Synthesis of readily accessible BODIPY-based fluorescence derivatization reagents for amines

Article abstract of DOI:10.3987/COM-10-12031

Two BODIPY-based fluorescence derivatization reagents (FDRs) for amines were synthesized in a simple manner with practically acceptable yields via five steps. Both FDRs showed prompt reactivity, even against sterically hindered amines, to afford the corresponding FDR-labeled products in good yields. The solvent effect upon the fluorescence intensity of the derivatized amine was also investigated in a methanol-water binary solvent system to demonstrate that the strong fluorescence intensity was maintained in the solvent with a 0-60% water ratio. The detection limits of the new FDRs were estimated to be 20 fmol, which makes it much more sensitive than commercially available FDRs. The Japan Institute of Heterocyclic Chemistry.

Full text of DOI:10.3987/COM-10-12031

HETEROCYCLES, Vol. 81, No. 11, 2010
2599
HETEROCYCLES, Vol. 81, No. 11, 2010, pp. 2599 - 2608. © The Japan Institute of Heterocyclic Chemistry
Received, 30th July, 2010, Accepted, 27th September, 2010, Published online, 28th September, 2010
DOI: 10.3987/COM-10-12031
SYNTHESIS
OF
READILY
ACCESSIBLE
BODIPY-BASED
FLUORESCENCE DERIVATIZATION REAGENTS FOR AMINES
Ryota Saito* and Eri Ito
Department of Chemistry, Toho University, 2-2-1 Miyama, Funabashi, Chiba,
274-8510 Japan
E-mail: saito@chem.sci.toho-u.ac.jp
Abstract – Two BODIPY-based fluorescence derivatization reagents (FDRs) for
amines were synthesized in a simple manner with practically acceptable yields via
five steps. Both FDRs showed prompt reactivity, even against sterically hindered
amines, to afford the corresponding FDR-labeled products in good yields. The
solvent effect upon the fluorescence intensity of the derivatized amine was also
investigated in a methanol-water binary solvent system to demonstrate that the
strong fluorescence intensity was maintained in the solvent with a 0-60% water
ratio. The detection limits of the new FDRs were estimated to be 20 fmol, which
makes it much more sensitive than commercially available FDRs.
The detection and quantification of trace amounts of amines and their derivatives in biological systems are
important in the field of medical diagnoses. High-performance liquid chromatography (HPLC) in
combination with fluorescence detection is known to be a powerful tool for the sensitive and expeditious
detection of trace amounts of biological molecules. Because most biologically important amines do not
contain necessary fluorophoric groups, a number of precolumn fluorescence derivatization reagents (FDRs)
have been developed containing fluorophores such as NBD,1 fluorescein,2 and borondipyrromethenes
(BODIPYs), all of which have different structures.3 Among these, BODIPYs have attracted considerable
attention owing to their high extinction and fluorescence efficiencies, excellent photostability, largely
red-shifted emission colors, and flexibility toward chemical modifications. Thus, a number of
amine-labeling agents with BODIPY skeletons have been developed. However, most commercially
available BODIPY-based FDRs are expensive and the methods used for their synthesis are complicated.
Therefore, it is still necessary to develop BODIPY-based FDRs that are easy to prepare.
We recently developed a BODIPY-based luminescent probe for reactive oxygen species using a short
number of reaction steps.4 The synthesis of this probe suggested that compounds 1 and 2, which consisted

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HETEROCYCLES, Vol. 81, No. 11, 2010
of a BODIPY and a succinimidyl group as the reactive unit for amines, can be synthesized quickly by
simply modifying the synthetic route employed in the previous study. Here, we describe the synthesis of
these compounds and their application as FDRs.
The synthetic routes used to obtain 1 and 2 are shown in Scheme 1. Amine 4 was prepared from
4-nitrobenzaldehyde and 3-ethyl-2,4-dimethylpyrrole via 3 as demonstrated previously.4, 5 Compound 4 was
subsequently reacted with isocyanates 5 and 6 to produce 7 and 8, respectively, with quantitative yields.
Saponification of 7 and 8 gave the corresponding carboxylic acids 9 and 10, respectively, with high yields.
A conventional procedure for preparing a succinimidyl ester from a carboxylic acid afforded the target
compound (1) from 9 with a good yield. Analog 2 was similarly obtained from 10 with a moderate yield.
The yield of 2 was lower than that of 1 because of repeated column chromatography carried out to remove a
byproduct, which had polarity similar to that of 2. Although the yield of 2 was slightly diminished, the
syntheses of 1 and 2 were achieved via five steps with practically acceptable yields.
X
NH2
NO2
OCN
CO2Et
NO2
5: X = (CH2)5
6: X =
1) TFA
2) chloranil
3) DIEA, BF3·OEt2
H2
Pd/C
H
N
+
CH2Cl2
MeOH/CH2Cl2
MeCN/CH2Cl2
N
N
B
N
N
B
O
H
F
F
F
F
3 (27%)
4 (73%)
O
O
O
O
X
X
X
O
HN
N
CO2Et
HN
N
CO2H
HN
N
N
H
H
H
O
O
WSC·HCl
HOSu
aq. LiOH
EtOH/CH2Cl2
THF/CH2Cl2
N
N
N
N
N
N
B
B
B
F
F
F
F
F
F
7: X = (CH2)5 (quant.)
9
1
2
: X = (CH2)5 (90%)
10
: X = (CH2)5 (80%)
8
: X =
(quant.)
: X =
(83%)
: X = (56%)
Scheme 1
For sensitive and rapid detection of analytes, it is important for FDRs to readily react with target
compounds. To confirm the reactivity of compounds 1 and 2 with amines, they were reacted with several
aliphatic amines at room temperature. Both 1 and 2 readily reacted with n-butylamine. Figure 1 shows a
representative time course of the reaction of 2 monitored by HPLC analysis. Although this reaction was
almost completed within 10 min to give the corresponding amide, it required more time for the yield to be
sufficient. The maximum and constant peak areas were attained after 20 min. In addition, 1 was reacted
with isopropylamine and tert-butylamine. The reaction times for all of the examinations are summarized in

HETEROCYCLES, Vol. 81, No. 11, 2010
2601
Table 1. Each compound exhibited excellent reactivity, and all of the reactions were completed within 35
min with an almost quantitative yield. It is important to note that 1 exhibited good reactivity even with
sterically hindered isopropylamine and tert-butylamine.
Table 1. Reactivity of 1 and 2 toward various aliphatic amines
O
O
O
H
R NH2
N
N
Ar
X
O
Ar
X
F
F
B
N
N
N
N
N
R
Ar =
N
H
H
H
H
O
O
DMF
O
FDR
1
R
Reaction time
35 min
Product
11a ( X = (CH2)5 )
Yield
80%
n-buthyl
2
n-buthyl
20 min
12a
(X =
)
quant.
1
1
i-propyl
tert-buthyl
30 min
30 min
11b ( X = (CH2)5 )
11c ( X = (CH2)5 )
92%
quant.
7 x 104
6 x 104
5 x 104
4 x 104
3 x 104
2 x 104
1 x 104
0
3.0 x 105
2.5 x 105
2.0 x 105
1.5 x 105
1.0 x 105
0.5 x 105
0
1000
800
600
400
200
0
UV
FL
450
500
550
600
650
0
10
20
30
40
50
60
Wavelength /nm
Reaction time /min
Figure 1. Effect of reaction time on the peak height
for the reaction of 2 with n-buthylamine in DMF at
room temperature monitored by HPLC.
Figure 2. UV-Vis absorption (UV) and
fluorescence (FL) spectra of 11a in MeOH at room
temperature.
Table 2. UV-Vis absorption maxima (abs), fluorescence maxima (FL), Stokes
shifts, and the fluorescence quantum yields (FL) of 1, 2, 11a, and 12a in MeOH.
Compound
Stokes shift/ nm
abs /nm FL /nm
FL
1
11a
2
522
522
522
522
535
535
534
534
13
13
12
12
0.73
0.70
0.72
0.70
12a

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HETEROCYCLES, Vol. 81, No. 11, 2010
The UV-Vis absorption and fluorescence (FL) spectra of 1 and 2 and their n-butylamine conjugates were
measured. The results are summarized in Table 2. Each compound exhibited the maximum absorption band
at 522 nm and strong fluorescence emission around 535 nm with a fluorescence quantum yield of about 0.7.
The selected UV-Vis and FL spectra of 11a are shown in Figure 2. The other compounds exhibited similar
behavior. It should be noted that the high fluorescence efficiency of 1 and 2 was maintained after the
derivatization reaction with the amine.
Generally, fluorescent detection of biomolecules is achieved in a medium containing water. Therefore, it is
important to investigate the effect of water concentration on the fluorescence intensity. Accordingly, the
fluorescence spectra of 11a were measured in aqueous MeOH containing various amounts of water. Figure 3
shows the plots of fluorescence maxima and the relative fluorescence intensity against the given water ratio.
The fluorescence intensity of 11a remained almost constant with the change in the water ratio from 0 to 60%
(v/v), but a rapid decrease occurred when the water ratio increased to > 70%. However, the fluorescence
maximum was not affected by the water ratio. A similar phenomenon was observed in the UV-Vis absorption
in which the absorbance decreased rapidly when the water ratio increased to above 70% (data not shown).
These results indicate that the observed fluorescence quenching may be caused by decomposition of the
fluorophore with water or by the poor solubility of 11a toward aqueous media. To determine the cause of this
quenching, methanol was added to the solution of 11a in the ratio 25% methanol:75% water so as to increase
the proportion of methanol to 85%. A subsequent measurement of the fluorescent spectra revealed that the
fluorescence intensity had recovered to almost the same levels as that separately measured for 11a in 85%
methanol at the same concentration (Figure 4).
550
540
530
520
510
500
1000
800
600
400
200
0
250
200
150
100
50
a (25% MeOH)
b (25%→85% MeOH)
c (85% MeOH)
FL
FL intensity
0
0
20
40
60
80
100
520
540
560
580
600
Water ratio (%)
Wavelength /nm
Figure 3. Effect of water ratio on the fluorescence
(FL) intensity and maximum (FL) of 11a (0.25
M) in aqueous MeOH.
Figure 4. Fluorescence spectra of 11a in (a) 25%
MeOH (0.25 M), (b) 85% MeOH prepared by
adding MeOH to the sample solution to measure the
spectrum a (50 nM), and (c) 85% MeOH (50 nM).

HETEROCYCLES, Vol. 81, No. 11, 2010
2603
Me
Me
Table 3. Chemical yields for the derivatization of n-butylamine
with several FDRs, excitation (Ex) and emission (Em) wavelengths
set for detecting the derivatized products, and detection limits for
detecting the amine-FDR conjugates in HPLCa).
N
Cl
N
N
O
SO2Cl
Dansyl-Cl
NO2
NBD-Cl
FDR
Yield Ex /nm Em /nm Detection limit
NCS
1
2
80%
quant.
Dansyl-Cl quant.
520
520
350
495
470
540
540
530
520
530
20 fmol
20 fmol
1.25 pmol
750 fmol
2.6 pmol
COOH
O
FITC
NBD-Cl
87%
34%
HO
O
FITC
a) Column: SHISEIDO Capcell Pack C18 SG120, 250 mm × 4.6
mm i.d.; Mobile phase: 90% MeOH; Flow rate: 1.0 mL/min.
These findings confirmed that the observed fluorescence quenching was due to the poor solubility of 11a
toward such polar protic media. To evaluate the sensitivity of 1 and 2 during HPLC analysis, the detection
limits of their n-butylamine adducts 11a and 12a, respectively, were estimated. For comparison, Dansyl-Cl,
FITC, and NBD-Cl, which are known to be effective FDRs detectable at the pico-mol order, were also
reacted with n-butylamine and the detection limits of the resulting amine adducts were estimated under the
same conditions. In all analyses, excitation and emission wavelengths for detection were set at the
maximum wavelengths. The results are summarized in Table 3. All FDRs reacted effectively with
n-butylamine except for NBD-Cl. The detection limits of 11a and 12a were estimated to be 20 fmol/1.0-L
injection (S/N = 2), which are much lower than those of other amine adducts. These results clearly indicate
that 1 and 2 are superior FDRs when compared to other FDRs.
In summary, highly sensitive FDRs for amines were prepared via a five-step synthetic route. The
fluorescent properties of the corresponding amine-FDR conjugates provided significant information
regarding the solubility of the BODIPYs in aqueous media. This information suggests interesting
possibilities for the fine detection of biomaterials using BODIPY-based fluorescent probes.
EXPERIMENTAL
General. All melting points were measured on MEL-TEMP apparatus (LABORATORY DEVICES Inc,
1
U.S.A.) in open capillary tubes; the values are uncorrected. H-NMR spectra were recorded on a
JNM-ECP400 spectrometer (JEOL Ltd., Japan). Chemical shifts () are reported in ppm and were measured
using tetramethylsilane or an undeuteriated solvent as an internal standard in the deuterated solvent used.
Coupling constants (J) are given in Hz. Chemical shift multiplicities are reported as s = singlet, d = doublet,
t = triplet, q = quartet, quint = quintet, and m = multiplet. Infrared (IR) spectra were obtained using
FT/IR-660plus spectrophotometers (JASCO Co., Ltd., Japan). Fast-atom-bombardment (FAB) mass spectra

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HETEROCYCLES, Vol. 81, No. 11, 2010
were measured on a JMS-600-H mass spectrometer (JEOL Ltd., Japan). Xenon was used as a bombardment
gas, and all the analyses were carried out in a positive mode with the ionization energy and accelerating
voltage set at 70 eV and 3 kV, respectively. A mixture of dithiothreitol (DTT) and -thioglycerol (TG) (1:1
or 1:2) was used as a liquid matrix. High-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 voltage was 0.5 kV. Absorption spectra were measured on a UV-550 spectrophotometer
(JASCO Co., Ltd., Japan), and combustion analyses were performed on a MT-6 analyzer (Yanaco New
Science Inc., Japan). Column chromatography was carried out on silica gel (particle size: 63–210 μm or
40–100 μm; Kanto Chemical Co.). Conventional chemicals used in the present study are commercially
available and were used as received.
Ethyl 6-[4-(2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl)phenyl]-
ureidohexanoate (7). Ethyl 6-isocyanatohexanoate (5) (0.07 ml, 0.39 mmol) was added to a solution of 45
(95.6 mg, 0.24 mmol) in anhydrous CH2Cl2/MeCN (1/1, 10 mL) at room temperature under an argon
atmosphere. The reaction mixture was then refluxed for 10 h, after which water (10 mL) was added and the
organic materials were extracted with CH2Cl2 (10 mL × 3). Next, the organic layer was dried over Na2SO4,
and the solvents were evaporated. Finally, the crude material was purified by column chromatography on a
silica gel using a hexane/EtOAc mixture as the eluent to give 7 as a red solid (quant.). mp 220–220.5 ºC. 1H
NMR (CDCl3, 400 MHz) /ppm 7.49 (2H, d, J = 8.1 Hz), 7.18 (2H, d, J = 8.1 Hz), 6.39 (1H, s), 4.87 (1H, t,
J = 7.0 Hz), 4.14 (2H, q, J = 7.3 Hz), 3.31 (2H, q, J = 7.0 Hz), 2.53 (6H, s), 2.34 (2H, t, J = 7.0 Hz), 2.30
(4H, q, J = 7.3 Hz) (6H, m), 1.67 (2H, quint, J = 7.0 Hz), 1.59 (2H, quint, J = 7.0 Hz), 1.41 (2H, quint, J =
7.0 Hz), 1.34 (6H, s), 1.27 (3H, t, J = 7.0 Hz), 0.98 (6H, t, J = 7.3 Hz). IR (KBr) max/cm–1 3343 (N–H),
2957, 2929 (C-H), 1733, (C=O), 1651 (C=O), 1315 (B–O), 1186, 978 (B–N). MS (FAB, DTT/TG =
1:2) m/z 581 [M+H]+, 561 [M–F]+. Anal. Calcd for C32H43BF2N4O3·0.6H2O: C, 64.99; H, 7.43; N, 9.48.
Found: C, 64.73; H, 7.22; N, 9.19.
Ethyl 4-[4-(2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl)phenyl]-
ureidobenzoate (8). Compound 8 was prepared in quantitative yield from 45 and ethyl
1
4-isocyanatobenzoate (6) by a procedure analogous to that used to obtain 5a. mp 257 ºC (decomp). H
NMR (CDCl3, 400 MHz) /ppm 8.00 (2H, d, J = 8.8 Hz), 7.54 (2H, d, J = 8.8 Hz), 7.47 (2H, d, J = 8.8
Hz), 7.20 (2H, d, J = 8.8 Hz), 7.20 (1H, s), 7.15 (1H, s), 4.37 (2H, q, J = 7.0 Hz), 2.53 (6H, s), 2.29 (4H, q,
J = 7.3 Hz), 1.40 (3H, t, J = 7.0 Hz), 1.33 (6H, s), 0.97 (6H, t, J = 7.3 Hz). IR (KBr) max/cm–1 3413
(N–H), 1723 (C=O), 1592 (C=O), 1313 (B–O), 1196, 982 (B–N). MS (FAB, DTT/TG = 1:2) m/z
587 [M+H]+, 567 [M–F]+. Anal. Calcd for C33H37BF2N4O3·0.3AcOEt: C, 67.02; H, 6.48; N, 9.14. Found:
C, 67.13; H, 6.34; N, 8.90.

HETEROCYCLES, Vol. 81, No. 11, 2010
2605
6-[4-(2,6-Diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl)phenyl]ureido-
hexanoic acid (9). To prepare 9, 1-M aqueous lithium hydroxide (1.50 ml) was added to a solution of 7 in
EtOH/CH2Cl2 (4 ml/9 ml), and the mixed solution was then stirred at room temperature for 24 h. After
completion of the reaction, water (35 ml) was added to the reaction mixture and the resulting solution was
acidified (pH 6) with 0.5 M HCl. The organic materials were then extracted with EtOAc (100 mL × 3), after
which the organic layer was dried over Na2SO4. Removal of the solvent gave 9 directly as a red solid (223
mg, 90%). mp 260 ºC (decomp). 1H NMR (CDCl3, 400 MHz) /ppm 7.49 (2H, d, J = 8.4 Hz), 7.18 (2H, d,
J = 8.8 Hz), 6.63 (1H, s), 4.88 (1H, t, J = 7.0 Hz), 3.29 (2H, q, J = 7.0 Hz), 2.52 (6H, s), 2.40 (2H, t, J = 7.0
Hz), 2.29 (4H, q, J = 7.3 Hz), 1.69 (2H, quint, J = 7.0 Hz), 1.59 (2H, quint, J = 7.0 Hz), 1.43 (2H, quint, J =
7.0 Hz), 1.34 (6H, s), 0.98 (6H, t, J = 7.3 Hz). IR (KBr) max/cm–1 3389 (O–H), 1708 (C=O), 1662
(C=O), 1317 (B–O), 1194, 981 (B–N). MS (FAB, DTT/TG = 1:1) m/z 553 [M+H]+, 533 [M–F]+; Anal.
Calcd for C30H39BF2N4O3·1.0H2O: C, 63.16; H, 7.07; N, 9.82. Found: C, 63.04; H, 6.92; N, 9.77.
6-[4-(2,6-Diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl)phenyl]ureido-
hexanoic acid O-succinimidyl ester (1). To produce 1, WSC·HCl (59.4 mg, 0.31 mmol) in CH2Cl2 (3
mL) was added to a mixture of 9 (112.4 mg, 0.20 mmol) and N-hydroxysuccinimide (35.3 mg, 0.31 mmol)
in THF (15 mL) at 0 °C under an argon atmosphere. The resulting mixture was then stirred at room
temperature for 7 h. Next, the reaction mixture was washed with water (20 mL) and extracted with EtAOc
(30 mL × 3). The organic phase was then washed with brine and dried over Na2SO4. Following the
evaporation of the solvent, the residue was purified by chromatography on silica gel using CH2Cl2 as an
eluent to give 1 as a red solid (105.4 mg, 80%). mp 193–194.5 ºC. 1H NMR (CDCl3, 400 MHz) /ppm 7.51
(2H, d, J = 8.4 Hz), 7.15 (2H, d, J = 8.4 Hz), 7.01 (1H, s), 5.22 (1H, t, J = 7.0 Hz), 3.35 (2H, q, J = 7.0 Hz),
2.94 (2H, s), 2.93 (2H, s), 2.69 (2H, t, J = 7.0 Hz), 2.52 (6H, s), 2.29 (4H, q, J = 7.3 Hz), 1.84 (2H, quint, J
= 7.0 Hz), 1.62 (2H, quint, J = 7.0 Hz), 1.58–1.52 (2H, m, overlapped with H2O protons), 1.35 (6H, s), 0.98
(6H, t, J = 7.3 Hz). IR (KBr) max/cm–1 3377 (N–H), 1740 (C=O), 1652 (N–H), 1542 (C=O), 1317
(B–O), 1194, 981 (B–N). MS (FAB, DTT/TG = 1:1) m/z 650 [M+H]+, 630 [M–F]+. Anal. Calcd for
C34H42BF2N5O5·0.3H2O: C, 62.35; H, 6.51; N, 10.70. Found: C, 62.16; H, 6.44; N, 10.45.
4-[4-(2,6-Diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl)phenyl]ureido-
benzoic acid O-succinimidyl ester (2). The precursor 10 was prepared from 8 by a procedure analogous to
that used to obtain 7 (83%); 1H NMR (DMSO-d6, 400 MHz) /ppm 12.6 (1H, s), 9.15 (1H, s), 9.07 (1H, s),
7.88 (2H, d, J = 8.5 Hz), 7.66 (2H, d, J = 8.5 Hz), 7.58 (2H, d, J = 8.5 Hz), 7.25 (2H, d, J = 8.5 Hz), 2.43
(6H, s), 2.30 (4H, q, J = 7.3 Hz), 1.34 (6H, s), 0.94 (6H, t, J = 7.3 Hz); IR (KBr) max/cm–1 3398 (O–H),
3376 (N–H), 1692 (C=O), 1594 (N–H), 1542 (C=O), 1313 (B–O), 1196, 982 (B–N). MS (FAB,
DTT/TG = 1:2) m/z 559 [M+H]+, 539 [M–F]+; This material was used in the next step without further

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HETEROCYCLES, Vol. 81, No. 11, 2010
purification. Next, 10 was converted into 2 by a procedure analogous to that used to obtain 1 in 56% yield.
mp 272 ºC (decomp). 1H NMR (CDCl3, 400 MHz) /ppm 7.93 (2H, d, J = 8.4 Hz), 7.73 (1H, s,), 7.62 (1H,
s), 7.56 (2H, d, J = 8.4 Hz), 7.44 (2H, d, J = 8.4 Hz), 7.13 (2H, d, J = 8.4 Hz), 2.97 (4H, s), 2.53 (6H, s),
2.29 (4H, q, J = 7.3 Hz), 1.34 (6H, s), 0.97 (6H, t, J = 7.3 Hz). IR (KBr) max/cm–1 3496 (N–H), 1737
(C=O), 1590 (N–H), 1539 (NC=O), 1316 (B–O), 1197, 983 (B–N). MS (FAB, DTT/TG = 1:1) m/z
656 [M+H]+, 636 [M–F]+. Anal. Calcd for C35H36BF2N5O5·2.5H2O: C, 60.00; H, 5.54; N, 10.00. Found: C,
59.90; H, 5.43; N, 9.82.
Derivatization reactions. To a solution of an FDR (21 mol) in DMF (0.7 ml) was added n-butylamine
(4.2 L, 25 mol) and stirred at room temperature for 3.5 h under argon atmosphere. After evaporation of
the solvent, the crude material was purified by column chromatography on silica gel using EtOAc as
eluents to give the corresponding FDR-derivatized amine.
N-Butyl-6-[4-(2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl)phenyl]-
ureidohexanamide (11a) was obtained in 80% yield; 1H NMR (CDCl3 400 MHz) /ppm 7.53 (2H, d, J =
8.4 Hz), 7.15 (2H, d, J = 8.4 Hz), 6.97 (1H, s), 5.56 –5.49 (2H, m), 3.33 (2H, q, J = 7.0 Hz), 3.29 (2H, q, J =
7.0 Hz), 2.52 (6H, s), 2.29 (4H, q, J = 7.3 Hz), 2.24 (2H, t, J = 7.0 Hz), 1.69 (2H, quint, J = 7.0 Hz),
1.61–1.47 (4H, m), 1.42 (0.98 (6H, t, J = 7.3 Hz), 0.92 (3H, t, J = 7.3 Hz); IR (KBr) max/cm–1 1708 (C=O),
1644 (C=O), 1543, 1477 (C=C, C=N, ring stretching ), 1316 (B–O), 1191, 979 (B–N); MS (FAB,
DTT/TG = 1:2) m/z 608 [M+H]+ and 588 [M–F]+.
N-n-Butyl-4-[4-(2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl)-
phenyl]ureidobenzamide (12a) was obtained quantitatively; 1H NMR (CDCl3, 400 MHz) /ppm 8.15 (1H,
s, NH), 8.13 (1H, s, NH), 7.65 (2H, d, J = 8.4 Hz, ArH), 7.61 (2H, d, J = 8.4 Hz, ArH), 7.39 (2H, d, J = 8.4
Hz, ArH), 7.19 (2H, d, J = 8.4 Hz, ArH), 6.32 (1H, t, J = 7.0 Hz, NH), 3.51 (2H, q, J = 7.0 Hz), 2.53 (6H, s),
2.29 (4H, q, J = 7.3 Hz), 1.65 (2H, quint., J = 7.0 Hz), 1.43 (2H, sex, J = 7.0 Hz), 1.34 (6H, s), 0.97 (6H, t, J
= 7.3 Hz), 0.96 (3H, t, J = 7.0 Hz); MS (FAB, DTT/TG = 1:1) m/z 614 [M+H]+ and 594 [M–F]+.
N-Isopropyl-4-[4-(2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl)-
phenyl]ureidohexanamide (11b) was obtained in 92% yield; 1H NMR (CDCl3, 400 MHz) /ppm 7.85 (1H,
s, NH), 7.56 (2H, d, J = 8.4 Hz), 7.12 (2H, d, J = 8.4 Hz), 5.86 (1H, t, J = 7.0 H), 5.73 (1H, d, J = 6.6 Hz),
4.09 (1H, octet, J = 6.6 Hz), 3.29 (2H, q, J = 7.0 Hz), 2.52 (6H, s), 2.28 (4H, q, J = 7.3 Hz), 2.20 (2H, t, J =
7.0 Hz), 1.67 (2H, quint, J = 7.0 Hz), 1.56 (2H, quint, J = 7.0 Hz), 1.40 (2H, quint, J = 7.0 Hz), 1.33 (6H, s),
1.17 (6H, d, J = 6.6 Hz), 0.96 (6H, t, J = 7.3 Hz); MS (FAB, DTT/TG = 1:1) m/z 594 [M+H]+ and 574
[M–F]+.
N-tert-Butyl-4-[4-(2,6-diethyl-4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacen-8-yl)-
phenyl]ureidobenzamide (11c) was obtained quantitatively; 1H NMR (CDCl3, 400 MHz) /ppm 7.79 (1H,

HETEROCYCLES, Vol. 81, No. 11, 2010
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s), 7.56 (2H, d, J = 8.4 Hz), 7.12 (2H, d, J = 8.4 Hz), 5.90 (1H, t, J = 7.0 Hz), 5.60 (1H, s), 3.28 (2H, q , J =
7.0 Hz), 2.52 (6H, s), 2.28 (4H, q, J = 7.3 Hz), 2.16 (2H, t, J = 7.0 Hz), 1.64 (2H, quint, J = 7.0 Hz), 1.56
(2H, quint, J = 7.0 Hz), 1.40 (2H, quint, J = 7.0 Hz), 1.37 (9H, s), 1.33 (6H, s), 0.97 (6H, t, J = 7.3 Hz); MS
(FAB, DTT/TG = 1:1) m/z 608 [M+H]+ and 588 [M–F]+. Dansyl-Cl, FITC and NBD-Cl were reacted with
n-butylamine by the same procedure that for obtaining 11a–c. The corresponding derivatized products,
N-n-butyl-5-(dimethylamino)naphthalene-1-sulfonamide (N-n-butyl-Dansyl)6, N-n-butyl-5-thioureido-
fluorescein (FTU-n-butyl),7 and 4-n-butylamino-7-nitrobenzo[c][1,2,5]oxadiazol (4-n-butylamino-NBD),8
were obtained in quantitative, 87% and 34% yields, respectively. The identification of these products was
achieved by comparing their spectral features with those of previously reported data.
Fluorescence measurements
Fluorescence spectra were collected using a JASCO F-777 Spectrofluorometer. The excitation wavelength
was set at 492 nm for each measurement. The fluorescence quantum yields were measured at 25 °C relative
to fluorescein in 0.1 M NaOH (FLexcited at 467 nm) and then calculated according to the
manufacturer’s instructions.
HPLC analysis
HPLC analysis was conducted using a Capcell Pak C18 SG120 column (particle size: 5 m, 250 mm × 4.6
mm id., SHISEIDO Co., Ltd., Japan) with an SSC-3461 pump (SENSHU SCIENTIFIC Co. Ltd., Japan), a
DG-1110 degasser (SENSHU SCIENTIFIC Co., Ltd., Japan), an 875-UV Intelligent UV/Vis detector
(JASCO Co., Ltd, Japan), and a FL SPECTRO PHOTO METER S-3370 (SOMA Co Ltd., Japan).
HPLC-grade methanol and water were used as the eluents. The analytes were eluted with 90% (v/v) MeOH
at a flow rate of 1.0 mL/min. The excitation wavelength of the detector was set at 520 nm and the
fluorescence was detected at 540 nm. Measurements were conducted at a temperature of 25 ºC, which was
maintained using a SSC-2300 column oven (SENSHU SCIENTIFIC Co. Ltd., Japan).
ACKNOWLEDGEMENTS
This work was financially supported by “High-Tech Research Center” Project for Private Universities:
matching fund subsidy from the MEXT (2005-2009).
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