Exhaustive syntheses of naphthofluoresceins and their functions

Article abstract of DOI:10.1021/jo300177b

Naphthofluorescein and/or seminaphthofluorescein derivatives possessing the additional benzene units to one or both sides of fluorescein were exhaustively constructed through Friedel-Crafts type reactions between corresponding aroylbenzoic acids and dihydroxynaphthalenes. Compound 4 works as a one-dye pH indicator, which shows red in strong acid condition and blue in basic solution. Compound 23 (diacetate of compound 4) shows good transitivity to the HEK 293 cells and acts as a fluorescent pigment for the living cell imaging. Compounds 5, 6, and 9 show fluorescent emission in the NIR region (>700 nm) and imply the potentialities of NIR fluorescent probes.

Full text of DOI:10.1021/jo300177b

Article
pubs.acs.org/joc
Exhaustive Syntheses of Naphthofluoresceins and Their Functions
Eriko Azuma,† Naoko Nakamura,† Kouji Kuramochi,† Takahiro Sasamori,‡ Norihiro Tokitoh,‡
Ikuko Sagami,† and Kazunori Tsubaki†,*
†Graduate School of Life and Environmental Sciences, Kyoto Prefectural University Shimogamo, Sakyo-ku Kyoto 606-8522, Japan
‡Institute for Chemical Research, Kyoto University Uji, Kyoto, 611-0011, Japan
S
* Supporting Information
ABSTRACT: Naphthofluorescein and/or seminaphthofluor-
escein derivatives possessing the additional benzene units to
one or both sides of fluorescein were exhaustively constructed
through Friedel−Crafts type reactions between corresponding
aroylbenzoic acids and dihydroxynaphthalenes. Compound 4
works as a one-dye pH indicator, which shows red in strong
acid condition and blue in basic solution. Compound 23
(diacetate of compound 4) shows good transitivity to the HEK
293 cells and acts as a fluorescent pigment for the living cell
imaging. Compounds 5, 6, and 9 show fluorescent emission in
the NIR region (>700 nm) and imply the potentialities of NIR fluorescent probes.
NIR. Furthermore, these compounds should have practical
benefits due to their structural similarities with fluorescein, as
fluorescein has a long history of modifications and applications.
In this paper, we introduce the exhaustive synthesis of
naphthofluoresceins 2−10 (Figure 1) and evaluate their
properties, including UV−vis and fluorescent spectra. Among
them, compounds 78 and 109 are known as fluorescent
materials. Several biochemical applications of 7 such as
hydrogen peroxide and superoxide radical anion imaging
probes have been reported.8b,8d
INTRODUCTION
■
Because of their significant contributions, fluorescent probes
have become indispensable tools in the fields of biology and
chemical biology.1 Advantages of fluorescent probes for
bioimaging include (1) less damage to living cells, (2) high
sensitivity, and (3) real-time tracing of target biological
materials. Among fluorescent probes, fluorescein2 and cyanine3
derivatives are the most common. Fluorescein derivatives have
a good transitivity into the cell and can increase lipophilicity by
introducing acyl groups on their two hydroxy groups.
Additionally, once the acyl groups are hydrolyzed by enzymes
in the cell, the active forms are well retained by the cell.4
Furthermore, a methodology to control the on/off fluorescent
properties based on Pet2c or d-Pet5 mechanism has also been
developed. However, their excitation and emission wavelengths
are around 500 nm and overlap with autofluorescence in
biological materials such as tryptophan and porphyrin. For
biological applications, avoidance of overlap with autofluor-
escence is desirable. Hence, probes with excitation and
emission wavelengths in the near-infrared region (NIR)
(>700 nm) are preferable. Fluorescent probes with excitation
and emission wavelengths in the NIR are also suitable to
visualize deeper tissues with less damage to the organism using
low energy light.6 Some cyanine derivatives have excitation and
emission wavelength around the NIR, and consequently, the
aforementioned autofluorescence problems do not occur.7
Although cyanine derivatives are designed for longer wave-
lengths, their chemical structures are complicated and often
cause changes that reduce cell transitivity.
RESULTS AND DISCUSSION
■
Synthesis of Compounds 2−10. Based on the synthetic
route to fluorescein (1),10 we initially examined a double
Friedel−Crafts reaction with 2 equiv of 2,7-dihydroxynaph-
thalene (11) and phthalic anhydride (12) in methanesulfonic
acid. The reactivity on the naphthalene ring for electrophilic
substitution is higher at the 1-position of 11 than at the 3-
position. Consequently, compound 2, which is due to a double
Friedel−Crafts reaction at 1-position, was generated in 6% yield
instead of 3 (4%) and 5 (1%). Additionally, a large amount of
thick paste was formed (Scheme 1).
An alternative and selective synthetic route for linear
naphthofluorescein 5 was also developed (Scheme 2).
Methoxymethyl (MOM)-protected bromonaphthalene 14,
which was produced from corresponding diol 1311 in 86%
yield, was treated with n-BuLi and allowed to react with 0.5
equiv of phthalic anhydride (12) to give 15 in 81% yield. The
phenolic MOM groups of 15 were removed by 4 N HCl in
dioxane to afford 16 in 73% yield. Intramolecular dehydration
Under such a background, we hypothesized that if the
benzene units could be expanded to one or both sides of the
dibenzopyran system in the fluorescein skeleton, then the
excitation and emission wavelengths could be elongated to the
Received: February 8, 2012
Published: March 19, 2012
© 2012 American Chemical Society
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Figure 1. Structures of fluorescein (1), naphthofluoresceins (2−7), and seminaphthofluoresceins (8−10).
Scheme 1. Synthetic Route for Naphthofluoresceins 2, 3, and
5
Scheme 2. Synthetic Route for Naphthofluorescein 5
Figure 2. Crystal structure of the naphthofluorescein 5.
Scheme 3. Synthetic Route for Naphthofluorescein 4 and 23
occurred under acidic conditions to give desired 5 in 77% yield.
Colorless crystals of 5 precipitated from toluene−ethyl acetate.
According to X-ray analysis, the crystals of 5 are monoclinic
and belong to the P21/c space group. One unit cell is composed
of two compound 5 molecules (molecule A and molecule B)
and two toluene molecules (Figure 2). Of particular interest is
that compared to a planar hexagonal shape (720°), the two
binaphthopyranes in molecules A and B show high planarities
based on the sum of the internal angles of the central pyrane
ring (719.33° and 719.27° for A and B, respectively).
Scheme 3 outlines the synthesis of compound 4. According
to the literature, methoxytetralone 17 was converted to 18 in
two steps.12 A methyl group of 18 was removed by BBr3 (19;
88% yield) and then two hydroxy groups of 19 were protected
by MOM to give 20 in 81% yield. Lithiated 20 was reacted with
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1 equiv of phthalic anhydride (12) to afford key intermediate 2-
aroyl-1-benzoic acid 21 in 61% yield. After deprotection of the
MOM group in HCl and dioxane (22; 96%), 22 was treated
with 2,7-dihydroxynaphthalene (11) to afford 4 in 76% yield.
For the living cell imaging, compound 4 was treated with acetic
anhydride to afford the diacetate 23 in 92% yield.
Aroylbenzoic acid 25 was constructed from 14 and phthalic
anhydride (12) in 68% yield, and subsequent deprotection of
the MOM group occurred in 67% yield. Using aroylbenzoic
acid 25, a variety of naphthofluoresceins were synthesized.
Thus, 25 was reacted with 2,7-dihydroxynaphthalene (11) to
give 3 (48%) and 5 (20%), with 1,6-dihydroxynaphthalene (26)
to afford 6 (47%), and with resorcinol to give 9 in 82% yield
(Scheme 4).
The aroylbenzoic acid route from 33, a 2,7-dihydroxynaph-
thalene possessing a keto acid in its 1 position, is a dead end.
For example, in the case of reaction between 33 and resorcinol
in methanesulfonic acid, unexpected retro Friedel−Crafts
reaction should have precedence over desired Friedel−Crafts
reaction to give 2,7-dihydroxynaphthalene (11) in 43% yield
and fluorescein (1) in 20% yield, which should be generated
from fragmented phthalic anhydride (12) and resorcinol
(Scheme 6).
Scheme 6. Unexpected Retro-Friedel−Crafts Reaction
Scheme 4. Synthetic Route for 3, 5, 6 and 9
Coloration of 2−10 under Various pH Conditions.
With the series of benzene-unit expanded fluoresceins 2−10 in
hand, we then scrutinized the coloration of 2−10 under various
pH conditions (−1 to 14). Figure 3 shows pictures and UV−vis
spectra of naphthofluoresceins 2−7 (see Supporting Informa-
tion Figure S-1 for seminaphthofluoresceins 8−10). The
compounds have low solubilities in aqueous solutions and
precipitate in the acidic region. Homogeneous solutions occur
in basic solutions or in extremely acidic solutions (i.e.,
methanesulfonic acid).
Compounds 2−10 display peaks near 490−580 nm in the
acidic region (red lines in each spectra) and near 595−708 nm
in the basic region (blue lines (pH = 11) in the corresponding
spectra). The data indicates that (1) the HOMO−LUMO gap
of dianionic form in a basic solution is narrower than that of in
cationic form in an acidic region (Figure 4, Figure S-2 and S-3
for the DFT calculations, Supporting Information), and (2)
linear naphthofluorescein 5 shows longer λmax wavelengths in
both acidic and basic solutions compared to other bent
naphthofluoresceins.
The aroylbenzoic acid route might be applicable to
synthesize one benzene-unit expanded seminaphthofluoresceins
8−10 (Scheme 5). Thus, aroylbenzoic acid 29,13 which was
synthesized from 27,14 was coupled with 2,7-dihydroxynaph-
thalene (11) to afford 8 and regioisomer 9 in yields of 30 and
27%, respectively. Additionally, coupling 29 with 1,6-dihydrox-
ynaphthalene (26) produced 10 in 61% yield.
Scheme 5. Synthetic Route for 8−10
Intriguingly, compounds 2 and 4 show a clear color contrast;
they are vibrant red in strongly acidic conditions and bright
blue in basic conditions. In particular, the absorbance for
compound 4 in strongly acidic conditions is almost same as that
in pH 10. Hence, we attempted a trial of compound 4 as a one-
dye pH-test paper. White filter paper was impregnated with
compound 4 and dried. Then the color responses against pH
were examined. As expected, the filter paper is suitable as a pH-
test paper. It turns red in acidic conditions and blue in basic
conditions (Figure 5).
Fluorescence Properties of 2−10 under Basic Con-
ditions (pH 11). Despite the UV−vis spectrum possessing
large absorptions at pH 10 and 11 near 700 nm, linear
naphthofluorescein 5 is a sickly green or blue-green color to the
naked eye because λmax is beyond the human visible range
(>700 nm). If the synthesized compounds provide a fluorescent
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Figure 3. Coloration of the naphthofluoresceins 2−7 under various pH conditions. Conditions: photo; [compound] = 1.0 × 10−4 M, UV−vis
spectra; [compound] = 2.0 × 10−5 M, pH = −1 (MeSO3H aq.), pH = 1 (HCl-KCl buffer), pH = 3, 5, 7 (citrate-phosphate buffer), pH = 8, 9 (Tris-
HCl buffer), pH = 10−14 (glycine-NaOH buffer). ppt = precipitates.
response, their excitation and emission wavelengths should be
in the NIR. Therefore, to investigate the potential of 2−10 as
probes in the chemical biology field, we examined their
fluorescent properties in aqueous glycine-NaOH buffer
conditions (pH 11).
Figure 6 shows the normalized fluorescent spectra of typical
compounds, while Table 1 summarizes the characteristic
parameters such as λmax of absorbance, excitation and emission
wavelengths, quantum yield (Φ), and Stokes shift of the
benzene unit expanded fluoresceins.
wavelengths of other compounds, all new compounds 4−6, and
9 (solid lines in Figure 6) have longer wavelengths than known
compounds 7, 10, and fluorescein (1) (dotted lines in Figure
6). Especially, the emission peaks of compounds 5, 6, and 9
exceed 700 nm and are in the NIR region. In addition,
compounds 6 (133 nm) and 9 (196 nm) exhibit quite large
Stokes shifts, which are advantageous in avoiding the overlap
between emission and scattering light.
For a preliminary living cell imaging experiment, we chose
compound 23 (diacetate of compound 4) as an appropriate
fluorophore under the condition of common fluorescence
microscopy and filters. HEK 293 or NIH 3T3 cells were
Compounds 2, 3, and 8 do not have meaningful emissions
under basic aqueous conditions. Comparing to the emission
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Figure 4. Proposed colored forms in acidic or basic conditions.
Figure 5. Naphthofluorescein 4 as one-dye pH indicator.
Figure 7. Raw and fluorescence images of HEK 293 cells. HEK 293
cell were incubated in 0.05 mM of compound 23 for 60 min. (a) Raw
image, (b) fluorescence image, and (c) overlapping image of (a) and
(b).
cells and released active compound 4, suggesting the clear
fluorescence response from the cells and the low cytotoxicity.
CONCLUSIONS
■
We have exhaustively constructed benzene unit expanded
fluoresceins, mainly through an aroylbenzoic acid route, and
elucidated their properties. Compound 4 works as a one-dye
pH indicator, which is red in strongly acid conditions and blue
in basic conditions. Although the quantum yields of 5, 6, and 9
are moderate, they have important functions, such as a simple
framework, and may aid in designing functional fluorescein
derivatives. In particular, compound 9 emits in the NIR with a
large Stokes shift and is water-soluble. We are currently
investigating these compounds for application for living cell
under NIR fluorescence microscopy conditions as well as
improvement of quantum yield based on increasing rigidity of
the framework.
Figure 6. Fluorescence spectra of fluorescein (1), naphthofluoresceins
(4−7), and seminaphthofluoresceins (9−10). Conditions; glycine-
NaOH buffer conditions (pH 11). exciting wavelength; 1 (491 nm), 4
(583 nm), 5 (715 nm), 6 (647 nm), 7 (591 nm), 9 (536 nm), 10 (536
nm).
incubated in PBS buffer containing 0.01−0.10 mM of
compound 23 for 15, 30, 60 min at rt. After replacing the
medium with PBS buffer, the cells were observed by a standard
fluorescence microscopy. Under all conditions examined, the
fluorescence images for both types of cells were obtained
without obvious cytotoxicity as shown in Figure 7. These data
indicated that compound 23 was smoothly transferred into the
Table 1. UV−Vis−NIR and FL Spectral Properties of Compounds 1−10
a
compound
λAbs. max (nm)
ε (λAbs. max
)
λex (nm)
ε (λex)
λem (nm)
Φ (%)
Stokes shift (nm)
22
b
1
2
491
645
691
610
708
650
595
540
536
538
78,000
9,800
491
78,000
513
97
3
15,000
30,000
57,000
35,000
44,000
20,000
29,000
44,000
4
583
715
647
591
25,000
56,000
34,000
43,000
674
790
780
670
0.23
0.17
91
75
5
6
0.4
133
79
b
7
14
8
9
536
536
29,000
44,000
732
629
0.24
196
93
10
35
a
The fluorescence quantum yields of 4−6 and 9−10 (Φ) were determined using a solution of compound 7 in glycine-NaOH buffer (pH 11) as a
b
reference standard (Φ = 0.14). Taken from ref 8a.
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304 (11), 284 (19). HRMS (EI+) Calcd for C22H20O7 (M+): 396.1209.
Found: 396.1210.
EXPERIMENTAL SECTION
■
General Procedure for the Protection of Phenolic Hydroxy
Group with MOMCl. The preparation of the MOM-protected
bromonaphthalene 14 is typical. To a stirred solution of 3-bromo-
naphthalene-2,7-diol (13, 9.0 g, 37.6 mmol) in dry DMF (50 mL),
NaH (60% dispersion in mineral oil, 4.5 g, 112.5 mmol, 3 equiv) was
added portionwise under ice-bath cooling. After 1 h, MOMCl (6.8 mL,
89.5 mmol, 2.4 equiv) was added dropwise to the suspension and
stirred for 4.5 h at room temperature. The reaction mixture was
poured into the mixed solvent of ethyl acetate and water. The organic
layer was separated and washed successively with water (twice) and
brine. After being dried over sodium sulfate, the solvent was
evaporated in vacuo to give a residue. The residue was purified by
column chromatography (SiO2; n-hexane to n-hexane/ethyl acetate =
10/1) to afford 14 (10.6 g, 32.4 mmol, 86%) as a white powder.
Compound 14: 86% yield; white powder; mp 50−51 °C; IR (neat)
2956, 1628, 1595, 1504, 1371, 1149 cm−1; 1H NMR (270 MHz,
CDCl3) δ 7.98 (s, 1H), 7.60 (d, J = 8.9 Hz, 1H), 7.39 (s, 1H), 7.29 (d,
J = 2.4 Hz, 1H), 7.10 (dd, J = 8.9, 2.4 Hz, 1H), 5.35 (s, 2H), 5.27 (s,
2H), 3.55 (s, 3H), 3.51 (s, 3H); 13C NMR (68 MHz, CDCl3) δ 155.5,
151.4, 134.4, 131.7, 128.0, 125.8, 117.8, 111.2, 110.2, 109.0, 95.0, 94.3,
56.3, 56.1; MS (EI+) m/z (rel. int.) 328 (M+, 75), 326 (M+, 75), 296
(13), 266 (17), 247 (12), 182 (23), 180 (23), 113 (100). HRMS (EI+)
Calcd for C14H1579BrO4 (M+): 326.0154. Found: 326.0159.
Compound 20: 81% yield from 19; colorless oil; IR (neat) 1624,
1585, 1500, 1363, 1236 cm−1; 1H NMR (270 MHz, CDCl3) δ 8.11 (d,
J = 8.9 Hz, 1H), 7.53 (d, J = 8.9 Hz, 1H), 7.40 (d, J = 8.9 Hz, 1H),
7.36 (d, J = 2.4 Hz, 1H), 7.26 (dd, J = 8.9, 2.4 Hz, 1H), 5.29 (s, 2H),
5.25 (s, 2H), 3.71 (s, 3H), 3.52 (s, 3H); 13C NMR (68 MHz, CDCl3)
δ 155.4, 150.6, 134.9, 130.4, 125.4, 124.5, 124.0, 119.5, 110.4, 109.8,
100.1, 94.3, 58.2, 56.1; MS (EI+) m/z (rel. int.) 328 (M+, 100), 326
(M+, 100), 298 (55), 296 (55). HRMS (EI+) Calcd for C14H1579BrO4
(M+): 326.0154. Found: 326.0150. Calcd for C14H1581BrO4 (M+):
328.0133. Found: 328.0130. Anal. Calcd for C14H15BrO4: C, 51.40; H,
4.62. Found: C, 51.18; H, 4.64.
Compound 31: 99% yield; colorless oil; IR (neat) 2902, 1628, 1510,
1360, 1151 cm−1; 1H NMR (270 MHz, CDCl3) δ 7.77 (d, J = 2.5 Hz,
1H), 7.71 (d, J = 8.9 Hz, 1H), 7.70 (d, J = 8.9 Hz, 1H), 7.29 (d, J = 8.9
Hz, 1H), 7.15 (dd, J = 8.9, 2.5 Hz, 1H), 5.35 (s, 2H), 5.34 (s, 2H),
3.57 (s, 3H), 3.54 (s, 3H); 13C NMR (68 MHz, CDCl3) δ 156.5,
152.1, 134.3, 129.7, 128.3, 126.2, 117.5, 114.6, 109.3, 109.0, 95.4, 94.5,
56.5, 56.3; MS (EI+) m/z (rel. int.) 328 (M+, 81), 326 (M+, 81), 298
(28), 296 (26), 268 (27), 266 (27), 153 (61), 136 (53), 106 (46), 89
(66), 77 (100). HRMS (EI+) Calcd for C14H15O479Br (M+): 326.0153.
Found: 326.0148.
General Procedure for the Aroylbenzoic Acid Derivatives.
The preparation of compound 21 is typical. A solution of n-BuLi (1.63
M n-hexane solution; 1.35 mL, 2.21 mmol) was added dropwise to a
solution of 20 (600 mg, 1.83 mmol) in dry THF (7 mL) under N2
atmosphere at −78 °C. After 1 h of stirring, the solution of lithiated 20
in THF was added dropwise to a solution of phthalic anhydride (12,
251.5 mg, 1.70 mmol) in dry THF (10 mL). The resultant solution
was stirred at −78 °C for 1 h and then allowed to warm to room
temperature with stirring for 2.5 h. The reaction mixture was poured
into the mixed solvent of ethyl acetate and 0.1 M aq. HCl. The organic
layers were successively washed with water and brine, dried over
Na2SO4, filtered, and concentrated under reduced pressure to dryness.
The residue was purified by column chromatography (SiO2; n-hexane-
EtOAc, 3:1 → 1:1) to afford 21 (437 mg, 61%) as a pale-yellow oil.
Compound 21. 61% yield; pale-yellow oil; IR (neat) 3444, 1718,
1664, 1620, 1469, 1240 cm−1; 1H NMR (270 MHz, CDCl3) δ 8.29 (d,
J = 8.9 Hz, 1H), 8.01−7.98 (m, 1H), 7.61−7.55 (m, 2H), 7.52 (d, J =
8.9 Hz, 1H), 7.46 (d, J = 8.9 Hz, 1H), 7.47−7.42 (m, 1H), 7.36 (d, J =
2.4 Hz, 1H), 7.27 (dd, J = 8.9, 2.4 Hz, 1H), 5.31 (s, 2H), 5.05 (s, 2H),
3.52 (s, 3H), 3.40 (s, 3H); 13C NMR (68 MHz, CD3OD) δ 192.4,
169.9, 158.7, 156.7, 144.2, 139.9, 132.6, 132.5, 130.8, 130.6, 129.1,
128.9, 127.2, 125.4, 123.6, 120.4, 110.6, 102.7, 95.3, 58.1, 56.5 (one
peak overlapped); MS (EI+) m/z (rel. int.) 396 (M+, 27), 334 (100),
Compound 24: 68% yield from 12 and 14; white powder; mp 132−
134 °C; IR (KBr) 2893, 1697, 1655, 1622, 1464, 1288 cm−1; 1H NMR
(270 MHz, CDCl3 40 °C) δ 8.16 (brs, 1H), 7.95 (d, J = 7.0 Hz, 1H),
7.76 (d, J = 8.9 Hz, 1H), 7.64−7.50 (m, 2H), 7.35 (d, J = 7.3 Hz, 1H),
7.35−7.25 (m, 2H), 7.10 (dd, J = 8.9, 2.2 Hz, 1H), 5.28 (s, 2H), 4.98
(s, 2H), 3.47 (s, 3H), 3.22 (s, 3H); 13C NMR (68 MHz, CD3OD) δ
194.6, 167.5, 156.6, 153.0, 143.7, 137.3, 132.0, 131.5, 130.9, 130.1,
129.3, 129.1, 127.1, 126.5, 123.3, 117.6, 109.0, 108.3, 93.8, 93.7, 55.8,
55.7; MS (EI+) m/z (rel. int.) 396 (M+, 13), 334 (50), 284 (77), 256
(55), 129 (64), 73 (100). HRMS (EI+) Calcd for C22H20O7 (M+):
396.1209. Found: 396.1202.
Compound 28: 60% yield from 12 and 27; pale-yellow oil; IR
(neat) 3458, 2966, 1714, 1651, 1601, 1261 cm−1; 1H NMR (270 MHz,
CD3OD) δ 7.96 (dd, J = 7.7, 1.4 Hz, 1H), 7.77 (d, J = 9.5 Hz, 1H),
7.59 (ddd, J = 7.7, 7.7, 1.4 Hz, 1H), 7.51 (ddd, J = 7.7, 7.7, 1.4 Hz,
1H), 7.25 (dd, J = 7.7, 1.4 Hz, 1H), 6.77−6.72 (m, 2H), 5.21 (s, 2H),
4.83 (s, 2H), 3.44 (s, 3H), 3.14 (s, 3H); 13C NMR (68 MHz, CDCl3)
δ 195.5, 170.5, 161.9, 158.2, 145.6, 132.9, 132.2, 129.7, 128.6, 128.3,
126.2, 121.1, 108.7, 103.1, 94.2, 94.1, 56.3, 56.1; MS (EI+) m/z (rel.
int.) 346 (M+, 34), 313 (16), 284 (100), 241 (13), 149 (12). HRMS
(EI+) Calcd for C18H18O7 (M+): 346.1053. Found: 346.1052.
Compound 32: 96% yield from 12 and 31; yellow powder; mp
102−103 °C; IR (KBr) 2952, 1705, 1651, 1514, 1242, 1147 cm−1; 1H
NMR (270 MHz, CDCl3) δ 7.87 (d, J = 7.3 Hz, 1H), 7.85 (d, J = 8.9
Hz, 1H), 7.72 (d, J = 8.9 Hz, 1H), 7.59−7.47 (m, 4H), 7.25 (d, J = 8.9
Hz, 1H), 7.14 (dd, J = 8.9, 2.4 Hz, 1H), 5.19 (s, 2H), 4.97 (s, 2H),
3.43 (s, 3H), 3.21 (s, 3H); 13C NMR (68 MHz, CDCl3) δ 196.7,
172.6, 156.5, 154.3, 142.0, 133.3, 132.6, 131.1, 130.8, 130.7, 129.5,
129.5, 129.1, 125.2, 122.0, 117.3, 113.3, 107.4, 94.6, 94.3, 56.2, 56.1;
MS (EI+) m/z (rel. int.) 396 (M+, 8), 334 (12), 284 (67), 256 (55),
129 (64), 73 (100). HRMS (EI+) Calcd for C22H20O7 (M+): 396.1209.
Found: 396.1198.
Compound 15: Compound 15 was synthesized in a similar manner
to that for 21 with exception of 2 equiv of 14; 73% yield; white foam;
IR (neat) 2956, 1765, 1633, 1504, 1466, 1431, 1377, 1223, 1146 cm−1;
1H NMR (270 MHz, CDCl3) δ 7.97 (dd, J = 7.5, 1.1 Hz, 1H), 7.82
(dd, J = 7.5, 1.1 Hz, 1H), 7.68 (s, 2H), 7.64 (ddd, J = 7.5, 7.5, 1.1 Hz,
1H), 7.57 (d, J = 8.9 Hz, 2H), 7.54 (ddd, J = 7.5, 7.5, 1.1 Hz, 1H), 7.33
(s, 2H), 7.27 (d, J = 2.4 Hz, 2H), 7.05 (dd, J = 8.9, 2.4 Hz, 2H), 5.26
(s, 4H), 4.95 (ABq, νAB = 3.5 Hz, JAB = 7.0 Hz, 4H), 3.50 (s, 6H),
3.02 (s, 6H); 13C NMR (68 MHz, CDCl3) δ 170.4, 155.8, 153.4,
152.4, 135.4, 133.5, 129.5, 128.9, 127.7, 127.4, 126.6, 125.5, 124.6,
124.3, 117.2, 109.5, 108.6, 94.4, 94.1, 90.6, 56.1, 55.9; MS (EI+) m/z
(rel. int.) 626 (M+, 9), 595 (3), 581 (85), 565 (100), 537 (11), 521
(12). HRMS (EI+) Calcd for C36H34O10 (M+): 626.2152. Found:
626.2181.
General Procedure for the Deprotection of MOM group. The
synthesis of 16 is typical. A solution of 4 M hydrogen chloride in 1,4-
dioxane (3.7 mL) was added dropwise to a solution of 15 (467.5 mg,
0.76 mmol) in 1,4-dioxane (5 mL) and stirred for 3 h at room
temperature. The reaction mixture was poured into a mixture of ethyl
acetate and 0.1 M aq. HCl. The organic layer was separated, washed
successively with water and brine, dried over Na2SO4 and evaporated
in vacuo to give a residue. The residue was purified by column
chromatography (SiO2; n-hexane/EtOAc = 1/2) to furnish 16 (246.3
mg, 73%) as a green foam.
Compound 16: 73% yield; green foam; IR (KBr) 3375, 1739, 1635,
1448, 1381, 1348, 1292, 1219 cm−1; 1H NMR (270 MHz, CD3OD) δ
7.93 (d, J = 7.6 Hz, 1H), 7.87 (d, J = 7.6 Hz, 1H), 7.69 (dd, J = 7.6, 7.6
Hz, 1H), 7.57−7.53 (m, 1H), 7.55 (s, 2H), 7.45 (d, J = 8.9 Hz, 2H),
6.91 (s, 2H), 6.87 (d, J = 2.4 Hz, 2H), 6.80 (dd, J = 8.9, 2.4 Hz, 2H);
13C NMR (68 MHz, CD3OD) δ 173.0, 157.1, 155.2, 154.3, 137.9,
134.6, 130.8, 129.8, 129.1, 127.7, 126.1, 125.7, 123.9, 116.5, 110.0,
107.6, 93.3 (one peak overlapped); MS (EI+) m/z (rel. int.) 450 (M+,
5), 432 (14), 387 (73), 371 (25), 358 (25), 329 (24), 300 (81), 104
(100). HRMS (EI+) Calcd for C28H18O6 (M+): 450.1103. Found:
450.1121.
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Compound 22: 96% yield from 21; brown oil; IR (neat) 3240,
Hz, 1H); 13C NMR (68 MHz, DMSO-d6) δ 169.3, 157.4, 156.6, 154.5,
150.3, 144.4, 135.9, 135.4, 132.8, 132.6, 131.1, 130.1, 126.3, 125.6,
125.4, 123.6, 123.5, 123.1, 122.5, 119.2, 116.8, 116.5, 114.7, 110.5,
109.4, 106.7, 106.4, 83.5; MS (EI+) m/z (rel. int.) 432 (M+, 13), 387
(63), 371 (100), 284 (90), 256 (53), 129 (40); HRMS (EI+) Calcd for
C28H16O5 (M+): 432.0998. Found: 432.1014.
Compound 3: 48% yield from 25 and 2,7-dihydroxynaphthalene
(11); pale-purple powder; mp 196−199 °C; IR (KBr) 3410, 1736,
1624, 1520, 1444, 1354, 1286 cm−1; 1H NMR (270 MHz, CD3OD) δ
8.21 (d, J = 7.6 Hz, 1H), 7.89 (d, J = 8.6 Hz, 1H), 7.73−7.61 (m, 2H),
7.69 (d, J = 8.8 Hz, 1H), 7.52 (s, 1H), 7.51 (d, J = 8.9 Hz, 1H), 7.29
(d, J = 8.9 Hz, 1H), 7.16 (s, 1H), 7.08−7.05 (m, 2H), 6.93 (dd, J = 8.8,
2.4 Hz, 1H), 6.88 (dd, J = 8.6, 2.2 Hz, 1H), 6.44 (d, J = 2.2 Hz, 1H);
13C NMR (100 MHz, CD3OD) δ 173.2, 159.1, 158.7, 157.9, 153.7,
149.6, 138.2, 137.8, 135.3, 134.9, 132.9, 131.8, 129.4, 128.5, 128.1,
127.7, 127.6, 125.1, 120.7, 120.3, 117.9, 116.8, 112.1, 108.9, 108.6, 86.1
(some peaks overlapped); MS (EI+) m/z (rel. int.) 432 (M+, 2), 387
(5), 371 (10), 284 (23), 129 (56), 73 (100). HRMS (EI+) Calcd for
C28H16O5 (M+): 432.0998. Found: 432.1001.
Compound 6: 47% yield from 25 and 1,6-dihydroxynaphthalene
(26); purple powder; mp 182−183 °C; IR (KBr) 3336, 1736, 1637,
1288, 1252 cm−1; 1H NMR (270 MHz, CD3OD) δ 8.50 (d, J = 8.9 Hz,
1H), 8.11−8.08 (m, 1H), 7.77−7.73 (m, 2H), 7.76 (s, 1H), 7.57 (d, J
= 8.9 Hz, 1H), 7.32 (d, J = 8.9 Hz, 1H), 7.32 (s, 1H), 7.25 (dd, J = 8.9,
2.4 Hz, 1H), 7.23−7.21 (m, 1H), 7.15 (d, J = 2.4 Hz, 1H), 7.12 (d, J =
2.4 Hz, 1H), 6.97 (dd, J = 8.9, 2.4 Hz, 1H), 6.67 (d, J = 8.9 Hz, 1H);
13C NMR (68 MHz, CD3OD) δ 171.5, 158.4, 158.0, 154.9, 150.0,
150.0, 148.7, 137.8, 137.5, 136.6, 131.0, 130.7, 129.0, 127.4, 126.6,
125.8, 125.0, 124.8, 124.7, 122.8, 119.6, 119.2, 118.4, 111.8, 110.6,
110.3, 108.3, 85.4; MS (EI+) m/z (rel. int.) 432 (M+, 17), 387 (70),
284 (100), 256 (60), 149 (28), 97 (35). HRMS (EI+) Calcd for
C28H16O5 (M+): 432.0998. Found: 432.1009.
1
1705, 1628, 1591, 1238 cm−1; H NMR (270 MHz, CD3OD) δ 8.26
(d, J = 8.9 Hz, 1H), 8.07 (dd, J = 7.6, 1.4 Hz, 1H), 7.68 (ddd, J = 7.6,
7.6, 1.4 Hz, 1H), 7.57 (ddd, J = 7.6, 7.6, 1.4 Hz, 1H), 7.36 (dd, J = 7.6,
1.4 Hz, 1H), 7.04 (dd, J = 8.9, 2.4 Hz, 1H), 6.95 (d, J = 2.4 Hz, 1H),
6.90 (d, J = 8.9 Hz, 1H), 6.84 (d, J = 8.9 Hz, 1H); 13C NMR (68 MHz,
CD3OD) δ 203.8, 168.5, 163.7, 160.7, 141.8, 141.1, 133.4, 131.3,
130.5, 130.3, 128.3, 128.0, 127.1, 119.8, 118.6, 117.9, 113.1, 110.4; MS
(EI+) m/z (rel. int.) 308 (M+, 59), 290 (91), 284 (100), 262 (82), 256
(51), 129 (40). HRMS (EI+) Calcd for C18H12O5 (M+): 308.0685.
Found: 308.0694.
Compound 25: 67% yield from 24; yellow powder; mp 221−222
1
°C; IR (KBr) 3354, 1697, 1641, 1597, 1448, 1329, 1211 cm−1; H
NMR (270 MHz, CD3OD) δ 8.14 (dd, J = 7.3, 1.4 Hz, 1H), 7.74
(ddd, J = 7.3, 7.3, 1.4 Hz, 1H), 7.67 (ddd, J = 7.3, 7.3, 1.4 Hz, 1H),
7.57 (s, 1H), 7.47 (d, J = 8.9 Hz, 1H), 7.46 (dd, J = 7.3, 1.4 Hz, 1H),
7.03 (s, 1H), 6.91 (d, J = 2.4 Hz, 1H) 6.83 (dd, J = 8.9, 2.4 Hz, 1H);
13C NMR (68 MHz, CD3OD) δ 204.3, 168.4, 160.2, 158.6, 141.8,
141.5, 136.6, 133.4, 132.5, 131.4, 130.8, 130.7, 128.6, 123.2, 120.7,
118.0, 110.5, 107.8; MS (EI+) m/z (rel. int.) 308 (M+, 33), 290 (55),
284 (100), 262 (58), 256 (51), 241 (30), 129 (65), 73 (100). HRMS
(EI+) Calcd for C18H12O5 (M+): 308.0685. Found: 308.0679.
Compound 29: 90% yield from 28; pale-yellow powder; mp 203−
205 °C; IR (KBr) 3398, 1714, 1624, 1356, 1282, 1227, 1122 cm−1; 1H
NMR (270 MHz, CD3OD) δ 8.08 (dd, J = 7.8, 1.4 Hz, 1H), 7.69
(ddd, J = 7.8, 7.8, 1.4 Hz, 1H), 7.61 (ddd, J = 7.8, 7.8, 1.4 Hz, 1H),
7.36 (dd, J = 7.8, 1.4 Hz, 1H), 6.92 (d, J = 8.9 Hz, 1H), 6.31 (d, J = 2.4
Hz, 1H), 6.21 (dd, J = 8.9, 2.4 Hz, 1H); 13C NMR (68 MHz, CD3OD)
δ 202.5, 168.5, 166.4, 166.4, 141.8, 135.9, 133.3, 131.3, 130.5, 130.4,
128.5, 114.8, 108.9, 103.5; MS (EI+) m/z (rel. int.) 258 (M+, 18), 256
(60), 213 (18), 129 (33), 73 (43). HRMS (EI+) Calcd for C14H10O5
(M+): 258.0528. Found: 258.0527.
Compound 33: 94% yield from 32; brown powder; mp 168−169
Compound 9: 82% yield from 25 and resorcinol; orange powder;
mp 185−187 °C; IR (KBr) 3354, 1732, 1624, 1444, 1240, 1165 cm−1;
1H NMR (270 MHz, DMSO-d6) δ 10.09 (s, 1H), 9.92 (s, 1H), 7.98
(d, J = 7.3 Hz, 1H), 7.74 (dd, J = 7.3, 7.3 Hz, 1H), 7.68 (dd, J = 7.3,
7.3 Hz, 1H), 7.59 (d, J = 8.9 Hz, 1H), 7.57 (s, 1H), 7.26 (d, J = 7.3 Hz,
1H), 7.25 (s, 1H), 7.05 (s, 1H), 6.87 (d, J = 8.9 Hz, 1H), 6.66 (s, 1H),
6.53 (d, J = 8.9 Hz, 1H), 6.48 (d, J = 8.9 Hz, 1H); 13C NMR (68 MHz,
DMSO-d6) δ 168.6, 159.6, 156.7, 152.4, 152.0, 148.6, 135.7, 135.6,
130.1, 129.9, 128.8, 127.8, 125.8, 124.8, 124.5, 123.9, 118.3, 117.3,
112.3, 110.2, 109.7, 107.1, 102.4, 82.8; MS (EI+) m/z (rel. int.) 382
(M+, 12), 337 (68), 284 (90), 256 (50), 207 (44), 129 (64), 73 (100);
HRMS (EI+) Calcd for C24H14O5 (M+): 382.0841. Found: 382.0837.
Compound 8: 30% yield from 29 and 2,7-dihydroxynaphthalene
(11); red powder; mp 178−179 °C; IR (KBr) 3255, 1732, 1628, 1522,
1
°C; IR (KBr) 3375, 1705, 1633, 1518, 1292, 1213 cm−1; H NMR
(270 MHz, CD3OD) δ 7.97 (dd, J = 7.4, 1.4 Hz, 1H), 7.85 (d, J = 8.6
Hz, 1H), 7.61 (d, J = 8.6 Hz, 1H), 7.58 (ddd, J = 7.4, 7.4, 1.4 Hz, 1H),
7.51 (ddd, J = 7.4, 7.4, 1.4 Hz, 1H), 7.17 (dd, J = 7.4, 1.4 Hz, 1H), 6.91
(d, J = 8.6 Hz, 1H), 6.87 (d, J = 2.4 Hz, 1H), 6.82 (dd, J = 8.6, 2.4 Hz,
1H); 13C NMR (68 MHz, CD3OD) δ 202.1, 170.3, 163.7, 158.1,
144.6, 137.7, 135.5, 132.8, 132.1, 131.4, 131.3, 131.0, 128.5, 124.5,
116.3, 116.1, 114.8, 109.3; MS (EI+) m/z (rel. int.) 308 (M+, 5), 284
(11), 160 (100), 104 (53). HRMS (EI+) Calcd for C18H12O5 (M+):
308.0685. Found: 308.0680.
General Procedure for the Benzene-ring Expanded Fluo-
rescein Derivatives. The synthesis of 5 is typical. A solution of 16
(127.5 mg, 0.28 mmol) in toluene (1.5 mL) and methanesulfonic acid
(0.8 mL) was stirred for 30 min at 65 °C. The reaction mixture was
poured into a mixture of ethyl acetate and water. The organic layer was
separated, washed successively with water (twice) and brine, dried over
Na2SO4 and evaporated in vacuo to give a residue. The residue was
purified by column chromatography (SiO2; toluene-EtOAc = 2:1) to
afford 5 (94.7 mg, 77%) as a pale-blue powder.
1
1446, 1240 cm−1; H NMR (270 MHz, CD3OD) δ 8.09 (dd, J = 5.7,
1.6 Hz, 1H), 7.81 (d, J = 8.9 Hz, 1H), 7.66−7.57 (m, 3H), 7.18 (d, J =
8.9 Hz, 1H), 7.00 (dd, J = 5.9, 1.6 Hz, 1H), 6.82 (dd, J = 8.6, 2.4 Hz,
1H), 6.63 (d, J = 1.4 Hz, 1H), 6.49 (dd, J = 9.2, 1.4 Hz, 1H), 6.45 (d, J
= 9.2 Hz, 1H), 6.32 (d, J = 2.4 Hz, 1H); 13C NMR (68 MHz,
CD3OD) δ 172.1, 160.4, 157.4, 156.3, 152.5, 151.6, 136.6, 134.2,
133.6, 131.8, 130.6, 129.3, 127.9, 127.1, 126.3, 124.3, 116.9, 115.7,
113.8, 112.1, 108.1, 107.7, 102.9, 86.4; MS (EI+) m/z (rel. int.) 382
(M+, 13), 337 (52), 321 (100), 284 (30), 256 (15). HRMS (EI+)
Calcd for C24H14O5 (M+): 382.0841. Found: 382.0845.
Preparation of the Benzene-ring Expanded Fluorescein
Derivatives 2, 3 and 5 through the Conventional Double
Friedel−Crafts Reaction. A solution of phthalic anhydride (150 mg,
1.01 mmol) and 2,7-dihydroxynaphthalene (322.4 mg, 2.03 mmol) in
methanesulfonic acid (2 mL) was stirred at 100 °C for 10 h. The
reaction mixture was poured into iced-water and a precipitate was
collected by filtration and dried under reduced pressure. A soluble
fraction from the thick paste was extracted with a mixed solvent of
chloroform and methanol (1:10) and evaporated in vacuo to give a
residue. The residue was purified by reverse phase column
chromatography (water/methanol = 1/3) to give a mixture of 3 and
5 and pure 2 (25.0 mg, 6% yield). The mixture of 3 and 5 was purified
by preparative TLC on silica gel (n-hexane/ethyl acetate = 2/1
Compound 5: 77% yield from 16; pale-blue powder; mp 291 °C
(decomp.); IR (KBr) 3423, 1736, 1637, 1506, 1441, 1346, 1290 cm−1;
1H NMR (270 MHz, CD3OD) δ 8.10 (dd, J = 6.5, 1.4 Hz, 1H), 7.85−
7.74 (m, 2H), 7.57 (s, 2H), 7.55 (d, J = 8.9 Hz, 2H), 7.37 (dd, J = 6.6,
1.4 Hz, 1H), 7.30 (s, 2H), 7.10 (d, J = 2.4 Hz, 2H), 6.95 (dd, J = 8.9,
2.4 Hz, 2H); 13C NMR (100 MHz, CD3OD) δ 172.3, 159.2, 155.0,
152.1, 138.8, 137.6, 132.3, 131.9, 129.4, 128.5, 127.3, 127.2, 126.1,
120.6, 120.0, 112.6, 109.3, 86.1; MS (EI+) m/z (rel. int.) 432 (M+, 10),
388 (52), 371 (15), 284 (23), 241 (23), 185 (27), 129 (55), 73 (100).
HRMS (EI+) Calcd for C28H16O5 (M+): 432.0998. Found: 432.0996.
Compound 4: 76% yield from 22 and 11; pale-pink powder; mp
1
>300 °C; IR (KBr) 3311, 1724, 1610, 1398, 1242 cm−1; H NMR
(270 MHz, DMSO-d6) δ 10.12 (s, 1H), 9.83 (s, 1H), 8.42 (d, J = 8.9
Hz, 1H), 8.16 (d, J = 7.3 Hz, 1H), 8.03 (d, J = 8.9 Hz, 1H), 7.79 (d, J
= 8.9 Hz, 1H), 7.72 (dd, J = 7.3, 7.3 Hz, 1H), 7.66 (dd, J = 7.3, 7.3 Hz,
1H), 7.53 (d, J = 8.9 Hz, 1H), 7.37 (d, J = 8.9 Hz, 1H), 7.26 (dd, J =
8.9, 2.2 Hz, 1H), 7.19 (d, J = 7.3 Hz, 1H), 7.11 (d, J = 2.2 Hz, 1H),
6.93 (dd, J = 8.9, 2.2 Hz, 1H), 6.51 (d, J = 8.9 Hz, 1H), 6.30 (d, J = 2.2
3498
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Article
developing about 10 times) to afford 3 (16.3 mg, 4% yield) and 5 (6.3
mg, 1% yield)
treatment was assessed by trypan blue staining. More than 90% cells
were still alive even after treatment with 100 μM dye for 60 min.
Compound 2. 6% yield; pale-pink powder; mp >300 °C; IR (KBr)
3421, 1720, 1637, 1616, 1442, 1406, 1238 cm−1; 1H NMR (270 MHz,
CD3OD) δ 8.22 (d, J = 7.3 Hz, 1H), 7.83 (d, J = 8.6 Hz, 2H), 7.66 (d,
J = 8.6 Hz, 2H), 7.63−7.55 (m, 2H), 7.26 (d, J = 8.6 Hz, 2H), 7.14 (d,
J = 7.6 Hz, 1H), 6.89 (dd, J = 8.6, 2.0 Hz, 2H), 6.76 (d, J = 2.0 Hz,
2H); 13C NMR (100 MHz, CD3OD) δ 174.4, 158.6, 156.7, 151.5,
137.6, 134.7, 133.2, 132.0, 131.4, 128.7, 127.0, 125.8, 117.5, 116.4,
110.4, 109.2, 89.0 (one peak overlapped); MS (EI+) m/z (rel. int.) 432
(M+, 1), 416 (11), 387 (13), 371 (18), 284 (23), 241 (23), 213 (21),
185 (27), 129 (55), 97 (26), 73 (100). HRMS (EI+) Calcd for
C28H16O5 (M+): 432.0998. Found: 432.1019. Anal. Calcd for
C28H16O5: C, 77.77; H, 3.73. Found: C, 77.57; H, 3.45.
ASSOCIATED CONTENT
■
S
* Supporting Information
Coloration of seminaphthofluoresceines 8−10 under various
pH conditions, calculated UV−vis spectrum, HOMO and
LUMO of the dianion and cation of compound 5, H and 13C
1
NMR spectra for all new compounds, and CIF of compound 5
(CCDC 862009). This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Compound 19: A solution of 1 M BBr3 in dichloromethane (4.6
mL) was added dropwise to a solution of 18 (1.05 g, 4.15 mmol) in
dichloromethane (7 mL) under ice-bath cooling, and stirred for 16 h at
room temperature. The reaction mixture was poured into a mixture of
chloroform and water. The organic layer was separated, washed with
brine, dried over Na2SO4 and evaporated in vacuo to give a residue.
The residue was purified by column chromatography (SiO2; n-hexane/
EtOAc = 5/1) to give 19 (0.87 g, 88%) as a white powder; mp 89 °C
Corresponding Author
*tsubaki@kpu.ac.jp
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
1
(decomp); IR (KBr) 3452, 1624, 1587, 1385, 1171 cm−1; H NMR
This study was partly supported by KAKENHI (No.
22390003), Kyoto Prefectural University Research Fund, A-
STEP from JST (No. 231Z03077), and a Grant-in-Aid for JSPS
Fellows to E.A.
(270 MHz, CD3OD) δ 8.03 (d, J = 8.9 Hz, 1H), 7.30 (d, J = 8.9 Hz,
1H), 7.03 (d, J = 8.9 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.98 (s, 1H);
13C NMR (68 MHz, CD3OD) δ 156.7, 150.4, 136.6, 130.6, 124.8,
121.7, 120.3, 118.6, 109.8, 101.8; MS (EI+) m/z (rel. int.) 240 (M+,
21), 238 (M+, 21), 153 (69), 136 (52), 107 (52), 89 (68), 77 (100),
58 (25). HRMS (EI+) Calcd for C10H7O279Br (M+): 237.9629. Found:
237.9622.
REFERENCES
■
(1) For recent reviews, see: (a) Kikuchi, K. Chem. Soc. Rev. 2010, 39,
2048−2053. (b) Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. L.;
Urano, Y. Chem. Rev. 2010, 110, 2620−2640. (c) Loudet, A.; Burgess,
K. Chem. Rev. 2007, 107, 4891−4932. (d) de Silva, A. P.; Gunaratne,
H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.;
Rademacher, J. T.; Rice, T. E. Chem. Rev. 1997, 97, 1515−1566.
(2) (a) Duan, Y.; Liu, M.; Sun, W.; Wang, M.; Liu, S.; Li, Q. X. Mini-
Rev. Org. Chem. 2009, 6, 35−43. (b) Baeyer, A. Ber. Dtsch. Chem. Ges.
1871, 4, 555−558. (c) Urano, Y.; Kamiya, M.; Kanda, K.; Ueno, T.;
Hirose, K.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 4888−4894.
(3) Mishra, A.; Behera, R. K.; Behera, P. K.; Mishra, B. K.; Behera, G.
B. Chem. Rev. 2000, 100, 1973−2011.
(4) (a) Kojima, H.; Urano, Y.; Kikuchi, K.; Higuchi, T.; Hirata, Y.;
Nagano, T. Angew. Chem., Int. Ed. 1999, 38, 3209−3212. (b) Izumi, S.;
Urano, Y.; Hanaoka, K.; Terai, T.; Nagano, T. J. Am. Chem. Soc. 2009,
131, 10189−10200.
(5) (a) Fujikawa, Y.; Urano, Y.; Komatsu, T.; Hanaoka, K.; Kojima,
H.; Terai, T.; Inoue, H.; Nagano, T. J. Am. Chem. Soc. 2008, 130,
14533−14543. (b) Mineno, T.; Ueno, T.; Urano, Y.; Kojima, H.;
Nagano, T. Org. Lett. 2006, 8, 5963−5966.
Compound 23: A solution of 4 (11.0 mg, 0.025 mmol) and acetic
anhydride (48.1 μL, 0.51 mmol) in acetic acid (29 μL) was stirred for
18 h at 100 °C. The reaction mixture was poured into a mixture of
chloroform and aqueous NaHCO3. The organic layer was separated,
washed with water (twice) and brine, dried over Na2SO4 and
evaporated in vacuo to give a powder. The powder was washed with a
mixed solvent of n-hexane/EtOAc = 10/1 to give 23 (12.0 mg, 92%)
as a white powder; mp 292−293 °C; IR (KBr) 1766, 1412, 1369, 1200
1
cm−1; H NMR (270 MHz, CDCl3) δ 8.59 (d, J = 8.9 Hz, 1H), 8.19
(dd, J = 7.3, 1.1 Hz, 1H), 7.96 (d, J = 8.9 Hz, 1H), 7.81 (d, J = 8.9 Hz,
1H), 7.65 (ddd, J = 7.3, 7.3, 1.1 Hz, 1H), 7.60 (d, J = 8.9 Hz, 1H), 7.59
(ddd, J = 7.3, 7.3, 1.1 Hz, 1H), 7.53 (d, J = 2.2 Hz, 1H), 7.41 (d, J =
8.9 Hz, 1H), 7.41 (dd, J = 8.9, 2.2 Hz, 1H), 7.10 (dd, J = 8.9, 2.2 Hz,
1H), 7.08 (dd, J = 7.3, 1.1 Hz, 1H), 6.89 (d, J = 2.2 Hz, 1H), 6.77 (d, J
= 8.9 Hz, 1H), 2.37 (s, 3H), 2.20 (s, 3H); 13C NMR (68 MHz,
CDCl3) δ 169.7, 169.1, 168.6, 154.7, 150.6, 150.0, 149.3, 144.7, 135.5,
134.5, 132.3, 131.8, 130.3, 129.8, 129.2, 126.5, 125.2, 123.8, 123.7,
123.6, 123.4, 121.8, 121.3, 119.5, 118.5, 117.9, 115.5, 113.3, 108.3,
83.4, 21.3, 21.2; MS (EI+) m/z (rel. int.) 516 (M+, 13), 429 (25), 413
(100), 387 (50), 371 (50), 300 (15), 97 (11), 57 (16). HRMS (EI+)
Calcd for C32H20O7 (M+): 516.1209. Found: 516.1205.
Treatment of Living Cells with Compound 23. NIH3T3 and
HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium
(DMEM) supplemented with penicillin (100 units/mL), streptomycin
(100 μg/mL), and 5 and 7.5% (v/v) fetal bovine serum, respectively,
in a humidified incubator containing 5% CO2 gas. The clear image of
cells was not obtained in a culture dish under our fluorescence
microscope. Therefore, the confluent cells were trypsinized, and then
the isolated 5 × 106 cells were treated with 1.0 mL of 1× PBS
containing 0.1% (v/v) glucose, 0.058% (v/v) L-glutamine and
compound 23 (final 10, 50, 100 μM) dissolved in DMSO (final less
than 1%) for 15, 30, and 60 min at room temperature. After the
treatment, the medium containing fluorescein derivative was removed
and the cells were suspended in 20 μL of 1× PBS containing 0.1% (v/
v) glucose and 0.058% (v/v) L-glutamine. Fluorescence images of the
treated cells were captured using an Axio Imager M1 (Carl Zeiss) with
a CCD camera, a Mercury Arc lamp with a G-365 nm excitation filter,
and a BP-445/50 nm emission filter, or a BP-545/25 nm excitation
filter, and a BP-625/53 nm emission filter. The cell viability after dye
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