole to give 4,
followed by successive treatment with chloranil and boron
trifluoride etherate to afford borondipyrromethene 5. The palla-
dium-catalyzed cross-coupling reaction of 5 with 2-amino-5-bro-
mopyrazine, prepared by the bromination of 2-aminopyrazine,
1)
Method A
N
O
B
O
B
H
1) DIEA
2) BF3 OEt2
O
O
TFA
2) p-Chloranil
HO
OH
O
B
.
B(OH)2
N
H
N
O
F
B
THF
CH2Cl2
CH2Cl2
OHC
N
N
F
OHC
(20% from 3)
(97%)
2
3
4
5
N
N
NH2
Br
1,4-dioxane
(69%)
Method B
Pd(PPh3)4
K2CO3
N
NH2
N
N
NH2
1)
Br
N
TFA
N
NH2
N
H
Pd(PPh3), K2CO3
N
N
F
B
1,4-dioxane
(88%)
2) p-Chloranil / CH2Cl2
3) DIEA / CH2Cl2
4) BF3 OEt2 / CH2Cl2
N
F
OHC
7
6
.
(13% from 2)
MeCOCl
pyridine
Ac2O
pyridine
CH2Cl2 (80%)
(80%)
O
Me
NH
N
O
Me
NH
N
N
N
N
F
B
N
F
8
9
OHC
Scheme 1.

R. Saito et al. / Tetrahedron 66 (2010) 583–590
585
Compound 6 was reacted with pyruvinaldehyde dimethyl acetal
to form 1 (Table 1). For the first trial, we adopted the most popular
reaction condition: the precursor aminopyrazines are reacted with
5.07, and 3.95 min, respectively (Table 2). This result suggests that 1
was the most lipophilic among the three compounds. In order to
describe this result more quantitatively, the n-octanol/water par-
tition coefficient (log Pow), which is the common lipophilicity scale
of chemicals, was estimated from the observed tR. Generally, tR
obtained by RP-HPLC is related to the capacity factor (k0) given by
the following equation:19
a
-ketoaldehydes or a-ketoalcetals in an alcohol in the presence of
catalytic amount of concentrated hydrochloric acid.8,16 The detailed
reaction conditions are described as run 1 in Table 1. A thin-layer
chromatographic analysis of the reaction mixture after 3 h at 80 ꢂC
indicated that multiple products were formed, none of them were
fluorescent, and target compound 1 was not obtained. This result
suggests that the borondipyrromethene moiety was unstable under
the condition employed in this reaction, i.e., heating in ethanol in
the presence of concentrated hydrochloric acid. To confirm this, 6
was treated with deuterium chloride in methanol-d4 and moni-
tored by 1H NMR spectroscopy. The time course of the spectral
change in the aromatic region is depicted in Figure 1. Obviously, the
signals exhibited downfield shift within 330 min. This indicates
that 6 was converted into another species. The direction of this
spectral shift was opposite to that of borondipyrromethene for-
mation from the corresponding dipyrromethene with boron tri-
fluoride. Moreover, a fast-atom-bombardment mass spectrum of
the converted product exhibited an m/z value at 426. This value
coincided with the value calculated for [C27H31N5þHþ] that corre-
sponded to the protonated form of 6. From these results, it could be
safely concluded that the borondipyrromethene moiety was
decomposed to give the corresponding deboronated dipyrrome-
thene under the conditions employed for run 1 in Table 1. Then, we
examined another experimental condition: 1,4-dioxane was
employed as a solvent (run 2, Table 1). This condition has also been
used to synthesize imidazopyrazinones.17 Further, it has been
reported that a BODIPY skeleton resisted a reaction involving
treatment with 4-M HCl/1,4-dioxane mixture during the synthesis
of a BODIPY-labeled sphingosine.18 Consequently, 6 was success-
fully converted into target imidazopyrazinone 1, as shown in Table 1.
These results provided not only a successful method for pre-
paring 1 but also useful information about handling BODIPY-
containing compounds under strong acidic conditions.
k0 ¼ ðtR ꢁ t0Þ=t0
(1)
where t0 is the dead time. log Pow of a test compound can be
obtained by substituting the calculated k0 value into the following
equation:19
log Pow ¼ a log k0 þ b
(2)
where a and b are linear regression coefficients, which can be
obtained by linearly regressing log Pow of reference substances
against their log k0. The k0 and log Pow values for 1, MCLA, and FCLA
were calculated using these equations and are listed after the tR
values in Table 2. As expected, FCLA was the most hydrophilic
among them with a negative log Pow value (ꢁ0.06), showing that
FCLA was miscible with water up to the same extent as methyl-
formamide (log Pow¼ꢁ0.06).20 The lipophilicity of MCLA
(log Pow¼1.18) was found to be almost the same as that of benzyl-
alcohol (log Pow¼1.1).20 log Pow of 1 was estimated to be 3.57, and it
was much larger than that of MCLA and FCLA. Judging from its
log Pow value, the lipophilicity of 1 was higher than that of benze-
noid hydrocarbons such as p-xylene (log Pow¼3.15)20 and naph-
tharene (log Pow¼3.37)21 and similar to that of propranolol
(log Pow¼3.56),22 a naphtharene-containing beta-adrenergic re-
ceptor blocking agent. Thus, it was confirmed that introducing the
BODIPY unit markedly increased the lipophilicity of the parent
molecule, and the lipophilicity of 1 was much higher than that of
the other two imidazopyrazinones.
2.3. Chemiluminescent property of BODIPY-linking
imidazopyrazinone (1)
Table 1
Reaction conditions for the synthesis of compound 1
First, in order to verify that the emission from 1 originated from
the BODIPY unit, we carried out the chemiluminescent reaction of 1
in aerated DMSO, which has been commonly used for performing
chemiluminescence measurements of imidazopyrazinones and for
affording brighter luminescence as compared to the reaction in
aqueous solutions.23 A chemiluminescence spectrum of 1 is shown
in Figure 2, along with the chemiluminescence spectra of MCLA and
FCLA measured under the same conditions. Compound 1 was
confirmed to emit yellow light with a luminescence maximum at
557 nm, while FCLA gave luminescence with a maximum at
542 nm. These luminescence peaks apparently originated from the
BODIPY moiety of 1 and the fluorescein moiety of FCLA, re-
spectively. It is noteworthy that the emission band of 1 was red-
shifted as compared to that of FCLA. These spectra revealed the
emission of a small amount of blue light at about 460 nm, in-
dicating that the luminescence energy produced at the MCLA
moiety was not completely transferred to the energy acceptors,
BODIPY, and fluorescein units during the chemiluminescent re-
action in DMSO. The BODIPY fluorophore in 1 was directly con-
Me
O
N
N
NH2
Me
O
MeO
MeO
N
N
N
H
HCl
N
F
B
N
N
F
F
B
6
1
N
F
Run
Solvent
Temperature
80 ꢂ
80 ꢂC
Reaction period
Yield
1
2
EtOH
1,4-Dioxane
C
3.5 h
5.5 h
(Decomp. of 6)
24%
2.2. Lipophilicity of BODIPY-attached imidazopyrazinone (1)
Lipophilicity is an important property that affects the trans-
membrane transport of chemical substances. Reversed-phase high
performance liquid chromatography (RP-HPLC) using a C18 ana-
lytical column is one of the commonly adopted experimental
methodologies for the measurement of lipophilicity of chemicals.
Thus, the lipophilicity of newly synthesized imidazopyrazinone 1
was investigated by means of RP-HPLC. For comparison, MCLA and
FCLA were also analyzed under the same conditions. As a result, the
retention time (tR) of 1, MCLA, and FCLA was observed to be 11.04,
nected to the imidazopyrazinone-phenyl group p conjugating
system, and therefore, we did not expect 1 to exhibit bimodal lu-
minescent behavior at first. Presumably, the phenyl ring and
BODIPY unit were twisted owing to the two methyl groups at the C1
and C7 positions of the BODIPY skeleton, and they were con-
jugatively disconnected.24
The chemiluminescence of 1 with Oꢀ2ꢁ was measured in a phos-
phate buffer using the hypoxanthine/xanthine oxidase system as an
Oꢀ2ꢁgenerator.8 Chemiluminescence spectra of MCLA and FCLA were

586
R. Saito et al. / Tetrahedron 66 (2010) 583–590
Figure 1. Time course of change in the 1H NMR spectrum of 6 (a) in the alkyl region (
d 0.8–2.5 ppm) and (b) in the aromatic region (d 7.3–8.7 ppm) measured in methanol-d4
containing deuterium chloride.
Table 2
luminescence maximum of 1 was observed at longer wavelength
than that of FCLA (532 nm), similar to the observation in DMSO.
Under this condition, either 1 or FCLA exhibited a single lumines-
cence peak; the absence of emission originating from the MCLA
moiety. This indicated that the luminescence energy generated at
the MCLA moiety was efficiently transferred to the fluorescent
chromophores under the Oꢀ2ꢁ-induced chemiluminescent reaction
condition. The reason for the difference in the emission intensities
from the MCLA moiety in DMSO and in buffer solution was unclear.
One possible reason was that the electronic structures of the light
emitters involved in the reaction of 1 and FCLA in DMSO were
different from those in the buffer solution. During the oxidative
chemiluminescent reaction, an imidazopyrazinone reacts with
a molecular oxygen or reactive oxygen species such as Oꢀ2ꢁ to afford
a singlet-excited amidopyrazine, which decays to the ground state,
accompanied by light emission, as shown in Scheme 2. The elec-
tronic structures of the singlet-exited amidopyrazines involved in
the reactions in DMSO have been confirmed to be of amide anion
Retention time (tR), capacity factor (k0), and log Pow of the imidazopyrazinones
(1, MCLA, and FCLA) obtained from the RP-HPLC analysis at 25 ꢂCa
Compound
tR/min
k0
log Pow
1
11.04
5.07
3.95
0.46
3.57
1.18
ꢁ0.06
MCLA
FCLA
ꢁ0.12
ꢁ0.42
a
Column: SHISEIDO Capcell Pak C18; id: 250 mmꢃ4.6 mm; mobile phase: 80%
MeOH; flow rate: 0.7 mL/min.
measured under the same conditions for comparison, and all of the
observed spectra are shown in Figure 3. As shown in the figure, the
chemiluminescence maximum of 1 was observed at 545 nm. This
luminescence was consistent with the fluorescence of 9, prepared
separately from 6 as shown in Scheme 1, proving the light emitter
involved in the chemiluminescent reaction of 1 was the corre-
sponding oxidized product 9 (see Supplementary data). The

R. Saito et al. / Tetrahedron 66 (2010) 583–590
587
forms,16b while those in the reaction with an Oꢀ2ꢁ-producing system
at physiological pH conditions are known to be neutral.8,25 Another
possible reason for the difference in the emission intensities was
that the emission was rather quenched in buffer solution. Re-
searches on the luminescent properties of amidopyrazines have
shown that their fluorescence intensity decreased in protic sol-
vents.26 This fluorescence quenching may be caused by the vibra-
tional deactivation of the singlet-excited energy through hydrogen
bonding. Therefore, the luminescence intensity for 1 as well as FCLA
was likely to reduce to levels where the emission originating from
the MCLA moiety could not be detected.
150
100
50
MCLA
FCLA
1
3. Conclusion
We have successfully synthesized BODIPY-attached imidazo-
pyrazinone 1. The synthetic investigation also revealed that the
BODIPY unit is unstable when the reaction is carried out using
hydrochloric acid in ethanol. The RP-HPLC analyses have clearly
demonstrated that the BODIPY unit substantially enhances the
lipophilicity of the imidazopyrazinones. Further, it may be worth
noting that this work provides the first quantitative data, log Pow
values, of the imidazopyrazinones. The chemiluminescence studies
have revealed that the luminescence energy produced at the imi-
dazopyrazinone moiety in 1 is effectively transferred to the BODIPY
unit to emit yellow light with a maximum wavelength longer than
that of FCLA. Overall, we may say that we have succeeded in de-
veloping a yellow-light-chemiluminescent Oꢀ2ꢁ probe with a rather
lipophilic character.
0
400
450
500
550
600
650
Wavelength /nm
Figure 2. Chemiluminescence spectra of MCLA (black line), FCLA (blue line), and 1 (red
line) in aerated DMSO at 25 ꢂC. The final concentration of each probes, 50
mM.
20
MCLA
FCLA
1
15
4. Experimental
4.1. General
10
5
All melting points were measured on MP-21 apparatus
(Yamato Scientific Co., Ltd., Japan) in open capillary tubes; the
values were uncorrected. 1H NMR spectra were recorded on
a JNM-ECP400 spectrometer (JEOL Ltd., Japan). Chemical shifts (d)
are reported in ppm and were measured using tetramethylsilane
or an undeuteriated solvent as an internal standard in the deu-
terated solvent used. Coupling constants (J) are given in Hz.
Chemical shift multiplicities are reported as s¼singlet, d¼doublet,
t¼triplet, q¼quartet, and m¼multiplet. Infrared (IR) spectra were
obtained using FT/IR-4100, FT/IR-460plus, or FT/IR-660plus spec-
trophotometers (JASCO Co., Ltd., Japan). Fast-atom-bombardment
(FAB) mass spectra were measured on a JMS-600-H mass spec-
trometer (JEOL Ltd., Japan). Xenon was used as a bombardment
gas, and all the analyses were carried out in a positive mode with
0
400
450
500
550
600
650
Wavelength /nm
Figure 3. Oꢀ2ꢁ-induced chemiluminescence spectra of MCLA (black line), FCLA (blue
line), and 1 (red line) in 10 mM phosphate buffer (pH 8.6) containing 100 mM KCl,
0.05 mM EDTA, 0.15 mM hypoxanthine, and 0.02 unit/mL xanthine oxidase at 25 ꢂC.
The final concentration of each probes, 50 mM.
R1
O
*
*
O
R1
O
R1
NH
or
or
O2
O2
O2
O2
N
N
N
N
N
N
–H+
in a buffer (pH <9)
R3
N
R2
R3
N
H
R2
R3
R2
in DMSO
(singlet-excited state)
(singlet-excited state)
light
light
O
R1
O
R1
NH
N
N
N
N
N
R3
R2
R3
R2
(ground state)
(ground state)
Scheme 2.

588
R. Saito et al. / Tetrahedron 66 (2010) 583–590
the ionization energy and accelerating voltage set at 70 eV and
3 kV, respectively. A mixture of dithiothreitol and -thioglycerol
The combined organic layer was evaporated and partially purified
by column chromatography on alumina (Merck, 300 mesh) with
hexane/ethyl acetate (7/3, v/v) as the eluent to give the target
a
(1:1 or 1:2) was used as a liquid matrix. High and low resolution
electron impact (EI) mass spectra were obtained with a JMS-AM II
50 mass spectrometer (JEOL Co., Ltd., Japan). The ionization energy
was 70 eV, and the accelerating voltages were 0.3 kV for the low
resolution and 0.5 kV for the high resolution analyses. Absorption
spectra were measured on a V-650 spectrophotometer (JASCO Co.,
Ltd., Japan), and combustion analyses were performed on a MT-6
analyzer (Yanaco New Science Inc., Japan). Column chromatogra-
product as a reddish powder. 1H NMR (DMSO-d6, 400 MHz)
d/ppm
7.80 (d, 2H, J¼7.7 Hz), 7.20 (d, 2H, J¼7.7 Hz), 4.14 (s, 6H), 3.79 (s, 6H),
2.23 (q, 4H, J¼7.4 Hz), 0.98 (s, 6H), 0.91 (t, 6H, J¼7.4 Hz). This ma-
terial was not further assigned.
4.2.3. 8-[4-(5-Aminopyrazin-2-yl)phenyl]-2,6-diethyl-4,4-difluoro-
1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (6) via method
phy was carried out on silica gel (particle size: 63–210
m
m; Kanto
A. To a solution of 2-amino-5-bromopyrazine (17.3 mg, 99.4
and 5 (50.0 mg, 102 mol) in 10 mL of 1,4-dioxane were succes-
sively added tetrakis(triphenylphosphine)palladium (8.9 mg,
7.7 mol) and 0.25 mL of 2-M K2CO3, and the mixture was refluxed
for 3 h under argon. After cooling to room temperature, the reaction
mixture was washed with water and dried over MgSO4. The
evaporation of the solvent gave the crude product, which was pu-
mmol)
Chemical Co.). Chemiluminescence spectra were collected with
a JASCO F-777 spectrofluorometer (excitation light was shut off
and only the light detector was used to measure the emitted light;
bandpass of the detector: 10 nm; scan speed: 1000 nm/min for
chemiluminescence in DMSO and 2000 nm/min for Oꢀ2ꢁ-induced
chemiluminescence).
m
m
MCLA, FCLA, and hypoxanthine were purchased from Tokyo
Chemical Industry Co., LTD. (Japan). HPLC-grade methanol and
water were obtained from Kanto Chemical Co. Inc. (Japan). Xan-
thine oxidase (from buttermilk) was purchased from EMB Bio-
science Inc. (CA, USA). Other conventional chemicals used in the
present study are commercially available and were used as
received.
rified by column chromatography on silica gel (46–50 mm) with
hexane/ethyl acetate (7/3, v/v) as the eluent to afford 6 as a red solid
(32.5 mg, 69%). Mp 258 ꢂC (decomp.); IR (KBr) nmax/cmꢁ1 3423,
3318 (
(C]C, C]N, ring stretching),1194, 979 (
(CDCl3, 400 MHz)
J¼1.4 Hz), 8.03 (d, 2H, J¼8.1 Hz), 7.38 (d, 2H, J¼8.1 Hz), 2.54 (s, 6H),
2.30 (q, 4H, J¼7.5 Hz), 1.35 (s, 6H), 0.98 (t, 6H, J¼7.5 Hz); MS (FABþ,
DTT/TG¼1/2) 473 [M]þ; HRMS (positive EI) calcd for C30H32BF2N5O
473.2562, found: 473.2539.
n
N–H), 2960, 2925, 2870 (
n
C–H), 1634 (
d
N–H), 1541, 1475
C–H); 1H NMR
n
B–N), 721 (
g
d/ppm 8.56 (d, 1H, J¼1.4 Hz), 8.11 (d, 1H,
4.2. Synthesis
4.2.1. 2,6-Diethyl-4,4-difluoro-1,3,5,7-tetramethyl-8-[4-(5,5-di-
methyl-1,3,2-dioxaborinan-2-yl)phenyl]-4-bora-3a,4a-diaza-s-in-
4.2.4. 8-[4-(5-Aminopyrazin-2-yl)phenyl]-2,6-diethyl-4,4-difluoro-
1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (6) via method
B. To a solution of 7 (50.4 mg, 0.253 mmol) and 2,4-dimethyl-3-
ethylpyrrole (73 mg, 0.59 mmol) in 150-mL dichloromethane was
added one drop of trifluoroacetic acid (TFA), and the mixture was
stirred overnight at room temperature under argon. After con-
firming the disappearance of 7 by TLC, p-chloranil (63.4 mg,
0.258 mmol) in dry CH2Cl2 was added to the stirred reaction mix-
ture over a 45-min time period. N,N-Diisopropylethylamine (DIEA,
0.22 g, 1.7 mmol) was added to the reaction mixture, and the
resulting mixture was stirred at room temperature for 20 min un-
der argon. Boron trifluoride–diethyl ether complex (0.23 g,
1.6 mmol) was added dropwise to the mixture. After stirring for
additional 1.5 h, the reaction mixture was washed with water and
dried over MgSO4. After evaporating the solvent, the obtained
crude product was purified by column chromatography on silica gel
dacene (5). To
a solution of 3 (401 mg, 1.84 mmol) and
2,4-dimethyl-3-ethylpyrrole (0.46 g, 3.7 mmol) in 200 mL of
dichloromethane was added one drop of trifluoroacetic acid (TFA),
and the mixture was stirred overnight at room temperature under
argon. After confirming the disappearance of 3 by thin-layer chro-
matography (TLC), p-chloranil (453 mg, 1.84 mmol) in dichloro-
methane (50 mL) was added to the reaction mixture over a 35-min
time period. N,N-Diisopropylethylamine (DIEA, 1.5 g, 11.5 mmol)
was then added to the mixture, which was stirred at room tem-
perature for another 30 min under argon. Boron trifluoride–diethyl
ether complex (1.9 g, 12 mmol) was added dropwise to the reaction
mixture, and stirring was continued for 2 h. The reaction mixture
was washed with water and dried over MgSO4. After evaporating
the solvent, the obtained crude materials were purified by column
chromatography on silica gel (46–50
orange solid (185 mg, 20%). Mp 247 ꢂC (decomp.); IR (KBr) nmax
cmꢁ1 2962, 2928, 2870 (
C–H), 1540, 1475 (C]C, C]N, ring
B–O), 1190, 977 ( B–N), 717 (
C–H); 1H NMR
mm, chloroform) to give 5 as an
/
(46–50 mm) with chloroform as the eluent to afford 6 as an orange
solid (5.2 mg, 43%).
n
stretching), 1318 (
n
n
g
(CDCl3, 400 MHz)
d
/ppm 7.89 (d, 2H, J¼8.4 Hz), 7.27 (d, 2H,
4.2.5. 2-Amino-5-(4-formylphenyl)pyrazine (7). To a solution of 2-
amino-5-bromopyrazine (80.3 mg, 0.461 mmol) and 2-(4-for-
mylphenyl)boronic acid (79.8 mg, 0.532 mmol) in 1,4-dioxane
(2.0 mL) was successively added tetrakis(triphenylphosphine)palla-
J¼8.4 Hz), 3.82 (s, 4H), 2.53 (s, 6H), 2.29 (q, 4H, J¼7.5 Hz), 1.26 (s,
6H),1.07 (s, 6H), 0.97 (t, 6H, J¼7.5 Hz); MS (FABþ, m-NBA) 492 [M]þ;
HRMS (positive EI) calcd for C28H36B2F2N2O2 492.2931, found:
492.2938.
dium (22.1 mg, 19.1 mmol) and 2-M K2CO3 (0.6 mL), and the reaction
mixture was refluxed for 3 h under argon. After cooling to room
temperature, chloroform (20 mL) and water (20 mL) were added to
the reaction mixture, and the organic phase was separated. The
aqueous layer was extracted with one portion of chloroform (20 mL),
and the combined organic phase was dried over MgSO4. After evap-
orating the solvent, the resulting brownish yellow solid was purified
by recrystallization from ethyl acetate/hexane to give 7 as a yellow
powder (45.3 mg, 88%). The residual filtrate was concentrated and
further purified by preparative TLC on silica gel to afford 7 as a yellow
powder (21.7 mg). The totalyieldof 7 was 67.0 mg (73%). Mp>360 ꢂC;
4.2.2. 3-Ethyl-5-((1Z)-(4-ethyl-3,5-dimethyl-2H-pyrrol-2-yli-
dene)(4-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)phenyl)methyl)-2,4-
dimethyl-1H-pyrrole (4). In order to confirm that 4 was formed
during the above synthesis of 5 from 3, the following experiment
was achieved: To a solution of 3 (251 mg, 1.15 mmol) and 3-ethyl-
2,4-dimethyl-1H-pyrrole (0.30 g, 2.5 mmol) in 100 mL of
dichloromethane was added one drop of trifluoroacetic acid (TFA),
and the mixture was stirred overnight at room temperature under
argon. After confirming the disappearance of 3 by TLC, p-chloranil
(285 mg, 1.16 mmol) in dichloromethane was added to the reaction
mixture. The mixture was further stirred for 1.5 h, and then, the
reaction was quenched with water. The organic layer was sepa-
rated, and the water layer was further extracted with chloroform.
IR (KBr) nmax/cmꢁ1 3407, 3317 (
nN–H), 2855, 2731 (nH–CO), 1687
(
(
nC]O), 1605 (dN–H), 1545, 1395 (C]C, C]N, ring stretching), 829
g
C–H); 1H NMR (CDCl3, 400 MHz)
d/ppm 10.05 (s, 1H), 8.54 (s, 1H),
8.09 (s, 1H), 8.06 (d, 2H, J¼8.4 Hz), 7.96 (d, 2H, J¼8.4 Hz); MS (FABþ,

R. Saito et al. / Tetrahedron 66 (2010) 583–590
589
m-NBA) 200 [MþH]þ; HRMS (positive EI) calcd for C11H9N3O
were eluted with 80% (v/v) methanol in water at a flow rate of
0.7 mL/min and were detected at 254 nm. Measurements were
performed at 25 ꢂC, controlled by a SSC-2300 column oven (SEN-
SHU SCIENTIFIC Co. Ltd., Japan). Tartrazin (purchased from Tokyo
Chemical Industry Co., LTD., Japan) was used as an unretained
compound to determine the dead time (t0).
199.0746, found: 199.0744.
4.2.6. 2-Acetamido-5-(4-formylphenyl)pyrazine (8). To a solution of
2-amino-5-(4-formyl)pyrazine (190.4 mg, 0.956 mmol) and pyri-
dine (10 mL, 0.124 mol) in chloroform (20 mL) was added dropwise
acetyl chloride (473 mg, 6.03 mmol), and the mixture was stirred
under argon for 40 h. After evaporation of the solvent, water was
added to the residue and the resulting precipitates were collected
by suction filtration. The obtained materials were washed with
water and methanol to afford the aimed compound as yellow
solids. (184.3 mg, 80%). Mp>360 ꢂC; IR (KBr) nmax/cmꢁ1 3476, 3285
A series of standard compounds including phenol (log Pow¼1.46),
benzene (log Pow¼2.13), bromobenzene (log Pow¼2.99), naphthar-
ene (log Pow¼3.37), biphenyl (log Pow¼3.98), and phenanthrene
(log Pow¼4.57) were analyzed to generate a calibration curve. Each
standard was injected three times, and the average log k0 values
obtained from the RP-HPLC analysis were plotted versus their known
literature log Pow values. The data points were subjected to least-
squares fitting to give a straight line (Fig. 4). Parameters a and b in Eq.
2 were determined from the slope and the intercept of this straight
line, respectively. The imidazopyrazinones (1, MCLA, and FCLA) were
also analyzed three times each to obtain their average log k0 values.
Substituting the obtained values for log k0 in Eq. 2 yielded the log Pow
values of the imidazopyrazinones. The variability in the de-
termination of the k0 value from the replicate injections of the ana-
lytes was between 2.3 and 5.3% (Table 3).
(
n
N–H), 3066 (
1508, 1468 (C]C, C]N, ring stretching), 837 (
(DMSO-d6, 400 MHz) /ppm 11.00 (1H, s), 10.08 (1H, s), 9.44 (1H, s),
n
C–H), 2846, 2741 (
n
H–CO), 1693 (
n
C]O), 1603, 1544,
g
CH); 1H NMR
d
9.13 (1H, s), 8.34 (1H, d, J¼7.7 Hz), 8.04 (1H, d, J¼7.7 Hz), 2.17 (3H,
s); MS (FABþ, DTT:TG¼1:1) 242 [MþH]þ; HRMS (positive EI) calcd
for C13H11N3O2 241.0851, found: 241.0852.
4.2.7. 6-[4-(2,6-Diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-
3a,4a-diaza-s-indacen-8-yl)phenyl]-2-methyl-3,7-dihy-
droimidazo[1,2-a]pyrazin-3(7H)-one (1). To a solution of 6 (15.6 mg,
33.0 mmol) and pyruvicaldehyde dimethyl acetal (32 mg, 273 mmol)
5
in 1,4-dioxane (1.5 mL), through which argon gas was passed for
30 min to drive out dissolving oxygen, was added two drops of 2-M
HCl, and the mixture was stirred at 80 ꢂC for 5.5 h. After cooling to
room temperature, the solvent was evaporated and crude materials
y = 4.3502x + 1.4659
R2 = 0.9895
4
3
2
1
were purified by chromatography on silica gel (46–50
chloroform/methanol (6/1, v/v) as the eluent to give 1 as a red solid
(4.0 mg, 24%). Mp 213 ꢂC (decomp.); IR (KBr) nmax/cmꢁ1 3422 (
N–
C]O), 1542, 1476 (C]C, C]N,
mm) with
n
H), 2962, 2926, 2869 (
ring stretching), 1193, 981 (
400 MHz)
7.92 (s, 1H), 7.52 (d, 2H, J¼8.0 Hz), 2.49 (s, 6H), 2.47 (s, 3H), 2.36 (q,
4H, J¼7.5 Hz), 1.39 (s, 6H), 1.00 (t, 6H, J¼7.5 Hz); MS (FABþ, DTT/
TG¼1/2) 528 [MþH]þ; HRMS (positive EI) calcd for C30H32BF2N5O
527.2668, found: 527.2666.
n
C–H), 1629 (
n
n
B–N), 712 (
g
C–H); 1H NMR (CD3OD,
d
/ppm 8.03 (s, 1H, 6-pyrazine), 7.95 (d, 2H, J¼8.0 Hz),
-0.2
0
0.2
0.4
0.6
0.8
1
4.2.8. 2-Acetamido-5-[4-(2,6-diethyl-4,4-difluoro-1,3,5,7-tetra-
log k'
methyl-4-bora-3a,4a-diaza-s-indacen-8-yl)phenyl]pyrazine (9). To
Figure 4. Calibration curve generating by plotting log Pow against log k0 obtained by
the RP-HPLC for a series of standards (phenol, benzene, bromobenzene, naththarene,
biphenyl, and phenanthrene).
a solution of 6 (59.7 mg, 126 mmol) in pyridine (1.0 mL) was added
acetic anhydride (389 mg, 3.81 mmol), and the mixture was stirred
at 50 ꢂC for 9 days. After cooling to room temperature, ethyl acetate
(15 mL) was added to the reaction mixture and washed with 5%
citric acid (15 mL, three times). The organic phase was dried over
sodium sulfate, and the solvent was removed under reduced
pressure. The resulting material was purified by column chroma-
Table 3
Precision of k0 value determination by RP-HPLC for 1, MCLA, FCLA, and standards
Compound
k0
%RSD
tography on silica gel (63–200
eluent to give 9 as a solid (52.3 mg, 80%). Mp 260 ꢂC (decomp.); IR
(KBr) nmax/cmꢁ1 2965, 2928, 2870 (
C–H), 1665 ( C]O), 1544, 1473
(C]C, C]N, ring stretching), 1192, 978 ( B–N), 758 (
C–H); 1H
NMR (CD3OD, 400 MHz)
/ppm 9.61 (s, 1H), 8.77 (d, 1H, J¼1.5 Hz),
mm, 34 g) with chloroform as an
1
3.04
0.86
0.45
0.90
1.59
2.27
2.64
3.69
5.11
3.2
3.3
3.5
5.3
0.0
4.0
0.0
2.3
3.4
MCLA
FCLA
Phenol
Benzene
Bromobenzene
Naphtharene
Biphenyl
Phenanthrene
n
n
n
g
d
8.14 (d, 2H, J¼8.4 Hz), 7.91 (s,1H), 7.43 (d, 2H, J¼8.4 Hz), 2.54 (s, 6H),
2.30 (q, 4H, J¼7.3 Hz), 2.30 (s, 3H),1.35 (s, 6H), 0.98 (t, 6H, J¼7.3 Hz);
MS (EIþ, DTT/TG¼1/2) 516 [MþH]þ; HRMS (positive EI) calcd for
C29H32BF2N5O 515.2668, found: 515.2678.
4.3. Determination of lipophilic parameter log Pow
by reversed-phase HPLC
4.4. Chemiluminescence measurement
An HPLC analysis was performed on a Capcell Pak C18 SG120
Chemiluminescent reactions in DMSO were achieved by mixing
1.0-mM solution of an imidazopyrazinone in methanol (50 L) and
DMSO containing 0.1 vol % of 10% NaOH (950 L) in a quartz cuvette
(path length: 1 cm) at 25 ꢂC. The chemiluminescence induced by
Oꢀ2ꢁ was measured in a mixture of 50-
L methanol solution of an
imidazopyrazinone (1 mM) and 930 L of 0.01 mol/L phosphate
column (particle size: 5
mm, id: 250 mmꢃ4.6 mm, SHISEIDO Co.,
m
Ltd., Japan) with an SSC-3461 pump (SENSHU SCIENTIFIC Co. Ltd.,
Japan), a DG-1110 degasser (SENSHU SCIENTIFIC Co. Ltd., Japan),
and an 875-UV Intelligent UV/VIS detector (JASCO Co., Ltd., Japan).
HPLC-grade methanol and water were used as eluents. Analytes
m
m
m

590
R. Saito et al. / Tetrahedron 66 (2010) 583–590
10. (a) Karolin, J.; Johansson, B. A.; Strandberg, L.; Ny, T. J. Am. Chem. Soc. 1994, 116,
7801–7806; (b) Qin, W.; Baruah, M.; Auweraer, M. V. d.; Schryver, F. C. D.;
Boens, N. J. Phys. Chem. A. 2005, 109, 7371–7384.
11. Ulrich, G.; Ziessel, R.; Harriman, A. Angew. Chem., Int. Ed. 2008, 47, 1184–1201.
12. Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New
York, NY, 2006.
13. Mason, W. T. Fluorescent and Luminescent Probes for Biological Activity: A Prac-
tical Guide to Technology for Quantitative Real-Time Analysis, 2nd ed.; Academic:
London, 1999.
14. Loudet, A.; Burgess, K. Chem. Rev. 2007, 107, 4891–4932.
15. DiCesare, N.; Lakowicz, J. R. Tetrahedron Lett. 2001, 42, 9105–9108.
16. (a) Shimomura, O.; Musicki, B.; Kishi, Y. Biochem. J. 1989, 261, 913–920; (b) Saito,
R.; Hirano, T.; Niwa, H.; Ohashi, M. Chem. Lett. 1998, 27, 95–96; (c) Nakamura,
H.; Aizawa, M.; Takeuchi, D.; Murai, A.; Shimomura, O. Tetrahedron Lett. 2000,
41, 2185–2188.
buffer (pH 8.6) containing 100-mM KCl, 0.05-mM EDTA, and 0.15-
mM hypoxanthine; light emission was started by adding xanthine
oxidase (1.0 unit/mL, 20 m
L) at 25 ꢂC.
Acknowledgements
This work was financially supported by ‘High-Tech Research
Center’ Project for Private Universities: matching fund subsidy from
the Ministry of Education, Culture, Sports, Science, and Technology
(2005–2009).
17. (a) Teranishi, K.; Isobe, M.; Yamada, T.; Goto, T. Tetrahedron Lett. 1992, 33, 1303–
1306; (b) Hirano, T.; Negishi, R.; Yamaguchi, M.; Chen, F. Q.; Ohmiya, Y.; Tsuji, F.
I.; Ohashi, M. J. Chem. Soc., Chem. Commun. 1995, 1335–1336; (c) Kuse, M.; Doi, I.;
Kondo, N.; Kageyama, Y.; Isobe, M. Tetrahedron 2005, 61, 5754–5762.
18. Peters, C.; Billich, A.; Ghobrial, M.; Ho¨genauer, K.; Ullrich, T.; Nussbaumer, P.
J. Org. Chem. 2007, 72, 1842–1845.
Supplementary data
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.tet.2009.11.086.
19. Ritter, S.; Hauthal, W. H.; Maurer, G. J. Chem. Eng. Data 1994, 39, 414–417.
20. ICSC No. 1087, National Institute for Occupational Safety and Health, In-
ternational Chemical Safety Cards (ICSCs) Database (U.S. National Version).
http://www.cdc.gov/niosh/ipcs/nicstart.html (accessed Apr 2007).
21. Ran, Y.; Xiao, B.; Huang, W.; Peng, P. a.; Liu, D.; Fu, J.; Sheng, G. J. Environ. Qual.
2003, 32, 1701–1709.
22. Adlard, M.; Okafo, G.; Meenan, E.; Camilleri, P. J. Chem. Soc., Chem. Commun.
1995, 2241–2243.
23. Goto, T.; Sugiura, S.; Nishikawa, K.; Isobe, M.; Abe, Y. Tetrahedron Lett. 1968, 9,
4035–4038.
References and notes
1. (a) Barrett, W. C.; DeGnore, J. P.; Keng, Y.-F.; Zang, Z.-Y.; Yim, M. B.; Boon Chock,
P. J. Biol. Chem. 1999, 274, 34543–34546; (b) Knapp, L. T.; Klann, E. J. Biol. Chem.
2000, 275, 24136–24145.
2. Sauer, H.; Wartenberg, M.; Hescheler, J. Cell. Physiol. Biochem. 2001, 11, 173–186.
3. (a) Jabs, T.; Dietrich, R. A.; Dangl, J. L. Science 1996, 273, 1853–1856; (b) Cai, J.;
Jones, D. P. J. Biol. Chem. 1998, 273, 11401–11404.
24. (a) Gabe, Y.; Urano, Y.; Kikuchi, K.; Kojima, H.; Nagano, T. J. Am. Chem. Soc. 2004,
126, 3357–3367; (b) Bro¨ring, M.; Kru¨ger, R.; Link, S.; Kleeberg, C.; Ko¨hler, S.; Xie,
X.; Ventura, B.; Flamigni, L. Chem.dEur. J. 2008, 14, 2976–2983.
25. Fujimori, K.; Nakajima, H.; Akutsu, K.; Mitani, M.; Sawada, H.; Nakayama, M.
J. Chem. Soc., Perkin Trans. 2 1993, 2405–2409.
26. (a) Saito, R.; Hirano, T.; Niwa, H.; Ohashi, M. J. Chem. Soc., Perkin Trans. 2 1997,
1711–1716; (b) Teranishi, K.; Hisamatsu, M.; Yamada, T. Luminescence 1999, 14,
297–302; (c) Teranishi, K.; Nishiguchi, T.; Ueda, H. Carbohydr. Res. 2003, 338,
987–993.
4. Halliwell, B.; Gutteridge, J. M. Reactive species as useful biomolecules. In Free
RadicalsinBiologyandMedicine, 3rded.;OxfordUniversity:Oxford,1999;Chapter6.
5. Nakano, M.; Kikuyama, M.; Hasegawa, T.; Ito, T.; Sakurai, K.; Hiraishi, K.;
Hashimura, E.; Adachi, M. FEBS Lett. 1995, 372, 140–143.
6. Suzuki, N.; Suetsuna, K.; Mashiko, S.; Yoda, B.; Nomoto, T.; Toya, Y.; Inaba, H.;
Goto, T. Agric. Biol. Chem. 1991, 55, 157–160.
7. Wang, J.; Xing, D.; He, Y.; Hu, X. FEBS Lett. 2002, 523, 128–132.
8. Saito, R.; Inoue, C.; Katoh, A. Heterocycles 2004, 63, 759–764.
9. Hinkeldey, B.; Schmitt, A.; Jung, G. ChemPhysChem 2008, 9, 2019–2027.